Alpha1-adrenoceptors in the rat cerebral cortex: new insights into the characterization of alpha1L- and alpha1D-adrenoceptors.
Among the three alpha(1)-adrenoceptor subtypes (alpha(1A), alpha(1B) and alpha(1D)) a peculiar intracellular localization and poor coupling to membrane signals of cloned alpha(1D)-adrenoceptor have been reported. In addition, the alpha(1L)-adrenoceptor (low affinity for prazosin), a functional phenotype of alpha(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 alpha(1)-adrenoceptor subtypes in a native tissue, the rat cerebral cortex. mRNA for the three subtypes was quantified by real-time RT-PCR (alpha(1D)>alpha(1B)>>alpha(1A)). alpha(1)-Adrenoceptors were also detected by immunoblotting, revealing alpha(1A)- and alpha(1B)-adrenoceptors to be predominantly expressed in the membrane fraction and the alpha(1D)-adrenoceptor to be localized in the cytosolic fraction. Competitive radioligand binding studies revealed the presence of alpha(1D)-adrenoceptor in tissue homogenates, whereas only alpha(1A)- and alpha(1B)-subtypes were detected in membranes. The proportion of alpha(1A)-adrenoceptor increased after treatment with noradrenaline, suggesting differences in agonist-mediated trafficking. Saturation experiments detected high- and low (alpha(1A/L))-prazosin binding sites, the latter of which disappeared on incubation with GppNHp. The alpha(1A/L)-adrenoceptor was heavily implicated in the inositol phosphate response, while the alpha(1D)-subtype did not play a relevant role. These results suggest that the predominant cytosolic localization of alpha(1D)-adrenoceptor lies behind its poor coupling to membrane signalling such as inositol phosphate pathway. The fact that the alpha(1L)-adrenoceptor detected in radioligand binding studies disappeared in the presence of GppNHp implies that it represents a conformational state of the alpha(1A)-adrenoceptor coupled to G-protein.
Departamento de Farmacología, Facultat de Farmàcia, Universitat de València, Avda. Vicent Andrés Estelles s/n, Valencia, Spain.
Vanessa
V
Segura
Nicla
N
Flacco
Eduardo
E
Oliver
Domingo
D
Barettino
Pilar
P
D'Ocon
Maria Dolores
MD
Ivorra
D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
C547721:Adra1d protein, rat; D007295:Inositol Phosphates; D012333:RNA, Messenger; D018340:Receptors, Adrenergic, alpha-1; D006160:Guanosine Triphosphate