Research Paper J Vase Res 1992:29:33-40
Department of Pharmacology, Smith Kline Beecham Pharmaceuticals. King of Prussia, Pa.. USA
Heterogeneity of Postjunctional 0i -Adrenoceptors in Mammalian Aortae: Subclassification Based on Chlorethylclonidine, W B 4101 and Nifedipine
Key Words
Abstract
cti-Adrenoceptors Receptor heterogeneity Aorta Chlorethylclonidine WB 4101 Nifedipine
The effects of chlorethylclonidine. WB 4101 and nifedipine on norepinephrine-induced con tractions of rat. guinea-pig. rabbit and dog aortae were investigated in order to characterize the ttpadrenoceptor subtype(s) present in the aortae of these different species. The putative a iA-adrenoceptor antagonist, WB 4101. was significantly more potent in the rat aorta com pared to the rabbit, guinea-pig and dog aortae which were not significantly different from each other. The calcium channel antagonist, nifedipine (1 pA/). had little or no effect on norepinephrine-induced contractions in aortic segments from the rabbit, guinea pig and dog: whereas in the rat aorta, nifedipine significantly inhibited the response to norepinephrine. Based on the studies with WB 4101 and nifedipine. cp-adrenoceptors in rat aorta would be tentatively classified as a |A-adrenoceptors, whereas those in the guinea-pig. rabbit and dog aortae would be of the «m-adrenoceptor subtype. The putative irreversible aiB-adrcnoceptor antagonist, chlorethylclonidine. inhibited the response to norepinephrine in aortae from all species, but to dramatically different degrees. The response to norepinephrine was inhibited by 500-fold and 450-fold by chlorethylclonidine in the rat and dog aortae. respectively, whereas in the guinea-pig and rabbit aortae, the potency of norepinephrine was reduced by only 3- and 20-fold, respectively. Thus, based on studies with chlorethylclonidine, a r adrenoceptors in the rat and dog aortae would be classified as ctm-adrenoceptors (i.e.. chlorethylclonidine-sensitive), whereas oi|A-adrenoceptors (chlorethylclonidine-insensitive) would pre dominate in the guinea-pig aorta, and possibly both a iA- and a iB-adrenoceptors would coexist in the rabbit aorta. It is apparent that the subclassification of a,-adrenoccptors based on chlorethylclonidine docs not agree with the subclassification based on WB 4101 and nifedi pine in these tissues. Specifically, studies with WB 4101 and nifedipine indicate that the rat aorta contains primarily aiA-adrenoceptors. whereas the high sensitivity to chlorethylcloni dine in the rat aorta would indicate the existence of (im-adrenoceptors. Similar discrepancies exist in the aortae from the other species. The results indicate clearly that significant hetero geneity exists in ai-adrenoceptors in mammalian aortae. Furthermore, our findings suggest that chlorethylclonidine and WB 4101 are not sufficiently reliable tools to use in subclassify ing ai-adrenoceptors in vascular smooth muscle, and the results obtained in the present study do not allow a,-adrenoceptors in mammalian aortae to be subclassified reliably as either a ,Aor am-adrenoceptors.
Received: February 4. 1991 Accepted: August 29. 1991
Robert R. RufFolo. Jr. Department o f Pharmacology Smith Kline Beecham Pharmaceuticals P.O. Box 1539. King of Prussia. PA 19406 (USA)
© 1992 S. Kargcr AG. Basel 1018-1172/9270291-0033 $2.75/0
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 6:59:13 AM
Mabayoje A. Oriowo Robert R. Rujfolo, Jr.
