Methodological considerations for the measurement of protein kinase C translocation in intact smooth muscle

This study investigated several potential artifacts that may influence agonist-induced distribution of protein kinase C (PKC) activity between cytosolic and membrane fractions of intact smooth muscle. Protein kinase C activity in the membrane fraction prepared from rat aorta exposed to phorbol myristate acetate (PMA) was only partially extracted by 0.2% Triton (T)X-100, while 1% TX-100, or repeated 0.2% TX-100 extractions completely extracted PKC activity. Extraction of PKC activity from the membrane fraction with TX-100 concentrations of 0.2% or higher was problematic, however, since TX-100 concentrations as low as 0.04% nearly abolished Ca(2+)+ phosphatidyserine+diolein-induced phosphorylation of histone substrate in the PKC assay. Substitution of PMA for diolein, however, restored histone phosphorylation to the level observed in the absence of TX-100. Triton X-100 concentrations as low as 0.025% also abolished Ca(2+)-induced histone phosphorylation, while Ca(2+)+ phosphatidylserine-induced phosphorylation was little affected. In contrast to our previous demonstration that exposure of rat aorta to phorbol ester increased PKC activity in the membrane fraction in aorta washed in Ca(2+)-free solution following phorbol ester exposure (Chuprun et al., Am J Physiol 261:C675-C684, 1991; Bazan et al., Eur J Pharmacol-Molec Pharmacol Section 227:343-348, 1992), PMA decreased PKC activity in the initial 0.2% TX-100 extraction of the membrane fraction in the absence of tissue wash in Ca(2+)-free solution following PMA exposure. This study, along with our previous reports, suggest that partial PKC extraction from the membrane, and Ca(2+)-dependent homogenization-induced translocation of PKC from the cytosol to the membrane fraction, may complicate measurements of agonist-induced PKC translocation. The reliability of PKC assays in crude fractions may be increased in the presence of TX-100, due to the ability of TX-100 to inhibit Ca(2+)-induced phosphorylation, and through the substitution of PMA for diolein, which maximally stimulates PKC in the presence of detergent.