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Atypical β-adrenoceptors of rat thoracic aorta
General Pharmacology 32 (1999) 557–562
Atypical b-adrenoceptors of rat thoracic aorta Massoumeh Shafiei, Massoud Mahmoudian * Department of Pharmacology, Faculty of Medicine, Iran University of Medical Sciences, P.O. Box 14155-6183, Noori Highway, Tehran, Iran Manuscript received April 15, 1998; accepted manuscript November 6, 1998
Abstract The possible existence of atypical b-adrenoceptors in vascular smooth muscle of rat isolated thoracic aorta was investigated. Isoprenaline (1028–1024 M) produced concentration-dependent relaxation of phenylephrine (1025 M) precontracted rings of endothelium-denuded rat aorta in vitro. Isoprenaline-induced relaxation was resistant to blockade by atenolol (1026 M). But, propranolol (2 3 1027 M) caused a non-competitive inhibition and SR 59230A (6.6 3 1026 M), a b3-adrenoceptor selective antagonist, failed to produce additional antagonism in presence of propranolol. BRL 37344 (1028–1024 M), a b3-selective agonist, did not relax ring segments precontracted with phenylephrine (1025 M) in the absence of endothelium. The non-conventional partial agonist (–)-cyanopindolol (5 3 1026–1024 M) induced a marked relaxation in phenylephrine (1025 M) precontracted aortic rings without endothelium. This vasodilation was resistant to blockade by propranolol (2 3 1027 M) and SR 59230A (1025 M). Salbutamol (1028–1024 M) produced concentration-dependent relaxation in isolated endothelium-denuded aortic rings precontracted with phenylephrine (1025 M). Propranolol (2 3 1027 M), but not atenolol (1026 M), inhibited this relaxant response. It is concluded that in endothelium-denuded thoracic aorta, salbutamol acts through b2-adrenoceptors whereas isoprenaline seems to activate both b2-adrenoceptors and an atypical b-adrenergic receptor. This atypical b-adrenoceptor is distinct from putative b3-adrenoceptor and maybe resembles the reported fourth cardiac b-adrenoceptor. 1999 Elsevier Science Inc. All rights reserved. Keywords: Rat aorta; Atypical b-adrenoceptors; Isoprenaline; Cyanopindolol; BRL 37344
There have been reports of existence of atypical b-adrenoceptors in several tissues such as rat adipose tissue (Arch et al., 1984; Wilson et al., 1984), rat colon (Bianchetti and Manara, 1990; Kirkham and Kelly, 1992) and guinea-pig ileum (Bond and Clarke, 1988). This atypical b-adrenoceptor, which is designated as b3, is relatively insensitive to antagonists possessing only high affinity for b1- and b2-adrenoceptors such as propranolol. In addition, evidence is accumulating for a fourth b-adrenoceptor that mediates cardiostimulation (Kaumann and Molenaar, 1996; Kaumann, 1997). The existence of this receptor is proposed in human ventricle and atrium, and rat atrium based on its pharmacologic properties. The characteristics of the fourth b-adrenoceptors and b3-adrenoceptors has been discussed by Kaumann (1997). Both the fourth cardiac b-adrenoceptor and b3-adrenoceptor can be stimulated by nonconventional partial agonists, such as compounds chemically related to pindolol, and both receptors are resistant to blockade by propranolol. However, only b3-adrenocep* Corresponding author. Tel.: 198-21-8278510; Fax: 198-218278514; E-mail: [email protected].
tors are selectively stimulated by b3-adrenoceptor-selective agonists such as BRL 34377 and blocked by b3renoceptor-selective antagonists such as SR 59230A. The existence of b3-adrenoceptors in vascular smooth muscle has been reported (Berlan et al., 1994; Shen et al., 1994). It appears that these receptors are involved in vasodilation of peripheral vessels. The present study was carried out to characterize b-adrenoceptors of vascular smooth muscle of rat thoracic aorta.
