Correspondence should be addressed to Pradip K. Sarkar;

Transcription

1 Journl of Thyroid Reserch Volume 213, Article ID , 9 pges Reserch Article Mechnisms of L-Triiodothyronine-Induced Inhibition of Synptosoml N + -K + -ATPse Activity in Young Adult Rt Brin Cerebrl Cortex Prdip K. Srkr, 1,2,3 Avijit Bisws, 2 Arun K. Ry, 3 nd Joseph V. Mrtin 2 1 Deprtment of Bsic Sciences, Prker University, 25 Wlnut Hill Lne, Dlls, TX 75229, USA 2 Center for Computtionl & Integrtive Biology, Rutgers University, 315 Penn Street, Cmden, NJ 812, USA 3 Deprtment of Moleculr Medicine, Bose Institute, P-1/12, CIT, Scheme VII-M, Clcutt 754, Indi Correspondence should be ddressed to Prdip K. Srkr; psrkr@prker.edu Received 29 June 213; Revised 19 September 213; Accepted 24 September 213 Acdemic Editor: Noriyuki Koibuchi Copyright 213 Prdip K. Srkr et l. This is n open ccess rticle distributed under the Cretive Commons Attribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. The role of thyroid hormones (TH) in the norml functioning of dult mmmlin brin is uncler. Our studies hve identified synptosoml N + -K + -ATPse s TH-responsive physiologicl prmeter in dult rt cerebrl cortex. L-triiodothyronine (T 3 ) nd L-thyroxine (T 4 )bothinhibitedn + -K + -ATPse ctivity (but not Mg 2+ -ATPse ctivity) in similr dose-dependent fshions, while other metbolites of TH were less effective. Although both T 3 nd the β-drenergic gonist isoproterenol inhibited N + -K + -ATPse ctivity in cerebrocorticl synptosomes in similr wys, the β-drenergic receptor blocker proprnolol did not counterct the effect of T 3. Insted, proprnolol further inhibited N + -K + -ATPse ctivity in dose-dependent mnner, suggesting tht the effect of T 3 on synptosoml N + -K + -ATPse ctivity ws independent of β-drenergic receptor ctivtion. The effect of T 3 on synptosoml N + -K + -ATPse ctivity ws inhibited by the α 2 -drenergic gonist clonidine nd by glutmte. Notbly, both clonidine nd glutmte ctivte G i -proteins of the membrne second messenger system, suggesting potentil mechnism for the inhibition of the effects of TH. In this pper, we provide support for nongenomic mechnism of ction of TH in neuronl membrne-relted energy-linked process for signl trnsduction in the dult condition. 1. Introduction Thyroid hormones (TH) exert mjor influences on the growth nd development of the mmmlin brin through specific nucler receptor-medited gene expression. Although severl different isoforms of nucler receptors for TH hve been described in dult mmmlin brin, their physiologicl function is quite uncler [1 4]. Still, dult onset of dysthyroidism develops number of functionl, neurologicl nd psychologicl mnifesttions in humns [5 7]. In contrst to the developing brin, most of the chnges resulting from hormone vritions in the dult condition re reversible with the proper djustment of circultory TH [5 7]. Recent evidence hs demonstrted tht L-triiodothyronine (T 3 ) is distributed, concentrted, nd metbolized in the synptosoml frction of dult rt cerebrl cortex [5, 8, 9]. Specific T 3 -binding sites hve lso been described in cerebrocorticl synptosomes [1, 11] nd grded binding of T 3 to its synptosoml receptor binding sites hs been correlted with the corresponding inhibition of the N + -K + - ATPse ctivities in dult rt brin [11]. TH rpidly lters in vitro phosphoryltion of synptosoml proteins in dosedependent fshion [12]. TH levels re lso ltered in dult rt brin in different thyroid conditions [9]. TH enhnces clcium entry in dult rt brin synptosomes [13 15], in hypothyroid mouse brin [16],nd in single rt myocytes[17]. However, there is lck of cler understnding of the mechnism(s) of ction of TH in the regultion of synptic functions in dult neurons. The present study investigtes the pthwys of T 3 -medited signling from its binding to the synptosoml membrne receptors to the subsequent

