2 SEPARATE PLASMA-MEMBRANE CA2-MEDIATED CA2+ INFLUX IN RAT HEPATOCYTES( CARRIERS PARTICIPATE IN RECEPTOR)

The plasma membrane Ca2+ carrier system involved in receptor-mediated Ca2+ entry was studied. Using the Ca2+ readdition protocol, the rate o f cytosolic free Ca2+ concentration ([Ca2+]i) increase in vasopressin- pretreated hepatocytes was significantly higher than in thapsigargin- or 2,5-di(tert-butyl)hpdroquinone-pretreate cells. The addition of Mn2 + to unstimulated hepatocytes resulted in a biphasic quench of fura-2 fluorescence. After an initial phase that was fast in rate but of shor t duration, the rate of fura-2 quench by Mn2+ became much slower and l asted until all the cellular fura-2 was quenched. Pretreatment of the cells with vasopressin only accelerated the rate of the latter phase b ut not of the initial one. In agonist-stimulated cells, acidification of the extracellular medium or the presence of ruthenium red, econazol e or SK&F 96365 decreased the rates of both [Ca2+](i) increase and Mn2 + entry upon addition of the respective cation. By contrast, neomycin and N-tosyl-L-phenylalanine chloromethyl ketone markedly decreased the rate of [Ca2+](i), increase upon Ca2+ readdition but had no effect on vasopressin-stimulated Mn2+ entry. None of the treatments affected th e ability of vasopressin and thapsigargin to mobilize the internal Ca2 + store. It is concluded that in hepatocytes the two pathways of recep tor-mediated Ca2+ entry control two distinct yet pharmacologically rel ated cation carriers.