1. Material and methods Sprague-Dawley rats of either sex weighing 180–200 g were used in this study. Each rat was anesthetized with chloroform and bled through a cut to carotid artery. The thoracic aorta was isolated and placed in Krebs’ solution at room temperature gassed with a 95% O2 and 5% CO2 mixture. The composition of the Krebs’ solution used in all cases was as follows (mmol/l): NaCl 118, KCl 4.8, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, glucose 10. The endothelium was removed mechanically by gentle rubbing of the intimal surface with a
0306-3623/99/$–see front matter 1999 Elsevier Science Inc. All rights reserved. PII: S0306-3623(98)00283-3
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stainless steel stick for 30–50 s. Thereafter ring segments (3–4 mm) were suspended in Krebs’ solution (378C) contained in organ bath. A resting tension of 1 g was applied and tissues were allowed to stabilize for about 90 min, the bathing solution being renewed every 15 min. Tension was recorded with isometric transducer connected to a Beckman polygraph. To obtain relaxant responses aortic rings were contracted with phenylephrine (1025 M). This concentration of phenylephrine usually produced about 75–80% of maximum contraction in this tissue. At the beginning of the experiments, the absence of the functional endothelium was confirmed by the inability of acetylcholine (1026 M) to induce a relaxation. Ascending concentrations of agonist were added cumulatively to generate a concentration-response curve. When antagonists were used each compound was added to the bath and tissue was allowed to equilibrate for 30 min. before the agonist concentration-response curve was repeated. In each individual preparation only one agonist/antagonist pair was tested. Antagonism was assumed to be noncompetitive when the slope of the Schild regression line was significantly different from 1. Agonist potency was expressed as the pD2 value. 1.1. Drugs Drugs used in this investigation included: acetylcholine bromide, DL-isoprenaline hydrochloride, DLpropranolol hydrochloride (all from Sigma Chemical Co, St. Louis, MO), phenylephrine hydrochloride, salbutamol sulfate, atenolol (ALPS Pharmac. Ind. Co. Ltd.), BRL 37344 (RR1SS)(4-[2-[2-(3-chlorophenyl)2-hydroxyethyl]phenoxyl]acetic acid (Tocris Cooksen Ltd., UK), (–)-cyanopindolol hemifumerate (ANAWA Trading SA, Switzerland), and SR 59230A (3-(2-ethylphenoxy)-1-[(1s)-1, 2, 3, 4-tetrahydronapht-1-ylamino]2s-2-propanol oxalate) was a gift from Dr. Luciano Manara (Sanofi Midy, Italy). 1.2. Statistics Results are presented as mean 6 SE of n number of experiments. Mean values are compared by Student’s ttest and differences between mean values were accepted as significant when p , 0.05. 2. Results 2.1. Relaxant effects of b-adrenergic agonists Phenylephrine (1025 M) caused a submaximal contraction in endothelium-denuded rings of rat thoracic aorta. Isoprenaline (1028–1024 M) and salbutamol (1028–1024 M) produced concentration-dependent relaxation in these ring segments (Fig. 1). The pD2 values for isoprenaline and salbutamol are shown in Table 1. Isoprenaline- and salbutamol-evoked maximal responses were 55.2 6 1.03% and 22.76 6 2.95% of initial tension,
Fig. 1. Concentration-response curves for isoprenaline (d) and salbutamol (j) in endothelium-denuded ring segments of rat thoracic aorta precontracted with phenylephrine (1025 M). Results are expressed as percentage relaxation of tone induced by phenylephrine (1025 M). Each point represents the mean 6 SE of 3–5 experiments.
respectively. BRL 37344 (1028–1024 M) did not elicit any relaxation in ring segments of the thoracic aorta after endothelium denudation. 2.2. Blockade of agonist-elicited relaxation by b-adrenergic antagonists Isoprenaline- and salbutamol-induced relaxation were resistant to blockade by atenolol (1026 M) in endothelium-denuded rings. Propranolol (2 3 1027 M) inhibited the relaxant response of isoprenaline in endotheliumdenuded aorta in a noncompetitive manner (Fig. 2). The Schild plot gave a straight line with a slope significantly (p , 0.05) less than unity (0.25), see Fig. 3. The relaxation elicited by salbutamol in endotheliumdenuded rings was completely abolished by propranolol (2 3 1027 M) pretreatment (Fig. 4). SR 59230A (6.6 3 1026 M) did not cause further rightward replacement of concentration-response curve of isoprenaline in presence of propranolol (2 3 1027 M). 2.3. Effect of nonconventional partial agonist (2)-Cyanopindolol (5 3 1026–1024 M) caused concentration-dependent relaxation of phenylephrine (1025 M) precontracted rings of rat endothelium-denuded aorta (Fig. 5). The pD2 value for (2)-cyanopindolol is shown in Table 1. The maximal relaxation produced by (2)-cyanopindolol was 76.2 6 10.2% of initial tension. (2)-Cyanopindolol-induced vasorelaxation was resisTable 1 Relaxant potencies of b-adrenoceptor agonists in rat thoracic aorta (mean 6 SE of 3–5 experiments) Agonist
pD2 values
Isoprenaline Salbutamol Cyanopindolol
5.83 6 0.1 5.34 6 0.1 4.61 6 0.01
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Fig. 2. Concentration-response curves of isoprenaline in the absence (d) and in the presence of propranolol 2 3 1027 M (s) in endothelium-denuded ring segments of rat thoracic aorta precontracted with phenylephrine (1025 M). Results are expressed as percentage relaxation of tone induced by phenylephrine (1025 M). Each point represents the mean 6 SE of 9 experiments.
