When 10(-5) M carbachol was added to parotid tissue slices incubated in buffer containing Ca++, watery vacuoles were formed in the cells. The percent volume density of vacuoles, as measured from 0.5-micron sections, increased from 0.64 +/- 0.15 SE (n = 7) to 3.09 +/- 0.99 (n = 5) in 10 min and, finally, to 7.27 +/- 1.88 (n = 4) in 30 min. In electron micrographs, most of the vacuoles appeared to arise from a location near the Golgi apparatus. Condensation of nuclear chromatin and a conformational change in mitochondria were also noted immediately after stimulation. The percent volume density values returned to basal levels with the addition of either 5 mM EGTA or 10(-6) M atropine after the addition of carbachol. Nuclei and mitochondria returned to normal configurations. In the presence of either 1 mM ouabain or high K+, or in the absence of added Ca++, carbachol failed to induce vacuole formation. However, low Na+ medium did not prevent the formation of vacuoles due to carbachol. Ultrastructural changes in nuclei and mitochondria were consistently associated with the appearance of vacuoles. Since both high K+ and ouabain blocked vacuole formation, it is unlikely that Na+ or K+ movements were important for the response. Rather, receptor-activated Ca++ influx, which is likely to be inhibited by depolarizing agents (such as high K+ or ouabain), is probably the more important factor in vacuole formation and other concomitant ultrastructural changes.

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