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


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

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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],



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

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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.


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


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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.



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

Heterogeneity of postjunctional alpha 1-adrenoceptors in mammalian aortae: subclassification based on chlorethylclonidine, WB 4101 and nifedipine.

The effects of chlorethylclonidine, WB 4101 and nifedipine on norepinephrine-induced contractions of rat, guinea-pig, rabbit and dog aortae were inves...
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