tant to blockade by propranolol (2 3 1027 M) and the b3adrenoceptor-selective antagonist SR 59230A (1025 M). 3. Discussion Many studies using selective agonists and antagonists have shown that vasodilation could result from activation of b1- or b2-adrenoceptor subtypes or both. For example, while b2-adrenoceptors mediate vasodilation in guinea-pig pulmonary artery (O’Donnell and Wanstall, 1985), human saphenous vein (Ikezono et al., 1987), and canine mesenteric vessels (Tiara et al., 1977; Daly and Levy, 1979), b1-adrenoceptors appear to be responsible for relaxation in human cerebral arteries (Edvinsson et al., 1976) and canine coronary artery (O’Donnell and Wanstall, 1985; Bernard et al., 1991). Coexistence of b1- and b2-adrenoceptors has been also demonstrated (Tiara et al., 1977; O’Donnell and Wanstall, 1984, 1985). In addition, there are reports concerning the involvement of atypical b-adrenoceptors in vasorelaxation of rat common carotid and aorta (Oriowo, 1994, 1995). Atypical pharmacological characteristics maybe mediated by b3-adrenoceptors, so the names b3- and atypical b-adrenoceptor have been extensively used interchangeably. However, it has been shown that atypical b-adrenoceptors in different tissues are not identical (Oriowo, 1995). Recent studies demonstrated that, in addition to b1- and b2-adrenoceptors, a third cardiostimulatory b-adrenoceptor is present in human and rat heart (Kaumann, 1989, 1996; Kaumann and Molenaar, 1996, 1997; Molenaar et al., 1997). Evidence indicates that this atypical cardiac b-adrenoceptor and the b3adrenoceptor are distinct (Kaumann and Molenaar, 1996; Malinowska and Schlicker, 1996; Kaumann et al., 1997), and to avoid confusion the former has been designated the fourth cardiac b-adrenoceptor (Kaumann,
Fig. 3. (A) Concentration-response curves for isoprenaline in the absence (d) and presence of propranolol 2 3 1028 M (j), 2 3 1027 M (s), and 2 3 1026 M (h) in endothelium-denuded ring segments of rat thoracic aorta precontracted with phenylephrine (1025 M). Results are expressed as percentage relaxation of tone induced by phenylephrine (1025 M). Each point represents the mean 6 SE of 5–9 experiments. (B) Schild plot with DRs measured at 50% of the maximum response. Each point represents the mean 6 SE of 5–9 experiments. Y 5 0.25X 1 3.42 (r2 5 0.969).
1997). Regarding the similarities and differences between the fourth cardiac b-adrenoceptor and the b3adrenoceptor, the following criteria are used to accept the involvement of b3-adrenoceptors in a physiological system (Arch and Kaumann, 1993; Kaumann and Molenaar, 1996; Molenaar and Kaumann, 1997): (1) the receptor should be selectively stimulated by b3-adrenoceptor-selective agonists; (2) the receptor should be stimulated by nonconventional partial agonists; (3) the receptor should be resistant to block by antagonists possessing only high affinity for b1- and b2-adrenoceptors; (4) the receptor should be blocked by b3-adrenoceptor-selective antagonists. For the fourth cardiac b-adrenoceptor, criteria (1) and (4) are not fulfilled. The presence of b3-adrenoceptors have been shown in canine cutaneous and fat vascular smooth muscles (Shen et al., 1994; Berlan et al., 1994). The stimulation of these receptors induces a sustained peripheral vasodilation. But, no reports have been implied the existence of the fourth subtype of b-adrenoceptors in vasculature as yet. The results obtained in the present study show that propranolol (2 3 1027 M) did not produce competitive
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Fig. 4. Concentration-response curves for salbutamol in the absence (d) and in the presence of propranolol 2 3 1027 M (s) in endothelium-denuded ring segments of rat thoracic aorta precontracted with phenylephrine (1025 M). Results are expressed as percentage relaxation of tone induced by phenylephrine (1025 M). Each point represents the mean 6 SE of 3 experiments.
antagonism of isoprenaline-induced relaxation of precontracted ring segments of rat aorta in the absence of endothelium. This concentration of propranolol, i.e., 2 3 1027 M, has been shown to block b1- and b2-adrenoceptors, but does not affect atypical adrenoceptors significantly (Kaumann and Molenaar, 1996). The Schild plot was a straight line with a slope of 0.25 which is significantly less than 1 (Fig. 3). These results are therefore consistent with the idea that isoprenaline interacts with two sites that have high and low affinities for propranolol probably correspond to b1- and b2-adrenoceptor subtype (high affinity) and atypical b-adrenoceptors (low affinity). Regarding the failure of atenolol to antagonize the relaxant response to isoprenaline in rat aorta, it seems unlikely that b1-adrenoceptors are involved in this response. This is inconsistent with some previous reports suggesting the existence of b1-adrenoceptors in rat aorta (O’Donnell and Wanstall, 1984, 1985). This maybe due to the difference between the preparation used in the present study and that used by O’Donnell and Wanstall (denuded or intact aorta). The presence of b2-, and not b1-adrenoceptors, in endothelial cells has been reported by Molenaar et al., 1988; Ahmad et al., 1990. With regard to salbutamol, despite being a potent b2adrenoceptor agonist in tissues such as lung (Schreurs et al., 1980) and uterine (Granger et al., 1985), its low potency in vascular preparations has been showed previously (Bernard et al., 1991; Gray and Marshal, 1992; Oriowo, 1994). The low pD2 value obtained in present study (Table 1) is therefore consistent with the previous reports. Salbutamol-induced relaxation of precontracted endothelium-denuded ring segments of rat aorta was completely inhibited by propranolol, but not prevented by atenolol, indicating activation of b2-adrenoceptors in rat aorta by salbutamol. The weak relaxation induced
Fig. 5. Concentration-response curve for cyanopindolol in endothelium-denuded ring segments of rat thoracic aorta precontracted with phenylephrine (1025 M). Results are expressed as percentage relaxation of tone induced by phenylephrine (1025 M). Each point represents the mean 6 SE of 5 experiments.