2 2 Journl of Thyroid Reserch ctivtion of second messenger system components tht ultimtely ffect the further downstrem effector molecule, the N + -K + -ATPse. In this pper, we hypothesize nongenomic mechnism of ction of TH in neuronl membrne-relted energy-linked process(es) for signl trnsduction in dult condition. We hve used α- nd β-drenoceptor gonists nd ntgonists for modultion of the ctivity of G s -ndg i - proteins of the membrne denylte cyclse system. Portions of this work hve ppered elsewhere in preliminry form [18, 19]. 2. Mterils nd Methods 2.1. Mterils. The following compounds were purchsed from Sigm Chemicl Compny, USA: bovine serum lbumin (BSA), clonidine hydrochloride (CLO), disodium-atp, isoproterenol hydrochloride (ISO), 2-mercptoethnol, oubin octhydrte, przosin hydrochloride (PRA), phenylephrine hydrochloride (PHE), proprnolol hydrochloride (PROP), sodium glutmte, 3,5,3 -L-triiodothyronine (T 3 ), L-thyroxine (T 4 ), 3,3,5 -L-triiodothyronine (reverse T 3 or r-t 3 ), 3,5-L-diiodothyronine (T 2 ), Tris-ATP, yohimbine hydrochloride (YOH), dibutyryl 3,5 -cyclic denosine monophosphte sodium slt (DB camp), nd sodium orthovndte Tretment of Animls. Adult mle Chrles Foster rts (3 months old) were housed t 25 ± 1 C in 12 h drk-12 h light conditions nd fed d libitum with stndrd rt diet nd wter. The nimls were scrificed by quick decpittion nd the brins were removed into ice-cold 25 mm sucrose solution. The cerebrl cortices were dissected out for synptosoml frction preprtion Preprtion of Synptosomes. The synptosomes from thecerebrl cortex were prepred s described previously [2]. Briefly, the cerebrl cortex ws homogenized (1% weight/volume) in.32 M sucrose nd centrifuged t 1 g for 1 minutes to remove cell debris nd nuclei. The superntnt ws collected nd recentrifuged t 1 g for nother 1 min. The resulting pellet ws discrded nd the superntnt ws lyered over 1.2 M sucrose nd centrifuged t 34, g for 5 min t 4 C. The frction collected between the.32mnd1.2msucroselyerwsdilutedt1:1.5with ice-cold bidistilled wter, further lyered on.8 M sucrose, nd gin centrifuged t 34, g for 3 min. The pellet thus obtined ws wshed nd repelleted t 2, g for 2 min. Synptosoml pellets were lysed by suspending in ice-cold bidistilled wter to relese the occluded N + -K + -ATPse ctivity Assy of Synptosoml N + -K + -ATPse Activity. Synptosoml N + -K + -ATPse ctivity ws ssyed s oubinsensitive ATP hydrolysis in rection mixtures of (i) 3 mm imidzole-hcl, 13 mm NCl, 2 mm KCl, nd 4 mm MgCl 2 nd (ii) 3 mm imidzole-hcl, 4 mm MgCl 2,nd1mM oubin, t ph 7.4. Both the rection medi (i) nd (ii) were first preincubted in vitro with or without simultneous ddition of vrious concentrtions of thyroid hormones (T 3, T 4 ) nd TH-nlogue (T 2 )(.1nMto1μM), drenergic drugs (1 nm for ISO, PRA, PHE nd YOH; 1 nm 1 nm for CLO nd PROP), glutmte (1 μm), DB camp (1 μm 5 mm), nd sodium orthovndte (1 nm 2 mm) followed by ddition of the synptosoml lystes, ech contining 2 5 μg synptosoml protein, t Cfor6minutesin drk. To get stedy-stte oubin binding, both the ssy medi (i) nd (ii), with nd without oubin, respectively, s described bove, were preincubted for 6 min t Cinthe drk, followed by 5-min incubtion t 37 Ctoequilibrte the temperture. The rection ws strted by dding 4 mm Tris-ATP nd incubted t 37 Cfor1min.Anliquotof 1 μl of 1% sodium dodecylsulfte ws dded to stop the enzymtic rection. The inorgnic phosphte (P i )formed ws determined in the rection mixture [21]. N + -K + -ATPse ctivity ws clculted s difference in the P i content between medi (i) nd (ii) nd expressed s μmols P i /h/mg protein [22]. The oubin-sensitive portion of the totl ATPse (N + - K + -Mg 2+ -ATPse) ws determined from the P i relesed in the medium (i) minus tht in medium (ii). The P i relesed from the rection medium (ii) ws used for determintion of the synptosoml Mg 2+ -ATPse ctivity. Synptosoml Mg 2+ -ATPse ctivity, therefore, ws ssyed s the oubininsensitive ATP hydrolysis Mesurement of Protein. Synptosoml protein content ws mesured using bovine serum lbumin s stndrd [23] Sttisticl Anlysis. Results re expressed s the men ± SEM of 3-4 seprte experiments or s mentioned. Ech experiment ws mde from six rts. The sttisticl nlysis of the dt ws performed by Student s t-test, considering P <.5 s the significnce level. The dt for multiple groups were lso nlyzed by one-wy ANOVA followed by Student Newmn-Keuls post-hoc comprisons using Sigmstt softwre. Nonliner regression nlysis ws performed using GrphPd Prism softwre. 