pituitrin and 2-aminoethoxydiphenyl-borate

An increase in the cytoplasmic free Ca2+ concentration in hepatocytes as a result of the release of Ca2+ from intracellular stores and Ca2+ inflow from the extracellular space is a necessary part of the mechanism by which bile acids are moved along the bile cannaliculus by contraction of the cannaliculus. 2-Aminoethyl diphenylborate (2-APB) is a recently discovered inhibitor of store-operated plasma membrane Ca2+ channels in hepatocytes. The aim of the present study was to test the ability of 2-APB to inhibit bile flow.. Bile flow was measured in the isolated perfused rat liver using cannulation of the common bile duct. Measurements were carried out in the presence or absence of 2-APB in either the presence of taurocholic acid (to enhance basal bile flow) or in the absence of taurocholic acid and in the presence of the hormones vasopressin and glucagon, which are known to stimulate bile flow.. In livers perfused in the presence of taurocholic acid, 2-APB reversibly inhibited bile flow with a slow time of onset. The time of onset of inhibition was reduced by prior addition of the endoplasmic reticulum (Ca(2+) + Mg2+)adenosine triphosphatase inhibitor, 2,5-di-t-butylhydroquinone. In livers perfused in the absence of taurocholate, 2-APB had little effect on the basal rate of bile flow, but inhibited the ability of vasopressin and glucagon to stimulate bile flow.. It is concluded that an inhibitor of hepatocyte plasma membrane Ca2+ channels can induce cholestasis. The results provide evidence that suggests that, over a period of time, the normal function of hepatocyte store-operated Ca2+ channels is required to maintain bile flow. Future strategies directed at the regulation of bile flow might include pharmacological or other interventions that modulate Ca2+ inflow to hepatocytes.

Topics: Animals; Bile; Boron Compounds; Calcium Channel Blockers; Calcium Channels; Calcium-Transporting ATPases; Cholestasis; Enzyme Inhibitors; Glucagon; Hepatocytes; Hydroquinones; Liver; Peptide Hormones; Rats; Taurocholic Acid; Vasopressins

The compound 2-aminoethyl diphenylborate (2-APB), an inhibitor of Ins(1,4,5)P(3) receptor action in some cell types, has been used to assess the role of Ins(1,4,5)P(3) receptors in the activation of store-operated Ca2+ channels (SOCs) [Ma, Patterson, van Rossum, Birnbaumer, Mikoshiba and Gill (2000) Science 287, 1647-1651]. In freshly-isolated rat hepatocytes, 2-APB inhibited thapsigargin- and vasopressin-stimulated Ca2+ inflow (measured using fura-2) with no detectable effect on the release of Ca2+ from intracellular stores. The concentration of 2-APB which gave half-maximal inhibition of Ca2+ inflow was approx. 10 microM. 2-APB also inhibited Ca2+ inflow initiated by a low concentration of adenophostin A but had no effect on maitotoxin-stimulated Ca2+ inflow through non-selective cation channels. The onset of the inhibitory effect of 2-APB on thapsigargin-stimulated Ca2+ inflow was rapid. When 2-APB was added to rat hepatocytes in the presence of extracellular Ca2+ after a vasopressin-induced plateau in the cytoplasmic free Ca2+ concentration ([Ca2+](cyt)) had been established, the kinetics of the decrease in [Ca2+](cyt) were identical with those induced by the addition of 50 microM Gd(3+) (gadolinium). 2-APB did not inhibit the release of Ca2+ from intracellular stores induced by the addition of Ins(1,4,5)P(3) to permeabilized hepatocytes. In the H4-IIE rat hepatoma cell line, 2-APB inhibited thapsigargin-stimulated Ca2+ inflow (measured using fura-2) and, in whole-cell patch-clamp experiments, the Ins(1,4,5)P(3)-induced inward current carried by Ca2+. It was concluded that, in liver cells, 2-APB inhibited SOCs through a mechanism which involved the binding of 2-APB to either the channel protein or an associated regulatory protein. 2-APB appeared to be a novel inhibitor of SOCs in liver cells with a mechanism of action which, in this cell type, is unlikely to involve an interaction of 2-APB with Ins(1,4,5)P(3) receptors. The need for caution in the use of 2-APB as a probe for the involvement of Ins(1,4,5)P(3) receptors in the activation of SOCs in other cell types is briefly discussed.

Topics: Animals; Boron Compounds; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Membrane Permeability; Cells, Cultured; Drug Interactions; Endoplasmic Reticulum; Fura-2; Inositol 1,4,5-Trisphosphate Receptors; Liver; Rats; Receptors, Cytoplasmic and Nuclear; Thapsigargin; Vasopressins