Evidence from various sources suggested that the Gonadotropin-Releasing Hormone (GnRH) neuron does not contain glutamate receptors. Northern analysis of the hypothalamus showed the presence of NMDAR1, GluR1, GluR4 and GluR6 mRNA, while the pituitary showed the presence of NMDAR1, GluR1 and GluR6 mRNA. Western blot analysis also showed the presence of NMDAR1 and GluR1 protein. Since there are relatively few GnRH neurons in the hypothalamus, and GT1-7 cells have been considered to be a GnRH neuronal cell line, GT1-7 cells were studied in detail. GT1-7 cells contained NMDAR1 mRNA levels as shown by Northern analysis but did not contain GluR1, GluR4, or GluR6 mRNA. They did not show the presence of NMDAR1 and GluR1 protein by Western analysis. In addition, GT1-7 cells showed no NMDA receptor binding using the competitive inhibitor CGP-39563 and the noncompetitive inhibitor MK-801. Likewise, no binding was detected for kainate receptors. However, a small amount of binding for AMPA receptors was found in GT1-7 cells. GT1-7 cells did not exhibit glutamate toxicity and NMDA failed to elicit inward currents using patch-clamp techniques, although GABA did induce currents in the cells. As a whole, these studies suggest that GT1-7 cells lack or possess only low levels of ionotropic glutamate receptors.

Keywords:
Excitatory amino acids, Excitatory amino-acid receptors, GT1-7 cells, Gonadotropin-releasing hormone, Kainate
1.
Braun DW, Mahesh VB: Excitatory amino acids: Evidence for a role in the control of reproduction and anterior pituitary hormone secretion. Endocr Rev 1997;18:678–700.
2.
Olney JW, Cicero TJ, Meyer E, De Gubareff T: Acute glutamate-induced elevations in serum testosterone and luteinizing hormone. Brain Res 1976;112:420–424.
3.
Ondo JG, Pass KA, Baldwin R: The effects of neurally active amino acids on pituitary gonadotropin secretion. Neuroendocrinolgy 1976;21:79–87.
4.
Price M, Olney J, Cicero T: Acute elevations of serum luteinizing hormone induced by kainic acid, N-methyl aspartic acid or homocysteic acid. Neuroendorcinology 1978;26:352–358.
5.
Urbanski HF, Ojeda SR: Activation of luteinizing hormone-releasing hormone release advances the onset of female puberty. Neuroendocrinology 1987;46:273–276.
6.
Brann DW, Mahesh VB: Endogenous excitatory amino acid involvement in the preovulatory and steroid-induced surge of gonadotropins in the female rat. Endocrinology 1991;128:1541–1542.
7.
Luderer U, Strobl FJ, Levine JE, Schwartz NB: Differential gonadotropin responses to N-methyl-D-aspartate (NMDA) in metestrous, proestrous, and ovariectomized rats. Biol Reprod 1993;48:857–866.
8.
Ondo JG, Wheller DD, Dom RM: Hypothalamic site of action for N-methyl-D-aspartate (NMDA) on LH secretion. Life Sci 1988;43:2283–2286.
9.
Abbud R, Smith MS: Differences in the luteinizing hormone and prolactin responses to multiple injections of kainate as compared to N-methyl-D,L-aspartate, in cycling rats. Endocrinology 1991;129:3254–3258.
10.
Ebling FJ, Hui Y, Mirakhur A, Maywood ES, Hastings MH: Photoperiod regulates the LH response to central glutamatergic stimulation in the male Syrian hamster. J Neuroendocrinol 1993;5:609–618.
11.
Urbanski H, Ojeda S: A role for N-methyl-D-aspartate (NMDA) receptors in the control of LH secretion and initiation of female puberty. Endocrinology 1990;126:1774–1776.
12.
