VOLUME-SENSITIVE CA INFLUX AND RELEASE FROM INTRACELLULAR POOLS IN GASTRIC PARIETAL-CELLS

The effects of osmotically induced changes in cell volume on cytoplasmic free Ca (Ca(i)) were studied in parietal cells from intact rabbit gastric glands using digital image processing of fura-2 fluorescence. In resting unstimulated cells, Ca(i) was unaffected by either cell swelling or shrinking when osmolarity was varied between 200 and 400 mosM (isotonicity 290 mosM). However, when cells were swelled in a 165 mosM solution (55% tonicity), a biphasic Ca increase was observed. On average, Ca(i) increased transiently from 80 to 218 nM before stabilizing at approximately 140 nM. The peak was due to release from intracellular pools because it was present in Ca-free solutions while the sustained elevation was dependent on external Ca. In carbachol-stimulated cells, Ca influx was most sensitive to cell shrinkage. For example, addition of 25 mM sucrose (108% tonicity) caused a 30% decrease in the sustained carbachol-stimulated Ca(i) increase (plateau). In contrast, carbachol-stimulated cells were relatively insensitive to cell swelling, with a 30% decrease in tonicity causing only a 15% increase in the plateau. However, as in the unstimulated cells, extreme (55% tonicity) swelling caused additional increases in Ca(i) levels. The carbachol-dependent effects of changes in cell volume on Ca(i) could be mimicked by treating cells with thapsigargin, an inhibitor of Ca pumps of intracellular membranes that also has been shown to stimulate Ca entry. Thus, although extreme swelling conditions (55% tonicity) could elicit Ca(i) increases in either the presence or absence of agonist, agonist was required to observe Ca(i) decreases due to cell shrinkage. The steep sensitivity of the agonist-stimulated Ca(i) plateau to elevated osmolarity suggests that cell shrinkage may play an important role in regulating Ca influx during stimulus-secretion coupling.