Effects of Captopril on Vascular Reactivity of SHR In Vivo and In Vitro MICHAEL J. ANTONACCIO, PH.D., BERNARD RUBIN, PH.D., AND DOROTHY KOTLER,

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1 Effects f Captpril n Vascular Reactivity f SHR In Viv and In Vitr MICHAEL J. ANTNACCI, PH.D., BERNARD RUBIN, PH.D., AND DRTHY KTLER, B.S. SUMMARY The effect f captpril treatment (100 mg/kg by muth daily fr up t 6 mnths) n pressr respnses t nrepinephrine (NE) and angitensin II (AH) was examined in spntaneusly hypertensive rats (SHR). Als, helical strips f rat arta were remved frm rats that had been similarly dsed. The artic strips were suspended fr ismetric recrding in mdified Krebs' slutin kept at 7 C and bubbled with 95% 0,-5%,. Pressr respnses f bth NE and AH in viv were inhibited by captpril in SHR treated fr all treatment perids. Respnses t NE were mre significantly and cnsistently inhibited than thse fr AH. Artic strips frm SHR previusly dsed with captpril shwed equivalent r greater cntractile respnses t ptassium chlride (KG) and NE, when cmpared with strips frm untreated age-matched cntrls. In artic strips frm untreated Sprague-Dawley rats incubated with captpril, 0 Mg/ m ' fr 1 hur (a cncentratin 6000 times higher than that needed t inhibit angitensin-cnverting enzyme by 50% in vitr), captpril had n effect n nitrglycerin-induced relaxatin r NE-induced cntractins, whereas ethacrynic acid (25 Mg/ml) reduced bth the NE cntractile respnse as well as the nitrglycerin-induced relaxatin. These results suggest that captpril has n direct effect n the ability f islated vascular smth muscle t cntract r relax despite causing a significant inhibitin f pressr respnses in viv. It is suggested that this effect is related t an interactin f captpril with bld-brne elements necessary fr the full expressin f vascnstrictin, but unrelated t angitensin-cnverting enzyme inhibitin. (Hypertensin (suppl II): , 1981) KEY WRDS angitensin cnverting enzyme islated arta vascnstrictin vasdilatin IN previus studies we have shwn that captpril is capable f inhibiting the pressr respnse t bth sympathetic nerve stimulatin and, at higher dses, t endgenus nrepinephrine (NE) and angitensin II (All) in spntaneusly hypertensive rats (SHR). 1 '' Inhibitin f the pressr respnse t NE after captpril was related nt nly t dse but als t duratin f therapy, that is, the higher the dse f captpril and the lnger the treatment perid, the greater the degree f inhibitin.* In vitr studies f cntractile respnses f vascular preparatins t NE, All, and sdium chlride (KC1) have given variable results in the presence f captpril. Several investigatrs reprted n effect f captpril in vitr n the cntractility f islated vascular preparatins t varius agents, 1 " 6 whereas thers bserved at least sme degree f inhibitin.*"* ne f the purpses f this study was t examine the effects f captpril dsage n the pressr respnses t NE and All in SHR in viv after varius perids f dsage, and in vitr respnses t NE and KC1 f islated SHR artae taken frm animals similarly treated. Captpril is a thil-cntaining cmpund capable f interacting either with itself r ther thil-cntain- Frm the Squibb Institute fr Medical Research, Princetn, New Jersey Address fr reprints Michael J Antnacci, Ph.D, Scheving- Plugh, 60 range Street, Blmfield, New Jersey ing cmpunds such as cysteine, when administered systemically. 10 ther cmpunds that interact with thil grups dramatically alter the ability f islated vascular smth muscle t respnd bth t vascnstrictr and vasdilatr agents. Ethacrynic acid, which presumably alklylates thil grups n smth muscle, decreased NE- and KCl-induced cntractins f rabbit ear artery 11 and vasdilatin f rabbit arta t nitrglycerin. 1 * Dithithreitl, a thil-reducing agent, decreased NE- and KCl-induced cntractin f islated arta frm bth SHR and Wistar-Kyt rats (WKY). 1S An additinal purpse f this study was t determine if the thil miety f captpril culd als interact with vascular smth muscle t alter either cntractile r dilatry respnses in vitr t NE and nitrglycerin, respectively. Methds We placed 12- t 14-week-ld male SHR (Tacnic Farms, Germantwn, New Yrk) n a nrmal chw diet and water ad libitum, and after giving them a daily ral dse f captpril (100 mg/kg), selected them at randm fr the measurement f mean arterial bld pressure (MAP), heart rate, and respnses t NE and All. The SHR were anesthetized with sdium pentbarbital (5 mg/kg, i.p.) after dsing with captpril s that respnses were btained 2 hurs after the last dse. Bld pressure was measured frm a cartid

