Cholesterol-mediated membrane surface area dynamics in neuroendocrine cells

How cholesterol, a key membrane constituent, affects membrane surface area dynamics in secretory cells is unclear. Using methyl-β-cyclodextrin (MβCD) to deplete cholesterol, we imaged melanotrophs from male Wistar rats in real-time and monitored membrane capacitance (Cm), fluctuations of which reflect exocytosis and endocytosis. Treatment with MβCD reduced cellular cholesterol and caused a dose-dependent attenuation of the Ca2+-evoked increase in Cm(IC50 =5.3 mM) vs. untreated cells. Cytosol dialysis of MβCD enhanced the attenuation of Cm increase (IC50=3.3 mM), suggesting cholesterol depletion at intracellular membrane sites was involved in attenuating exocytosis. Acute extracellular application of MβCD resulted in an immediate Cm decline, which correlated well with the cellular surface area decrease, indicating the involvement of cholesterol in the regulation of membrane surface area dynamics. This decline in Cm was three-fold slower than MβCD-mediated fluorescent cholesterol decay, implying that exocytosis is the likely physiological means for plasma membrane cholesterol replenishment. MβCD had no effect on the specific Cm and the blockade of endocytosis by Dyngo 4a, confirmed by inhibition of dextran uptake, also had no effect on the time-course of MβCD-induced Cm decline. Thus acute exposure to MβCD evokes a Cm decline linked to the removal of membrane cholesterol, which cannot be compensated for by exocytosis. We propose that the primary contribution of cholesterol to surface area dynamics is via its role in regulated exocytosis.