The intention of the present proposal is to describe in isolated rat liver cells the operation of the Na/Ca exchanger and to determine whether the combined operation of this exchanger and that of the Na/H Exchanger System can contribute to the increased [Ca]i which is observed after the action of substances which promote calcium entry into the hepatocyte. Normally, the Na/Ca anti-porter seems to operate in the liver by exchanger intracellular sodium for extracellular calcium. It has been estimated that in rat liver slices about 60% of the resting calcium flux across the cell membrane would occur via the exchanger. This fact would make the Na/Ca anti-porter an important target for effectors which affect [Ca]i such as the calcium modeling hormones and hematotoxins like ethanol. In isolated hepatocytes liver slices and the perfused liver, these hormones and ethanol are known to increase [Ca]i and calcium uptake. However, the role of the Na/Ca Exchanger on the calcium entry across the cell membrane induced by these dependent calcium influx into the cell, and that in isolated rat hepatocytes the cytosolic calcium concentration markedly rises when intracellular sodium is increased by the use of ouabain, a well known inhibitor of the Sodium Pump (see preliminary data). It is also known that increases in intracellular calcium may constitute an important link in the development of cell pathologies and cell death. Using the isolated hepatocyte, it is specifically proposed to kinetically describe, in terms of Km and Vmax, the Na/Ca as well as to determine the kinetic characteristics of the exchanger function as a calcium extruder by studying the [Ca]i-dependent 22Na uptake at different [Ca]i and [Na]o. The stoichiometry of the anti-porter will be determined by analyzing the dependence of sodium-efflux on [Ca]o. In addition, the actions of ethanol on the activity and kinetics of the Na/Ca Exchanger will be analyzed and the effect of the calcium-mobilizing hormones, phenylephrine, vasopressin, angiotensin II, and glucogen on the activity and kinetic parameters of the Na/Ca anti-porter, will be determined by analyzing the dependence of sodium-efflux on [Ca]o. In addition, the actions of ethanol on the activity and kinetics of the Na/Ca Exchanger will be analyzed and the effect of the calcium-mobilizing hormones, phenylephrine, vasopressin, the effects of ethanol on the calcium-mobilizing activity of these hormones through the Na/Ca Exchanger will be studying 45Ca uptake and [Ca]i increases in cells at normal or at low pHi [proportionate- loaded) in the presence or absence of specific inhibitors of the Na/Ca, or Na/H exchangers, respectively.
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