We investigate cellular signaling cascades and secretion in neuroendocrine cells, with special emphasis on the interactions between plasma membrane electrical events and receptor-controlled pathways. The majority of neuroendocrine cells exhibit spontaneous firing of action potentials and calcium transients, and hormonal stimulation leads to up- or down-regulation of electrical activity and voltage-gated calcium influx-dependent secretion through a complex cascade of events. We are currently studying the biophysical basis of pituitary cell type-specific calcium signaling-secretion coupling, the metabolism and roles of cyclic nucleotides in regulation of electrical activity and calcium signaling, and the expression pattern and molecular properties of calcium-conducting P2X receptor channels. ? ? Our recent experiments in anterior pituitary cells indicated that both calcium influx inhibitable and insensitive adenylyl cyclase (AC) subtypes contributed to the basal cAMP production, and that soluble guanylyl cyclase (sGC) was exclusively responsible for basal cGMP production. Inhibition of basal AC activity but not sGC activity reduced prolactin (PRL) release. In contrast, forskolin stimulated cAMP and cGMP production as well as pacemaking, calcium influx, and PRL secretion. Elevation of cAMP and cGMP levels by inhibition of phosphodiesterase activity was also accompanied by increased PRL release. The AC inhibitors attenuated forskolin-stimulated cyclic nucleotide production, calcium influx, and PRL release. Cell-permeant 8-Br-cAMP stimulated firing of action potentials and PRL release, and rescued hormone secretion in cells with inhibited ACs in an extracellular calcium-dependent manner, whereas 8-Br-cGMP and 8-pCPT-2Me-cAMP were ineffective. Protein kinase A inhibitors did not change spontaneous and forskolin-stimulated pacemaking, calcium influx, and PRL release. Hyperpolarization-activated (HCN) channels are members of cyclic nucleotide-gated channels and may thus mediate the action of cAMP on pacemaking activity in pituitary cells. Consistent with this hypothesis, our RT-PCR analysis revealed the presence of mRNA transcripts for HCN2, HCN3 and HCN4 subunits in these cells. Furthermore, hyperpolarization of the membrane potential below -60 mV elicited a slowly activating voltage-dependent and caesium and ZD7288-sensitive inward current (Ih) in the majority of tested cells. Receptor- and non-receptor-mediated activation of AC and sGC, and the addition of the membrane permeant cAMP analogue, 8-Br-cAMP, did not affect Ih. Inhibition of basal AC activity, but not basal sGC activity, caused reduction of the peak amplitude and a leftward shift in the activation curve of Ih. Such inhibition of current was reversed by stimulation of AC with forskolin and by the addition of 8-Br-cAMP. Application of caesium had no significant effect on the resting membrane potential or electrical activity, whereas ZD7288 exhibited complex and Ih-independent effects on spontaneous electrical activity, calcium signalling, and prolactin release. These results indicate that HCN channels in pituitary cells are under tonic activation by the basal level of cAMP and are not critical for spontaneous firing of action potentials. ? ? Lipid membranes are virtually impermeable to cyclic nucleotides and their extrusion against a concentration gradient is consistent with operation of an ATP-dependent and probenecid-sensitive transport mediated by plasma membrane proteins, termed cyclic nucleotide efflux pumps. Such energy-dependent cyclic nucleotide cellular efflux is operative in numerous eukaryotic cells and could be mediated by the multidrug resistance proteins MRP4, MRP5, and MRP8. Our recent experiments indicated that cellular efflux of cyclic nucleotides was also detectable in normal and immortalized pituitary cells under resting conditions and was increased after concurrent stimulation of cAMP and cGMP production. In resting and stimulated cells, the efflux pumps transported the majority of de novo produced cGMP, limiting its intracellular accumulation to a concentration range of 1-2 ?M. In contrast, only a small fraction of cAMP was released and there was a time- and concentration-dependent accumulation of this messenger in the cytosol, ranging from 1 to 100 ?M. Stimulation and inhibition of cGMP production alone did not affect cAMP efflux, suggesting the operation of two different transport pathways in pituitary cells. The rates of cAMP and cGMP effluxes were comparable and both pathways were blocked by probenecid and progesterone. Normal pituitary cells expressed mRNA transcripts for MRP4, MRP5, and MRP8, whereas immortalized GH3 pituitary cells expressed only transcripts for MRP5. Down-regulation of MRP5 expression in GH3 cells decreased cGMP release without affecting cAMP efflux. These results indicate that cyclic nucleotide cellular efflux has a critical role in elimination of intracellular cGMP but not cAMP in pituitary cells and that such selectivity is achieved by expression of MRP5.? ? 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. To measure the inter-subunit interaction in living cells, the efficient transfer of bioluminescent resonance energy between luciferase and fluorescent proteins attached to the N- or C-subunit termini of these subunits was used. The constitutive interactions between the full-length C-termini of P2X2 receptor were detected by a significant increase in fluorescence/luminescence intensity ratio compared with negative controls. Moreover, interactions between C-termini, and between C- and N-termini of adjacent subunits, were significantly enhanced in homomeric and heteromeric receptors containing P2X2b or P2X2e subunits. Finally, deletion of two amino acids at the splicing junction, but not at the C-terminal end of the P2X2b receptor, caused enhancement of channel desensitization and luminescence resonance energy transfer. These results indicate that C-terminal structure has a critical role in the cytoplasmic intersubunit interactions, and suggest that the extent of subunit interactions before ATP application could contribute to the subsequent channel activity and conformation changes associated with agonist-dependent desensitization. We also progressed in characterizing the expression and electrophysiological properties of P2X receptors in pituitary gonadotrophs from embryonic, neonatal, and adult rats. In cells from all three age groups, the calcium-mobilizing agonist GnRH induced oscillatory, hyperpolarizing, non-desensitizing, and slow deactivating currents. In contrast, ATP induced non-oscillatory, depolarizing, slowly desensitizing, and rapidly deactivating current, indicating that these cells express cation-conducting P2X channels but not calcium-mobilizing P2Y receptors. The amplitudes of P2X current response and the rates of receptor desensitization were dependent on ATP concentration. The biophysical and pharmacological properties of P2X currents were consistent with the expression of the P2X2 subtype of channels in these cells. ATP-induced rapid depolarization of gonadotrophs lead to initiation of firing in quiescent cells, an increase in the frequency of action potentials in spontaneously active cells, and a transient stimulation of LH release. ATP also influenced GnRH-induced current and membrane potential oscillations, as well as LH release, in an extracellular Ca2+-dependent manner. These results indicate that P2X receptors in gonadotrophs could operate as pacemaking channels and modulators of GnRH-regulated electrical activity and secretion.
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