SKF-104078

The alpha-2 adrenoceptor subtype and its signal transduction pathway mediating vascular relaxation in rats were studied in vitro using rings of superior mesenteric arteries. Removal of endothelium or incubation with NG-nitro-L-arginine completely blocked relaxant responses to UK14,304, suggesting endothelium-derived nitric oxide mediates relaxation. The order of potency for full (F) or partial (P) agonists causing relaxation was guanabenz (P) > UK14,304 (F) > clonidine (P) > epinephrine (F) > norepinephrine (F). Affinities (Ka) of alpha-2 adrenoceptor subtype-selective drugs for blocking relaxation were obtained in side-by-side experiments comparing rat mesenteric arteries with pig coronary arteries. Relaxation of pig coronary arteries is known to be mediated by the alpha-2A adrenoceptor subtype. Ka values in nM for rauwolscine (19), WB-4101 (265), SKF-104078 (197), spiroxatrine (128), and prazosin (1531) for blocking relaxation in rat arteries were consistent with their affinities for binding at the alpha-2D adrenoceptor subtype. Ka values for rauwolscine and WB-4101, drugs distinguishing the alpha-2D from the alpha-2A adrenoceptor subtype, were significantly higher in blocking relaxation of rat arteries compared with pig arteries, suggesting the alpha-2D adrenoceptor subtype mediates NO-induced relaxation in rat arteries. We used forskolin to oppose alpha-2 adrenoceptor-mediated inhibition of cAMP formation by directly stimulating cAMP formation in endothelium. Forskolin did not affect the relaxant response to UK14,304, suggesting that cAMP is not involved in the coupling of alpha-2 adrenoceptors to nitric oxide-induced vascular relaxation.

Functional studies have shown that 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4,5-tetrahydro-3- benzazepine (SKF 104078) has very low affinity for prejunctional alpha 2-adrenoceptors (alpha 2-AR) in the guinea pig atrium. In this study, we have cloned guinea pig homologues of the human alpha 2-C10, alpha 2-C4 AR subtypes and have studied them in isolation by heterologous expression in cultured mammalian cells. Oligonucleotide primers, designed from conserved areas of the human alpha 2-ARs were used in a polymerase chain reaction (PCR) with template cDNA synthesized from guinea pig atrial mRNA. Three PCR products were obtained that shared identity with the three human alpha 2-AR subtypes. A guinea pig (gp) genomic library was screened with a cDNA clone encoding a portion of the gp-alpha 2A, and genes containing the complete coding sequences of the guinea pig alpha 2A, alpha 2B, and alpha 2C AR subtypes were obtained. These guinea pig genes were subcloned into a eukaryotic expression plasmid and were expressed transiently in COS-7 cells. The binding of the alpha 2-selective antagonist [3H]MK-912 to membranes prepared from these cells was specific and of high affinity with Kd values of 810 pM for gp-alpha 2A, 2700 pM for gp-alpha 2B and 110 pM for gp-alpha 2C. Competition for the binding of [3H]MK-912 by SKF 104078 indicated that it was of moderately high affinity (approximately 100 nM) but that it was not selective for any of the guinea pig alpha 2-AR subtypes. Co-expression of guinea pig alpha 2-AR subtypes with a cyclicAMP-responsive chloramphenicol acetyltransferase (CAT) reporter gene resulted in agonist-dependent modulation of CAT activity. For the gp-alpha 2 A, a biphasic response was obtained with low concentrations of noradrenaline (NE) decreasing forskolin-stimulated CAT activity and high concentrations causing a reversal. For the gp-alpha 2B, NE produced mostly potentiation of forskolin-stimulated activity, and for the gp-alpha 2C, NE caused mainly inhibition. Overall, the pharmacology of the cloned guinea pig alpha 2-AR subtypes was in agreement with data obtained for the native guinea pig receptors and was functionally similar to that of the cloned human alpha 2-AR subtypes.