Somatostatin release from dispersed hypothalamic cells - effects of membrane depolarization, calcium and glucose...

We have developed a new short term in vitro system to examine hypothalamic somatostatin (SRIF) release. Hypothalamic cells were obtained from normal rats after trypsin or collagenase aided dispersion and released immuno-reactive (IR) SRIF which eluted in 3 molecular weight (MW) forms on gel chromatography. The smallest MW form, which constituted the major peak, co-eluted with synthetic cyclic 1-14 SRIF on gel and reverse phase high pressure liquid chromatography (HPLC). After 24 h in culture in medium containing heat inactivated fetal calf serum, cell viability was demonstrated by two techniques, (1) vital staining with trypan blue, and (2) incorporation of 32Pi into phospholipids. SRIF release was also studied at this time which was optimal in terms of responsivity of the cells to depolarizing stimuli. SRIF release increased in a time dependent manner, over 3 h. Membrane depolarization, induced either by potassium chloride 56 mM or ouabain (the Na+, K+-ATPase inhibitor) 10(-6) M or greater, markedly stimulated SRIF release. Incubation at 4 degrees C, or in the presence of EDTA 0.05 M or verapamil, the calcium channel blocker, 50 microM abolished these stimulatory effects. Glucose deprivation was induced by the addition of 2-deoxy-D-glucose (2-DG) to the experimental medium. 2-DG, at concentrations of up to 200 mg%, had no significant effect on SRIF release during incubation periods of up to 1 h.