Subplasmalemmal hydrogen peroxide triggers calcium influx in gonadotropes

Abstract

Gonadotropin-releasing hormone (GnRH) stimulation of its eponymous receptor on the surface of endocrine anterior pituitary gonadotrope cells (gonadotropes) initiates multiple signaling cascades that culminate in the secretion of luteinizing and follicle-stimulating hormones, which have critical roles in fertility and reproduction. Enhanced luteinizing hormone biosynthesis, a necessary event for ovulation, requires a signaling pathway characterized by calcium influx through L-type calcium channels and subsequent activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK). We previously reported that highly localized subplasmalemmal calcium microdomains produced by L-type calcium channels (calcium sparklets) play an essential part in GnRH-dependent ERK activation. Similar to calcium, reactive oxygen species (ROS) are ubiquitous intracellular signaling molecules whose subcellular localization determines their specificity. To investigate the potential influence of oxidant signaling in gonadotropes, here we examined the impact of ROS generation on L-type calcium channel function. Total internal reflection fluorescence (TIRF) microscopy revealed that GnRH induces spatially restricted sites of ROS generation in gonadotrope-derived αT3-1 cells. Furthermore, GnRH-dependent stimulation of L-type calcium channels required intracellular hydrogen peroxide signaling in these cells and in primary mouse gonadotropes. NADPH oxidase and mitochondrial ROS generation were each necessary for GnRH-mediated stimulation of L-type calcium channels. Congruently, GnRH increased oxidation within subplasmalemmal mitochondria, and L-type calcium channel activity correlated strongly with the presence of adjacent mitochondria. Collectively, our results provide compelling evidence that NADPH oxidase activity and mitochondria-derived hydrogen peroxide signaling play a fundamental role in GnRH-dependent stimulation of L-type calcium channels in anterior pituitary gonadotropes.

Keywords: NADPH oxidase; calcium imaging; cell signaling; mitochondria; reactive oxygen species (ROS).

Figure 1.

GnRH and exogenous hydrogen peroxide induce localized L-type Ca²⁺ channel influx in primary mouse gonadotrope cells. A, representative TIRF images and traces showing time courses of localized Ca²⁺ influx at the circled sites before (left) and after (right) application of GnRH (10 nm). B, plot of nP_s (left) and plot of mean ± S.E. Ca²⁺ sparklet densities (Ca²⁺ sparklet sites/μm²; right) before and after GnRH in the absence (n = 15) and the continuous presence of the L-type Ca²⁺ channel antagonist nicardipine (10 μm; n = 19; raw data not shown). The horizontal dashed gray line marks the threshold for high-activity Ca²⁺ sparklet sites (nP_s ≥ 0.2; see “Experimental procedures”). C, representative TIRF images and traces showing time courses of localized Ca²⁺ influx at the circled sites before (left) and after (right) application of H₂O₂ (100 μm). D, plot of nP_s (left) and plot of mean ± S.E. Ca²⁺ sparklet densities (Ca²⁺ sparklet sites/μm²; right) before and after H₂O₂ in the absence (n = 10) and the continuous presence of nicardipine (10 μm; n = 21; raw data not shown). Error bars represent S.E. *, p < 0.05; ns, not significantly different.

Figure 2.

Exogenous hydrogen peroxide induces localized L-type channel Ca²⁺ influx in gonadotrope-derived αT3-1 cells. A, representative TIRF images and traces showing time courses of localized Ca²⁺ influx at the circled sites before (left) and after (right) application of H₂O₂ (100 μm). B, plot of nP_s (left) and plot of mean ± S.E. Ca²⁺ sparklet densities (Ca²⁺ sparklet sites/μm²; right) before and after H₂O₂ (n = 9). C, representative TIRF images and traces showing time courses of localized Ca²⁺ influx at the circled sites before (left) and after (right) application of H₂O₂ (100 μm) in the continuous presence of the L-type Ca²⁺ channel antagonist nicardipine (10 μm). D, plot of nP_s (left) and plot of mean ± S.E. Ca²⁺ sparklet densities (Ca²⁺ sparklet sites/μm²; right) before and after H₂O₂ in the continuous presence of the L-type Ca²⁺ channel antagonist nicardipine (10 μm; n = 12). Error bars represent S.E. *, p < 0.05; ns, not significantly different.

Figure 3.