by salbutamol in endothelium-denuded rings may be due either to the present of scanty number of b2-adrenoceptors in vascular smooth muscle or to the activation of b2-adrenoceptors in residual endothelium remained after denudation. It should be mentioned that in some studies the stimulation of both b1-adrenoceptors (Bernard et al., 1991) and atypical b-adrenergic receptors (Oriowo, 1995) by salbutamol have been reported. Cyanopindolol is a nonconventional partial agonist, i.e., high affinity blocker of both b1- and b2-adrenoceptors, known to exert also stimulant effect through b3adrenoceptors in rat colon (Kaumann and Molenaar, 1996) and through an atypical cardiostimulant b-adrenoceptor in rat and human atrium (Kaumann and Molenaar, 1996; Sarsero et al., 1997). In the present study, cyanopindolol behaved as a full agonist producing greater maximal relaxation of precontracted endothelium-denuded aortic rings compared to isoprenaline, however, it has a lower potency than isoprenaline (Table 1). The relaxant effect of cyanopindolol was not antagonized by propranolol (2 3 1027 M) which points to the interaction of cyanopindolol with atypical b-adrenoceptors in thoracic aorta. The relaxation was slow in onset and took a much longer time, compared to isoprenaline, to attain a peak response. The selective b3-adrenoceptor agonist BRL 37344 is a potent stimulant of lipolysis in both brown and white adipose tissues of the rat (Arch et al., 1984; Wilson et al., 1984) and a relaxant of smooth muscles of gastrointestinal tract including the rat proximal colon (Bianchetti and Manara, 1990), distal colon (McLaughlin and McDonald, 1990; Kirkham and Kelly, 1992; Oriowo, 1995), guinea-pig colon (Coleman et al., 1987) and ileum (Bond and Clarke, 1988). Results obtained in the present study showed that BRL 37344 did not relax precontracted endothelium-denuded rings of the thoracic aorta. Although it has been reported that BRL 37344
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produced concentration-dependent vasorelaxation in rat carotid artery and thoracic aorta (Oriowo, 1994, 1995), pretreatment with this agonist, in the same experiment, did not induce desensitization of the aorta and carotid artery to isoprenaline. Since cross-desensitization to isoprenaline is a common occurrence in tissues containing b3-adrenoceptors pretreated with BRL 37344, e.g., rat distal colon (McLaughlin and McDonald, 1990; Oriowo, 1995), it could be suggested that there is no common site of action for isoprenaline and BRL 37344 in rat carotid artery and aorta. On the other hand, it has been demonstrated that isoprenaline, as a nonselective b-adrenoceptor agonist, could activate b3adrenoceptors in different tissues such as adipose tissue (Murphy et al., 1993; Shih and Taberner, 1995; D’Allaire et al., 1995), distal colon and fundic strip (Oriowo, 1995), and heart (Gauthier et al., 1995, 1996). So, it seems unlikely that b3-adrenoceptors exist in the above mentioned vascular tissues as it was confirmed by the results of the present study. Moreover, at concentrations higher than that competed for b3-adrenoceptors of the rat colon (Kaumann and Molenaar, 1996) the b3adrenoceptor-selective antagonist SR 59230A failed to block the relaxant effects of isoprenaline and cyanopindolol. 4. Conclusion The results would show that in rat endotheliumdenuded thoracic aorta, b2-adrenoceptors mediate vasodilation caused by salbutamol. In the case of isoprenaline, the relaxation seems to be partially mediated by b2-adrenoceptors. Additionally, atypical b-adrenoceptors are involved in the latter response. To characterize these atypical b-adrenoceptors the criteria mentioned above (Arch and Kaumann, 1993; Kaumann and Molenaar, 1996; Molenaar and Kaumann, 1997) were used. For agonist-induced relaxation of rat endotheliumdenuded aorta, criteria (1) and (4) were not fulfilled. It is therefore concluded that atypical b-adrenoceptor population in the smooth muscle of rat thoracic aorta is distinct from the population of putative b3-adrenoceptors. It needs to be clarified whether this receptor resembles the fourth cardiac b-adrenoceptor. References Ahmad, S., Chretien, P., Daniel, E.E., Shen, S.H., 1990. Characterization of b-adrenoceptors on cultured endothelial cells by radioligand binding. Life Sci 47, 2365. Arch, J.R.S., Kaumann, A.J., 1993. b3-Adrenoceptors and atypical b-adrenoceptors. Med Res Rev 48, 663–729. Arch, J.R., Ainsworth, A.T., Cawthorne, M.A., Piercy, V., Sennitt, M.V., Thody, V.E., 1984. Atypical b-adrenoceptor on brown adipocytes as target for anti-obesity drugs. Nature 309, 163–165. Bianchetti, A., Manara, L., 1990. In vitro inhibition of intestinal motility by phenylethanolaminotetralines: evidence of atypical b-adrenoceptors in rat colon. Br J Pharmacol 100, 831–839.
