Peptidase Regulation of Gonadotropin‐Releasing Hormone Levels During In Vitro Incubations of the Rat Hypothalamus

Abstract

The fate of gonadotropin-releasing hormone (GnRH) was examined by a GnRH radioimmunoassay during in vitro incubations of the rat medial basal hypothalamus (MBH). There was a progressive disappearance of exogenous GnRH during MBH incubations. The GnRH degradation could be explained by the release of peptidases from the MBH into the incubation medium. The cytoplasmic marker lactic dehydrogenase (LDH) was also released into the incubation medium. The peptide antibiotic bacitracin produced a dose-dependent inhibition of GnRH degradation during MBH incubations; 1 mM-bacitracin completely inhibited exogenous GnRH degradation during 4-h incubations. Bacitracin also produced dose-dependent increases in the recovery of endogenous GnRH released from the MBH under basal conditions or when stimulated with the depolarizing agent veratrine. Veratrine also was found to decrease the GnRH peptidase activity significantly but not the LDH activity during MBH incubations. The present results indicate that peptidase activity can be an important regulator of endogenous GnRH released from the hypothalamus during in vitro incubations.

In vitro Release of LHRH from the Hypothalamus of Female Rats during Prepubertal Development

“…In vitro incubation experiments of hypothalamic tissue have shown their usefulness in studies on LHRH release in adult rats [3,4,14,19,20,22,23]. In the present study we describe in vitro release of LHRH from MBH of developing rats.…”

Differential Effects of an Luteinizing-Hormone-Releasing Hormone (LHRH) Antagonist Analogue on Lordosis Behavior Induced by LHRH and the LHRH Fragment Ac-LHRH^5–1⁰

“…The demonstration that a synthetic fragment of LHRH is capable of enhancing lordotic posture suggests that peptidase activity may also be important in the modulation of mating behavior. When exogenous LHRH was incubated with hypothalamic tissue, a significant disappearance of the molecule was observed within 30 min [17], After 2 h, 75% of the LHRH had disappeared. En hancement of mating behavior following infusion of LHRH into the VMH has been reported to occur between 60 and 90 min after infusion [1], which is a sufficient amount of time for significant degradation to have occurred.…”

Correlation of LH, FSH and GnRH Release during Various Brain-Pituitary Coincubations

“…Veratrine depolarizes nerve tissue through an activation of sodium channels sensitive to tetrodotoxin [19,27], and causes a calciumdependent release of numerous neurohor mones [18,20,21], We have found veratrine to release GnRH from the MBH at concen trations which release little LH from hemipituitaries and which increase the sensitivity of the pituitary to GnRH [20], The properties of veratrine permitted its use in brain-pituitary coincubations to determine whether LH and/or FSH releasing factors other than GnRH could be released from brain tissue.…”

“…GnRH levels are constant during hemipituitary incubations with synthetic GnRH, however, we have demonstrated that GnRH immunoreactivity linearly increases during 4-hour MBH incubations in the presence of veratrine [21]. The first 30 min of GnRH release occurs in the absence of a hemipituitary using the coincubation technique em ployed; thus, the mean GnRH concentration the hemipituitary is exposed to should equal 55% of the concentration measured at the end of the coincubation.…”

“…and gassed with 95% O j/5% CO2 (pH 7.4) was used. Unless noted otherwise, bacitracin (Sig ma), a peptidase inhibitor [15], was added to the medium to give a concentration of 1 rnM: this concen tration of bacitracin completely inhibits GnRH degra dation by peptidases during 4-hour hypothalamic in cubations [21], Veratrine (Sigma) was initially dissolved in 0.1 M HC1 and then diluted with 0.9% saline containing 0.1 % bovine serum albumin and 5 mM HCI (pH 3.0). Solid state synthesized GnRH (Beckman Instrument Co., Bioproducts Div" Palo Alto, Calif.) was dissolved in the same solution.…”