ANGIOTENSIN II SIGNALING TO PHOSPHOLIPASE D IN A MODEL OF GENETIC HYPERTENSION. Bradley T. Andresen. BS, Hope College, 1998

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1 ANGIOTENSIN II SIGNALING TO PHOSPHOLIPASE D IN A MODEL OF GENETIC HYPERTENSION by Brdley T. Andresen BS, Hope College, 1998 Submitted to the Grdute Fculty of the School of Medicine, Deprtment of Phrmcology in prtil fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2002

2 We hve red this disserttion entitled Angiotensin II Signling to Phospholipse D in Model of Genetic Hypertension by Brdley T. Andresen, nd recommend tht it be ccepted towrds the prtil fulfillment of the requirement for the degree of Doctor of Philosophy. Mtthew F. Muldoon, M.D., M.P.H. Associte Professor Deprtment of Medicine Committee Member Gregg E. Homnics, Ph.D. Associte Professor Deprtment of Anesthesiology Committee Member Dniel L. Altschuler, Ph.D. Assistnt Professor Deprtment of Phrmcology Committee Member Peter A. Friedmn, Ph.D. Professor Deprtment of Phrmcology Committee Chir Guillermo G. Romero, Ph.D. Associte Professor Deprtment of Phrmcology Co-Advisor Edwin K. Jckson, Ph.D. Professor Deprtment of Phrmcology Co-Advisor ii

3 ANGIOTENSIN II SIGNALING TO PHOSPHOLIPASE D IN A MODEL OF GENETIC HYPERTENSION Brdley T. Andresen, Ph.D. University of Pittsburgh, 2002 In spontneously hypertensive rts (SHR) the hypersensitivity of the renl vsculture to ngiotensin II (Ang II), compred to Wistr-Kyoto rts (WKY), ppers to be the determining fctor in the development nd progression of hypertension. Recent evidence indictes tht the ERK cscde nd NAD(P)H oxidse genertion of superoxide re involved in smooth muscle contrction, nd Phospholipse D (PLD) genertion of phosphtidic cid is involved in ctivtion of ERK nd NAD(P)H. Importntly, Ang II-medited PLD ctivity is greter in ortic smooth muscle from SHR compred with WKY; however, this signling pthwy hs not been exmined in the kidney vsculture. The purpose of these studies were to define Ang II-medited signl trnsduction mechnism(s) involved in PLD regultion in WKY nd SHR preglomerulr smooth muscle cells (PGSMCs). The gols of this study were to determine: 1) whether Ang II-medited PLD ctivity is greter in SHR; 2) the Ang II signling pthwy(s) responsible for regulting PLD ctivity, nd whether they re ltered in SHR; nd 3) whether PLD-medited genertion of phosphtidic cid is involved in Ang II-induced ctivtion of the ERK cscde. The dt indictes tht the mechnisms leding to ctivtion of PLD re similr in WKY nd SHR nd PLD is required for Ang II ctivtion of ERK; however, Ang II more potently ctivtes PLD in SHR. Further nlysis indictes tht the AT 2 receptor inhibits AT 1 receptor/rhoa-dependent ctivtion of PLD through nitric oxide/cgmp-dependent iii

4 phosphoryltion of RhoA t serine 188, which promotes RhoGDI inhibition of RhoA. These experiments expose two key differences between WKY nd SHR PGSMCs: 1) SHR hve n incresed AT 1 /AT 2 receptor rtio; nd 2) SHR re less sensitive to nitric oxide nd cgmp. Therefore, the hypersensitivity of the SHR to Ang II my be due to n imblnce in Ang II receptors nd/or impired AT 2 receptor-medited signling within the kidney vsculture. iv

5 FOREWARD I must first cknowledge those whom hve supported me throughout my eductionl endevors. Foremost, my prents Dr. Normn A. Andresen nd Sndr L. Andresen for their loving support throughout my schooling. My undergrdute dvisors Dr. Christopher C. Brney nd Dr. Mri Burntowsk-Hledin whom encourged me to conduct well controlled reserch experiments tht nswer pertinent physiologicl questions t the whole niml nd moleculr level. My grdute dvisors Dr. Guillermo G. Romero nd Dr. Edwin K. Jckson whom directed my further scientific development, nd tught me how to investigte the cellulr mechnisms underlying bsic biologicl functions utilizing phrmcology. Lstly I would like to cknowledge the fculty of the deprtment of phrmcology t the University of Pittsburgh nd my friends nd coworkers whom supported me in innumerble wys. To ll thnks, nd I could not hve progressed this fr without your help, guidnce, nd cre. Mny people hve sked why I hve gone into reserch, nd especilly question my dediction to the cdemic life due to the lower finicl wrd compred to other occuptions. My nswer hs lwys been two fold. First, to ddress the science question, I hve lwys wnted to be n explorer nd discovering the mechnisms underlying how life functions is s exciting s trip to the moon in my mind s eye. Second, to ddress the cdemic choice, I m hedstrong individul thus I desire to be s independent s possible, which leds to the cdemic life. Unfortuntely, these two nswers hve lwys sounded hollow to my ers; thus I offer third reson from Emily Dickinson, nd other quotes tht my better explin my choices. v

6 If I cn stop one hert from breking, We find in life exctly wht we put into it. I shll not live in vin. - Emerson If I cn ese one life the ching, Or cool one pin, Or help one finting robin A thing of beuty is joy forever; Unto his nest gin, Its loveliness increses; it will never I shll not live in vin. Pss into nothingness. - Emily Dickinson - Kets Lives of gret men ll remind us, For out of the old fieldes, s men sithe we cn mke our lives sublime, Cometh l this new corne for yere to yere, And, deprting, leve behind us, And out of old bookes, in good fithe, Footprints on the snds of time. Cometh l this new science tht men lere. - Longfellow - Chucer Dost thou love life? Then wste not time; Employ thy time well, if thou menest to For time is the stuff tht life is mde of. Gin leisure; nd since thou rt not sure of - Benjmin Frnklin minute, throw not wy n hour. - Benjmin Frnklin I hope I shll possess firmness nd virtue enough to mintin in wht I consider the most envible of ll titles, tht of n Honest Mn. - George Wshington How poor re they tht hve not ptience - Shkespere Those whom shve quickly, nick themselves. - Brdley Andresen vi

7 PREFACE One chpter of this disserttion hs been published Chpter 3: Andresen,B.T.; Jckson,E.K.; Romero,G.G Angiotensin II Signling to Phospholipse D in Renl Microvsculr Smooth Muscle Cells in SHR. Hypertension 37: This chpter hs been modified from the originl mnuscript to provide greter detil nd consistent formt throughout the disserttion. vii

8 TABLE OF CONTENTS Pge Chpter 1: Introduction Generl Introduction Animl nd Orgn Physiology Cellulr Physiology AT1 Receptors AT2 Receptors Internliztion of AT1 nd AT2 Receptors Physiologicl Significnce Signl Trnsduction Pthwys Involved in Hypertension Rho/Rho Kinse MEK/ERK Cscde Superoxide Integrted Pthwys Phospholipse D PLD nd Rho/Rho Kinse PLD nd the MEK/ERK Cscde PLD nd Superoxide Sttement of Problem nd Hypothesis 20 Chpter 2: Experimentl Methods Mterils Used Cell Culture Trnsfection of PGSMCs Phospholipse D Assys I-Sr 1 -Ile 8 -Ang II Binding 25 viii

9 2.6 Quntifiction of mrna Cloning of RhoA from WKY nd SHR PGSMCs Genertion of S188A RhoA MAPK Assys Confocl Imgining Experiments Dt nd Sttisticl Anlysis 32 Chpter 3: Mechnism of Angiotensin II Stimultion of PLD Introduction Results Angiotensin II Stimultes PLD Through the AT 1 Receptor Identifiction of the PLD Activted by Ang II Role of PI3K nd PKC in Ang II-Medited PLD Activity Role of Smll G Proteins in Ang II-Medited PLD Activity Role of Rho Kinses in Ang II-Medited PLD Activity Discussion Conclusions 51 Chpter 4: Chrcteriztion of Ang II Receptors Present on WKY nd SHR PGSMCs nd their Effects on Ang II-Medited PLD Activity Introduction Results Pertussis Toxin cts similrly to PD-123, Determintion of Angiotensin II Receptor Expression Effect of the AT 2 R on Ang II-Medited PLD Activity Discussion Conclusions 64 Chpter 5: Mechnisms of AT 2 R Inhibition of AT 1 R-Medited Activtion of PLD Introduction Results 66 ix

10 5.2.1 Effect of Inhibition PLA 2 nd NOS Addition of NO Inhibits Ang II-Medited PLD Activity Soluble Gunylte Cyclse Genertion of cgmp Medited AT 2 R Inhibition of AT 1 R Induced PLD Activity The AT 2 R Inhibits Ang II-Medited PLD Activity Through Phosphoryltion of RhoA Discussion Conclusion 84 Chpter 6: Role of PLD in Ang II-Medited Activtion of MEK nd ERK Introduction Results Ang II Stimultes ERK1/ Ang II Stimultion of PLD2 nd Subsequent Genertion of PA is Required for Activtion of MEK nd ERK1/ Cellulr Locliztion of Activted MEK nd ERK1/2 fter Stimultion with Ang II Discussion Conclusion 97 Chpter 7: Discussion Ang II Regultion of PLD Role of PLD in Ang II-Medited ERK Activtion Potentil Clinicl Significnce Conclusions 103 Appendix A: Effect of PKG nd PKA Inhibitors on Ang II-Medited PLD Activity 106 Appendix B: WKY nd SHR RhoA cdna Sequence 110 Appendix C: Role of c-src in Ang II-Medited PLD nd ERK1/2 Activtion 111 Appendix D: Experimentl Procedures 115 D.1: PLD Assy 116 x

11 D.2: Rdiolbeled Binding Assy 117 D.3: Preprtion of Cells for Imging 118 Bibliogrphy 119 xi

12 LIST OF TABLES Pge Tble 1.1: Differences Between the Angiotensin Receptors 7 Tble 1.2: NAD(P)H Subunits in Vsculr Smooth Muscle Cells 15 Tble 2.1: List of Agonists nd Antgonists 22 Tble 2.2: Primers used for RT-PCR 29 Tble 2.3: Primers used for Sequencing nd Genertion of S188A RhoA 30 Tble 2.4: Antibodies nd Fluorophores used for Confocl Imging Experiments 32 Tble 4.1: AT 1 R/AT 2 R Rtios 56 Tble 5.1: Comprison of Ang II-Medited PLD Activity EC 50 Vlues in SHR, WKY, nd WKY expressing S188A RhoA PGSMCs 79 xii

13 LIST OF FIGURES Pge Figure 1.1: Angiotensin II Mobiliztion of Clcium in PGSMCs 4 Figure 1.2: Digrm of the RhoA GDP/GTP Cycle 8 Figure 3.1: Simplified Flow Digrm oh how GPCR cn Activte PLD 35 Figure 3.2: Ang II-Medited PLD Activity Concentrtion-Response Curve 36 Figure 3.3: Ang II-Medited PA Production 37 Figure 3.4: Ang II Activtes PLD by Acting on the AT 1 R 38 Figure 3.5: All PLD Isoforms re Present nd Ang II Activtes PLD2 40 Figure 3.6: Ang II-Medited PLD Activity is not Medited through PI3K 41 Figure 3.7: Ang II-Medited PLD Activity is not Medited through PKC 42 Figure 3.8: The PKC Inhibitors Block PMA-Induced PLD Activity 43 Figure 3.9: Differentil Effects of Inhibiting ARF GEFs 45 Figure 3.10: Effects of dn-g proteins on Ang II-Medited PLD Activity 46 Figure 3.11: Ang II-Medited PLD Activity is not Medited by Rho Kinse 48 Figure 3.12: Rho Kinse Inhibitors Block the Synergy between UK-14,304 nd Ang II-Medited PLD Activity in SHR 49 Figure 4.1: Effects of Pertussis Toxin nd PD-123,319 on Ang II-Medited PLD Activity 54 Figure 4.2: Semi-Quntittive RT-PCR of WKY nd SHR AT 1 Rs nd AT 2 Rs 55 Figure 4.3: Identifiction of AT 1 R nd AT 2 R Number on WKY nd SHR PGSMCs 57 Figure 4.4: Ang II-Medited PLD Activity Concentrtion-Response Curve 60 Figure 4.5: Effect of PD-123,319 on Ang II-Medited PLD Activtion 61 Figure 4.6: Effect of CGP-42112A on Ang II-Medited PLD Activtion 62 Figure 5.1: Effects of Inhibiting PLA 2 nd NOS on Ang II-Medited PLD Activity 67 Figure 5.2: NO Inhibits Ang II-Medited PLD Activity 69 xiii

14 Figure 5.3: NO nd CGP-42112A Similrly Inhibit Ang II-Medited PLD Activity in WKY PGSMCs 71 Figure 5.4: NO but not CGP A Reduces the Efficcy of Ang II-Medited PLD Activity in SHR PGSMCs 72 Figure 5.5: Differentil Effects of NO on Ang II-Medited PLD Activity in WKY nd SHR PGSMCs 73 Figure 5.6: Stimultion of sgc Inhibits Ang II-Medited PLD Activity 74 Figure 5.7: Effects of sgc/cgmp on Ang II-Medited PLD Activity 75 Figure 5.8: Expression of S188A RhoA Prevents NO-Medited Inhibition of 1µM Ang II-Medited PLD Activity 77 Figure 5.9: Expression of S188A RhoA Elimintes NO-Medited Inhibition of Ang II-Medited PLD Activity nd Shifts the Concentrtion- Response Curve Towrds the SHR PGSMCs Concentrtion- Response Curve 78 Figure 5.10: Expression of S188A RhoA Elimintes AT 2 R-Medited Inhibition of Ang II-Medited PLD Activity 80 Figure 5.11: Ang II Signl Trnsduction Involved in PLD Regultion 82 Figure 6.1: Ang II Activtion of ERK1/2 is Greter in SHR thn WKY PGSMCs 87 Figure 6.2: dnpld2 Reduces Ang II-Medited PLD Activity 88 Figure 6.3: dnpld2 Attenutes Ang II-Medited MEK nd ERK1/2 Phosphoryltion 90 Figure 6.4: PA Prtilly Recovers dnpld2-medited Inhibition of Ang II- Medited ERK1/2 Activtion 91 Figure 6.5: EGF Stimultes ERK1/2 but not PLD 92 Figure 6.6: Confocl Imges of WKY PGSMCs 93 Figure 6.6b: Confocl Imges of SHR PGSMCs 94 Figure 6.7: Phospho-ERK1/2 Loclizes to Actin Filments After Stimultion with Ang II 96 Figure A.1: Effect of PKG Inhibition on Ang II-Medited PLD Activity 108 Figure A.2: Effect of PKA Inhibition on Ang II-Medited PLD Activity 109 Figure C.1: Effect of c-src Inhibition on Ang II-Medited PLD Activity 113 Figure C.2: Inhibiting c-src Reduces Ang II-Medited ERK1/2 Phosphoryltion 114 xiv

15 ABBREVIATIONS ACE Ang II ARF AT 1 R AT 2 R BFA BP C 2+ camp cgmp DAG dn EGF EGFP EGFR GAP GDI GDP GEF GPCR GRK GTP GTP IP 3 MAPK ngiotensin converting enzyme ngiotensin II ADP ribosyltion fctor ngiotensin II receptor type I ngiotensin II receptor type II Brefeldin A blood pressure Clcium denosine 3',5'-cyclic monophosphte cyclic gunosine 3',5'-monophosphte dicylglycerol dominnt negtive/ctlyticlly inctive epiderml growth fctor enhnced green flourescent protein EGF receptors GTPse-ctivting protein gunine nucleotide dissocition inhibitor gunine nucleotide diphosphte GTP exchnge fctor G-protein coupled receptor G-protein coupled receptor kinse gunine nucleotide triphosphte gunine nucleotide triphosphte inositol 1,4,5-trisphosphte mitogen ctivted protein kinse xv

16 MMP NO NOS PA PC PGSMCs PH PI3Kγ PIP3 PKA PKC PKG PLA 2 PLC PLD PtdEtOH PX RAS RTK sgc SHR WKY mtrix metlloproteinse nitric oxide nitric oxide synthse phosphtidic cid phosphtidyl choline preglomerulr smooth muscle cells pleckstrin homology phosphoinositide 3-kinse gmm phosphtidylinositol 3,4,5-trisphosphte protein kinse A protein kinse C protein kinse G phospholipse A2 phospholipse C phospholipse D phosphtidylethnol Phox homology renin-ngiotensin system receptor tyrosine kinse soluble gunylte cyclse spontneously hypertensive rt Wistr-Kyoto rt xvi

17 CHAPTER 1 INTRODUCTION 1.1 GENERAL INTRODUCTION Hypertension is defined s systolic blood pressure (BP) of 140 mm Hg or greter, or distolic BP of 90 mm Hg or greter. This condition, though generlly symptomtic, ffects 25% of the dult US popultion. In ddition to the lrge number of hypertensive Americns, it is estimted tht the verge Americn hs 90 percent chnce of developing hypertension before deth (1). Importntly, hypertension is leding cuse of stroke, end-stge renl disese, congestive hert filure, coronry rtery disese, nd peripherl vsculr disese nd, ccording to Americn Hert Assocition sttistics, hypertension killed 42,565 nd contributed to 210,000 deths in the US in Consequently, hypertension nd hypertension-induced/relted illnesses re, nd will continue to be, lrge public helth concern. Development of interventions to prevent or tret hypertension requires n understnding of the pthophysiology of essentil hypertension, yet tht understnding remins elusive. Anticipting the need to discern the bsic biology of essentil hypertension, reserchers t the Kyoto School of Medicine, Okmoto, Jpn in the lte 1950s nnounced tht rodent model for humn essentil hypertension ws developed through selective breeding of rts for incresed blood pressure. These rts developed hypertension spontneously nd were thus nmed the spontneously hypertensive rt (SHR), which ws bred from the Wistr-Kyoto line (WKY). Since the estblishment of the SHR, over 12,772 ppers hve been published 1

