Ryanodine-induced calcium release from hepatic microsomes and permeabilized hepatocytes

Abstract

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] is a second messenger that releases Ca2+ from hepatocyte microsomes. The toxic alkaloid ryanodine modulates Ca2+ release via a receptor (RyR) identified in a variety of cell systems, but its regulation and functional significance in liver are undefined. Similarly, the role in hepatocyte Ca2+ regulation of adenosine 5'-cyclic diphosphate-ribose (cADPR), which is the putative endogenous ligand for RyR in other cell systems, has not been defined. Utilizing microsomes and permeabilized cells, we have investigated Ca2+ regulation in hepatocytes and, in particular, effects of ryanodine, cADPR, and other putative modulators on Ca2+ release and compared these with Ins(1,4,5)P3-induced Ca2+ release. Ryanodine at > or = 50 microM released 20% of microsomal Ca2+, and, in contrast to Ins(1,4,5)P3, no potentiation was observed with guanosine 5'-triphosphate and polyethylene glycol. Ins(1,4,5)P3-induced Ca2+ release was demonstrable after maximal ryanodine-induced Ca2+ release, suggesting that distinct Ca2+ stores are involved. cADPR (5 microM) did not induce Ca2+ release, alone or in combination with calmodulin or hepatic cytosol, nor did it influence ryanodine-induced release, in microsomes or permeabilized hepatocytes (in which ryanodine released 25% of the sequestered Ca2+). Ryanodine-induced Ca2+ release in microsomes was not influenced by 20 mM caffeine, which itself did not mobilize Ca2+, but was prevented by 500 microM tetracaine, which was shown to induce Ca2+ release. We conclude that ryanodine is capable of mobilizing Ca2+ in the hepatocyte from microsomal stores that are distinct from those that can be regulated by Ins(1,4,5)P3 but that cADPR has no such effect. These data suggest that cADPR does not serve as the endogenous ligand for RyR in liver cells or that the site of action of ryanodine in hepatocyte microsomes is distinct from that in other cell types.