Dennis W. Waring

Functional cross-talk between receptors for peptide and steroid hormones

Communication between a cell surface peptide hormone receptor and an intracellular steroid hormone receptor can take various routes, as dictated by the physiology of a particular cell type. There is increasing evidence for a novel route which requires that a peptide hormone receptor pathway converge on a steroid hormone receptor, leading to its activation. One consequence of such a process can be signal amplification for the peptide hormone receptor agonist. This is exemplified by the self-potentiating action of GnRH, which is a critical component in events leading to a surge in LH secretion and ovulation. One signaling pathway stimulated by the GnRH receptor may entail a phosphorylation cascade resulting in progesterone-independent modulation of progesterone receptor activity.

Characteristics of the adenohypophyseal Ca2+-phospholipid-dependent protein kinase

The Ca2+-phospholipid-dependent protein kinase, initially described by Takai et al. (J. Biol. Chem. 254, 3692-3695, 1979), has been identified in the anterior pituitary gland of the rat and sheep. The enzyme is essentially undetectable in initial cell extracts but marked activity is manifest following DEAE chromatography, suggesting the potential presence of an endogenous inhibitor of this enzyme. Two forms of this protein kinase exist in both sheep and rat anterior pituitary gland, both of which are similarly dependent upon Ca2+, phosphatidyl serine and diacylglycerol. Several endogenous substrates for this protein kinase have been observed in both the pars distalis and pars tuberalis of the sheep adenohypophysis.

MODULATION OF STIMULUS-SECRETION COUPLING IN SINGLE RAT GONADOTROPHS

1 Exocytosis and intracellular [Ca2+] were determined simultaneously in single anterior pituitary gonadotrophs from ovariectomized female rats. Dispersed cells were cultured for 2–4 days with or without 0.2 nm oestradiol‐17 β (E2) before use. Cells were stimulated with either gonadotrophin releasing hormone (GnRH) or by membrane depolarization. Exocytosis was determined from the change in membrane capacitance (Cm) using the perforated‐patch whole‐cell recording technique. Intracellular [Ca2+] was measured using fura‐2 fluorescence. 2 The exocytotic response to 1 nm GnRH was characterized by a wide spectrum of responses, ranging from exocytotic bursts to relatively slow, graded increases that were dependent on the evoked intracellular Ca2+ pattern. A kinetic model is presented that incorporates the observed steep dependence of exocytosis on measured intracellular [Ca2+]; simulated exocytosis reasonably approximated observed exocytotic responses, both kinetically and quantitatively. The model also suggests that the modulatory effects of E2 are brought about either by a change in the Ca2+ sensitivity of exocytosis or by a preferential clustering of docked‐secretory granules close to sites of Ca2+ release. The results suggest that in gonadotrophs an oscillatory Ca2+ signal is sensed by the exocytotic apparatus in a modified form of digital encoding. 3 Exocytosis in E2‐treated cells was 3‐fold greater than in non‐treated cells for GnRH‐evoked secretion, and 38% greater for depolarization; however, there was no effect of E2 on the intracellular Ca2+ response to either stimulus. The results show that maximum expression of the effect of E2 on exocytosis requires activation of GnRH‐dependent pathways.

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.

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 LβT2 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.

Endocrinology of the Single Cell: Tools and Insights

Whether all cells ascribed to the same type are indeed identical and the related question, “How are signals integrated at tissue and cell levels?” are fundamental issues in biological research and are responsible, in part, for the acceleration of activity in single-cell research. Also contributing are the major advances in single cell methodology. The capability to interrogate, manipulate with spatiotemporal specificity, and acquire data on multiple activities in the same cell can be powerful for understanding normal function and also dysfunction and drug targeting. Reviewed here are examples of how the emergent field of single-cell analysis and its tools are being used to understand the dynamics of the multiple components in regulatory networks for genes, intracellular signaling molecules, and secretion. For more in-depth consideration, the pancreatic β-cell and the pituitary gonadotrope are reviewed as parallel examples for application of single-cell analysis in the examination of one component of regulated secretion.