We have previously demonstrated that heterogeneity exists in postjunctional a|-adrenoceptors in mammalian aortae [1-4], where differences of over 200-fold were observed in the affinities of clonidine and yohimbine for a|-adrenoceptors in the rat aorta at one extreme, and the guinea-pig and rabbit aortae at the other. Aortae from the cat. dog and hamster fell between these extremes into yet a third distinct group. Additional studies using a variety of agonists further confirmed that differences exist be tween a|-adrenoceptors in the rat and rabbit aortae [4], Thus, for a series of imidazoline agonists, the rank order of agonist affinities in the rat aorta was clonidine > tetrahydrozoline > tramazoline > oxymetazoline, whereas, in the rabbit aorta, the rank order of agonist affinities was exactly reversed [4], Furthermore, while clonidine had a 10-fold higher affinity than oxymetazoline in the rat aor ta, oxymetazoline had an approximately 500-fold higher affinity than clonidine in the rabbit aorta. Based on these studies, it was concluded that ai-adrenoceptors in mam malian aortae represented a heterogeneous population of at least three distinct subtypes. Recently, chlorethylclonidine. a reactive analog of clo nidine [5], has been shown to inactivate selectively a sub population of ai-adrenoceptors in several rat tissues [6], Based in part on the sensitivity to chlorethylclonidine, d r adrenoceptors have been classified into two subtypes, namely d iA (chlorethylclonidine-insensitive) and Oib (chlorethylclonidine-sensitive) [7], The division of d|-adrenoceptors into the d |A and Oib subtypes with chlorethylclonidine has lead to additional characterization of these two putative di-adrenoceptor subtypes. Thus, UiA-adrenoceptors are proposed to me diate responses through the translocation of extracellular calcium and involve a guanine nucleotide regulatory pro tein (G-protein) that is sensitive to inhibition by pertussis toxin [6, 8], By virtue of the dependence of d |A-adrenoceptors on the translocation of extracellular calcium, responses mediated by diA-adrenoceptors are markedly sensitive to inhibition by calcium channel blockers [6, 8], In contrast, U|B-adrenoceptors are postulated to mediate responses through the activation of phospholipase C and the ultimate formation of inositol phosphates which mo bilize intracellular stores of calcium [6, 8], Thus, d |Badrenoceptor-mediated responses are not sensitive to inhibition by calcium channel antagonists. Furthermore. aiB-adrenoceptors appear to require a pertussis toxininsensitive G-protein to activate a second messenger sys tem [6],
34
Oriowo/Ruffolo
WB 4101 has been proposed to be a selective 2 h) washing of the tissues. The response of norepinephrine was markedly inhib ited in the rat and dog aortae by chlorethylclonidine. such that approximately 500- and 450-fold rightward shifts were observed in the concentration-response curves to norepinephrine, respectively. Chlorethylclonidine also produced a rightward shift in the concentration-response curve to norepinephrine in the rabbit aorta, but only by approximately 20-fold. In guinea-pig aortic ring seg ments. chlorethylclonidine treatment produced only a 2to 3-fold rightward shift in the concentration-response curve to norepinephrine. Following treatment with chlorethylclonidine. the re sidual responses to norepinephrine in the rat and canine aortae were not antagonized by prazosin (0.1 \iM), and were therefore not mediated by a|-adrenoceptors. Thus, chlorethylclonidine completely and irreversibly inhibited all functional, prazosin-sensitive ai-adrenoceptor-me diated responses in the rat and dog aortae. However, in
ott-Adrenoceptor Fleterogeneity
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 6:59:13 AM
ity of WB 4101 is highest in the rat aorta, and is approxi mately one order of magnitude lower in the rabbit, guin ea-pig and dog aortac. Although the dissociation constant in the rat aorta is significantly higher than in the other tissues, all values fall within the range reported for ot|adrenoceptors, and the -log KB value of 9.3 ± 0.1 obtained for WB 4101 in the rat aorta is nearly identical to that value reported recently by Mir and Fozard [15] in the same tissue. The calcium channel antagonist, nifedipine (1 p.V/), did not inhibit responses to norepinephrine in aortic ring segments from the rabbit, guinea pig and dog (fig. 2). However, the same concentration of nifedipine did signif icantly inhibit the response of norepinephrine in the rat aorta, consistent with previous observations [ 16. 17], In all mammalian aortae, the selective, irreversible «iB-adrenoceptor antagonist, chlorethylclonidine (100 \iM), displaced the norepinephrine concentration-re sponse curves to the right without depressing their maxi mum responses (fig. 3), consistent with previous observa tions [18-20]. It is not apparent why the maximum responses to norepinephrine were not depressed by chlor ethylclonidine. as would be characteristic of an irrevers ible antagonist [11], However, this unusual pattern of
Fig. 3. The effects of chlorethylclonidine (CEC) at a concentration of 100 \iM on norepinephrine-induced responses in aortae from rat, rabbit, guinea pig and dog.
