Comparison of human α1-adrenoceptor subtype coupling to protein kinase C activation and related signalling pathways

The coupling of human α1-adrenoceptor subtypes to protein kinase C (PKC) and PKC-related signalling events were investigated in rat-1 fibroblasts stably expressing α1A-, α1B- or α1D-adrenoceptors at densities of 1328 ± 200, 5030 ± 703 and 150 ± 14 fmol/mg protein respectively. In functional assays the α1-adrenoceptor agonist phenylephrine significantly stimulated PKC (assessed as increased activity in the membrane fraction) in cells expressing α1A- or α1B- but not α1D-adrenoceptors. In immunoblot assays phorbol ester treatment enhanced membrane-associated immunoreactivity of PKCα, PKCδ and PKCɛ to a similar extent in all three cell lines. Stimulation of α1A- and α1B-adrenoceptors also increased immunoreactivity of PKCα, PKCδ and PKCɛ in the membrane fraction, while α1D-adrenoceptor stimulation yielded only very small and inconsistent alterations. Immunoreactivity of PKCζ was not consistently affected by phorbol ester or phenylephrine in any of the cell lines. Stimulation of all three α1-adrenoceptors time- and concentration-dependently increased inositol phosphate formation. Maximum activation occurred with the order α1A≈α1B > α1D. Phenylephrine also concentration dependently elevated free intracellular [Ca2+] in all three cell lines with the order of efficacy α1A > α1B > α1D. In the presence of ethanol, phenylephrine stimulated phosphatidylethanol formation in α1A- and α1B-adrenoceptor-expressing cells time and concentration dependently but only weakly and inconsistently in α1D-adrenoceptor-expressing cells. The efficacy of phenylephrine (100 μM) relative to that of noradrenaline (100 μM) for stimulation of phosphatidylethanol formation was similar (≥ 75%) for all three subtypes. The alkylating agent phenoxybenzamine concentration dependently reduced α1A-adrenoceptor density and phenylephrine-stimulated Ca2+ elevations to levels seen with α1D-adrenoceptors but reductions of phenylephrine-stimulated phosphatidylethanol formation were weaker. We conclude that human α1A- and α1B-adrenoceptors expressed in rat-1 cells couple to activation of PKCα, PKCδ and PKCɛ but not PKCζ; this may involve stimulation of phospholipases C and D and intracellular Ca2+ elevations. Activation of these pathways by α1D-adrenoceptors appears to be much weaker and was not detected consistently; this was not fully explained by weak partial agonism of phenylephrine at this subtype or by lower receptor densities. Overall the α1A-adrenoceptor may have the highest efficiency of stimulus-response coupling among human α1-adrenoceptor subtypes.

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