2 PRCEEDINGS/INTERAMERICAN SCIETY SUPP II, HYPERTENSIN, VL, N 6, NV/DEC, 1981 artery and drugs administered thrugh a jugular vein catheter. Heart rate was measured with a carditachmeter triggered by the systlic bld pressure pulse. All measurements were recrded n a Beckman Dyngraph. Thracic rat artae were excised frm SHR and Sprague-Dawley male rats sacrificed by a blw n the head. The artae were cut spirally int strips 14 and then suspended in 10 ml tissue baths cntaining mdified Krebs' slutin 18 bubbled with 95% 0,-5% i at 7 C. Cntractile activity was recrded ismetrically under 1 g tensin via Grass FT.0 frce displacement transducers cupled t a Beckman Dyngraph. Apprximately 1 hur equilibratin was used befre any drugs were added. In the SHR arta preparatin, NE bitartrate in cncentratins ranging frm 10~ n M t 10 7 M, r KC1 in cncentratins ranging frm X 10"' M t 10~! M, were added at apprximately 20-minute intervals. Each cncentratin was allwed t reach maximum cntractile effect fr 10 minutes, and then the preparatin was washed and relaxed with drug-free Krebs' slutin fr anther 10 minutes. Grups f five t six artic strips were used frm rats either dsed with captpnl r frm nndsed age-matched cntrls. All changes are shwn as grams f tensin. In the artic preparatins taken frm Sprague- Dawley rats, fur grups f six rats were emplyed, each tissue being cntracted by 50 ng/ml f 1-NE bitartrate fr 10 minutes, and then subsequently treated with cumulative relaxant cncentratins f nitrglycerin ranging frm 1 t 1000 ng/ml, each at 5- minute intervals. Thereafter, each f the fur grups f tissues was treated with Krebs' slutin fr 1 hur with either captpril (0 jig/ml), ethacrynic acid (10 r 25 Mg/ml), r drug-free Krebs' slutin, and then subjected t the NE-nitrglycerin treatment utlined Q) 40 0 D m 10 T abve. Changes in tensin are shwn in either abslute frm (Ag) r in relative frm (% relaxatin). Mean changes in cntractile r relaxant effects were cnsidered significant at p 0.05 in unpaired / tests and in paired t tests where apprpriate. Results Pressr Respnses t Angltensin II and Nrepinephrine in Pithed SHR Pressr respnses t bth All and NE were significantly inhibited in SHR pretreated with captpril (fig. 1). The respnses t NE were mre inhibited than thse t All. In additin, the inhibitin was greater after chrnic therapy than acute therapy. Respnses f Artae frm Captpril-Treated SHR t Nrepinephrine and Ptassium Chlride Artic strips frm SHR predsed with captpril shwed greater r equivalent cntractile respnses t NE and t KC1 when cmpared with strips frm untreated age-matched SHR (fig. 2). Artic Respnses t Nitrglycerin and Nrepinephrine after Incubatin with Either Captpril r Ethacrynic Acid In artic strips frm Sprague-Dawley rats incubated with captpril (0 fig/ml fr 1 hur), there was little r n effect n NE induced cntractins r nitrglycerin-induced relaxatins as cmpared with the drug-free Krebs' cntrls series (fig. ). n the ther hand, the artic strips incubated with ethacrynic acid (25 Mg/ml) significantly decreased the cntractile effect f NE, and significantly and markedly reduced the relaxant effect f nitrglycerin (fig. ). Ethacrynic acid at 10 Mg/ml, hwever, did nt significantly affect either the NE-induced cntractile respnse r the D) X 0) (I) * p0 05 **p0.01 Saline (12) 2hrs (6) 2 wks ms (8) 6 ms 0- Saline (12) 2hrs (6) 2 wks ms (8) 6 ms. FIGURE I. Effects f captpril (Cap.) dsage, 100 mg/kg p daily n pressr respnses t angitensin II (left) and nrepinephrine (right) (1 ng/kg) in pithed SHR. All respnses are 2 hurs after the last dse f captpril. Parentheses indicate number f SHR in each grup.

