Protein kinase C distribution and translocation in rat myocardium: Methodological considerations

Introduction: Protein kinase C (PKC) is an important modifier of several cardiovascular phenomena, including cardioprotection, apoptosis, and hypertrophy. Although pharmacological activation of PKC is often assessed by translocation, the effects of isolation procedures on left ventricular (LV) PKC distribution have not been systematically examined. Accordingly, we sought to determine whether homogenization methods (Polytron, glass-glass tissue grinder), detergent selection and concentration, or centrifugation protocols affect PKC (α, ε) distribution or phorbol-12-myristate-13-acetate (PMA)-induced translocation. Methods: Hearts of male F344 or Wistar rats were Langendorff perfused with either 100 nM PMA or vehicle, and LV cytosolic and particulate PKC (α, ε) distributions were assessed by differential centrifugation and Western blotting. Results: Following 100 000 ×g centrifugation of the homogenate, resuspension of the pellet (P₁) in 0.1% sodium dodecyl sulfate (SDS) increased electrophoretic mobility of PKC (α, ε) such that PKCε comigrated with a nonspecific band. Resuspension of P₁ in Triton X-100 (TX) did not affect mobility but decreased P₁ PKC (α, ε) levels in a TX-concentration-dependent manner; however, this decrease was found to be due to differential protein solubilization. Decreased levels of PKC (α, ε) were also noted in soluble and P₂ (supernatant of 100 000 ×g centrifugation of P₁) fractions due to increased Polytron burst and total homogenization times. Interestingly, the P₂ fraction also revealed Polytron-dependent decreases (47% vs. glass-glass tissue grinder; p<0.05) in PKCε following an initial 1000 ×g centrifugation and an increased PMA-dependent translocation of PKC (α, ε; 2.4-fold and 1.6-fold, respectively, vs. P₁; p<0.05). Discussion: Taken together, these results suggest that PKC isolation procedures should be carefully considered when designing or comparing LV PKC studies due to the potential effects isolation may have on PKC distribution and translocation.