3. Results 3.1. Effects of T 3 nd Metbolites on N + -K + -ATPse Activity. In vitro ddition of vrious doses of T 3 to the synptosoml frction (which is devoid of cell nuclei) confirmed our previous observtion [11] nd showed nerly the sme trend of dose-dependent inhibition (IC 5 = ± 55. pm; mximl inhibition = 63.2 ± 3.4% t 95% confidence levels) of N + -K + -ATPse ctivity. No significnt effect of T 3 ws noticed on the Mg 2+ -ATPse specific ctivity (Figure 1). T 4 hd similr inhibitory effect s T 3 on N + -K + -ATPse ctivity (IC 5 = 77.2 ± 31.8 pm; mximl inhibition = 66.5 ± 7.2%), while T 2 hd miniml effects (Figure 2). Furthermore, thesmerngeofdoses( M) of r-t 3 did not inhibit either N + -K + -ATPse or Mg +2 -ATPse ctivities (dt not shown) Effect of T 3 nd β-adrenergic Agonists/Antgonists on N + -K + -ATPse Activity. Equimolr doses (1 nm) of T 3 nd

3 Journl of Thyroid Reserch ATPse ctivity (% control) Log [T 3 ] N + -K + -ATPse Mg 2+ -ATPse mg protein 1 ) N + -K + -ATPse (μmols P i h , b, c Figure 1: Inhibitory effect of vrious doses (.1 nm 1 nm) of T 3 on synptosoml N + -K + -ATPse or Mg 2+ -ATPse ctivity, in vitro. The dt re represented s men ± SEM of ten seprte experiments, tking six nimls in ech group. The verticl lines denote SEM. Filled circles indicte N + -K + -ATPse while filled tringles indicte Mg 2+ -ATPse ctivity. N + -K + -ATPse specific ctivity (% control) Control 1 nm T 3 1 nm ISO 1 nm PROP 1 nm PROP 1 nm PROP Tretment Figure 3: Effect of T 3 on synptosoml N + -K + -ATPse ctivity nd its modultion by β-drenergic receptor gonist (ISO) nd βdrenergic receptor ntgonist (PROP) in vitro. A hlf-mximlly effective dose of T 3 (1 nm) ws chosen from the dose-response curve for T 3 in Figure 1. The dt re represented s men ± SEM of five seprte experiments tking six nimls in ech group. P <.1, compred to the control group. b P <.1 nd c P <.5,compred to T 3 (1 nm) + PROP (1 nm) group (one-wy ANOVA followed by Newmn-Keuls test). The verticl lines denote SEM. 1 nm ISO + 1 nm PROP 1 nm T nm PROP 1 nm T nm PROP 1 nm T nm PROP Log [compound] L-T 4 L-T 2 differences in the potentition of the T 3 effect (1 nm) by PROP on N + -K + -ATPse ctivity were noticed between 1 nm nd 1 nm (P <.1) nd between 1 nm nd 1 nm (P <.5) doses (Figure 3). Figure 2: Inhibitory effect of vrious doses (.1 nm 1 nm) of T 4 or T 2 on synptosoml N + -K + -ATPse ctivity, in vitro.thedtre represented s men ± SEM of four seprte experiments, tking six nimls in ech group. The verticl lines denote SEM. Filled circles indicte effects of T 4 on N + -K + -ATPse ctivity while filled squres indicte effects of T 2. the nonselective β-drenergic gonist ISO were dded seprtely in vitro,inhibitedthen + -K + -ATPse enzyme ctivity by 41.3% nd42.6%, respectively (Figure3). The nonselective β-drenergic ntgonist PROP lone did not lter the enzyme ctivity t different doses (1 nm, 1 nm, nd 1 nm). The inhibitory ction of ISO (1 nm) on the N + -K + -ATPse ctivity ws countercted by PROP (1 nm), wheres PROP could not block T 3 -medited inhibition of the enzyme ctivity. Insted PROP potentited the T 3 -medited inhibition of the enzyme ctivity in dose-dependent mnner. Significnt 3.3. Effects of T 3 nd α-adrenergic Agonists/Antgonists on N + -K + -ATPse Activity. The effects of in vitro ddition of 1 nm doses of PHE (selective α 1 -drenergic receptor gonist) nd PRA (α 1 -drenergic receptor ntgonist) on synptosoml N + -K + -ATPse ctivity or Mg 2+ -ATPse ctivity were miniml (Figure 4). Furthermore, 1 nm doses of PHE or PRA did not lter the inhibitory effect of 1 nm T 3 on N + -K + - ATPse ctivity, nor did it chnge the Mg 2+ -ATPse ctivity, in vitro (Figure 4). Similrly, in vitro ddition of CLO (α 2 -drenergic gonist) t different doses did not elicit significnt chnges in the synptosoml N + -K + -ATPse ctivity (Figure 5). However, when CLO ws dded in the presence of n equimolr dose of T 3, the inhibitory effect of T 3 on the N + -K + - ATPse ctivity ws completely countercted. The effect of T 3 on the enzyme ctivity remined prominent t 1 nm dose of T 3 (1 nm T 3 : 1.29 ±.2 μmols P i /h/mg protein;