Discussion
Minneman [6] has proposed a subclassification of a r adrenoceptors based on differences in sensitivities to selective antagonists, such as chlorethylclonidine and WB 4101. as well as differences in signal transduction pro cesses. such as the mobilization of intracellular calcium or the translocation of extracellular calcium. An aiA-adrenoceptor is proposed to mediate a response that involves a pertussis toxin-sensitive G-protein and the ultimate trans location of extracellular calcium [8]. ciiA-Adrenoceptors have been postulated to be sensi tive to inhibition by WB 4101 and resistant to inhibition by chlorethylclonidine. Based on the proposed depen dence of aiA-adrenoceptor-mediated responses on the translocation of extracellular calcium. a iA-adrenoceptors are also suggested to be sensitive to inhibition by calcium channel blockers, such as nifedipine [8]. In contrast. a,B-adrenoceptors mediate responses that are insensitive to inhibition by pertussis toxin and which involve the activation of phospholipase C, with the gener ation of inositol phosphates and the ultimate mobiliza tion of intracellular stores of calcium [8], ctiB-Adrenoceptors are sensitive to inhibition by chlorethylclonidine and
37
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 6:59:13 AM
aortic ring segments from the rabbit and guinea pig. the residual responses to norepinephrine after chlorethylclo nidine treatment were significantly antagonized by prazo sin. and with similar dissociation constants as obtained in control tissues that were not pretreated with chlorethyl clonidine (table I ). Thus, in the rabbit and guinea-pig aor tae, the residual contractions to norepinephrine were me diated by prazosin-sensitive a r adrenoceptors that were insensitive to inhibition by chlorethylclonidine. As was the case with prazosin, the residual responses to norepinephrine that existed the rat and dog after com plete alkylation by chlorethylclonidine were resistant to antagonism by WB 4101, whereas the residual responses to norepinephrine in the rabbit and guinea-pig aorta were inhibited by WB 4101. and with similar dissociation con stants as obtained in control tissues that were not treated with chlorethylclonidine.
38
Oriowo/Ruffolo
antagonized by prazosin, indicating the presence of a pra zosin-sensitive, chlorethylclonidine-resistant a r adrenoceptor population. Thus, in the rabbit aorta, both chlorethylclonidine-sensitive and chlorethylclonidine-insensitive ai-adrenoceptors exist, which could possibly indicate the coexistence of both
Department of Pharmacology, Smith Kline Beecham Pharmaceuticals. King of Prussia, Pa.. USA
Heterogeneity of Postjunctional 0i -Adrenoceptors in Mammalian Aortae: Subclassification Based on Chlorethylclonidine, W B 4101 and Nifedipine
Key Words
Abstract
cti-Adrenoceptors Receptor heterogeneity Aorta Chlorethylclonidine WB 4101 Nifedipine
The effects of chlorethylclonidine. WB 4101 and nifedipine on norepinephrine-induced con tractions of rat. guinea-pig. rabbit and dog aortae were investigated in order to characterize the ttpadrenoceptor subtype(s) present in the aortae of these different species. The putative a iA-adrenoceptor antagonist, WB 4101. was significantly more potent in the rat aorta com pared to the rabbit, guinea-pig and dog aortae which were not significantly different from each other. The calcium channel antagonist, nifedipine (1 pA/). had little or no effect on norepinephrine-induced contractions in aortic segments from the rabbit, guinea pig and dog: whereas in the rat aorta, nifedipine significantly inhibited the response to norepinephrine. Based on the studies with WB 4101 and nifedipine. cp-adrenoceptors in rat aorta would be tentatively classified as a |A-adrenoceptors, whereas those in the guinea-pig. rabbit and dog aortae would be of the «m-adrenoceptor subtype. The putative irreversible aiB-adrcnoceptor antagonist, chlorethylclonidine. inhibited the response to norepinephrine in aortae from all species, but to dramatically different degrees. The response to norepinephrine was inhibited by 500-fold and 450-fold by chlorethylclonidine in the rat and dog aortae. respectively, whereas in the guinea-pig and rabbit aortae, the potency of norepinephrine was reduced by only 3- and 20-fold, respectively. Thus, based on studies with chlorethylclonidine, a r adrenoceptors in the rat and dog aortae would be classified as ctm-adrenoceptors (i.e.. chlorethylclonidine-sensitive), whereas oi|A-adrenoceptors (chlorethylclonidine-insensitive) would pre dominate in the guinea-pig aorta, and possibly both a iA- and a iB-adrenoceptors would coexist in the rabbit aorta. It is apparent that the subclassification of a,-adrenoccptors based on chlorethylclonidine docs not agree with the subclassification based on WB 4101 and nifedi pine in these tissues. Specifically, studies with WB 4101 and nifedipine indicate that the rat aorta contains primarily aiA-adrenoceptors. whereas the high sensitivity to chlorethylcloni dine in the rat aorta would indicate the existence of (im-adrenoceptors. Similar discrepancies exist in the aortae from the other species. The results indicate clearly that significant hetero geneity exists in ai-adrenoceptors in mammalian aortae. Furthermore, our findings suggest that chlorethylclonidine and WB 4101 are not sufficiently reliable tools to use in subclassify ing ai-adrenoceptors in vascular smooth muscle, and the results obtained in the present study do not allow a,-adrenoceptors in mammalian aortae to be subclassified reliably as either a ,Aor am-adrenoceptors.
