352 Agonist-Indued Endothelium-Dependent in Rat Thorai Aorta May Be Mediated through Robert M. Rapoport and Ferid Murad From the Departments of Mediine and Pharmaology, Stanford University Shool of Mediine, Palo Alto Veterans Administration Medial Center, Palo Alto, California SUMMARY. The present study investigates the hypothesis that endothelium-dependent relaxation of vasular smooth musle may be mediated through the formation of. of the rat thorai aorta to aetylholine, histamine, and Ca ++ ionophore A23187 was assoiated with inreased levels of in a time- and onentration-dependent manner, whereas AMP levels were unaltered. Removal of the endothelium prevented relaxation to these agents and prevented the inreased levels of. Removal of the endothelium after exposure to aetylholine only partially dereased the elevated levels of, suggesting that the hanges in ourred within the smooth musle ells. Eiosatetraynoi aid, an inhibitor of lipoxygenase and ylooxygenase, and quinarine, an inhibitor of phospholipase, prevented and reversed aetylholine-indued relaxation, respetively, and inhibited aetylholine-indued inreased levels of. In ontrast, sodium nitroprusside-indued relaxation and inreased levels of were independent of the presene of the endothelium, exposure to eiosatetraynoi aid, and quinarine. The present results support the hypothesis that vasular smooth musle relaxation indued by some agents is dependent on the presene of the endothelium and is mediated through the formation of an endothelial fator that inreases levels in smooth musle. (Cir Res 52: 352-357, 1983) Downloaded from http://ahajournals.org by on January 22, 219 RELAXATION of numerous vasular smooth musle preparations by various pharmaologial agents has been shown to be dependent upon the presene of the endothelium (Furhgott and Zawadski, 198; Lee, 198; Chand and Altura, 1981; Altura and Chand, 1981; De Mey and Vanhoutte, 1981; Furhgott, 1981; Van de Voorde and Leusen, 1982; Lee, 1982; Singer and Peah, 1982). However, the mehanism by whih the endothelium leads to relaxation of the smooth musle is not known. It has been proposed (Furhgott and Zawadski, 198) that exposure of the endothelial ells to various agents indues the release of a substane whih, following a series of events, relaxes the smooth musle. The substane released is thought to be arahidoni aid or some other unsaturated fatty aid, sine quinarine, an inhibitor of phospholipase A2, inhibits the endothelium-dependent agonist-indued relaxation (Furhgott and Zawadski, 198). It has also been hypothesized that the fatty aid may be oxidized, sine anoxia, 5,8,11,14-eiosatetraynoi aid (ETYA) and nordihydroguaiareti aid, inhibitors of ylooxygenase and/or lipoxygenase, inhibited the relaxation (Furhgott and Zawadski, 198; Furhgott, 1981). The oxidized produt may be a labile hydroperoxide or free radial, sine hydroquinone, an antioxidant and free radial savenger, inhibited the relaxation. The effets of nitroprusside, nitroglyerin, and other nitro-vasodilators on smooth musle relaxation are thought to be mediated through the ativation of guanylate ylase and formation (Katsuki and Murad, 1977; Katsuki et al., 1977; Shultz et al., 1977). These agents probably lead to the formation of nitri oxide whih ativates guanylate ylase (Katsuki et al., 1977; Arnold et al., 1977). Guanylate ylase an also be ativated with hydroxyl free radial (Mittal and Murad, 1977), unsaturated fatty aids (Walah and Pastan, 1976; Glass et al., 1977), and lipid peroxides (Hidaka and Asano, 1977). The purpose of our study was to test the hypothesis that relaxation of vasular smooth musle with other agents, whih are dependent upon the endothelium, may also be mediated through the formation of within the smooth musle. The effet of aetylholine, histamine, A23187, and sodium nitroprusside on relaxation and the levels of and AMP in the presene and absene of