3 DISTINCT BINDING-SITES FOR [H-3] PRAZOSIN IN THE RAT CEREBRAL-CORTEX

1 The putative alpha-1-adrenoceptor subtypes of rat cerebral cortex membranes were characterized in binding experiments with [H-3]-prazosin. 2 Specific binding of [H-3]-prazosin was saturable between 20-5000 pM. Scatchard plots of the binding data were non-linear, indicating the presence of two distinct affinity sites for prazosin (pK(D, high) = 10.18, R(high) = 308 fmol mg-1 protein; pK(D, low) = 8.96, R(low) = 221 fmol mg-1 protein). 3 In the membranes pretreated with chlorethylclonidine (CEC) two affinity sites for prazosin were also observed: the affinities were similar to those without CEC pretreatment, but the maximum numbers of binding sites were reduced by CEC pretreatment to 23 and 62% for prazosin-high (R(high)) and low affinity sites (R(low)), respectively. 4 The prazosin-high affinity sites were further subdivided into two subclasses by WB4101(2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane) and phentolamine; the low affinity sites for WB4101 and phentolamine were more potently inactivated by CEC as compared with high affinity sites. On the other hand, prazosin, HV723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)-amino)-propyl)benzeneacetonitrile fumarate) and yohimbine inhibited [H-3]-prazosin binding to prazosin-high affinity sites monophasically. 5 In addition to the high affinity sites, the prazosin-low affinity sites were labelled at high concentrations of [H-3]-prazosin. Thus, prazosin and WB4101 showed shallow displacement curves. On the other hand, HV723 and yohimbine did not discriminate between prazosin-high and low affinity sites. 6 Two distinct alpha-1-adrenoceptor subclassifications have been recently proposed (alpha-1A, alpha-1B subtypes and alpha-1H, alpha-1L, alpha-1N subtypes). According to the criteria defined with competitive antagonists in both the subclassifications, the present results indicate that the alpha-1-adrenoceptors of rat cerebral cortex consist of three different subtypes, presumably alpha-1A, alpha-1B and alpha-1L, and suggest that the alpha-1A and alpha-1B subtypes are identified as a single affinity site for prazosin (alpha-1H). The results also indicate that care must be taken in the use of CEC for alpha-1-adrenoceptor subclassification because of its low selectivity.