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 < 10nM), 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 µM TTX. This current was abolished by ionic Ca2+ channel blockade. In the presence of nifedi-pine 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 µM 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 Ca2+-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 10nM 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, Ca2+-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.
- Gonadotropin-releasing hormone
- Patch clamp