Role of chloride ions in progesterone production by chicken granulosa cells

Abstract

The importance of chloride ions in luteinizing hormone (LH)-stimulated progesterone production by chicken granulosa cells from the two largest preovulatory follicles was investigated in vitro. Reduction of the extracellular chloride concentration from 147.8 mM to 2.8 mM, by substitution with equimolar concentrations of non-permeant glutamate and aspartate, inhibited the ability of LH to stimulate progesterone production and cAMP accumulation during a 4 h incubation. LH-stimulated granulosa cell progesterone production was also suppressed in a concentration-dependent manner by the chloride channel blockers 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonic acid (SITS; 10 −8−5 × 10 −5 M) or 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS; 10 −8−5 × 10 −5 M). The inhibitory effect was observed within 30 min of the addition of the blockers and was irreversible. DIDS appeared to act at a site(s) proximal to the generation of cAMP, since concentrations of DIDS (10 −8−10 −6 M) which inhibited LH- and human chorionic gonadotropin-stimulated progesterone production, did not affect progesterone production stimulated by dibutyryl cAMP, 8-bromo cAMP or forskolin. In addition, concentrations of DIDS (10 −8−10 −6 M) which attenuated LH-stimulated progesterone production also reduced the accumulation of extracellular cAMP. These studies suggest that chloride ions may play an important role in the stimulatory action of LH on chicken granulosa cell progesterone production.