The coupling of human alpha1-adrenoceptor subtypes to protein kinase C (PKC) and PKC-related signalling events were investigated in rat-1 fibroblasts stably expressing alpha1A-, alpha1B- or alpha1D-adrenoceptors at densities of 1328+/-200, 5030+/-703 and 150+/-14 fmol/mg protein respectively. In functional assays the alpha1-adrenoceptor agonist phenylephrine significantly stimulated PKC (assessed as increased activity in the membrane fraction) in cells expressing alpha1A- or alpha1B- but not alpha1D-adrenoceptors. In immunoblot assays phorbol ester treatment enhanced membrane-associated immunoreactivity of PKCalpha, PKCdelta and PKCepsilon to a similar extent in all three cell lines. Stimulation of alpha1A- and alpha1B-adrenoceptors also increased immunoreactivity of PKCalpha, PKCdelta and PKCepsilon in the membrane fraction, while alpha1D-adrenoceptor stimulation yielded only very small and inconsistent alterations. Immunoreactivity of PKCzeta was not consistently affected by phorbol ester or phenylephrine in any of the cell lines. Stimulation of all three alpha1-adrenoceptors time- and concentration-dependently increased inositol phosphate formation. Maximum activation occurred with the order alpha1A approximately = alpha1B > alpha1D. Phenylephrine also concentration dependently elevated free intracellular [Ca2+] in all three cell lines with the order of efficacy alpha1A > alpha1B > alpha1D. In the presence of ethanol, phenylephrine stimulated phosphatidylethanol formation in alpha1A- and alpha1B-adrenoceptor-expressing cells time and concentration dependently but only weakly and inconsistently in alpha1D-adrenoceptor-expressing cells. The efficacy of phenylephrine (100 microM) relative to that of noradrenaline (100 microM) for stimulation of phosphatidylethanol formation was similar (> or = 75%) for all three subtypes. The alkylating agent phenoxybenzamine concentration dependently reduced alpha1A-adrenoceptor density and phenylephrine-stimulated Ca2+ elevations to levels seen with alpha1D-adrenoceptors but reductions of phenylephrine-stimulated phosphatidylethanol formation were weaker. We conclude that human alpha1A- and alpha1B-adrenoceptors expressed in rat-1 cells couple to activation of PKCalpha, PKCdelta and PKCepsilon but not PKCzeta; this may involve stimulation of phospholipases C and D and intracellular Ca2+ elevations. Activation of these pathways by alpha1D-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 alpha1A-adrenoceptor may have the highest efficiency of stimulus-response coupling among human alpha1-adrenoceptor subtypes.