Mechanism of Action of Carbocyclic Thromboxane A 2 and Its Interaction with Prostaglandin I 2 and Verapamil in Isolated Arteries

Transcription

1 Circulatin Research An Official Jurnal f the American Heart Assciatin DECEMBER 1982 VOL. 51 NO. 6 Mechanism f Actin f Carbcyclic Thrmbxane A 2 and Its Interactin with Prstaglandin I 2 and Verapamil in Islated Arteries Nbru Tda Frm the Department f Pharmaclgy, Shiga University f Medical Sciences, Seta, Ohtsu , Japan SUMMARY. Carbcyclic thrmbxane A2 (1CT 9 t 10~ 7 M) prduced a cncentratin-dependent cntractin f helical strips f dg cerebral, crnary, mesenteric, renal, and femral arteries and f mnkey cerebral, crnary, and mesenteric arteries. Cntractins induced by lw cncentratins f carbcyclic thrmbxane A2 tended t be greater in cerebral arterial strips. Even after 60 minutes f expsure t Ca ++ -free media, apprximately ne-half f the cntractile respnse f dg cerebral and mesenteric arteries t 10~ 8 M carbcyclic thrmbxane A2 was retained. The cntractile respnse was attenuated by diphlretin phsphate, a prstaglandin antagnist, and was ptentiated by aspirin. In dg cerebral arterial strips cntracted with carbcyclic thrmbxane, the relaxant respnse t prstaglandin I2 was less than the respnse seen in mesenteric and crnary arteries, whereas, in cntrast, the respnse t verapamil was greater in cerebral arteries. Cncentratin-relaxatin curves fr papaverine did differ appreciably. It may be cncluded that cntractins induced by carbcyclic thrmbxane are assciated with the release f Ca ++ frm intracellular strage sites and, in additin, the increase in transmembrane Ca ++ influx. Greater susceptibility f cerebral arteries t verapamil may indicate that the Ca ++ antagnist is f use t relieve the persistent cntractin f cerebrarterial smth muscle. Prstaglandin I2 appears t cunteract effectively the actin f ptent vascnstrictrs, such as thrmbxane A2 and its carbcyclic analg, in varius vascular beds. (Circ Res 51: , 1982) THROMBOXANE A 2 (TxA 2 ) was first intrduced by Hamberg et al. (1975) as an unstable intermediate, which is cnverted frm prstaglandin (PG) endperxides by catalysis f an enzyme cntained in the micrsmal fractin f human and hrse platelets. TxA 2 in minute amunts prduces a cntractin f islated rabbit artae (Needleman et al., 1976), rabbit celiac and mesenteric arteries (Bunting et al., 1976), bvine cerebral and crnary arteries (Ellis et al., 1977), prcine crnary arteries (Svenssn and Hamberg, 1976), and guinea pig crnary vasculature (Terashita et al., 1978) and als a platelet aggregatin (Hamberg et al., 1975). Therefre, TxA 2 is regarded as ne f the endgenus substances respnsible fr crnary and cerebral vasspasm. Hwever, the rapid degradatin f this substance in buffer slutins [the half life f abut 30 secnds (Hamberg et al., 1975)] makes the analysis f its vascular actins difficult. Recently, Lefer et al. (1980) have synthesized a stable analg f TxA 2, carbcyclic TxA 2, (ctxa 2 ), which is a ptent crnary vascnstrictr but des nt shw a platelet-aggregating activity. The present study was undertaken t cmpare the actin f ctxa 2 n islated dg cerebral, crnary, mesenteric, renal, and femral arteries f similar size and n islated mnkey cerebral, crnary, and mesenteric arteries, t analyze the mechanism f actin f ctxa 2 in the dg arteries, and t determine different effectiveness f vasdilatr agents, such as PGI2, verapamil, and papaverine, in a variety f dg arteries cntracted with ctxa 2. Methds Mngrel dgs f either sex, weighing 8-15 kg, were anesthetized with intraperitneal injectins f sdium thipental (50 mg/kg) and killed by bleeding frm the cartid arteries. Japanese mnkeys (Macacca hiscata) f either sex, weighing 6-11 kg, were anesthetized with intramuscular injectins f ketamine (25-40 mg/kg) and sacrificed. The brain, heart, and kidneys were rapidly remved. Basilar and middle cerebral arteries ( mm utside diameter in dgs; mm in mnkeys) were islated frm the brain, ventral interventricular, and circumflex branches f the left crnary artery ( mm in dgs; mm in mnkeys) were islated frm the heart, and intrarenal, interlbar branches f the renal artery ( mm in dgs) were islated frm the kidneys. Distal prtins f superir mesenteric ( mm in dgs; mm in mnkeys) and femral arteries ( mm in dgs) als were islated. The arteries were helically cut int strips, apprximately 20 mm lng (Tda, 1981). The specimen was vertically fixed