endothelium was investigated. Furthermore, the effets of quinarine and ETYA on relaxation and levels indued by aetylholine and nitroprusside were investigated. Some of these studies have been reported in abstrat form (Rapoport et al., 1983). Methods Studies Rats (Sprague-Dawley, male, 2-3 g) were deapitated, and their thorai aortas were removed and ut into spiral strips (approximately 2 mm X 1 m)'. Strips were mounted in organ baths ontaining Krebs-Ringer biarbonate solution whih was gassed with 95% CV5% CO2 and had the following omposition (HIM): NaCl, 118.5; KC1, 4.74; MgSO 4, 1.18; KH2PO4, 1.18; CaCl 2, 2.5; NaHCO 3, 24.9; gluose, 1. Resting tension of.4 g - fore was maintained
Rapoport and Murad/Endothelium-Dependent and 353 Downloaded from http://ahajournals.org by on January 22, 219 throughout the experiment. Tissues were allowed to equilibrate for 2 hours prior to the addition of any drugs. Responses to.1 fim norepinephrine then were eliited, followed by the addition of aetylholine, histamine, A23187, or sodium nitroprusside. The intimal surfae of some of the strips then was rubbed gently with a salpel, whih removed the endothelial layer and left the internal elasti lamina intat, as onfirmed by means of sanning eletron mirosopy. Following reequilibration (3-6 min), subsequent ontratile responses to.1jum norepinephrine were unaffeted by the rubbing proedure. Aetylholine, histamine, A23187, or nitroprusside then was added. Other strips were pretreated with ETYA (.1 DIM) for 3 minutes, followed by the addition of norepinephrine and the smooth musle relaxants. Still other tissues were exposed to quinarine (.1 ITIM) for 3 seonds after relaxations to aetylholine, histamine, A23187, and nitroprusside had been eliited. and AMP Measurements Rubbed or unrubbed spiral strips were prepared as desribed above and were allowed to equilibrate in flasks ontaining Krebs-Ringer biarbonate solution gassed with 95% O 2-5% CO2. Various times after exposure to the agents indiated, the tissues were frozen in liquid nitrogen, and yli nuleotide levels were assayed as previously desribed (Katsuki and Murad, 1977). Briefly, frozen tissues were homogenized in 6% trihloroaeti aid and samples were entrifuged. Supernatant frations were extrated with ether and radioimmunoassayed for yli nuleotides (Steiner et al., 1972; Kimura et al., 1974). Similar results were obtained when tissues were mounted in organ baths and exposed to the agents indiated (Rapoport and Murad, unpublished observation). Protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. Signifiane was aepted at the.5 level of probability, using Student's r-test unless otherwise indiated. Materials Aetylholine-HCl, 1-norepinephrine-HCl, histamine- HC1, sodium nitroprusside, and quinarine-hcl were obtained from Sigma, 6S-[6a(2S\ 3S*), 8/?(R*), 9/3, lla]-5- (methylamino)-2-[[3,9,ll - trimethyl-8- [l-methyl-2-oxo-2- (lh-pyrrol-2-yl)ethyl] - 1,7 - dioxaspiro[5.5] - unde-2-yl] methyl]-4-benzoxazolearboxyli aid (A23187) from Calbiohem and 5,8,11,14-eiosatetraynoi aid (ETYA) was a gift from Hoffman-La Rohe. Other materials were obtained as previously desribed (Kimura et al., 1974; Kimura et al., 1975; Mittal and Murad, 1977). Studies Results The results of the relaxation studies are similar to those of other investigators (Furhgott and Zawadski, 198; Furhgott, 1981; Singer and Peah, 1982; Van de Voorde and Leusen, 1982) and, thus, most of the relaxation data are not presented. The time ourse for relaxation indued by maximally effetive onentrations of aetylholine, histamine, and A23187 is shown in Figures 1, 2, and 3, respetively. to aetylholine and histamine ourred within 5 seonds of their addition, whereas relaxation to A23187 TABLE 1 Effet of Sodium Nitroprusside on Levels of in Rat Thorai Aorta with and without Endothelium Treatment Rubbed Nitroprusside (1 /IM) Rubbed + nitroprusside (1/1M) 2.2 ±.5.5 ±.2* 66.5 ± 12.1f 82.6 ± 18.1 (%) 1 1 Results are expressed as mean ± SE; n = 7 in eah ase. Aortas were rubbed (see Methods) or allowed to remain intat. Tissues then were exposed to.1 /IM norepinephrine for 5 minutes, followed by 1 )IM nitroprusside for 1.5 minutes, frozen, and assayed for (see Methods). * Signifiantly less than ontrol. t Signifiantly greater than ontrol. ourred within 1 seonds of addition. Neither relaxation nor ontration to these agents was observed in tissues whose endothelium had been removed and then were ontrated with.1 JUM norepinephrine (data not shown). In ontrast, relaxation indued by a maximally effetive onentration of nitroprusside (1 JUM) ompletely relaxed both rubbed and ontrol tissues (Table 1). Maximal relaxation ourred within 1.5 minutes of addition of nitroprusside. Pretreatment with ETYA (.1 ITIM) for 3 minutes prevented relaxation indued by maximally effetive onentrations of aetylholine, histamine, and A23187 (Table 2; some data not shown). However, relaxation indued by 1 JUM nitroprusside was unaffeted by ETYA (Table 2). Pretreatment with ETYA (.1 mm) dereased the ontratile response to nor- TABLE 2 Effet of ETYA on Aetylholine- and Sodium Nitroprusside- Indued and Inreased Levels of in Rat Thorai Aorta Addition Experiment 1 ETYA (.1 mm) Aetylholine (1 /IM) ETYA (.1 mm) + aetylholine (1 /IM) Experiment 2 ETYA (.1 mm) Nitroprusside (1 /IM) ETYA (.1 mm) + nitroprusside (1 fim).8 ±.1.5 ±.1* 11.3 ± 2.5f.8 ±.1 2.4 ± 1.7.4 ±.1* 52.6 ± 3.5f 29.9 ± 6.t^ (%) 85.6 ± 3.8 1 1 Results are expressed as mean ± SE; n = 4 in eah experiment. Tissues were exposed to.1 mm ETYA for 3 minutes followed by.1 /IM norepinephrine for 5 minutes. Other tissues remained unexposed to ETYA. Aetylholine (1 /IM) or nitroprusside (1 /IM) was then added for 1 and 1.5 minutes, respetively. Tissues then were frozen and assayed for (see Methods). * Signifiantly less than ontrol. t Signifiantly greater than ontrol. % Signifiantly less than nitroprusside-treated tissues.
354 Cirulation Researh/Vol. 52, No. 3, Marh 1983 Downloaded from http://ahajournals.org by on January 22, 219 epinephrine (.1 /IM) by 18.6 ± 4.5% (mean ± SE; P <.5, paired r-test; n = 9 in eah ase). ETYA (.1 miu) added following the relaxation aused only a small reversal. indued by maximally effetive onentrations of aetylholine and histamine was partially reversed by quinarine (36% and 3%, respetively; Table 3). In ontrast, relaxations indued by.3 JUM A23187 and 1 JUM nitroprusside were not reversed by.1 mm quinarine. Quinarine (.1 mmj itself aused omplete relaxation of strips ontrated with.1 /IM norepinephrine within approximately 8 minutes of its addition. Effet of Aetylholine, Histamine, A23187, and Nitroprusside on Cyli Nuleotide Levels The inrease in levels of indued by aetylholine (1 fim), histamine (1 mm) and A23187 (.3 JUM) was abolished in tissues that were rubbed (Figs. 1-3). When endothelium was present, signifiant inreases in ourred within 5 seonds of addition of aetylholine (1 JUM) and histamine (1 nun) and within 1 seonds of addition of A23187 (.3 ftm). AMP levels were unaltered by exposure to aetylholine (Fig. 1), histamine, or A23187 (data not shown). Basal levels of and AMP were dereased in those tissues that were rubbed. With endothelium present, the inrease in and relaxation were related to the onentration of aetylholine (Table 4). In ontrast, rubbing the intimal surfae did not alter the elevated levels of indued with nitroprusside (Table 1). Norepinephrine (.1 JUM) had no effet upon levels of (data not shown). TABLE 3 Effet of Quinarine on Aetylholine- and Sodium Nitroprusside-Indued and Inreased Levels of in Rat Thorai Aorta Addition Experiment 1 Quinarine (.1 mm) Aetylholine (1 /IM) Aetylholine (1 /JM) + quinarine (.1 mm) Experiment 2 Quinarine (.1 mm) Nitroprusside (1 /IM) Nitroprusside (1 /IM) + quinarine (.1 mm).7 ±.1.8 ±.1 25.4 ± 6.6* 