Plasma converting enzyme activity during the development and maintenance of experimental and spontaneous in rats

Transcription

1 Atlanta University Center W. Wdruff Library, Atlanta University Center ETD Cllectin fr AUC Rbert W. Wdruff Library Plasma cnverting enzyme activity during the develpment and maintenance f experimental and spntaneus in rats Walter Len Salters Atlanta University Fllw this and additinal wrks at: Part f the Bilgy Cmmns Recmmended Citatin Salters, Walter Len, "Plasma cnverting enzyme activity during the develpment and maintenance f experimental and spntaneus in rats" (1976). ETD Cllectin fr AUC Rbert W. Wdruff Library. Paper This Thesis is brught t yu fr free and pen access by DigitalCmmns@Rbert W. Wdruff Library, Atlanta University Center. It has been accepted fr inclusin in ETD Cllectin fr AUC Rbert W. Wdruff Library by an authrized administratr f DigitalCmmns@Rbert W. Wdruff Library, Atlanta University Center. Fr mre infrmatin, please cntact cwiseman@auctr.edu.

2 PLASMA CONVERTING ENZYME ACTIVITY DURING THE DEVELOPMENT AND MAINTENANCE OF EXPERIMENTAL AND SPONTANEOUS HYPERTENSION IN RATS A THESIS SUBMITTED TO THE FACULTY OF ATLANTA UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY WALTER LEON SALTERS DEPARTMENT OF BIOLOGY ATLANTA, GEORGIA DECEMBER 1976

3 ABSTRACT BIOLOGY SALTERS, WALTER LEON B.S., Claflin Cllege, 1957 M.S., Atlanta University, 1964 Plasma Cnverting Enzyme Activity During the Develpment and Maintenance f Experimental and Spntaneus Hypertensin in Rats Advisr: Dr. Jseph B. Myers Dctr f Philsphy degree cnferred May 16, 1977 Thesis dated December 1976 The precise identificatin f the frm f angitensin recvered 'frm in vitr incubatin fr plasma renin activity (PRA) determinatins has becme increasingly imprtant with chemical means fr quantisatin such as radiimmunassay. The ability t discriminate A I and A II quantitatively in mixtures f these peptides als prvides means fr quantitative assessment f angitensin cnverting enzyme activity. Cnsequently, measurement f the rati f A I and A II recvered as the 37 C incubatin prduct in plasma by radiimmunassay has been used t examine plasma cnverting enzyme activity (PCEA). The antibdy capture methd was used fr the determinatin f A II cncentratins in plasma frm 3 experimentally induced hypertensive mdels f male Sprague-Dawley rats. This methd prvides fr the capture f A II frmed by the actin f the plasma cnverting enzyme by use f an excess f A II antiserum during the 37 C incubatin prcess. Simul taneus measurements f A I cncentratins which demnstrates PRA's were als determined. Whenever an increase r decrease in PRA was bserved, iii

4 similar activities were demnstrated by the plasma cnverting enzyme. As bld pressure increased in ne mdel f hypertensive rats that had the left renal artery cnstricted with simultaneus remval f the right kidney, an increase in bth PRA and PCEA was bserved. Increases in these enzymatic activities, apparently caused by renal artery cn strictin, are assumed t be due t a negative sdium balance. Sdium depletin f this type is believed t be due t an excessive lss f s dium frm the circulating bld by the kidney tubules. These physilgic phenmena apparently result in increased renal enzymatic activities. In cntrast t these findings, similar PRA and PCEA activities were bserved in ne kidney nephrectmized cntrl rats at 10 days. Hwever, decreases in PRA and PCEA at 20 and 30 days indicate that sme animals with nly ne functinal kidney presumably have the ability t aut-regulate, and thereby maintain hmestasis. These results indicate an active physi lgical rle f the cnverting enzyme in the maintenance f bld pressure. Increases r decreases in PCEA result in either increased r decreased cncentratins f A II, the vascnstrictr hrmne f the renin-angitensin system. The same enzymatic activities were significantly reduced in a grup f rats given 1.5% saline t drink fr 30 days. The results were assumed t be caused by suppressin f the renin-angitensin system due t chrnic salt ingestin. In anther grup f rats that were administered injectins (ip) f 5-hydrxytryptphan simultaneusly with injectins (sc) f estradil benzate, PRA and PCEA decreased by 100% and 131.2%, respectively, frm that f the cntrl animals. The sertnin hrmne precursr, 5-HTP, apparently has a nephrtxic effect n the kidney, thereby causing iv

5 decreased enzymatic activities. The ability f the radiimmunassay prcedures used t detect very lw cncentratins f A I and All was determined prir t the assay n experimental plasma samples. Plasma frm 36 hr 2-kidney nephrectmized rats and plasma frm a grup f SHR's was assayed t make these deter minatins. PRA and PCEA were significantly decreased in plasma frm the 2-kidney nephrectmized rats due t the remval f the surce f the renal enzyme (renin). PRA was 60 times greater in the SHR's plasma than that bserved in the 2-kidney nephrectmized rats. In the meantime PCEA was increased by apprximately 700%. While these results shw the va lidity and effectiveness f the radiimmunassay, they simultaneusly implicate the cnverting enzyme (CE) as an imprtant factr in regulating the levels f A II in plasma which subsequently regulate bld pressure. These results further imply that it is imprtant t measure cnverting enzyme activity (CEA) under cnditins in which the renin-angitensin system is suspected f playing a rle. This is evident frm these results since regulatin f CEA might nt be reflected in measured PRA. Under these cnditins the imprtance f CEA is a better index f hypertensin f a renal rgin than renin, because the actin f the CE is the last step in the enzymatic reactins f the renin-angiten sin system, with the subsequent liberatin f A II.