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Atypical b-adrenoceptors of rat thoracic aorta Massoumeh Shafiei, Massoud Mahmoudian * Department of Pharmacology, Faculty of Medicine, Iran University of Medical Sciences, P.O. Box 14155-6183, Noori Highway, Tehran, Iran Manuscript received April 15, 1998; accepted manuscript November 6, 1998
Abstract The possible existence of atypical b-adrenoceptors in vascular smooth muscle of rat isolated thoracic aorta was investigated. Isoprenaline (1028–1024 M) produced concentration-dependent relaxation of phenylephrine (1025 M) precontracted rings of endothelium-denuded rat aorta in vitro. Isoprenaline-induced relaxation was resistant to blockade by atenolol (1026 M). But, propranolol (2 3 1027 M) caused a non-competitive inhibition and SR 59230A (6.6 3 1026 M), a b3-adrenoceptor selective antagonist, failed to produce additional antagonism in presence of propranolol. BRL 37344 (1028–1024 M), a b3-selective agonist, did not relax ring segments precontracted with phenylephrine (1025 M) in the absence of endothelium. The non-conventional partial agonist (–)-cyanopindolol (5 3 1026–1024 M) induced a marked relaxation in phenylephrine (1025 M) precontracted aortic rings without endothelium. This vasodilation was resistant to blockade by propranolol (2 3 1027 M) and SR 59230A (1025 M). Salbutamol (1028–1024 M) produced concentration-dependent relaxation in isolated endothelium-denuded aortic rings precontracted with phenylephrine (1025 M). Propranolol (2 3 1027 M), but not atenolol (1026 M), inhibited this relaxant response. It is concluded that in endothelium-denuded thoracic aorta, salbutamol acts through b2-adrenoceptors whereas isoprenaline seems to activate both b2-adrenoceptors and an atypical b-adrenergic receptor. This atypical b-adrenoceptor is distinct from putative b3-adrenoceptor and maybe resembles the reported fourth cardiac b-adrenoceptor. 1999 Elsevier Science Inc. All rights reserved. Keywords: Rat aorta; Atypical b-adrenoceptors; Isoprenaline; Cyanopindolol; BRL 37344
There have been reports of existence of atypical b-adrenoceptors in several tissues such as rat adipose tissue (Arch et al., 1984; Wilson et al., 1984), rat colon (Bianchetti and Manara, 1990; Kirkham and Kelly, 1992) and guinea-pig ileum (Bond and Clarke, 1988). This atypical b-adrenoceptor, which is designated as b3, is relatively insensitive to antagonists possessing only high affinity for b1- and b2-adrenoceptors such as propranolol. In addition, evidence is accumulating for a fourth b-adrenoceptor that mediates cardiostimulation (Kaumann and Molenaar, 1996; Kaumann, 1997). The existence of this receptor is proposed in human ventricle and atrium, and rat atrium based on its pharmacologic properties. The characteristics of the fourth b-adrenoceptors and b3-adrenoceptors has been discussed by Kaumann (1997). Both the fourth cardiac b-adrenoceptor and b3-adrenoceptor can be stimulated by nonconventional partial agonists, such as compounds chemically related to pindolol, and both receptors are resistant to blockade by propranolol. However, only b3-adrenocep* Corresponding author. Tel.: 198-21-8278510; Fax: 198-218278514; E-mail: [email protected].
tors are selectively stimulated by b3-adrenoceptor-selective agonists such as BRL 34377 and blocked by b3renoceptor-selective antagonists such as SR 59230A. The existence of b3-adrenoceptors in vascular smooth muscle has been reported (Berlan et al., 1994; Shen et al., 1994). It appears that these receptors are involved in vasodilation of peripheral vessels. The present study was carried out to characterize b-adrenoceptors of vascular smooth muscle of rat thoracic aorta.
