title: Inward membrane currents and electrophysiological responses to GnRH in ovine gonadotropes.
abstract: We have used conventional whole-cell patch-clamp to investigate the membrane currents of ovine anterior pituitary gonadotropes, and nystatin-perforated whole-cell patch-clamp to record the membrane potential changes elicited by the natural hypothalamic secretagogue, gonadotropin-releasing hormone (GnRH). A large basal inward current found by voltage clamp was blocked by tetrodotoxin (TTX) (ED50 < 10 nM), identifying it as a Na+ current. Slowly inactivating inward current, activated at potentials more positive than -30 mV, remained in Na(+)-free medium or in the presence of 1 microM TTX. This current was abolished by ionic Ca2+ channel blockade. In the presence of nifedipine about 70% of this high voltage-activated Ca2+ current was abolished, leaving a slowly inactivating component. No transient Ca2+ current was found. The nifedipine-insensitive slowly inactivating inward current was eliminated by 1 microM omega-conotoxin GVIA (CGTX), consistent with the presence of N-type channels. Outward K+ currents sensitive to membrane voltage and intracellular Ca2+ concentration ([Ca2+]i) were present. The resting membrane potential lay between -20 and -75 mV (mean = -43 +/- 1.5) with spontaneous TTX-sensitive action potentials occurring in 34% of cells. GnRH had concentration-dependent effects on gonadotrope membrane potential. Application of 100 nM GnRH resulted in a rapid hyperpolarization, followed by a gradual depolarization during which action potentials returned briefly. This was followed by protracted electrical quiescence. Application of 1 or 10 nM GnRH led to hyperpolarization, followed by gradual depolarization, upon which rhythmic hyperpolarizations were superimposed, giving membrane potential oscillations. During the depolarising stage of each oscillation, a burst of action potentials occurred. Action potentials, then oscillations, ceased after 5-15 min. Depolarization was then maintained (at -20 to -35 mV) for up to 1 h. Apamin, the SK-type Ca(2+)-dependent K+ channel blocker, prevented the hyperpolarizing oscillations and produced membrane depolarisation, but Ca2+ channel blockade did not. Microfluorimetric detection of [Ca2+]i showed that 10 nM GnRH induced [Ca2+]i oscillations. We conclude that Ca2+ derived from intracellular pools is involved in producing the membrane potential oscillations. The [Ca2+]i fluctuations may activate the apamin-sensitive, Ca(2+)-dependent SK-type K+ channel, and entrain TTX-sensitive action potentials to a bursting pattern of generation following GnRH stimulation. In the absence of T-type currents, the Na+ current spikes may be crucial for activation of the nifedipine- and CGTX-sensitive high-voltage-activated Ca2+ channels.
authors.affiliation: Prince Henry's Institute of Medical Research, Clayton, Vic., Australia.
authors.forename: P M
authors.initials: PM
authors.lastname: Heyward
authors.forename: C
authors.initials: C
authors.lastname: Chen
authors.forename: I J
authors.initials: IJ
authors.lastname: Clarke
mesh_terms: D000818:Animals; D001030:Apamin / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004594:Electrophysiology; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology*; D007473:Ion Channels / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D009761:Nystatin / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010902:Pituitary Gland / Q000187:drug effects / Q000378:metabolism*; D012756:Sheep; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013757:Tetraethylammonium Compounds / Q000494:pharmacology; D013779:Tetrodotoxin / Q000494:pharmacology
chemical_list: D015220:Calcium Channels; D007473:Ion Channels; D015222:Sodium Channels; D013757:Tetraethylammonium Compounds; D009761:Nystatin; D001030:Apamin; D007987:Gonadotropin-Releasing Hormone; D013779:Tetrodotoxin