2 676 Circulatin Research/V/. 51, N. 6, December 1982 between hks in the muscle bath cntaining the nutrient slutin, which was maintained at 37 ± 0.3 C and aerated with a mixture f 95% O2 and 5% CO2. The hk anchring the upper end f the strips was cnnected t the lever f a frce-displacement transducer (Nihnkden Kgy C., Tky, Japan). The resting frce was adjusted t 1.5 g fr dg arteries (Tda et al., 1978) and t 1.0 g fr mnkey arteries (Tda, 1981). Cnstituents f the slutin were as fllws (HIM): Na +, 140; K +, 5.4; Ca ++, 2.2; Mg ++, 1.0; Cl~, 131.8; HCO3", 20.0; and dextrse, 5.6. The ph f the slutins was Befre the start f experiments, all the arterial strips were allwed t equilibrate in bathing media fr minutes, during which time the bathing media were replaced every minutes. Ismetric cntractins and relaxatins were displayed n an ink-writing scillgraph (Nihnkden Kgy C.). The cntractile respnse t 30 mm K + was btained first, then the preparatins were repeatedly washed and equilibrated fr minutes. Respnses t ctxa2 were btained in arterial strips under resting cnditins, and thse t vasdilatr drugs, such as PGI2, verapamil, and papaverine, were btained in the strips previusly cntracted with 10~ 7 M ctxa2. The cncentratin-repnse curve was btained by adding these drugs directly t the bathing media in cumulative cncentratins. At the end f each series f experiments with the vasdilatr drugs, papaverine in a cncentratin f 10~ 4 M was added t attain the maximum relaxatin (Tda, 1974a), and relaxatins relative t thse induced by papaverine are presented. Preparatins had been treated fr minutes with diphlretin phsphate (DPP), aspirin, phentlamine, chlrpheniramine, and cinanserin befre the respnse t ctxa2 was btained. Sme f dg cerebral and mesenteric arterial strips were studied fr the dependence f ctxa2-induced cntractins upn extracellular Ca ++. After the cntractile respnse t 10~ 8 M ctxa2 was btained in nrmal media, the strips were repeatedly washed with drug-free slutins and expsed t Ca ++ -free media fr 60 minutes, during which time the slutins were replaced twice every 20 minutes. ctxa2 (10~ 8 M) then was added. After the ctxa2-induced cntractin leveled ff, Ca ++ (2.2 and 4.4 mm) was added. The results shwn in the text, figures, and tables are expressed as mean values ± SEM. Statistical analyses were made using the Tukey's methd after the ne-way analysis f variance (Wallenstein et al., 1980) (Figure 1 and Tables 1, 2, and 3) r the Student's paired and unpaired t-test. Drugs used were carbcyclic thrmbxane A2 (ctxa2>, prstaglandin (PG) I2 sdium salt, diphlretin phsphate (DPP) (On Pharmaceutical C.) d/-verapamil hydrchlride, papaverine hydrchlride, acetylsalicylicacid (aspirin), phentlamine mesylate, d-chlrpheniramine maleate, uabain ctahydrate (E. Merck) and cinanserin. ctxa2 was synthesized in On Pharmaceutical C. by the methd reprted by Ohuchida et al. (1979). This substance was disslved in abslute alchl t make a stck slutin (10~ 3 M) and diluted with distilled water befre use. Results Cntractile Respnse t ctxa2 f a Variety f Arteries In helically cut strips f dg cerebral, crnary, mesenteric, renal, and femral arteries, the additin f ctxa2 in cncentratins in a range between 1CT 10 and 10~ 7 M caused a dse-related cntractin (Fig. 1, left). Further increase in the cncentratin t 10~ 6 M prduced nly a slight r n additinal cntractin ct>a2 I M ) MONKEY ARTERY Ctrcbrl ( 14 I O Crnary ( 8 ) X Mtstntcric ( 8) I0* 10 Cne, r ct«fl2 I M ) FIGURE 1. Cncentratin-respnse curves fr ctxai in strips f a variety f arteries islated frm dgs (left figure) and mnkeys (right). Cntractins induced by 30 miw K + were taken as %; mean abslute values in dg cerebral, crnary, mesenteric, renal, and femral arteries were 2153 ± 216 mg (n = 17), 1788 ± 206 mg (n = 18), 2588 ± 298 mg (n = 13), 2662 ± 213 mg (n = 14) and 2227 ± 210 mg (n = 12), respectively, and the values in mnkey cerebral, crnary and mesenteric arteries were 1059 ± 172 mg (n = 14), 1465 ± 129 mg (n = 8) and 1314 ± 266 mg (n = 8), respectively. Numbers in parenthesis indicate the number f preparatins used. Vertical bars represent SEM. a. Significantly different frm values in cerebral arteries, P < 0.02; b, P< 0.05; c, significantly different frm values in crnary arteries, P < Typical recrdings f the respnse f a cerebral arterial strip are shwn in Figure 2. In lw cncentratins (1CT 10 and 10~ 9 M), the cntractins f cerebral arteries relative t thse induced by 30 mm K + tended t be greater than thse f the ther arteries, whereas in cntrast in the higher cncentratins, extracerebral arteries cntracted t a greater extent. Mean values f the apparent median effective cncentratin (ED50) f ctxa2 are summarized in Table 1. The values in different dg arteries did nt differ significantly. Mnkey cerebral, crnary, and mesenteric arterial strips als cntracted dse dependently in respne t ctxa2 in cncentratins ranging frm 10~ 9 t 1CT 7 M (Fig. 1, right). Cntractins f mesenteric arteries at a lw cncentratin (1CT 9 M) were less than thse f FIGURE 2. Respnses f dg basilar arterial strips t ctxa?, PCI 2, and verapamil. Tw strips were btained frm the same dg. The hrizntal line just left f the lwer tracing represent the level prir t the additin f 10~ 7 M CTXAI. Verap. = verapamil; PA = 10~ 4 M papaverine.