18 concerning the SHR. Judging from the ttention the SHR hs received the SHR is the most studied model in hypertension reserch. However, s with humn essentil hypertension, the precise cuse of hypertension in the SHR is unknown. 1.2 ANIMAL AND ORGAN PHYSIOLOGY Exhustive studies hve demonstrted tht the SHR develops hypertension erly in life by mechnism tht is dependent on the renin-ngiotensin system (RAS). In vivo ngiotensinogen is cleved by renin to form the decpeptide ngiotensin I tht is then cleved by ngiotensin converting enzyme (ACE) to form ngiotensin II (Ang II) tht then cts on AT 1 (AT 1 R) nd AT 2 (AT 2 R) receptors. In the SHR, inhibition of ny component of the RAS reduces blood pressure (2-4) nd normlizes blood pressure of the SHR to tht of the WKY. In kidney trnsplnttion experiments, hypertensive rts receiving kidneys from normotensive rts become normotensive, wheres normotensive recipients of hypertensive kidneys become hypertensive irrespective of genetic bckground (5-7). These experiments hve been conducted in other models of genetic hypertension besides the SHR with similr results, thus indicting tht the kidney is the orgn responsible for hypertension (reviewed in (8)). Ang II is more potent renl vsoconstrictor in SHR thn WKY (9-11). Since the renl vsculture genertes the gretest contrctile response to Ang II (12) nd the kidney is responsible for hypertension in the SHR, it is not surprising tht the renl vsculture of the SHR is hypersensitive to Ang II compred to WKY (11). These dt suggest tht the defect leding to hypertension in the SHR is ltered Ang II-medited signl trnsduction leding to enhnced vsoconstriction in the renl vsculture. 2

19 1.3 CELLULAR PHYSIOLOGY As mentioned bove, there re two types of Ang II receptors: AT 1 R nd AT 2 R (13). Rodents hve two subtypes of AT 1 receptors: AT 1A nd AT 1B ; however, s of yet, no differences in the signl trnsduction of the AT 1A nd AT 1B receptors hve been described. Unlike rodents, humns do not hve multiple AT 1 R subtypes; therefore, for the reminder of this review we will consider the signling properties of the AT 1A nd AT 1B receptors s singulr AT 1 R AT 1 RECEPTORS: AT 1 Rs re heterotrimeric G-protein coupled receptors (GPCR) tht cn pprently couple to ll Gα subunits except Gα 13 (14). Although these coupling dt re bsed on chimeric Gα s constructs in COS cells trnsfected with the AT 1 R (15) nd thus only indicte wht G-proteins my bind to the AT 1 R, the diversity of signl trnsduction cscdes ctivted by AT 1 Rs (reviewed in (16)) strongly suggests tht AT 1 Rs my couple to mny Gα subunits in vivo; however, the precise coupling my be cell nd environment dependent. Specific signl trnsduction mechnisms of the AT 1 R tht my be responsible for hypertension re reviewed below. However, it ppers tht the min ction of AT 1 R is the ctivtion of phospholipse C (PLC) through Gα q/11 -medited pthwy (16). This pthwy results in the formtion of inositol trisphosphte (IP 3 ) tht then ctivtes C 2+ chnnels on the endoplsmic reticulum resulting in n increse in cytosolic C 2+. Simultneous genertion of dicylglycerol (DAG) by PLC lso leds to the ctivtion of protein kinse C (PKC). Although the IP 3 -C 2+ /PKC pthwy is known to led to vsoconstriction, Ang II-medited C 2+ trnsients re identicl in SHR nd WKY preglomerulr smooth muscle cells (Fig. 1.1) (17). Thus, this pthwy is unlikely to be the custive pthwy of hypertension. 3

20 Mximum C 2+ Response (Arbitrry Units) SHR WKY Log[Angiotensin II (M)] Figure 1.1: Angiotensin II Mobiliztion of Clcium in PGSMCs. Angiotensin II increses free [C 2+ ] i in WKY nd SHR PGSMCs. Cells were loded with 5µM of the rtiometric dye Fur-2 cetoxymethyl t 37 C for 30 min, nd the 345 nm/375 nm rtio of fluorescence ws quntified for 5 cells t ech concentrtion. Importntly, ANOVA nlysis indictes tht the curves re not significntly different from one nother. Dt obtined from Mokkptti R. et l. (17) with ssistnce from K.E. Dineley nd I.J. Reynolds. 4

21 1.3.2 AT 2 RECEPTORS: Like the AT 1 Rs, AT 2 Rs re lso heterotrimeric G-protein coupled receptors, but unlike AT 1 Rs, AT 2 Rs couple selectively to Gα i/o subunits s demonstrted by reconstitution nd immunoprecipittion experiments (18;19). The AT 2 R is considered fetl gene due to high expression of AT 2 Rs during embryogenesis nd significntly lower expression nd distribution in the dult; due to this there is much less informtion regrding AT 2 R signling compred to AT 1 R signling (reviewed in (20)). In generl the AT 1 R leds to vsoconstriction, wheres the AT 2 R countercts the AT 1 R through three min pthwys: 1) genertion of the vsodiltor nitric oxide (NO) through yet undetermined mechnisms (21;22); 2) ctivtion of phospholipse A2 (PLA 2 ), presumbly both mpla 2 nd cpla 2, through unexmined mechnisms nd subsequent formtion of rchidonic cid metbolites (23;24); nd 3) ctivtion of vriety of phosphtses (25;26) INTERNALIZATION OF AT 1 AND AT 2 RECEPTORS: The AT 1 R rpidly internlizes upon stimultion (t ½ = 5 min (27)) through β-rrestin 1- medited clthrin-dependent pthwy (28). On the other hnd, the AT 2 R does not internlize fter stimultion with Ang II (29;30). This difference ppers to be due to n ltered sequence in the C-terminl til of the AT 2 R. The AT 1 R hs conserved sequence from serine 326 to serine 338 cross vriety of species. However, the AT 2 R shres only 4 residues in common with this sequence nd muttion of the conserved AT 1 R residues brogtes Ang II-medited internliztion (31). Furthermore, deletion experiments confirm tht the conserved sequence in the AT 1 R is involved in internliztion, nd is not involved in signling to PLC, mesured by C 2+ trnsients, ERK, or gonist induced desensitiztion of the receptor (32). These differences 5

22 re importnt becuse Ang II ctivtion of AT 1 Rs is ttenuted by receptor internliztion nd recycling nd degrdtion of Ang II in the lysosome (30), wheres AT 2 Rs cn ct s constitutively ctive receptors, conclusion supported by observtions of the physiologicl effects of renl AT 2 Rs (22). Thus, the blnce between AT 1 Rs nd AT 2 Rs on ny given cell determines the signl trnsduction nd physiologicl outcome of Ang II stimultion PHYSIOLOGICAL SIGNIFICANCE: The min differences between the AT 1 R nd AT 2 R re within their coupling nd internliztion (Tble 1.1). Physiologiclly, these differences cn be best illustrted in Ang II receptor knockout mice. AT 2 R knockout mice hve incresed blood pressure compred to controls nd hve enhnced responses to exogenously dded Ang II (33;34). Conversely, AT 1A R/AT 1B R double knockout mice hve lower blood pressure compred to controls nd lck the typicl increse in blood pressure fter exogenously dded Ang II (35). Additionlly, the AT 2 R cts s vsodepressor gent in the hypertensive (mren-2)27 trnsgenic rt (36). Furthermore, similr experiments conducted in rts tht re not geneticlly modified, utilizing AT 1 R ntgonists nd AT 2 R gonists nd ntgonists indicte tht the AT 2 R indeed countercts the AT 1 R nd ctively lowers blood pressure (37-39). In conclusion, the AT 1 R medites the contrctile hypertensive properties of Ang II, wheres AT 2 Rs counterct the AT 1 R through vsodiltory mechnisms. 6

23 Tble 1.1: Differences Between the Angiotensin Receptors Description AT 1 R AT 2 R All Gα subunits Coupling except Gα 13 Gα i, nd Gα o Agonist-Medited Internliztion t ½ = 5 min Internliztion deficient Vsculr Expression Abundnt Low, not on ll vsculr beds Physiology Contrction Diltion 1.4 SIGNAL TRANSDUCTION PATHWAYS INVOLVED IN HYPERTENSION RHO/RHO KINASE: The Rho fmily is group of smll G-proteins tht re ctivted by vriety of stimuli (reviewed in (40)). The bsic cycle of Rho ctivity is shown in Figure 1.2. Smll G-proteins re inctive when bound to GDP nd re ctivted by GTP exchnge fctor proteins (GEFs) tht initite exchnge of bound GDP for GTP. This results in ctivted G-proteins, which bind to their respective downstrem trgets. Inctivtion requires the hydrolysis of bound GTP. Smll G-proteins hve slow intrinsic hydrolysis rte (41), nd therefore inctivtion of the smll G- protein is ctlyzed by GTPse-ctivting proteins (GAPs), which enhnces/increses the intrinsic GTPse ctivity of the G-protein. The third component in this cycle is the gunine nucleotide dissocition inhibitor (GDI). In the cse of RhoA, the GDI binds to serine 188 when this residue is phosphorylted by either protein kinse A (PKA) (42) or protein kinse G (PKG) (43;44). Binding of Rho GDI to either GDP- or GTP-bound RhoA inhibits nucleotide exchnge, hydrolysis of GTP, nd GTP-bound RhoA signling (45). Additionlly, the GDI msks the cysteine 190 prenyltion site (C20 gernylgernyl) (46;46) tht is crucil for proper interctions 7

24 When Phosphorylted GEF + GTP S Inctive Rho GDP bound GEF + GDP GEF/GAP + GTP Inctive Rho GDP bound PO 4 -S188 GDI P Pi + GAP Active Rho GTP bound GAP S Figure 1.2: Digrm of the RhoA GDP/GTP Cycle. RhoA cycles between the inctive GDP bound stte nd ctive GTP bound stte with the ssistnce of GEFs nd GAPs. Structurlly, RhoA contins consensus PKA/PKG phosphoryltion site t S188 represented by the S in the spiked circle, nd RhoA is gernylgernylted t C190 represented by the squiggly line originting ner the S188 site. When RhoA is phosphorylted the GDI binds to RhoA inhibiting intrinsic GTPse ctivity, nd GEF nd GAP function nd msking the gernylgernyltion moiety (box). 8

25 with RhoGAPs (47). Non-phosphorylted RhoA resides on membrnes due to interctions of the prenyltion group with the lipid membrne; however, GDI msking of the prenyltion group llows RhoA to move to the cytoplsm where it no longer intercts with effectors. Importntly, RhoA is more ctive in SHR thorcic ort nd thorcic ortic smooth muscle cells thn in WKY (48), suggesting tht RhoA my ply role in hypertension. Although RhoA is typiclly viewed s signl trnsduction protein involved primrily in cytoskeleton reorgniztion (reviewed in (49)), RhoA lso ctivtes vriety of kinses tht medite cell signling. One of these, p160rock, is ctivted by RhoA nd is involved in smooth muscle contrction (50-52). p160rock contributes to contrction by phosphorylting, nd thereby inctivting, the regultory subunit of myosin phosphtse (53;54). This process llows greter percentge of the myosin to remin phosphorylted, which is hypersensitive to C 2+, thereby resulting in incresed contrctile responses to C 2+ (55). Thus, p160rock, nd potentilly other members of the Rho kinse fmily, my ply role in hypertension. In support of this hypothesis, Y-27632, n inhibitor of Rho ssocited kinses, reduces blood pressure in mny hypertensive rt models (56); therefore, Rho kinse inhibitors my be novel group of nti-hypertensive drugs (57) MEK/ERK CASCADE: Although the clssicl mechnisms leding to ERK ctivtion through receptor tyrosine kinses (RTKs) re well estblished (reviewed in (58)), the mechnisms by which GPCRs ctivte the ERK mitogen-ctivted protein kinse (MAPK) cscde re still debted. In brief, tyrosine kinse receptors recruit Shc, Grb2, nd SOS, phosphotyrosine binding protein, dpter protein, nd Rs-GEF respectively. SOS engges Rs, smll G-protein, which in turn ctivtes 9

26 Rf-1. Rf-1 is kinse tht ctivtes, through phosphoryltion, the kinse MEK, which is the ctivtor, through phosphoryltion, of the kinses ERK1 nd ERK2. Phosphorylted ERK1/2 trnsloctes to the nucleus nd ctivtes, through phosphoryltion, vrious trnscription fctors (59-61). This pthwy lso hs mny supporting plyers tht llow for the cscde to loclize nd trffic to the correct res of the cell nd thus function properly. Numerous studies demonstrte tht Ang II stimultes the ERK MAPK pthwy (reviewed in (62)); moreover, in SHR smooth muscle cells Ang II more potently ctivtes ERK, compred to WKY, suggesting tht ERK is involved in hypertension (63). However, the mechnism for ctivtion of the ERK MAPK cscde initited by Ang II is unknown. There re three prominent theories regrding GPCR-medited ctivtion of MAPK: 1) RTK trnsctivtion/phospho-tyrosine scffolds; 2) β-rrestin scffolding/internliztion; nd 3) direct stimultion of Rf. The RTK trnsctivtion model is bsed on the ide tht n EGF-like lignd (prohb- EGF) is present within the mtrix surrounding cells (64). Upon ctivtion of AT 1 Rs, ccording to this model, the trget cell secretes mtrix metlloproteinse(s) (MMP) tht cleve prohb-egf relesing HB-EGF, which cts in n utocrine fshion ctivting EGF receptors (EGFR) (65). Evidence supporting this theory includes use of MMP inhibitors, the dependence of trnsctivtion on C 2+, which is required for the secretion of the MMP, nd the inhibition of Ang II-medited phosphoryltion with high concentrtions of EGFR kinse inhibitors (66). However, competing theories regrding trnsctivtion re proposed tht involve cytosolic mechnisms for phosphoryltion of EGFR. Evidence for n intrcellulr mechnism, or t lest n intrcellulr component, include the requirement of c-src nd rective oxygen species, bsed on experiments utilizing inhibitors, in Ang II medited phosphoryltion of the EGFR (67). 10

27 Furthermore, recent evidence indictes tht Ang II phosphoryltes cveolin-1 thereby relesing the EGFR; the relesed trnsctivted EGFR, phosphorylted t tyrosine 845, c-src consensus site (68), co-loclizes to focl dhesion complexes (69). An lterntive hypothesis for trnsctivtion is tht Ang II medites EGFR re-locliztion from cveolin-1 rich res to focl dhesion points where the focl dhesions phosphorylte the EGFR through c-src dependent mechnism. Evidence for this hypothesis is tht depletion of cholesterol with β-cyclodextrin inhibits Ang II-medited trnsctivtion of the EGFR tht is reversed by dding bck cholesterol (69). Recent evidence from our lbortory indictes tht removl of cholesterol from the membrne with β-cyclodextrin destroys membrne lipid rfts tht hve been proposed to be importnt microdomins for loclizing signl trnsduction proteins to the plsm membrne. When these rfts re disrupted, the rft-bound proteins re distributed throughout the membrne nd cese to signl properly. Thus, since EGFR is ssocited with cveolin-1 (70), nd β-cyclodextrin removes cveole structures while lso inhibiting EGFR trnsctivtion but not Ang II-medited C 2+ mobiliztion (69), logicl conclusion is tht Ang II-medited re-locliztion of the EGFR is the key mechnism by which Ang II trnsctivtes EGFR. The forementioned lterntive hypothesis implies tht focl dhesion complexes my ply role in Ang II-medited ctivtion of ERK. Specificlly, the focl dhesion kinses PYK2 nd FAK, which re ctivted through Rho/Rho kinse-dependent mechnism (71), re recruited to nd phosphorylte integrins (72), nd both focl dhesion kinses nd integrin ctivtion/enggement cn led to ctivtion of ERK MAPK (73;74). The mechnisms for integrin-medited ctivtion of the ERK MAPK cscde independent of focl dhesion kinses re not fully understood. However, FAK ctivtion of the ERK MAPK cscde is proposed to 11

28 be medited through GRB2/SOS. GRB2 s SH2 domin binds to phospho-tyrosine 925 of FAK, nd SOS is ssocited with FAK through its ssocition with GRB2 (75); therefore, the integrins nd FAK cn ct s phosphotyrosine scffolds for the recruitment nd ctivtion of some of the components of the ERK MAPK cscde. Furthermore, c-src is recruited to nd ctivted in focl dhesion complexes (74), thus leding to c-src mechnisms of ctivtion of the ERK MAPK cscde including the forementioned RTK trnsctivtion. GPCR desensitiztion nd internliztion re often medited by G-protein receptor kinses (GRKs) (reviewed in (76)). Recent dt indicte tht β-rrestin, which binds phosphorylted G-protein coupled receptors, cts s scffolding protein for MAPKs (77). Therefore, β-rrestin nd potentilly other β-rrestin like proteins my ct s the scffold required for Ang II ctivtion of the ERK MAPK cscde. Although AT 1 R internliztion is independent of GRK2-medited phosphoryltion, s demonstrted by GRK2 phosphoryltion site deficient mutnt (78), β-rrestin ssocition with the AT 1 R is correlted with AT 1 R internliztion (28), nd internliztion, in some systems, is requirement for ctivtion of the ERK MAPK cscde (79;80). Correspondingly, the forementioned mutnt AT 1 R (78), tht cn internlize, still ctivtes ERK1/2, but the role of internliztion in Ang II-medited ERK1/2 phosphoryltion ws not exmined. Additionlly, PKC ws demonstrted to phosphorylte the mutnt receptor, which in some systems cn medite internliztion (81). Therefore, the role of internliztion of Ang II receptors in the ctivtion of the ERK MAPK cscde is still not fully known. Direct ctivtion of the ERK MAPK cscde my occur through two pthwys: PKC or c-src (82). Ang II, s previously mentioned, cn ctivte PKC nd PKC cn directly ctivte Rf-1 through phosphoryltion of multiple serine residues (83;84). However, for some PKC 12

