Stimulation and inhibition of secretion in Paramecium: role of divalent cations.

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Abstract
The effects of Ca2+ and Mg2+ on exotytosis in P. tetraurelia cells were examined with light microscopy, freeze fracture (FEM) and transmission electron microscopy (TEM) of thin-sectioned embedded cells. Picric acid-Ca2+ induced secretion in wild type (wt) cells were captured by quick fixation with OsO4, and TEM demonstrated membrane fusion occurring before trichocyst matrix (tmx) expansion. Cells stimulated with picric acid in the presence of high extracellular Mg2+ showed very few sites of membrane fusion and no tmx expansion, suggesting that Ca2+ is required for membrane fusion and tmx expansion. Further information was obtained by comparing secretory responses of wt cells with a temperature-sensitive secretory mutant, nd 9. These cells, when grown at the permissive temperature (18.degree. C), possess normal rosettes at the secretory site and secrete in response to picric acid-Ca2+, but when grown at 27.degree. C they lack rosettes and do not secrete. Quantitation of picric acid-Ca2+-induced secretion revealed that: the number of tmx secreted by wt and nd 9 cells was independent of their cultural growth phase, wt cells secreted the same number of tmx when grown either at 18.degree. or 27.degree. C, and nd 9 18.degree. C cells secreted the same number of tmx as wt 18.degree. or 27.degree. C cells. Wild type and nd 9 cells had the same frequencies of occupied and unoccupied secretory sites as determined by quantitative analyses of freeze-fracture replicas. After stimulation with divalent cation ionophore A23187 [calcimycin] and Ca2+, wt cells showed a significant reduction in the frequency of occupied sites. FEM and TEM studies revealed that A23187-Ca2+ induced tmx expansion and normal fusion of the plasma and trichocyst membranes in wt and nd 9 18.degree. C cells, but induced tmx expansion without concomitant membrane fusion in nd 9 27.degree. C cells. The lack of membrane fusion in nd 9 27.degree. C cells suggests that the molecules represented by rosette particles are required specifically for membrane fusion.