INOSITOL 1,4,5-TRISPHOSPHATE ACTIVATES RECEPTOR-MEDIATED CALCIUM-ENTRY BY 2 DIFFERENT PATHWAYS IN HEPATOCYTES

The quenching of fura-2 fluorescence by the influx of extracellular Mn2+ was measured to indicate the flux rates through receptor-operated calcium channels in the plasma membrane of rat hepatocytes. Neomycin, an inhibitor of phospholipase C, inhibited the vasopressin-induced influx of Mn2+. Thus, the agonist-induced entry of extracellular calcium into hepatocytes is Linked to a phospholipase C-generated second messenger. Microinjection of inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P-4], inositol 1,4,5-trisphosphate [Ins(1,4,5)P-3] or 3-deoxy-3-fluoro-Ins(1,4,5)P-3 revealed that Ins(1,4,5)P, rather than Ins(1,3,4,5)P-3 is responsible for calcium entry. The activation of phospholipase C by vasopressin produced an influx of Mn2+ independent of the depletion of intracellular calcium stores if this depletion was delayed by the Ins(1,4,5)P-3 receptor antagonist heparin or by the use of a low agonist concentration. Thapsigargin, an inhibitor of the store calcium pump, leading to an Ins(1,4,5)P-3-independent emptying of stores, gave a short living signal (less than 3 min) for calcium entry. We propose that Ins(1,4,5)P-3 is able to stimulate calcium entry by two pathways. (a) Ins(1,4,5)P-3 activates receptor-operated calcium channels in a direct manner. The calcium entry resulting from this is followed (b) by the Ins(1,4,5)P-3-induced depletion of calcium stores, producing a store-dependent entry.