6 ACKNOWLEDGMENTS I wish t express eternal thanks t my mther and father, wh inspired in me the value f the necessity t btain an educatin. I am als grateful t my graduate advisr, Dr. Jseph B. Myers, fr guidance and instructin thrughut this perid f study at Atlanta University. Finally, but nne in the least d I express gratitude and thanks t my wife, Grace, and daughter, Damita, fr their patience and encurage ment during the pursuit f this study. This investigatin was supprted (in part) by the Natinal Institutes f Health MARC Fellwships N. 5-F14-GM and 1-F32-GM frm the Natinal Institute f General Medical Sciences. VI

7 LIST OF FIGURES Figure Page 1. Terminal mean bld pressures f experimentally induced hypertensive rats, cmpared t their cntrls and spntaneusly hypertensive rats Terminal mean bld pressures f 10, 20,and 30- day ne kidney Gldblatt and salt-induced hypertensive rats, cmpared t their cntrls Plasma cnverting enzyme activity f cntrl and experimental plasma samples, expressed as a functin f generated and endgenus A II cncentratins Generated A II cncentratins f experimental plasma samples, cmpared t their cntrls, 36 hr tw kidney nephrectmized and spntaneusly hypertensive rats Endgenus A II cncentratins f experimental plasma samples, cmpared t their cntrls, 36 hr tw kidney nephrectmized and spntaneusly hypertensive rats Plasma renin activity f cntrl and experimental plasma samples,expressed as a functin f generated and endgenus A I cncentrati ns Generated A I cncentratins f experimental plasma samples, cmpared t their cntrls, 36 hr tw kidney nephrectmized and spntaneusly hypertensive rats Endgenus A I cncentratins f experimental plasma samples, cmpared t their cntrls, 36 hr tw kidney nephrectmized and spntaneusly hypertensive rats Plasma cnverting enzyme activity f 10, 20, and 30- day ne kidney Gldblatt and salt-induced experimental plasma samples, cmpared t their cntrls Generated A II cncentratins f 10, 20, and 30-day ne kidney Gldblatt and salt-induced experimental plasma samples, cmpared t their cntrls Endgenus A II cncentratins f 10, 20, and 30-day ne kidney Gldblatt and salt-induced experimental piasma samples, cmpared t their cntrls 47 vii

8 12. Plasma renin activity f 10, 20, and 30-day ne kidney Gldblatt and salt-induced plasma samples, cmpared t their cntrls Generated A I cncentratins f 10, 20, and 30-day ne kidney Gldblatt and salt-induced experimental plasma samples, cmpared t their cntrls Endgenus A I cncentratins f 10, 20, and 30- day ne kidney Gldblatt and salt-induced experimental plasma samples, cmpared t their cntrl s 53 vm

9 LIST OF TABLES Table Page 1. Radiimmunassay prtcl fr angitensin II (A II) determinatins Statistical data cmparing ne kidney Gldblatt mdel t their cntrls, and values frm 36 hr tw kidney nephrectmized and spntaneusly hypertensive rats Statistical data cmparing salt-induced mdel t their cntrls, and values frm 36 hr tw kidney nephrectmized and spntaneusly hypertensive rats Statistical data cmparing 5-hydrxy-tryptphan mdel t their cntrls, and values frm 36 hr tw kidney nephrectmized and spntaneusly hypertensive rats 35 IX

10 TABLE OF CONTENTS ABSTRACT 111 Page ACKNOWLEDGMENTS vi LIST OF FIGURES vii LIST OF TABLES 1x CHAPTER I. INTRODUCTION 1 II. REVIEW OF LITERATURE 6 III. MATERIALS AND METHODS 21 Materials 21 Methds f Prcedure 22 Bld pressure measurement 22 Surgical prcedures 23 Cllectin f bld samples 23 One kidney Gldblatt hypertensive mdel 23 Salt-induced hypertensive mdel 24 Malignant hypertensive mdel 24 Spntaneusly hypertensive mdel 25 Radiimmunassay f angitensin I 25 Radiimmunassay f angitensin II 27 IV. RESULTS 30 Bld pressure measurements 30 Validity f radiimmunassay fr determinatin f A I and A II cncentratins 36 Plasma cnverting enzyme activities in the experimental hypertensi ve mdels 40 x

11 Generated and endgenus A II plasma cncentratins 45 PIasma reni n acti vi ty 48 Generated and endgenus A I cncentratins 50 V. DISCUSSION AND CONCLUSION 54 VI. SUMMARY 68 LITERATURE CITED 72 xi

12 CHAPTER I INTRODUCTION The renin-angitensin system has been under investigatin fr many years. It has been established that the renal enzyme, renin, acts n the renin substrate, antigensingen. This reactin results in the frmatin f the decapeptide angitensin I (A I), generally believed t be bilgically inactive. T achieve bilgical activity, A I must be cleaved by a cnverting enzyme in the circulatin. This reactin pr duces the bilgically active ctapeptide angitensin II (A II). Based n these bservatins, the cnverting enzyme is the apparent crnerstne in the renin-angitensin system, yet little is knwn abut its physi lgical activity. The discvery and descriptin f the A I cnverting enzyme frm plasma may be dated frm the early wrk f Skeggs et al. (1954,1956) and that f Helmer (1957). Except fr the wrk f these investigatrs, and especially Ng and Vane (1968), wh indicated that mst f the angi tensin cnversin ccurs in the lungs, little is knwn abut the cnvert ing enzyme. Early studies with this enzyme implied that the primary site whereby A I was cnverted t the active frm, A II, was lcalized in plasma. It was sn realized hwever, that the rate f cnversin by the plasma enzyme was nt sufficient t accunt fr the rapid rise in bld pressure fllwing intravenus injectin. The tissue respnsible fr the primary cnversin remained unknwn until the wrk f Ng and Vane (1968). They fund that the primary site f cnversin appeared t be 1

13 2 the lungs. Supprt fr this site was als reprted by Huggins and Thampi (1968) wh bserved that under in vitr cnditins, lungs cn tained a relatively high cntent f cnverting enzyme. Cushman and Cheung (1969) reprted studies n the purificatin f A I cnverting enzyme frm dg lung hmgenates and described sme f its kinetic pr perties. Oparil et al. (1970) have shwn a rapid pulmnary cnversin f tritium-labeled A I under in viv cnditins. The first methd fr measuring plasma cnverting enzyme activity (PCEA) was reprted by Skeggs et al. (1956) and was based n an eighttube cuntercurrent distributin prcedure fr the separatin f A I frm A II. Helmer (1955) used a spirally cut strip f rabbit arta which respnded t A II but nt t A I, in rder t measure the capacity f human plasma t frm A II. Mre recently, A I labeled at the C- terminal amin acid was used by Huggins and Thampi (1968) fr the mea surement f A I cnverting enzyme activity. Bucher et al. (1970) used a catin exchange adsrptin technique fr the same purpse. The pr cedure cnsisted f the elutin f A I and II adsrbed n ne clumn f 50W-X8 (NH4+) Dwex resin int a secnd clumn. A I was adsrbed n the secnd clumn f 50W-X8 Dwex resin, while A II went thrugh and was cllected in a si 1 icnized flask and later lyphilized. The amunt f A II frmed by the actin f the cnverting enzyme n the A II substrate after a 20 min incubatin perid at 47 C was measured by bilgical assay using nephrectmized rats. Reprducibility was within a range f 15%. All these methds essentially measure A II nce it has been separated frm A I, r they measure A II in the presence f A I n the assumptin that the radiimmunassays are highly specific fr bth angitensins.

