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 microg/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 microM) 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.
Aguirre
C
C
Department of Internal Medicine, National Science Foundation Center for Biological Timing, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Jayes
F C
FC
Veldhuis
J D
JD
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D005260:Female; D007986:Luteinizing Hormone / Q000494:pharmacology*; D010507:Periodicity; D037342:Pertussis Toxin; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D015290:Second Messenger Systems; D015398:Signal Transduction; D013552:Swine; D013799:Theca Cells / Q000187:drug effects / Q000378:metabolism; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D002614:Chelating Agents; D004963:Estrenes; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D010566:Virulence Factors, Bordetella; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D003035:Cobalt; D004533:Egtazic Acid; D007986:Luteinizing Hormone; D037342:Pertussis Toxin; D010738:Type C Phospholipases; C018021:cobaltous chloride; D002118:Calcium