METHODOLOGICAL CONSIDERATIONS FOR THE MEASUREMENT OF PROTEIN-KINASE-CTRANSLOCATION IN INTACT SMOOTH-MUSCLE

This study investigated several potential artifacts that may influence agonist-induced distribution of protein kinase C (PKC) activity betwe en cytosolic and membrane fractions of intact smooth muscle. Protein k inase C activity in the membrane fraction prepared from rat aorta expo sed 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 extrac tions completely extracted PKC activity. Extraction of PKC activity fr om 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 Ca2+ + phosphatidylserine + diolein-induced phosphory lation of histone substrate in the PKC assay. Substitution of PMA for diolein, however, restored histone phosphorylation to the level observ ed in the absence of TX-100. Triton X-100 concentrations as low as 0.0 25% also abolished Ca2+-induced histone phosphorylation, while Ca2+ phosphatidylserine-induced phosphorylation was little affected. In con trast to our previous demonstration that exposure of rat aorta to phor bol ester increased PKC activity in the membrane fraction in aorta was hed in Ca2+-free solution following phorbol ester exposure (Chuprun et al., Am I 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 Ca2+-free solution following PMA. exposure. This study, along with our previous reports, suggest that partial PKC extraction from the membrane, and Ca2+-dependent homogenization-induc ed translocation of PKC from the cytosol to the membrane fraction, may complicate measurements of agonist-induced PKC translocation. The rel iability of PKC assays in crude fractions may be increased in the pres ence of TX-100, due to the ability of TX-100 to inhibit Ca2+-induced p hosphorylation, and through the substitution of PMA for diolein, which maximally stimulates PKC in the presence of detergent. (C) 1996 Elsev ier Science Inc.