14 3 The develpment f radiimmunassay techniques, with their applicatin t lw mlecular weight peptides, has permitted the measure ment f A I and A II (Waite, 1972b). In general, it appears that there has been little difficulty in raising antisera f high specificity and avidity t bth A I and A II. These antibdies have been raised against immungens prepared in a variety f ways. The immungen cmplexes have cnsisted f angitensin with prcine gamma glbulin, rat serum albumin (Gdfriend et al., 1964), ply-l-lysine (Haber et al., 1965) and micrparticles f carbn (Peart, 1969). These varius immungens have been emplyed in different animals, and with varied immunizatin schedules. The animal mst ften used has been the rabbit. With the presence f tyrsine in the cmpund, the preparatin f labeled A I with 125I r IJII f high specific activity has been achieved with cmparative ease using the chlramine-t methd f Hunter and Greenwd (1962). The develpment f suitable extractin prcedures has in recent years permitted the measurement f the cncentratin f A II in bld r plasma. Until recently, hwever, the radiimmunassay f A I has been limited t the measurement f plasma renin cncentratin. Mre recently, suitable techniques fr the extractin f A I have been develped and the circulating levels f A I have been measured. Using the assay tech nique f Brwn et al. (1966) t measure plasma renin cncentratin, and the technique f Dusterdieck and McElwee (1971) t measure plasma A II, Waite (1972a) fund a significant relatinship between plasma renin and plasma A II cncentratins and demnstrated that there is a strng psitive crrelatin between A I and A II. Current enzyme assays are mst ften perfrmed by allwing the

15 4 enzyme t act n its substrate under standard cnditins with subse quent analysis f the prduct. This principle is als used when the prduct is measured by radiimmunassay; the antibdy against the prduct is added after the enzymatic incubatin has taken place. Pulsen (1971) intrduced a simplified methd fr the radiimmunassay f enzyme systems. His prcedure intrduced a new applicatin fr radiimmunassay. The principle cnsists f the presence f an excess f radiimmunassay anti bdy at the start f the enzymatic incubatin. The antibdies can cap ture an intermediate prduct such as angitensin frmed with time, and thereby, prtect it against enzymatic degradatin. Subsequent t the enzymatic reactin, the incubatin mixture is diluted and labeled angi tensin is added simultaneusly. Finally, free angitensin is remved with charcal, and cunting f the antibdy-bund angitensin gives the amunt f captured angitensin with an accuracy f ±0.04. The methd is simple and the whle prcedure, except fr cunting, is perfrmed in the same tube. This assay, if used fr A II, can be used with an analgus assay fr A I in rder t ascertain the activities f the cnverting enzyme. At present it is pssible t btain highly specific affinity antibdies against bth hrmnes (A I and A II) with less than 5% crss-reactivity. Therefre, the majr purpse f this investigatin was t study the ac tivity f the cnverting enzyme f the renin-angitensin system during the develpment and maintenance f hypertensin in ne mdel f spn taneusly hypertensive rats (SHR) and three mdels f experimentally induced hypertensive rats. The hypertensive mdels are as fllws: 1) ne kidney Gldblatts, 2) salt-induced, 3) 5-Hydrxy-DL-tryptphan

16 5 induced, and 4) SHR's. The antibdy capture technique fr A II prpsed by Pulsen (1971) was used t elucidate these phenmena, with subsequent radiimmunassay analysis f the A I and A II hrmnes in the plasma f each hypertensive mdel.

17 CHAPTER II REVIEW OF LITERATURE Varius aspects f the renin-angitensin system have been studied fr many years. The first bservatins were made by Tigerstedt and Bergman in 1898, when they extracted renin frm rabbit kidneys and shwed that it wuld raise the bld pressure in dgs and that its activity culd be destryed by biling. Little interest was taken in these bservatins until 1934 when Gldblatt and his clleagues shwed that the applicatin f a clip t ne renal artery wuld raise the bld pressure in the experimental animal (Gldglatt et al., 1934). Cnse quently, fr abut 36 years after its discvery renin was nly a curi sity and a matter f dispute because n particular attentin was paid t the enzyme's pssible significance since the idea f primary (nn-renal) hypertensin was dminant. Accrding t Berman and Vertes (1973) it is n exaggeratin t call Gldblatt's experiment ne f the mst imprtant in mdern science fr it nt nly reestablished the frgtten enzyme, but als led t sme f the mdern cncepts f hypertensive disrders. After Gldblatt's experiment many investigatrs began t lk again at the pssibility f release f pressr substances frm such a kidney, and renin was rediscvered in kidney extracts (Pickering and Prinzmetal, 1938). The subsequent wrk f Page and Helmer (1940) and Braun-Menedez et al, (1939,1940) led t the discvery that renin was an enzyme which acted n a substrate in the plasma t prduce pressr activity called, initially, angitnin by Page and his assciates and hypertensin by 6