1. Material and methods Sprague-Dawley rats of either sex weighing 180–200 g were used in this study. Each rat was anesthetized with chloroform and bled through a cut to carotid artery. The thoracic aorta was isolated and placed in Krebs’ solution at room temperature gassed with a 95% O2 and 5% CO2 mixture. The composition of the Krebs’ solution used in all cases was as follows (mmol/l): NaCl 118, KCl 4.8, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, glucose 10. The endothelium was removed mechanically by gentle rubbing of the intimal surface with a
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stainless steel stick for 30–50 s. Thereafter ring segments (3–4 mm) were suspended in Krebs’ solution (378C) contained in organ bath. A resting tension of 1 g was applied and tissues were allowed to stabilize for about 90 min, the bathing solution being renewed every 15 min. Tension was recorded with isometric transducer connected to a Beckman polygraph. To obtain relaxant responses aortic rings were contracted with phenylephrine (1025 M). This concentration of phenylephrine usually produced about 75–80% of maximum contraction in this tissue. At the beginning of the experiments, the absence of the functional endothelium was confirmed by the inability of acetylcholine (1026 M) to induce a relaxation. Ascending concentrations of agonist were added cumulatively to generate a concentration-response curve. When antagonists were used each compound was added to the bath and tissue was allowed to equilibrate for 30 min. before the agonist concentration-response curve was repeated. In each individual preparation only one agonist/antagonist pair was tested. Antagonism was assumed to be noncompetitive when the slope of the Schild regression line was significantly different from 1. Agonist potency was expressed as the pD2 value. 1.1. Drugs Drugs used in this investigation included: acetylcholine bromide, DL-isoprenaline hydrochloride, DLpropranolol hydrochloride (all from Sigma Chemical Co, St. Louis, MO), phenylephrine hydrochloride, salbutamol sulfate, atenolol (ALPS Pharmac. Ind. Co. Ltd.), BRL 37344 (RR1SS)(4-[2-[2-(3-chlorophenyl)2-hydroxyethyl]phenoxyl]acetic acid (Tocris Cooksen Ltd., UK), (–)-cyanopindolol hemifumerate (ANAWA Trading SA, Switzerland), and SR 59230A (3-(2-ethylphenoxy)-1-[(1s)-1, 2, 3, 4-tetrahydronapht-1-ylamino]2s-2-propanol oxalate) was a gift from Dr. Luciano Manara (Sanofi Midy, Italy). 1.2. Statistics Results are presented as mean 6 SE of n number of experiments. Mean values are compared by Student’s ttest and differences between mean values were accepted as significant when p , 0.05. 2. Results 2.1. Relaxant effects of b-adrenergic agonists Phenylephrine (1025 M) caused a submaximal contraction in endothelium-denuded rings of rat thoracic aorta. Isoprenaline (1028–1024 M) and salbutamol (1028–1024 M) produced concentration-dependent relaxation in these ring segments (Fig. 1). The pD2 values for isoprenaline and salbutamol are shown in Table 1. Isoprenaline- and salbutamol-evoked maximal responses were 55.2 6 1.03% and 22.76 6 2.95% of initial tension,
Fig. 1. Concentration-response curves for isoprenaline (d) and salbutamol (j) in endothelium-denuded ring segments of rat thoracic aorta precontracted with phenylephrine (1025 M). Results are expressed as percentage relaxation of tone induced by phenylephrine (1025 M). Each point represents the mean 6 SE of 3–5 experiments.
respectively. BRL 37344 (1028–1024 M) did not elicit any relaxation in ring segments of the thoracic aorta after endothelium denudation. 2.2. Blockade of agonist-elicited relaxation by b-adrenergic antagonists Isoprenaline- and salbutamol-induced relaxation were resistant to blockade by atenolol (1026 M) in endothelium-denuded rings. Propranolol (2 3 1027 M) inhibited the relaxant response of isoprenaline in endotheliumdenuded aorta in a noncompetitive manner (Fig. 2). The Schild plot gave a straight line with a slope significantly (p , 0.05) less than unity (0.25), see Fig. 3. The relaxation elicited by salbutamol in endotheliumdenuded rings was completely abolished by propranolol (2 3 1027 M) pretreatment (Fig. 4). SR 59230A (6.6 3 1026 M) did not cause further rightward replacement of concentration-response curve of isoprenaline in presence of propranolol (2 3 1027 M). 2.3. Effect of nonconventional partial agonist (2)-Cyanopindolol (5 3 1026–1024 M) caused concentration-dependent relaxation of phenylephrine (1025 M) precontracted rings of rat endothelium-denuded aorta (Fig. 5). The pD2 value for (2)-cyanopindolol is shown in Table 1. The maximal relaxation produced by (2)-cyanopindolol was 76.2 6 10.2% of initial tension. (2)-Cyanopindolol-induced vasorelaxation was resisTable 1 Relaxant potencies of b-adrenoceptor agonists in rat thoracic aorta (mean 6 SE of 3–5 experiments) Agonist
pD2 values
Isoprenaline Salbutamol Cyanopindolol
5.83 6 0.1 5.34 6 0.1 4.61 6 0.01
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Fig. 2. Concentration-response curves of isoprenaline in the absence (d) and in the presence of propranolol 2 3 1027 M (s) in endothelium-denuded ring segments of rat thoracic aorta precontracted with phenylephrine (1025 M). Results are expressed as percentage relaxation of tone induced by phenylephrine (1025 M). Each point represents the mean 6 SE of 9 experiments.