4 4 Journl of Thyroid Reserch N + -K + -ATPse specific ctivity (μmols/h/mg protein) Mg 2+ -ATPse specific ctivity (μmols/h/mg protein) 2 1 Con Phe Pr Phe + Pr T 3 T 3 + Phe T 3 + Pr Tretment Con Phe Pr Phe + Pr T 3 T 3 + Phe T 3 + Pr Tretment () (b) Figure 4: Modultion of the T 3 ction on synptosoml N + -K + -ATPsectivitybyselectiveα 1 -drenergic gonist (PHE) nd selective α 1 -ntgonist (PRA) in vitro. A hlf-mximlly effective dose of T 3 (1 nm) ws chosen from the dose-response curve for T 3 in Figure 1.The doses for PHE nd PRA used for the in vitro experiment were 1 nm in ech cse. The dt re represented s men ± SEM of five seprte experiments, tking six nimls in ech group. The verticl lines denote SEM. Control: ±.2 μmols P i /h/mg protein) long with 1 nm CLO (1 nm T 3 +1nMCLO:15.23 ±.4 μmols P i /h/mg protein); however, 1 nm CLO ttenuted the effect of T 3 (1 nm) by 32% more towrds the control vlue (dt not shown grphiclly). The α 2 -drenergic receptor ntgonist YOH lso inhibited synptosoml N + -K + -ATPse ctivity (Figure 5). Inhibition of the enzyme ctivity in the presence of both 1 nm T 3 nd 1 nm YOH ws found to be intermedite between the levels of inhibition by either compound lone, lthough there were no significnt differences between these groups (Figure 5) Effect of T 3 nd Glutmte on N + -K + -ATPse Activity. In vitro ddition of 1 μm glutmte lone did not lter the synptosoml N + -K + -ATPse ctivity compred to control vlues, wheres, ddition of 1 μm glutmte showed complete ttenution of T 3 (1 nm)-medited inhibition of synptosoml N + -K + -ATPse ctivity in dult rt cerebrl cortex (Figure 6). A higher dose of T 3 (1 nm) ws chosen, in order to test the effect of glutmte ginst greter inhibitory ction on the N + -K + -ATPse ctivity Effect of DB camp nd T 3 on N + -K + -ATPse Activity. TostudytheeffectofDBcAMPonmodultionofN + -K + - ATPse ctivity by T 3, first dose response experiment with vriousconcentrtionsofdbcamp(.1mmto5mm)ws performed. In vitro ddition of DB camp showed typicl sigmoidl curve with grdul decrese in the N + -K + -ATPse ctivity to mximl inhibition t.2 mm (Figure 7()). From this stndrdiztion, we chose to use.2 mm finl concentrtion of DB camp for further experiments. In vitro ddition of DB camp (.2 mm) with nd without vrious doses of T 3 (.1 nm 1 nm) ws exmined for effects on N + -K + -ATPse ctivity (Figure 7(b)). T 3 -induced inhibition of synptosoml N + -K + -ATPse ctivity ws further depressed in the presence of.2 mm DB camp. However, the twocurvespperedtoconvergetthehighestdosesoft Influence of Sodium Orthovndte on Modultion of N + -K + -ATPse Activity by T 3. The in vitro effect of sodium orthovndte, protein tyrosine phosphtse inhibitor, ws exmined in cerebrocorticl synptosomes. The cerebrocorticl synptosomes were treted with fixed dose of T 3 (1 nm) with or without different doses of sodium o-vndte (Figure 8).AhigherdoseofT 3 (1 nm) ws chosen from the T 3 dose-response curve, considering its greter inhibitory ction on the N + -K + -ATPse ctivity. T 3 cused n inhibition of N + -K + -ATPse specific ctivity, nd this effect ws enhnced by sodium orthovndte in dose-dependent wy. In generl, the effects of sodium orthovndte nd T 3 ppered to be dditive until the N + -K + -ATPse specific ctivity ws completely inhibited. 4. Discussion The objective of the present investigtion ws to serch for possible mechnisms for the inhibition by TH of synptosoml N + -K + -ATPse ctivity in dult rt cerebrl cortex. Initil studies exmined the specificity of the effect ccording to the pttern of iodintion of the hormone derivtives (Figures 1 nd 2). In vitro inhibitory effect of T 3 on synptosoml N + -K + -ATPse ctivity supported our previous observtion nd showed nerly the sme trend of dose-dependent inhibition of N + -K + -ATPse ctivity [11]. In ddition to our erlier report, the current study showed n insignificnt effect of T 3 on the synptosoml Mg 2+ -ATPse specific ctivity (Figure 1). In vitro ddition of T 4 lso indicted similr pttern of inhibitory influence on the synptosoml N + -K + -ATPsectivity,liketheeffectof T 3,withnosignificntchngesontheMg 2+ -ATPse ctivity. The effects of TH on N + -K + -ATPse ctivity seemed to be

