Abstracts
1998 Digestive Disease Week
#1069
CALCIUM ACCENTUATES GASTRIC INJURY INDUCED BY ETHANOL. E.R. Kokoska, G.S. Smith, T.A.Miller, Saint Louis University Health Sciences Center. St. Louis, MO.
The mechanism(s) by which ethanol (Et) induces cellular cytotoxicity is poorly understood. Our objective was to determine the role of calcium (Ca++) in gastric mucosal injury elicited by Et (%=v/v). Changes in [Ca++]i (nM) and cell mortality (relative fluorescence) were measured in human gastric cells (AGS) with Fluo-3 (5µM) and Ethidium homodimer-2 (5µM), respectively. In separate studies, extracellular Ca++ influx was evaluated with manganese (Mn++) uptake as monitored by the quenching of Fluo-3 fluorescence. Apical to basolateral clearance (pl/min/cm2) of Texas Red Albumin (Alb) was also determined. Injury induced by Et (5 to 16%) was concentration dependent and preceded in time by Ca++ accumulation (R2=0.79, p<0.01). Studies assessing Mn++ entry suggested that Et initially caused Ca++ release from internal stores followed by sustained extracellular Ca++ influx. Sustained Ca++ influx and Mn++ entry were both blocked with the store operated Ca++ channel (SOCC) blocker lanthanum (La+++; 25 µM) but not the voltage operated Ca++ channel blockers verapamil (20 µM) or nifedepine (10 µM). Cells were then treated with 12% Et for 10 min. either following La+++ pretreatment (+La+++) or with Ca++ free buffer (-[Ca++]e) to determine the role of extracellular Ca++ in inducing injury. To investigate the role of intracellular Ca++, separate cells were pretreated with thapsigargin (+Thap; 500 nM) to deplete intracellular Ca++ stores and subsequently exposed to 12% Et in -[Ca++]e for 10 min. Results are shown below as mean ± SEM (n=6-12/group).
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Control
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12% Et
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+La+++
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-[Ca++]e
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+Thap/-[Ca++]e
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[Ca++]i
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104 ± 7
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446 ± 24*
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234 ± 9*_
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216 ± 10*_
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134 ± 5*_§
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Injury
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410 ± 5
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789 ± 17*
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603 ± 14*_
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589 ± 11*_
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534 ± 7*_§
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Clearance
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21 ± 1
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102 ± 3*
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62 ± 5*_
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53 ± 4*_
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35 ± 4*_§
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12% Et increased [Ca++]i, cellular injury, and Alb clearance (*p<0.01). These viability and permeability changes were attenuated when extracellular Ca++ influx was prevented (_p<0.01) and further decreased when intracellular Ca++ stores were depleted (§p<0.01). However, adding supplemental Ca++ to buffer (up to 5 mM) did not increase the observed changes in [Ca++]i, injury, or clearance induced by 12% Et. These data suggest that intracellular Ca++ accumulation induced by Et is a result of initial Ca++ release from internal stores followed by sustained extracellular Ca++ influx through SOCCs. Furthermore, Et induced gastric mucosal injury, under in vitro conditions, appears to be, at least in part, mediated by Ca++ accumulation.
Supported by NIH Grant DK25838.
Copyright 1996 - 1998, SSAT, Inc. Revised 29 June 1998.
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