tant to blockade by propranolol (2 3 1027 M) and the b3adrenoceptor-selective antagonist SR 59230A (1025 M). 3. Discussion Many studies using selective agonists and antagonists have shown that vasodilation could result from activation of b1- or b2-adrenoceptor subtypes or both. For example, while b2-adrenoceptors mediate vasodilation in guinea-pig pulmonary artery (O’Donnell and Wanstall, 1985), human saphenous vein (Ikezono et al., 1987), and canine mesenteric vessels (Tiara et al., 1977; Daly and Levy, 1979), b1-adrenoceptors appear to be responsible for relaxation in human cerebral arteries (Edvinsson et al., 1976) and canine coronary artery (O’Donnell and Wanstall, 1985; Bernard et al., 1991). Coexistence of b1- and b2-adrenoceptors has been also demonstrated (Tiara et al., 1977; O’Donnell and Wanstall, 1984, 1985). In addition, there are reports concerning the involvement of atypical b-adrenoceptors in vasorelaxation of rat common carotid and aorta (Oriowo, 1994, 1995). Atypical pharmacological characteristics maybe mediated by b3-adrenoceptors, so the names b3- and atypical b-adrenoceptor have been extensively used interchangeably. However, it has been shown that atypical b-adrenoceptors in different tissues are not identical (Oriowo, 1995). Recent studies demonstrated that, in addition to b1- and b2-adrenoceptors, a third cardiostimulatory b-adrenoceptor is present in human and rat heart (Kaumann, 1989, 1996; Kaumann and Molenaar, 1996, 1997; Molenaar et al., 1997). Evidence indicates that this atypical cardiac b-adrenoceptor and the b3adrenoceptor are distinct (Kaumann and Molenaar, 1996; Malinowska and Schlicker, 1996; Kaumann et al., 1997), and to avoid confusion the former has been designated the fourth cardiac b-adrenoceptor (Kaumann,
Fig. 3. (A) Concentration-response curves for isoprenaline in the absence (d) and presence of propranolol 2 3 1028 M (j), 2 3 1027 M (s), and 2 3 1026 M (h) in endothelium-denuded ring segments of rat thoracic aorta precontracted with phenylephrine (1025 M). Results are expressed as percentage relaxation of tone induced by phenylephrine (1025 M). Each point represents the mean 6 SE of 5–9 experiments. (B) Schild plot with DRs measured at 50% of the maximum response. Each point represents the mean 6 SE of 5–9 experiments. Y 5 0.25X 1 3.42 (r2 5 0.969).
1997). Regarding the similarities and differences between the fourth cardiac b-adrenoceptor and the b3adrenoceptor, the following criteria are used to accept the involvement of b3-adrenoceptors in a physiological system (Arch and Kaumann, 1993; Kaumann and Molenaar, 1996; Molenaar and Kaumann, 1997): (1) the receptor should be selectively stimulated by b3-adrenoceptor-selective agonists; (2) the receptor should be stimulated by nonconventional partial agonists; (3) the receptor should be resistant to block by antagonists possessing only high affinity for b1- and b2-adrenoceptors; (4) the receptor should be blocked by b3-adrenoceptor-selective antagonists. For the fourth cardiac b-adrenoceptor, criteria (1) and (4) are not fulfilled. The presence of b3-adrenoceptors have been shown in canine cutaneous and fat vascular smooth muscles (Shen et al., 1994; Berlan et al., 1994). The stimulation of these receptors induces a sustained peripheral vasodilation. But, no reports have been implied the existence of the fourth subtype of b-adrenoceptors in vasculature as yet. The results obtained in the present study show that propranolol (2 3 1027 M) did not produce competitive
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Fig. 4. Concentration-response curves for salbutamol in the absence (d) and in the presence of propranolol 2 3 1027 M (s) in endothelium-denuded ring segments of rat thoracic aorta precontracted with phenylephrine (1025 M). Results are expressed as percentage relaxation of tone induced by phenylephrine (1025 M). Each point represents the mean 6 SE of 3 experiments.
antagonism of isoprenaline-induced relaxation of precontracted ring segments of rat aorta in the absence of endothelium. This concentration of propranolol, i.e., 2 3 1027 M, has been shown to block b1- and b2-adrenoceptors, but does not affect atypical adrenoceptors significantly (Kaumann and Molenaar, 1996). The Schild plot was a straight line with a slope of 0.25 which is significantly less than 1 (Fig. 3). These results are therefore consistent with the idea that isoprenaline interacts with two sites that have high and low affinities for propranolol probably correspond to b1- and b2-adrenoceptor subtype (high affinity) and atypical b-adrenoceptors (low affinity). Regarding the failure of atenolol to antagonize the relaxant response to isoprenaline in rat aorta, it seems unlikely that b1-adrenoceptors are involved in this response. This is inconsistent with some previous reports suggesting the existence of b1-adrenoceptors in rat aorta (O’Donnell and Wanstall, 1984, 1985). This maybe due to the difference between the preparation used in the present study and that used by O’Donnell and Wanstall (denuded or intact aorta). The presence of b2-, and not b1-adrenoceptors, in endothelial cells has been reported by Molenaar et al., 1988; Ahmad et al., 1990. With regard to salbutamol, despite being a potent b2adrenoceptor agonist in tissues such as lung (Schreurs et al., 1980) and uterine (Granger et al., 1985), its low potency in vascular preparations has been showed previously (Bernard et al., 1991; Gray and Marshal, 1992; Oriowo, 1994). The low pD2 value obtained in present study (Table 1) is therefore consistent with the previous reports. Salbutamol-induced relaxation of precontracted endothelium-denuded ring segments of rat aorta was completely inhibited by propranolol, but not prevented by atenolol, indicating activation of b2-adrenoceptors in rat aorta by salbutamol. The weak relaxation induced
Fig. 5. Concentration-response curve for cyanopindolol in endothelium-denuded ring segments of rat thoracic aorta precontracted with phenylephrine (1025 M). Results are expressed as percentage relaxation of tone induced by phenylephrine (1025 M). Each point represents the mean 6 SE of 5 experiments.
