Intracellular Signaling In Endocrine Cells

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications & trials

Abstract

Our recent work was focused on secretion of prolactin, a hormone that controls lactation, by pituitary lactotrophs and the role of dopamine D2 receptors and hypotonicity in this process. We also investigated the expression and role of gamma-amino butyric acid (GABA)-A receptor-channels in lactotrophs and gonadotrophs and provided structural and functional characterization of recombinant ATP-gated P2X4 receptor-channels that we cloned from pituitary cells. The work with dopamine D2 receptors revealed that their activation in pituitary lactotrophs leads to inhibition of prolactin release. It has been suggested by others that this inhibition occurs through the Gi/o-alpha protein-mediated inhibition of cAMP production and/or Gi/o-beta/gamma dimer-mediated activation of inward rectifier potassium channels and inhibition of voltage-gated calcium channels. We show that the dopamine agonist-induced inhibition of spontaneous calcium influx and release of pre-stored PRL was preserved when cAMP levels were elevated by forskolin treatment. We further observed that dopamine agonists inhibited both spontaneous and depolarization-induced calcium influx in untreated but not in pertussis toxin-treated cells. This inhibition was also observed in cells with blocked inward rectifier potassium channels, suggesting that dopamine effects on voltage-gated calcium channel gating are sufficient to inhibit spontaneous calcium influx. However, agonist-induced inhibition of prolactin release was only partially relieved in pertussis-treated cells, indicating that dopamine receptors also inhibit exocytosis downstream of voltage-gated calcium influx. The pertussis toxin-insensitive step in agonist-induced inhibition of prlactin release was not affected by the addition of wortmannin, an inhibitor of PI3-kinase, and lithium, an inhibitor of GSK-3, but was attenuated in the presence of phorbol ester PMA, which inhibits Gz signaling pathway in a protein kinase C-dependent manner. These results indicate for the first time that dopamine inhibits basal prolactin release not only by blocking voltage-gated calcium influx through the pertussis toxin-sensitive-signaling pathway but also by desensitizing calcium-secretion coupling through the pertussis toxin-insensitive and protein kinase C-sensitive signaling pathway. In collaboration with Dr. Zorecs group, we studied the release of the pituitary hormone prolactin by hypotonicity, because this hormone also contributes to osmoregulation. In perifused rat lactotrophs, hypotonicity resulted in a transient increase followed by a sustained depression of prolactin release, as monitored by radioimmunoassay. In single cells imaged by confocal microscopy, hypotonicity elicited discharge of the fluorescently-labeled atrial natriuretic peptide cargo from 2% of vesicles/cell, which synchronously loaded the styryl dye FM 4-64 through the same fusion pores. In contrast, high potassium-induced depolarization resulted in a response of 10% of vesicles/cell, with different unloading/loading time-course of the two fluorescent probes. In cell-attached studies, discrete changes in the membrane capacitance were recorded in both unstimulated and stimulated conditions, reflecting single vesicle fusion/fissions with the plasma membrane. In stimulated cells, the probability of occurrence of full fusion events was low and unchanged, whereas over 95% of fusion events were transient, with the open fusion pore probability, the average pore dwell-time, the frequency of occurrence, and the fusion pore conductance increased. Hypotonicity only rarely elicited new fusion events in silent membrane patches. The results indicate that, in hypotonicity-stimulated lactotrophs, rapidly releasable vesicles appear pre-fused and release hormone in a kiss-and-run mode. During the last year, we also studied the expression of GABA-A receptor-channels in pituitary cells, their distribution within the secretory anterior pituitary cell types, and nature (stimulatory or inhibitory) of actions. Our results show that mRNAs for all GABAA receptor subunits are expressed in pituitary cells and that alpha1/beta1 subunit proteins are present in all secretory cells. In voltage-clamped gramicidin-perforated cells, GABA induced dose-dependent increases in current amplitude that were inhibited by bicuculline and picrotoxin and facilitated by diazepam and zolpidem in a concentration-dependent manner. In intact cells, GABA and the GABA-A receptor agonist muscimol caused a rapid and transient increase in intracellular calcium, whereas the GABA-B receptor agonist baclofen was ineffective, suggesting that chloride-mediated depolarization activates voltage-gated calcium channels. Consistent with this finding, RT-PCR analysis indicated high expression of NKCC1, but not KCC2 cation/chloride transporter mRNAs in pituitary cells. Furthermore, the GABA-A channel reversal potential for chloride ions was positive to the baseline membrane potential in most cells and the activation of ion channels by GABA resulted in depolarization of cells and modulation of spontaneous electrical activity. These results indicate that secretory pituitary cells express functional GABA-A receptor-channels that are depolarizing. In a work on structural-functional characterization of recombinant P2X4 receptor that was cloned from pituitary cells, the focus on investigations was on identification of residues contributing to allosteric regulation of these channels by ivermectin, a large macrocyclic lactone that specifically enhances P2X4 receptor-channel function by interacting with residues of transmembrane helices in the open conformation state. The sensitivity of this receptor to iveremectin was also used as an indicator in identifying residues in transmembrane regions that face the pore of the channel. For these purposes, we used cysteine-scanning mutagenesis of rat P2X4 transmembrane regions. The receptor function was unchanged by mutations in 29 different residues, and among them the ivermectin effects were altered in Gln36, Leu40, Val43, Val47, Trp50, Asn338, Gly342, Leu346, Ala349, and Ile356 mutants. The substitution-sensitive Arg33 and Cys353 mutants could also be considered as ivermectin-sensitive hits. The pattern of these 12 residues was consistent with helical topology of both transmembrane regions, with every third or fourth amino acid affected by substitution. These predominantly hydrophobic-nonpolar residues are also present in the ivermectin-sensitive Schistosoma mansoni P2X subunit. They lie on the same side of their helices, and could face lipids in the open conformation state and provide the binding pocket for IVM. In contrast, the IVM-independent hits Met31, Tyr42, Gly45, Val49, Gly340, Leu343, Ala344, Gly347, Thr350, Asp354, and Val357 map on the opposite side of their helices, probably facing the pore of receptor or protein and playing important roles in gating. Once the receptor architecture becomes available from crystal studies, we will know the real topology of these functionally important residues.