29 isotypes, PKC-medited ctivtion of ERK does not involve phosphoryltion of Rf-1, but proceeds through other unknown pthwys (85). Interestingly, the mjority of theories regrding AT 1 R-medited ctivtion of the ERK MAPK cscde propose the involvement of c-src. Furthermore, c-src is involved in AT 1 R-medited ctivtion of ERK1/2 in SHR smooth muscle cells (86) nd plys role in the elevted levels of phospho-erk1/2 in hypertensive ptients (87). c-src my potentilly medite GPCR-induced ctivtion of ERK MAPK by: 1) recruitment to focl dhesion complexes (74), where: ) c-src my be involved in RTK trnsctivtion (66), nd b) c-src cn further ctivte other kinses ssocited with focl dhesions tht then led to phosphoryltion of Rf-1 (88); 2) intercting with β-rrestin (89); nd 3) direct phosphoryltion of Rf-1 t tyrosine 341 (82). Additionlly, recent dt suggest tht the AT 1 R directly couples to c-src in the C-terminl til, independent of Gα-subunits nd trditionl AT 1 R signling (90), which suggests tht Ang II cn ctivte the ERK MAPK cscde through direct ctivtion of c- Src. Regrdless of how the ERK1/2 MAPK cscde is ctivted, it plys key role in smooth muscle contrction s demonstrted by experiments with the specific MEK inhibitor PD98059 (91-93). In primry smooth muscle cells, Ang II induces greter contrctile responses in SHR compred to WKY nd inhibition of MEK reduces the contrction in both SHR nd WKY cells. Importntly, PD98059 normlizes the contrctile responses of SHR cells to Ang II to those of the WKY (91). However, there is evidence tht MEK does not ply role in Ang II medited vsculr contrction of isolted smooth muscle from the thorcic ort (94), indicting tht the role of MEK in contrction my not be physiologiclly relevnt. To ddress this disprity, PD98059 ws used in vivo, where it reduced Ang II-medited increses in men rteril blood pressure in normotensive rts (92) nd lowered blood pressure in deoxycorticosterone cette 13

30 (DOCA)-slt-induced hypertensive rts (93). Therefore, PD98059 reduces smooth muscle contrctions in physiologiclly relevnt systems, but the mechnism by which MEK medites smooth muscle contrction is unknown SUPEROXIDE: Recent dt indicte tht hormones, including Ang II, cting through GPCRs, induce superoxide genertion in smooth muscle cells (95;96), nd superoxide genertion is linked to hypertension (reviewed in (97)). Formtion of superoxide clssiclly occurs through NAD(P)H oxidse in leukocytes in order to destroy phgocytosed pthogens (reviewed in (98)). Therefore, NAD(P)H oxidse or homologue of NAD(P)H oxidse should be present in vsculr smooth muscle cells. Recently, fmily of proteins similr to some of the clssicl NAD(P)H oxidse subunits, clled Nox, were found in the kidney nd smooth muscle cells long with n ssortment of the trditionl NAD(P)H oxidse subunits (Tble 1.2) (99-101). The p47 phox subunit, which is required to ctivte the oxidse subunits, is present in both leukocyte nd smooth muscle cells (96) suggesting tht the signl trnsduction pthwys leding to ctivtion of the ctlytic oxidse subunit gp91 phox /Nox1 re similr in the two systems. The leukocyte p67 phox nd p47 phox NAD(P)H oxidse subunits re wekly ctivted by DAG, modertely ctivted by phosphtidic cid (PA), strongly ctivted by rchidonic cid, nd mximlly ctivted by the synergistic ctions of PA nd DAG (102). Although the p67 phox subunit hs yet to be identified nd is most likely not present in smooth muscle, Ang II ctivtes ll the phospholipses, A, C, nd D. Thus, Ang II hs the potentil to ctivte fully NAD(P)H oxidse, indeed, Ang II cting through the AT 1 R genertes superoxide primrily by NADH-medited mechnism (95;103). 14

31 SHR hve higher levels of superoxide compred to the WKY (104). However, these observtions could be consequence not cuse of hypertension; to ddress this, normotensive rts were mde similrly hypertensive with Ang II nd norepinephrine. Only in Ang II-medited hypertension did superoxide dismutse mimetics reduce blood pressure. Correspondingly, elevted superoxide levels were observed only in the Ang II-medited hypertensive rts (105). Therefore, Ang II-medited production of superoxide is prtilly responsible for incresed blood pressure in rts chroniclly infused with Ang II. Moreover, the AT 1 R medites superoxide production (103), AT 1 R medites hypertension in the SHR (2), nd superoxide dismutse lowers blood pressure in SHR (106). These fcts strongly indicte tht hypertension is prtilly dependent on superoxide. Tble 1.2: NAD(P)H Subunits in Vsculr Smooth Muscle Cells Subunit mrna references Protein references p22 phox (107;108) (108) p47 phox (96) (96) p67 phox Not present in smooth muscle nox-1 (99;100) Not exmined Cytochrome b 558 α-subunit (107) Not exmined Rc * Not exmined (96;109) * Rc is prt of the functionl NAD(P)H complex. However, the precise role of Rc in the complex remins to be determined. 15

32 1.5 INTEGRATED PATHWAYS Ang II, cting through the AT 1 R, is mjor contributing fctor to hypertension in the SHR nd Ang II ctivtes ll the three signling mechnisms discussed thus fr. Therefore, n importnt question tht remins to be nswered is: Cn ll the forementioned vsoctive signling pthwys be tied together into one common pthwy? Here we would like to propose tht there is common signl trnsduction pthwy involving Ang II stimultion of phospholipse D, thereby generting phosphtidic cid (PA) PHOSPHOLIPASE D Ang II-medited phospholipse D (PLD) ctivity, through the AT 1 R (110;111), is incresed in smooth muscle from SHR compred to WKY (112;113), thus suggesting tht PLD my ply role in the hypertensive phenotype of the SHR. There re two subtypes of PLD, PLD1 (114) nd PLD2 (115). PLD1, which hs two splice vrints PLD1 nd PLD1b (116), is predomintely expressed on the Golgi nd endoplsmic reticulum (115;117). Recent evidence suggests tht PLD1 cn lso be found on the plsm membrne (118;119). On the other hnd, PLD2 is expressed predomintely on the plsm membrne (115), nd recent studies utilizing ctlyticlly inctive PLD2 indicte tht, in most cses, PLD2 medites hormone-induced PLD ctivtion. Thus, PLD2 is n enzyme involved in signl trnsduction (120;121). Agonists induce PLD ctivity primrily through two generl pthwys, PKC nd smll G-proteins of the ADP ribosyltion fctor (ARF) nd Rho fmilies. PKC ctivtes PLD in vriety of systems (122;123) nd cn synergize with other fctors (124;125). Recent evidence indictes tht PKC cn phosphorylte nd ctivte PLD (126). However, PKC cn lso ctivte PLD through kinse-independent mechnisms (127). Furthermore, PKC-dependent ctivtion of 16

33 PLD is prtly medited by smll G-proteins (128;129) presumbly through ctivtion of Rho (128) nd ARF (130) GEFs. Therefore, PKC cn directly nd indirectly ctivte PLD through vriety of mechnisms. The requirement for G-proteins is supported by originl observtions indicting tht cytosolic fctors re required for PKC-medited PLD ctivity in system where PKC lone did not ctivte PLD (131). ARF proteins were the first smll G-proteins found to ctivte PLD (132;133), nd subsequent studies show tht ll ARF fmily clsses cn ctivte PLD in vriety of systems (120;134;135). Additionlly, ARF is the strongest single gent for ctivting PLD in vitro (115), suggesting tht ARF my be the primry regultor of PLD. The second type of G-protein found to ctivte PLD is Rho (136;137). Additionlly, other members of the Rho fmily nd similr smll G-proteins ctivte PLD (138;139). As with ARF, mny systems signl through Rho to ctivte PLD. However, Rho is poor ctivtor of PLD in vitro suggesting tht mechnisms other thn the direct interction of Rho with PLD re involved (134). The function of PLD is to remove the choline hed group from phosphtidylcholine (PC) by trnsphosphtidyltion rection resulting in the formtion of PA nd free choline. This rection requires both of PLD s HKD domins (140) nd phosphtidylinositol 4,5-bisphosphte (PIP 2 ) (141). The HKD domins re criticl for coordinting nd ctlyzing the rection (142). PIP 2 binding, not through the consensus PH domin locted ner the mino-terminus PX domin (117;143), but through second PIP 2 binding domin between the two HKD domins (144) is essentil for ctlysis. The physiologicl role of PLD is genertion of PA. PLD1-medited genertion of PA is predomintely involved in the trfficking of vesicles to nd from the vrious Golgi comprtments nd ER ( ), presumbly through genertion of negtive curvture in the membrne inititing the fission event (148). PLD2-medited genertion of PA is lso 17

34 involved with vesicle formtion/endocytosis (149), gin presumbly by generting negtive curvture t the membrne nd by recruiting dynmin through dynmin s PH domin to the budding vesicle (150). Since PLD2 is ctivted by vriety of gonists (120;121), it most likely plys vriety of roles in signl trnsduction. However, due to the high bsl ctivity of PLD2 (134) nd lck of specific inhibitors, little is known bout the physiologicl functions of PLD PLD AND RHO/RHO KINASE RhoA hs been implicted in Ang II-medited ctivtion of PLD (151). In thorcic smooth muscle, thrombin-medited RhoA ctivity is incresed in SHR compred to WKY (48). This suggests tht the incresed ctivtion of RhoA my be responsible for the incresed Ang IImedited PLD ctivity observed in SHR compred to WKY (112). As mentioned, PLD2, which cnnot be directly ctivted by Rho (143), is the min Ang II-ctivted PLD (120). Therefore, other intermediry protein(s) re probbly involved in conveying the signl from RhoA to PLD2. It hs been reported tht muscrinic cetylcholine M3 receptor-medited ctivtion of PLD occurs through Rho kinse dependent mechnism bsed on the use of Y (152). However, the effect of Rho kinse inhibitors on Ang II-medited ctivtion of PLD2 hs not been studied PLD AND THE MEK/ERK CASCADE The work of our lbortory nd others hs shown tht the coupling of the Rs/Rf-1/ERK cscde is criticlly dependent on the locliztion of the components of the cscde in specific loci (121;153;154). Rf-1 ctivtion requires its trnsloction to the membrne, nd Rf-1 membrne interctions re medited in prt by the binding of Rf-1 to specific lipids including 18

35 PA (155). PLD is the mjor source of PA in gonist-stimulted cells. In fct, over-expression of ctlyticlly inctive form of PLD2 inhibits gonist-dependent PLD ctivity nd simultneously impirs ERK1/2 phosphoryltion tht cn be recovered by the ddition of PA (120;121). Ang II-dependent ERK1/2 phosphoryltion is lso inhibited by expression of ctlyticlly inctive PLD2 in A10 cells (120). Therefore, PLD nd PA ply n importnt role in Ang II-dependent ERK1/2 phosphoryltion PLD AND SUPEROXIDE PLD1 is responsible for generting the oxidtive burst exhibited by leukocytes through the forementioned PA-medited ctivtion of NAD(P)H oxidse (102), indicting tht PLD my ply role in genertion of superoxide in smooth muscle. Although PA lone is moderte ctivtor of NAD(P)H oxidse, Ang II stimultes PLC nd PLD resulting in the ccumultion of DAG nd PA tht cn, through synergism, mximlly ctivte NAD(P)H oxidse (102). The role of PLD in this pthwy hs been demonstrted by inhibiting PLD with D-erythrosphingosine, dihydro- (DESD) in vsculr smooth muscle cells (156). Unfortuntely, PLD inhibitors re nonspecific nd generlly function s PLX inhibitors, where X is D, C, nd/or A. For exmple, DESD inhibits PLA 2 (157) nd clphostin C, clssic PKC inhibitor, lso inhibits PLD (158). Furthermore, DESD lso inhibits PKC (159) nd ctivtes ERK1/2 (unpublished observtions), indicting tht DESD is not specific inhibitor of PLD. However, inhibition of Ang II-medited genertion of superoxide with DESD ws rescued by ddition of PA, indicting tht PLD my indeed ply role in Ang II-medited genertion of superoxide. A second rgument tht lends strength to the ide tht PLD is involved in formtion of superoxide is the time course of gonist-medited production of PA nd superoxide. Insulin-medited genertion 19

36 of PA peks within 2 minutes (121) nd Ang II-medited ctivtion of NAD(P)H oxidse peks between 3 to 4 minutes (95). This indictes tht gonists stimulte PLD before NAD(P)H oxidse. However, the time course of Ang II-medited PLD ctivity remins to be determined. Therefore, the current dt indicte tht PLD is most likely ctivted before NAD(P)H oxidse nd, thus, cn be involved in genertion of superoxide. Additionlly, Ang II-medited PLD ctivity is incresed in SHR compred to WKY (112), nd SHR hve incresed superoxide levels compred to WKY (104). Tken together, these dt indicte tht PLD leds to genertion of superoxide. This suggests tht PLD is required for Ang II-medited genertion of superoxide, thus prticipting in the pthophysiology of hypertension. 1.6 STATEMENT OF PROBLEM AND HYPOTHESIS An integrted signling pthwy cn be formed by linking Ang II to the PLD pthwy. However, the mechnism of Ang II-medited ctivtion of PLD is unknown. Previous reports suggest tht RhoA nd c-src re involved (151), nd tht Ang II more potently ctivtes PLD in SHR compred to WKY thorcic ortic smooth muscle cells (112). As mentioned, kidney vsculture is the tissue most likely to contribute to hypertension but no studies hve exmined Ang II-medited PLD ctivtion in this tissue. Therefore, the objective of these studies is to define Ang II-medited signl trnsduction mechnism(s) leding to ctivtion of PLD in preglomerulr smooth muscle cells (PGSMCs). Due to the forementioned differences between the SHR nd WKY, the hypothesis underlying the objective is tht Ang II-medited PLD ctivity is greter in SHR compred to WKY PGSMCs due to n ltertion in signl trnsduction pthwy. 20

37 Chpter 2 EXPERIMENTAL METHODS 2.1 MATERIALS USED: Angiotensin II ws obtined from Sigm. All gonists nd ntgonists used long with their vendors nd reported IC 50 vlues for the ntgonists re reported in Tble 2.1. [ 3 H]plmitte nd 125 I-Sr 1 -Ile 8 -Ang II were obtined from NEN/Perkin Elmer (Boston, MA). All primry ntibodies were obtined from Cell Signling Technology (Beverly, MA), nd the corresponding secondry ntibodies were obtined from Jckson Immuno Lbortories (West Grove, PA). Alex 488 conjugted phlloidin ws obtined from Moleculr Probes (Eugene, OR). All moleculr biology enzymes were obtined from New Englnd Biolbs (Beverly, MA), nd the lipids were obtined from Avnti Polr Lipids (Albster, AL). The ctlyticlly inctive (dominnt negtive, dn) mutnts T31N ARF1, T27N ARF6, nd T17N RhoA were used to inhibit specific G-protein functions; E156K ARNO, mutnt of n ARF GEF tht inhibits nucleotide exchnge, ws lso used to exmine G-protein functions. Ctlyticlly inctive K898R PLD1 nd K758R PLD2 were used to investigte PLD function. All constructs, except the dnarno were previously subcloned s EGFP fusion proteins s described elsewhere (120;121;160). 2.2 CELL CULTURE: All cell culture regents were obtined from Invitrogen/GibcoBRL (Crlsbd, CA). Thirteen to 15 week old SHR nd WKY rts from Tconic Frms (Germntown, NY) were used to obtin the PGSMCs. PGSMCs were obtined by forcefully injecting greter-thn 1% Fe 2 O 3 21

38 Tble 2.1: List of Agonists nd Antgonists. Nme Vendor Loction Description IC 50 Refs PMA Sigm SintLouis, MO DAG nlogue, PKC ctivtor - - UK-14,304 Sigm/RBI Sint Louis, MO Specific α 2 -drenergic gonist - (161) CGP-42112A Sigm/RBI Sint Louis, MO Specific AT 2 R gonist - (162;163) SNAP Moleculr Probes Eugene, OR Instnt Nitric Oxide donor - (164) DEANO Moleculr Probes Eugene, OR Nitric Oxide donor, t ½ = 2 min - (165) 8-Br-cGMP Clbiochem Sn Diego, CA Non-hydrolizble cgmp nlogue - (164;165) YC-1 Alexis Corp. Crlsbd, CA Nitric Oxide independent stimulnt of soluble gunylte cyclse - (166) L-158,809 Sigm/RBI Sint Louis, MO Specific AT 1 R ntgonist 0.3 nm (167;168) PD-123,319 Sigm/RBI Sint Louis, MO Specific AT 2 R ntgonist 3 nm (169;170) LY Clbiochem Sn Diego, CA PI3K inhibitor 1.4 µm (171;172) Wortmnnin Clbiochem Sn Diego, CA PI3K inhibitor 1 to 5 nm ( ) Gö-6983 Clbiochem Sn Diego, CA Inhibits PKCα (7 nm), β (7 nm), γ (6 nm), δ (10 nm), ζ (60 nm), nd µ (20 µm). (IC 50 vlues) (174) Ro Clbiochem Sn Diego, CA Inhibits PKCα (8 nm), βi (8 nm), βii (14 nm), γ (13 nm), nd ε (39 nm). (IC 50 vlues) (175) Sturosporine Clbiochem Sn Diego, CA Non-specific PKC inhibitor. Inhibits PKCα (28 nm), βi (13 nm), βii (11 nm), γ (32 nm), δ (28 nm), ε (25 nm), nd ξ (>1.5 µm). (IC 50 vlues) (176) Brefeldin A Sigm Sint Louis, MO Inhibits the interction between mny ARF GEFs nd ARF ~3 µg/ml (177) Y Tocris Bllwin, MO Inhibits Rho kinses 140 nm (56) HA-1077 Clbiochem Sn Diego, CA Inhibits Rho kinses ~300 nm (178) Pertussis Toxin Sigm Sint Louis, MO Inhibits signling through Gα i ~0.01 ng/ml (179) OBAA Tocris Bllwin, MO Inhibits PLA 2 ~70 nm (180) NNA Sigm Sint Louis, MO Competes with L-rginine to inhibit NOS. (181) ODQ Alexis Corp. Crlsbd, CA Inhibits Nitric Oxide sensitive gunylte cyclse. ~1 µm (182;183) See next pge for tble legend. 22