5 Journl of Thyroid Reserch 5 N + -K + -ATPse (μmols P i h 1 mg protein 1 ) Control T 3 (1 nm) T 3 (1 nm) T 3 (1 nm) CLO (1 nm) CLO (1 nm) CLO (1 nm) Tretment Figure 5: Modultion of the T 3 ction on synptosoml N + -K + - ATPse ctivity by n α 2 -drenergic gonist (CLO) nd n α 2 - drenergic ntgonist (YOH) in vitro. The dt re represented s men ± SEM of six seprte experiments tking six nimls in ech group. P <.1,compredtothecontrolgroup(one-wyANOVA followed by Newmn-Keuls test). The verticl lines denote SEM. YOH (1 nm) T 3 (1 nm) + CLO (1 nm) T 3 (1 nm) + CLO (1 nm) T 3 (1 nm) + CLO (1 nm) T 3 (1 nm) + YOH (1 nm) N + -K + -ATPse (μmols P i h 1 mg protein 1 ) Control T 3 (1 nm) Tretment Glutmte (1 μm) T 3 (1 nm) + glutmte (1 μm) Figure 6: Modultory effect of glutmte on T 3 -induced inhibition of synptosoml N + -K + -ATPse ctivity in cerebrl cortex, in vitro. A higher dose of T 3 (1 nm) ws chosen from the T 3 dose-response curve, considering its greter inhibitory ction on the N + -K + - ATPse ctivity nd to observe the effect of 1 μm glutmte on this T 3 -induced inhibition. The dt re represented s men ± SEM of four seprte experiments tking six nimls in ech group. P<.1, compred to the control group (one-wy ANOVA followed by Newmn-Keuls test). specific for compounds with 2 iodine toms on the inner ring, s T 2 nd r-t 3 were without ctivity in the current studies. T 3 wslesspotentthnt 4.Itisconsistentwith reports of the reltive ffinities of the two compounds for cell surfce receptor, integrin α v β 3 known to medite vriety of nongenomic effects of THs [24]. Binding of T 4 to integrin α v β 3 cuses internliztion of the receptor nd nongenomiclly promotes phosphoryltion of mitogen-ctivted protein kinse/extrcellulr regulted kinse 1 nd 2 (MAPK/ERK 1/2 ) in the CV-1 line of monkey fibroblsts [24]. A similr mechnism seems likely in chick choriollntoic membrne [25]. Following the internliztion of the integrin α v β 3,theα v monomer is trnslocted to the nucleus, where it my trnscriptionlly regulte expression of protein [26]. TH cuses lungs to rpidly (within hours) increse lveolr fluid clernce [27] nd to express incresed N + -K + -ATPse protein by MAPK/ERK 1/2 -dependent pthwy [28]. Note, however, tht the current finding of n immedite effect to decrese N + -K + -ATPse ctivity could not be due to mechnism involving trnscriptionl regultion, since the synptosoml preprtion is devoid of cell nuclei. It is lso suggested tht some of the effects of T 3 stimultion of the integrin α v β 3 could be more direct thn the nucler interction [29]. A potentil mechnism for the inhibitory effects of TH in the present study might be the regultion of phosphoryltion of N + -K + -ATPse or modultory molecule. It is well known tht ctecholmine-medited phosphoryltion of N + -K + -ATPse inhibits enzymtic ctivity in Chinese hmster ovry (CHO) cells, but not through process of internliztion of the enzyme [3 32]. Intriguingly in this respect, one of the proteins found to be phosphorylted t thetyrosylresidueinsynptosomestretedfor5swithth hd moleculr weight of 95 kd [12],mtchingthesizeofαsubunitofN + -K + -ATPse [33]. The significnt inhibition of the synptosoml N + - K + -ATPse ctivity in vitro by T 3 confirmed our previous in vivo observtions [22]. In order to chrcterize this inhibitory influence of THs on the synptosoml membrne, we intended to study the effect of drenergic receptor gonists nd ntgonists which regulte gunine nucleotide binding proteins (G-proteins) vi their ctivting nd inhibitory ctions. Both T 3 nd ISO (β-drenergic receptor gonist) showed n nlogous but independent (prllel) inhibitory effect on the enzyme ctivity (Figure 3). ISOinduced inhibition of N + -K + -ATPse ctivity ws blocked by PROP (β-drenergic receptor blocker) indicting tht the synptosoml membrne interction with ISO ws likely

