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.