A Comparative Study of the Effects of Nitric Oxide and Carbon Monoxide on the in vivo Release of Gonadotropin-Releasing Hormone and Neuropeptide Y from Rat Hypothalamus during the Estradiol-Induced Luteinizing Hormone Surge: Estimation by Push-Pull Perfusion

Abstract

Recent evidence suggests that nitric oxide (NO), a free radical gas, plays an important role in regulating the function of a variety of neuroendocrine systems. With respect to the hypothalamo-pituitary-gonadal axis, a stimulatory effect of NO on the release of gonadotropin-releasing hormone (GnRH) from rat hypothalamus has been demonstrated in vitro. However, no previous study has reported NO-stimulated secretion of GnRH from in vivo hypothalamus, and also the precise cellular site of action of NO within the GnRH neuronal system remains to be elucidated. In the present study, utilizing the push-pull perfusion technique of rat hypothalamus, we examined the effect of L-arginine (L-Arg), an NO donor, on the release of GnRH, neuropeptide Y and cyclic GMP (c-GMP), which is a pivotal second messenger molecule of the NO system. For comparison, we also examined the effect of carbon monoxide (CO), another putative gaseous neurotransmitter, using hematin, a CO donor. During the period of 11.00–18.00 h, we collected blood and hypothalamic perfusates from ovariectomized adult rats that had been implanted with an estradiol capsule 2 days before. During the entire period of observation, L-Arg (1.0 or 10 mM), hematin (10 or 100 µM) or artificial cerebrospinal fluid alone (as the control) was infused into the medial preoptic area (MPOA) where there are cell bodies of GnRH neurons, or the median eminence-arcuate nucleus complex (ME-ARC) where axon terminals of GnRH neurons are localized. Although 10 mM of L-Arg significantly stimulated GnRH and c-GMP, but not neuropeptide Y, levels in both the MPOA and ME-ARC, GnRH and c-GMP in the ME-ARC were already increased by 1.0 mM of L-Arg. By contrast, both concentrations of hematin were without effect at either site of the hypothalamus. This study is the first to demonstrate that NO is capable of stimulating GnRH release from rat hypothalamus in vivo. Our data also suggests that both cell bodies and axon terminals of GnRH neurons may be sites of action of NO. Our data do not support a previous study by other investigators that reported a stimulatory effect of CO on the GnRH release.

Carbon Monoxide: Endogenous Production, Physiological Functions, and Pharmacological Applications

“…The secretion of gonadotropin releasing hormone seems to be stimulated by CO in vitro (Lamar et al, 1996). However, this observation cannot be confirmed by in vivo study during the estradiol-induced luteinizing hormone surge (Kohsaka et al, 1999).…”

In vivo Release of Prolactin-Releasing Peptide in Rat Hypothalamus in Association with Luteinizing Hormone and Prolactin Surges

“…Coordinates for the ME-ARC were 0.0 mm anterior to and 0.5 mm lateral to the bregma and 9.8 mm ventral from the dura. The PPP cannulae used were the same as described in our previous studies [14, 15, 16, 17, 18]. The device was fixed onto the skull with anchor screws and dental cement.…”

“…The device was fixed onto the skull with anchor screws and dental cement. Two days prior to PPP, while under light ether anesthesia, the animals were implanted with a jugular vein catheter filled with heparin solution and also implanted subcutaneously with a single Silastic capsule containing 300 µg/ml of estradiol-17β (Sigma Chemical Company, St. Louis, Mo., USA) in a manner similar to the technique used in our previous studies [14, 16, 18]. …”

“…Thereafter, artificial cerebrospinal fluid, with the same composition as in our previous reports [14, 15, 16, 17, 18], was infused through the push cannula and collected from the pull cannula at a flow rate of 15 µl/min. The dead space of the pull system (from the tip of the guide cannula to the distal end of the pull tubing) was adjusted to 225 µl (corresponding to a 15-min period of perfusion), so that each blood sample could be drawn in the middle of each time period.…”

Evidence for a Spontaneous Nitric Oxide Release from the Rat Median Eminence: Influence on Gonadotropin-Releasing Hormone Release*

“…However, in the hypothalamus, the GnRH neurons have been shown, thus far, to be devoid of NOS immunoreactivity (18) and, further supporting this observation, they do not exhibit NADPH diaphorase activity (V. Prevot, personal communication) but do secrete their neurohormone in a pulsatile manner into the portal blood capillaries (37,38). Additionally, NO-donors have been shown, by push-pull perfusion, to directly affect GnRH release (39). Taken together, we surmise that a hypothalamic source of NO, exogenous to GnRH neurons, might control their secretion.…”