6. ± 1.6*f.8 ±.3.8 ±.1 94.4 ± 23.7* 98.8 ±25.1* (%) 86.7 ± 8.4 55.5 ± 6.2f 1 1 Results are expressed as mean ± SE; n = 7 and 4 in experiments 1 and 2, respetively; n = 3 in all relaxation studies. Tissues were exposed to.1 fim norepinephrine for 5 minutes and then either 1 JIM aetylholine for 1 minute pr 1 /IM aetylholine for 3 seonds, followed by.1 mm quinarine for 3 seonds. Other tissues were exposed to norepinephrine followed by either 1 /IM nitroprusside for 1.5 minutes or 1 fim nitroprusside for 1 minute followed by.1 mm quinarine for 3 seonds. Additional tissues were exposed to norepinephrine and then.1 nun quinarine for 3 seonds. Tissues were then frozen and. assayed for (see Methods). * Signifiantly greater than ontrol. t Signifiantly less than aetylholine-treated tissues. Time (se) 9 12 FIGURE 1. Effet of aetylholine on levels of CMP, AMP, and relaxation in rat thorai aorta. Aortas were rubbed ( ; see Methods) or allowed to remain intat ( ). Tissues then were exposed to.1 fim norepinephrine for 5 minutes, followed by 1 fim aeryihoiine for various times, frozen and assayed for yli nudeotides [(9); n varied from 6 to 12]. to 1 \IM aetylholine (O) was reorded in another set of tissues ontrated with.1 [IM norepinephrine (n = 8 in eah ase). Aetylholine (1 JIM) did not relax tissues that had been rubbed previously. and AMP levels of rubbed tissues were signifiantly less than those of unrubbed tissues. * Signifiantly greater than ontrol at time. Effet of Quinarine on Levels Whereas quinarine (.1 mm) partially reversed the elevated levels of indued by aetylholine, it had no effet on the inreased levels of indued _ 3 O a -1 2 1 o u 5 1 2 3 6 Time (se) FIGURE 2. Effet of histamine on levels of and relaxation in rat thorai aorta. Aortas were rubbed ( ; see Methods) or allowed to remain intat ( ). Tissues then were exposed to.1 fim norepinephrine for 5 minutes, followed by 1 mm histamine for various times, frozen and assayed for [(9); n varied from 3 to 6]. to 1 min histamine (O) was reorded in another set of tissues ontrated with.1 fim norepinephrine (n = 5 in eah ase). Histamine (1 mm) did not relax tissues that were previously rubbed. levels of rubbed tissues were signifiantly less than those of unrubbed tissues. * Signifiantly greater than ontrol at time. 9 o X J? ID q s JS CD CC
Rapoport and Murad/Endothelium-Dependent and 355 Downloaded from http://ahajournals.org by on January 22, 219 Q> o a O) "5 1 5 1 3 6 Time (se) 9 12 FIGURE 3. Effet of A23187 on levels of CMP and relaxation in rat thorai aorta. Aortas were rubbed ( ; see Methods) or allowed to remain intat ( ). Tissues were then exposed to.1 JUM norepinephrine for 5 minutes, followed by.3 p.m A23187 for various times, frozen and assayed for. [(9); n varied from 8 to 12J. to.3 /IM A23187 (O) was reorded in another set of tissues ontrated with.1 jim norepinephrine (n 5 in eah ase). A23187 (.3 \IM) did not relax tissues that were previously nibbed. levels were signifiantly less than those of unrubbed tissues. * Signifiantly greater than ontrol at time. by nitroprusside (Table 3). Quinarine itself did not alter basal levels of. Effet of 5,8,11,14-Eiosatetraynoi Aid on Levels Pretreatment with ETYA (.1 HIM) ompletely prevented the inreased levels of indued by aetylholine and only partially lowered those indued by nitroprusside (Table 2). ETYA also dereased basal levels of. Lower onentrations of ETYA did not alter the relaxant effets of aetylholine, and levels were not examined. Effet of Rubbing on Levels after Exposure to Aetylholine Intat strips were inubated with 1 JUM aetylholine for 3 seonds followed by rubbing for 5 seonds in order to remove the endothelium of some tissues. Under these onditions, the levels in the residual (rubbed) strips remained elevated (Table 5). 8 7 6 5 4 3 2 TABLE 4 Effet of Aetylholine on Levels of and in Rat Thorai Aorta Addition Aetylholine (.1 JIM) Aetylholine (1 J*M) Aetylholine (1 (IM).8 3.3 12. 