A putative adrenoceptor distinct from currently defined alpha- and beta-subtypes

Research was undertaken to examine the proposal that phenylethylamine based agonists and agonists of the imidazoline or imidazolidine structure might interact differently with alpha-adrenoceptors. Previous studies with competitive alpha2- adrenoceptor antagonists in guinea-pig ileum and rat vas deferens have shown that the phenylethylamines often fail to interact competitively whereas the imidazolines and imidazolidines appear to obey competitive kinetics. Quantitative experiments measuring inhibition of longitudinal smooth muscle contraction in the isolated transmurally stimulated guinea-pig ileum were undertaken to examine the interaction of (a) the phenylethylamine, norepinephrine; (b) the imidazoline, UK-14,304-18 (5-bromo-6-[2-imidazolin-2yl-amino]-quinoxaline); and (c) the imidazolidine, clonidine; with the competitive alpha2- adrenoceptor antagonists, rauwolscine, idazoxan, and St 587 (2-[2-chloro-5-trifluoro-methyl-phenyliminoJ- imidazolidine). Norepinephrine did not interact in a competitive manner with the antagonists whereas clonidine and UK-14,304-18 obeyed competitive kinetics. The discrepancy between the two groups of agonists could not be attributed to non-equilibrium conditions. Experiments with the irreversible alpha-adrenoceptor antagonist, benextramine, also differentiated between norepinephrine and imidazoli(di)ne agonists. Concentrations of benextramine which completely abolished the inhibitory responses of clonidine and UK-14,304-18 only partially attenuated the inhibitory responses to norepinephrine. This difference could not be explained on the basis of a receptor reserve for norepinephrine. Remaining responses to norepinephrine after treatment with benextramine were resistant to antagonism by phentolamine, rauwolscine, propranolol, and sulpiride. Thus alphap adrenoceptors, alpha2-adrenoceptors, beta-adrenoceptors and dopamine receptors were ruled out as mediators of the resistant response to norepinephrine. The results with competitive and non-competitive antagonists suggested that norepinephrine might activate a second site for which clonidine and UK-14,304-18 lacked affinity. Experiments were undertaken to characterize the putative site using histamine induced contractions of guinea-pig ileum. The putative site was found to exhibit an agonist rank order of potency for phenylethylamine based compounds consistent with activation of beta-adrenoceptors: BRL 37344 (4-[2-amino(2-hydroxy-2-(3'-chloro-phenyl)ethyl)- propyl]-phenoxyacetic acid > isoproterenol > norepinephrine > epinephrine > fenoterol. However the putative site was not blocked by concentrations of nadolol or propranolol which fully saturate beta-adrenoceptors. Experiments with the (+) and (-) isomers of both norepinephrine and isoproterenol revealed that the proposed site was capable of recognizing optical isomers. Furthermore, nadolol was shown to exhibit a low affinity for the site, yielding pA2 values of 4.4 to 4.7 versus five different agonists. These results provide evidence to suggest that the putative site is a functionally active adrenoceptor which is pharmacologically distinct from the currently defined alpha- and beta-subtypes.