title: Recruitment of individually (all-or-none) responding cells, rather than amplitude enhancement, is the single-cell mechanism subserving the dose-responsive activation of intracellular calcium second messenger signaling by the human luteinizing-hormone receptor.
abstract: We have investigated at the single-cell level how the human LH receptor mediates a dose-responsive increase in intracellular free calcium-ion concentrations ([Ca2+]i). In human embryonic kidney cells (293 cells) stably transfected with the full-length human LH receptor cDNA. Intact dimeric LH, but not LH beta- or alpha-subunits, evoked specific [Ca2+]i signals. High-resolution fluorescence (fura-2) video-microscopy demonstrated cell-to-cell variability in [Ca2+]i signaling responses in individual cells, viz., an all-or-none spike (9%), spike-and-plateau (25%), or plateau (52%) types of temporal signal. Oscillatory [Ca2+]i responses were observed in 12-14% of LH-stimulated cells unrelated to LH concentration. The LH dose-response originated by higher concentrations of LH recruiting more individually responding cells (rather than altering [Ca2+]i signal amplitude), and eliciting a [Ca2+]i rise more rapidly, i.e., at reduced latency. Cobalt did not abolish the LH-stimulated [Ca2+]i spike-and-plateau response, but decreased the percentage of cells with a plateau pattern. Quench experiments demonstrated influx of Mn2+ following the [Ca2+]i spike, thus directly documenting divalent cation inflow during the plateau phase. Adenylyl-cyclase activation with forskolin or treatment with a cAMP analog failed to elicit the biphasic [Ca2+]i response, and pertussis toxin (PTX) did not alter LH-stimulated [Ca2+]i signaling. However, overnight preincubation with LH reduced the percentage of [Ca2+]i-responding cells following re-exposure to LH to 5.7% (vs 72% in control), suggesting LH-induced desensitization of the LH-receptor directed [Ca2+]i signal. In summary, the present studies of human LH receptor signal transduction at the single-cell level show that increasing concentrations of LH achieve a dose-dependent intracellular Ca2+ signaling response by recruiting an increasing number of [Ca2+]i-responding cells, while concomitantly decreasing the temporal latency of the biphasic [Ca2+]i signal without altering the amplitude of its spike phase. Prolonged exposure to LH appears to desensitize the LH receptor-driven [Ca2+]i signal.
authors.affiliation: Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
authors.forename: F
authors.initials: F
authors.lastname: Sun
authors.forename: D J
authors.initials: DJ
authors.lastname: Berry
authors.forename: D A
authors.initials: DA
authors.lastname: Leong
authors.forename: J D
authors.initials: JD
authors.lastname: Veldhuis
mesh_terms: D037361:Adenylate Cyclase Toxin; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000502:physiology; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D007986:Luteinizing Hormone / Q000008:administration & dosage / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D037342:Pertussis Toxin; D011974:Receptors, LH / Q000235:genetics / Q000502:physiology*; D015290:Second Messenger Systems; D015398:Signal Transduction; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D037361:Adenylate Cyclase Toxin; D011974:Receptors, LH; D010566:Virulence Factors, Bordetella; D008345:Manganese; D007986:Luteinizing Hormone; D000242:Cyclic AMP; D037342:Pertussis Toxin; D002118:Calcium