In the last few years, nitric oxide (NO) has emerged as an important intra- and intracellular messenger involved in the control of hypothalamic-pituitary function. The present experiments were undertaken in order to evaluate the pituitary component in the modulatory action of NO on gonadotropin secretion, as well as the second messenger pathway(s) involved. In a first step, we assessed LH and FSH secretion by hemipituitaries incubated in the presence of increasing concentrations of sodium nitroprusside (SNP), a potent NO donor, and cyclic guanosin monophosphate (cGMP), the second messenger for a wide range of NO actions. In addition, given that SNP induces the release of NO and cyanide ions, the response to SNP was tested in the presence of hemoglobin (an NO scavenger) or rhodanese + sodium thiosulfate (inactivators of cyanides) in order to ensure that the effects of SNP on gonadotropin secretion were mediated by the release of NO. SNP (10–4–10–3 M) stimulated gonadotropin secretion in our incubation system, whereas cGMP, at all doses tested, was ineffective. Similar results were obtained using dispersed pituitary cells. The stimulatory action of SNP is attributable to its ability to induce NO release since it was blocked by hemoglobin, but preserved after incubation with rhodanese + sodium thiosulfate. In further experiments, we aimed to identify the mechanism(s) underlying SNP-induced gonadotropin secretion. First, to evaluate the involvement of calcium (Ca2+), the effects of SNP were analyzed in a calcium-free medium, after depletion of Ca2+ stores by caffeine, in the presence of the Ca2+ chelator ethylene glycol bis (p-aminoethyl ether) N,N-tetra-acetic acid (EGTA), and after incubation with the Ca2+ channel blockers verapamil and nifedipine. Second, to confirm that cGMP is not involved in the stimulatory action of SNP, the effects of the latter on gonadotropin secretion were tested in the presence of the antagonists of the guanylyl cyclases oxadiazoloquinoxaline and LY 83,583. Our results showed that the stimulatory action of SNP on gonadotropin release is blunted in Ca2+-free medium and after incubation with EGTA, verapamil, nifedipine, and caffeine. On the contrary, the effect of SNP remained unaltered after antagonization of guanylyl cyclases. We conclude that NO, acting at the pituitary level, stimulates gonadotropin secretion through a calcium-dependent, cGMP-independent mechanism.

Keywords:
Nitric oxide, Gonadotropins, cGMP, Ca2+
1.
Brann DW, Bhat GK, Lamar CA, Mahesh VB: Gaseous transmitters and neuroendocrine regulation. Neuroendocrinology 1997;65:385–395.
2.
Palmer RMJ, Ashton DS, Moncada S: Vascular endothelial cells synthesise nitric oxide from L-arginine. Nature 1988;333:664–666.
3.
Bredt DS, Hwang PM, Snyder SH: Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature 1990;347:768–770.
4.
Bhat GK, Mahesh VB, Lamar CA, Ping L, Aguan K, Brann DW: Histochemical localization of nitric oxide neurons in the hypothalamus: Association with gonadotropin-releasing hormone neurons and colocalization with N-methyl-D-aspartate receptors. Neuroendocrinology 1995;62:187–197.
5.
Bredt DS, Hwang PM, Glatt CE, Lowenstein C, Reed RR, Snyder SH: Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature 1991;351:714–718.
6.
Ceccatelli S, Hulting AL, Zhang X, Gustafsson L, Villar M, Hökfelt T: Nitric oxide synthase in the rat anterior pituitary gland and the role of nitric oxide in regulation of luteinizing hormone secretion. Proc Natl Acad Sci USA 1993;90:11292–11296.
7.
Moretto M, López FJ, Negro-Vilar A: Nitric oxide regulates luteinizing hormone-releasing hormone secretion. Endocrinology 1993;133:2399–2402.
8.
Rettori V, Belova N, Dees WL, Nyberg CL, Gimeno M, McCann SM: Role of nitric oxide in the control of luteinizing hormone-releasing hormone release in vivo and in vitro. Proc Natl Acad Sci USA 1993;90:10130–10134.