3 Tda/Vascular Actin f Carbcyclic Thrmbxane A crnary arteries. Cmpared with dg arteries, mnkey arterial strips respnded with a greater cntractin t 1CT 9 M ctxa2. Apparent ED50 values in mnkey cerebral and crnary arteries (Table 1) were significantly less than thse in the dg arteries. In dg cerebral arteries expsed fr 60 minutes t Ca ++ -free media, ctxa 2 (10~ 8 M) prduced a tnic cntractin, the magnitude f which was 48.9 ± 7.9% (n = 8) f the cntractin seen in nrmal media. When the ctxa 2 -induced cntractin stabilized, the additin f 2.2 ITIM Ca ++ elicited a marked, transient cntractin fllwed by a relaxatin and a persistent cntractin (Fig. 3). Change in the Ca ++ cncentratin frm 2.2 t 4.4 mm prduced a relaxatin. The persistent cntractin stabilized during the expsure t Ca ++ fr 30 t 60 minutes at a level similar t that attained by ctxa 2 in the nrmal media. Quantitative data are presented in Figure 4, upper panel. Similar results were btained with mesenteric arteries expsed t Ca ++ -free media (Fig. 4, lwer), althugh the transient cntractin was nt s evident as that seen in cerebral arteries (123% vs. 172%, relative t cntractins induced by 10~ 8 M CTXA 2 in nrmal media). In cerebral arterial strips, the Ca ++ (2.2 mm)-induced cntractin was ptentiated by treatment with 2 X 10~ 7 M uabain; the transient relaxatin was ablished and the cntractin persisted fr 20 minutes r lnger (stabilized cntractins f 195 ± 24.2%, n = 7, relative t cntractins induced by ctxa 2 in nrmal media, cf. 103 ± 8.0% shwn as B in Figure 4, upper panel), as was the respnse t Ca ++ in the arteries expsed t Ca ++ -free media and deplarized by excess K + (Tda, 1974b). When the Ca ++ -induced cntractin was stabilized, the further additin f 2.2 mm Ca ++ prduced an additinal cntractin in the presence f uabain (n = 6). Treatment with verapamil (10~ 7 and 10" 6 M) ablished the phasic cntractin and relaxatin induced by Ca ++ and attenuated the TABLE 1 Mean Values f the Apparent Median Effective Cncentratin (ED) f ctxa 2 in Dg and Mnkey Arterial Strips Artery Dg Cerebral Crnary Mesenteric Renal Femral Mnkey Cerebral Crnary Mesenteric n (X 10~ 9 M) 5.09 ± ± ± ± ± ± 0.50* 1.34 ± 0.48f 4.10 ± 0.99 * Significantly different frm the value with dg cerebral arteries, P < t Significantly different frm the value with dg crnary arteries, P < F ratis btained by the analysis f variance (6.21 fr dg arteries and 3.96 fr mnkey arteries) are greater than the P = and 0.05 critical values, respectively. Hwever, there was n significant difference between ED50 values in different dg arteries and thse in mnkey arteries. et.a2 L C**-frw nwdja FIGURE 3. Respnses f a dg basilar arterial strip t ctxa 2 in the presence and absence f external Ca**. Left tracing = cntractin induced by 10~ 8 M CTXAZ in nrmal media; right tracing = cntractins induced by 10' e M CTXAI in Ca**-free media, and respnses' t the additin f Ca** (2.2 and 4.4 mm). tnic cntractin in a cncentratin-dependent manner; mean values f the tnic cntractin induced by ctxa 2 plus Ca ++ were 68.6 ± 8.5% (n = 5) and 26.8 ± 3.1% (n = 6), respectively (cf. cntrl value f 103 ± 8.0%, n = 8). Aspirin (5 X 10~ 5 M) did nt alter the triphasic respnse t Ca ++ (n = 5). Cerebral art. 200 c I c O c O Mesenteric art. 50 CTXA2 L ctxa 2 A B Ca*2.2mM Ca*-free FIGURE 4. Cntractile respnses t ctxa 2 and Ca ** f dg cerebral and