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-2) videomicroscopic imaging of single thecal cells in vitro. Stimulation of 388 cells with LH (5 μg/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 periodicities of 0.5 to 4.5 min^-1 (63 ± 4.5%), 2) a [Ca²⁺]_i spike followed 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 not the amplitude or frequency, of LH-induced [Ca²⁺]_i oscillations in thecal cells was dependent on the agonist concentration. Reduced availability of extracellular Ca²⁺ induced by treatment with EGTA or cobaltous chloride did not block the initiation, but reversibly abolished ongoing [Ca²⁺]_i oscillations (72% of cells) or increased the mean [Ca²⁺]_i interspike periodicity from 1.09 ± 0.16 to 0.59 ± 0.07 min^-1 (P < 0.05). Putative phospholipase C inhibition with U-73122 (10 μM) also abolished or frequency-damped LH-driven [Ca²⁺]_i oscillations in 95 ± 4.7% of cells. [Ca²⁺]_i oscillations in thecal cells were not abrogated by overnight pretreatment with pertussis toxin. We conclude that 1) thecal cells (unlike earlier findings in granulosa cells) manifest a diverse array of [Ca²⁺]_i signaling responses to LH at the single cell level; 2) LH can dose dependently recruit an increasing number of individually [Ca²⁺]_i oscillating thecal cells; 3) extracellular Ca²⁺ is required for LH to sustain (but not initiate) frequent and high amplitude [Ca²⁺] oscillations in thecal cells; and 4) these signaling actions of LH are mediated via phospholipase C, but not a pertussis-toxin sensitive mechanism. Accordingly, the present data extend the apparent complexity of LH-induced [Ca²⁺]_i second messenger signaling and identify at the single cell level LH's dose-responsive drive of [Ca²⁺]_i oscillations in gonadal cells.