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Nitrendipine and omega-conotoxin modulate gonadotropin release and gonadotrope [Ca2+]i
M Blotner1, G A Shangold, E Y Lee
1Department of Pharmacological, University of Chicago Pritzker School of Medicine, IL 60637.
Abstract:
We have examined the pharmacology of the voltage-sensitive Ca2+ channels (VSCCs) that mediate gonadotropin secretion from primary cultures of rat pituitary cells, stimulated by either cell depolarization or by binding of gonadotropin-releasing hormone (GnRH). We also measured single-cell [Ca2+]i transients using fura-2 in gonadotropes identified by a reverse hemolytic plaque assay employing an antiserum to luteinizing hormone (LH). Cell depolarization evoked by either 50 mM K+ or 30 microM veratridine induced 2- to 6-fold increases in gonadotropin secretion over basal levels. GnRH caused 6- to 20-fold increases in follicle-stimulating hormone (FSH) and LH secretion, respectively, with maximal stimulation at 100 nM GnRH. K(+)- or GnRH-induced FSH release was largely prevented by co-incubation with 1 mM CdCl. Tetrodotoxin (TTX, 5 microM) prevented the veratridine-, but not the K(+)- or GnRH-induced, stimulation of FSH secretion. Nitrendipine (Ntd, 1 microM) produced 35-50% inhibition (NS) of both FSH and LH release stimulated by either 50 mM K+ or 100 nM GnRH. Ntd also inhibited the K(+)-induced [Ca2+]i rise (greater than 90%), as well as the secondary, plateau phase of the GnRH-induced elevation of [Ca2+]i (100% inhibition). Omega-conotoxin (omega-CgTx, 100 nM) partially suppressed FSH and LH release (NS) due to both K+ (33% each) and GnRH (44% and 18%, respectively). omega-CgTx showed variable effects on [Ca2+]i transients evoked by K+ or GnRH ranging from clear inhibition to no effect. We conclude that influx of extracellular Ca2+ is one of several fundamental events underlying the depolarization- or receptor-activated release of LH and FSH, and that this influx can be inhibited by dihydropyridine-sensitive ('L') Ca2+ channels. Two classes of L-channels may exist in gonadotropes, that differ in their sensitivity to omega-CgTx.
Insights
Voltage-sensitive calcium channels (VSCCs) in rat pituitary cells control gonadotropin secretion. Dihydropyridine-sensitive L-type channels are key, with potential subtypes differing in omega-conotoxin sensitivity.
Area of Science:
- Endocrinology
- Neuroscience
- Cell Biology
Background:
- Gonadotropin secretion from pituitary cells is crucial for reproductive function.
- Voltage-sensitive calcium channels (VSCCs) play a critical role in regulating hormone release.
- Gonadotropin-releasing hormone (GnRH) stimulates the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH).
Purpose of the Study:
- To investigate the pharmacology of VSCCs involved in gonadotropin secretion.
- To elucidate the role of different calcium channel types in GnRH- and depolarization-induced hormone release.
- To characterize the subtypes of L-type calcium channels in rat gonadotropes.
Main Methods:
- Primary cultures of rat pituitary cells.
- Measurement of gonadotropin secretion (FSH and LH) via radioimmunoassay or similar.
- Single-cell calcium transients ([Ca2+]i) using fura-2.
- Identification of gonadotropes using a reverse hemolytic plaque assay with anti-LH antiserum.
- Pharmacological manipulation using K+ depolarization, veratridine, GnRH, CdCl2, tetrodotoxin (TTX), nitrendipine (Ntd), and omega-conotoxin (omega-CgTx).
Main Results:
- Both K+ depolarization and GnRH significantly increased FSH and LH secretion.
- Cadmium chloride (CdCl2) largely blocked K+- and GnRH-induced FSH release.
- Nitrendipine inhibited K+- and GnRH-stimulated FSH and LH release, and K+-induced [Ca2+]i rise, as well as the plateau phase of GnRH-induced [Ca2+]i elevation.
- Omega-conotoxin partially suppressed FSH and LH release evoked by K+ and GnRH, with variable effects on [Ca2+]i transients.
- Tetrodotoxin blocked veratridine-induced, but not K+- or GnRH-induced, FSH secretion.
Conclusions:
- Extracellular calcium influx is essential for depolarization- and GnRH-activated LH and FSH release.
- Dihydropyridine-sensitive ('L') calcium channels mediate this critical calcium influx.
- Evidence suggests the existence of at least two subtypes of L-type calcium channels in gonadotropes, potentially differing in their sensitivity to omega-conotoxin.