mesenteric arteries expsed previusly t nrmal and Ca **- frce media. Cntractins induced by 10~ s M CTXA 2 in nrmal media were taken as %; mean abslute values in cerebral and mesenteric arteries were 1880 ± 208 (n = 8) and 2772 ± 522 mg (n = 9), respectively. A = initial, transient cntractin induced by 2.2 mm Ca ** in Ca **-free media, in additin t the cntractin induced by ctxav B = persistent cntractin induced by 2.2 mm Ca** plus ctxaz-induced cntractin (see Fig. 3). Number f preparatins used: eight cerebral and nine mesenteric arterial strips, a, Significantly different frm cntrl ctxai-'mduced cntractins, P <

4 678 Mdificatin by Antagnists f the Respnse t ctxa2 f Dg Arteries Cntractins induced by 10" 7 M CTXA2 were nt cmpletely reversed by repeated washing f preparatins at intervals f minutes during an bservatin perid f 3-4 hurs. Hwever, the cntractins induced at the lwer cncentratins (10~ 8 M r lwer) were cmpletely reversed by repeated washing, althugh it tk minutes t attain the stabilized level cmparable t that prir t the additin f ctxa 2. When the cntractins were cmpletely reversed, the respnse t 10~ 8 M CTXA2 were reprducible three times. Therefre, the influence f antagnists n the respnse t a single cncentratin (10~ 8 M) f ctxa2 was tested. Cntractile respnses f cerebral and mesenteric arteries t ctxa 2 (10~ 8 M) were attenuated by treatment fr 30 min with diphlretin phsphate (DPP, 3 X 10~ 6 and 10~ 5 M) in a dse-dependent manner (Fig. Circulatin Research/V/. 51, N. 6, December 1982 Cerebral art. 50 w O Mesenteric art. ctxa 2 IO" 8 M Cerebral art. h IO" 8 M ctxag IO" 7 M ctxa 2 50 Z 50 O 0 L Mesenteric art. h Z 50 0 Cntrl DPP 3XI0" 6 M DPP IO" 5 M FIGURE 5. Mdificatin by diphlretin phsphate (DPP) f the cntractile respnse f dg cerebral and mesenteric arteries t ctxa 2. Cntractins induced by 10' a M CTXA 2 befre treatment with DPP were taken as %; mean abslute values in cerebral and mesenteric arteries were 2318 ± 280 mg (n = 8) and 2652 ± 227mg (n = 6), respectively. When the cntractile respnse t 10~ a M ctxaz was suppressed by 10~ b M DPP, the higher cncentratin (10~ 7 M) fctxa 2 was added. Number f preparatins used: eight cerebral and six mesenteric arterial strips, a, Significantly different frm cntrls, P < 0.002; b, P < L Cntrl Aspirin Wash FIGURE 6. Mdificatin by aspirin f the cntractile respnse f dg cerebral and mesenteric arteries t ctxa 2. Cntractins induced by 10~ a M ctxa 2 befre treatment with 5 X 2O~ 5 M aspirin were taken as %; mean abslute values in cerebral and mesenteric arteries were 1764 ± 251 mg (n = 7) and 1992 ± 304 mg (n = 8), respectively. "Wash" indicates remval f aspirin frm bathing media. Number f preparatins used: seven cerebral and eight mesenteric arterial strips, a, Significantly different frm cntrls, P < 0.001; b, P < ). Even when the respnse t 10~ 8 M f the drug was markedly suppressed by 10" 5 M DPP, the preparatins respnded with a mderate cntractin t 10 M ctxa 2. The attenuatins by DPP f the respnse f cerebral and mesenteric arteries did nt differ significantly. Treatment with these cncentratins f DPP did nt inhibit the cntractile respnse t sertnin in fur f fur cerebral arteries r the respnse t nrepinephrine in fur f fur mesenteric arteries. Treatment fr 20 minutes with 5 X 10~ 5 M aspirin significantly ptentiated the cntractins f cerebral and mesenteric arteries induced by 10~ 8 M CTXA 2 (Fig. 6). The ptentiatin was reversed by repeated washing f preparatins. Cntractile respnses t ctxa 2 were nt significantly influenced by treatment with 10~ 6 M phentlamine, 10~ 6 M cinanserin, and 10~ 6 M chlrpheniramine; as cmpared with cntractins befre the treatment, percent changes averaged 0.7 ± 6.4 (n = 3), +3.3 ± 3.3 (n = 4), and -2.0 ± 0.9 (n = 5), respec-