3 CAPTPRIL AND VASCULAR REACT!V1TY/Antnaca et al SHR - NN-DSED AGE-MATCHED NTRL RATS - DSED, CAPTPRIL 100 MG/KG/DAY P N=5-6/PINT = p0 05 E z c cr EC I WEEKS 10 X X10 2 MNTHS 6 MNTHS KCl NCENTRATIN (M) E z c t DC I 1 0_ WEEKS AV - A MNTHS 6 MNTHS 10" i '"1'1 10 ' 10 ' ' 10"' u 10" M 10" 10 ' 10 ' ' 10 n-9 m-8,n~f NREPINEPHRINE NCENTRATIN (M) FIGURE 2 Cntractile respnses f artic strips t KCl and nrepinephrine frm captpril-treated SHR and frm untreated, age-matched SHR. Each pint n thefigurerepresents the mean ± SEM. *p relaxant effect f nitrglycerin (nt shwn). Representative tracings fr each f the fur grups are shwn in figure 4. Discussin In the present study, captpril caused inhibitin f pressr respnses t bth NE and All in SHR treated daily fr 1 day r up t 6 mnths. Similar results have been bserved in WKY. 1 " Als, marked inhibitin f pressr respnses t sympathetic stimulatin has been bserved in cmparably treated SHR." Inhibitin in this study was greater and mre cnsistent n the NE than All pressr respnse and became mre bvius with increased duratin f dsage. It is unlikely that captpril therapy in SHR interfered with the intrinsic ability f rat artic strips t either cnstrict r relax. Islated artic strips frm SHR treated with captpril in the same manner as fr the in viv studies shwed n inhibitin f cntractile respnses t either NE r KCl. In fact, many tissues frm captpril-treated rats cntracted significantly mre t bth NE and KCl than did cntrls. This was bserved in artic strips frm SHR taken after weeks, and 6 mnths f daily therapy. In previus studies, captpril administered in vitr was shwn t have n effect n cntractile respnses f vascular tissues t NE, KCl, r AII,'~ S althugh ther studies 6 "" have shwn sme degree f inhibitin after rather high cncentratins f captpril. Fr example, superir mesenteric arteries f Wistar rats perfused at a cnstant rate with Krebs' slutin cntaining captpril (-81 Mg/ m ') prduced a cncentratin-dependent inhibitin f the pressr respnse f NE as well as KCl.' Artic rings frm nrmtensive rats and tw-kidney, tw wrapped hypertensive rats dsed previusly with captpril (48 mg/kg/day fr 5 weeks) shwed decreased cntractile respnses t NE, phenylephrine, and KCl. 7 Similarly, pretreatment f artic rings with a cncentratin f captpril at 2 X 10" 4 M (4 fig/m\) inhibited the cntractile respnse t bth NE and KCl.' ther investigatrs have perfused the excised kidney f Sprague-Dawley rats with Tyrdes' dextran slutin at a cnstant rate. The perfusin slutin,

4 PRCEEDINGS/INTERAMERICAN SCIETY SUPP II, HYPERTENSIN, VL, N 6, NV/DEC, NANG/ML NREPINEPHRINE-INDUCED TENSIN 97±57 MG 980±67 MG 100, T 746±54 MG I V 46± MG 1000 "1 (NANG/ML) (NANG/ML) HR ETMACRYNIC ACID 25^0/ML FIGURE Relaxatin by mtrglycerin () f nrepmephrine-induced cntractin fsprague-dawley rat artic strips Six strips each were used in (a) Krebs cntrl, (b) captpril 0 ng/ml, (c) ethacrynic acid 25 ng/'ml. Each pint n the figure represents the mean ± SEM *p 0.05 which cntained as much as 100 fig f captpril/min/ml, resulted in an inhibitin f the pressr respnses t NE and t All but nt t sertnin. 