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.
Highlights
Gonadotropin-releasing hormone (GnRH) stimulation of its eponymous receptor on the surface of endocrine anterior pituitary gonadotrope cells initiates multiple signaling cascades that culminate in the secretion of luteinizing and folliclestimulating hormones, which have critical roles in fertility and reproduction
To test our hypothesis that reactive oxygen species (ROS) signaling contributes to localized Ca2ϩ influx through L-type Ca2ϩ channels in gonadotropes exposed to GnRH, we formulated four requisite experimental criteria
To continue our investigation of L-type Ca2ϩ channel signaling in gonadotropes, we once again used a combination of electrophysiology and Total internal reflection fluorescence (TIRF) microscopy to visualize subplasmalemmal Ca2ϩ influx with high temporal and spatial resolution [4]
Summary
GnRH, gonadotropin-releasing hormone; [Ca2ϩ]i, intracellular Ca2ϩ concentration; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DCF, 2Ј,7Ј-dichlorodihydrofluorescein diacetate; ERK, extracellular signal-regulated kinase; FSH, follicle-stimulating hormone; H2O2, hydrogen peroxide; IP3, inositol 1,4,5-trisphosphate; IQR, interquartile range; LH, luteinizing hormone; nPs, Ca2ϩ sparklet site activity; PKC, protein kinase C; ROS, reactive oxygen species; TIRF, total internal reflection fluorescence; Alexa 555–WGA, wheat germ agglutinin–Alexa Fluor. Recent evidence suggests that, in gonadotropes exposed to GnRH, Ca2ϩ- and PKC-dependent NADPH oxidase activity increases ERK signaling and gonadotropin gene expression [6]. Together, these observations evoke the hypothesis of functional coupling between GnRH-dependent ROS generation and Ca2ϩ signaling in gonadotropes. Our data support a model where GnRH-dependent stimulation of L-type Ca2ϩ channels incorporates a localized H2O2 signaling mechanism generated by NADPH oxidase and subplasmalemmal mitochondria
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