Identification of α 1 ‐adrenoceptor subtypes in the rat vas deferens: binding and functional studies

1 The α 1 ‐adrenoceptor subtypes of the prostatic and epididymal portion of rat vas deferens were characterized in binding and functional experiments. 2 In saturation experiments, [ 3 H]‐prazosin bound to two distinct affinity sites in the epididymal portion of rat vas deferens (p K D = 10.1 ± 0.13 and 9.01 ± 0.15, B max = 507 and 1231 fmol mg −1 protein, respectively). In the prostatic portion [ 3 H]‐prazosin bound to a single affinity site (p K D = 9.82 ± 0.04, B max = 924 fmol mg −1 protein). 3 In the displacement experiments, unlabelled prazosin displaced biphasically the binding of 200 p m [ 3 H]‐prazosin to the epididymal portion; the resulting two p K I values were consistent with the affinity constants obtained in the saturation experiments. WB4101 (2‐(2,6‐dimethoxy‐phenoxyethyl)‐aminomethyl‐1,4‐benzodioxane) and benoxathian also discriminated the two affinity sites in the epididymal portion and the population of low affinity sites for the three antagonists was approximately 40%. On the other hand, the prostatic portion predominantly showed a single affinity site for prazosin, WB4101 and benoxathian, although the presence of a small proportion (less than 10%) of the low affinity site could be detected. HV723 (α‐ethyl‐3,4,5‐trimethoxy‐α‐(3‐((2‐(2‐methoxyphenoxy)ethyl)‐amino)‐propyl) benzeneacetonitrile fumarate) displaced the [ 3 H]‐prazosin binding monophasically with a low affinity in both halves. 4 Pretreatment with chlorethylclonidine (CEC) at concentrations higher than 1 μ m inhibited 700 p m [ 3 H]‐prazosin binding to the prostatic portion by approximately 50%. However, the inhibition in the epididymal portion was much less (approximately 21% at 50 μ m CEC). 5 In the functional study, the contractile response to noradrenaline was competitively inhibited by prazosin, WB4101, benoxathian and HV723 with similar and low affinities (p K B value ranging from 8.0 to 9.0) in the epididymal portion of rat vas deferens. In the prostatic portion of rat vas deferens, noradrenaline also produced a contraction, but the maximal amplitude of contraction developed was approximately one‐fourth of that in the epididymal portion. Prazosin and WB4101 also inhibited the contractile response of the prostatic portion with the p K B values similar to those obtained in the epididymal portion. The contractions to noradrenaline in both portions were potently attenuated by 1 μ m nifedipine but were not affected by pretreatment with 10 μ m CEC. 6 Under conditions where P 2x ‐purinoceptors and prejunctional α 2 ‐adrenoceptors were blocked, electrical transmural stimulation produced a rapidly developing phasic contraction and a subsequent tonic contraction in the epididymal portion of rat vas deferens. The phasic and tonic contractions were inhibited in a concentration‐dependent manner by prazosin (IC 50 = 25.7 and 25.9 n m , respectively), WB4101 (IC50 = 7.27 and 7.58 n m ), benoxathian (IC 50 = 10.9 and 8.66 n m ) and HV723 (IC 50 = 15.9 and 14.9 n m ). Nifedipine selectively attenuated the tonic contraction induced by electrical stimulation, and the residual phasic response was inhibited by the antagonists mentioned above with similar affinities to those in the absence of nifedipine. CEC (10 μ m ) had little effect on the adrenergic neurogenic contractions. 7 The present results indicate the presence of two distinct α 1 ‐adrenoceptor subtypes in the rat vas deferens, which show respectively high and low affinities for each of prazosin, WB4101 and benoxathian, and presumably correspond to putative α 1A and α 1L subtypes according to the recent α 1 ‐adrenoceptor subclassifications. The contractions induced by exogenous and endogenous noradrenaline seem to be predominantly mediated through the α 1L subtype. The heterogeneous distribution of the low affinity sites (α 1L subtype) may well explain differences in functional responsiveness between the two portions of rat vas deferens.