PURINERGIC RECEPTOR-MEDIATED INTRACELLULAR CA2-CELLS( OSCILLATIONS INCHICKEN GRANULOSA)

These studies were designed to investigate the effects of extracellula r ATP on intracellular calcium ion concentration ([Ca2+](i)) and proge sterone secretion in granulosa cells obtained from the two largest pre ovulatory follicles (F-1 and F-2) of hens. [Ca2+](i) was measured in c ells loaded with the Ca2+-responsive fluorescent dye fura-2. The resti ng [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 withi n 5 sec of adding a maximally stimulatory concentration (100 mu M) of extracellular ATP. The initial spike was followed by [Ca2+](i) oscilla tions 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 mu M UTP, UDP, GTP, GDP, ADP, and the nonhydrolyzable analog ATP gamma S. A denosine, AMP, GMP, and UMP (all at 100 mu M) were ineffective. The lo west ATP concentration to trigger a [Ca2+](i) response was 1 mu M. The sustained oscillatory phase of the response, but not the initial spik e, was inhibited by incubating the cells in Ca2+-free medium containin g 2 mM EGTA. The nucleotide-triggered [Ca2+](i) oscillations were not affected by adding the dihydropyridine Ca2+ channel blockers verapamil (100 mu M), methoxy-verapamil (D600; 100 mu M), or nifedipine (10 mu M), before or during the response. However, the oscillations, but not the initial spike, were prevented by pretreating the cells with a gene ral Ca2+ channel blocker, lanthanum (1 mM) or cobalt (5 mM). Lanthanum and cobalt also promptly stopped the [Ca2+](i) oscillations when adde d during the oscillatory phase. The nucleotide-triggered [Ca2+](i) res ponse was also abolished by pretreating the cells with an inhibitor of inositol phospholipid hydrolysis, neomycin (1.5 mM). In 3-h incubatio ns, adenosine (100 mu M) or ATP (100 mu M) did not affect basal or LH (20 or 100 ng/ml)stimulated progesterone production. These studies dem onstrate that chicken granulosa cells display P-2 purinergic receptors on their surfaces. Activation of these receptors triggers [Ca2+](i) o scillations that follow the release of Ca2+ from internal stores and d epend on Ca2+ influx through dihydropyridine-insensitive Ca2+ channels . The physiological function(s) of P-2 purinergic receptors on granulo sa cells is not known.