Elevation of intracellular Ca 2+ concentration by protein kinase C stimulation in isolated single rabbit sino-atrial node cells

Abstract

1. Effects of phorbol esters (stimulators of protein kinase C, PK-C) on the L-type Ca 2+ current ( I Ca) and the intracellular Ca 2+ level ([Ca] i) were investigated using single rabbit sino-atrial (SA) node cells. 2. In whole-cell patch-clamp experiments, both phorbol esters (1 μM). 4-β-phorbol-12, 13-dibutyrate (PDB) and 12- O-tetradecanoyl-phorbol-13-acetate (TPA), inhibited the I Ca. Phorbol esters did not affect the voltages of half-maximum activation and inactivation for I Ca. 3. The time-course of inactivation phase for I Ca was two exponentials. The fast component was decreased, whereas the slow component was increased by phorbol esters. 4. Addition of H-7 (10 μM), an inhibitor of PK-C, did not antagonize, but rather increased both components. In contrast, 4-α-phorbol-12, 13-didecanoate (PDD), an inactive analog, did not affect the I Ca amplitude and the inactivation process. 5. The phorbol esters elicited a transient inward current by repetitive clamp pulses. 6. In SA node cells loaded with fura-2 (a Ca 2+-sensitive fluorescent dye), both TPA and PDB at 0.3 μM in the presence of isoproterenol (ISP, 0.2 μM) elevated the [Ca] i. PDD did not affect [Ca] i. 7. These results indicate that PK-C stimulation by phorbol esters elevates [Ca] i (which is potentiated by ISP), possibly resulting in cellular calcium overload.