Purinergic receptor-mediated intracellular Ca2+ oscillations in chicken granulosa cells

Abstract

These studies were designed to investigate the effects of extracellular ATP on intracellular calcium ion concentration ([Ca2+]i) and progesterone secretion in granulosa cells obtained from the two largest preovulatory follicles (F1 and F2) of hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. The resting [Ca2+]i in these cells was 99 +/- 7 nM (n = 22). There was a 5.7 +/- 0.7-fold increase in [Ca2+]i in all (n = 140) of the cells within 5 sec of adding a maximally stimulatory concentration (100 microM) of extracellular ATP. The initial spike was followed by [Ca2+]i oscillations that returned to the resting level between spikes. The frequency and amplitude of the [Ca2+]i oscillations were varied and persisted for 1-40 min. [Ca2+]i oscillations were also triggered by 100 microM UTP, UDP, GTP, GDP, ADP, and the nonhydrolyzable analog ATP gamma S. Adenosine, AMP, GMP, and UMP (all at 100 microM) were ineffective. The lowest ATP concentration to trigger a [Ca2+]i response was 1 microM. The sustained oscillatory phase of the response, but not the initial spike, was inhibited by incubating the cells in Ca(2+)-free medium containing 2 mM EGTA. The nucleotide-triggered [Ca2+]i oscillations were not affected by adding the dihydropyridine Ca2+ channel blockers verapamil (100 microM), methoxy-verapamil (D600; 100 microM), or nifedipine (10 microM), before or during the response. However, the oscillations, but not the initial spike, were prevented by pretreating the cells with a general Ca2+ channel blocker, lanthanum (1 mM) or cobalt (5 mM). Lanthanum and cobalt also promptly stopped the [Ca2+]i oscillations when added during the oscillatory phase. The nucleotide-triggered [Ca2+]i response was also abolished by pretreating the cells with an inhibitor of inositol phospholipid hydrolysis, neomycin (1.5 mM). In 3-h incubations, adenosine (100 microM) or ATP (100 microM) did not affect basal or LH (20 or 100 ng/ml)-stimulated progesterone production. These studies demonstrate that chicken granulosa cells display P2 purinergic receptors on their surfaces. Activation of these receptors triggers [Ca2+]i oscillations that follow the release of Ca2+ from internal stores and depend on Ca2+ influx through dihydropyridine-insensitive Ca2+ channels. The physiological function(s) of P2 purinergic receptors on granulosa cells is not known.