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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 LH¹

Division of Endocrinology, Department of Internal Medicine, National Science Foundation Center for Biological Timing, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Dr. J. D. Veldhuis, Division of Endocrinology, Department of Internal Medicine, Box 202, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908. E-mail: jdv{at}virginia.edu

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.

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