Exogenous hydrogen peroxide and GnRH increase subplasmalemmal ROS in αT3-1 cells. A, representative TIRF images showing subplasmalemmal DCF fluorescence (indicating intracellular oxidation) in a cell before (top) and after application of H₂O₂ (100 μm; bottom). Yellow circles indicate DCF fluorescence sites that satisfy the statistical criteria for ROS puncta designation (see “Experimental procedures”). B, plot of the normalized mean ± S.E. of the average cell (left) and ROS puncta (right) DCF fluorescence (arbitrary units (AU)) before and after H₂O₂ (n = 7). C, plot of ROS puncta densities (ROS puncta sites/μm²) before (open circles) and after H₂O₂ (closed circles; n = 7). D, representative TIRF images showing subplasmalemmal DCF fluorescence in cells before (top) and after application of GnRH (10 nm; bottom) in the presence (left) and absence (right) of external Ca²⁺ (2 mm). E, plot of the normalized mean ± S.E. of the average cell (gray, bottom) and ROS puncta (black, top) DCF fluorescence before and after GnRH in the presence (left; n = 17) and absence (right; n = 8) of external Ca²⁺. F, plot of ROS puncta densities before (open circles) and after GnRH (closed circles) in the presence (left; n = 17) and absence (right; n = 8) of external Ca²⁺. Error bars represent S.E. *, p < 0.05; ns, not significantly different.

Figure 4.

NADPH oxidase and endogenous hydrogen peroxide contribute to localized GnRH-dependent ROS generation in αT3-1 cells. A and D, representative TIRF images showing punctate DCF fluorescence in αT3-1 cells before (left) and after GnRH (right) in the continuous presence of the NADPH oxidase inhibitor apocynin (25 μm) (A) and the cell-permeant H₂O₂-decomposing enzyme PEG-catalase (500 units/ml) (D). B and E, plots of the normalized mean ± S.E. of the average cell (gray, bottom) and ROS puncta (black, top) DCF fluorescence (arbitrary units (AU)) before (left) and after GnRH (right) in the continuous presence of apocynin (n = 14) (B) and PEG-catalase (n = 14) (E). C and F, plots of ROS puncta densities before (open circles) and after GnRH (closed circles) in the presence of apocynin (n = 14) (C) and PEG-catalase (n = 14) (F). Error bars represent S.E. *, p < 0.05; ns, not significantly different.

Figure 5.

NADPH oxidase and endogenous hydrogen peroxide contribute to localized GnRH-dependent Ca²⁺ influx in αT3-1 cells. A–C, representative TIRF images and traces showing time courses of localized Ca²⁺ influx at the circled sites before (left) and after (right) GnRH (10 nm) in a control cell (A) and in the continuous presence of the Nox1-selective NADPH oxidase inhibitor ML171 (B; 1 μm) and the H₂O₂-decomposing enzyme catalase (C; 500 units/ml). D, plot of nP_s before and after GnRH in control cells (n = 20) and in the continuous presence of the Nox1-selective NADPH oxidase inhibitor ML171 (n = 6), the nonselective NADPH oxidase inhibitor apocynin (representative data not shown; n = 19), and the H₂O₂-decomposing enzyme catalase (n = 19). E, plot of mean ± S.E. Ca²⁺ sparklet densities (Ca²⁺ sparklet sites/μm²) before and after GnRH in control cells (n = 15) and in the continuous presence of ML171 (n = 6), apocynin (representative data not shown; n = 19), and the H₂O₂-decomposing enzyme catalase (n = 19). Error bars represent S.E. *, p < 0.05; ns, not significantly different.

Figure 6.

GnRH promotes oxidation within subplasmalemmal mitochondria in αT3-1 cells. A and B, representative TIRF images showing subplasmalemmal MitoSOX-Red fluorescence (indicating oxidation within mitochondria) in cells before (left) and after exposure (right) to GnRH (10 nm) (A) and the mitochondrial electron transport chain complex III inhibitor antimycin (500 nm) (B). C, representative TIRF images showing subplasmalemmal MitoSOX-Red fluorescence in a cell incubated with the mitochondrial uncoupler CCCP (1 μm; 10 min) before (left) and after exposure (right) to GnRH (10 nm). D, representative TIRF images showing subplasmalemmal MitoSOX-Red fluorescence in a cell incubated with the mitochondria-targeted antioxidant mitoTEMPO (25 nm; 10 min) before (left) and after (right) exposure to GnRH (10 nm). E, representative TIRF images showing subplasmalemmal MitoSOX-Red fluorescence in a cell incubated with the nonselective NADPH oxidase inhibitor apocynin (25 μm; 10 min) before (left) and after exposure (right) to GnRH (10 nm). F, plot of normalized mean ± S.E. MitoSOX-Red fluorescence intensity (arbitrary units (AU)) in αT3-1 cells before and after GnRH (n = 9), before and after antimycin (n = 7), before and after GnRH (n = 6) and antimycin (representative data not shown, n = 5) in the continuous presence of CCCP, and before and after GnRH in the continuous presence of mitoTEMPO (n = 12) and apocynin (n = 5). Error bars represent S.E. *, p < 0.05; ns, not significantly different.