39 Tble 2.1: LEGEND Ref indictes pproprite references tht describe the function of the drug nd resonble concentrtions for the experiments conducted. PMA is Phorbol 12-myristte 13-cett, SNAP is S- nitroso-n-cetylpenicillmine, DEANO is diethylmine nitric oxide, OBAA is 4-(4-Octdecyl)-4- oxobenzenebutenoic cid, NNA is N 5 -(Nitromidino)-L-2,5-diminopentnoic cid, nd ODQ is 1H- [1,2,4]Oxdizole[4,3-]quinoxlin-1-one. Dulbecco s Modified Egle s Medium (DMEM) solution contining 20 U of penicillinstreptomycin into isolted kidneys through the renl rtery. The iron-loded kidney ws removed from the rt, the cortex minced, nd wshed multiple times in 1% collgense IV solution. During the wshes the vessels were isolted from the solution with mgnet, llowing the solution to be decnted without loosing the vessels. After multiple wshes, enough to visibly seprte the remining tissue from the vessels, the vessels were plted with Dulbecco s Modified Egle s/f12 medium (DMEM/F12) supplemented with 10% fetl bovine serum (FBS) nd 20 U penicillin-streptomycin. After the cells grew to confluence, differentil pltting ws performed by the following procedure: the cells were trypsinized nd pssed into fresh plte, fter 20 min the medi from the new plte ws removed nd plced on second plte, fter second 20 min intervl the medi ws removed nd plced on third plte. The few remining cells were PGSMCs t pssge 1 (184). All experiments were conducted between pssge 4 nd 10, nd the PGSMCs were grown in DMEM/F12 supplemented with 10% FBS, 100 U/mL penicillin G, 100 µg/ml streptomycin, nd 0.25 µg/ml mphotericin B. 2.3 TRANSFECTION OF PGSMCS: PGSMCs were grown to 50-75% confluence in 60-mm dishes nd trnsfected using Lipofectmine Plus following the mnufcturer s protocol, or grown to 100% confluence nd 23

40 trnsfected with Lipofectmine 2000 following the mnufcturer s protocol (Invitrogen/GibcoBRL). The following dy the cells were wshed nd fresh medi ws dded. Since ll constructs used were subcloned s EGFP fusion proteins or co-trnsfected with EGFP, the trnsfection efficiency ws determined by the frction of green fluorescent cells. Cells were serum strved the evening fter trnsfection nd the experiments were conducted the following dy. Therefore, cells expressed the plsmid(s) for 36 to 48 hr before experiments. 2.4 PHOSPHOLIPASE D ASSAYS: PGSMCs were serum strved for t lest 15 hours in 2 ml of DMEM/F12 with [ 3 H]plmitte (5 µci/ml). PGSMCs were incubted for 12 min. in DMEM/F12 with 0.5% ethnol (EtOH medi) to tke dvntge of the trnsphosphtidyltion rection (160). All ntgonists used were included in the EtOH medi. Cells were then stimulted with gonist(s) for 20 min., wshed with ice cold PBS, scrped on ice, nd centrifuged t 16,000 x g for 1 min. The resulting superntnt ws decnted nd the cell pellet ws resuspended in 200 µl PBS, vortexed, then 800 µl of 1:1 chloroform:methnol solution ws dded, vortexed, nd the smples plced on ice. After 5 min, the smples were centrifuged t 16,000 x g for 12 min t 4 C, then the upper queous phse ws removed nd the lower orgnic phse ws concentrted using rotry evportor. The concentrted orgnic frction, which contined the lipids, ws resuspended in 20 µl chloroform nd spotted onto plstic-bcked silic gel 60 TLC pltes (VWR). Ech smple ws spiked with cold phosphtidylethnol (PtdEtOH), nd PA control ws run on ech plte (Avnti Polr Lipids). The TLC pltes were resolved using ethyl cette:trimethylpentne:cetic cid (9:5:2) s the solvent, nd seprtion of PtdEtOH from PA ws determined by developing the plte in n iodine chmber. The lipid spots corresponding to 24

41 PtdEtOH nd PA were circled with pencil nd the iodine ws llowed to disperse overnight. The re round the circles ws spryed with En 3 Hncer Spry (NEN/Perkin Elmer). After the En 3 Hncer dried slightly, the plte ws wrpped in Srn Wrp nd exposed to Fuji X-ry film for 4-7 dys t -80 C. After developing the film, the utordiogrm ws compred to the circled spots to verify where the PtdEtOH generted by PLD resides on the plte. Once determined, the precise loction of the PtdEtOH spot, the PtdEtOH spot nd origin spot (totl unrected phospholipid) were scrpped, mixed with 5 ml EcoLite(+) (ICN), nd the mount of PtdEtOH nd totl unrected phospholipid ws determined by β-counting. PLD ctivity is expressed s the rtio of PtdEtOH to totl unrected phospholipid. To mesure PA produced fter ddition of Ang II, the bove protocol ws followed with the following chnges: no ethnol ws dded to the smple, ll use of PBS ws replced with ice cold deionized wter nd the smples were spiked with PA. Additionlly, Ang II ws left on the cells for vrious mounts of time. For nlysis, the PA spot ws locted nd nlyzed utilizing the sme procedures for PtdEtOH I-SAR 1 -ILE 8 -ANG II BINDING: PGSMCs were grown to confluence in 6-well pltes, then they were wshed, serum strved overnight, nd the next dy wshed twice for 5 min in 1.5 ml binding buffer (50 mm N 2 HPO 4, 150 mm NCl, 10 mm MgCl 2, nd 0.05% bovine serum lbumin ph = 7.1). Next, 1 ml binding buffer with pproximtely 30 pm 125 I-Sr 1 -Ile 8 -Ang II ws dded to the PGSMCs. Then vrious mounts of Ang II, L-158,809, or PD-123,319 were dded t room temperture for 60 min. Next, the solution ws decnted nd the PGSMCs were quickly wshed with 1 ml 25

42 binding buffer then lysed in 10% SDS. Protein concentrtion ws determined by the BCA method (Pierce), nd the mount of 125 I-Sr 1 -Ile 8 -Ang II bound ws determined by γ-counting. To nlyze the binding dt, the number of cells in ech smple ws determined s follows. Multiple 100-mm pltes were trypsinized nd resuspended in 5 ml serum free medi, 100 µl of this cell solution ws plced into 20 ml Isoton solution nd the cells were counted on Coulter Counter. Ech smple ws counted 6 times, the two outlying numbers were discrded, nd the remining 4 numbers were verged nd the number of cells in the smple ws determined by the following formul: Coulter counts x 40 (the dilution fctor) = cell number. This ws repeted for 5 seprte 100 µl liquots. Protein concentrtion ws determined utilizing the BCA protocol on six 100 µl liquots of the sme cell solution. The mount of cells in 1 µg of protein ws then clculted by dividing the verge number of cells in the 100 µl liquot by the verge µg protein in the 100 µl liquot. 2.6 QUANTIFICATION OF MRNA: Totl RNA ws isolted from confluent 100-mm pltes utilizing the Rnesy Mini Kit protocol (Qigen, Vlenci, CA), nd 10 µl of totl RNA ws used in the Clontech Advntge RT-for-PCR Kit. The resulting cdna ws used to mplify PLD1, PLD2, AT 1 R, AT 2 R, nd glycerldehyde-3-phosphte dehydrogense (GAPDH). The primers nd expected sizes re listed in Tble 2.2. All primers used were designed with Primer Designer 2.0 from Scientific & Eductionl Softwre (Durhm, NC). GAPDH ws used s n internl control in ech rection. The primers for the AT 1 R were designed to mplify both AT 1A nd AT 1B generting the sme size bnd, nd the PLD1 primers were designed to mplify both PLD1 nd PLD1b generting two seprte bnds. The mplifiction cycles were designed so tht ll the rections would occur 26

43 simultneously (nneling t 60 C for 45 sec nd elongtion t 72 C for 60 sec). This cycle ws repeted for totl of 30 times. The PLD PCR products were run on 3% grose gel stined with ethidium bromide, wheres the Ang II receptors were run on seprte 2% grose gel stined with ethidium bromide. A digitl picture ws tken of the gels nd the negtives were nlyzed utilizing Moleculr Anlyst for Windows NT (Moleculr Dynmics). 2.7 CLONING OF RHOA FROM WKY AND SHR PGSMCS: Totl RNA ws isolted nd cdna generted s for RT-PCR. RhoA ws mplified from the cdna vi PCR with the primers in Tble 2.3. Restriction sites for EcoRI nd BmHI respectively were plced in the 5 re of the primers (before the dsh) for future cloning nd restriction nlysis of colonies. Deep Vent polymerse ws used to mplify RhoA due to its bility to generte blunt ends, which is needed for the cloning kit. The mplifiction cycles were: nneling 68 C for 45 sec, elongtion 72 C for 1 min, nd denturing 94 C for 1 min. The PCR products were run on 2% grose gel, nd DNA isoltion ws performed using GENECLEAN Turbo (Q-BIOgene, Crlsbd, CA). The isolted RhoA cdna ws inserted into the Zero Blunt TOPO PCR Cloning Kit for Sequencing (Invitrogen) ccording to the mnufcturer s instructions. Since the pcr4blunt-topo plsmid is designed to grow only if there is n insert, only 8 colonies from the LB plte were screened by restriction nlysis with EcoRI nd BmHI. Colonies tht resulted in 600bp bnd were sent to the University of Pittsburgh core sequencing fcilities with primers to the T3 nd T7 priming sites, which flnk the insert region on pcr4blunt-topo. 27

44 2.8 GENERATION OF S188A RHOA: The S188A RhoA mutnt ws generted from WKY RhoA DNA by PCR with the primers listed in Tble 2.3. The muttion ws introduced in the second primer t (C), which is normlly n A nd produces n lnine t position 188 insted of the endogenous serine. Restriction sites XhoI nd PstI respectively were plced in the 5 re of the primers (before the dsh) for future cloning nd restriction nlysis of colonies. The PCR products were seprted on 2% gel nd the 600bp bnd ws extrcted vi the GENECLEAN protocol. The isolted bnd long with the EGFP-C1 plsmid (Clontech) ws digested for 2 hours with XhoI nd PstI then the PCR product ws ligted into the cut EGFP plsmid overnight t 4 C. The ligtion rection ws then trnsformed into DH5α cells (Invitrogen/GibcoBRL) nd plted onto LB pltes with 30 µg/ml knmycin (Sigm). The resulting colonies were screened for 615bp bnd on 2% grose gel fter digestion with XhoI nd PstI. Colonies tht resulted in the 615bp bnd were sent to the sequencing fcility. 2.9 MAPK ASSAYS: PGSMCs were grown to confluence in 60-mm dishes nd serum strved overnight. The next dy, the cells were stimulted with Ang II for 5 min, wshed with ice cold PBS, scrped on ice, nd centrifuged t 16,000 x g for 1 min. The resulting superntnt ws decnted nd the cell pellet ws resuspended in 220µL lysis buffer (0.5% Triton-X-100, 50 mm HEPES, 10 mm MgCl 2, 5 mm MnCl 2, 1 mm PMSF, 1 mm vndte, nd 10 ug/ml Leupeptin, ph 7.4). The smples were then vortexed nd 20 µl of ech smple ws removed for protein concentrtion determintion by the BCA method. After removl of 20 µl, 62.5 µl of 4x SDS smple buffer (0.2 M Tris ph 6.8, 4% SDS, 4% 2-Mercptoethnol, 0.8% bromo-phenolblue, nd 40% 28

45 Tble 2.2: Primers used for RT-PCR. Forwrd Reverse Primers for PLD1 5 -CGTGAACCACAGAACCAATG-3 5 -TCTCACGGCAGCATCAGTAG-3 Expected Sizes PLD1 = 527bp PLD1b = 413bp Primers for PLD2 Forwrd 5 -CAGGAGGCGGTTGAGGTAAT-3 Reverse 5 -AGTTGCACATGGAGCCAGAT-3 Primers for AT 1 R Forwrd 5 -GGAAACAGCTTGGTGGTG-3 Reverse 5 -CTGAATTTCATAAGCCTTCTT-3 Primers for AT 2 R Forwrd 5 -AGTGCATGCGGGAGCTG-3 Reverse 5 -GACAACAAAACAGTGAG-3 Primers for GAPDH Forwrd 5 -TACTCCTTGGAGGCCATGTA-3 Reverse 5 -CGTGGAGTCTACTGGCGTCT-3 Expected Size 467bp Expected Size 555bp Expected Size 309bp Expected Size 723bp The reverse primers were designed to bind to the complement strnd of cdna. 29

46 Tble 2.3: Primers used for Sequencing nd Genertion of S188A RhoA. Forwrd Reverse Forwrd Reverse Primers for Cloning RhoA 5 -GACGAATTCA-ATGGCTGCCATCAGGAAG-3 5 -GGATCCTAC-TGAGGCTGCGTTCACAAG-3 Primers for S188A RhoA 5 -TACTCGAGCT-ATGGCTGCCATCAGGAAGAA-3 5 -ATCTGCAG-TCACAAGATGAGGCACCCCG(C)CTTTTT-3 Size 631bp Size 630bp The reverse primers were designed to bind to the complement strnd of cdna. The (C) in S188A RhoA represents the introduced muttion. glycerol) ws dded nd boiled for 5 min. The smples were then run on 10% SDS-PAGE gel, trnsferred to Nitrobond nitrocellulose (VWR) nd probed for the proteins in the MAPK cscde. To determine ERK1/2 nd MEK ctivity the following protocol ws utilized. The specific nti-phospho-erk1/2, E10, ntibody ws used to determine the level of phopho-erk1/2 by densitometry of the Western blot. The E10 nd secondry ntibody were stripped from the nitrocellulose with stripping buffer (62.5 mm Tris-HCl 2% SDS, nd 100 mm 2- mercptoethnol ph = 6.8), nd the membrne ws reprobed with the specific Erk1/2 ntibody. Totl ERK1/2 levels were determined by densitometry. ERK1/2 ctivtion ws expressed s the rtio of phospho-erk1/2 to totl Erk1/2. Identicl procedures were used to determine MEK ctivtion. In order to confirm tht PLD genertion of PA is involved ERK1/2 ctivtion, the protocol for mesuring ctivtion of ERK1/2 ws used with the following dditions. 20 mm PA ws dded to DMEM/F12 contining 1 mg/ml BSA. The PA solution ws sonicted t 40 units on Het Systems Sonifier Cell Disrupter (Plinview, NY) while on ice for 10 sec then llowed 30

47 to sit on ice for 10 sec. This sequence ws repeted 10 times. Immeditely following the lst soniction 20 µl of the PA solution ws dded to the cells resulting in finl concentrtion of 200 µm PA. The cells were incubted with the PA for 5 min t 37 C nd 5% CO 2, then 1 µm Ang II ws dded to the cells for 5 min. The reminder of the experimentl procedure for ccessing the levels of ctive ERK1/2 ws followed CONFOCAL IMAGINING EXPERIMENTS: PGSMCs were plted on 25-mm dimeter poly-l-lysine coted glss coverslips (Fisher Scientific, Pittsburgh, PA) in 6-well pltes. Cells were trnsfected with SuperFect (Qigen), following the mnufcturer s protocol, nd stimulted with 1 µm Ang II or 100 ng/ml EGF for 5 min. Following stimultion the coverslips were wshed twice with ice-cold PBS nd 1.5 ml fresh 3% prformldehyde in PBS ws pplied for 30 min t 4 C to fix the cells. After fixtion, the cells were wshed with PBS, permebilized with 1.5 ml 0.1% Triton X-100 in PBS for 2 min, wshed three times in PBS, nd blocked with 2 ml 3% BSA in PBS for 30 min. After blocking, the primry ntibodies (Tble 2.4) were dded t 1:500 dilution in 2 ml 3% BSA in PBS for 60 min. The cells were then wshed twice with PBS, then the secondry ntibodies (Tble 2.4) were dded t 3 µg/ml nd 2.6 µg/ml for Cy3- nd Cy5-conjugted ntibodies respectively in 2 ml 3% BSA in PBS for 60 min. Ten units of the Alex 488-conjugted phlloidin ws dded with the secondry ntibodies. The cells were wshed fter immunostining twice with PBS then once with deionized wter. The coverslips were then mounted, cells down, on Fisherbrnd Superfrost plus microscope slides with Gel/Mount (Biomed, Foster City, CA) mounting gel nd seled with Revlon cler nil polish. Confocl 31

48 imges were tken with Zeiss LSM5 PASCAL confocl microscope AxioVert 200 stge (Crl Zeiss Inc., Jen, Germny) equipped with 1.4 NA 63x oil immersion objective DATA AND STATISTICAL ANALYSIS: For ll mthemticl opertions contining two independent dt sets with mesurble error, the following error propgtion formuls were pplied. If f nd g re two mens nd fe nd ge re their respective error, then the error for f/g is [fe*g-f*ge]/g 2 nd the error for f±g is fe+ge. For multiple comprisons, the dt were nlyzed by ANOVA with Fisher s LSD post hoc test. For individul comprisons, t-test ws used to determine significnce. Dt points re ssumed to be significnt only if P < Sttisticl nlysis ws conducted using the NCSS 2000 (Kysville, UT) softwre pckge. Dose response curves were nlyzed using the curve fit routines of GrphPd Prism (Sn Diego, CA). Tble 2.4: Antibodies nd Fluorophores used for Confocl Imging Experiments. Position Nme Conjugte Origin Epitope 1 E10 - Mouse Rbbit Phospho- ERK1/2 Phospho- MEK Absorption Pek (nm) Emission Pek (nm) Assigned Color EGFP dnpld Blue - phlloidin Alex Binds F-ctin Blue 2 AffiniPure Cy5 Donkey Anti-Mouse Red 2 AffiniPure Cy3 Donkey Anti-Rbbit Green 32