5 Tda/ Vascular Actin f Carbcyclic Thrmbxane A TABLE 2 Mean Values f the Maximum Relaxatin Induced by PGI2 and Verapamil Relative t the Papaverine (10~ 4 M)-Induced Relaxatin in Dg Arterial Strips Maximum relaxatin (9i Artery n PGI2 P< n Verapamil P< Cerebral ± ± 2.72 Crnary ± ± Mesenteric ± ± Renal ± ± n = number f preparatins used. F-ratis btained frm the analysis f variance (6.75 fr PGI 2 and 14.6 fr verapamil) are greater than the P = critical value. Only the values significantly different by Tukey's methd are included. tively, in cerebral arteries, and +2.0 ± 0.9 (n = 4), +5.6 ± 3.1 (n = 5) and -0.4 ± 3.0 (n = 5), respectively, in mesenteric arteries. Respnses t Vasdilatr Drugs f Dg Arteries Cntracted with ctxa2 In cerebral, crnary, mesenteric, and renal arterial strips cntracted with 10~ 7 M CTXA2, the effect f vasdilatr drugs, including PGI2, verapamil, and papaverine, were cmpared. PGI2 rapidly relaxed the arterial strips (Fig. 2). The additin f PGI2 in cncentratins ranging frm 10~ 9 t 10~ 6 M caused a dsedependent relaxatin (Fig. 7, left). The relaxant respnse f cerebral arteries was less than the respnse f the ther arteries (Table 2). Relaxatins f mesenteric and crnary arteries were greater; apparent ED50 values f these arteries were significantly less than the value f cerebral arteries (Table 3). Verapamil caused a slwly-develping relaxatin, which leveled ff within 10 t 30 min (Fig. 2). In cntrast t PGI2, verapamil relaxed cerebral arteries t a greater extent than the ther arteries (Fig. 7, middle). Althugh the maximum relaxatin f cerebral arteries induced by 10~ 5 M verapamil was greater than that f the ther arteries (Table 2), apparent ED50 values did nt significantly differ in these arteries (Table 3). Papaverine (10~ 7 t 10~ 4 M) relaxed the arterial strips in a dse-dependent manner. Increase in the cncentratin t 5 X 10~ 4 M did nt prduce an additinal relaxatin, as previusly reprted (Tda, 1974a). Therefre, the relaxatin induced by 10~ 4 M papaverine was taken as %. Relaxant respnses t (Fig. 7, right) and apparent ED50 values f papaverine (Table 3) in cerebral, crnary, mesenteric, and renal arteries did nt differ significantly. TABLE 3 Mean Values f the Apparent Median Effective Cncentratin (ED50) f PGI2, Verapamil and Papaverine in Dg Arterial Strips Artery Cerebral Crnary PGI2 (X 10~ 8 M) 8.17 ± 1.43 (15) 1.91 ± 0.43 (12) P< 0.05 EDs Verapamil (X 10" 7 M) 1.00 ± 0.13 (16) 1.80 ± 0.24 (14) Papaverine (X 10" 6 M) 2.86 ± 0.36 (13) 4.38 ± 0.59 (14) Mesenteric 2.27 ± 0.50 (10) ± 0.38 (10) 3.19 ± 0.55 (9) Renal 4.60 ± 1.50 (11) 2.20 ± 0.46 (12) 4.58 ± 0.69 (10) Numbers in parentheses indicate the number f preparatins used. F ratis btained by the analysis f variance (6.75 fr PGI2 and 3.04 fr verapamil) are greater than the P = and 0.05 critical values, respectively. Only the values significantly different by the Tukey's methd are included. The F rati fr papaverine (2.40) is less than the P = 0.05 critical value.