Received: February 4. 1991 Accepted: August 29. 1991
Robert R. RufFolo. Jr. Department o f Pharmacology Smith Kline Beecham Pharmaceuticals P.O. Box 1539. King of Prussia. PA 19406 (USA)
© 1992 S. Kargcr AG. Basel 1018-1172/9270291-0033 $2.75/0
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 6:59:13 AM
Mabayoje A. Oriowo Robert R. Rujfolo, Jr.
We have previously demonstrated that heterogeneity exists in postjunctional a|-adrenoceptors in mammalian aortae [1-4], where differences of over 200-fold were observed in the affinities of clonidine and yohimbine for a|-adrenoceptors in the rat aorta at one extreme, and the guinea-pig and rabbit aortae at the other. Aortae from the cat. dog and hamster fell between these extremes into yet a third distinct group. Additional studies using a variety of agonists further confirmed that differences exist be tween a|-adrenoceptors in the rat and rabbit aortae [4], Thus, for a series of imidazoline agonists, the rank order of agonist affinities in the rat aorta was clonidine > tetrahydrozoline > tramazoline > oxymetazoline, whereas, in the rabbit aorta, the rank order of agonist affinities was exactly reversed [4], Furthermore, while clonidine had a 10-fold higher affinity than oxymetazoline in the rat aor ta, oxymetazoline had an approximately 500-fold higher affinity than clonidine in the rabbit aorta. Based on these studies, it was concluded that ai-adrenoceptors in mam malian aortae represented a heterogeneous population of at least three distinct subtypes. Recently, chlorethylclonidine. a reactive analog of clo nidine [5], has been shown to inactivate selectively a sub population of ai-adrenoceptors in several rat tissues [6], Based in part on the sensitivity to chlorethylclonidine, d r adrenoceptors have been classified into two subtypes, namely d iA (chlorethylclonidine-insensitive) and Oib (chlorethylclonidine-sensitive) [7], The division of d|-adrenoceptors into the d |A and Oib subtypes with chlorethylclonidine has lead to additional characterization of these two putative di-adrenoceptor subtypes. Thus, UiA-adrenoceptors are proposed to me diate responses through the translocation of extracellular calcium and involve a guanine nucleotide regulatory pro tein (G-protein) that is sensitive to inhibition by pertussis toxin [6, 8], By virtue of the dependence of d |A-adrenoceptors on the translocation of extracellular calcium, responses mediated by diA-adrenoceptors are markedly sensitive to inhibition by calcium channel blockers [6, 8], In contrast, U|B-adrenoceptors are postulated to mediate responses through the activation of phospholipase C and the ultimate formation of inositol phosphates which mo bilize intracellular stores of calcium [6, 8], Thus, d |Badrenoceptor-mediated responses are not sensitive to inhibition by calcium channel antagonists. Furthermore. aiB-adrenoceptors appear to require a pertussis toxininsensitive G-protein to activate a second messenger sys tem [6],
34
Oriowo/Ruffolo
WB 4101 has been proposed to be a selective 2 h) washing of the tissues. The response of norepinephrine was markedly inhib ited in the rat and dog aortae by chlorethylclonidine. such that approximately 500- and 450-fold rightward shifts were observed in the concentration-response curves to norepinephrine, respectively. Chlorethylclonidine also produced a rightward shift in the concentration-response curve to norepinephrine in the rabbit aorta, but only by approximately 20-fold. In guinea-pig aortic ring seg ments. chlorethylclonidine treatment produced only a 2to 3-fold rightward shift in the concentration-response curve to norepinephrine. Following treatment with chlorethylclonidine. the re sidual responses to norepinephrine in the rat and canine aortae were not antagonized by prazosin (0.1 \iM), and were therefore not mediated by a|-adrenoceptors. Thus, chlorethylclonidine completely and irreversibly inhibited all functional, prazosin-sensitive ai-adrenoceptor-me diated responses in the rat and dog aortae. However, in
ott-Adrenoceptor Fleterogeneity