8 n the ther hand, ther investigatrs fund that captpril had little r n acute inhibitry effect n the cntractile respnses t NE, KC1, r ther agnists. Fr example, excised strips f thracic rabbit arta, rat prtal vein, as well as several nnvascular tissues bathed in mdified Krebs' slutin shwed n reductins in the usual mechanical respnse t NE, All, and dpamine, as well as t a number f ther agnists after pretreatment with captpril (. 100 jzg/ml).' Similar negative results against All-induced cntractins were btained by thers in cat superir mesenteric artery strips bathed in 5 Mg/ m l f tpril, 4 as well as in captpril-treated artic strips f SHR cntracted with NE, All, r sertnin. 8 Regardless f these results btained with the use f captpril in vitr, it seems clear frm this study that even very high dses f captpril in SHR fr up t 6 mnths des nt alter the ability f their subsequently islated vascular smth muscle t cntract in vitr t NE r KC1, despite inhibitin f pressr respnses t NE and AH in viv after the same dsage. These data suggest that captpril may interfere with a bldbrne substance(s) that is nrmally necessary fr the full pressr respnse t vascnstrictr agents t be expressed. Furthermre, the thil grup f captpril seems t be invlved rather than simple angitensincnverting enzyme inhibitin since the angitensincnverting enzyme inhibitr MK-421 had n effect f pressr respnses either t sympathetic nerve stimulatin, NE, r All. 17 There are, f curse, ther pssible alternative explanatins such as the ability f captpril t interact with sulfhydryl grups n plasma prteins. Mre wrk is needed t explre these pssibilities. Captpril has been shwn t frm disulfides with itself, glutathine, and, in particular, cysteine. 10 It seems pssible that such an interactin with these r ther substances in bld may be smehw respnsible fr the in viv inhibitin f pressr respnses bserved after captpril but nt MK-421. It shuld als be pinted ut that artic tissue f SHR dsed with captpril at the same dse used in this study (100 mg/kg/day) fr 6 weeks had a reduced sdium and ptassium cntent althugh the sdium/ptassium rati was unchanged. 1 " The significance and relevance f these findings t the present bservatins are uncertain but d nt seem t explain the lack f captpril's in vitr effects. T determine if the thil grup f captpril might interact directly with vascular smth muscle, we examined its effects n the vasdilatr actins f nitrglycerin n artic strips cntracted with NE. Previus studies have shwn that ethacrynic acid, an alkylating agent fr thil grups, decreased rabbit artic sensitivity t nitrglycerin as well as ther vasdilatrs. 11 Dithithreitl, a thil-reducing agent which has essentially n angitensin-cnverting enzyme inhibitry activity, ' u decreased NE- and KCl-induced cntractins f artic strips frm SHR and WKY. 18 In this study, we cnfirmed the ability f ethacrynic acid t markedly attenuate the vasdilatin caused by nitrglycerin. Als, ethacrynic acid significantly inhibited the cntractin t NE, agreeing with the results f Neenng and Glver" but differing frm thse f Needleman et al. 11 In any case, captpril at a cncentratin f 0 Mg/ml neither inhibited the cntractin f artic strips t NE nr the vasdilatin t nitrglycerin. This cncentratin f kaptpril exceeds the IC M fr its angitensin-cnverting enzyme inhibitry activity by sme 6000-fld. Thus, it seems highly unlikely that captpril has any direct interactin with thils r entities n vascular smth muscle that are necessary fr either cntractin r dilatin.