9.
Bonavera JJ, Sahu A, Kalra PS, Kalra SP: Evidence that nitric oxide may mediate the ovarian steroid-induced luteinizing hormone surge: Involvement of excitatory amino acids. Endocrinology 1993;133:2482–2487.
10.
Aguila MC: Growth hormone-releasing factor increases somatostatin release and mRNA levels in the rat periventricular nucleus via nitric oxide by activation of guanylate cyclase. Proc Natl Acad Sci USA 1994;91:782–786.
11.
Karanth S, Lyson K, McCann SM: Role of nitric oxide in interleukin 2-induced corticotropin-releasing factor release from incubated hypothalami. Proc Natl Acad Sci USA 1993;90:3383–3387.
12.
Yu WH, Walczewska A, Karanth S, McCann SM: Nitric oxide mediates leptin-induced luteinizing hormone-releasing hormone (LHRH) and LHRH and leptin-induced LH release from the pituitary gland. Endocrinology 1997;138:5055–5058.
13.
Kato M: Involvement of nitric oxide in growth hormone (GH)-releasing hormone-induced GH secretion in rat pituitary cells. Endocrinology 1992;131:2133–2138.
14.
Tena-Sempere M, Pinilla L, Gonzalez D, Aguilar E: Involvement of endogenous nitric oxide in the control of pituitary responsiveness to different elicitors of growth hormone release in prepubertal rats. Neuroendocrinology 1996;64:146–152.
15.
Duvilanski BH, Zambruno C, Seilicovich A, Pisera D, Lasaga M, Diaz MC, Belova N, Rettori V, McCann SM: Role of nitric oxide in control of prolactin release by the adenohypophysis. Proc Natl Acad Sci USA 1995;92:170–174.
16.
Bonavera JJ, Sahu A, Kalra PS, Kalra SP: Evidence in support of nitric oxide (NO) involvement in the cyclic release of prolactin and LH surges. Brain Res 1994;660:175–179.
17.
Bonavera JJ, Kalra PS, Kalra SP: L-Arginine/nitric oxide amplifies the magnitude and duration of the luteinizing hormone surge induced by estrogen: Involvement of neuropeptide Y. Endocrinology 1996;137:1956–1962.
18.
Mahachoklertwattana P, Black SM, Kaplan SL, Bristow JD, Grumbach MM: Nitric oxide synthesized by gonadotropin-releasing hormone neurons is a mediator of N-methyl-D-aspartate (NMDA)-induced GnRH secretion. Endocrinology 1994;135:1709–1712.
19.
Rettori V, Belova N, Kamat A, Lyson K, Gimeno M, McCann SM: Blockade by interleukin-1-alpha of nitricoxidergic control of luteinizing hormone-releasing hormone release in vivo and in vitro. Neuroimmunomodulation 1994;1:86–91.
20.
Fehsel K, Jalowy A, Qi S, Burkart V, Hartmann B, Kolb H: Islet cell DNA is a target of inflammatory attack by nitric oxide. Diabetes 1993;42:496–500.
21.
Lara B, López MG, Villaroya M, Gandia L, Cleeman L, Morad M, Garcia AG: A caffeine-sensitive Ca2+ store modulates K+-evoked secretion in chromaffin cells. Am J Physiol 1997;272:C1211–1221.
22.
Greenwood FC, Hunter WM, Glover JS: The preparation of 131I-labelled human growth hormone of high specific radioactivity. Biochem J 1963;89:114–123.
23.
Murad F: The nitric oxide-cyclic GMP signal transduction system for intracellular and intercellular communication. Rec Prog Horm Res 1994;49:239–249.
24.
Bhat G, Mahesh VB, Aguan K, Brann DW: Evidence that brain nitric oxide synthase is the major nitric oxide synthase isoform in the hypothalamus of the adult female rat and that nitric oxide potently regulates hypothalamic cGMP levels. Neuroendocrinology 1996;64:93–102.
25.