21.2 ±.1 ±.5* ±2.3* ± 1.9* 1 52.1 79.7 87.2 ± ± ± 8.6 5.9 4.7 Results are expressed as mean ± SE; n = 4 and 11 in and relaxation experiments, respetively. Rat thorai aorta was exposed to.1 im norepinephrine for 5 minutes, followed by different onentrations of aetylholine for 3 seonds. Tissues then were assayed for (see Methods). The isometri ontratile responses were reorded in other strips. * Signifiantly greater than ontrols. Relaxatio TABLE 5 Effet of Aetylholine ion Levels of in Rat Thorai Aorta Treatment Rubbed Aetylholine (1 HM) Aetylholine (1 fjm) + rubbed.7 ±.1 2.7 ±.8* 27.7 ± 7.1* 16.1 ± 6.3*f Results are expressed as mean ± SE; n = 4 in eah ase. Aortas were exposed to.1 fim norepinephrine for 5 minutes followed by 1 fim aetylholine for 35 seonds and then were frozen or exposed to 1 pm aetylholine for 3 seonds, and then rubbed (see Methods) for 5 seonds while in Krebs-Ringer biarbonate solution ontaining 1 /IM aetylholine and.1 fim norepinephrine. Tissues then were assayed for (see Methods). * Signifiantly greater than ontrol. t Signifiantly less than non-rubbed tissues exposed to 1 JIM aetylholine using paired f-test. Disussion The present results support the hypothesis that vasular smooth musle relaxation indued by various hormones and agents, whih is dependent upon the presene of the endothelium (Furhgott and Zawadski, 198; Furhgott, 1981), is mediated through the formation of. It has been proposed that the substane, or preursor of the substane, whih is responsible for the relaxation of smooth musle, is released from the endothelium as a result of ativation of phospholipase. The present results demonstrate that quinarine, an inhibitor of phospholipase A2, partially reversed the relaxation indued by aetylholine and histamine, as well as the inreased levels of indued by aetylholine. Presumably, the endothelial-derived substane an be released by A23187 independently of quinarine, sine quinarine did not effet relaxation indued by A23187. Furhgott (1981) also observed that quinarine had no effet on relaxation indued by A23187 in rabbit aorta. Vallee et al. (1979) suggested that quinarine is an indiret inhibitor of phospholipase A2 and ats by preventing Ca ++ ativation of phospholipase A2. These authors observed that quinarine had no effet on phospholipase A 2 ativated by A23187 in platelets. The relaxation and inreased levels of aused by nitroprusside were independent of quinarine, sine nitroprusside ativates guanylate ylase within the smooth musle ell, probably through the generation of nitri oxide-free radial and diret ativation of guanylate ylase (Arnold et al., 1977). Quinarine also had no effet on nitroprusside-indued inreased levels of in rat dutus deferens (Spies et al., 198). The substane that indues relaxation is thought to have been oxidized by lipoxygenase (Furhgott and Zawadski, 198). The present results demonstrate that an inhibitor of both lipoxygenase and ylooxygenase, ETYA, inhibited relaxation indued by aetylholine, histamine, and A23187, as well as the inreased levels of indued by aetylholine. ETYA partially dereased the nitroprusside-indued inreased levels
356 Cirulation Researh/VoJ. 52, No. 3, Marh 1983 Downloaded from http://ahajournals.org by on January 22, 219 of, although not to the extent of the inhibition of the aetylholine-indued inrease. Sine basal levels of were also dereased by ETYA (.1 min), perhaps some ontinual release and metabolism of an unsaturated fatty aid plays a role in basal levels and smooth musle tone. Lower onentrations of ETYA did not inhibit the relaxation indued by aetylholine. Spies et al. (198) also observed that ETYA (.1 mm) and nordihydroguaiareti aid (.1 mm), an inhibitor of lipoxygenase, lowered both basal and nitroprusside-indued inreased levels of in rat dutus deferens. These authors suggested that these results may