Figure 7.

Mitochondria contribute to GnRH-dependent ROS and Ca²⁺ microdomain signaling in αT3-1 cells. A, representative TIRF images showing punctate DCF fluorescence before (left) and after GnRH (10 nm; right) in cells incubated with mitoTEMPO (25 nm; 10 min). B, plot of ROS puncta densities (ROS puncta sites/μm²) before (open circles) and after GnRH (closed circles) in the continuous presence of mitoTEMPO (n = 12). C, representative TIRF images and traces showing time courses of localized Ca²⁺ influx at the circled sites before (left) and after (right) application of the mitochondrial electron transport chain complex III inhibitor antimycin (500 nm). D, representative TIRF images and traces showing time courses of localized Ca²⁺ influx at the circled sites before (left) and after (right) application of GnRH (10 nm) in cells incubated with the mitochondria-targeted antioxidant mitoTEMPO (25 nm; 10 min). E, plot of nP_s (top) and plot of mean ± S.E. Ca²⁺ sparklet densities (Ca²⁺ sparklet sites/μm²; bottom) before and after GnRH (left; n = 14; raw data not shown), before and after antimycin (middle; n = 14), and before and after GnRH in the continuous presence of mitoTEMPO (right; n = 7). Error bars represent S.E. *, p < 0.05; ns, not significantly different.

Figure 8.

Subcellular distribution of mitochondria in αT3-1 cells. A, representative confocal z projection images showing the plasma membrane (panel 1; Alexa 555–WGA fluorescence, red) and mitochondria (panel 2; MitoTracker fluorescence, green) in a single αT3-1 cell. In panel 3, an overlay of panels 1 and 2, subplasmalemmal mitochondria (≤0.5 μm from the plasma membrane) are colored yellow for emphasis. B, plot of the mean ± S.E. mitochondrial and nonmitochondrial volumes (percentage of total cell volume; n = 6). C, plot of the mean ± S.E. nonperipheral (>0.5 μm from the plasma membrane) and subplasmalemmal (≤0.5 μm) mitochondrial volumes (percentage of total mitochondrial volume; n = 6). Error bars represent S.E.

Figure 9.

GnRH-dependent L-type Ca²⁺ channel activity correlates with the presence of subplasmalemmal mitochondria in αT3-1 cells. A, representative TIRF images showing subplasmalemmal mitochondria (MitoTracker fluorescence, panel 1; thresholded MitoTracker fluorescence, panel 2), GnRH-dependent (10 nm) L-type channel Ca²⁺ influx (fluo-5F fluorescence; panel 3), and an overlay of panels 2 and 3 (panel 4). Yellow circles in panels 3 and 4 indicate sites of L-type Ca²⁺ channel sparklet activity. B, distance map showing cumulative distribution functions representing the distance of observed Ca²⁺ sparklet site peaks from mitochondria (solid black line; n = 13) and 130 randomly distributed points (10 points per cell) within the visible TIRF footprint (dashed black line). Solid red lines are best fits of the cumulative distributions with a single exponential function (see “Experimental procedures”). The vertical dashed gray line marks the distance separating mitochondrial-associated (≤0.5 μm) and nonassociated (>0.5 μm) Ca²⁺ sparklet sites. C, plot of nP_s at sites >0.5 and ≤0.5 μm from the nearest thresholded MitoTracker signal (n = 13). *, p < 0.05.

Figure 10.

Working model of GnRH-dependent hydrogen peroxide and Ca²⁺ microdomain signaling in anterior pituitary gonadotropes. Our data, in conjunction with previous reports (2–4), support a localized mechanism of oxidant-dependent L-type Ca²⁺ channel signaling in gonadotropes. GnRH receptor stimulation promotes the generation of NADPH oxidase-dependent H₂O₂ microdomains functionally coupled (via PKC) to L-type Ca²⁺ channels. Colocalized L-type channel Ca²⁺ influx, in turn, promotes ERK activation and ultimately an increase in gonadotropin biosynthesis. Furthermore, our data suggest that mitochondria-dependent ROS-induced ROS release (RIRR) serves as an associated amplification mechanism that is necessary for the generation of functionally relevant colocalized Ca²⁺ and H₂O₂ signaling microdomains. GnRHR, gonadotropin releasing hormone receptor; LTCC, L-type Ca²⁺ channel; ETC, mitochondrial electron transport chain. See text for further details.