5 CAPTPRIL AND VASCULAR REACTIVITY/Antnacci et al CAPTPRIL NE50 1 LJ 1 I W W 1Hr T Treatment 1gn i KREBS NTRL 1 Hr Treatment 5 mln NE50 NE50 I I I I I I W W ETHACRYNIC ACID - T 1 Hr I Treatment 1gm ETHACRYNIC ACID 1 Hr Treatment?? T I I I I I NE FIGURE 4. Relaxatin by nitrglycenn () f nrepinephrine (NE)-induced cntractins f Sprague- Dawley rat artic strips. Representative tracings f effects fcaptpril, Krebs cntrl, and ethacrynic acid Cncentratins f and NE are in ng/ml. These data further supprt the suggestin that the inhibitry effects fcaptpril n vascular respnses in viv are indirect and prbably related t interactins with bld-brne substances. It shuld be nted that, in cmparable studies, captpril caused inhibitin f cntractin f arta frm WKY t NE and KC1 1 ' althugh the inhibitin was incnsistent and unrelated t the duratin f dsage. This may reflect differences amng hypertensive and nrmtensive animal mdels with respect t the actins f captpril n vascular smth muscle, and demnstrates the cautin required in reaching any universal statements abut vascular smth muscle respnsivity after captpril administratin. References 1 Antnacci MJ, Kcrwin L Evidence fr prejunctinal inhibitin f nrepinephrine release by captpril in spntaneusly hypertensive rats. Eur J Pharmacl 68: 209, Antnacci MJ, Kerwin L/ Pre- and pst-junctinal inhibitin f vascular sympathetic functin by captpril in SHR. Hypertensin, : (suppl I). 1-54, 1981 Rubin B, Laffan RJ, Ktler DG, 'Keefe EH, DeMai DA, Gldberg ME. SQ 14,225 (D--mercapt-2-methylprpanlyl- L-prline), a nvel rally active inhibitr f angitensin I- cnverting enzyme J Pharmacl Exp Ther 204: 271, Trachte GJ, Lefer AM: Beneficial actin f a new angitensincnverting enzyme inhibitr (SQ 14,225) in hemrrhagic shck in cats. re Res 4: 576, Lai FM, Tanikella T, Herzlinger H, Chan DS, Cervni P Studies n the mechanism f the antihypertensive activity f captpril by a diuretic in spntaneusly hypertensive rats. Fed Prc 9: 496, kun T SQ 14,225 attenuates the vascular respnse t nrepinephnne in the rat mesenteric arteries Life Sci 25: 14, Kikta DC, Fregly MJ. Effect f chrnic treatment with captpril n reactivity f artic rings frm nrmtensive and hypertensive rats (abstr). Physilgist 2: 126, Kikta DC, Fregly MJ Captpnl-induced changes in vascular reactivity during acute administratin in vitr (abstr) Fed Prc 9: 1191, Casellas D, Mimran A, Dupnt M, Chevillard C. Attenuatin by SQ 14,225 (captpril) f the vascular respnse t nradrenalinc in the rat islated kidney Br J Clin Pharmacl 10: 621, Knpalani KJ, McKinstry DN, Singhvi SM, Willard DA, Vukvich RA, Migdalf BH. Dispsitin fcaptpril in nrmal subjects Clin Pharmacl Ther 27: 66, Needleman P, Jakschick B, Jhnsn EM Jr. Sulfhydryl requirement fr relaxatin f vascular smth muscle. J Pharmacl Exp Ther 187: 24, Neenng IR, Glver WE The rle f sulfhydryl grups in cntractin f vascular smth muscle. J Pharmacl Exp Ther 208: 5, Mushlin PS, Rama Sastry BV, Berth RC, Surber MJ, Landn EJ Dithithreitl-induced alteratins f bld pressure, vascular reactivity and artic micrsmal calcium uptake in spntaneusly hypertensive rats J Pharmacl Exp Ther 207: 1, Furchgtt RF Spiral-cut strips f rabbit arta fr in vitr studies f respnses t arterial smth muscle In Methds f Medical Research, vl 8, edited by Bruner HD Chicag. The Yearbk Publishers, 1960, p Handschumacher RE, Vane JR The relatinship between the penetratin f tryptamine and 5-hydrxytryptamine int smth muscle and the assciated cntractins. Br J Pharmacl 29: 105, Rubin B, Ktler DG, Antnacci MJ Effects f captpril (C) n vascular reactivity f excised rat arta t KG, nrepinephrine (NE) and nitrglycenn () (abstr) Fed Prc 40: 276, Antnacci MJ, Kerwin L, Rubin B, Ktler D Effects fcaptpril n pre- and pst-junctinal sympathetic functin in SHR and WKY rats Interamer Sc Hypertensin In press 18 H K, Kike H, Miyamt M, Urakawa N Lng-term blckade f angitensin cnverting enzyme alters passive in transprt f vascular smth muscle. Life Sci 26: 102, Cushman DW, Cheung HS Spectrphtmetnc assay and prperties f angitensin-cnverting enzyme f rabbit lung Bichem Pharmacl 20: 167, 1971