This project addresses the cellular signaling cascade in endocrine and neuroendocrine cells operated by G protein-coupled receptors, and the interactions between plasma membrane electrical events and receptor-mediated signaling. Current emphasis is on the functional role of calcium influx through voltage-sensitive calcium channels (VSCC) in pituitary gonadotrophs and hypothalamic GnRH neurons. An increase in calcium influx through these channels is associated with several distinct positive effects on cellular functions. In gonadotrophs, calcium influx modulates InsP3-induced calcium oscillations via a direct effect on InsP3 receptors and indirectly, by affecting the level of repletion of the endoplasmic reticulum calcium pool. The coupling of InsP3-induced calcium oscillations and calcium influx during plasma membrane voltage spikes is accomplished through calcium-controlled calcium entry. Experimental observations and a mathematical model of this process indicate that calcium in the vicinity of the plasma membrane acts on calcium-activated potassium channels and calcium pumps in the plasma membrane to regulate calcium entry through VSCC. In addition to apamin-sensitive potassium channels, electrophysiological investigations indicate that these cells express an additional calcium-activated apamin-insensitive potassium current. This novel channel also participates in the sensing and refilling of calcium content in the ER stores by remodulation of spontaneous and agonist-induced electrical activity. Voltage-gated calcium entry in non-activated and agonist-stimulated gonadotrophs participates in the control of gonadotropin secretion through a wortmannin-sensitive step in calcium-controlled exocytosis. Several lines of evidence suggest that calcium influx-controlled secretion occurs through a calmodulin-myosin light chain kinase-dependent mechanism, which is affected by wortmannin in a micromolar concentration-range. Action potential-driven calcium influx also controls neuropeptide secretion from immortalized GnRH neurons. In these cells, voltage-gated calcium entry and protein kinase C act in an independent but cooperative manner to regulate phospholipase D activity, which contributes to the secretory response in GnRH neurons. Thus, the electrical activity of GnRH-secreting neurons participates in the functional coupling between calcium-mobilizing receptors and the phospholipase D pathway. These observations provide the basis for understanding the interactions between the plasma membrane and endoplasmic reticulum calcium channels, as well as enzymatic activity and exocytosis. Moreover, they emphasize the complexity in the positive feedback role of calcium in agonist-induced signaling and hormone secretion.
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