Luteinizing hormone (LH) drives diverse intracellular calcium second messenger signals in isolated porcine ovarian thecal cells: Preferential recruitment of intracellular Ca2+ oscillatory cells by higher concentrations of LH

The present study examines Ca2+ second messenger signaling driven by LH in isolated porcine thecal cells. To this end, we implemented semiquantitative fluorescent (fura-a) videomicroscopic imaging of single thecal cells in vi tro. Stimulation of 388 cells with LH (5 mu g/ml) elicited an intracellular Ca2+ ([Ca2+](i)) signal in 85 +/- 5.3% of individual thecal cells (n = 11 experiments). Among 337 LH-responsive cells, we identified four predominant temporal modes of [Ca2+](i) signaling: 1) [Ca2+](i) oscillations with peri odicities of 0.5 to 4.5 min(-1) (63 +/- 4.5%), 2) a [Ca2+](i) spike followe d by a sustained plateau (17 +/- 2.6%), 3) a [Ca2+](i) spike only (5.8 +/- 2.6%); and 4) a [Ca2+](i) plateau only(3.8 +/- 1.5%). The prevalence, but n ot the amplitude or frequency, of LH-induced [Ca2+](i) oscillations in thec al cells was dependent on the agonist concentration. Reduced availability o f extracellular Ca2+ induced by treatment with EGTA or cobaltous chloride d id not block the initiation, but reversibly abolished ongoing [Ca2+](i) osc illations (72% of cells) or increased the mean [Ca2+](i) interspike periodi city from 1.09 +/- 0.16 to 0.59 +/- 0.07 min(-1) (P < 0.05). Putative phosp holipase C inhibition with U-73122 (10 mu M) also abolished or frequency-da mped LH-driven [Ca2+](i) oscillations in 95 +/- 4.7% of cells. [Ca2+](i) os cillations in thecal cells were not abrogated by overnight pretreatment wit h pertussis toxin. We conclude that 1) thecal cells (unlike earlier finding s in granulosa cells) manifest a diverse array of [Ca2+](i) signaling respo nses to LH at the single cell level; 2) LB can dose dependently recruit an increasing number of individually [Ca2+](i) oscillating thecal cells; 3) ex tracellular Ca2+ is required for LH to sustain (but not initiate) frequent and high amplitude [Ca2+] oscillations in thecal cells; and 4) these signal ing actions of LH are mediated via phospholipase C, but not a pertussis-tox in sensitive mechanism. Accordingly, the present data extend the apparent c omplexity of LH-induced [Ca2+](i) second messenger signaling and identify a t the single cell level LH's dose-responsive drive of [Ca2+](i) oscillation s in gonadal cells.