The Lipid Products of Phosphoinositide 3-Kinase Contribute to Regulation of Cholangiocyte ATP and Chloride Transport

Abstract

ATP stimulates Cl؊ secretion and bile formation by activation of purinergic receptors in the apical membrane of cholangiocytes. The purpose of these studies was to determine the cellular origin of biliary ATP and to assess the regulatory pathways involved in its release. In Mz-Cha-1 human cholangiocarcinoma cells, increases in cell volume were followed by increases in phophoinositide (PI) 3-kinase activity, ATP release, and membrane Cl ؊ permeability. PI 3-kinase signaling appears to play a regulatory role because ATP release was inhibited by wortmannin or LY294002 and because volume-sensitive current activation was inhibited by intracellular dialysis with antibodies to the 110 kDa-subunit of PI 3-kinase. Similarly, in intact normal rat cholangiocyte monolayers, increases in cell volume stimulated luminal Cl ؊ secretion through a wortmannin-sensitive pathway. To assess the role of PI 3-kinase more directly, cells were dialyzed with the synthetic lipid products of PI 3-kinase. Intracellular delivery of phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate activated Cl ؊ currents analogous to those observed following cell swelling. Taken together, these findings indicate that volume-sensitive activation of PI 3-kinase and the generation of lipid messengers modulate cholangiocyte ATP release, Cl ؊ secretion, and, hence, bile formation.

Regulation of volume-regulated outwardly rectifying anion channels by phosphatidylinositol 3,4,5-trisphosphate in mouse ventricular cells

“…Similar results were obtained in cells loaded with another PI3K inhibitor, wortmannin (300 nM) (data not shown). These results are in agreement with the previous studies, in which the effect of these inhibitors on VRAC current was examined in rabbit ventricular cells (5,26), cultured smooth muscle cells (33) and hepatoma cells (11,12 (8), and PIP3 dic8 (Echelon Biosciences) (4) were added to the pipette solution. PIP2 and PIP3 were directly dissolved in the control pipette solution at a concentration of 10 μM (4,8).…”

“…In addition, although PIP3 alone without osmotic stimulation could not induce VRAC current in our study (Fig. 2C), a spontaneous activation of VRAC-like current was observed in cultured epithelial cells derived from intrahepatic bile duct of the rat when they were dialyzed with PIP3 under isotonic conditions (11). The reasons for these discrepancies are unknown.…”

“…PI3K/reactive oxygen species (ROS) (26) or PI3K/serum-and glucocorticoid-inducible kinase (SGK) (33) cascade, to activate VRAC. However, the role of PIs, which should strongly depend on PI3K activity, in the activation process of VRAC is not necessarily clear (11,18). In the present study, we examined the effect of intracellular PIs (PIP2 and PIP3) on VRAC current in mouse cardiac cells, in an attempt to elucidate the role of these compounds in VRAC activation.…”

“…The modified KB solution for cell storage contained (mM): 70 potassium glutamate, 20 KCl, 1.0 MgCl 2 , 10 KH 2 PO 4 , 10 taurine, 10 EGTA, 10 glucose, 0.1% albumin, 10 β-hydroxybutric acid and 10 Hepes; pH 7.2 with KOH; 300 mOsm with mannitol. For VRAC currents recording, the isotonic extracellular solutions (ISO) contained (mM): 80 NaCl, 20 TEA-Cl, 1.0 MgCl 2 , 0.5 CaCl 2 , 0.5 BaCl 2 , 0.5 CdCl 2 , 0.01 GdCl 3 , 5.5 glucose, 10 HEPES, 0.01 nicardipine; pH 7.4 with CsOH: 310 mOsm with PI3K (11,20,33), and that the inhibition of PI3K attenuates VRAC current in several cell types (5,12,28,32,33). These results may indicate the involvement of PI3K in the activation of VRAC, while the activated PI3K may trigger some downstream signaling cascades, e.g.…”

Ca2+-Evoked Serotonin Secretion by Parafollicular Cells: Roles in Signal Transduction of Phosphatidylinositol 3′-kinase, and the γ and ζ Isoforms of Protein Kinase C

“…It has been suggested that the CaR may enable neurons to respond to changes in the extracellular concentration of Ca 2ϩ and, by activating Ca 2ϩ -dependent Cl Ϫ channels, to participate in regulating their Cl Ϫ equilibrium potential (Brown et al, 1993). The lipid products of PI3Ј-kinase contribute to regulation of Cl Ϫ transport (Feranchak et al, 1999), and we have found that the CaR activates PI3Ј-kinase. Figure 6.…”