by salbutamol in endothelium-denuded rings may be due either to the present of scanty number of b2-adrenoceptors in vascular smooth muscle or to the activation of b2-adrenoceptors in residual endothelium remained after denudation. It should be mentioned that in some studies the stimulation of both b1-adrenoceptors (Bernard et al., 1991) and atypical b-adrenergic receptors (Oriowo, 1995) by salbutamol have been reported. Cyanopindolol is a nonconventional partial agonist, i.e., high affinity blocker of both b1- and b2-adrenoceptors, known to exert also stimulant effect through b3adrenoceptors in rat colon (Kaumann and Molenaar, 1996) and through an atypical cardiostimulant b-adrenoceptor in rat and human atrium (Kaumann and Molenaar, 1996; Sarsero et al., 1997). In the present study, cyanopindolol behaved as a full agonist producing greater maximal relaxation of precontracted endothelium-denuded aortic rings compared to isoprenaline, however, it has a lower potency than isoprenaline (Table 1). The relaxant effect of cyanopindolol was not antagonized by propranolol (2 3 1027 M) which points to the interaction of cyanopindolol with atypical b-adrenoceptors in thoracic aorta. The relaxation was slow in onset and took a much longer time, compared to isoprenaline, to attain a peak response. The selective b3-adrenoceptor agonist BRL 37344 is a potent stimulant of lipolysis in both brown and white adipose tissues of the rat (Arch et al., 1984; Wilson et al., 1984) and a relaxant of smooth muscles of gastrointestinal tract including the rat proximal colon (Bianchetti and Manara, 1990), distal colon (McLaughlin and McDonald, 1990; Kirkham and Kelly, 1992; Oriowo, 1995), guinea-pig colon (Coleman et al., 1987) and ileum (Bond and Clarke, 1988). Results obtained in the present study showed that BRL 37344 did not relax precontracted endothelium-denuded rings of the thoracic aorta. Although it has been reported that BRL 37344
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produced concentration-dependent vasorelaxation in rat carotid artery and thoracic aorta (Oriowo, 1994, 1995), pretreatment with this agonist, in the same experiment, did not induce desensitization of the aorta and carotid artery to isoprenaline. Since cross-desensitization to isoprenaline is a common occurrence in tissues containing b3-adrenoceptors pretreated with BRL 37344, e.g., rat distal colon (McLaughlin and McDonald, 1990; Oriowo, 1995), it could be suggested that there is no common site of action for isoprenaline and BRL 37344 in rat carotid artery and aorta. On the other hand, it has been demonstrated that isoprenaline, as a nonselective b-adrenoceptor agonist, could activate b3adrenoceptors in different tissues such as adipose tissue (Murphy et al., 1993; Shih and Taberner, 1995; D’Allaire et al., 1995), distal colon and fundic strip (Oriowo, 1995), and heart (Gauthier et al., 1995, 1996). So, it seems unlikely that b3-adrenoceptors exist in the above mentioned vascular tissues as it was confirmed by the results of the present study. Moreover, at concentrations higher than that competed for b3-adrenoceptors of the rat colon (Kaumann and Molenaar, 1996) the b3adrenoceptor-selective antagonist SR 59230A failed to block the relaxant effects of isoprenaline and cyanopindolol. 4. Conclusion The results would show that in rat endotheliumdenuded thoracic aorta, b2-adrenoceptors mediate vasodilation caused by salbutamol. In the case of isoprenaline, the relaxation seems to be partially mediated by b2-adrenoceptors. Additionally, atypical b-adrenoceptors are involved in the latter response. To characterize these atypical b-adrenoceptors the criteria mentioned above (Arch and Kaumann, 1993; Kaumann and Molenaar, 1996; Molenaar and Kaumann, 1997) were used. For agonist-induced relaxation of rat endotheliumdenuded aorta, criteria (1) and (4) were not fulfilled. It is therefore concluded that atypical b-adrenoceptor population in the smooth muscle of rat thoracic aorta is distinct from the population of putative b3-adrenoceptors. It needs to be clarified whether this receptor resembles the fourth cardiac b-adrenoceptor. References Ahmad, S., Chretien, P., Daniel, E.E., Shen, S.H., 1990. Characterization of b-adrenoceptors on cultured endothelial cells by radioligand binding. Life Sci 47, 2365. Arch, J.R.S., Kaumann, A.J., 1993. b3-Adrenoceptors and atypical b-adrenoceptors. Med Res Rev 48, 663–729. Arch, J.R., Ainsworth, A.T., Cawthorne, M.A., Piercy, V., Sennitt, M.V., Thody, V.E., 1984. Atypical b-adrenoceptor on brown adipocytes as target for anti-obesity drugs. Nature 309, 163–165. Bianchetti, A., Manara, L., 1990. In vitro inhibition of intestinal motility by phenylethanolaminotetralines: evidence of atypical b-adrenoceptors in rat colon. Br J Pharmacol 100, 831–839.
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