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Activation of Cl^– Channels by Human Chorionic Gonadotropin in Luteinized Granulosa Cells of the Human Ovary Modulates Progesterone Biosynthesis

Centro de Estudios Moleculares de la Célula and Instituto de Ciencias Biomédicas (P.O., E.L.-S., L.D., A.S.), Facultad de Medicina, Universidad de Chile, 838-0453 Santiago, Chile; Centro de Investigación en Biología Reproductiva (P.O.), Escuela de Medicina, Universidad de Valparaíso, 236-3070 Valparaíso, Chile; and Instituto de Investigaciones Materno Infantil (IDIMI) (L.D.), Hospital San Borja Arriarán and Departamento de Obstetricia y Ginecología, Universidad de Chile, 836-0160 Santiago, Chile

Address all correspondence and requests for reprints to: Andrés Stutzin Centro de Estudios Moleculares de la Célula, Facultad de Medicina Universidad de Chile, 838-0453 Independencia, Santiago, Chile. E-mail: astutzin{at}bitmed.med.uchile.cl.

Chloride permeability pathways and progesterone (P4) secretion elicited by human chorionic gonadotropin (hCG) in human granulosa cells were studied by electrophysiological techniques and single-cell volume, membrane potential and Ca²⁺_i measurements. Reduction in extracellular Cl^– and equimolar substitution by the membrane-impermeant anions glutamate or gluconate significantly increased hCG-stimulated P4 accumulation. A similar result was achieved by exposing the cells to hCG in the presence of a hypotonic extracellular solution. Conversely, P4 accumulation was drastically reduced in cells challenged with hCG exposed to a hypertonic solution. Furthermore, conventional Cl^– channel inhibitors abolished hCG-mediated P4 secretion. In contrast, 25-hydroxycholesterol-mediated P4 accumulation was unaffected by Cl^– channel blockers. In human granulosa cells, hCG triggered the activation of a tamoxifen-sensitive outwardly rectifying Cl^– current comparable to the volume-sensitive outwardly rectifying Cl^– current. Exposure of human granulosa cells to hCG induced a rapid 4,4'-diisothiocyanatostilbene-2,2-disulphonic acid-sensitive cell membrane depolarization that was paralleled with an approximately 20% decrease in cell volume. Treatment with hCG evoked oscillatory and nonoscillatory intracellular Ca²⁺ signals in human granulosa cells. Extracellular Ca²⁺ removal and 4,4'-diisothiocyanatostilbene-2,2-disulphonic acid abolished the nonoscillatory component while leaving the Ca²⁺ oscillations unaffected. It is concluded that human granulosa cells express functional the volume-sensitive outwardly rectifying Cl^– channels that are activated by hCG, which are critical for plasma membrane potential changes, Ca²⁺ influx, and P4 production.