18 7 Braun-Menendez and his cwrkers. The name f this pressr substance was later changed by agreement and called angitensin (Page, 1975). It has subsequently been shwn that this activity is due t the prductin f a decapeptide which is readily changed t an ctapeptide in the circulatin (Skeggs et al., 1955,1956; Ellitt and Peart, 1956,1957). Early effrts t islate and characterize angitensin were hampered by the presence f angitensinase (peptidase) in renin and renin substrate preparatins. The prblem was vercme by using techniques which destryed peptidase activity by acidificatin (Pulsen, 1969a) and by charcal ad srptin f angitensin as sn as it was frmed, thus rendering it inaccessible t peptidase activity r by preliminary purificatin t ex clude angitensinase frm the system (Peart, 1955,1956; Byd et al., 1969). First, hrse angitensin (Skeggs et al., 1955), then bvine (Ellitt and Peart, 1956,1957), hg (Bumpus et al., 1957) and human (Arakawa et al., 1967) angitensin I (A I) were islated. By the use f several amin acid analysis techniques, bth Skeggs et al. (1955) and Ellitt and Peart (1956,1957) were able t cncurrently identify the amin acid sequence in A I; the sequence was Asp.Arg.Val.Try.lie.His.Pr.Phe. His.Leu, respectively. The first pure A I was btained by Skeggs et al. (1954) frm the incubatin f pig renin with renin substrate btained frm hrse plasma. The material was shwn t be a decapeptide, with nine different amin acids and with an iselectric pint f 7.7. Its amin acid cmpsitin was substantiated by Lentz et al. (1956) when they demnstrated the amin acid cmpsitin f hypertensin II and shwed its bichemical relatinship t hypertensin I. Cnfirmatin f the sequence has since been made thrugh synthesis f the peptide by

19 8 Bumpus et al. (1957) and Rittel et al. (1957) during the perid when the Merrifield slid-phase methd f synthesis was unknwn (Page, 1975). Human, hrse and hg A I are identical in their amin acid sequence, but bvine A I differs in that valine replaces isleucine in the five-carbn psitin. Skeggs et al. (1954) discvered tw frms f angitensin that culd be separated by a 50 tube cuntercurrent distributin methd. One f the prducts (A I) was the decapeptide prduced by the actin f renin n the renin substrate (angitensingen). The secnd prduct (A II) was the ctapeptide frmed after the remval f the terminal histidyl-leucine frm A I. In the absence f plasma, A I exhibited n significant vas cnstrictr prperties, either n islated rabbit artic strip (Helmer, 1957) r the perfused islated rat kidney (Ng and Vane, 1968) whereas, A II was highly active. Such bservatins led t further awareness f an enzyme in plasma that cnverts the decapeptide t an ctapeptide. The wrk f Page and Helmer (1940) and f Braun-Menendez and his clleagues (1940) demnstrated that renin was nt, by itself, a pressr agent and that sme factr in plasma was necessary t prduce its pressr activity. Bth grups, therefre, suspected the existence f an activa tr, c-factr, r a substrate in plasma. Wrk by Plent et al. (1943) demnstrated that this c-factr was the renin substrate and that it culd be characterized as a prtein cntained largely in the a2-glbulin fractin f plasma. Subsequently, in a series f imprtant experiments, Skeggs et al. (1964) prvided cnsiderable infrmatin n the nature f renin substrate. This grup was able t degrade hrse renin substrate by treatment with trypsin and thereby btain a 14-amin acid residue

20 9 plypeptide. This tetradecapeptide yielded A I upn incubatin with renin. The first 10 amin acids frm the N-terminal grup were identi cal with thse fund in A I. The structure f the tetradecapeptide was later cnfirmed by synthesis and it was established that renin acts n this substrate at the leucyl-leucyl bnd t liberate A I and a tetrapeptide. The rgan surce f the circulating renin substrate (angitensingen) has nt been established unequivcally but a hepatic surce seems likely. Observatins by Page et al. (1941) and Lelir et al. (1942) indicate that hepatectmy reduces renin substrate. Stu dies by Drury et al. (1951) indicate that hepatectmy als eliminates respnses t injected renin. In supprt f these studies are ther b servatins indicating that plasma renin substrate may be very lw r at times even undetectable in patients with liver disease (Ayers, 1967). Renin substrate has nt been identified in extracts f liver, spleen, lungs r heart (Munz et al., 1940). Hwever, mre subsequent studies indicate that hepatic tissue cultures can generate renin substrate. The current state f knwledge f the cmpnents f the reninangitensin system wherein renin, after its release int the circulatin, acts n its substrate (angitensingen) t prduce the decapeptide (A I), and its subsequent cnversin by the cnverting enzyme t the bilgically active ctapeptide (A II), is due t the wrk f Bumpus et al. (1961), Peart (1965) and Brwn et al. (1966). Accrding t Peart (1969) it is apparent that the amunt f substrate present in the system affects the rate f reactin, and the rate f cnversin f deca- t ctapeptide will limit the amunt f bilgically active end prduct appearing in circulatin. The wrk f Ng and Vane (1968) demnstrated that large

21 10 amunts f A I are cnverted t A II in transit during a single passage thrugh the lungs rather than in the circulatin itself. While in circulatin A II is subject nt nly t the degradative actin f the varius angitensinases present in bld, but als t clearance in transit thrugh tissues. It is therefre likely that mst f the meta blic clearance is carried ut within the tissues rather than in the bld. Accrding t Vane (1969) renin has a lng half-life in the circula tin (15 min) in cmparisn with a single circulatin time f 15 t 30 sec. It therefre circulates again and again, and the gradual decrease in its cncentratin is due t liver inactivatin. By its actin n angitensingen, renin generates A I especially n the venus side f circulatin and it is relatively stable. Hwever, A I is subsequently cn verted t the mre active A II as it passes thrugh the lungs. A I frmed n the arterial side remains uncnverted and mst f it (50 t 70%) disappears in the peripheral vascular beds withut being cnverted t A II (Ng and Vane, 1968). The 30 t 50% f A I which survives pas sage thrugh peripheral vascular beds mixes with that which is cnverted t A II in the lungs. It is nt knwn hw quickly the angitensinases in the bld degrade A I but any such degradatin is prbably slw. A II frmed in the lungs is stable in bld (half-life, 3 min) cmpared with its disappearance (50-60%) in a single passage thrugh peripheral vascular beds. That which passes thrugh the peripheral vascular beds recirculates, hwever, there is n remval f A II in pulmnary circulatin (Hdge et al., 1967). The renin-angitensin system is evidently an enzyme system in which

