Calcium Signaling Is Involved in EthanolInduced Volume Decrease and Gap Junction Closure in Cultured Rat Gastric Mucosal Cells

Abstract

Ethanol is a well-established "barrier breaker" in gastric mucosa, but its detailed effects at the cellular level remain unclear. We have previously shown that the intracellular free calcium concentration is increased, gap junctions are closed, and cell volume is decreased after exposure to 5% (v/v) ethanol in primarily cultured rabbit gastric epithelial cells. Rat gastric mucosal (RGM) cells were grown to confluence on a coverslip or on a filter membrane. Gap junctional diffusion was measured in 5-carboxyfluorescein-loaded cells by bleaching a small area with a laser and measuring the recovery with confocal microscope. Intracellular calcium was measured spectrofluorometrically in fura-2-loaded cells. For cell volume measurements the cell monolayer was loaded with calcein and imaged along the Z-axis with a confocal microscope. The changes in fluorescence intensity were intercepted as a measure of cell volume change. TMB-8 was used to inhibit intracellular calcium release and lanthanum to block plasma membrane calcium selective ion channels, while BABTA served as an intracellular calcium chelating agent. Results showed that ethanol (7.5%, v/v) exposure increased intracellular calcium from 69 +/- 7 to 142 +/- 11 nM (N = 5; P < 0.05), decreased cell volume by -23 +/- 5% (N = 8; P < 0.05), and induced gap junction closure (fluorescence recovery from 37 +/- 9 to 15 +/- 3%; N = 6; P < 0.05). A serosal potassium channel blocker, quinine, almost completely prevented the ethanol-induced cell volume decrease (from -23 +/- 5 to -3 +/- 3%), suggesting that opening of basolateral potassium channels underlies cell shrinkage. BABTA inhibited completely (from 35 +/- 3 to 39 +/- 4 nM; N = 6; P < 0.05), and TMB-8 + lanthanum partially (from 60 +/- 6 to 92 +/- 12 nM; N = 6; P < 0.05), the ethanol-induced intracellular calcium increase. BABTA also abolished the ethanol-induced volume decrease (from -23 +/- 5 to 1 +/- 4%; N = 6; P < 0.05), while TMB-8 + lanthanum had a lesser effect on it (from -23 +/- 5 to -11 +/- 3%; N = 9; P < 0.05). They also abolished the closure of gap junctions induced by ethanol (fluorescence recovery, 38 +/- 5% for BABTA and 30 +/- 4% for TMB-8 + lanthanum). We conclude that luminal ethanol opens basolateral calcium-dependent potassium selective channels with resultant shrinkage of the cells and blocks the intercellular gap junctions. These actions are mediated by intracellular calcium signaling.

Ethanol induced NF-κB activation protects against cell injury in cultured rat gastric mucosal epithelium

“…membrane structure. We have previously shown that the plasma membrane first becomes permeable to small marker molecules and thereafter to larger molecules, such as LDH, during the first minutes of ethanol exposure, probably demonstrating a direct fluidizing influence of ethanol on plasma membranes (17). The present findings indicate that during exposure to low concentrations of ethanol (1-5%), cell membrane damage also increases dose-dependently in a heat shockpretreated epithelium.…”

“…We have recently shown that ethanol exposure moderately increases intracellular free calcium concentration in primary cultured rabbit gastric surface epithelial cells (18) and in immortal RGM-1 cells (17). In these studies, inhibition of calcium signaling either totally with BAPTA or partially with specific inhibitors [3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester to inhibit intracellular calcium release and lanthanum to block plasma membrane calcium channels] also inhibited the target actions under investigation in these studies, i.e., opening of basolateral potassium ion channels with resultant cell volume shrinkage and closure of gap-junctional channels.…”

Taurocholate Potentiates Ethanol-Induced NF-κB Activation and Inhibits Caspase-3 Activity in Cultured Rat Gastric Mucosal Cells

“…Indirect mechanism of EtOH-induced damage was also suggested in our previous studies, as EtOH-induced calcium signaling led to opening of basolateral potassium channels and shrinkage of cell volume [16,17].…”

Analysis of gap junctional intercellular communications using a dielectrophoresis-based microchip

“…Then, a mixture of cells (10 4 cells from each population) was prepared and loaded into the DEPArrayª cartridge to follow the dye coupling. Some experiments were carried out in the presence of 2 mM ethanol, a conventional inhibitor of GJIC (Chiba et al, 1994, Dillon et al, 2013, Mustonen et al, 2005, Noritake, et al, 2015.…”