GnRH evokes localized subplasmalemmal calcium signaling in gonadotropes

Abstract

The binding of GnRH to its receptor initiates signaling cascades in gonadotropes, which result in enhanced LH and FSH biosynthesis and secretion. This process is necessary for follicular maturation and ovulation. Calcium influx activates MAPKs, which lead to increased transcription of LH and FSH genes. Previous research suggests that two MAPK signaling pathways, ERK and jun-N-terminal kinase, are activated by either calcium influx through L-type calcium channels or by global calcium signals originating from intracellular stores, respectively. Here we continued this investigation to further elucidate molecular mechanisms transducing GnRH receptor stimulation to ERK activation. Although it is known that GnRH activation of ERK requires calcium influx through L-type calcium channels, direct evidence supporting an underlying local calcium signaling mechanism was lacking. Here we used a combination of electrophysiology and total internal reflection fluorescence microscopy to visualize discrete sites of calcium influx (calcium sparklets) in gonadotrope-derived αT3-1 cells in real time. GnRH increased localized calcium influx and promoted ERK activation. The L-type calcium channel agonist FPL 64176 enhanced calcium sparklets and ERK activation in a manner indistinguishable from GnRH. Conversely, the L-type calcium channel antagonist nicardipine inhibited not only localized calcium sparklets but also ERK activation in response to GnRH. GnRH-dependent stimulation of L-type calcium channels was found to require protein kinase C and a dynamic actin cytoskeleton. Taken together, we provide the first direct evidence for localized L-type calcium channel signaling in αT3-1 cells and demonstrate the utility of our approach for investigating signaling mechanisms and cellular organization in gonadotropes.

Figure 1.

GnRH induces localized Ca²⁺ influx in αT3–1 cells. A, Schematic illustrating the electrophysiological and TIRF imaging method used to visualize Ca²⁺ influx in αT3–1 cells. B, Representative TIRF images showing localized Ca²⁺ influx in αT3–1 cells before and after GnRH (3 nM). C, Traces showing the time course of localized Ca²⁺ influx at the four circled sites before and after GnRH. The scale bar provides reference for changes in [Ca²⁺]_i during the traces (see Materials and Methods). D, Plot of Ca²⁺ sparklet site activities (nP_s; see Materials and Methods) before and after GnRH (n = 32 cells); solid gray lines are the arithmetic means of each group, and dashed lines mark the threshold for high-activity Ca²⁺ sparklet sites [nP_s ≥ .2; (10)]. E, Plot of mean ± SEM Ca²⁺ sparklet site densities (Ca²⁺ sparklet sites/μm²) before and after GnRH (n = 32 cells). *, P < .05. cont, control.

Figure 2.

GnRH-induced Ca²⁺ sparklets are mediated by L-type Ca²⁺ channels and promote ERK activation. A and C, Representative traces showing time courses of Ca²⁺ influx in an αT3–1 cell before and after application of the L-type Ca²⁺ channel agonist FPL 64176 (500 nM) (A) and GnRH (3 nM) (C) in the presence of the L-type Ca²⁺ channel antagonist nicardipine (10 μM). B and D, Plots of Ca²⁺ sparklet site activities (nP_s) and mean ± SEM Ca²⁺ sparklet site densities (Ca²⁺ sparklet sites per square micrometers) before and after FPL 64176 (n = 12 cells) (B) and GnRH in the presence of nicardipine (n = 7 cells) (D). E, Western blot analysis of ERK activation (as measured by ERK phosphorylation) in αT3–1 cells exposed to GnRH (3 nM) or FPL 64176 (500 nM) in the presence of nicardipine (10 μM) for 5 or 10 minutes (n ≥ 3 independent experiments). *, P < .05 vs nicardipine; †, P < .05 vs nicardipine and P < .05 vs GnRH. cont, control; nic, nicardipine.

Figure 3.

GnRH-induced L-type Ca²⁺ channel sparklets and ERK activation require PKC. A and C, Representative traces showing time courses of Ca²⁺ influx in an αT3–1 cell before and after application of the PKC activator PDBu (50 nM) (A) and GnRH (3 nM) (C) in the presence of the broad-spectrum PKC inhibitor GFX (1 μM). B and D, Plots of Ca²⁺ sparklet site activities (nP_s) and mean ± SEM Ca²⁺ sparklet site densities (Ca²⁺ sparklet sites per square micrometer) before and after PDBu (n = 11 cells) (B) and GnRH in the presence of GFX (n = 9 cells) (D). E, Western blot analysis of ERK activation (as measured by ERK phosphorylation) in αT3–1 cells exposed to PDBu (50 nM) or PDBu with nicardipine (1 μM) for 5 minutes and αT3–1 cells exposed to GnRH (3 nM) or FPL 64176 (500 nM) for 5 minutes in the presence or absence of GFX (1 μM; n ≥ 3 independent experiments). *, P < .05. cont, control; nic, nicardipine.

Figure 4.

Actin stabilization disrupts GnRH-dependent Ca²⁺ sparklet stimulation. A, Representative traces showing time courses of Ca²⁺ influx in αT3–1 cells exposed to GnRH (3 nM) in the presence or absence of jasplakinolide (100 nM for 5 min). B and C, Plots of Ca²⁺ sparklet site activities (nP_s) and mean ± SEM Ca²⁺ sparklet site densities (Ca²⁺ sparklet sites per square micrometer) for GnRH (3 nM; n = 9 cells in each group) with or without jasplakinolide (100 nM for 5 min). D, Western blot analysis of ERK activation (as measured by ERK phosphorylation) in αT3–1 cells exposed to GnRH (3 nM) with or without jasplakinolide (100 nM for 5 min; n ≥ 3 independent experiments). *, P < .05; †, P < .05 vs jasplakinolide and P < .05 vs GnRH. cont, control; jas, jasplakinolide.

Figure 5.

Actin stabilization does not disrupt Ca²⁺ sparklet stimulation by direct activation of L-type Ca²⁺ channels or PKC. A and B, Representative traces showing time courses of Ca²⁺ influx in αT3–1 cells exposed to the L-type Ca²⁺ channel agonist FPL 64176 (500 nM) (A) or the PKC activator PDBu (50 nM) (B) with or without jasplakinolide (100 nM for 5 min). C and D, Plots of Ca²⁺ sparklet site activities (nP_s) and mean ± SEM Ca²⁺ sparklet site densities (Ca²⁺ sparklet sites per square micrometer) for FPL 64176 (500 nM; n = 8 cells in each group) and PDBu (50 nM; n = 11 cells in each group) with or without jasplakinolide (100 nM for 5 min). E, Western blot analysis of ERK activation (as measured by ERK phosphorylation) in αT3–1 cells exposed to FPL 64176 (500 nM) and PDBu (50 nM) with or without jasplakinolide (100 nM for 5 min; n ≥ 3 independent experiments). *, P < .05. cont, control; jas, jasplakinolide.