22 11 renin is the enzyme and angitensin the prduct f the enzymatic reactins (Skeggs et al., 1959). A II is respnsible fr all the knwn effects f the system. It is the mst effective pressr substance and influences the salt and water balance thrugh its effect n the kidneys and adrenals. The interest in the system is due t the unslved questin whether r nt the renin-angitensin system is active in the prductin f the varius types f hyperten sin (Pulsen, 1970). Renin is a prtelytic enzyme that is synthesized, stred and secreted mainly by the kidney. Oparil and Haber (1974) reprted that renin-like enzymes have been extracted frm a variety f rgans, including the uterus, placenta, fetal membranes, amnitic fluid, brain adrenal glands, and the submixillary glands f the white muse. Extrarenal surces have been used t explain the ccasinal finding f renin activity in the bld f anephric subjects (Capelli et al., 1968), but there is yet n evidence that these extrarenal surces have any physi lgic rle in bld pressure regulatin r that the enzymes are identical with renal renin. Althugh the enzyme is fund in high cncentratins in varius r gans, renin is cncentrated mailny in the epithelial cells f the juxtaglmerular apparatus in the kidney (Fraup, 1968). The nly knwn place in which all f the cmpnents f the renin-angitensin system are fund tgether is in plasma. Pulsen (1970) reprted that the half-life f renin is hr whereas that f the cnverting enzyme is in the range f 2-10 min, and the peptide enzymes (angitensinases) apprxi mately 5 min. Based n these data renin is the rate-limiting enzyme.

23 12 In spite f this, the cncentratin in plasma f the bilgically active ctapeptide A II, is nt slely determined by the cncentratin f renin. Observatin f the actins f the angitensins led t the study f the enzymes invlved in the metablism f the liberated peptides, including the A I cnverting enzyme. The A I cnverting enzyme was discvered by Skeggs and his assciates in the mid 1950's when they nticed that hrse plasma cntains an enzyme that cnverts A I t A II (Skeggs et al., 1954,1956; Lentz et al., 1956). They reprted that renin releases the decapeptide (A I) frm angitensingen and that this decapeptide is in turn cnverted t the ctapeptide (A II) when the cnverting enzyme cleaves a histdylleucine dipeptide frm the C- terminal end f A I. The enzyme requires chlride ins and is inhibited by ethylenediamine tetraacetic acid (EDTA) r ther chelating agents such as dimercaprl (BAL) r 8-hydrxyquinline. These bservatins strngly suggest that the enzyme is a metallenzyme. Arund the same time Helmer (1955) als bserved the existence f a factr in plasma that activated angitensin preparatins. After these discveries, the matter lay drmant fr a lng time, because A I was nt available in pure r synthetic frm in substantial quantities. The issue f cnversin was kept alive, hwever, because f the difference between the effects f A I and A II n islated smth muscle preparatins. It was bserved that A I must be cnverted t A II befre it becmes active in mst bilgical systems in vitr (Skeggs et al., 1956). By means f a spirally cut strip f rabbit thracic arta Helmer (1957) demnstrated that angitensin exists in tw frms.

24 13 One frm, A II, causes a cntractin f the strip. The ther, A I, is inactive. They are equally pressr when injected intravenusly in ani mals. An enzyme in plasma cnverts the inactive frm t the active frm. The identical pressr activity can be explained by the excess f the cn verting enzyme in the plasma f the intact animal which rapidly cnverts A I t A II. Bumpus et al. (1961) used the islated rat uterus fr the same purpse. Andersn (1967) reprted a similar biassay technique. Several chrmatgraphic prcedures fr separating A I and II have been described. A I labeled at the C-terminal amin acid was used by Huggins and Thampi (1968) fr the measurement f A I cnverting enzyme in rat plasma, heart, brain, liver, diaphragm, kidney, lung, arta and uterus. Helmer (1957) als reprted that sme patients with hypertensin have a greater cntent f the cnverting enzyme in their plasma than is fund in nrmtensive subjects. It was suggested that in additin t the cn verting enzyme, ther factrs in plasma may enhance the ability f angitensin (i.e., catechlamines, prstaglandins, nrepinephrine, etc.) t induce cnstrictin f tissues in biassays. These factrs may sensitize the mechanisms in the muscle tissue which set up the prcess f cntractin. Erds (1975) reprted in a review article that the need fr such cnversin in viv is nt that nticeable because the tw peptides have similar effects n systematic bld pressure after intravenus injectin due t the rapid cnversin f A I in the bdy. It has been difficult t measure the cnversin f A I t A II by biassay, because tissues which cntain the cnverting enzyme als inactivate the released peptide. Estimatin f cnversin in vitr gave nly semiquantitative data (Bumpus et al., 1961) until radiactive substrates became available.

25 14 The measurement f renin activity, cncentratin and substrate has been widely perfrmed by biassay f the A I generated during incubatin f plasma samples in vitr (Bucher et al., 1964; Pickens et al., 1965; Helmer and Judsn, 1963). Recent studies in human beings (Skinner et al., 1969) and rats (Menard et al., 1970) have demnstrated that measurements f all three parameters cncerned with renin activity in viv plasma renin activity (PRA), plasma renin cncentratin (PRC), and plasma renin substrate (PRS) are necessary fr cmplete evaluatin f changes in the renin-angitensin system. In small animals such as the rat, simul taneus measurement f these parameters has nt been pssible due t the vlume f plasma required fr use with the biassay (Menard and Catt, 1972). Radiimmunassays fr A I have been applied t the measurement f PRA in man, giving results which appear t crrelate well with thse btained by biassay (Byd et al., 1969; Haber et al., 1969; Waite et al., 1972a). As radiimmunassay f A I is cnsiderably mre sensitive than biassay methds, Menard and Catt (1972) applied it t the assay f PRA, PRC, and PRS during studies in the rat, and develped methds which allw all three measurements t be perfrmed n small bld samples. These methds are applicable t the assay f renin parameters in the plasma f ther species. They als allw fr the simultaneus measurement f all three parameters in the renin-angintensin system. Regardless f the methd used in many situatins, Byd et al. (1967, 1969) cncluded that the biassay methds available fr the esti matin f A II in human plasma did nt pssess sufficient sensitivity fr this hrmne in varius physilgical and pathlgical states, therefre,

