STUDIES SUGGESTING THE PARTICIPATION OF PROTEIN-KINASE-A IN 1,25(OH)(2)-VITAMIN-D-3-DEPENDENT PROTEIN-PHOSPHORYLATION IN CARDIAC-MUSCLE

We have previously established that the secosteroid hormone 1 alpha,25 -dihydroxy-vitamin D-3 [1,25(OH)(2)D-3] rapidly stimulates dihydropyri dine-sensitive calcium channel-mediated Ca2+ influx in chick cardiac m uscle by a non-genomic action which is accompanied by phosphorylation of microsomal proteins. In the present study, we investigated the part icipation of the cyclic AMP/protein kinase A (PKA) signalling pathway in hormone-induced changes on protein phosphorylation in chick heart t issue. A major increase in the phosphorylation of a microsomal protein of 45 kDa, and, to a lesser extent, of a protein of 70 KDa, was obser ved after incubation with [gamma-P-32]ATP of membranes isolated from h eart thin slices (HTS) pretreated for 1-5 min with 1,25(OH)(2)D-3. Thi s effect was dose-and time-dependent, reaching a maximum after 3 min a nd at the physiological concentrations of 10(-10) and 10(-11) M. 1,25( OH)(2)D-3 steadily increased cellular cAMP levels as a function of the dose (10-(12)-10(-9) M). The specific agonist of PKA, Sp-cAMPS and th e PM catalytic subunit stimulated the phosphorylation of the same memb rane proteins as the hormone. The 1 alpha,25-dihydroxy-vitamin D-3-dep endent changes in microsomal protein phosphorylation were diminished b y the specific PKA inhibitor, Rp-cAMPS. In addition, the PKA activity ratio (-cAMP/+cAMP) increased 60% above the control after treatment of HTS with 10(-11) M 1,25(OH)(2)D-3. The data obtained clearly indicate that activation of the cAMP/PKA signalling pathway mediates the stimu lation of protein phosphorylation by 1 alpha,25-dihydroxy-vitamin D-3 in chick cardiac muscle. (C) 1998 Academic Press Limited.