Lopez F, Donoso AO, Negro-Vilar A: Endogenous excitatory amino acid neurotransmission regulates the estradiol-induced LH surge in ovariectomized rats. Endocrinology 1990;126:1771–1773.
13.
Brann DW, Ping L, Mahesh VB: Possible role of non-NMDA receptor mediated neurotransmission in steroid induced and preovulatory gonadotropin surges in the rat. Mol Cell Neurosci 1993;4:292–297.
14.
Zuo Z, Mahesh VB, Zamorano PL, Brann DW: Decreased gonadotropin-releasing hormone neurosecretory response to glutamate agonists in middle-aged rats on proestrus afternoon: A possible role in reproductive aging. Endocrinology 1996;137:2334–2338.
15.
Donoso AO, Lopez FJ, Negro-Vilar A: Glutamate receptors of the non-N-methyl-D-aspartic acid type mediate the increase in luteinizing hormone-releasing hormone release by excitatory amino acids in vitro. Endocrinology 1990;126:414–420.
16.
Lopez FJ, Donoso AO, Negro-Vilar A: Endogenous excitatory amino acids and glutamate receptor subtypes involved in the control of hypothalamic luteinizing hormone-releasing hormone secretion. Endocrinology 1992;130:1986–1992.
17.
Urbanski HF: Excitatory amino acids and the control of seasonal breeding; in Brann DW, Mahesh VB (eds): Excitatory Amino Acids: Their Role in Neuroendocrine Function. Boca Raton, CRC Press, 1996, pp 253–280.
18.
Urbanski HF, Daschel M, Lew NM, Fahy MM: N-Methyl-D-aspartate receptor gene expression in the hamster hypothalamus and in immortalized luteinizing hormone releasing hormone neurons. J Reprod Fertil 1994;100:5–9.
19.
Abbud R, Smith S: Do GnRH neurons express the gene for the NMDA receptor. Brain Res 1995;690:117–120.
20.
Eyigor O, Jennes L: Identification of glutamate receptor subunit mRNAs in gonadotropin-releasing hormone neurons in rat brain. Endocrine 1996;4:133–139.
21.
Lee W, Abbud K, Hoffman GE, Smith MS: Effects of N-methyl-D-aspartate receptor activation on c-fos expression in luteinizing hormone-releasing hormone neurons in female rats. Endocrinology 1993;133:2248-2254.
22.
Gore AC, Wu TJ, Roserberg JJ, Roberts JL: Gonadotropin-releasing hormone and NMDA receptor gene expression and colocalization change during puberty. J Neurosci 1996;16:5281–5289.
23.
Urbanski HF, Kohama SG, Garyfallou VT: Mechanisms mediating the response of GnRH neurons to excitatory amino acids. Rev Reprod 1996;1:173–181.
24.
Mellon PL, Windle JJ, Goldsmith PC, Padula CA, Roberts JL, Weiner RI: Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 1990;5:1–10.
25.
Mahachoklertwattana P, Sanchez J, Kaplan S, Grumbach M: N-methyl-D-aspartate (NMDA) receptors mediate the release of gonadotropin-releasing hormone (GnRH) by NMDA in a hypothalamic GnRH neuronal cell line (GT1-7). Endocrinology 1994;134:1023–1030.
26.
Spelberg PJ, Krsmanovic L, Stojilkovic S, Catt K: Glutamate modulates calcium and gonadotropin-releasing hormone secretion in immortalized hypothalamic GT1-7 neurons. Neuroendocrinology 1994;134:1023–1030.
27.
Peduto JC, Mahesh VB: Effects of progesterone on hypothalamic and plasma LHRH. Neuroendocrinology 1985;40:238–245.
28.
Chomczynshi P: A reagent for the single-step simultaneous isolation of RNA, DNA and protein from cell and tissue samples. Biotechniques 1993;15:532–535.
29.
