Influence of the muscarinic agonist carbachol on intracellular Ca2+ in chicken granulosa cells. I. Dependence on follicular maturation

The present study addressed the influence of follicular development on carbamylcholine chloride (Cch)-induced Ca2+ transients and inositol 1,4,5-trisphosphate (IP3) production in granulosa cells isolated from the first (F1), third (F3), and fifth and sixth (F5,6) largest follicles. Intracellular free calcium, [Ca2+]i, was measured in fura-2-loaded cells about 20-36 h after their isolation. The percentage of cells responding to a maximal stimulatory concentration of Cch (0.2 mM) was higher in the F1 (89%) granulosa cells than in cells from the F3 (68%) and the F5,6 (72%) follicles. Most of the Ca2+ transients that were elicited in F1 granulosa cells were characterized by large (696 +/- 119 nM), fast (260 +/- 55 nM/sec) increases in [Ca2+]i, followed by a slow, uneven decrease in [Ca2+]i, to the resting concentration. In contrast, Cch-induced changes in [Ca2+]i in F3 and F5,6 granulosa cells were generally both smaller (154 +/- 37 nM and 165 +/- 37 nM, respectively) and slower (36 +/- 25 nM/sec and 46 +/- 16 nM/sec, respectively) than those observed in cells from the largest follicle. Removal of external Ca2+ did not alter the large, fast increases in [Ca2+]i; however, it nearly blocked the slow responses observed in F3 and F5,6 cells. IP3 production was elevated in 3H-myo-inositol-loaded F1 granulosa cells after 1 min of Cch (0.2 mM) treatment, whereas inositol bisphosphate (IP2) production and inositol monophosphate (IP) production were elevated only after longer incubations. Both Cch (0.2 mM) and acetylcholine (0.2 mM) elevated the synthesis of IP, IP2, and IP3 in cultures containing F1 or F5,6 granulosa cells, although the response of the latter was consistently lower. In conclusion, the present studies demonstrate that the response of the IP3-Ca2+ signaling system to muscarinic stimulation is dependent on the stage of follicular development.