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

Abstract

Episodic release of luteinizing hormone (LH) by the pituitary gland is controlled by hypothalamic gonadotropin-releasing hormone (GnRH). In the period leading up to the preovulatory surge of LH, estrogen increases the number of pituitary receptors for GnRH and sensitises the gonadotropes to GnRH. The postreceptor events that are responsible for the increase in responsiveness to GnRH are not clearly delineated, but LH release is known to be Ca²⁺ dependent. The present study addressed the question as to whether or not estrogen may act to modify voltage-dependent Ca²⁺ entry in normal gonadotropes. Primary cultures enriched in gonadotropes or somatotropes were produced from anestrous female sheep. Conventional whole-cell patch-clamp recording was used to measure inward membrane current in the absence of GnRH treatment, with and without 10 nM estradiol-17β (E₂) treatment for 0 to 36 h. Nystatin-perforated whole-cell patch-clamp recording was used to record membrane voltage responses to GnRH. Ca²⁺ current density (Ic_a, pA/pF) began to increase after 2 h exposure to E₂ and reached peak values of about 200% of control by 16-20 h (p < 0.005), then declined. If E₂ was withdrawn at 24 h, I_ca returned towards control values by 36 h. If E₂ treatment was continued beyond 24 h, however, Ic_a fell to about 75% of control by 36 h (p < 0.005). Actinomycin D prevented the enhancement of Ic_a. E₂ was without effect on Na⁺ current density in gonadotropes, or on Ic_a in somatotropes. The proportion of Ic_a carried by L-type and N-type channels in gondadotropes was not changed by E₂. Ovine gonadotropes respond to GnRH with membrane potential fluctuations driven by periodic activation of Ca²⁺-dependent K⁺ channels, and synchronised action potential generation. This response was found to be sensitive to E₂. Responses were categorised according to the pattern of activity evoked by 10 nM GnRH. Without E₂ treatment, 11/14 cells responded with oscillations and 3/14 cells responded with spiking (hyperpolarizations following single action potentials). After 20 h 10 nM E₂, just 1/14 cells responded with hyperpolarizing oscillations while 13/14 cells showed spiking activity. The predominance of the spiking pattern in E₂-treated cells is consistent with the increased Ca²⁺ flux, and with enhanced LH release. We conclude that E₂ has a transient effect on gonadotropes to enhance voltage-gated Ca²⁺ channel function. The time-course and biphasic nature of the influence of E₂ on I_Ca may be physiologically appropriate to the preovulatory LH surge. Enhanced Ca²⁺ influx may participate in increased Ca²⁺-dependent hormone release, while the delayed inhibitory action of E₂ on I_ca may serve to limit the duration of the surge.

Components of the Neuronal Exocytotic Machinery in the Anterior Pituitary of the Ovariectomised Ewe and the Effects of Oestrogen in Gonadotropes as Studied with Confocal Microscopy

“…The fact that we were able to detect synaptotagmin I in ovine gonadotropes is in agreement with previous findings which show that the secretion of LH from the ovine adenohypophysis is dependent upon Ca 2+ [5]. Synaptotagmin I is a Ca 2+ dependent isoform which has been suggested to be a calcium sensor that triggers final membrane fusion.…”

“…This has been shown to involve an increase in the calcium channel conductance of gonadotropes [5] and an increase in potassium channels as well [6]. In other species, such as the rat, the action of estrogen to increase the responsiveness of the gonadotropes to GnRH at the time of the pro-oestrous surge in luteinising hormone (LH) is associated with an increase in the methylation of phospholipids [7] and an increase in the activity of protein kinase C [8, 9].…”

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

“…This may play a fundamental role in the way that E acts to enhance the responsiveness of these cells to GnRH [5, 6, 27], but the exact physiological relevance of the change is not yet established. Previous studies from this laboratory [21]have shown that there is increased Ca 2+ conductance in the membranes of E-treated gonadotropes, as well as altered membrane potential changes in response to GnRH and we have suggested that these factors are important in the mechanisms causing increased GnRH responsiveness. In the present study, we hypothesised that this would be associated with a concomitant alteration in the function of delayed rectifier K + current and this is indeed the case.…”

“…E pretreatment of gonadotropes causes GnRH to elicit a more pronounced hyperpolarisation followed by more frequent action potentials [21]. After E treatment, cells must be able to hyperpolarise at a faster rate so that Na + and Ca 2+ channels can be more rapidly reactivated to allow further action potentials to be generated.…”

“…Hyperpolarisation in rat gonadotropes is mainly caused by an increase in K + conductance although this channel has not been fully characterised [20]. In ovine cells E causes an increase in Ca 2+ current density, but does not change Na + current [21]. The more profound hyperpolarisation that is seen in E-primed ovine gonadotropes in response to GnRH is probably due to increased efflux of positive ions, although it might also be due to decreased influx of positive ions (Na + ) or greater influx of negative ions (Cl – ).…”