Increased cytosolic calcium stimulates exocytosis in bovine lactotrophs: direct evidence from changes in membrane capacitance

Abstract

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 µF/cm². Discrete C_m steps of 2-10 ƒF were recorded, which most likely reflect single fusion and retrieval events of prolactin-containing granules (0.2-0.6 µm in diameter). High Ca ²⁺ 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 Ca⁺ through the membrane. This is supported by the following findings: addition of Co ²⁺ 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 Ca ²⁺ influx is proposed; this is shown to provide adequate control of basal and triggered secretion monitored by C_m.