49 Chpter 3 MECHANISM OF ANGIOTENSIN II STIMULATION OF PLD 3.1 INTRODUCTION Angiotensin II importntly contributes to the pthophysiology of hypertension in SHR. Blood pressure in SHR is normlized by ngiotensin converting enzyme inhibitors (3), AT 1 R ntgonists (2), nd ctive immuniztion ginst renin (4). However, Ang II production is pprently not elevted in SHR, either systemiclly (185) or loclly (186), compred to WKY. On the other hnd, renl trnsplnttion studies demonstrte tht the kidneys medite hypertension in SHR (7;187;188). A working hypothesis to explin these observtions is tht hypertension in SHR is due to enhnced renovsculr sensitivity to Ang II (10;11;189). The mechnism of enhnced renl sensitivity to Ang II is uncler, but my involve multiple signling pthwys. Previous studies demonstrted tht Ang II-induced PLD ctivity is incresed in thorcic smooth muscle cells from SHR compred to WKY (112). PLD genertes PA, lipid involved in the ctivtion of MAPK (121;153). Becuse MAPK ctivity is elevted in vsculture from the SHR (63), nd MAPK ppers to be involved in vsculr smooth muscle cell contrction (91;190), it is possible tht PLD medites, in prt, the enhnced renovsculr response to Ang II in SHR. In order to describe the incresed PLD ctivity in SHR, the mechnisms of PLD ctivtion must first be described. Since the underlying hypothesis is tht incresed PLD ctivity my be involved in hypertension, PGSMCs from SHR nd WKY were utilized to determine: 1) the effect of Ang II on PLD ctivity; 2) the reltive roles of Ang II receptor subtypes in the stimultion of PLD; 3) the reltive contributions of PLD isoforms to 33

50 Ang II- dependent PLD ctivity; nd 4) the signling pthwys tht medite Ang II-induced stimultion of PLD (Fig. 3.1). 3.2 RESULTS ANGIOTENSIN II STIMULATES PLD THROUGH THE AT 1 RECEPTOR. As shown in Figure 3.2, Ang II incresed PLD ctivity in both SHR nd WKY PGSMCs in concentrtion-dependent fshion, but the EC 50 for Ang II in SHR PGSMCs (6.0 x 10-9 M) ws significntly less (P < 0.05) thn tht for WKY PGSMCs (7.2 x 10-8 M). Two-fctor ANOVA nlysis indicted tht there is significnt shift to the left of the SHR dose response when compred with the WKY response curve (Fig. 3.2). However, the time course of Ang IImedited genertion of PA is similr in WKY nd SHR PGSMCs (Fig. 3.3). The PA genertion time course indictes tht PA is generted within 40 sec nd mximl concentrtions rise before 3 min. Thus, Ang II-medited increse in PA levels fit the kinetics of other erly signl trnsduction compounds. To determine the Ang II receptor tht is responsible for ctivting PLD, the specific AT 1 R nd AT 2 R ntgonists, L-158,809 nd PD-123,319 respectively, were dministered t 5x their IC 50 vlues. As shown in Figure 3.4, 1.5 nm L-158,809 inhibited Ang II-induced PLD ctivity in SHR nd WKY PGSMCs by 92 ± 9 nd 63 ± 5 percent, respectively, nd 15 nm PD- 123,319 did not significntly ttenute Ang II-medited PLD ctivity. Becuse only L-158,809 decresed Ang II-dependent PLD ctivtion in WKY nd SHR, PLD ctivtion is downstrem of the AT 1 R in PGSMCs s previously shown in thorcic ortic smooth muscle cells (110). Further nlysis of the receptors is presented in Chpter 4 34

51 PI3Kγ PKC PIP3 ARNO PLD 1 βγ Gαq Gαi R Gα12/13 ARF PLD 2 Rho Figure 3.1: Simplified Flow Digrm of how GPCR cn Activte PLD. PLD cn be ctivted by vriety of mechnisms. However, ll mechnisms described thus fr involve PI3K, PKC, ARF, or Rho. The (R) indictes the GPCR. 35

52 PLD Activity [PtdEtOH (% of totl lipid)] WKY EC 50 = 7.2 x 10-8 ± 4.3 x 10-8 M SHR EC 50 = 6.0 x 10-9 ± 1.3 x 10-9 M * Log [Ang II (M)] * * Figure 3.2: Ang II-Medited PLD Activity Concentrtion Response Curve. Ang II stimultes PLD in WKY nd SHR PGSMCs; however, Ang II is more potent ctivtor of PLD in SHR, compred to WKY PGSMCs. Dt re expressed s Men ± SEM, n = 6; * indictes tht the SHR is significntly greter thn WKY (P < 0.05) t the respective concentrtion. 36

53 WKY SHR PA (% totl lipid) Time (min) Figure 3.3: Ang II-Medited PA Production. Stimultion of WKY nd SHR PGSMCs with 1 µm Ang II leds to rpid ccumultion of PA. Dt re expressed s Men ± SEM, n 3; SHR is not significntly different thn WKY (P = ). 37

54 PLD Activity (Fold over control) WKY SHR b c b b c 0 Control 1 µm Ang II AT 1 + AT 2 + Control 1 µm Ang II AT 1 + AT 2 + Figure 3.4: Ang II Activtes PLD by Acting on the AT 1 R. 1 µm Ang II-medited PLD ctivity is significntly ttenuted by the AT 1 R ntgonist L-158,809 (AT 1 ) in both WKY nd SHR PGSMCs, nd the AT 2 R ntgonist PD-123,319 (AT 2 ) does not inhibit Ang II-medited PLD ctivity in WKY nd SHR PGSMCs. Dt re expressed s Men ± SEM, n = 3; brs with different letters re significntly different (P < 0.05). 38

55 3.2.2 Identifiction of the PLD Activted by Ang II. RT-PCR demonstrted the presence of ll PLD isoforms in both SHR nd WKY PGSMCs (Fig. 3.5, top pnel). Trnsfection of cells with dnpld2, but not dnpld1, blocked Ang II-induced PLD ctivity (Fig. 3.5, bottom pnel) in both SHR nd WKY PGSMCs. These dt suggest tht PLD2 is the min signling isoform in Ang II-medited PLD ctivity in WKY nd SHR PGSMCs, nd the more potent Ang II-medited PLD ctivity in SHR is not due to incresed quntity of PLD ROLE OF PI3K AND PKC IN ANG II-MEDIATED PLD ACTIVITY. As shown in Figure 3.6, neither 7 µm LY nor 100 nm wortmnnin bolished Ang II-medited PLD ctivity. There is slight inhibition in WKY PGSMCs by wortmnnin. However, the degree of inhibition does not mtch wht is expected for 100 nm wortmnnin nd LY does not inhibit signling in the WKY, thus indicting tht the significnt decrese observed is not biologiclly significnt. Therefore, PI3K ctivity is not essentil for the ctivtion of PLD by Ang II. 390 nm Ro , 1 µm Go-6983, nd 250 nm sturosporine did not inhibit Ang II-medited PLD ctivity suggesting tht the effects of Ang II on PLD re independent of PKC ctivity (Fig. 3.7). Combining Ro nd Go-6983 slightly inhibited Ang II-medited PLD2 ctivity only in SHR PGSMCs. However, the degree of inhibition ws less thn 50%, which is not wht would be expected with the concentrtions of Ro nd Go-6983 used becuse t these concentrtions PKC signling should be nerly bolished (Fig. 3.8). To verify tht: 1) the PKC inhibitors re functionl in this experimentl system, nd 2) the inhibition of PLD by combining Ro nd Go-6983 is n rtifct nd not due to PKC inhibition, 500nM PMA served s positive control for the PKC inhibitors (Fig. 3.8). All PKC 39

56 Bnd Intensity (PLD/GAPDH) WKY SHR GAPDH PLD 1 PLD 1b WKY SHR PLD PLD 1 PLD 1b PLD 2 PLD Activity (Fold over Control) b c d b c d 0.0 Control 1 µm Ang II dnpld1 + dnpld2 + Control 1 µm Ang II dnpld1 + dnpld2 + Figure 3.5: All PLD Isoforms re Present nd Ang II ctivtes PLD2. RT-PCR (Top) indictes tht ll PLD isoforms re present nd similrly expressed in WKY nd SHR PGSMCs. Trnsfection with dnplds (Bottom) indictes tht 1 µm Ang II ctivtes PLD2. Dt re expressed s Men ± SEM, n = 3; brs with different letters re significntly different (P < 0.05). 40

57 PLD Activity [PtdEtOH (% of totl lipid)] WKY SHR b c d e b b c 0.0 Control LY Wort AII LY + AII Wort + AII Control LY Wort AII LY + AII Wort + AII Figure 3.6: Ang II-Medited PLD Activity is not Medited through PI3K. Inhibition of PI3K with 7 µm LY (LY) nd 100 nm wortmnnin (Wort) does not inhibit 1 µm Ang II-medited PLD ctivity. Dt re expressed s Men ± SEM, n = 3; brs with different letters re significntly different (P < 0.05). 41

58 PLD Activity [PtdEtOH (% of totl lipid)] Control Ro WKY SHR b Go b Ro + Go b c c Str AII Ro + AII c Go + AII c Ro + Go + AII c Str + AII Control,b Ro Go Ro + Go b,c d Str AII Ro + AII d c,d,e Go + AII b,e Ro + Go + AII c,d,e Str + AII Figure 3.7: Ang II-Medited PLD Activity is not Medited through PKC. Inhibition of PKC with 390 nm Ro (Ro), 1 µm Go-6983 (Go), nd 250 nm sturosporine (Str) does not inhibit 1 µm Ang II-medited PLD ctivity. Dt re expressed s Men ± SEM, n = 3; brs with different letters re significntly different (P < 0.05). 42

59 PLD Activity [PtdEtOH (% of totl lipid)] WKY SHR * * * * * * * * * 0.0 Control + Wort + Ro +Go PMA +Ro +Go + Str Control + Wort + Ro +Go PMA +Ro +Go + Str Figure 3.8: The PKC Inhibitors Block PMA-Induced PLD ctivity. 500 nm PMA stimultes PLD in WKY nd SHR PGSMCs. PMA stimultion of PLD is gretly reduced by ll PKC inhibitors used (390 nm, 1 µm, nd 250 nm for Ro, Go, nd Str, respectively), however, 100 nm wortmnnin hs little effect on PMA-medited PLD ctivtion. Dt re expressed s Men ± SEM, n = 3; * indictes tht the br is significntly less thn control PMA (P < 0.05). 43

60 inhibitors, but not the negtive control wortmnnin, significntly nd similrly blocked PMAinduced PLD ctivity in WKY nd SHR PGSMCs. Therefore, the PKC inhibitors re functionl in this experimentl system, nd due to the similrity of inhibition between the vrious inhibitors, the effect of Ro nd Go-6983 in combintion in SHR PGSMCs is most likely n rtifct. Additionlly, the fct tht wortmnnin hd little effect on PMA-induced PLD ctivity indictes tht PKC is not non-specificlly inhibited by the ddition of ny compound ROLE OF SMALL G PROTEINS IN ANG II-MEDIATED PLD ACTIVITY. Previous dt from A10 smooth muscle cells indicte tht ARF is involved in Ang IImedited PLD ctivtion (120). Therefore, nlysis of the G proteins involved in Ang IImedited PLD ctivity ws conducted in two steps: 1) nlysis of ARF ctivtion by ARF GEFs, nd 2) the role of the smll G proteins ARF nd RhoA. Trnsfection of PGSMCs with E156K ARNO (dnarno), which inhibits BFA-insensitive ARF GEFs, hd no effect on Ang IImedited PLD ctivity in WKY PGSMCs, but enhnced Ang II-medited PLD ctivity in SHR PGSMCs (Fig. 3.9, top pnel). Addition of 50 µg/ml BFA completely blocked Ang II-medited PLD ctivity in SHR PGSMCs, but only reduced by 40 ± 0.4 % in WKY PGSMCs (P < 0.05 compred with inhibition of SHR PGSMCs) (Fig. 3.9, bottom pnel). These dt suggest tht BFA-sensitive GEF ctivtes ARF tht then ctivtes PLD in SHR PGSMCs, nd tht there is different mechnism for ctivtion of PLD in WKY PGSMCs. To exmine further these conclusions, WKY nd SHR PGSMCs were trnsfected with ctlyticlly inctive mutnts of the smll G proteins (Fig. 3.10). T17N RhoA significntly reduced Ang II-medited PLD ctivity in WKY nd SHR PGSMCs. However, T31N ARF1 nd T26N ARF6 did not reduce Ang II-induced PLD ctivity. The inhibition of Ang II-medited 44

61 PLD Activity [PtdEtOH (% of totl lipid)] WKY SHR b b b c d 0.0 Con dnarno Ang II dnarno + Ang II Con dnarno Ang II dnarno + Ang II PLD Activity [PtdEtOH (% of totl lipid)] b c c b,c 0.0 Control BFA Ang II BFA + Ang II Control BFA Ang II BFA + Ang II Figure 3.9: Differentil Effects of Inhibiting ARF GEFs. Trnsfection of dnarno (top) does not inhibit 1 µm Ang II-medited PLD ctivity. However, dministrtion of 50 µg/ml BFA (bottom) inhibits 1 µm Ang II-medited PLD ctivity to greter extent in SHR compred to WKY PGSMCs. Dt re expressed s Men ± SEM, n = 3; brs with different letters re significntly different (P < 0.05). 45

62 PLD Activity [PtdEtOH (% of totl lipid)] WKY SHR b c b d,e b,e c b,d d e 0.0 Con dnarf1 dnarf6 dnrhoa AII dnarf1 + AII dnarf6 + AII dnrhoa + AII Con dnarf1 dnarf6 dnrhoa AII dnarf1 + AII dnarf6 + AII dnrhoa + AII Figure 3.10: Effects of dn-g proteins on Ang II-Medited PLD Activity. Only dnrhoa significntly inhibits 1 µm Ang II-medited PLD ctivity. Con nd AII cells were trnsfected with empty EGFP plsmid. Dt re expressed s Men ± SEM, n = 3; brs with different letters re significntly different (P < 0.05). 46

63 PLD ctivity by T17N RhoA corresponds with the observed trnsfection efficiency of 50% nd the ARF mutnts increse PLD ctivity only in SHR indicting tht RhoA medites Ang IIinduced PLD ctivtion in both WKY nd SHR PGSMCs. Thus, the results with the smll G protein mutnts do not confirm the previous conclusion regrding BFA nd ARF in SHR PGSMCs. Therefore, the BFA dt re either n rtifct or there is non-arf GEF trget for BFA in SHR PGSMCs ROLE OF RHO KINASES IN ANG II-MEDIATED PLD ACTIVITY. Rho cnnot directly ctivte PLD2 (143). However, Rho kinse hs been implicted in gonist-medited ctivtion of PLD (152). Therefore, the role of Rho kinse in Ang II-medited PLD ctivity ws exmined with two Rho kinse inhibitors Y nd HA Neither Y nor HA-1077 inhibited Ang II-medited PLD ctivity (Fig. 3.11), yet the effectiveness of the inhibitors in this experimentl system is unknown. To determine if the inhibitors block Rho kinse under these specific conditions, the effect of the inhibitors on the interction of UK- 14,304, n α 2 -drenergic gonist, nd Ang II ws exmined in SHR PGSMCs becuse previous studies indicted tht UK-14,304 nd Ang II synergisticlly constrict renl blood vessels in SHR (191). UK-14,304 nd Ang II synergisticlly ctivte PLD nd the Rho kinse inhibitors block the synergism without inhibiting the Ang II component of PLD ctivtion (Fig. 3.12). Therefore, the Rho kinse inhibitors function in this experimentl system, nd the inhibitors do not ffect Ang II-medited PLD ctivtion. 47

64 PtdEtOH (% of totl lipid) WKY SHR b b b Con Y HA Con Y HA Ang II b b b Con Y HA Con Y HA Ang II Figure 3.11: Ang II-Medited PLD Activity is not Medited by Rho Kinse. Inhibition of Rho Kinses with 1 µm Y (Y) nd 1 µm HA-1077 (HA) does not inhibit 1 µm Ang II-medited PLD ctivity. Dt re expressed s Men ± SEM, n = 3; brs with different letters re significntly different (P < 0.05). 48

65 PtdEtOH (% of totl lipid) WKY SHR b b c c 0.0 Drug Y HA Y HA Ang II UK b c d e Figure 3.12: Rho Kinse Inhibitors Block the Synergy between UK-14,304 nd Ang IImedited PLD Activity in SHR. Stimultion with 1 µm UK-14,304 (UK) leds to enhnced 1 µm Ang II-medited PLD ctivity only in SHR nd this synergy is blocked 1 µm of the Rho kinse inhibitors Y nd HA The dotted gry line indictes the level of ctivtion by 1 µm Ang II in SHR. Dt re expressed s men nd SEM, n = 3; brs with different letters re significntly different (P < 0.05). 49