6 680 Circulatin Research/V/. 51, N. 6, December 1982 Cerebral ( 15) O Crnary ( 12 ) X Mesenteric ( 10) a Renal (II) Cerebral ( 16) Crnary ( 14 ) Mesenteric ( IO) Renal ( 12) Cerebral ( 13) O Crnary ( 14) X Mesenteric ( 9) A Renal ( 10) IO" 9 I0" 8 IO" I0 7 I0" 6 Cne, f PGI 2 ( M) IO" 8 IO" 7 IO" 6 IO" S Cne, f verpatntl { M ) I0" 7 IO" 6 10" 5 Cne, f papaverine ( M ) i- 4 FIGURE 7. Cncentratin-relaxatin curves fr PGh (left figure), verapamil (middle), and papaverine (right) f a variety f dg arteries cntracted with IO' 1 M CTXA 2. Relaxatins induced by 10~ 4 M papaverine were taken as %; mean abslute values in cerebral, crnary, mesenteric, and renal arteries in the left figure were 2572 ± 220 mg (n = 15), 2132 ± 291 mg (n = 12), 2379 ± 322 mg (n = 10), and 3124 ± 357 mg (n = 11), respectively, the values in the middle figure were 2704 ± 230 mg (n = 16), 1955 ± 194 mg (n = 14), 2383 ± 318 mg (n = 10), and 2654 ± 316 mg (n = 12), respectively, and the values in the right figure were 1992 ± 265 mg (n = 13), 1899 ± 248 mg (n = 14), 2218 ± 250 mg (n = 9), and 2125 ± 288 mg (n = 10), respectively. Numbers in parentheses indicate the number f preparatins used. Discussin The additin f ctxa2 elicited a cncentratin-related, persistent cntractin in dg cerebral, crnary, mesenteric, renal, and femral arteries and mnkey cerebral, crnary, and mesenteric arteries; the cntractins seen at lw cncentratins tended t be greater in cerebral arteries. Maximum cntractins induced by 10~ 7 M CTXA 2 were identical (dg cerebral arteries) t r greater than thse induced by 30 mm K + (ther arteries used), whereas maximum cntractins induced by IO" 5 M PGF 2tt and E 2 are reprtedly less than the K + -induced cntractins [apprximately 60 and 70%, respectively, in dg cerebral arteries (Tda and Miyazaki, 1978)]. ctxa 2 is ne f the mst ptent vascnstrictrs in islated dg and mnkey arteries. TxA 2, pssibly generated by the additin f PGH 2 (255 nui) t particulate fractin f human platelets, prduces a marked cntractin in bvine cerebral arterial strips (apprximately 150% f the cntractin induced by 40 mm K + ), but prduces nly a mderate cntractin in bvine crnary arteries and prcine crnary and renal arteries (30-50% f the cntractin) (Ellis et al., 1977). Cntractins induced by IO" 8 M ctxa 2 in dg cerebral and mesenteric arteries expsed fr 60 minutes t Ca ++ -free media were 49 and 53%, respectively, f the cntractins btained in nrmal bathing media. Therefre, increase in the transmembrane influx f Ca ++ as well as the release f Ca ++ frm intracellular strage sites is invlved in the ctxa 2 -induced cntractin. Since prlnged expsure f the arteries t Ca ++ -free media is cnsidered t reduce intracellular Ca ++, such an invlvement f Ca ++ release wuld be underestimated. On the ther hand, cntractile respnses t PGF 2Q and sertnin in Ca ++ -free media relative t thse in nrmal media are apprximately 25 and 15%, respectively, in cerebral arteries, and 14 and 8%, respectively, in mesenteric arteries (Tda, 1982a). Thus, the ability f ctxa 2 t release intracellularly stred Ca ++ appears t be greater than that f PGF 2a and sertnin. In dg cerebral arteries expsed t Ca ++ -free media and stimulated by ctxa 2, Ca ++ caused a triphasic respnse, a transient cntractin, relaxatin, and sustained cntractin. Similar respnses are btained in dg cerebral arterial strips placed under the same experimental cnditins, except fr the fact that PGF 2(1 and K + are used as stimulants (Tda, 1974b; Tda, 1982a). The relaxatin was nt influenced by aspirin, suggesting that the invlvement f vasdilatr PG's is excluded. Vasdilatr PGI 2 is reprtedly frmed frm PGH 2 in the wall f dg cerebral and extracerebral arteries (Tda, 1980) and is released frm the arterial wall by activatin f angitensin II receptrs (Tda and Miyazaki, 1981). Ouabain ptentiated the Ca ++ - induced cntractin and ablished the relaxatin in dg cerebral arteries stimulated by ctxa 2 (present study) as well as PGF^ (Tda, 1982a) and K + (Tda, 1974b). On the ther hand, the transient cntractin and relaxatin were ablished by treatment with Ca ++ antagnists, including verapamil (present study) and nicardipine (Tda, unpublished data). These findings suggest that the rapid transmembrane influx f Ca ++ triggers the relaxatin, which may be assciated with the mechanism sensitive t uabain. Webb and Bhr (1978) have pstulated that the Ca ++ -induced relaxa-