Kutsuwada T, Kashiwabuchi N, Mori H, Sakimura K, Kushiya E, Arabi K, Meguro H, Masaki H, Kumanishi T, Arakawa M, Mishima M: Heterogenic diversity of the NMDA receptor channel. Nature 1992;358:36–41.
30.
Monyer H, Sprengel R, Schaepler K, Herb A, Higuchi M, Lomeli H, Burnashev N, Sakmann B, Seeburg PH: Heteromeric NMDA receptor: Molecular and functional distinction of subtypes. Science 1992;256:1217–1221.
31.
Hollmann M, O’Shea-Greenfield A, Rogers SW, Heinemann S: Cloning by functional expression of a member of the glutamate receptor family. Nature 1989;342:643–648.
32.
Keinamen K, Urisden W, Sommer B, Werner P, Herb A, Verdoorn TA, Sakmann B, Seeburg PA: A family of AMPA-selective glutamate receptor. Science 1990;249:556–560.
33.
Bettler B, Boulter S, Hermans-Borgmeyer I, O’Shea-Greenfield A, Deneris ES, Moll C, Borgmeyer U, Hollmann M, Heinemann S: Cloning of a novel glutamate receptors subunit, GluR5: Expression in the nervous system during development. Neuron 1990;5:583–595.
34.
Werner P, Voigt M, Keinanen K, Wisden W, Seeburg PA: Cloning of a putative high affinity kainate receptor expresses predominantly in hippocampal CA3 cells. Nature 1991;351:742–744.
35.
Egebjer J, Bettler B, Hermans-Borgmeyer I, Heinemann S: Cloning of a cDNA for a glutamate receptor subunit activated by kainate but not AMPA. Nature 1991;351:745–748.
36.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–275.
37.
Laemmli UK: Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 1970;227:680–685.
38.
Murphy D, Hutchison A, Hurt S, Williams M, Sills M: Characterization of the binding of [3H]CGS 19755: A novel N-methyl-D-aspartate antagonist with nanomolar activity in rat brain. Br J Pharmacol 1988;95:932–938.
39.
Sills MA, Fagg G, Pozza M, Angst C, Brundish DE, Hunt SD, Wilazz EJ, Williams M: [3H]-CGP.39563: A new N-methyl-D-aspartate antagonist radioligand with low nanomolar affinity in rat brain. Eur J Pharmacol 1991;192:19–24.
40.
London ED, Coyle JT: Specific binding of [3H]-kainic acid to receptor sites in rat brain. Mol Pharmacol 1978;15:492–505.
41.
Toceo G, Short TJ, Baudry M, Thompson RF: Selective increase of AMPA binding to the AMPA quisqualate receptor in the hippocampus in response to acute stress. Brain Res 1991;559:168–171.
42.
Scatchard G: The attraction of protein for small molecules and ions. Am NY Acad Sci 1949;51:660–672.
43.
Younkin DP, Tang CM, Hardy M, Reddy UR, Shi QY, Pleasure SJ, Lee VM, Pleasure D: Inducible expression of neuronal glutamate receptor channels in the NT2 human cell line. Proc Natl Acad Sci USA 1993;90:2174–2178.
44.
Hamil OP, Marty A, Neher E, Sakmann B, Sigworth FJ: Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 1981;391:85–100.
45.
Hales TG, Sandrson MJ, Charles AC: GABA has excitatory actions on GnRH-secreting immortalized hypothalamic (GT1-7) neurons. Neuroendocrinology 1994;59:297–308.
46.
Ping L, Mahesh VB, Bhat GK, Brann DW: Regulation of gonadotropin-releasing hormone and luteinizing hormone secretion by AMPA receptors. Neuroendocrinology 1997;66:246–253.
47.
Ebling FJP, Cronin AS, Hastings MH: Resistance of GnRH neurons to glutamatergic neurotoxicity. Brain Res Bull, in press.
48.
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 co-localization with N-methyl-D-aspartate receptors. Neuroendocrinology 1995;62:187–197.
49.
