Nitric oxide modulates the release of corticotropin-releasing hormone from the rat hypothalamus in vitro

Abstract

There is now considerable evidence that nitric oxide (NO) is an important neuroregulatory agent, but there has been very little investigation of the possible role of NO in neuroendocrine mechanisms. We have previously shown that acute rat hypothalamic explants can be used to study the regulation of hypothalamic neuropeptide release, and we have now utilised this experimental approach to investigate the putative involvement of NO in the control of the principal corticotropin-releasing hormone, CRH. We studied the direct effects of the NO precursor l-arginine ( l-ARG), as well as the NO donors molsidomine and sodium nitroprusside, on both the basal and stimulated release of CRH; the stimuli used were non-specific depolarisation with potassium chloride (KCl) and the specific cytokine, interleukin-1β (IL-1β; 100 U/ml). l-ARG was tested in each experimental condition with and without contemporaneous addition of its competitive antagonist N G-monomethyl- l-arginine ( l-NMMA). IL-1β-induced CRH release was also investigated in the presence of d-arginine ( d-ARG), which is not active as a precursor to NO, and ferrous hemoglobin (Hb), a substance which is a potent inactivator of NO. None of the NO precursors ( l-ARG, molsidomine, sodium nitroprusside) or antagonist ( l-NMMA or Hb) was able to affect basal CRH release. However, l-ARG 10 and 100 μM were found to significantly inhibit the release of CRH induced by 40 mM KCl; CRH fell to 45% of its stimulated level at the higher dose of l-ARG. This effect was attenuated in the presence of l-NMMA at a ten-fold higher dose. Equally, molsidomine dose-dependently attenuated both 28 mM and 40 mM KCl-induced CRH release. A significant inhibitory effect on KCl 40 mM-induced CRH secretion was also shown by 100 μM and 10 μM sodium nitroprusside. The cytokine IL-1β 100 U/ml significantly stimulated the release of hypothalamic CRH compared to control incubation vials; this stimulation was significantly prevented by both 100 μM and 10 μM l-ARG. The CRH response to IL-1β was, however, fully restored by the addition of either l-NMMA or 10 μM Hb. Both sodium nitroprusside 1 μM and molsidomine 100 μM also significantly inhibited IL-1β-induced CRH release, while the addition of 10 μM Hb was found to antagonize both molsidomine and sodium nitroprusside effects. The l-ARG stereoisomer, d-ARG, was without effect on IL-1β-induced CRH secretion. Thus, NO appears to directly and specifically inhibit the stimulated release of CRH from rat hypothalamic explants in vitro, while leaving basal CRH secretion unaffected. These findings are the first direct evidence that NO may be involved in neuroendocrine regulation, most specifically, that it may modulate the release of a hypothalamic neurohormone. The blockade of the effect of IL-1β by NO also suggests that certain neuroendocrine-immune interactions may be modulated by NO.