6 6 Journl of Thyroid Reserch 1 1 N + -K + -ATPse specific ctivity (μmols/h/mg protein) ATPse ctivity (% control) Log [DB camp] () Log [T 3 ] T 3 DB camp + T 3 (b) Figure 7: Influence of DB camp nd T 3 on synptosoml N + -K + -ATPse ctivity, in vitro. () Inhibitory effect of vrious doses of DB camp on synptosoml N + -K + -ATPse ctivity, in vitro.thedtrerepresentedsmen± SEM of four seprte experiments, tking six nimls in ech group. The verticl brs denote SEM. (b) Interction of the effects of of DB camp nd T 3 on synptosoml N + -K + -ATPse ctivity, in vitro. Filled squres indicte effects of grded doses of T 3 (.1 nm 1 nm) lone on N + -K + -ATPse ctivity while filled tringles indicte effects of the.2 mm dose of DB camp with grded doses of T 3 (.1 pm 1 μm). β-drenoceptor-medited event, potentilly coupled to G s - protein. However, PROP ws completely unble to block T 3 -medited inhibition of synptosoml N + -K + -ATPse ctivity. This clerly indicted tht T 3 -medited inhibition of the enzyme ctivity ws not coupled to β-drenoceptor, but rther, my hve hd similr effect through nother kind of receptor. The ugmenttion of the T 3 effect by PROP ppered to be type of synergistic ction, the mechnism ofwhichreminsunclertpresent.incresedctivityof denylte cyclse cused by THs, independent of proprnolol blockde, hs been shown in cultured cerebrl cells from embryonic mice, suggesting tht the effect of T 3 ws not medited through β-drenergic-dependent system [34]. The T 3 -induced increse in sodium current in neontl rt myocytes lso could not be blocked by PROP, wheres it ws ntgonized by miodrone, nonspecific blocker of βdrenoceptor, suggesting tht the effects were not medited through β-drenergic signling pthwys [35]. However, βdrenoceptor blockde by chronic subcutneous delivery of PROP for 14 dys hs been shown to downregulte levels of TH receptor TR α 1 -mrna nd β 1 -mrna in mouse hert, which my influence the genomic effect of the hormone [36]. Next, we wnted to check for the role of n α 1 - drenergic receptor gonist nd ntgonist. Agonists for the α 1 -drenergic receptor medite their ctions through G q proteinfollowedbyctivtionofphospholipsecnd subsequent production of the second messengers inositol triphosphte nd dicylglycerol, n ctivtor of protein kinse C[37]. Neither PHE (selective α 1 gonist) nor PRA (α 1 ntgonist) hd n influence on N + -K + -ATPse ctivity. Furthermore, neither compound intercted with the effects of T 3. Mg 2+ -ATPse ctivity remined unltered when treted with either of these α 1 -drenergic drugs (gonist nd ntgonist) nd T 3, lone or in combintion (Figure 4). These results suggest tht the effects of T 3 on N + -K + -ATPse ctivity do not shre common mechnisms with α 1 -receptors. On the other hnd, CLO, n α 2 -drenergic receptor gonist (Figure 5), nd glutmte (Figure 6), possibly cting vi metbotropic glutmte receptor (mglur), blocked T 3 -induced inhibition of N + -K + -ATPse ctivity. Neither CLOnorglutmteshowednysignificnteffectonthe N + -K + -ATPse ctivity in rt hippocmpus nd frontl cortex homogentes [38]. One possibility would be tht the counterction of the effect of T 3 on synptosoml N + -K + - ATPse by CLO nd glutmte might be medited through the inhibition of denylte cyclse ctivity with the ctivtion of inhibitory G-protein (G i ) followed by the inhibition of camp synthesis nd the protein phosphoryltion cscde mechnism. It is well known tht α 2 -drenergic gonists ct through stimultion of G i -protein [18, 19, 39, 4]. Assocition of the glutmte trnsporter with N + - K + -ATPseinsynptosomeshsbeenimplictedbytheir correlted regultion vi protein kinses [41]. Glutmte lso hs been reported to inhibit denylte cyclse ctivity in rt hippocmpl synptosomes [39, 4, 42, 43], s well s in stritl nd cerebrocorticl neurons, both in intct cells nd membrnes [4] vimetbotropicglutmte receptors (mglurs), which re coupled to effector systems through GTP binding proteins. In fct, in the nucleus trctus solitrius of dult brin, it ws shown tht n ntibody to the G i inhibited the effects of mglurs [44]. mglur 1 nd mglur 5 subtypes re coupled to phosphtidyl inositol hydrolysis/c 2+ -signl trnsduction. mglur 1 hs lso been shown to stimulte relese of rchidonic cid nd to increse camp formtion. The mglur 2,mGluR 3,mGluR 4, nd mglur 5 subtypes pper to be coupled to inhibition

