Influence of the Muscarinic Agonist Carbachol on Intracellular Ca2+ in Chicken Granulosa Cells: I. Dependence on Follicular Maturation1

Abstract

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. IP, 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of Tumor Necrosis Factor Alpha on Intracellular Ca2+ in Hen Granulosa Cells in Vitro during Follicular Development1

“…Consequently, we attribute the inability of this portion of the granulosa cell population to mobilize intracellular Ca 2+ to their failure to access and/or fill Ca 2+ stores. We propose that TNFa facilitates 1) the filling of these stores by either activation of Ca 2+ -ATPases on Ca 2 + stores with a concomitant influx of extracellular Ca 2 + or via an influx of extracellular Ca 2+ directly into these Ca 2 + stores as suggested by the model of capacitive Ca 2 + entry [25] or 2) Cch-induced IP 3 production, as we have previously demonstrated that fast Ca 2+ transients in F1 cells are mediated by IP 3 [23]. Determining which (if any) of the mechanisms proposed above accurately describe the mechanisms behind the TNFa-Cch interaction will require further experimentation.…”

“…Furthermore, it has been demonstrated that Ach increases ovarian oxytocin and progesterone secretion in vivo in sheep [40] and bovine granulosa cell oxytocin and progesterone production in vitro [41]. Previous studies from our laboratory have demonstrated that muscarinic input has at least two mechanisms of action in hen granulosa cells: increasing [Ca 2+ ]i [22,23] and intracellular pH [42]. Moreover, data from the present study has demonstrated that the nature and magnitude of the Cchinduced Ca 2 + transient are modified by pretreatment with TNFa, and that this TNFa effect increases with follicular development.…”

“…This is in contrast to the quick response observed with Cch-induced Ca 2+ transients that occurs within the first 10 sec of exposure to the agonist at a much greater rate of rise. In contrast to the mode of action of TNFa, regulation of Ca 2+ signaling by muscarinic agonists appears to involve both mobilization of Ca 2+ from intracellular stores and transmembrane influx of Ca 2+ , presumably via G-protein mediated channel activation [22,23].…”

“…Although the precise mode of this interaction in the induction of Ca 2 + mobilization from intracellular stores is unclear, the present findings suggest participation of a complex regulatory mechanism(s) upstream of the Ca 2+ surge. The fact that pretreatment with TNFa only increased the magnitude of slow Cch-induced Ca 2 + transients is interesting, as it implies that this portion of the granulosa cell population failed to maximally respond to Cch challenge despite the fact that higher concentrations of Cch will not induce larger Ca 2+ transients in these slow responding cells [23]. Consequently, we attribute the inability of this portion of the granulosa cell population to mobilize intracellular Ca 2+ to their failure to access and/or fill Ca 2+ stores.…”

Prostaglandins Mediate the Stimulation of Deoxyribonucleic Acid Synthesis by Transforming Growth Factor α in Hen Granulosa Cells during Ovarian Follicular Development1

“…In addition, the ability of PGE 2 to override the suppression of [ 3 H]thymidine incorporation into DNA by PLA 2 inhibitor was greater in cells at the earlier than at the later stage of follicular maturation. It would be of interest to determine whether these follicular stage-dependent differences are the result of programmed changes in granulosa cell PG receptor abundance and/or production or activation of factor X, as has been previously suggested [33,34].…”