be due to a diret inhibitory effet of ETYA and nordihydroguaiareti aid on soluble guanylate ylase. Rubbing the intimal surfae dereased basal levels of and AMP. Whether this derease is the result of the loss of the endothelial ells or the effet of some substane on the smooth musle is not known. It is interesting to note that removal of the endothelium over a 5-seond period and then freezing the tissue resulted in levels greater than basal levels (Table 5). However, after some equilibration, basal levels were dereased (Figs. 1-3 and Table 1). One might speulate that rubbing the intimal surfae aused the release of some substane (arahidoni aid or some metabolite) that ativates guanylate ylase in the smooth musle omponent. It is also interesting to note that an equilibration of approximately 3-6 minutes is required following rubbing before sensitivity to ontratile agents returns to ontrol levels (Furhgott and Zawadski, 198). This equilibration time may be required in order to return levels and/or subsequent steps indued by to basal levels. indued by aetylholine, histamine, and A23187 was maintained over a time period in whih the elevated levels of delined. A similar relationship was observed between sodium nitroprusside-indued relaxation and levels of (Rapoport and Murad, unpublished observation). The reason for this is not lear; however, several explanations are possible: (1) extrusion of into the surrounding media may have ourred; (2) phosphodiesterase ativity may have inreased; (3) a step(s) following the formation of whih is involved in the relaxation mehanism may have a slower turnover rate than. In this regard, sodium nitroprusside-indued relaxation and protein phosphorylation were maintained over a period in whih elevated levels delined (Rapoport et al., 1982). Furthermore, the sodium nitroprusside-indued protein phosphorylation profile was mimiked by 8-bromo-. Contratile agents have also been shown to inrease levels of in a variety of smooth musle preparations (Dunham et al., 1974; Clyman et al., 1975; Diamond and Blisard, 1976; Katsuki and Murad, 1977; Kukovetz et al., 1982). Several explanations of these results are possible: (1) a ell type other than smooth musle may be responsible for the inrease in levels; (2) funtionally distint pools of may exist; and (3) may, in some instanes, inrease seondarily to ontration and at as a feedbak inhibitor of ontration. However, in ontrast to what has been reported with other blood vessels and/or speies (Furhgott and Zawadski, 198; Lee, 198; Chand and Altura, 1981; Singer et al., 1982), aetylholine did not eliit a ontration in rubbed or unrubbed rat thorai aorta. The inreased levels of observed in these studies presumably ourred within the smooth musle ells, sine inreased levels were still observed when the endothelium was removed during exposure to aetylholine. These studies support the hypothesis that the relaxant effets of agents whih require the presene of endothelium may also be mediated through the aumulation of, as appear to be the effets of nitrovasodilators. The nitrovasodilators may, in fat, at through bypassing part of the endothelium-dependent vasodilator system and diretly ativating guanylate ylase in smooth musle. Altered formation may also play an important role in vasular ontration and spasm in various pathophysiologial states with endothelial injury. The skillful tehnial assistane of Gilberto A. Martinez is gratefully appreiated. We would also like to thank Dr. Jon Kosek for the sanning eletron mirosopi studies and Kathleen M- Fadden for help in preparation of the manusript. This work was supported by Researh Grants AM 3787 and HL 28474 from the National Institutes of Health, grants from The Counil for Tobao Researh-U.S.A., In., and from the Veterans Administration, and by National Researh Servie Award GM 7673 (to R.M. 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