66 3.3 DISCUSSION Preglomerulr microvsculr smooth muscle cells modulte preglomerulr vsculr resistnce nd thus regulte renl blood flow, glomerulr filtrtion rte nd, indirectly, renl sodium excretion (192). Since renl function determines long-term rteril blood pressure (193), ltered responses of PGSMCs to vsoctive fctors re likely to be importnt in the pthophysiology of genetic hypertension. Severl studies suggest tht the preglomerulr microcircultion is more responsive to Ang II in SHR compred with WKY rts (10;11;189), nd the bsis for this hyperresponsiveness to Ang II is under intense investigtion. PLD is n importnt regultory enzyme ctivted by severl vsoctive gents including Ang II nd endothelin-1 (120). Recent work hs demonstrted tht PA, the product of PLDctlyzed hydrolysis of phosphtidylcholine, plys centrl role in the regultion of the MAPK cscde (121;153). In A10 cells, vsculr smooth muscle cell line, the stimultion of MAPK phosphoryltion by Ang II is inhibited in the bsence of PLD ctivity, nd ddition of PA restores the effects of Ang II on MAPK phosphoryltion (120). Recent dt indicte tht MAPK inhibition; 1) reduces Ang II-induced primry smooth muscle cell contrction, nd 2) normlizes the Ang II contrctile response of the SHR smooth muscle cells (91). Additionlly, MAPK hs been implicted in the phosphoryltion of cldesmon leding to incresed smooth muscle contrction (190). Therefore, ltertions in the regultion of PLD ctivity by vsoctive peptides my ply n importnt role in the development of hypertension. These experiments demonstrte tht Ang II stimultes PLD ctivity in PGSMCs, nd tht SHR PGSMCs re significntly more sensitive to Ang II thn WKY PGSMCs. In order to estblish the biochemicl bsis for these differences, the mechnism of Ang II-medited ctivtion of PLD ws exmined. Previous studies on thorcic ort vsculr smooth muscle cells show tht AT 1 receptors trnsduce signls to PLD (110). SHR nd WKY PGSMCs re not 50

67 different, since the selective AT 1 ntgonist L-158,805 inhibited the effects of Ang II on PLD. This finding is consistent with previous in vivo experiments demonstrting tht the enhnced renovsculr response to Ang II in the SHR kidney is medited by the AT 1 R (9). Semiquntittive PCR shows tht SHR nd WKY PGSMCs express similr mounts of PLD1 nd PLD2, thus Ang II could ctivte either or both PLDs. To determine the PLD isoform(s) ctivted by Ang II, PGSMCs were trnsfected with dnpld1 nd dnpld2. The dt indicte tht PLD2 is the min signling isoform, thus confirming previous findings from our lbortory (120;121). G-protein coupled receptors (GPCR) cn regulte PLD ctivity by severl mechnisms (194). For instnce, GPCRs ctivte PI3K (195), nd PIP 2 nd/or PIP 3 pper to be required for the ctivity of PLD (141). Likewise, mny GPCRs ctivte PKC, which hs been shown to be n importnt regultor of PLD ctivity (196). Additionlly, the smll G proteins ARF nd RhoA cn be ctivted by GPCRs nd consequently ctivte PLD (120;137). Furthermore, RhoA ctivtes Rho kinses tht then ctivte PLD (152). Phrmcologicl inhibition of PI3K, PKC, nd Rho kinse(s) nd dn-mutnts of the smll G proteins were used to determine the pthwy Ang II utilizes to ctivte PLD. Since only dnrhoa inhibited Ang II-medited PLD ctivity in both SHR nd WKY PGSMCs, the dt from these experiments led to the conclusion tht PLD is ctivted through RhoA-dependent mechnism, nd tht Rho kinse is not involved in Ang II/RhoA-medited ctivtion of PLD. 3.4 CONCLUSIONS Ang II stimultes PLD2 ctivity through the AT 1 R nd RhoA in WKY nd SHR PGSMCs. However, the sensitivity of SHR PGSMCs to Ang II-medited PLD2 ctivity is incresed by ten fold compred to WKY PGSMCs. 51

68 Chpter 4 CHARACTERIZATION OF ANG II RECEPTORS PRESENT ON WKY AND SHR PGSMCS AND THEIR EFFECTS ON ANG II- MEDIATED PLD ACTIVITY 4.1 INTRODUCTION The AT 2 R is trditionlly thought to be expressed during development nd wound heling, s well s sprsely in few orgns of the dult orgnism (20), but not the renl vsculture ( ). However, physiologicl studies utilizing specific gonists nd ntgonists suggest tht the AT 2 R is present in the renl vsculture (37;200;201). Additionlly, binding studies indicte tht there re AT 2 Rs in the vsculture of rts up to 8 weeks of ge (202) nd immunohistochemicl stining indictes tht there re AT 2 Rs present in the vsculture of the humn kidney (203). The experiments in Chpter 3 with the AT 2 R specific ntgonist PD- 123,319 indicte tht there re AT 2 Rs on cultured PGSMCs. The role of the AT 2 R in Ang IImedited PLD ctivtion ppers to be opposite in WKY nd SHR PGSMCs (Fig 3.4). Therefore, the dt suggest tht there re differences in the expression nd/or signl trnsduction of the AT 2 R between cultured WKY nd SHR PGSMCs. The purpose of the following experiments ws to exmine the levels of ngiotensin II receptors on WKY nd SHR PGSMCs, nd to define the role of the AT 2 R in Ang II-medited PLD ctivity. 52

69 4.2 RESULTS PERTUSSIS TOXIN ACTS SIMILARLY TO PD-123,319. Due to the pprent potentition of Ang II-medited PLD ctivity in SHR PGSMCs by 15 nm PD-123,319, 10 nm Ang II-medited PLD ctivity ws exmined in WKY nd SHR PGSMCs. Becuse the AT 2 R signls through Gα i/o proteins (18;19), AT 2 R signling cn be inhibited with pertussis toxin s well s PD-123,319. As shown in Figure 4.1, 1 ng/ml pertussis toxin cts similrly to 15 nm of PD-123,319 in WKY nd SHR PGSMCs. Importntly inhibition of AT 2 R signling potentites 10 nm Ang II-medited signling in WKY but not SHR PGSMCs. Thus there is n pprent discrepncy in tht inhibition of the AT 2 R potentites t low concentrtions of Ang II signling in WKY PGSMCs nd t high concentrtions of Ang II in SHR PGSMCs. Therefore, the role of the AT 2 R in Ang II-medited PLD ctivity wrrnts further investigtion DETERMINATION OF ANGIOTENSIN II RECEPTOR EXPRESSION. The mount of AT 1 R nd AT 2 R mrna present in PGSMCs from WKY nd SHR ws determined by RT-PCR with GAPDH s n internl control (Fig. 4.2). As indicted by the Ang II receptor ntgonists nd pertussis toxin, WKY nd SHR PGSMCs express both receptor subtypes. Importntly, SHR PGSMCs contin significntly more AT 1 R messge nd significntly less AT 2 R messge compred to WKY PGSMCs. Furthermore SHR PGSMCs contin more AT 1 R thn AT 2 R messge, wheres WKY PGSMCs contin similr mounts of AT 1 R nd AT 2 R messge. 53

70 PtdEtOH (% of totl lipid) WKY SHR b,b c c,b c c b 0.0 Con PD PTX 10 nm Ang II PD + PTX + Con PD PTX 10 nm Ang II PD + PTX + Figure 4.1: Effects of Pertussis Toxin nd PD-123,319 on Ang II-Medited PLD Activity. Ang II-medited PLD ctivity is significntly ttenuted by the AT 2 R ntgonist PD-123,319 (PD) nd Pertussis toxin (PTX) in WKY PGSMCs, but PD nd PTX enhnce Ang II-medited PLD ctivity in SHR PGSMCs. The difference between the WKY nd SHR with PD nd PTX is significntly different (P < 0.05). Dt re expressed s Men ± SEM, n = 3; brs with different letters re significntly different (P < 0.05). 54

71 2.5 Bnd Intensity (Ang II receptor/gapdh) WKY SHR * * 0.0 AT 1 AT 2 FIGURE 4.2: Semi-Quntittive RT-PCR of WKY nd SHR AT 1 Rs nd AT 2 Rs. Levels of AT 1 R mrna re greter in SHR compred to WKY PGSMCs. SHR hve 4-fold greter levels of AT 1 R mrna compred to AT 2 R mrna, wheres WKY PGSMCs hve similr mrna levels for AT 1 Rs nd AT 2 Rs, nd WKY PGSMCs hve 2 fold more AT 2 R mrna thn SHR PGSMCs. Dt re expressed s Men ± SEM, n = 3; * indictes tht the SHR is significntly different (P < 0.05) compred to WKY by t-test. 55

72 Receptor messge does not necessrily directly correlte with bundnce of receptor protein. To determine the mount of receptor vilble for ctivtion by Ang II series of binding experiments with 125 I-Sr 1 -Ile 8 -Ang II were conducted. Displcement of 125 I-Sr 1 -Ile 8 - Ang II with unlbeled Ang II (Fig. 4.3) indictes tht there re more Ang II receptors present on SHR PGSMCs compred to WKY PGSMCs, yet the ffinity for Ang II is similr between the SHR nd WKY PGSMCs. Displcement of 125 I-Sr 1 -Ile 8 -Ang II with L-158,809 (Fig. 4.3b) indictes tht there re significntly more AT 1 receptors on SHR compred to WKY PGSMCs, nd displcement with PD-123,319 (Fig. 4.3c) indictes tht SHR PGSMCs hve significntly fewer AT 2 receptors thn WKY PGSMCs. Interestingly, the ffinities for L-158,809 nd PD- 123,319 re similr in WKY nd SHR PGSMCs, indicting tht the only difference between WKY nd SHR PGSMCs is the reltive mount of receptors. Tble 4.1 illustrtes tht the rtios of AT 1 Rs to AT 2 Rs re similr in the RT-PCR nd lignd binding studies. Therefore, SHR PGSMCs express more AT 1 R thn AT 2 R nd hve n incresed AT 1 R to AT 2 R rtio compred to WKY PGSMCs. This suggests tht the ltered responses observed in blood pressure control in the SHR re due to n imblnce of Ang II rtios. Tble 4.1: AT 1 R/AT 2 R Rtios WKY SHR RT-PCR 1.00 ± ± 0.90 Binding 0.74 ± ±

73 A [ 125 I]-Sr 1 -Ile 8 -Ang II Binding/cell (Totl - non-specific) I-Ang II + Cold Ang II WKY SHR log[cold Ang II (M)] B [ 125 I]-Sr 1 -Ile 8 -Ang II Binding/cell (Totl - non-specific) I-Ang II + L-158,809 WKY SHR C [ 125 I]-Sr 1 -Ile 8 -Ang II Binding/cell (Totl - non-specific) I-Ang II + PD-123,319 WKY SHR log[l-158,809 (M)] log[pd-123,319 (M)] Figure 4.3: Identifiction of AT 1 R nd AT 2 R Number on WKY nd SHR PGSMCs. Displcement of 125 I-Sr 1 -Ile 8 -Ang II with cold Ang II (A) indictes tht SHR PGSMCs hve 50% more Ang II receptors thn the WKY, however WKY nd SHR PGSMCs disply similr ffinities for Ang II. Displcement of 125 I-Sr 1 -Ile 8 -Ang II with L-158,809 (B) indictes tht SHR PGSMCs hve 2-fold more AT 1 receptors thn WKY, nd displcement with PD-123,319 (C) indictes tht WKY PGSMCs hve 2-fold more AT 2 receptors thn SHR. 57

74 4.2.3 EFFECT OF THE AT 2 R ON ANG II-MEDIATED PLD ACTIVITY. Becuse the AT 2 R generlly inhibits the functions of the AT 1 R nd the AT 1 R is the receptor tht medites Ang II ctivtion of PLD, the incresed sensitivity of SHR PGSMCs, compred to WKY PGSMCs, to Ang II-medited PLD ctivity my be due to the ltered AT 1 R/AT 2 R rtio. To exmine the hypothesis tht the ltered receptor rtio is responsible for the different effects of Ang II-medited PLD ctivity in WKY nd SHR PGSMCs, Ang II-medited PLD ctivtion concentrtion-response curves were generted in the presence of 15 nm PD- 123,319 nd 100 nm CGP-42112A, n AT 2 R gonist. As reported in Chpter 3, Ang II-medited PLD ctivity hs pproximtely 10-fold greter potency in SHR compred to WKY PGSMCs, EC 50 = 4 nm nd 47 nm respectively (Fig. 4.4). The efficcy of Ang II-medited PLD ctivity is significntly decresed by 30% in SHR compred to WKY PGSMCs due to higher bsl levels of PLD ctivity in SHR PGSMCs. Additionlly, two-fctor ANOVA nlysis indictes tht there is significnt shift to the left of the SHR concentrtion-response curve when compred with the WKY response curve. As predicted by the imblnce in receptor hypothesis, the WKY concentrtion-response curve is normlized to the SHR with ddition of PD-123,319, EC 50 = 4 nm nd 24 nm for the WKY nd SHR respectively (Fig. 4.5). Interestingly, there is no significnt chnge in the SHR concentrtion-response curve with the ddition of PD-123,319 (Fig. 4.5b). However, the efficcy of Ang II-medited PLD ctivity is incresed to tht of WKY PGSMCs. Addition of PD-123,319 significntly shifted the WKY concentrtion-response curve leftwrd, indicting tht the AT 2 R ttenutes the AT 1 R signling to PLD (Fig. 4.5c). Thus, these dt support the hypothesis tht decresed mounts of AT 2 Rs on SHR PGSMCs compred to WKY PGSMCs re 58

75 responsible for incresed sensitivity of PLD to Ang II in SHR PGSMCs, nd AT 2 Rs ttenute Ang II-medited PLD ctivity. The hypothesis tht the AT 2 R ttenutes Ang II-medited PLD ctivity cn be exmined with the AT 2 R gonist. Use of CGP-42112A should result in little effect in the SHR, but should shift the WKY concentrtion-response curve rightwrds indicting tht the AT 2 R is ttenuting Ang IImedited PLD ctivity. Interestingly, CGP-42112A significntly ltered the Ang II-medited PLD ctivity concentrtion-response curves of WKY nd SHR PGSMCs (Fig. 4.6). The WKY concentrtion-response curve is significntly shifted downwrds resulting in 60% decrese in efficcy (Fig. 4.6b). The SHR curve is shifted significntly rightwrd resulting in n EC 50 = 31 nm, compred to n EC 50 = 4 nm without CGP-42112A, without ltering the efficcy of Ang IImedited PLD ctivity (Fig. 4.6c). Thus, the SHR hs functionl AT 2 Rs, but too few to inhibit PLD ctivtion s seen in WKY PGSMCs. Therefore, the mount of AT 2 Rs pper to be the primry difference in Ang II-medited PLD ctivity between SHR nd WKY PGSMCs. 4.3 DISCUSSION Although the mjority of reports indicte tht there re no AT 2 Rs in the renl vsculture (198;199;204), the experiments presented here indicte tht there re AT 2 Rs on cultured WKY nd SHR PGSMCs. Furthermore, there re more AT 2 Rs on WKY compred to SHR PGSMCs, nd the rtio of AT 1 Rs to AT 2 Rs is incresed by pproximtely 3-fold in SHR PGSMCs compred to WKY PGSMCs. Importntly, the chnge in receptor rtio is due to both n increse in SHR AT 1 R levels, s well s to decrese in SHR AT 2 R levels compred to WKY PGSMCs. Becuse the AT 2 R generlly ttenutes AT 1 R-medited responses, the effect of the AT 2 R on Ang II/AT 1 R-medited PLD ctivity ws exmined. As expected, the AT 2 R ttenutes 59

76 PtdEtOH (% of totl lipid) WKY SHR * * * * * log[angiotensin II (M)] Figure 4.4: Ang II-Medited PLD Activity Concentrtion Response Curve. Ang II stimultes PLD in WKY nd SHR PGSMCs. However, Ang II is more potent ctivtor of PLD in SHR compred to WKY PGSMCs. Dt re expressed s Men ± SEM, n 3. * indictes tht the SHR is significntly greter thn WKY (P < 0.05) t the respective concentrtion. 60

77 A PtdEtOH (% of totl lipid) WKY + PD SHR + PD log[angiotensin II (M)] B C PtdEtOH (% of totl lipid) SHR SHR + PD PtdEtOH (% of totl lipid) WKY WKY + PD log[angiotensin II (M)] log[angiotensin II (M)] Figure 4.5: Effect of PD-123,319 on Ang II-Medited PLD Activtion. (A) Addition of the AT 2 R ntgonist, PD-123,319 (PD), normlizes the Ang II-medited PLD ctivity concentrtion-response curve of WKY PGSMCs to tht of SHR PGSMCs. (B) PD-123,319 increses the efficcy of the SHR concentrtion-response curve. However, the WKY concentrtionresponse curve (C) is significntly shifted leftwrd. Dt re expressed s Men ± SEM, n = 3. 61

78 A PtdEtOH (% of totl lipid) WKY + CGP SHR + CGP log[angiotensin II (M)] B C PtdEtOH (% of totl lipid) SHR SHR + CGP PtdEtOH (% of totl lipid) WKY WKY + CGP log[angiotensin II (M)] log[angiotensin II (M)] Figure 4.6: Effect of CGP-42112A on Ang II-Medited PLD Activtion. (A) Addition of the AT 2 R gonist, CGP-42112A (CGP), significntly lters the WKY nd SHR concentrtion-response curves. The SHR concentrtion-response curve is significntly shifted to the right decresing the potency of Ang II (B). However, the WKY concentrtionresponse curve (C) is significntly shifted downwrds, determined by ANOVA nlysis, thus decresing the efficcy of Ang II-medited PLD ctivity. Dt re expressed s Men ± SEM, n = 3. 62

