Intracellular Signaling In Endocrine Cells

Child Health And Human Development

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Abstract

Numerous plasma membrane channels have been characterized in pituitary cells, but the mechanism underlying their electrical activity is still not known. Our current efforts focus on the role of sodium-conducting channels in the control of electrical activity in pituitary cells. In these cells, removal of sodium reversibly hyperpolarized the membrane potential and suppressed calcium oscillations, followed by a reduction in the level of intracellular calcium concentration to near steady-state levels. In contrast, the blockade by tetrodotoxin of voltage-dependent sodium channels, which are expressed in these cells, was ineffective. One group of the channels that could account for such effects of removal of extracellular sodium are hyperpolarization-activated channels, which have a distinct role in the control of membrane excitability in spontaneously active cardiac and neuronal cells. Using molecular biology and electrophysiological tools, we identified these channels in pituitary cells. Our results further indicated that these channels were under tonic activation by the basal level of cAMP. We also combined experiments and theory to clarify the mechanisms underlying spontaneous and receptor-controlled electrical activity using pituitary somatotrophs as a cell model. Experiments support the role of a sodium-conducting and tetrodotoxin-insensitive channel in controlling spontaneous and growth hormone-releasing hormone-stimulated pacemaking, the latter in a cAMP-dependent manner; an opposing role of spontaneously active inwardly rectifying potassium (Kir) channels and G-protein-regulated Kir channels in somatostatin-mediated inhibition of pacemaking; as well as a role of voltage-gated calcium channels in spiking and large conductance (BK-type) calcium-activated potassium channels in plateau bursting. The mathematical model is compatible with a wide variety of experimental data involving pharmacology and extracellular ion substitution and supports the importance of constitutively active tetrodotoxin-insensitive sodium and Kir channels in maintaining spontaneous pacemaking in pituitary somatotrophs. The model also suggests that these channels are involved in the up- and down-regulation of electrical activity by growth hormone-releasing hormone and somatostatin. In the model, the plateau bursting is controlled by two functional populations of BK channels, characterized by distance from the voltage-gated calcium channels. The rapid activation of the proximal BK channels is critical for the establishment of the plateau, whereas slow recruitment of the distal BK channels terminates the plateau.[unreadable] Spontaneous electrical activity of pituitary cells is stimulated and/or inhibited by numerous hormones. Our ongoing work is focused on the role of endothelins, ATP, and androgens in control of voltage-gated calcium influx and hormone secretion. Endothelins are produced by pituitary cells and functional endothelin-A receptors are expressed in all five major secretory cell types. In gonadotrophs, stimulation of these receptors leads to activation of the Gq/11 signaling pathway, accompanied by the oscillatory calcium release from intracellular pools and gonadotropin secretion and facilitation of voltage-gated calcium influx. However, in somatotrophs and lactotrophs endothelins inhibit spontaneous voltage-gated calcium influx through the Gi/o signaling pathway. These observations raised the possibility that multiple receptor subtypes are generated by alternative RNA splicing of the pituitary endothelin-A receptor and exhibit comparable binding characteristics but are coupled to different G proteins and intracellular signaling. Consistent with this hypothesis, we reported recently on the isolation of cDNAs of endothelin-A transcripts from rat anterior pituitary, which are generated by alternative RNA splicing. Deletion of exon 2 and insertion of fragments from intron 1 and 2 accounted for formation of three misplaced proteins, whereas the insertion of a fragment from intron 6 resulted in generation of a functional plasma membrane receptor, termed the endothelin-A-C13 receptor. In this splice variant, the C-terminal 382S-426N sequence of the wild type receptor was substituted with a shorter 382A-399L sequence, resulting in alteration of the putative domains responsible for coupling to Gq/11 and Gs proteins and the endocytotic recycling, as well as in deletion of the predicted protein kinase C/casein kinase 2 phosphorylation sites. The mRNA transcripts for the splice receptor were identified in normal and immortalized pituitary cells and several other tissues. The pharmacological profiles of recombinant wild type and spliced receptors were highly comparable, but the coupling of the splice receptor to the calcium-mobilizing signaling pathway was attenuated, causing a rightward shift in the agonist potency. Furthermore, the efficacy of the spliced receptor to stimulate adenylyl cyclase signaling pathway and to internalize was significantly reduced. These results indicate for the first time the presence of a novel endothelin-A splice receptor, which could contribute to the functional heterogeneity among secretory pituitary cell types. [unreadable] P2X receptors are a family of ligand-gated cation channels composed of two transmembrane domains, with N- and C-termini located intracellularly and a large extracellular loop containing the ATP binding domain. We progressed in identifying the residues important for agonist binding and gating, using ivermectin (IVM), an alosteric modulator. All experiments were done with enhanced green fluorescent protein-tagged receptors to identify cells expressing receptors for electrophysiological recordings and to visualize the subcellular distribution of receptors by confocal microscopy. In the presence of IVM, all low or non-responsive mutants responded to agonists in a dose-dependent manner. The results further indicated that lysines 67 and 313 and arginine 295 have a critical role in forming the proper three-dimensional structure of P2X4 receptor for agonist binding and/or channel gating. To study the roles of the sequence downstream of ATP binding domain, we mutated the numerous conserved residues in the Lys313-Ile333 ectodomain sequence. The rates of wild type channel opening and closing in the presence of ATP, but not the rate of washout-induced closing, were dependent on agonist concentration. All mutants other than I317A were expressed in the plasma membrane at comparable levels. The majority of mutants showed significant changes in the peak amplitude of responses and the EC50 values for ATP. When stimulated with the supramaximal ATP concentration, mutants also differed in the kinetics of their activation, deactivation, and/or desensitization. The results suggest a critical role of the Lys313 residue in receptor function other than coordination of the phosphate group of ATP and a possible contribution of the Tyr315 residue to the agonist-binding module. The pattern of changes of receptor function by mutation of other residues was consistent with the operation of the Gly316-Ile333 sequence as a signal transduction module between the ligand binding domain and the channel gate in the second transmembrane domain. Finally, to characterize IVM binging site at P2X4 receptors we generated several chimeric and single-point mutants. Experiments with chimeric receptors revealed that the Val49-Val61 but not the Val64-Tyr315 ectodomain sequence is important for the effects of IVM on channel deactivation. Receptor-specific mutations placed in the Gly29-Val61 and Asp338-Leu358 regions showed the importance of Trp50, Val60, and Val357 residues in IVM regulation of the rate of channel deactivation, but not on the maximum current amplitude. These results suggest that the transmembrane domains and the nearby ectodomain region contribute to the eff