26 15 they cnsidered the use f radiimmunassay techniques. A majr difficulty with attempts t apply the radiimmunassay technique t A II had been the prblem f develping a suitable antibdy. A II was fund t be an ctapeptide with a small mlecular weight f apprximately 1,000 and antigenically weak. Sme success had pre viusly been achieved by immunizing with angitensin which had been cvalently linked t larger carrier mlecules (Haber et al., 1965; Catt et al., 1967). Recently, radiimmunassays fr angitensin III (A III), based n such antibdies, have been reprted frm tw separate labratries (Vallttn et al., 1967; Catt et al., 1967). In an effrt t achieve maximum avidity and specificity f the antibdy t A II, Byd et al. (1969) reprted success in their attempts t raise antibdies nly against the free angitensin mlecule itself. These investigatrs raised antibdies in rabbits by immunizing with Val -A II amide adsrbed nt micr-particles f carbn. Accrding t the methd f Peart (1955), carbn was used as the adsrbent because f its high affinity fr A II and its ability t prtect the mlecule against the actin f the plasma angitensinases. The subsequent use f such high titre antibdies in the radiimmunassay f A II in human circulating plasma has been reprted (Byd et al., 1967, 1969). The test used in these experiments was able t detect 30 pg amunts f A II and was nt influenced significantly by A I. Since 1967 ther investigatrs (Vallttn et al., 1967; Catt et al., 1967; Sundsfjrd, 1970; Dusterdieck et al., 1971; McBride et al., 1971) described radiimmunassays fr A II which detect small amunts f the peptide, but lengthy extractin and cncentratin prcedures were

27 16 required. Reprts by Vallttn et al. (1967) and Gdfriend et al. (1968) als indicated a lack f agreement between measurements f A II (generated in vitr by incubatin with renin) btained by the rat pressr assay and that measured by radiimmunassay f the same sample. Gcke et al. (1968) described the range f cncentratin f A II in nrmal human subjects and dcumented the variatins in A II cncentratins with changes in sdium intake. These wrkers als crrelated the varia tins with renin activity. Their findings were btained using a simple rapid radiimmunassay prcedure capable f measuring A II directly frm 0.1 ml f human plasma. The validity f the measurement was substantiated by the ability f the immunassay t duplicate results btained by the pressr biassay f the A II generated in vitr by incubatin with either endgenus r added renin. In experiments perfrmed by Gcke et al. (1968), A II was generated by incubatin at 37 C with endgenus renin fr 24 hr r with increments f highly purified human renin fr 30 min. Excellent agreement between biassay and immunassay determinatins was btained ex cept when M EDTA was present in the renin-generatin step. In this case, less than 1% f the A II fund by biassay was detected by the radi immunassay. This was mst likely caused by the inhibitin f the cnver sin f A I t A II by the presence f EDTA. In mst radiimmunassay pr cedures reprted, the antibdy shws very little crss-reactin with the decapeptide. Thus, the antibdy fails t recgnize the A I frmed. Hwever, n injectin f the sample int the rat fr biassay, A I is rapidly and quantitatively cnverted t the pressr substance, A II. When the plasma was dialyzed t remve the EDTA prir t the incubatin step full agreement between the tw assay methds was btained. This crrelatin f immunassay

28 17 and biassay measurements prvided imprtant verificatin that bth methds measure the same substance and ruled ut the pssibility that degradatin prducts r ther factrs in plasma crss-react with the antibdy t any significant extent. Sensitive and reprducible biassays have been used t elucidate much f what is knwn f the renin-angitensin system. Mst f the prcedures were based n the ability f A II t cause smth muscle cntractin. Bilgic respnse was assayed either directly r after an incubatin step in which the renin-substrate reactin was allwed t take place under standardized cnditins, thus generating angitensin and giving a measure f renin activity. The bilgic respnse was either cntractin f rabbit arta (Helmer, 1957) r rat cln (Needleman et al., 1972) r the systematic pressr effect in an intact nephrectnrized rat (Bucher et al., 1961). Respnses were related t cntrl injectins f standard A II. Althugh precise and reprducible measurements f plasma renin activity have been btained by biassay in many research labratries, the wide clinical ap plicatin f this apprach has been limited by incnsistent standardizatin and reprducibility, undue time requirement fr sample prcessing, and frequently a lack f sensitivity and specificity (Oparil and Haber, 1974). Radiimmunassay fr A II and A I became pssible because highly purified synthetic peptides fr use as antigens became available. By cupling angitensin t a macrmlecular carrier such as ply-l-lysine (Haber et al., 1965), a prtein (Dedhar, 1960; Gdfriend et al., 1964) r finely divided charcal (Byd and Peart, 1968) an effective immungen was created. The hapten-carrier cmplex gave rise t specific high affinity antibdies. Studies with peptide analgues and degradatin pr ducts f A I and II shwed that there was less than 5% crss-reactivity

29 18 between mst anti- A I and anti- A II antibdies (Haber et al., 1965). Furthermre, except fr the carbxyl hepta-peptides and hexa-peptides, the degradatin prducts f A II were nnreactive with mst antiangitensin antibdies (Dietrich, 1967). Vallttn (1971) reprted that since the structural requirements fr bilgic and immunlgic ac tivity in the A II mlecule are similar, fragments f A II d nt impair t any great degree the usefulness f the A II radiimmunassay in circu lating plasma. Labeled peptides f high specific activity fr the radiimmunassay prcedures were btained with I by the chlramine-t technique f Hunter and Greenwd (1962). A mdificatin f the prcedures made it pssible fr the prductin f a mnidinated peptide by Nielsen et al. (1971) that is stable fr many mnths. Separatin f bund frm free antigen is generally dne by a mdificatin f the charcal methd f Herbert et al. (1965), r by the antibdy-capture technique f Pulsen (1971). Radiimmunassay prcedures fr A II were develped befre thse fr A I because f the earlier availability f the ctapeptide as an antigen. The A II assays are sensitive enugh t measure circulating levels f A II in nrmal man (Byd et al., 1967; Catt et al., 1967; Gcke et al., 1968; Gdfriend et al., 1968; Page et al., 1969; Sundsfjrd, 1970). Oparil and Haber (1974) reprted that the apparent discrepancies between methds can be attributed t differences in sdium intake and psture f the subjects and a variability in extractin techniques. Accrding t Dietrich (1967) and Haber et al. (1969) levels f A II crrelate well with renin activity. The use f the A II assay t determine renin activity has been less