7 Journl of Thyroid Reserch 7 N + -K + -ATPse specific ctivity (μmols/h/mg protein) C Log [vndte] Control T 3 Figure 8: Modultion of the T 3 ction on synptosoml N + -K + - ATPse ctivity by sodium orthovndte. A higher dose of T 3 (1 nm) ws chosen from the T 3 dose-response curve considering its greter inhibitory ction on the N + -K + -ATPse ctivity nd to observe the effect of grded doses (1 nm 2 mm) of sodium orthovndte on this T 3 -induced inhibition. The dt re represented s men ± SEM of four seprte experiments, tking six nimls in ech group. The verticl brs denote SEM. Open circles indicte men vlues for set of control incubtions without T 3. Filled tringles indicte the results of set of incubtions with 1 nm T 3. of camp synthesis, but differ in their gonist selectivity. mglur 2 nd mglur 3 mrnas re highly expressed in the cerebrl cortex [4, 42, 43]. Activtion of mglur hs been shown to counterct β-drenoceptor-medited inhibition of fterhyperpolriztion in hippocmpl neurons of the CA1 re. This hs been suggested to be by mglur-medited ctivtion of protein kinse C, which inhibited denylte cyclse pthwys [42, 43]. The physiologicl functions of these mglurs re still being clrified. Thus, T 3 ction in dult rt synptosoml membrne, ultimtely to inhibit the effector molecule N + -K + -ATPse, might be medited through G s stimultion. mglu receptors my then hve some regultory roles in countercting T 3 -induced ction. Our observtion showed tht DB camp ( nonhydrolyzble form of camp nd ctivtor of camp-dependent protein kinses) hd T 3 -like effect on N + -K + -ATPse ctivity (Figure 7). Furthermore, the in vitro ddition of incresed doses of T 3 lowered the slope of the dose-response curve for DB camp. Such finding might be consistent with relted mechnism for the effects of DB camp nd T 3,nd would not represent merely dditive effects of two distinct mechnisms. Our previous observtions suggested tht the phosphoryltion sttus of certin synptosoml proteins couldbemeditedvicamp-nd/orc 2+ -dependent pthwys [12, 45]. A differentil stoichiometry of phosphoryltion of the α-subunit of the N + -K + -ATPse by protein kinse A ndproteinkinsechsbeenshowntoinhibitthisenzymtic ctivity in shrk rectl glnd, rt renl cortex, nd bsolterl membrne vesicles from rt renl cortex [46]. The effect of the protein tyrosine phosphtse inhibitor sodium orthovndte [47] ppered to be dditive to the effect of T 3, implying tht there could be seprte mechnismofctionofthetwocompounds(figure8). Since vndte is blocker of tyrosine phosphtse ctivity, it lso could be speculted tht T 3 -induced inhibition of N + -K + - ATPse ctivity is further suppressed by synergistic ction by vndtevikeepingtheenzymeinitsphosphoryltedform, cusing inhibition of its ctivity. A point to note here is tht the α-subunit is the ctlytic subunit nd its phosphoryltion cuses inhibition of this enzyme [46]. T 3 ppers not to hve the inhibitory effect on N + -K + -ATPse ctivity by n influence on phosphtse ctivity. 