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 6:59:13 AM
ity of WB 4101 is highest in the rat aorta, and is approxi mately one order of magnitude lower in the rabbit, guin ea-pig and dog aortac. Although the dissociation constant in the rat aorta is significantly higher than in the other tissues, all values fall within the range reported for ot|adrenoceptors, and the -log KB value of 9.3 ± 0.1 obtained for WB 4101 in the rat aorta is nearly identical to that value reported recently by Mir and Fozard [15] in the same tissue. The calcium channel antagonist, nifedipine (1 p.V/), did not inhibit responses to norepinephrine in aortic ring segments from the rabbit, guinea pig and dog (fig. 2). However, the same concentration of nifedipine did signif icantly inhibit the response of norepinephrine in the rat aorta, consistent with previous observations [ 16. 17], In all mammalian aortae, the selective, irreversible «iB-adrenoceptor antagonist, chlorethylclonidine (100 \iM), displaced the norepinephrine concentration-re sponse curves to the right without depressing their maxi mum responses (fig. 3), consistent with previous observa tions [18-20]. It is not apparent why the maximum responses to norepinephrine were not depressed by chlor ethylclonidine. as would be characteristic of an irrevers ible antagonist [11], However, this unusual pattern of
Fig. 3. The effects of chlorethylclonidine (CEC) at a concentration of 100 \iM on norepinephrine-induced responses in aortae from rat, rabbit, guinea pig and dog.
Discussion
Minneman [6] has proposed a subclassification of a r adrenoceptors based on differences in sensitivities to selective antagonists, such as chlorethylclonidine and WB 4101. as well as differences in signal transduction pro cesses. such as the mobilization of intracellular calcium or the translocation of extracellular calcium. An aiA-adrenoceptor is proposed to mediate a response that involves a pertussis toxin-sensitive G-protein and the ultimate trans location of extracellular calcium [8]. ciiA-Adrenoceptors have been postulated to be sensi tive to inhibition by WB 4101 and resistant to inhibition by chlorethylclonidine. Based on the proposed depen dence of aiA-adrenoceptor-mediated responses on the translocation of extracellular calcium. a iA-adrenoceptors are also suggested to be sensitive to inhibition by calcium channel blockers, such as nifedipine [8]. In contrast. a,B-adrenoceptors mediate responses that are insensitive to inhibition by pertussis toxin and which involve the activation of phospholipase C, with the gener ation of inositol phosphates and the ultimate mobiliza tion of intracellular stores of calcium [8], ctiB-Adrenoceptors are sensitive to inhibition by chlorethylclonidine and
37
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 6:59:13 AM
aortic ring segments from the rabbit and guinea pig. the residual responses to norepinephrine after chlorethylclo nidine treatment were significantly antagonized by prazo sin. and with similar dissociation constants as obtained in control tissues that were not pretreated with chlorethyl clonidine (table I ). Thus, in the rabbit and guinea-pig aor tae, the residual contractions to norepinephrine were me diated by prazosin-sensitive a r adrenoceptors that were insensitive to inhibition by chlorethylclonidine. As was the case with prazosin, the residual responses to norepinephrine that existed the rat and dog after com plete alkylation by chlorethylclonidine were resistant to antagonism by WB 4101, whereas the residual responses to norepinephrine in the rabbit and guinea-pig aorta were inhibited by WB 4101. and with similar dissociation con stants as obtained in control tissues that were not treated with chlorethylclonidine.
38
Oriowo/Ruffolo
antagonized by prazosin, indicating the presence of a pra zosin-sensitive, chlorethylclonidine-resistant a r adrenoceptor population. Thus, in the rabbit aorta, both chlorethylclonidine-sensitive and chlorethylclonidine-insensitive ai-adrenoceptors exist, which could possibly indicate the coexistence of both
