α1-Adrenoceptors in the rat cerebral cortex: New insights into the characterization of α1L- and α1D-adrenoceptors

Abstract Among the three α 1 -adrenoceptor subtypes (α 1A , α 1B and α 1D ) a peculiar intracellular localization and poor coupling to membrane signals of cloned α 1D -adrenoceptor have been reported. In addition, the α 1L -adrenoceptor (low affinity for prazosin), a functional phenotype of α 1A , has been described. The purpose of this work was to analyze the expression, cellular localization and coupling to membrane signalling (inositol phosphate accumulation) of α 1 -adrenoceptor subtypes in a native tissue, the rat cerebral cortex. mRNA for the three subtypes was quantified by real-time RT-PCR (α 1D > α 1B  ≫ α 1A ). α 1 -Adrenoceptors were also detected by immunoblotting, revealing α 1A - and α 1B -adrenoceptors to be predominantly expressed in the membrane fraction and the α 1D -adrenoceptor to be localized in the cytosolic fraction. Competitive radioligand binding studies revealed the presence of α 1D -adrenoceptor in tissue homogenates, whereas only α 1A - and α 1B -subtypes were detected in membranes. The proportion of α 1A -adrenoceptor increased after treatment with noradrenaline, suggesting differences in agonist-mediated trafficking. Saturation experiments detected high- and low (α 1A/L )-prazosin binding sites, the latter of which disappeared on incubation with GppNHp. The α 1A/L -adrenoceptor was heavily implicated in the inositol phosphate response, while the α 1D -subtype did not play a relevant role. These results suggest that the predominant cytosolic localization of α 1D -adrenoceptor lies behind its poor coupling to membrane signalling such as inositol phosphate pathway. The fact that the α 1L -adrenoceptor detected in radioligand binding studies disappeared in the presence of GppNHp implies that it represents a conformational state of the α 1A -adrenoceptor coupled to G-protein.