79 Ang II-medited PLD ctivity, s shown with the AT 2 R ntgonist. However, the results with the AT 2 R gonist did not conform to the initil hypothesis tht the curve would be shifted to the right. Insted, ddition of the AT 2 R gonist decresed the efficcy of Ang II-medited PLD ctivity in WKY PGSMCs nd hd smll but significnt effect in SHR PGSMCs. The effects seen with the AT 2 R gonist might be explined by the differences in internliztion of the AT 1 R nd AT 2 R. As previously mentioned, the AT 1 R rpidly internlizes upon stimultion (27), but the AT 2 R does not undergo gonist-dependent internliztion (29). When the AT 1 R is internlized, Ang II is trfficked to the lysosome nd the receptor is recycled to the plsm membrne (30). Therefore, ddition of Ang II results in degrdtion of the gonist through internliztion of AT 1 Rs, s well s ecto-peptidses, but ddition of the AT 2 R gonist results in degrdtion only through the ecto-peptidse pthwy. Thus, ddition of fixed mount of the AT 2 R gonist does not result in competitive interction during the durtion of stimultion in these experiments (20 min) but genertes wht ppers to be constitutively ctive receptor. Using the nlogy of constitutively ctive receptor, n inhibitory receptor would turn off the observed response, which would pper s downwrd shift of, or complete elimintion of the concentrtion response curve. This is wht is seen in the WKY PGSMCs, nd the response tht is generted from Ang II (40% of control) is most likely due to the kinetics of inhibition of the AT 1 R response. As shown in Fig. 3.3, genertion of PA is rpid nd results in doubling of bsl PA levels. This prllels the results seen when nlyzing PLD ctivity, suggesting tht PLD is responsible for Ang II genertion of PA. Therefore, if the kinetics of inhibition re slower thn ctivtion of PLD, then some PLD ctivity will be observed. Although the nlogy to the constitutively ctive receptor explins the dt for Ang IImedited PLD ctivity in WKY PGSMCs, it fils to explin the observtions in SHR PGSMCs. 63

80 Since SHR PGSMCs fil to respond to the AT 2 R ntgonist, the smll yet significnt shift in the concentrtion-response curve in the presence of the AT 2 R gonist indictes tht SHR PGSMCs hve functionl AT 2 Rs. In ddition to hving few receptors, there ppers to be defect in AT 2 R signl trnsduction since the concentrtion-response curve in the presence of the AT 2 R gonist is shifted slightly to the right insted of slight decrese in efficcy. 4.4 CONCLUSIONS There re AT 1 Rs nd AT 2 Rs on cultured PGSMCs. AT 1 Rs re responsible for Ang IImedited PLD ctivtion, wheres AT 2 Rs inhibit AT 1 R-medited PLD ctivtion. The incresed potency of Ang II-medited PLD ctivity in SHR PGSMCs compred to WKY PGSMCs is due to n ltered rtio of AT 1 to AT 2 receptors. 64

81 Chpter 5 MECHANISM OF AT 2 R INHIBITION OF AT 1 R-MEDIATED ACTIVATION OF PLD 5.1 INTRODUCTION Multiple physiologicl studies indicte tht AT 2 Rs ntgonize AT 1 R-medited responses (37;200;201). Furthermore, AT 2 R knockout mice hve incresed blood pressure compred to controls nd disply enhnced responses to exogenous Ang II (33;34). Conversely, AT 1A R/AT 1B R double knockout mice hve lower blood pressure compred to controls nd lck the typicl increse in blood pressure fter exogenous Ang II (35). These dt suggest tht ltertions in the levels of Ang II receptors cn ffect bsl blood pressure nd Ang II-medited chnges in blood pressure. Therefore, the previous observtions tht the AT 2 R inhibits AT 1 R- medited PLD ctivity (Chpter 4) is not surprising, nd elevted PLD ctivity my serve s biomrker for the enhnced physiologicl responses to Ang II in hypertension. To understnd the mechnism of AT 2 R-medited inhibition of AT 1 R-medited PLD ctivtion, the signl trnsduction of the AT 2 R must be exmined. Unfortuntely, the signl trnsduction initited by the AT 2 R is poorly understood in comprison to the AT 1 R. However, the AT 2 R hs been shown to couple to Gα i/o proteins (18;19) nd signl vi inhibiting denylyl cyclse through Gα i/o, stimulting NOS (21;22), ctivting PLA 2 (23;24), nd ctivting vriety of phosphtses (25;26). Since no kinses hve been described in Ang II-medited PLD ctivity in PGSMCs (Chpter 3), AT 2 R-medited inhibition of Ang II-medited PLD ctivity most likely does not occur through phosphtse. Interestingly, in plnts, PLD cn be inhibited by the ddition of lysophosphtidylethnolmine (LPE) (205), which cn be produced through 65

82 PLA 2 pthwy. Therefore, one mechnism of AT 2 R-medited inhibition of PLD could be through ctivtion of PLA 2 resulting in the formtion of LPE nd subsequent inhibition of PLD. PKA nd PKG shre consensus phosphoryltion sites, nd, when this site is phosphorylted RhoA is inctivted due to interctions with Rho GDI (Fig 1.2). Since the AT 2 R couples to Gα i nd inhibits denylyl cyclse thus reducing camp levels, it is unlikely tht the AT 2 R is inhibiting AT 1 R-medited ctivtion of PLD through camp-pka mechnism. However, genertion of NO nd subsequent cgmp cn stimulte PKG ctivity nd, thus, my provide pthwy for the AT 2 R to inhibit AT 1 R-medited PLD ctivity by inctivting RhoA, which is required for the AT 1 R to ctivte PLD (Fig. 3.10). Therefore, the two likely cndidte pthwys for inctivtion of AT 1 R-medited PLD ctivity re through PLA 2 or NOS. The purpose of the following experiments ws to identify nd chrcterize the pthwy utilized by the AT 2 R to inhibit AT 1 R-medited PLD ctivity. 5.2 RESULTS EFFECT OF INHIBITING PLA 2 AND NOS. Since inhibition of the AT 2 R increses PLD ctivity t low concentrtions of Ang II in WKY PGSMCs, 10n M Ang II ws used to exmine the role of PLA 2 nd NOS. If either PLA 2 or NOS re involved in AT 2 R inctivtion of PLD, then ddition of 700 nm OBAA, PLA 2 inhibitor, or 1 µm NNA, NOS competitive ntgonist, should mimic PD-123,319 nd pertussis toxin. As shown in Figure 5.1, only NNA cted similrly to inhibiting the AT 2 R in WKY PGSMCs. There ws little effect of ny ntgonist in SHR PGSMCs most likely due to no significnt AT 2 R-medited signling t 10 nm Ang II (Fig. 4.5). Therefore, the dt indicte tht 66

83 PtdEtOH (% of totl lipid) WKY SHR b,c,b b,c,b c d d d,b,b,c b b d,e d d,e,c 0.0 Con OBAA NNA PD PTX Con + 10 nm Ang II OBAA + NNA + PD + PTX + Con OBAA NNA PD PTX Con + 10 nm Ang II OBAA + NNA + PD + PTX + Figure 5.1: Effects of Inhibiting PLA 2 nd NOS on Ang II-Medited PLD Activity. 10 nm Ang II-medited PLD ctivity ws significntly incresed by 1 µm NNA, 15 nm PD- 123,319 (PD) nd 1 ng/ml pertussis toxin (PTX) in WKY, but not SHR PGSMCs. 700 nm OBAA does not increse 10 nm Ang II-medited PLD ctivity to the extent of NNA, PD, nd PTX in WKY PGSMCs. Dt re expressed s Men ± SEM, n 3; brs with different letters re significntly different (P < 0.05). 67

84 AT 2 R stimultion of NOS medites AT 2 R inhibition of AT 1 R-medited PLD ctivity ADDITION OF NO INHIBITS ANG II-MEDIATED PLD ACTIVITY. If AT 2 R stimultion of NOS is responsible for AT 2 R inhibition of AT 1 R-medited PLD ctivity, then co-dministrtion of Ang II nd NO should decrese Ang II-medited PLD ctivity. As shown in Figure 5.2, ddition of NO with either 200 µm of the NO donors SNAP or DEANO decresed Ang II-medited PLD ctivity in WKY nd SHR PGSMCs. Use of the two structurlly different NO donors indicte tht the observed decrese is not due to the donor molecule. This is supported by ddition of DEANO incubted overnight t 37 C (DEA) thus losing most of the NO. Although DEA reduced PLD ctivity in WKY PGSMCs, DEA did not reduce PLD ctivity to the extent tht DEANO reduced PLD ctivity, nd DEA hd no effect in SHR PGSMCs indicting tht the effects of DEANO re due to NO nd not the donor molecule. NO reduced Ang II-medited PLD ctivity in WKY nd SHR PGSMCs, thus supporting the erlier observtion tht the primry defect in PLD regultion is the ltered AT 1 /AT 2 receptor rtio. The imblnce in receptor hypothesis ws confirmed by concentrtion-response curves with n AT 2 R ntgonist nd gonist (Fig. 4.5 nd 4.6). Addition of NO to the Ang II concentrtion-response curve should mimic the effects of the AT 2 R gonist CGP-42112A in the WKY. Additionlly, NO dontion bypsses the AT 2 R. Thus, the effects of NO on the concentrtion-response curve should be equl in WKY nd SHR PGSMCs. As shown in Figures 5.3 nd 5.4, ddition of NO decreses the efficcy of Ang II-medited PLD ctivity in both WKY nd SHR PGSMCs. In WKY PGSMCs the concentrtion-response curve generted in the presence of DEANO is indistinguishble from the concentrtion-response curve generted in the presence of CGP-42112A (Fig. 5.3). In SHR PGSMCs the concentrtion-response curve 68

85 PtdEtOH (% of totl lipid) WKY SHR b c d b,c b c c b 0.0 Con SNAP DEANO 1 µm Ang II Con + SNAP + DEANO + DEA + Con SNAP DEANO Con + 1 µm Ang II SNAP + DEANO + DEA + Figure 5.2: NO inhibits Ang II-Medited PLD Activity. 1 µm Ang II-medited PLD ctivity is significntly ttenuted by 200 µm of the structurlly distinct NO donors SNAP nd DEANO in both WKY nd SHR PGSMCs. 200 µm DEA, DEANO tht ws llowed to incubte overnight t 37 C thus losing most of the NO, does not ttenute Ang IImedited PLD ctivity. Dt re expressed s Men ± SEM, n = 3; brs with different letters re significntly different (P < 0.05). 69

86 generted in the presence of DEANO is significntly different thn the concentrtion-response curve generted in the presence of CGP-42112A (Fig. 5.4) due to bypssing the AT 2 R. Although these dt support the hypothesis tht the defect in the regultion of PLD is the imblnce of receptors, SHR PGSMCs re less sensitive to NO thn WKY PGSMCs (Fig. 5.5) SOLUBLE GUANYLATE CYCALSE GENERATION OF CGMP MEDIATES AT 2 R INHIBITION OF AT 1 R INDUCED PLD ACTIVITY. NO is known stimulnt of soluble gunylte cyclse (sgc). Therefore, the role of sgc in AT 2 R-medited inhibition of AT 1 R-medited ctivtion of PLD ws exmined with 10 µm YC-1, NO independent stimulnt of sgc, nd 10 µm ODQ, n inhibitor of sgc. Following the erlier prdigm, YC-1 should reduce Ang II-medited PLD ctivity. As shown in Figure 5.6, YC-1 inhibits 1 µm Ang II-medited PLD ctivity in WKY nd SHR PGSMCs. Additionlly, DEANO nd YC-1 hve equivlent effects in WKY PGSMCs suggesting tht they inhibit PLD through common pthwy. However, in SHR PGSMCs YC-1 hs little effect nd synergizes with DEANO suggesting tht greter stimultion of sgc is required in SHR PGSMCs to observe reductions of Ang II-medited PLD ctivity similr to WKY PGSMCs. Although there re mrked differences between WKY nd SHR PGSMCs, the dt indicte tht NO is cting through sgc to inhibit Ang II-medited PLD ctivity. To confirm this, 10 nm Ang II ws dministered in the presence of ODQ (Fig. 5.7, top). ODQ incresed Ang II-medited PLD ctivity similrly to PD-123,319 in WKY but not SHR PGSMCs, which is wht ws observed for NNA nd pertussis toxin (Fig. 5.1). To confirm tht sgc genertion of cgmp is responsible for AT 2 R inhibition of AT 1 R-medited PLD ctivity, 100 µm of the non-hydrolyzble cgmp 70

87 % Chnge in PLD ctivity (from Control) Ang II + DEANO + CGP log[ang II (M)] Figure 5.3: NO nd CGP-42112A Similrly Inhibit Ang II-Medited PLD Activity in WKY PGSMCs. Ang II ctivtes PLD resulting in mximl ctivity of pproximtely 2-fold over bsl in WKY PGSMCs. Addition of 100 nm CGP-42112A (CGP) or NO from 200 µm DEANO significntly reduces the efficcy of Ang II-medited PLD ctivity by pproximtely 40%. Dt re expressed s Men ± SEM, n 3. 71

88 % Chnge in PLD ctivity (from Control) Ang II + DEANO + CGP log[ang II (M)] Figure 5.4: NO but not CGP-42112A Reduces the Efficcy of Ang II-Medited PLD Activity in SHR PGSMCs. Ang II ctivtes PLD resulting in mximl ctivity of pproximtely 2-fold over bsl in SHR PGSMCs. Addition of NO from 200 µm DEANO significntly reduces the efficcy of Ang II-medited PLD ctivity by pproximtely 30%; however, this reduction ppers to be ttributed to 30% decrese in bsl PLD ctivity. Addition of 100 nm CGP-42112A (CGP) hs no effect on the efficcy of ANG IImedited PLD ctivity. Dt re expressed s Men ± SEM, n 3. 72

89 % Chnge in PLD ctivity (from Control) 120 SHR Ang II SHR + DEANO WKY Ang II WKY + DEANO log[ang II (M)] Figure 5.5: Differentil Effects of NO on Ang II-Medited PLD Activity in WKY nd SHR PGSMCs. Ang II ctivtes PLD resulting in mximl ctivtion of PLD t 1µM Ang II in both WKY nd SHR PGSMCs. Addition of NO significntly (P < 0.05) reduces the pprent efficcy of Ang II-medited PLD ctivity in WKY nd SHR PGSMCs s determined by ANOVA nlysis. However, the mgnitude of reduction in WKY PGSMCs is significntly greter (P = ) thn in SHR PGSMCs determined by t-test. However, unlike WKY PGSMCs the reduction in efficcy in SHR PGSMCs is equl to the reduction in bsl PLD ctivity. 73

90 PtdEtOH (% of totl lipid) WKY SHR,b c,b b,b b c d c,d b Con YC-1 1µM Ang II DEANO Y + D Con + YC-1 + DEANO + Y & D + 1µM Ang II Con YC-1 DEANO Y + D Con + YC-1 + DEANO + Y & D + Figure 5.6: Stimultion of sgc Inhibits Ang II-Medited PLD Activity. 1 µm Ang II-medited PLD ctivity is significntly ttenuted by 10 µm the NO independent stimulnt of sgc, YC-1, in both WKY nd SHR PGSMCs. In WKY PGSMCs YC-1 nd 200 µm DEANO in combintion (Y & D) does not further ttenute Ang II-medited PLD ctivity compred to YC-1 or DEANO lone. However, in SHR PGSMCs, the ddition of YC-1 nd DEANO is required to obtin similr mgnitude of inhibition of Ang II-medited PLD ctivity observed in WKY PGSMCs indicting tht there is defect in NO signling in SHR PGSMCs. Dt re expressed s Men ± SEM, n = 3; brs with different letters re significntly different (P < 0.05). 74

91 PtdEtOH (% of totl lipid) ,b WKY SHR b c c b b,c c 0.0 Con ODQ PD 10 nm Ang II Con + ODQ + PD + Con ODQ PD 10 nm Ang II Con + ODQ + PD b PtdEtOH (% of totl lipid) c b b Con cgmp 1 µm Ang II Con + cgmp + Con cgmp 1 µm Ang II Con + cgmp + Figure 5.7: Effects of sgc/cgmp on Ang II-Medited PLD Activity. 10 nm Ang II-medited PLD ctivity is significntly enhnced by 10 µm of the sgc inhibitor ODQ similrly to 15 nm PD-123,319 (PD) in WKY, but not SHR PGSMCs (top). Conversely, ddition of 100 µm 8-Br-cGMP (cgmp) ttenutes 1 µm Ang II-medited PLD ctivity in WKY, but not SHR PGSMCs (bottom). Dt re expressed s Men ± SEM, n 3; brs with different letters re significntly different (P < 0.05). 75

92 nlogue, 8-Br-cGMP, ws used to inhibit Ang II-medited PLD ctivity (Fig. 5.7, bottom). 8- Br-cGMP inhibited Ang II-medited PLD ctivity in WKY, but not SHR PGSMCs. Therefore, the AT 2 R is signling through n sgc-dependent mechnism to inhibit AT 1 R-medited PLD ctivity, nd SHR PGSMCs pper to be less sensitive to sgc stimulnts nd cgmp THE AT 2 R INHIBITS ANG II-MEDIATED PLD ACTIVITY THROUGH PHOSPHORYLATION OF RHOA. To exmine the role of phosphoryltion of RhoA in AT 2 R-medited inhibition of AT 1 R- medited PLD ctivity, the PKA/PKG phosphoryltion-deficient mutnt S188A RhoA ws stbly trnsfected into WKY PGSMCs to exmine the role of phosphoryltion of RhoA in AT 2 R- nd NO-medited inhibition of AT 1 R-medited PLD ctivity. Since S188A RhoA cnnot be inctivted by phosphoryltion, NO should not reduce Ang II-medited PLD ctivity in cells expressing S188A RhoA. As shown in Figure 5.8, fter ddition of 200 µm DEANO there is slight reduction in PLD ctivity in the S188A RhoA cells, compred to control. This is most likely due to inhibition of endogenous RhoA. These dt were confirmed by generting Ang IImedited PLD ctivity concentrtion-response curves with S188A RhoA expressing WKY PGSMCs in the presence nd bsence of 200 µm DEANO. As shown in Figure 5.9, DEANO hd no effect on the Ang II medited PLD ctivity concentrtion-response curve (Fig. 5.9, top). Thus, NO inhibits Ang II-medited PLD ctivity through phosphoryltion of RhoA t S188A, which inctivtes RhoA by promoting ssocition of Rho GDI with RhoA. Importntly, the concentrtion-response curve from the S188A RhoA cells is shifted to the left of the WKY PGSMCs nd more closely resembles the SHR concentrtion-response curve (Fig. 5.9, bottom). 76

