Optimized amplification and single-cell analysis identify GnRH-mediated activation of Rap1b in primary rat gonadotropes.

Identifying the early gene program induced by GnRH would help understand how GnRH-activated signaling pathways modulate gonadotrope secretory response. We previously analyzed GnRH-induced early genes in LßT2 cells, however these lack GnRH self-potentiation, a physiological attribute of gonadotropes. To minimize cellular heterogeneity, rat primary pituitary cultures were enriched for gonadotropes by 40-60% using a sedimentation gradient. Given the limited number of gonadotropes, RNA was amplified prior to microarray analysis. Thirty-three genes were up-regulated 40 min after GnRH stimulation. Real-time PCR confirmed regulation of several transcripts including fosB, c-fos, egr-2 and rap1b, a small GTPase and member of the Ras family. GnRH stimulated rap1b gene expression in gonadotropes, measured by a sensitive single cell assay. Immunocytochemistry revealed increased Rap1 protein in GnRH-stimulated gonadotropes. These data establish rap1b as a novel gene rapidly induced by GnRH and a candidate to modulate gonadotropin secretion in rat gonadotropes.

Ca2+-activated K+ channels in gonadotropin-releasing hormone-stimulated mouse gonadotrophs.

GnRH receptor activation elicits release of intracellular Ca(2+), which leads to secretion and also activates Ca(2+)-activated ion channels underlying membrane voltage changes. The predominant Ca(2+)-activated ion channels in rat and mouse gonadotrophs are Ca(2+)-activated K(+) channels. To establish the temporal relationship between GnRH-induced changes in intracellular [Ca(2+)] ([Ca(2+)](i)) and membrane current (I(m)), and to identify specific Ca(2+)-activated K(+) channels linking GnRH-induced increase in [Ca(2+)](i) to changes in plasma membrane electrical activity, we used single female mouse gonadotrophs in the perforated patch configuration of the patch-clamp technique, which preserves signaling pathways. Simultaneous measurement of [Ca(2+)](i) and I(m) in voltage-clamped gonadotrophs revealed that GnRH stimulates an increase in [Ca(2+)](i) that precedes outward I(m), and that activates two kinetically distinct currents identified, using specific toxin inhibitors, as small conductance Ca(2+)-activated K(+) (SK) current (I(SK)) and large (big) conductance voltage- and Ca(2+)-activated K(+) (BK) current (I(BK)). We show that the apamin-sensitive current has an IC(50) of 69 pM, consistent with the SK2 channel subtype and confirmed by immunocytochemistry. The magnitude of the SK current response to GnRH was attenuated by 17beta-estradiol (E(2)) pretreatment. Iberiotoxin, an inhibitor of BK channels, completely blocked the residual apamin-insensitive outward I(m), substantiating that I(BK) is a component of the GnRH-induced outward I(m). In contrast to its suppression of I(SK), E(2) pretreatment augmented peak I(BK). SK or BK channel inhibition modulated GnRH-stimulated LH secretion, implicating a role for these channels in gonadotroph function. In summary, in mouse gonadotrophs the GnRH-stimulated increase in [Ca(2+)](i) activates I(SK) and I(BK), which are differentially regulated by E(2) and which may be targets for E(2) positive feedback in LH secretion.

Estradiol inhibition of voltage-activated and gonadotropin-releasing hormone-induced currents in mouse gonadotrophs.

We report the first study of voltage-activated and GnRH-induced plasma membrane currents and their modulation by estradiol (E2) in mouse gonadotrophs. In consideration of the pleiotropic effects of E2 on gonadotrophin secretion and the relationship between plasma membrane electrical excitability and secretion, our objective was to determine the role of E2 in modulating gonadotroph plasma membrane currents. We measured total voltage-activated and GnRH-induced currents using the perforated-patch configuration of the patch-clamp technique, which preserves signaling pathways, including GnRH-induced Ca2+ oscillations. We show that female mouse gonadotrophs are similar to those from other species in that the voltage-activated net current response exhibits an inward fast activating current that is inhibited by tetrodotoxin, which is characteristic of a Na+ current, and a larger magnitude outward current with a profile suggesting the presence of multiple K+ currents. Furthermore, in voltage-clamped mouse gonadotrophs, GnRH activates large amplitude current oscillations that are apamin sensitive and have a reversal potential of -90 mV, consistent with Ca2+-activated K+ currents. Significantly, E2 pretreatment for 2-5 d decreased the density of both the peak outward voltage-activated current and the peak GnRH-induced current. The specific linkage between the observed E2 effects on membrane currents and, ultimately, gonadotroph function remains to be established. However, because decreased K+ current density is associated with an increase in membrane electrical excitability, we postulate increased excitability is one of the modes of action of E2 in sensitizing the gonadotroph to GnRH, an event central to the regulation of cyclic gonadotrophin secretion.

Differential expression and regulation of progesterone receptor isoforms in rat and mouse pituitary cells and LbetaT2 gonadotropes.

Manipulation of endogenous progesterone receptor (PR) does not produce equivalent physiological effects in mouse and rat pituitary cells. To test whether this may be due in part to difference in PR isoform expression, we examined hormonally regulated pituitary PR-A and PR-B mRNA levels using quantitative real-time PCR. The LbetaT2 mouse gonadotrope line or pituitary cells from adult, ovariectomized rats or mice were cultured with or without 0.2 nM 17beta-estradiol (E(2)) for 3 days. PR-A was the predominant form expressed for all groups. For mouse cells, E(2) led to an increase in both isoforms without a change in the A:B ratio; for rat cells, the PR-B response to E(2) was more robust resulting in a decrease in the A:B ratio. Exposure of E(2)-treated pituitary cells to 200 nM progesterone for 6 h decreased both PR-A and PR-B levels in rat cells, but had no effect on PR isoform expression in mouse cells even when exposure was extended to 12 h. The low level of PR expression found in LbetaT2 gonadotropes was unaffected by E(2), alone or with progesterone. The weak PR expression and lack of responsiveness of LbetaT2 cells cannot be explained by a male phenotype as was shown by the more than tenfold higher PR mRNA level in primary cultures of male mouse pituitary cells, which responded to E(2) stimulation with a proportional increase in PR isoforms similar to female cells. Functionally, E(2)-stimulated changes in PR mRNA isoform ratios in rat, mouse or LbetaT2 cells correlated with the degree of progesterone augmentation of GnRH-stimulated LH secretion in these models. These results are consistent with the hypothesis that robust GnRH priming and progesterone augmentation of LH secretion in the rat compared to these events in the mouse are a consequence, in part, of differences in the E(2)-modulated ratio of PR isoforms.