Regulation of membrane currents in chicken ovarian granulosa cells
Abstract
Ion channels play an important role in the regulation of granulosa cell functions. Whole-cell patch clamp technique was used to study membrane currents in granulosa cells. In freshly isolated differentiated granulosa cells a fast activating and inactivating outward current carried by Cl− could be activated by removal of bath Ca2+. It could not be activated by Ca2+ channel blocker cobalt or by the inclusion of EGTA or BAPTA in intracellular solution, suggesting Ca2+ act at the external side of the plasma membrane. Differentiated granulosa cells cultured on intact basal lamina for 24 hours had smaller membrane capacitance than that on plastic. A slowly activating and inactivating inward current showed to be a Ca2+-activated Cl− current. Its average amplitude and current density in cells cultured on basal lamina were significantly lower than that on plastic. The density of an outward delayed rectifier K+ current was not significantly influenced by basal lamina, thus the effect of basal lamina on membrane currents is specific. Differentiated granulosa cells cultured for 24 hours expressed both T and L-type Ca2+ currents. The average amplitude and current density of both Ca2+ currents in cells cultured on basal lamina were also decreased. In addition, fibronectin, laminin and collagen IV exhibited similar effects on Ca2+ currents as basal lamina. Cycloheximide and actinomycin D completely inhibited the expression of Ca2+-activated Cl− current without significant effect on T and L-type Ca2+ currents. Basal lamina stored for 12 months and solubilized form of basal had similar effects on membrane currents. Intracellular MgATP kept the delayed rectifier K+ current activating and intracellular NaATP activated a current, possibly a cation current in differentiated granulosa cells. Estradiol and progesterone did not acutely influence membrane currents in granulosa cells at various developmental stages. Extracellular ATP, GnRH, arginine vasopressin, PGE2 and PGF2α also had no obvious acute effects on membrane currents in differentiated granulosa cells. Free oxygen radicals suppressed delayed rectifier K+ current in differentiated granulosa cells. Based on the discovery by this study, a proposal for future research was raised to study the effects of basal lamina on Ca2+ image in granulosa cells.
Degree
Ph.D.
Advisors
Asem, Purdue University.
Subject Area
Anatomy & physiology|Animals|Cellular biology
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