title: Cholesterol-mediated membrane surface area dynamics in neuroendocrine cells.
abstract: How cholesterol, a key membrane constituent, affects membrane surface area dynamics in secretory cells is unclear. Using methyl-beta-cyclodextrin (MbetaCD) to deplete cholesterol, we imaged melanotrophs from male Wistar rats in real-time and monitored membrane capacitance (C(m)), fluctuations of which reflect exocytosis and endocytosis. Treatment with MbetaCD reduced cellular cholesterol and caused a dose-dependent attenuation of the Ca(2+)-evoked increase in C(m) (IC50 = 5.3 mM) vs. untreated cells. Cytosol dialysis of MbetaCD enhanced the attenuation of C(m) increase (IC50 = 3.3 mM), suggesting cholesterol depletion at intracellular membrane sites was involved in attenuating exocytosis. Acute extracellular application of MbetaCD resulted in an immediate C(m) 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 C(m) was three-fold slower than MbetaCD-mediated fluorescent cholesterol decay, implying that exocytosis is the likely physiological means for plasma membrane cholesterol replenishment. MbetaCD had no effect on the specific C(m) and the blockade of endocytosis by Dyngo 4a, confirmed by inhibition of dextran uptake, also had no effect on the time-course of MbetaCD-induced C(m) decline. Thus acute exposure to MbetaCD evokes a C(m) 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.
authors.affiliation: Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, Zaloska 4, 1000 Ljubljana, Slovenia.
authors.forename: Bostjan
authors.initials: B
authors.lastname: Rituper
authors.forename: Helena Haque
authors.initials: HH
authors.lastname: Chowdhury
authors.forename: Jernej
authors.initials: J
authors.lastname: Jorgacevski
authors.forename: Jens R
authors.initials: JR
authors.lastname: Coorssen
authors.forename: Marko
authors.initials: M
authors.lastname: Kreft
authors.forename: Robert
authors.initials: R
authors.lastname: Zorec
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D002784:Cholesterol / Q000032:analysis* / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D034281:Dynamins / Q000378:metabolism; D004705:Endocytosis; D005089:Exocytosis; D008297:Male; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism; D008564:Membrane Potentials; D055099:Neuroendocrine Cells / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D047392:beta-Cyclodextrins / Q000378:metabolism
chemical_list: D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol; D034281:Dynamins