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 < 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.

A Transient Effect of Estrogen on Calcium Currents and Electrophysiological Responses to Gonadotropin-Releasing Hormone in Ovine Gonadotropes

“…The method of partial purification of dispersed sheep pituitary' cell suspensions to produce gonadotrope-enriched monolayer cul tures by Percoll density-gradient centrifugation, and immunocytochemical identification of cells containing LH, have been described in full [25,15], For the present work, pituitaries were obtained from female sheep during the nonbreeding season. Examination of the ovaries at slaughter confirmed that the animals were anestrous.…”

“…Responses to GnRH were recorded using turbulence-free microperifusion within the plastic culture dishes, as previously described [ 15). A laminar flow perifusion system (Scintilla, Melbourne, Austra lia) was used to provide rapid complete changes of bathing medium, with continuous perifusion by peristaltic pump.…”

“…BES 5. glucose 10,95%02/5% CO: saturated. To record responses to GnRH with minimal disturbance to intracellular biochemistry, nystatin was used in the pipette-filling solution to perforate the cell membrane under the recording pipette tip, as previously described [15]. Nystatin (0.5 mg/ml)-filled pipettes contained (in mV/) KC1 55, K 2S04 75, MgSO" 8, HEPES 10.…”

Estrogen Transiently Increases Delayed Rectifier, Voltage-Dependent Potassium Currents in Ovine Gonadotropes

“…Gonadotropes are excitable cells and exhibit spontaneous action potentials [11, 12, 13, 14, 15, 16]. Treatment of gonadotropes with GnRH stimulates formation of IP 3 which stimulates Ca 2+ release from intracellular stores [reviewed in refs.…”

“…17, 18]. This causes hyperpolarisation of the cell, followed by oscillations in membrane potential with bursts of action potentials generated at depolarised membrane potentials [12, 13, 16]. Ca 2+ enters the cell during action potentials [12, 19]to replenish intracellular stores, and to increase intracellular free Ca 2+ levels which causes hormone secretion [19].…”