7 Tda/ Vascular Actin f Carbcyclic Thrmbxane A2 681 tin in islated rat tail arteries is dependent n the activity f Na + -, K + -activated ATPase, since the relaxatin is attenuated by uabain, lw external Na +, reduced temperature, and lw external K +. The specific inhibitin f Ca ++ channels by Ca ++ has been pstulated in guinea pig ileum (Hurwitz et al, 1982); hwever, the evidence that Ca ++ relaxed the arteries t the level lwer than that prir t the additin f Ca ++ (Fig. 3) is nt explained nly by this assumptin. Relaxatins induced by increasing the cncentratin f Ca ++ frm 2.2 t 4.4 HIM in cerebral arterial strips expsed t Ca ++ -free media were reversed t cntractins after treatment with uabain. Whether this reversal is due t an ablitin f the electrgenic Na + pump activated by Ca ++ (Webb and Bhr, 1978), r t a pssible increase in the Ca ++ permeability (Tda, 1982a) caused by a membrane deplarizatin, remains t be determined. Hwever, the latter alternative may be supprted by the finding that, after cntractins induced by the first intrductin f Ca ++ (2.2 mm) leveled ff, the secnd additin f Ca ++ prduces a relaxatin in dg cerebral arterial strips expsed previusly t Ca ++ -free media and deplarized by 30 mm K + (Hayashi and Tda, 1977) but, in cntrast, a cntractin in the arteries deplarized by K + in cncentratins f 50 mm r higher (Tda, unpublished data). As cmpared with vascular effects f ther PCs, including PGAi, A 2, D 2, Ei, E 2, and F 2a, cntractins induced by ctxa 2 persisted lnger even after extensive washing f preparatins. ctxa 2 may bind t receptive sites f vascular smth muscle in vitr mre tightly than thse PCs. The cntractile respnse f cerebral and mesenteric arteries t ctxa2 was attenuated dse dependently by DPP, an analg f a PG antagnist plyphlretin phsphate (PPP) (Sanner, 1974), in cncentratins insufficient t inhibit the cntractins induced by sertnin and nrepinephrine (present study). Similar attenuatin by DPP as well as PPP has been bserved in cntractins induced by PGF 2<I, E 2, and D 2 (Tda and Miyazaki, 1978; Tda, 1982b; unpublished data with DPP). On the ther hand, treatment with aspirin ptentiated the cntractile respnse t ctxa 2 as did the respnse t PGF 2a, E 2, and D 2 (Tda and Miyazaki, 1978; Tda, 1982b). The release f vasdilatr PCs frm the vascular wall may partly interfere with the vascnstrictr actin. These findings suggest that ctxa 2, PGF 2a, PGE 2, and PGD 2 share the mechanism f vascular actins. ctxa2-induced cntractins were influenced neither by phentlamine, cinanserin, and chlrpheniramine in dg arterial strips (present study) nr by phentlamine, phenxybenzamine, and saralasin in islated, perfused cat crnary arteries (Smith et al., 1981), suggesting that a-adrenergic, sertnergic, histaminergic Hi, and angitensin Il-related mechanisms are nt invlved. Dg arterial strips cntracted with ctxa 2 respnded with a relaxatin t PGI2, verapamil, and papaverine in a cncentratin-dependent manner. The relaxatin induced by PGI 2 was in the rder f crnary and mesenteric > renal > cerebral arteries. Cerebral arteries partially cntracted with PGF2,, are als less sensitive t PGI 2 than the ther arteries (Tda, 1980). Such a hetergeneity f the PGI2 actin in a variety f dg arteries may be assciated with different ppulatin r sensitivity f receptive sites r with different rates f degradatin f PGI 2, since a nnselective vasdilatr, papaverine, prduced a similar extent f relaxatins in a variety f dg arteries. Endgenus PGI 2 may participate in attenuating smth muscle cntractins f crnary arteries, such as in the case f variant angina (Tada et al., 1981), mre effectively than thse f cerebral arteries. Relaxant respnses t verapamil were in the rder f cerebral > crnary and renal > mesenteric arteries. Greater relaxatins by nimdipine, a Ca ++ antagnist, f islated rabbit basilar arteries than saphenus arteries cntracted with ctxa 2 have als been demnstrated (Twart and Perzbrn, 1981). Susceptibility f cerebral arteries t Ca ++ antagnists, such as verapamil, diltiazem, and nifedipine, is greater than that f mesenteric arteries when cntracted with PGF 2a (Shimizu et al., 1980). The fact that the prfund cntractin induced by high cncentratins f ctxa2 was reversed by verapamil predminantly in cerebral and crnary arteries suggest