McCann SM, Rettori V: The role of nitric oxide in reproduction. Proc Soc Exp Biol Med 1996;211:7–15.
50.
Mahachoklertwattana P, Black S, Kaplan S, Bristoe J, Grumbach M: 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.
51.
Grossman A, Rossmanith W, Kabigting E, Cadd G, Clifton D, Steiner R: The distribution of hypothalamic nitric oxide synthase mRNA in relation to gonadotrophin-releasing hormone neurons. J Endocrinol 1994;140:R5–R8.
52.
Herbison AE, Simonian SX, Norris J, Emson PC: Relationship of neuronal nitric oxide synthase immunoreactivity to GnRH neurons in the ovariectomized and intact female rat. J Neuroendocrinol 1996;8:73–82.
53.
Bhat G, Mahesh V, 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.
54.
Moretto M, Lopez FJ, Negro-Vilar A: Nitric oxide regulates luteinizing hormone-releasing hormone secretion. Endocrinology 1993;133:2399–2402.
55.
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.
56.
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.
57.
Aguan K, Mahesh VB, Ping L, Bhat G, Brann DW: Evidence for a physiological role for nitric oxide in the regulation of the LH surge: Effect of central administration of antisense oligonucleotides to nitric oxide synthase. Neuroendocrinology 1997;64:449–455.
58.
Pu S, Xu B, Kalra SP, Kalra PS: Evidence that gonadal steroids modulate nitric oxide efflux in the medial preoptic area: Effects of N-methyl-D-aspartate and correlation with luteinizing hormone secretion. Endocrinology 1996;137:1949–1955.
59.
Pu S, Kalra PS, Kalra SP: Ovarian steroid-independent diurnal rhythm in cyclic GMP/nitric oxide efflux in the medial preoptic area: Possible role in preovulatory and ovarian steroid-induced LH surge. J Neuroendocrinol 1998;10:617–625.
60.
Bhat GK, Mahesh VB, Chu ZW, Chorich LP, Zamorano PL, Brann DW: Localization of the N-methyl-D-aspartate R1 receptor subunit in specific anterior pituitary hormone cell types of the female rat. Neuroendocrinology 1995;62:178–186.
61.
Zanisi M, Galbiati M, Messi E, Martini L: The anterior pituitary gland as a possible site of action of kainic acid. Proc Soc Exp Biol Med 1994;206:431–437.
62.
Yoshioka A, Ikegaki N, Williams M, Pleasure D: Expression of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor genes in neuroblastoma, medulloblastoma, and other cell lines. J Neurol Res 1996;46:164–172.
63.
Jung N, Sun W, Lee H, Cho S, Shim C, Kim K: Gonadotropin-releasing hormone (GnRH) gene regulation by N-methyl-D-aspartic acid in GT1-1 neuronal cells: Differential involvement of c-fox and c-jun protooncogenes. Mol Brain Res 1998;61:162–169.
64.
Favit A, Wetsel WC, Negro-Vilar A: Differential expression of γ-aminobutyric acid receptors in immortalized luteinizing hormone-releasing hormone neurons. Endocrinology 1993;133:1983–1989.
65.
Shi B, Mahesh VB, Bhat GK, Ping L, Brann DW: Evidence for a role of bradykinin neurons in the control of gonadotropin-releasing hormone secretion. Neuroendocrinology 1998;67:209–218.
66.
Negro-Vilar A, Conte D, Valenca M: Transmembrane signals mediating neural peptide secretion: Role of protein kinase C activators and arachidonic acid metabolites in luteinizing hormone-releasing hormone secretion. Endocrinology 1986;119:2796–2802.
67.
King T, Kang I, Javors M, Schenken R: Phorbol esters stimulate in vitro GnRH release from the hypothalamus of the estrogen-primed, ovariectomized rat: Inhibitory effets of cocaine. Neuroendocrinology 1992;56:526–532.
1999
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