ADENOSINE A(1) STIMULATION ACTIVATES DELTA-PROTEIN KINASE-C IN RAT VENTRICULAR MYOCYTES

By making use of immunoblotting and immunocytochemical analysis, we ex plored whether stimulation of adenosine A(1) receptors would promote t he activation of delta-protein kinase C (delta-PKC) immunolabeled with a polyclonal antibody. Immunoblot analysis of Triton X-100-soluble ce ll membrane and cytosolic fractions revealed the presence of a specifi c 75-kD band reactive to the delta-PKC polyclonal antibody. In freshly isolated rat cardiac myocytes, 28% of the total immunoreactive delta- PKC was associated with the membrane fraction, whereas 72% was associa ted with the soluble fraction. Under stimulation with the tumor-promot ing phorbol 12-myristate 13-acetate (PMA, 500 nmol/L) used as a positi ve control, delta-PKC translocated to the cell membrane, with the memb rane fraction representing 88% and the cytosolic fraction representing 12% of the total immunoreactive delta-PKC. Transverse optical section s performed with confocal laser microscopy showed that immunostaining with anti-delta-PKC antibody was distributed in the cytosol of unstimu lated cells but accumulated in the cell membrane under PMA stimulation . In the membrane fraction of cells pretreated with adenosine (100 mu mol/L) or with the adenosine A(1) agonist (-)-N-6-(2-phenylisopropyl)- adenosine (R-PIA, 1 mu mol/L), the 75-kD band corresponding to delta-P KC increased by 57% and 66%, respectively, when compared with nonstimu lated cells processed under the same experimental conditions. In cells exposed to either of the purine agonists, specific fluorescence stain ing decorated the cell membrane, a pattern that was not observed in co ntrol cells. Activation of membrane gamma-PKC produced either by adeno sine itself or by its analogue R-PIA was fully antagonized by the spec ific A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 mu mol/L). From these data, we conclude that adenosine A(1) stimulation activates delta-PKC in freshly isolated rat ventricular myocytes.