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 lutinizing-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 β- or α-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-lateau 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 resoonse, 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.