93 PtdEtOH (% of totl lipid) Control S188A b c d b b e f 0.00 Control 1µM Ang II DEANO 1µM Ang II + DEANO Figure 5.8: Expression of S188A RhoA Prevents NO-Medited Inhibition of 1µM Ang II-Medited PLD Activity. DEANO inhibits Ang II-medited PLD ctivity in WKY PGSMCs; however, upon expression of S188A RhoA elimintes the effects of DEANO hs no effect on Ang II-medited PLD ctivity. Dt re expressed s Men ± SEM, n = 3; brs with different letters re significntly different (P < 0.05). 77

94 PtdEtOH (% of totl lipid) 1.10 S188A S188A + DEANO log[ang II (M)] % Chnge in PLD ctivity (from Control) WKY WKY S188A RhoA SHR log[ang II (M)] Figure 5.9: Expression of S188A RhoA Elimintes NO-Medited Inhibition of Ang II- Medited PLD Activity nd Shifts the Concentrtion-Response Curve Towrds the SHR PGSMCs Concentrtion-Response Curve. Expression of S188A RhoA elimintes the NO-medited decrese in efficcy (top), nd shifts the Ang II-medited PLD ctivity concentrtion-response curve leftwrds in such wy tht it resembles the SHR curve more so thn the WKY curve (bottom). Dt re expressed s Men ± SEM. 78

95 The EC 50 vlues for the four curves re reported in Tble 5.1. The EC 50 vlues for WKY PGSMCs expressing S188A RhoA re similr to SHR, not WKY PGSMCs. In order to confirm tht the AT 2 R-medited phosphoryltion of RhoA t S188 is responsible for AT 2 R-medited inhibition of Ang II-medited PLD ctivity, the effects of PD- 123,319 nd CGP-42112A on Ang II-medited PLD ctivity in WKY PGSMCs expressing S188A RhoA ws compred to WKY PGSMCs. Inhibition of the AT 2 R in WKY PGSMCs incresed 10 nm Ang II-medited PLD ctivity by 2-fold, wheres there ws no effect of AT 2 R inhibition in WKY PGSMCs expressing S188A RhoA (Fig. 5.10, top). Likewise, stimultion of the AT 2 R receptor hs no effect in WKY PGSMCs expressing S188A RhoA, but reduces Ang IImedited PLD ctivity in WKY PGSMCs (Fig. 5.10, bottom). Thus, the dt indicte tht the AT 2 R leds to phosphoryltion, nd, thus, inctivtion of RhoA t S188. Tble 5.1: Comprison of Ang II-medited PLD Activity EC 50 Vlues in SHR, WKY, nd WKY expressing S188A RhoA PGSMCs. Curve: WKY* SHR* WKY S188A WKY S188A + DEANO EC 50 : 128 nm 10 nm 11 nm 13 nm * indictes EC 50 obtined from trnsformed dt (Fig. 5.9, bottom). 5.3 DISCUSSION The signl trnsduction mechnisms nd physiologicl effects of the AT 2 R re poorly chrcterized. However, use of AT 2 R drugs in physiologicl experiments indicte tht the AT 2 R countercts AT 1 R-medited effects (37;200;201). Furthermore, the AT 2 R knockout mice hve incresed blood pressure compred to litter mtes tht express the AT 2 R (33). The precise mechnisms by which the AT 2 R inhibits AT 1 R-medited effects re uncler. There re 4 79

96 Fold Chnge in PLD Activity by PD-123, WKY WKY S188A b 0.0 Control 10 nm Ang II Fold Chnge in PLD Activity by CGP-42112A b 0.00 Control 100 nm Ang II Figure 5.10: Expression of S188A RhoA Elimintes AT 2 R-Medited Inhibition of Ang II-Medited PLD Activity. PD-123,319 increses Ang II-medited PLD ctivity in WKY PGSMCs but hs no effect in WKY PGSMCs expressing S188A RhoA (top). Similrly, CGP-42112A fils to inhibit Ang II-medited PLD ctivity in the presence of S188A RhoA (bottom). Dt re expressed s Men ± SEM of AT 2 R drug/control, n = 3; gry dotted line represents expected vlues if the AT 2 R drug hs no effect. Brs with different letters re significntly different (P < 0.05). 80

97 seprte signl trnsduction pthwys identified thus fr tht the AT 2 R ctivtes tht my inhibit AT 1 R-medited effects. As presented in Chpter 4, the AT 2 R inhibits AT 1 R-medited PLD ctivity. Since AT 2 R-medited inhibition of denylyl cyclse nd ctivtion of phosphtses does not fit into the pthwy of Ang II-medited ctivtion of PLD (Chpter 3), the role of PLA 2 nd NOS were exmined s possible mechnisms for AT 2 R-medited inhibition of AT 1 R- medited ctivtion of PLD. The dt indicte tht AT 2 R inhibition of Ang II-medited PLD ctivity is due to signling through NOS nd sgc, presumbly through ctivtion of PKG, nd subsequent phosphoryltion of RhoA t S188. As depicted in Figure 5.11, Ang II ctivtion of PLD2 is medited by the AT 1 R nd requires RhoA. The AT 2 R inhibits PLD ctivity through phosphoryltion of RhoA resulting in inhibition of RhoA regrdless of the ctivtion stte of RhoA (45). This signl trnsduction scheme is supported by the dt presented here, except for the role of PKG. Since cgmp is known ctivtor of PKG (206) nd PKG phosphoryltes S188 of RhoA (44), the most compelling conclusion is tht PKG medites phosphoryltion of RhoA. However, NO/cGMP cn led to the ctivtion of PKA ( ). Interestingly, inhibition of PKA with 1 µm H89 incresed 10 nm Ang II-medited PLD ctivity in WKY PGSMCs, wheres in SHR PGSMCs H89 reduced bsl PLD ctivity, nd 10 nm Ang II-medited PLD ctivity (Appendix A). However, in SHR PGSMCs, the reduction of bsl nd 10 nm Ang II-medited PLD ctivity is similr, indicting tht H89 is not inhibiting Ang II-medited PLD ctivity in SHR PGSMCs. These dt suggest tht PKA my be involved in AT 2 R-medited inhibition of PLD through phosphoryltion of RhoA. Since PKA nd PKG phosphorylte the sme sequence in RhoA (43), ctivtion of either kinse will result in the sme effect on Ang II-medited PLD ctivity. 81

98 Ang II AT 2 NOS sgc ** S GAP PKG or PKA Active Rho GTP bound GAP + Pi Inctive Rho GDP bound GEF ** PO GEF/GAP 4-S188 S GDI P Rho GDP or GTP bound GEF AT 1 + GTP? + GDP PLD Figure 5.11: Ang II Signl Trnsduction Involved in PLD Regultion. A digrm of how Ang II ctivtes PLD through AT 1 Rs nd inctivtion of the AT 1 R-medited PLD ctivity through AT 2 R-medited signling resulting in inctivtion of RhoA. 82

99 Therefore, regrdless of which kinse phosphoryltes RhoA, the dt presented here indicte tht the AT 2 R leds to phosphoryltion of RhoA t S188 through NO/cGMP medited mechnism. Additionlly, these dt provide second hypothesis to explin the results obtined with CGP-42112A in WKY PGSMCs. As depicted in Figure 5.11, ctivtion of PLD by the AT 1 R my occur in t lest 3 steps fter receptor ctivtion: ctivtion of Rho GEF, ctivtion of RhoA, nd finlly RhoA ctivtion of PLD. The AT 2 R medited inhibition of RhoA occurs in more steps with miniml step count of 4 fter receptor ctivtion: ctivtion of NOS, genertion of NO nd subsequent ctivtion of sgc, genertion of cgmp nd subsequent ctivtion of PKG, nd finlly phosphoryltion of RhoA. Thus, the kinetics of ctivtion of RhoA nd subsequently PLD my be quicker thn the kinetics of inctivtion of RhoA. Evidence for this hypothesis is tht there is slight increse in PLD ctivity in the presence of the AT 2 R gonist (Fig. 4.6c) due to RhoA signling to PLD before RhoA cn be inctivted through phosphoryltion. This hypothesis is further supported by the DEANO nd YC-1 experiments tht indicte tht t mximl reduction of PLD ctivity there is still some PLD ctivity tht cn be explined by time dely to inctivtion. However, cvet in the NO dt is tht lthough NO is further downstrem thn the receptor nd should therefore ct quicker thn CGP-42112A, the exogenously NO must be relesed from the chemicl donor nd diffuse through the medi nd cell to its trget proteins tht then led to the inctivtion of RhoA. Unfortuntely, the kinetics of NO dontion by DEANO nd subsequent NO ctivtion of sgc re unknown in this system. Thus, direct kinetic mesurements would hve to be mesured for AT 2 R medited inhibition of RhoA/PLD to determine if the hypothesis regrding the kinetics of ctivtion is vlid. 83

100 5.4 CONCLUSIONS AT 2 Rs inhibit AT 1 R-medited PLD ctivtion through NO/cGMP-dependent mnner, most likely resulting in phosphoryltion of RhoA t S188. The ltered rtio of AT 1 to AT 2 receptors nd insensitivity to NO nd cgmp in SHR PGSMCs contribute to the incresed PLD ctivity observed in SHR compred to WKY PGSMCs. 84

101 Chpter 6 ROLE OF PLD IN ANG II-MEDIATED ACTIVATION OF MEK AND ERK 6.1 INTRODUCTION Hypertension in the SHR is disese primrily of the kidney vsculture (7;8;189) tht involves incresed responses to Ang II with regrds both to growth (212) nd contrctility of renl vsculr smooth muscle cells (9;11). Recent evidence indictes tht PD98059, MEK inhibitor, reduces Ang II-medited vsculr smooth muscle cell contrction in WKY nd SHR, nd normlizes the contrction of SHR to the WKY (91). PD98059, however, does not ttenute Ang II-medited vsculr contrction of isolted thorcic ort (94) indicting tht MEK my not be involved in contrction. To ddress this pprent disprity, in vivo experiments with PD98059 were conducted. In these experiments, PD98059 reduced blood pressure of hypertensive rts (93) nd ttenuted Ang II-medited increses in men rteril blood pressure in normotensive rts (92). Therefore, the dt indicte tht MEK is involved in Ang II-medited vsoconstriction. Becuse MEK is component of the ERK MAPK cscde, nd MAPK is involved in cell growth (213), MEK ctivtion could ply role in both the incresed contrctility nd cell growth observed in SHR renl vsculr smooth muscle cells. Therefore, elucidting the signl trnsduction mechnism mediting Ang II-induced MEK nd ERK ctivtion in WKY nd SHR PGSMCs my led to novel therpies for hypertension. Previous studies indicted tht in some systems PLD2 genertion of PA is required for trnsloction of Rf-1 to membrnes, nd disruption of Rf-1 trnsloction ttenutes gonistmedited signling to ERK (121;153). Furthermore, Ang II-induced ctivtion of ERK requires 85

102 PLD2 genertion of PA in rt smooth muscle cell line (120). Interestingly, PLD genertion of PA is incresed in SHR cells when compred to WKY (112)(Fig. 3.2), nd Ang II-medited ERK ctivtion is incresed in smooth muscle cells from SHR compred to WKY (86). These dt suggest tht PLD leds to ctivtion of ERK in PGSMCs, nd incresed ctivtion of PLD by Ang II in SHR my be responsible for the observed increses in Ang II-medited ERK ctivtion. Therefore, the purpose of this study ws to test the hypothesis tht PLD2 is required for Ang II-medited ctivtion of MEK nd ERK in WKY nd SHR PGSMCs. 6.2 RESULTS ANG II STIMULATES ERK1/2. As shown in Figure 6.1, 10 nm Ang II leds to greter ctivtion of ERK1/2 in SHR compred to WKY PGSMCs. These dt re similr to wht hs been previously reported (86) ANG II STIMULATION OF PLD2 AND SUBSEQUENT GENERATION OF PA IS REQUIRED FOR ACTIVATION OF MEK AND ERK1/2. As previously indicted (Fig. 3.5), WKY nd SHR PGSMCs trnsfected with dnpld2 reduced Ang II-medited PLD ctivity by pproximtely 50% (Fig. 6.2), figure tht correltes with the observed trnsfection efficiency. These dt confirm tht PLD2 is the PLD isoform ctivted by Ang II. To test the hypothesis tht PLD2 is involved in Ang II-medited ctivtion of MEK nd ERK1/2, dnpld2 ws trnsfected into WKY nd SHR PGSMCs nd the bility of 1 µm Ang II to ctivte MEK nd ERK1/2 ws ssessed by Western blot. After stimultion with Ang II, phospho-mek levels were significntly decresed by pproximtely 50% in the presence 86

103 ERK Activtion (PO 4 -ERK/Totl ERK) WKY SHR b c 0.0 Control 10 nm Ang II Figure 6.1: Ang II Activtion of ERK1/2 is Greter in SHR thn WKY PGSMCs. 10 nm Ang II ctivtes ERK1/2 nerly 2-fold more in SHR compred to WKY PGSMCs. Dt re expressed s Men ± SEM, n = 3; brs with different letters re significntly different (P < 0.05). 87

104 PtdEtOH (% of totl lipid) WKY SHR b b c c Control dnpld2 1 µm Ang II 1 µm Ang II + dnpld2 Figure 6.2: dnpld2 Reduces Ang II-Medited PLD Activity. Ang II-medited PLD ctivity is medited by PLD2 in WKY nd SHR PGSMCs. Dt re expressed s Men ± SEM, n 6; brs with different letters re significntly different (P < 0.05). 88

105 of dnpld2 (Fig. 6.3). Similrly, phospho-erk1/2 levels were significntly decresed by pproximtely 50% in the presence of dnpld2 (Fig. 6.3b). Since reduction in Ang II-medited PLD ctivity nd trnsfection efficiency ws pproximtely 50%, these dt indicte tht dnpld2 is required for Ang II-medited phosphoryltion of MEK nd ERK1/2. In order to confirm tht the expression of dnpld2 did not inhibit Ang II-medited ctivtion of ERK through PA independent mechnism, PA ws dded bck to the cells to recover Ang IImedited phosphoryltion of ERK1/2 (Fig. 6.4). As predicted, ddition of PA prtilly recovered Ang II-medited phosphoryltion of ERK1/2 in the presence of dnpld2 in WKY PGSMCs, but did not significntly increse ERK1/2 phosphoryltion in SHR PGSMCs. Although not significnt, PA did increse Ang II-medited phosphoryltion of ERK1/2 in the presence of dnpld2 in SHR PGSMCs. Therefore, exmining the WKY nd SHR together indicte tht PLD2 by generting PA plys key role in the ctivtion of MEK nd ERK1/2 in SHR nd WKY PGSMCs CELLULAR LOCALIZATION OF ACTIVATED MEK AND ERK1/2 AFTER STIMULATION WITH ANG II. In order to confirm tht dnpld2 inhibits Ang II signling to MEK nd ERK1/2, confocl imges of fixed cells stined for phospho-mek nd phospho-erk1/2 were compred to cells trnsfected with dnpld2 nd control cells. Since EGF stimultes ERK1/2 without stimulting PLD (Fig. 6.5), EGF ws used s positive control for stimultion of MEK nd ERK. As shown in Figure 6.6, essentilly no phospho-mek or phospho-erk1/2 is seen in control cells (1 st row); tretment with 1µM Ang II for 5 min (2 nd row) significntly increses stining compred to the control nd cells trnsfected with dnpld2 (3 rd row), wheres tretment with EGF show bundnt 89

106 PO 4 - MEK WKY SHR Shm dnpld2 Shm dnpld2 PO 4 - ERK 1/2 b WKY SHR Shm dnpld2 Shm dnpld2 MEK ERK 1/2 % MEK Phosphoryltion WKY * SHR * % ERK Phosphoryltion WKY * SHR * Figure 6.3: dnpld2 Attenutes Ang II-Medited MEK nd ERK1/2 Phosphoryltion. 1 µm Ang II-medited phosphoryltion of MEK () nd ERK1/2 (b) ws exmined in WKY nd SHR PGSMCs shm-trnsfected or trnsfected with dnpld2. Phospho-MEK ws decresed in the presence of dnpld2 (, upper pnel); repliction of the experiment indictes tht dnpld2 significntly ttenuted Ang II signling to MEK in WKY nd SHR PGSMCs (, lower pnel) (n = 3). dnpld2 lso decresed Ang II-medited phosphoryltion of ERK1/2 (b, upper pnel); repliction of the experiment indictes tht dnpld2 significntly ttenuted ERK phosphoryltion in WKY nd SHR PGSMCs (b, lower pnel) (n = 3), * indictes P < 0.05, control versus dnpld2. 90

107 WKY SHR Totl ERK1/2 Totl ERK1/2 PO 4 - ERK1/2 PO 4 - ERK1/ µM Ang II dnpld2 200 µm PA ERK1/2 Activtion (PO 4 -ERK1/2/totl ERK) b c b b WKY µm Ang II dnpld2 200 µm PA + SHR Figure 6.4: PA Prtilly Recovers dnpld2-medited Inhibition of Ang II-Medited ERK1/2 Activtion. Addition of 200 µm PA significntly increses Ang II-medited ctivtion of Erk1/2 in the presence of dnpld2 (top). Repliction of the experiments (bottom) indictes tht PA only prtilly recovers ERK1/2 ctivity, nd the increse in ctivity is only significntly incresed in WKY, not SHR PGSMCs. Dt re expressed s Men ± SEM, n = 3; brs with different letters re significntly different (P < 0.05). 91