30 19 successful. When an assay fr A II is used t measure renin activity, in vitr cnversin is necessary. Cnditins favrable fr the actin f plasma cnverting enzyme als favr angitensinase actin causing simul taneus generatin and destructin f A II (Oparil and Haber, 1974). Use f a specific anti- A II antibdy eliminates the cnversin step and permits a mre direct accurate measurement f renin activity (Haber et al., 1969; Byd et al., 1969; Hllemans et al., 1969; Lehfeldt and Hutchens, 1971; Chen et al., 1971). The inhibitrs f cnverting enzymes that are added t the incubatin mixture als inhibit angitensinase activity, ensuing imprved recvery f the generated peptide. All f the cmmnly used radiimmunassay prcedures fr renin ac tivity emply an initial incubatin step in which endgenus renin and substrate react t generate A I. The reactin is then stpped usually by freezing. 125I labeled A I and specific anti- A I antibdy are added at a later time and allwed t equilibrate with the generated A I in a secnd incubatin step. Bund and free frms f 125I - A I are then separated by cnventinal techniques and quantitated by gamma r scintillatin cunting. The amunt f A I present is determined by a standard curve, accrding t the methds f Bernsn and Yallw (1968). Oparil and Haber (1974) main tained that there are three main areas in which variatins in prcedure have been intrduced: ph f the initial incubatin, chice f enzyme in hibitrs, and duratin f the initial incubatin. Detailed evaluatins and cmparisns f methds fr measuring renin activity are currently being carried ut in a number f labratries. It is hped that the standardizatin f renin and angitensin preparatins, incubatin cndi tins fr generating angitensin, and units f expressing renin activity will facilitate cmparisn f renin activity measurement frm labratry

31 20 t labratry (Oparil and Haber, 1974). Variability in incubatin cnditins fr generating angitensin and in angitensin standards has made cmparisn between biassay and radi immunassay f plasma renin activity difficult t interpret. In ne study cmparing nrmal values btained in a number f labratries, values fr renin cncentratin btained by radiimmunassay in reference t an internatinal renin standard were lwer than biassay values (Haas and Gldblatt, 1972). With the use f the same plasma extracts fr assay, A I cncentratin has been reprted t be cnsistently lwer by radi immunassay than by biassay (Chen et a!., 1971; Menard and Catt, 1972), r cmparable t biassay values (Ktchen et al., 1973). Oparil and Haber (1974) suggested that the reslutin will require measurement f angitensin samples generated under identical cnditins by bth biassay and radiimmunassay with the same angitensin standard in bth pr cedures. It is difficult t measure the cncentratin f circulating A I with accuracy. In vitr reactin f renin with its substrate, particularly prminent in plasma that cntains large amunts f renin r has been allwed t becme warm, cause false elevatin f A I levels in unincubated samples. Sme antisera react with plasma cmpnents ther than A I and give falsely high results with unincubated plasmas (Page et al., 1971). Apprpriate selectin f antisera will prvide fr the eliminatin f this prblem, since nt all antibdies manifest such nnspecific crssreactivity (Oparil and Haber, 1974).

32 CHAPTER III MATERIALS AND METHODS Materials Male Sprague-Dawley and Wistar spntaneusly hypertensive rats (SHR's) used in this investigatin were btained frm Charles River Breeding Labratries, New Yrk, M. Y. Systlic bld pressures f all animals were mnitred using a Prgrammed Electr-Sphygmmanmeter (mdel PE-300). This instrument was cnnected t a desk mdel Physigraph recrder (mdel DMP-48), frm which readings were made. Bth instruments were purchased frm Marc Bi-Systems, Inc., (Hustn, Texas). The fllwing chemicals were purchased frm Sigma Chemical C. (St. Luis, M.); 3-estradil-3-benzate, 5-hydrxy-DL-tryptphan, nemycin sulfate, bacitracin, Tris (hydrxymethyl) aminmethane (Trizma base), Tris (hydrxymethyl) aminmethane hydrchlride (Trizma HC1), ethylenediamine tetraacetic acid (EDTA), diidprpylfurphsphate (DFP) and bvine serum albumin (BSA). Materials used in the radiimmunassay prcedures fr the measure ment f angitensin I (A I) and angitensin II (A II) were purchased frm New England Nuclear Bimedical Assay Labratries (Wrcester, Mass.); A I (5-L Isleucine) [tyrsyl-125i], A II (5-L-valine)[tyrsyl-125I] amide, A I antiserum, A II antiserum, maleate buffer cncentrate, A I standards, 8-hydrxyquinline sulfate, dimercaperl (BAL) and Riaflur (liquid scintillatr). 21

33 22 Radiimmunassay grade charcal and Dextran T-70 were purchased frm Schwartz/Mann (Orangeburg, New Yrk). Synthetic human A I and II were btained frm Beckman Biprducts, (Pal Alt, Cal.). Sdium pentabarbital (Nembutal, 60 mg/ml) used fr anesthetizatin was purchased frm Msher, Inc., (Atlanta, Ga.). All reagents and stck slutins were prepared in distilled r deinized water. Gamma emissins frm radiactive (mnidinated) A I and II samples were cunted in a Beckman Liquid Scintillatin Cunter (mdel LS-230, Beckman Instruments, Inc., Atlanta, Ga.). Centrifugatins were dne in a Lurdes refrigerated centrifuge. Methds f Prcedure All animals were fed Purina rat chw and tap water ad libitum unless specified therwise. Apprximately 125 rats (initial avg. wt g) were divided int 2 grups f cntrls, 1 grup f dubly nephrectmized rats, 3 experimental (induced) hypertensive grups and 1 grup f spntaneusly hypertensive rats. Bld pressure measurement Systlic bld pressures f all animals were mnitred 0-24 hr prir t any experimental prcedure. The tail cuff bld pressure methd was used, wherein a pneumatic cuff was placed arund the base f the tail, A pneumatic bulb cnnected t a pulse transducer was then secured (taped) ver the caudal artery. In this system, the animals were enclsed in a warming chamber in rder t increase bld circulatin which usually resulted in gd pulsatile waves in the caudal artery. At all times during the mnitring f bld pressures, the animals were secured in specially built rat restrainers in rder t minimize mvement. A

