PHARMACOLOGICAL CHARACTERIZATION OF ALPHA(1)-ADRENOCEPTOR SUBTYPES INRAT-HEART - A BINDING STUDY

1 The alpha(1)-adrenoceptor subtypes of rat heart were characterized i n binding experiments performed with [H-3]-prazosin as the radiolabel. The specific binding to the alpha(1)-adrenoceptors was determined wit h 0.3 mu M prazosin, because phentolamine (10 mu M) was insufficient t o inhibit completely the specific binding of high concentrations of [H -3]-prazosin. 2 In saturation experiments, [H-3]-prazosin bound to two distinct affinity sites (pK(D) = 10.39 and 8.19). The proportion of t he low affinity sites was approximately 84% of total specific binding. Membranes pretreated with chloroethylclonidine (CEC, 10 mu M) also sh owed two distinct affinity sites for [H-3]-prazosin, although the maxi mum numbers of high and low affinity sites were reduced by 86 and 64%, respectively. 3 In competition experiments, [H-3]-prazosin (100 pM) b inding was inhibited by WB4101 methoxy-phenoxyethyl)aminomethyl-1,4-be nzodioxane) and 5-methylurapidil. The inhibition curves displayed shal low slopes which could be subdivided into high and low affinity compon ents (pK(1) = 10.43 and 8.36 for WB4101, 8.62 and 6.61 for 5-methylura pidil). However, unlabelled prazosin or HV723 oxyphenoxy)-ethyl)amino) propyl)benzeneacetonitrile fumarate) competed for [H-3]-prazosin bindi ng monophasically (pK(I) = 10.34 and 8.28, respectively). In CEC-pretr eated membranes, prazosin, WB4101, 5-methylurapidil and HV723 antagoni zed the [H-3]-prazosin (100 pM) binding monophasically (pK(1) = 9.70, 9.56, 8.60 and 8.82, for each antagonist). 4 On the other hand, 1000 p M [H-3]-prazosin binding was inhibited by unlabelled prazosin biphasic ally (pK(1) = 10.49 and 8.49). HV723 did not discriminate both prazosi n-high and low affinity sites (pK(1) = 8.18). 5 These results suggest the presence of at least three distinct alpha(1)-adrenoceptor subtypes in rat hearts (two prazosin-high affinity sites and one prazosin-low affinity site). According to the recent alpha(1)-adrenoceptor subclass ifications, one of the former two sites corresponds to the alpha(1B) s ubtype with low affinities for WB4101 and 5-methylurapidil and sensiti ve to CEC, while another site with relatively high affinities for WB41 01 and 5-methylurapidil may be classical alpha(1A), cloned alpha(1C), alpha(1D) subtypes or their mixture. The prazosin-low affinity site co rresponds to putative alpha(1L) subtype with low affinity for HV723, w hich may be predominantly involved in the positive inotropic response to phenylephrine.