Ionic Channels Involved in the LHRH and SRIF Release from Rat Mediobasal Hypothalamus

Abstract

A superfusion system was used in order to investigate the ionic requirements of luteinizing hormone releasing hormone (LHRH) and somatostatin (SRIF) release from mediobasal hypothalamic (MBH) slices of male adult rats. Slices were superfused with Hepes-buffered Locke medium at 37 °C in an atmosphere of Ch 95%–CO2 5% for 1 h. Bacitracin (2 × 10^–5M) was added to the medium to prevent enzymatic degradation of neuropeptides. Depolarizing agents such as potassium (K⁺) or veratridine stimulated LHRH and SRIF release in a dose-dependent manner. Maximal effect was obtained with K⁺ 56 mM and veratridine 50 µM. The depolarizing effect of K⁺ 56 mM was specific and not due to the hypertonicity of the medium used. Neither Mg²⁺ nor chlorine was needed for the spontaneous or K⁺-evoked release of LHRH and SRIF. The amplitude of the secretory response to K⁺ 56 mM was related to Ca²⁺ concentration tested in the range of 0.2–8.8 mM; maximal responses were obtained between 0.8 and 1.8 mM. Removal of Ca²⁺ from the medium with or without replacement by Mg²⁺, as well as administration of voltage-sensitive Ca²⁺ channel blockers (D-600 10^–4M, Mn²⁺ 3 mM) blocked both K⁺ and veratridine induced neuropeptide release. When sodium channels and the ‘early’ calcium channels were blocked by tetrodotoxin (5 × 10^–7M) the stimulatory effect of veratridine was completely blocked whereas the stimulation of K⁺ was unaffected. These experiments indicate that: (1) both K⁺ and veratridine induced-LHRH and SRIF release is a Ca2⁺-dependent process; (2) Ca²⁺ concentration is critical for the amplitude of the secretory response; (3) the main Ca²⁺ channel involved in neuropeptide release corresponds to the voltage-sensitive Ca²⁺ channel, and (4) neither magnesium or chlorine is needed for either spontaneous or evoked release of LHRH and SRIF.

In vitro Secretion of Gonadotropin-Releasing Hormone (GnRH) and [Hydroxyproline⁹]GnRH from the Rat Hypothalamus Exhibits a Differential Sensitivity to Castration and Second Messengers

“…As already shown for GnRH [10, 12, 15, 31]and GAP [15], repeated stimulation did not significantly deplete tissue stores of HypGnRH. Repeated challenges with several secretagogues did not alter the molecular composition of the released material.…”

“…In order to propose a neurohormonal role for HypGnRH, it was necessary to establish whether endogenous HypGnRH, as GnRH itself, is secreted by the Ht in a calcium-dependent manner [10, 11, 12, 13]. It was of further interest to compare its regulation with that of GnRH release.…”

Effects of Noradrenaline on GnRH-Secreting Immortalized Hypothalamic (GT1-7) Neurons.

“…Noradrenaline depolarized the membrane potential, and increased [Ca2+]i, and then increased GnRH release. Our results are partially consistent with those of a study of mediobasal hypothalamic slices by Drouva et al [12], who reported that depolarizing agents such as potassium or veratridine stimulated GnRH release in a dosedependent manner and that removal of Ca2+ from the medium and administration of voltage-sensitive Ca2+ channel blockers (D-600, Mn2+) blocked potassiumand veratridine-induced release of GnRH. In our study, NA-induced [Ca2+]i increases in GT1-7 cells were also inhibited by the absence of extracellular calcium.…”

Calcium signaling and episodic secretion of gonadotropin-releasing hormone in hypothalamic neurons.

“…First, activation of VSCCs by high K+-induced depolarization (15) in cultured hypothalamic neurons and GT1-1 neuronal cells leads to increased GnRH release, as in perifused hypothalamic explants (32,33 …”