34 23 systlic bld pressure abve 150 mm Hg in unanesthetized rats was regarded as hypertensive (O'Steen et al., 1969). Surgical prcedures All prcedures invlving surgery were perfrmed after anesthetiza tin with sdium pentabarbital (Nembutal, 40 mg/kg, ip). Operatins were dne under aseptic cnditins. Surgery invlved pening f the peritneal cavity by making a mid-line incisin alng the linea alba ( cm in length). These prcedures invlved either placing a clip arund the left renal artery and remval f the right kidney (1- kidney Gldblatt), remval f the right kidney leaving the left kid ney intact (1-kidney nephrectmized), r remval f bth kidneys (2- kidney nephrectmy). After surgery the abdminal cavity was clsed by suturing with size 0-4 silk Ethicn surgical suture. The uter incisin was then clsed with metal wund clips. Cllectin f bld samples Bld samples were cllected at 10,20 and 30-day intervals after each rat was decapitated. The bld was allwed t flw int 200 ml beakers that had been rinsed with a 0.3% sdium citrate slutin and placed in an ice water bath (0-4 C). The samples were then measured and pured int 10 ml plyprpylene centrifuge tubes cntaining sdium citrate (final cncentratin, 0.3%). Subsequent t cllectin, the samples were centrifuged at 1,500 rpm in a refrigerated centrifuge (0-4 C). Plasma samples were then decanted and stred in 7 ml si 1 icnized vacutainers at -20 C until assayed fr A I and A II cncentratins. One kidney Gldblatt hypertensive mdel Frty-five male Sprague-Dawley rats were weighed, anesthetized and

35 24 the right kidney remved. The left renal artery at the site where it jins the drsal arta was dissected ut f its tissue bed and a clip ( mm, i. d.) placed arund the expsed artery. The clip was a mdificatin f that used by Gldblatt et al. (1934) made frm silver wire. The internal diameter f the clip varied accrding t the bdy weight f the animals. The 0.20 mm clip was used fr rats weighing up t 200 g (initial avg. wt.), and fr thse weighing abve 200 g a 0.25 mm clip was used. Cntrls fr this mdel were 21 rats that had the right kidney remved and the left kidney intact. Nine experimental and 7 cntrl rats were sacrificed at each designated time interval. Salt-induced hypertensive mdel The right kidney f 27 rats fr this mdel was remved with the left kidney left intact. After a 3-day recvery perid, all animals in this grup were given 1.5% saline t drink. Cntrls cnsisted f 21 rats that had the right kidney remved, but given tap water t drink. Nine salt-laded and 7 cntrl rats were sacrificed at each 10, 20, and 30-day interval, and plasma samples cllected. Malignant hypertensive mdel Fifteen rats were administered an intraperitneal (ip) injectin f 5-Hydrxy-DL-Tryptphan (50 mg/kg) disslved in 0.9% saline and a subcutan eus (sc) injectin f S-estradil-3-benzate in sesame il (40 yg/0.1 ml) simultaneusly. Cntrls fr this grup were given a sham injectin f 0.9% saline (ip) and 0.1 ml sesame il (sc). Injectins were made n day 1, 7, 14, 21, 42 and 47 with all surviving animals being sacrificed n day 50 and plasma samples cllected. Bld pressures f experimental and cntrl animals were recrded at each time interval prir t injectins

36 25 and n the day the experiment was terminated. Spntaneusly hypertensive mdel The spntaneusly hypertensive Wistar rats were direct descendants f the Wistar strain develped by Okamt and Aki (1963). Twelve f these rats ( g) were sacrificed and plasma samples cllected (apprx. age, wks.). Radiimmunassay f antitensin I The radiimmunassay prcedures used fr the determinatin f A I cncentratins in all plasma samples were based n prtcls published by New England Nuclear Bimedical Assay Labratries. The assay system is an adaptatin f the radiimmunassay methds f Haber et al. (1965,1969) and Gdfriend et al. (1968). The assay cnsisted f 0.1 ml aliqut each f a generated, and an endgenus sample f plasma. These samples cn sisted f 1.0 ml f plasma, 2.0 ml f 0.2 M maleate buffer (ph 6.0), and 0.1 ml f an inhibitr f bth the plasma cnverting enzyme and the angitensinases. The inhibitry mixture cntained 5.0 mm EDTA, 0.32 M 8- hydrxyquinline sulfate, 0.05 M DFP and 0.2% bactracin in distilled water. After vrtexing, 1.0 ml f each plasma sample was incubated in a water bath at 37 C fr 1 hr, the rest f the sample was left at 0-4 C in an ice water bath. T 0.1 ml f each f these plasma samples were added: rabbit A I antiserum in tris-acetate buffer (0.1 M, ph 7.4, 0.5 ml) and 125I tyrsyl labeled 5-L isleucine A I (apprx. 8 pg/0.1 ml, specific activity yci/mg, cunts/min), 0.1 ml. The final vlume f all samples was 0.7 ml and each sample was assayed in duplicate at 0-4 C. Endgenus A I plasma samples were kept at 0-4 C at all times during

37 26 the assay, while samples f generated A I were incubated at 37 C fr 1 hr. Generatin f the 37 C samples were terminated by immersin f the sample int an ice water bath at 0-4 C. After an hr incubatin perid f all samples at 0-4 C, free and bund A I were separated using a dextran T-70 treated Nrit A charcal suspensin (ph 7.4). A ttal vlume f 1.0 ml f the charcal suspensin was added t each plasma sample and the antibdy bund and free A I separated by centrifugatin fr 20 min at 4 C (5,000 rpm). The term endgenus refers t the actual amunt f A I in a plasma sample, while the term generated refers t the amunt f A I measured after incubatin in a water bath at 37 C fr a perid f 1 hr. All samples were assayed in 3.0 ml plystyrene test tubes (silicne treated). After charcal filtratin f free frm antibdy bund A I, gamma emmissins f the supernatant were cunted in a well type liquid scin tillatin cunter. The prcess invlved the use f 10.0 ml f Raiflur in 15 ml scintillatin vials int which the plasma samples were added. Standards cnsisted f 0.1, 0.25, 0.5, 1.0, 2.5, and 5.0 ng/ml f lyphilized synthetic human A I (0.1 ml aliquts). Standards were assayed in the same manner as endgenus and generated plasma samples. Prcedures fr calculating the values fr each set f duplicate standards and the unknwn A I cncentratins f 37 and 4 C plasma samples were thse suggested in the prtcls fr radiimmunassay f A I published by New England Nuclear Bimedical Assay Labratries. The net cunts per min are expressed as a percentage f the ttal cunt value by use f the fllwing equatin: