title: GABAA receptor mediated elevation of Ca2+ and modulation of gonadotrophin-releasing hormone action in alphaT3-1 gonadotropes.
abstract: gamma-amino butyric acid (GABA) is the major inhibitory neurotransmitter in the CNS, mediating fast inhibitory synaptic transmission, by activating GABAA receptors. However, these GABA-gated Cl- channels can also be excitatory, causing depolarization, and increasing Ca2+ entry via voltage-operated Ca2+ channels (VOCCs). Evidence exists for excitatory ionotropic GABA receptors in anterior pituitary cells, including gonadotropes, but these have not been directly characterized and their pharmacology remains controversial. Here we have measured the cytosolic Ca2+ concentration ([Ca2+]i) in alphaT3-1 gonadotropes, to test for expression of excitatory GABA receptors. The GABAA agonists, GABA and muscimol, both caused rapid, robust and dose-dependent increases in [Ca2+]i (EC50 values 2.7 and 1 microM), whereas the GABAB agonist, baclofen, did not. The GABAA antagonist, bicuculline, inhibited muscimol's effect, whereas the GABAB antagonist, phaclofen, did not. The neuroactive steroid 5alpha-pregnan-3alpha-ol-11,20-dione (an allosteric activator of GABAA receptors) increased [Ca2+]i, and this effect, like that of muscimol, was inhibited by picrotoxin. The muscimol effect on [Ca2+]i was blocked by the VOCC antagonist, nifedipine, or by Ca2+-free medium. When cells were pretreated with muscimol this increased the spike phase of the [Ca2+]i response to subsequent stimulation with gonadotropin-releasing hormone (GnRH). Similar amplification was seen in muscimol-pretreated cells stimulated with GnRH in Ca2+-free medium, but not when cells were pretreated with muscimol in Ca2+-free medium. The amplification was not, however, GnRH receptor-specific, because the spike response to ionomycin was also increased by muscimol pretreatment. These data provide the first direct evidence for expression of excitatory GABAA receptors, and the first demonstration of acute steroid effects, on GnRH-responsive pituitary cells. They also reveal a novel mechanism by which GABAA activation modulates GnRH action, raising the possibility that this may also influence gonadotrophin secretion from non-immortalized gonadotropes.
authors.affiliation: University of Bristol, Division of Medicine, Bristol, UK.
authors.forename: B
authors.initials: B
authors.lastname: Williams
authors.forename: M
authors.initials: M
authors.lastname: Bence
authors.forename: H
authors.initials: H
authors.lastname: Everest
authors.forename: W
authors.initials: W
authors.lastname: Forrest-Owen
authors.forename: S L
authors.initials: SL
authors.lastname: Lightman
authors.forename: C A
authors.initials: CA
authors.lastname: McArdle
mesh_terms: D000777:Anesthetics / Q000494:pharmacology; D000818:Animals; D001418:Baclofen / Q000494:pharmacology; D001640:Bicuculline / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D018755:GABA Agonists / Q000494:pharmacology; D018756:GABA Antagonists / Q000494:pharmacology; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D051379:Mice; D009118:Muscimol / Q000494:pharmacology; D009543:Nifedipine / Q000494:pharmacology; D010852:Picrotoxin / Q000494:pharmacology; D010902:Pituitary Gland / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D011277:Pregnanediones / Q000494:pharmacology; D011963:Receptors, GABA-A / Q000378:metabolism*; D018080:Receptors, GABA-B / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D000777:Anesthetics; D001418:Baclofen; D001640:Bicuculline; D002118:Calcium; D002121:Calcium Channel Blockers; D002712:Chlorides; D018755:GABA Agonists; D018756:GABA Antagonists; D007987:Gonadotropin-Releasing Hormone; D009118:Muscimol; D009543:Nifedipine; D010852:Picrotoxin; D011277:Pregnanediones; D011963:Receptors, GABA-A; D018080:Receptors, GABA-B; D005680:gamma-Aminobutyric Acid; C006477:alphaxalone