Estradiol plays a critical role in the feedback regulation of reproduction, in part by modulating the neurosecretory activity of gonadotropin-releasing hormone (GnRH) neurons. While indirect effects of estradiol on GnRH neurons have been clearly demonstrated, direct actions are still controversial. In the current study, we examined direct effects of 17β-estradiol upon the expression of receptors for afferent signals at the level of the GnRH neuron, using immortalized GT1-7 cells. Using RT-PCR, we confirmed the expression of mRNA for the adrenergic receptors (AR) α1A-, α1B-, α1D-, α2A-, α2C-, and β1-AR, and showed for the first time that mRNAs for α2B-, β2- and β3-AR, for kisspeptin and its receptor GPR54 and for the novel estrogenic receptor GPR30 are expressed in GT1-7 cells. After treatment with 10 nM 17β-estradiol, α1B-AR mRNA was significantly increased (14-fold) after 6 h as determined by real-time PCR, while α1B- and α1D-AR mRNA were significantly increased (19- and 23-fold, respectively) after 24 h. The expression of KiSS-1 and GPR54 mRNAs were also significantly increased (8- and 6-fold, respectively) after 24 h treatment of GT1-7 cells with estradiol. GPR30 mRNA expression was not affected by estradiol. Our data also showed that kisspeptin-10 (1–10 nM) can significantly stimulate GnRH release and GnRH mRNA expression in GT1-7 cells. These results suggest that the complex physiologic effects of estradiol on the function of the reproductive axis could be mediated partly through direct modulation of the expression of receptors for afferent signals in GnRH neurons.
Regulation of reproduction and energy homeostasis are linked, although our understanding of the central neural mechanisms subserving this connection is incomplete. Gonadotrophin-inhibiting hormone (GnIH) is a neuropeptide that negatively regulates reproduction and stimulates food intake. Neuropeptide Y (NPY) and products of the pro-opiomelanocortin (POMC) precursor (β-endorphin melanocortins) are appetite regulating peptides produced in the neurones of the arcuate nucleus; these peptides also regulate reproduction. In the present study, we determined the effects of GnIH on NPY and POMC neurones. Using brain slices from mice with transgenes for fluorescent tags in the two types of neurone and patch clamp electrophysiology, a predominant inhibitory effect of GnIH was observed. GnIH (100 nM) inhibited the firing rate in POMC cells, confirming the results of previous studies and consistent with the stimulatory effect of GnIH on food intake. Paradoxically (i.e. because both GnIH and NPY stimulate food intake), GnIH also had a predominantly inhibitory effect on action potential activity in NPY cells. GnIH also inhibited the secretion of NPY and α-melanocyte-stimulating hormone secretion in incubated hypothalamic blocks. GnIH (100 ng) injected into the cerebral ventricles of mice did not increase the number of NPY cells that were positively immunostained for c-Fos. Finally, dual label immunocytochemistry showed that 20% of NPY neurones had close contacts from GnIH fibres/varicosities. In conclusion, we confirm a negative effect of GnIH on POMC cells and demonstrate a paradoxical reduction of electrophysiological and functional activity in NPY cells.
The neurotransmitter γ-aminobutyric acid (GABA) is an important modulator of gonadotropin-releasing hormone (GnRH), and consequently of reproduction. GABA, acting via ionotropic GABAA receptors, exerts a biphasic effect on GnRH secretion in immortalized GnRH cells. The initial increase in GnRH secretion is triggered by a sharp rise in [Ca2+]i, while the progressive decline of GnRH levels that follows is paralleled by reduced levels of intracellular cAMP. The experiments described here were designed to explore the potential signaling pathways involved in this novel GABAA ionotropic inhibition of cAMP synthesis in GT1–7 cells. Using RT-PCR and real-time PCR, we found that GT1–7 cells express 8 of 9 known membrane adenylyl cyclase (AC) isoforms, including a large proportion of AC3 and AC9, as well as AC5 and AC6, all of which are negatively regulated by increases in [Ca2+]i. In contrast, isoforms of AC that are positively regulated by [Ca2+]i were barely detectable (AC1) or undetectable (AC8). Pharmacological activation of L-type voltage-operated calcium channels with BayK 8644 produced a decrease in [cAMP]i similar to that induced by GABA, while blocking these calcium channels with verapamil reversed the effect of GABA on cAMP synthesis. Furthermore, blocking calcineurin with deltamethrin, FK-506 or cyclosporin A blocked the inhibitory effect of GABA on [cAMP]i, supporting the involvement of AC9 in this effect. In addition, blocking Ca2+/calmodulin-dependent protein kinase II (CamKII) with KN-62 partially reversed the action of GABA, suggesting that AC3 may also be involved in this effect. Finally, GABA increased phosphatase activity in a calcium-dependent manner, an effect blocked by calcineurin inhibitors. Collectively, our results show that the ionotropic action of GABA via the activation of GABAA receptors can decrease AC activity in immortalized GnRH neurons, and that the effect of GABA appears to be mediated by a transient increase in [Ca2+]i followed by activation of calcineurin and CamKII, leading to dephosphorylation of AC9 and phosphorylation of AC3, respectively, and subsequently reducing the synthesis of cAMP.
sind direkt an die Sdiriffleitung zu richten, sie dürfen nidit gleichzeitig anderen Blättern zulu Abdruck angeboten werden. -Mit der Annahme des Manuskriptes erwirbt der Verlag das ausschließliche Redit der Vervielfältigung, Verbreitung und Ubersetzung der in dieser Zeitschrift zum Abdruck gelangten Beiträge, insbesondere audi des Recht, die Herstellung von fotornechanisdien Vertielfältigungen in gewerblichen Unternehmen zum innerbetrieblichen Gebrauch nach Maßgabe des zwischen dem Börsenverein des Deutschen Buchhandels und dem Bundesverband der Deùtschen Industrie abgeschlossenen Rabmenabkommens zu genehmigen. Der Verlag erwirbt weiter das Redit der Verwendung des Manuskriptes für fremdsprachliche Ausgaben. Kein Teil dieser Zeitschrift darf in irgendeiner Form, auch nicht durch Fotokopie, Mikrofilm oder irgendein anderes Verfahren ohne schriftliche Genehmigung des Vertiges reproduziert *erden; Jedoch wird gewerblichen Unternehmen die Anfertigung eines fotoxnechanischen Vervielfältigung (Fotokopie, Mikrokopie) für den innerbetrieblichen Gebrauch nach Maßgabe des zwischen dem Börsenverein des Deutschen Buchhandels und dem Bundesverband der Deutschen Industrie abgeschlossenen Rahmenabkommens gestattet. Werden die Gebühren durch Wertmarken entrichtet, so ist für Jedes Fotokopierblatt eine Marke 1m Betrag von DM 0.10 zu verwenden. -Die Aufnahme dieser Zeitschrift in Lesezirkel ist nicht gestattet g50 40 30 .2O 10 Im Rahmen der kardiologischen Tätigkeit an unserem Kran-Tab. 1. Sthwangersthaftsunterbrediungen an der Universitätskenhaus werden wir immer wieder mit dem Problem Herzfehler und Gravidität" konfrontiert. Deshalb haben wir uns seit einigen Jahren mit dieser Frage intensiver beschäftigt. Auch neuere von Schubert und Napp erarbeitete statistische Unterlagen für den Raum Hamburg bzw. die Universitätsfrauenklinik Eppendorf zeigen, daß eine weitere Stellungnahme gerechtfertigt ist.t
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