Three distinct binding sites for [3H]‐prazosin in the rat cerebral cortex

1 The putative α1‐adrenoceptor subtypes of rat cerebral cortex membranes were characterized in binding experiments with [3H]‐prazosin. 2 Specific binding of [3H]‐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 (pKD, high = 10.18, Rhigh = 308 fmol mg−1 protein; pKD, low = 8.96, Rlow = 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 (Rhigh) and low affinity sites (Rlow), 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 the high affinity sites. On the other hand, prazosin, HV723 (α‐ethyl‐3,4,5‐trimethoxy‐α‐(3‐((2‐(2‐methoxyphenoxy)ethyl)‐amino)‐propyl)benzeneacetonitrile fumarate) and yohimbine inhibited [3H]‐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 [3H]‐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 α1‐adrenoceptor subclassifications have been recently proposed (α1A, α1B subtypes and α1H, α1L, α1N subtypes). According to the criteria defined with competitive antagonists in both the subclassifications, the present results indicate that the α1‐adrenoceptors of rat cerebral cortex consist of three different subtypes, presumably α1A, α1B and α1L, and suggest that the α1A and α1B subtypes are identified as a single affinity site for prazosin (α1H). The results also indicate that care must be taken in the use of CEC for α1‐adrenoceptor subclassification because of its low selectivity.