5. Conclusion Our results regrding T 3 ction in reltion to the inhibition of synptosoml N + -K + -ATPse re consistent with T 3 - synptosoml membrne component binding site interction sensitive to the ctivtion of G i -protein. Such membrne binding component might interct with G s -protein, resulting in incresed synthesis of camp. The membrne N + - K + -ATPse is involved in severl spects of physiologicl processes. In the neuron, its inhibition is linked with neurotrnsmitter relese [46]. Hence, the present study provides further evidence of nongenomic membrne-relted ction of T 3 in the mture mmmlin synptosome. Understnding of the mechnism of ction of TH in dult mmmlin brinhsmjorimplictionsinthehighermentlfunctions nd in the regultion of severl neuropsychitric disorders developed in thyroid dysfunctions in dult humns. Acknowledgments Finncil support ws from the Council of Scientific & Industril Reserch, Indi, nd the Deprtment of Science & Technology, Government of Indi, to Prdip K. Srkr, nd NSF grnt IBN to Prdip K. Srkr nd Joseph V. Mrtin. References [1] J. H. Dussult nd J. Ruel, Thyroid hormones nd brin development, Annul Review of Physiology, vol. 49, pp , [2] J. Puymirt, Thyroid receptors in the rt brin, Progress in Neurobiology,vol.39,no.3,pp ,1992. [3] J. H. Oppenheimer, Evolving concepts of thyroid hormone ction, Biochimie,vol.81,no.5,pp , [4] J. Zhng nd M. A. Lzr, The mechnism of ction of thyroid hormones, Annul Review of Physiology, vol. 62, pp , 2. [5] M.B.DrtmnndJ.T.Gordon, Thyroidhormonessneurotrnsmitters, Thyroid,vol.6,no.6,pp ,1996. [6] W. N. Henley nd T. J. Koehnle, Thyroid hormones nd the tretment of depression: n exmintion of bsic hormonl

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Lin et l., Modifiction of survivl pthwy gene expression in humn brest cncer cells by tetriodothyrocetic cid (tetrc), Cell Cycle, vol. 8, no. 21, pp , 29. [27] M. Bhrgv, M. R. Runyon, D. Smirnov et l., Triiodo-Lthyronine rpidly stimultes lveolr fluid clernce in norml nd hyperoxi-injured lungs, Americn Journl of Respirtory nd Criticl Cre Medicine,vol.178,no.5,pp ,28. [28] J. Lei, C. N. Mrish, M. Bhrgv, E. V. Wttenberg, nd D. H. Ingbr, T 3 increses N-K-ATPse ctivity vi MAPK/ERK1/2-dependent pthwy in rt dult lveolr epithelil cells, Americn Journl of Physiology Lung Cellulr nd Moleculr Physiology, vol. 294, no. 4, pp. L749 L754, 28. [29] H. Y. Lin, H. Y. Tng, F. B. Dvis et l., Nongenomic regultion by thyroid hormone of plsm membrne ion nd smll molecule pumps, Discovery Medicine,vol.14,pp ,212. [3] X. Cheng, J. Höög, A. C. Nirn, P. Greengrd, nd A. 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9 Journl of Thyroid Reserch 9 [38] M. B. Conto nd M. A. Venditti, In vitro studies of the influence of glutmtergic gonists on the N +,K + -ATPse nd K + - p-nitrophenylphosphtse ctivities in the hippocmpus nd frontl cortex of rts, Journl of Negtive Results in BioMedicine, vol. 11, p. 12, 212. [39] W. L. Sthl nd W. E. Hrris, N +,K + -ATPse: structure, function, nd interctions with drugs, Advnces in Neurology, vol. 44, pp , [4] A. Levitzki, Signl trnsduction in hormone-dependent denylte cyclse, Cell Biophysics, vol. 12, pp , [41] E.M.Rose,J.C.P.Koo,J.E.Antflick,S.M.Ahmed,S.Angers, nd D. R. Hmpson, Glutmte trnsporter coupling to N,K- ATPse, The Journl of Neuroscience, vol. 29, no. 25, pp , 29. [42]M.A.Musgrve,M.A.Mdign,B.M.Bennett,ndJ.W. Goh, Stimultion of postsynptic nd inhibition of presynptic denylyl cyclse ctivity by metbotropic glutmte receptor ctivtion, Journl of Neurochemistry, vol.62,no.6,pp , [43]R.Nournifr,R.D.Blitzer,T.Wong,ndE.Lndu, Metbotropic glutmte receptors limit denylyl cyclse-medited effects in rt hippocmpus vi protein kinse C, Neuroscience Letters, vol. 244, no. 2, pp , [44] T. Endoh, Chrcteriztion of modultory effects of postsynptic metbotropic glutmte receptors on clcium currents in rt nucleus trctus solitrius, Brin Reserch, vol. 124, no. 1-2, pp , 24. [45] P. K. Srkr, l-triiodothyronine differentilly nd nongenomiclly regultes synptosoml protein phosphoryltion in dult rt brin cerebrl cortex: role of clcium nd clmodulin, Life Sciences,vol.82,no.17-18,pp ,28. [46] A. M. Bertorello, A. Aperi, S. I. Wls, A. C. Nirn, nd P. Greengrd, Phosphoryltion of the ctlytic subunit of N +,K + -ATPse inhibits the ctivity of the enzyme, Proceedings of the Ntionl Acdemy of Sciences of the United Sttes of Americ, vol. 88, no. 24, pp , [47] J. A. Gordon, Use of vndte s protein-phosphotyrosine phosphtse inhibitor, Methods in Enzymology, vol. 21, pp , 1991.

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