INCREASED CYTOSOLIC CALCIUM STIMULATES EXOCYTOSIS IN BOVINE LACTOTROPHS - DIRECT EVIDENCE FROM CHANGES IN MEMBRANE CAPACITANCE

The patch-clamp technique has been used to measure changes in membrane capacitance (C(m)) of bovine lactotrophs in order to monitor fluctuations in cell surface area associated with exo- and endocytosis. Cells were prepared by an enrichment procedure and cultured for up to 14 d before use. Under whole-cell recording, cell cytoplasm was dialyzed with various Ca2+-containing solutions. The resting C(m) of 6.05 +/- 1.68 pF was found to correlate well with squared cell radius, suggesting a specific C(m) of 0.8-mu-F/cm2. Discrete C(m) steps of 2-10 fF were recorded, which most likely reflect single fusion and retrieval events of prolactin-containing granules (0.2-0.6-mu-m in diameter). High Ca2+ resulted in a C(m) increase of 20-50% from the resting value, demonstrating a role for [Ca2+]i in stimulus-secretion coupling. Spontaneous C(m) changes have also been recorded, which presumably reflect prolactin secretion supported by a tonic influx of Ca2+ through the membrane. This is supported by the following findings: addition of Co2+ diminished or reversed the spontaneous C(m) changes and decreased resting [Ca2+]i; and membrane depolarization increased C(m), indicating the role of voltage-activated channels in stimulus-secretion coupling. As bovine lactotrophs have been found to be largely devoid of spontaneous electrical activity, a mechanism involving modulation of a tonic Ca2+ influx is proposed; this is shown to provide adequate control of basal and triggered secretion monitored by C(m).