that Ca ++ antagnists may be effective in the treatment f cerebral and crnary vasspasm. In fact, the latter spasm is knwn t respnd well t the Ca ++ antagnist therapy (Braunwald, 1981); in cntrast, the effectiveness n delayed cerebral vasspasm is still cntrversial. / thank M. Yamamt fr his excellent technical assistance. Carbcyclic thrmbxane A% prstaglandin 12 sdium salt, and diphlretin phsphate were kindly prvided by On Pharmaceutical C., Osaka, Japan. This wrk was supprted in part by Scientific Research Fund frm the Ministry f Educatin, Science, and Culture f Japan, and by Japan Heart Fundatin and Tki Marine Research Grant fr Address fr reprints: Dr. Nbru Tda, Department f Pharmaclgy, Shiga University f Medical Sciences, Seta, Ohtsu , Japan. Received April 19, 1982; accepted fr publicatin August 19, References Braunwald E (1981) Crnary artery spasm as a cause f mycardial ischemia. J Lab Clin Med 97: Bunting S, Mncada S, Vane JR (1976) The effects f prstaglandin endperxides and thrmbxane A2 n strips f rabbit celiac artery and certain ther muscle preparatins. Br J Pharmacl 57: 462P-463P Ellis EF, Nies AS, Oates JA (1977) Cerebral arterial smth muscle cntractin by thrmbxane A 2. Strke 8: Hamberg M, Svenssn J, Samuelssn B (1975) Thrmbxanes: a new grup f bilgically active cmpunds derived frm prstaglandin endperxides. 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8 682 Circulatin Research/VJ. 51, N. 6, December 1982 carbcyclic thrmbxane A 2, a stable analg f thrmbxane A2. Prc Natl Acad Sci USA 77: Needleman P, Mncada S, Bunting S, Vane JR (1976) Identificatin f an enzyme in platelet micrsmes which generates thrmbxane A 2 frm prstaglandin endperxides. Nature 261: Ohuchida S, Hamanaka N, Hayashi M (1979) Synthesis f thrmbxane A 2 analg, dl-(9,ll) / (ll,12)-didexa-(9 / ll),(ll,12)-dimethylene thrmbxane A 2. Tetrahedrn Letters 28: Sanner JH (1974) Substances that inhibit the actins f prstaglandins. Arch Intern Med 133: Shimizu K, Ohta T, Tda N (1980) Evidence fr greater susceptibility f islated dg cerebral arteries t Ca antagnists than peripheral arteries. Strke 11: Smith EF, Lefer AM, Niclau KC (1981) Mechanism f crnary vascnstrictin induced by carbcyclic thrmbxane A2. Am J Physil 240: H493-H497 Svenssn J, Fredhlm B (1977) Vascnstrictr effect f thrmbxane A 2. Acta Physil Scand 101: Tada M, Kuzuya T, Inue M, Kdama K, Mishima M, Yamada M, Inui M, Abe H (1981) Elevatin f thrmbxane B 2 levels in patients with classic and variant angina pectris. Circulatin 64: Terashita Z, Fukui H, Nishikawa K, Hirata M, Kikuchi S (1978) Crnary vasspastic actin f thrmbxane A 2 in islated, wrking guinea pig hearts. Eur J Pharmacl 53: Tda N (1974a) The actin f vasdilating drugs n islated basilar, crnary and mesenteric arteries f the dg. J Pharmacl Exp Ther 191: Tda N (1974b) Respnsiveness t ptassium and calcium ins f islated cerebral arteries. Am J Physil 227: Tda N (1980) Respnses t prstaglandins H 2 and I 2 f islated dg cerebral and peripheral arteries. Am J Physil 238: Hlll- H117 Tda N (1981) Respnse f islated mnkey crnary arteries t catechlamines and t transmural electrical stimulatin. Circ Res 49: Tda N (1982a) Ptentiatin by uabain f the respnse t vascnstrictr agents f islated dg cerebral and mesenteric arteries saked in Ca ++ -free media. J Cardivasc Pharmacl 4: Tda N (1982b) Different respnsiveness f a variety f islated dg arteries t prstaglandin D 2. Prstaglandins 23: Tda N, Miyazaki M (1978) Respnses f islated dg cerebral and peripheral arteries t prstaglandins after applicatin f aspirin and plyphlretin phsphate. Strke 9: Tda N, Miyazaki M (1981) Angitensin-induced relaxatin in islated dg renal and cerebral arteries. Am J Physil 240: H247- H254 Tda N, Hatan Y, Hayashi S (1978) Mdificatins by stretches f the mechanical respnse f islated cerebral and extracerebral arteries t vasactive agents. Pfluegers Arch 374: Twart R, Perzbrn E (1981) Nimdipine inhibits carbcyclic thrmbxane-induced cntractins f cerebral arteries. Eur J Pharmacl 69: Wallenstein S, Zucker CL, Fleiss JL (1980) Sme statistical methds useful in Circulatin Research. Circ Res 47: 1-9 Webb RC, Bhr DF (1978) Mechanism f membrane stabilizatin by calcium in vascular smth muscle. Am ] Physil 235: C227- C232 INDEX TERMS: Cerebral artery Carbcyclic thrmbxane A 2 Vascular actin f prstaglandin I 2 Vascular actin f verapamil