Intracellular Signaling In Endocrine Cells

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Studies on calcium signaling and cellular regulation are focused on the biophysical basis of pituitary cell type-specific calcium signaling-secretion coupling, the roles of G protein-coupled and tyrosine kinase receptors in control of signaling, hormone secretion and gene expression, and the molecular dissection of ATP-gated P2X receptor channels. Our earlier studies revealed that cultured pituitary cells, including lactotrophs, frequently exhibit larger membrane potential oscillations, on top of which the depolarizing plateau and bursts of action potentials are generated, with spikes that usually do not reach the reverse potential. In lactotrophs such spontaneous electrical activity and the associated voltage-gated calcium influx are sufficient to maintain high prolactin release. The finding that normal and immortalized pituitary cells express calcium-inhibitable adenylyl cyclases prompted us to examine the hypothesis that cyclic nucleotides may have a role in spontaneous pacemaking and basal prolactin release in these cells by controlling hyperpolarization-activated cation channels and/or cyclic nucleotide-gated channels. Consistent with this, we found that stimulation of adenylyl cyclases by forskolin initiates firing of action potentials in quiescent lactotrophs and increases the spiking frequency in spontaneously active cells. This in turn facilitates voltage-gated calcium influx and prolactin secretion. Inhibition of phosphodiesterases by 3-isobutyl-1-methylxanthine also stimulates cyclic nucleotide accumulation and prolactin release. Conversely, MDL-12330A inhibits basal and forskolin-stimulated cyclic nucleotide production in a concentration-dependent manner, as well as electrical activity, calcium transients, and prolactin secretion. Basal cyclic AMP production is augmented by removal of extracellular calcium and is attenuated by facilitation of voltage-gated calcium influx. These results suggest that the intrinsic activity of calcium-inhibitable adenylyl cyclases contributes to the control of spontaneous pacemaking activity. Our results further indicate that endothelin receptors inhibit voltage-gated calcium influx-dependent prolactin release. However, these receptors inhibit secretion downstream of voltage-gated calcium influx and in a phospholipase C and tyrosine kinase-independent manner. We also found that endothelin receptors are coupled to both pertussis toxin-sensitive and insensitive Gi proteins. Finally, we discovered that the coupling of endothelin receptors to the Gz signaling pathway accounts for inhibition of prolactin secretion downstream of voltage-gated calcium influx. Sustained inhibition of secretion is achieved through down-regulation of the adenylyl cyclase signaling cascade, whereas rapid inhibition also occurs at elevated cAMP levels regardless of the status of phospholipase C, tyrosine kinases, and protein kinase C. These results indicate that the coupling of seven transmembrane domain receptors to Gz proteins provides a pathway that effectively blocks hormone secretion for a prolonged time without interfering with pacemaking activity and calcium influx-dependent cellular functions. We previously found that the purinergic signaling system is operative in normal and immortalized anterior pituitary cells. These cells release ATP under resting conditions and in response to activation of calcium mobilizing receptors. However, there is no correlation between the rate of basal hormone and ATP release, suggesting that ATP is not co-secreted with hormones by regulated exocytosis. Experiments in progress are directed toward the characterization of a pathway responsible for ATP release. These cells also express ecto-nucleotidases, which hydrolyze ATP, resulting in formation of the respective nucleoside and free phosphate. The transcripts for ecto-nucleotidase eNTPDase 1-3 were found in pituitary cells. The products of this hydrolytic cascade, ADP and adenosine, also act as extracellular messengers by activating distinct plasma membrane receptors. These receptors are termed purinergic and belong to two groups: P1 and P2 receptors. P2X receptors are a family of ligand-gated cation channels composed of two transmembrane domains, N- and C-termini located intracellularly, and a large extracellular loop containing the ATP binding domain. To identify regions important for binding and gating, our experimental work with recombinant channels is focused on chimeras and point mutagenesis of conserved ectodomain residues. Mutant channels were expressed in human embryonic kidney 293 cells and mouse gonadotropin-releasing hormone-secreting GT1 neurons and analyzed using calcium imaging and patch clamp techniques. Experiments with chimeric P2XRs helped in characterization of gating and ionic conduction, deactivation of receptors, structural determinants of receptor desensitization and recovery from desensitization. To identify regions important for ATP binding we used the known sequence and secondary structure similarities between the Lys180-Lys326 ectodomain region of P2X4 and the class II aminoacyl-tRNA synthetases as a guide to generate a three-dimensional model of the receptor-binding site and to design mutants. The interplay between homology modeling and site-directed mutagenesis suggested that the Asp280 residue of P2X4R coordinates ATP binding via the magnesium ion, Phe230 coordinates the binding of the adenine ring of ATP, and Lys190, His286 and Arg278 coordinate the actions of negatively charged alpha, beta, and gamma phosphate groups, respectively. Until the crystal structure of the channel is solved, this model could provide a useful approach for future studies on identification of ATP binding domain and gating of P2XRs.