An important role of plasma membrane is to maintain flexible barrier between intracellular and extracellular space. Cells undergo volume changes associated with secretion of material stored in secretory vesicles. Swelling of secretory vesicles precedes exocytosis, so we hypothesized, that cell swelling stimulated exocytosis is induced and mediated by direct biophysical effect of hypotonicity. Our previous results showed, that in contrast to natural ß-cells and INS-1 cell line, INS-1E cells do not release insulin in response to cell swelling in spite of good response to glucose stimulation. One of possible explanations of this phenomenon is a special role of calcium; surprisingly, perifusion with Ca²⁺-depleted medium showed distinct secretory response of INS-1E cells to hypotonicity while that of INS-1 cells was partially inhibited. To analyze the mechanism, we tested the effect of blockers of Ca²⁺ channels on hypotonicity-induced insulin secretion: they did not prevent stimulation of secretion from isolated pancreatic islets or INS-1 cells, secretion from INS-1E cells appeared. Tetanus toxin, a metalloprotease inactivating soluble SNARE proteins, in presence of Ca²⁺ blocks hypotonicity-induced secretion from pancreatic islets. Unexpectedly, tetanus toxin in presence of calcium and Ca²⁺ channel blockers (nifedipine, w-agatoxin and mibefradil) did not prevent swelling-induced insulin secretion from INS-1E cells. Another participating mechanism of special behavior of INS-1E cells may be a change of membrane fluidity dependent on cholesterol content; INS-1E cells have significantly higher cholesterol content than INS-1 cells at basal condition. To define the role of membrane cholesterol in glucose- and swelling-induced insulin secretion from isolated Langerhans islets and insulinoma cell lines INS-1 and INS-1E we added 2-hydroxypropyl-ß-cyclodextrin or carboxymethyl-ß-cyclodextrin (agents removing membrane cholesterol) to preincubation medium (2 hours). Glucose- and hypotonicity- induced insulin secretion from freshly isolated rat Langerhans islets was prevented by preincubation with ß-cyclodextrins (2% w/v). Both glucose and swelling stimulated insulin secretion were not changed in insulinoma cell lines at this concentration. Higher concentration of ß-cyclodextrins inhibited swelling-induced insulin secretion from INS-1, but stimulated secretion from INS-1E cells. These data demonstrated different sensitivity of secretory mechanism to changes of cholesterol content in two tumor cell lines and pancreatic islets.

Conclusion: 

Mechanism of exocytosis from INS-1E seems to differ from that in pancreatic islets or INS-1 cells. Ca²⁺ and membrane cholesterol participate in the mechanism of this difference.

Supported by: 2/0094/09 (VEGA), APVV 0235-06, APVV VVCE-0064-07, APVV RPEU 0007-06 and CE SAV CENDO.