INFLUENCE OF THE MUSCARINIC AGONIST CARBACHOL ON INTRACELLULAR CA2-CELLS .1. DEPENDENCE ON FOLLICULAR MATURATION( IN CHICKEN GRANULOSA)

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 t he first (F1), third (F3), and fifth and sixth (F5,6) largest follicle s. Intracellular free calcium, [Ca2+](i), was measured in fura-2-loade d cells about 20-36 h after their isolation. The percentage of cells r esponding to a maximal stimulatory concentration of Cch (0.2 mM) was h igher 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 el icited in F1 granulosa cells were characterized by large (696 +/- 119 nM), fast (260 +/- 55 nM/sec) increases in [Ca2+](i) followed by a slo w, uneven decrease in [Ca2+](i) to the resting concentration. In contr ast, Cch-induced changes in [Ca2+](i) in F3 and F5,6 granulosa cells w ere generally both smaller (154 +/- 37 nM and 165 +/- 37 nM, respectiv ely) and slower (36 +/- 25 nM/sec and 46 +/- 16 nM/sec, respectively) than those observed in cells from the largest follicle. Removal of ext ernal Ca2+ did not alter the large, fast increases in [Ca2+](i); howev er, it nearly blocked the slow responses observed in F3 and F5,6 cells . IP3 production was elevated in H-3-myo-inositol-loaded F1 granulosa cells after 1 min of Cch (0.2 mM) treatment, whereas inositol bisphosp hate (IP2) production and inositol monophosphate (IP) production were elevated only after longer incubations. Both Cch (0.2 mM) and acetylch oline (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 la tter was consistently lower. In conclusion, the present studies demons trate that the response of the IP3-Ca2+ signaling system to muscarinic stimulation is dependent on the stage of follicular development.