Naor Z, Catt KJ: Independent actions of gonadotropin releasing hormone upon cyclic GMP production and luteinizing hormone release. J Biol Chem 1980;255:343–344.
26.
Nakano H, Fawcett CP, Kimura F, McCann SM: Evidence for the involvement of guanosine 3′,5′-monophosphate in the regulation of gonadotropin release. Endocrinology 1978;103:1527–1533.
27.
McArdle CA, Poch A, Käppler K: Cyclic guanosine monophosphate production in the pituitary: Stimulation by C-type natriuretic peptide and inhibition by gonadotropin-releasing hormone in αT3-1 cells. Endocrinology 1993;132:2065–2072.
28.
Abou-Samra AB, Catt KJ, Aguilera G: Synthetic atrial natriuretic factors (ANFs) stimulate guanine 3′,5′-monophosphate production but not hormone release in rat pituitary cells: peptide contamination with a gonadotropin-releasing hormone agonist explains luteinizing hormone-releasing activity of certain ANFs. Endocrinology 1987;120:18–24.
29.
Brunner F, Stessel H, Kukovetz WR: Novel guanylyl cyclase inhibitor, ODQ, reveals role of nitric oxide, but not of cyclic GMP in endothelin-1 secretion. FEBS Lett 1995;376:262–266.
30.
Sun P, Kanthasamy A, Yim GK, Isom GE: Inhibiton of potassium-stimulated dopamine release by the nitric oxide generator isosorbide dinitrate. Neuropharmacology 1995;34:205–210.
31.
Conn PM, Marian J, McMillian M, Stern J, Rogers D, Hamby M, Penna A, Grant E: Gonadotropin-releasing hormone action in the pituitary: A three step mechanism. Endocr Rev 1981;2:174–185.
32.
Stojilkovic SS, Tomic M: GnRH-induced calcium and current oscillations in gonadotrophs. Trends Endocrinol Metab 1996;7:379–384.
33.
Conn PM, Rogers DC, Sandhu FS: Alteration of intracellular calcium levels stimulates gonadotropin release from cultured anterior pituitary cells. Endocrinology 1979;105:1122–1127.
34.
Conn PM, Rogers DR: Gonadotropin release from pituitary cultures following activation of endogenous ion channels. Endocrinology 1980;107:2133–2134.
35.
Conn PM, Kilpatrick D, Kirshner N: Ionophoretic Ca2+ mobilization in rat gonadotropes and bovine adrenomedullary cells. Cell Calcium 1980;1:129–133.
36.
Stojilkovic SS, Catt KJ: Calcium oscillations in anterior pituitary cells. Endocr Rev 1992;13:256–280.
37.
Stojilkovic SS, Izumi SI, Catt KJ: Participation of voltage-sensitive calcium channels in pituitary hormone release. J Biol Chem 1986;15:13954–14061.
38.
Stutzin A, Stojilkovic SS, Catt KJ, Rojas E: Characteristics of two types of calcium channels in rat pituitary gonadotrophs. Am J Physiol 1989;257:C865–874.
39.
Laffranchi R, Gogvadze V, Richter C, Spinas GA: Nitric oxide (nitrogen monoxide, NO) stimulates insulin secretion by inducing calcium release from mitochondria. Biochem Biophys Res Commun 1994;217:584–591.
40.
Willmott NJ, Galione A, Smith PA: Nitric oxide induces intracellular Ca2+ mobilization and increases secretion of incorporated 5-hydroxytryptamine in rat pancreatic beta-cells. FEBS Lett 1995;371:99–104.
41.
Ciranna L, Feltz P, Schlichter R: Selective inhibition of high voltage-activated L-type and Q-type Ca2+ currents by serotonin in rat melanotrophs. J Physiol (Lond) 1996;490:595–609.
42.
Kuryshev YA, Childs GV, Ritchie AK: Corticotropin-releasing hormone stimulates Ca2+ entry through L-and P-type Ca2+ channels in rat corticotropes. Endocrinology 1996;137:2269–2277.
1998
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