Calcium-Prolactin Secretion Coupling in Rat Pituitary Lactotrophs Is Controlled by PI4-Kinase Alpha.

The role of calcium, but not of other intracellular signaling molecules, in the release of pituitary hormones by exocytosis is well established. Here, we analyzed the contribution of phosphatidylinositol kinases (PIKs) to calcium-driven prolactin (PRL) release in pituitary lactotrophs: PI4Ks - which control PI4P production, PIP5Ks - which synthesize PI(4, 5)P2 by phosphorylating the D-5 position of the inositol ring of PI4P, and PI3KCs - which phosphorylate PI(4, 5)P to generate PI(3, 4, 5)P. We used common and PIK-specific inhibitors to evaluate the strength of calcium-secretion coupling in rat lactotrophs.

Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel.

P2X2 receptors (P2X2R) exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes.

GnRH Induces ERK-Dependent Bleb Formation in Gonadotrope Cells, Involving Recruitment of Members of a GnRH Receptor-Associated Signalosome to the Blebs.

We have previously described a signaling complex (signalosome) associated with the GnRH receptor (GnRHR). We now report that GnRH induces bleb formation in the gonadotrope-derived LβT2 cells. The blebs appear within ~2 min at a turnover rate of ~2-3 blebs/min and last for at least 90 min.

The relationship between basal and regulated Gnrhr expression in rodent pituitary gonadotrophs.

Hypothalamic GnRH together with gonadal steroids and activins/inhibin regulate its receptor gene (Gnrhr) expression in vivo, which leads to crucial changes in GnRHR numbers on the plasma membrane. This is accompanied by alterations in the gonadotroph sensitivity and responsiveness during physiologically relevant situations. Here we investigated basal and GnRH-regulated Gnrhr expression in rodent pituitary gonadotrophs in vitro.

Spontaneous and CRH-Induced Excitability and Calcium Signaling in Mice Corticotrophs Involves Sodium, Calcium, and Cation-Conducting Channels.

Transgenic mice expressing the tdimer2(12) form of Discosoma red fluorescent protein under control of the proopiomelanocortin gene's regulatory elements are a useful model for studying corticotrophs. Using these mice, we studied the ion channels and mechanisms controlling corticotroph excitability. Corticotrophs were either quiescent or electrically active, with a 22-mV difference in the resting membrane potential (RMP) between the 2 groups.

Calcium signaling properties of a thyrotroph cell line, mouse TαT1 cells.

TαT1 cells are mouse thyrotroph cell line frequently used for studies on thyroid-stimulating hormone beta subunit gene expression and other cellular functions. Here we have characterized calcium-signaling pathways in TαT1 cells, an issue not previously addressed in these cells and incompletely described in native thyrotrophs. TαT1 cells are excitable and fire action potentials spontaneously and in response to application of thyrotropin-releasing hormone (TRH), the native hypothalamic agonist for thyrotrophs.

Role of PI4K and PI3K-AKT in ERK1/2 activation by GnRH in the pituitary gonadotropes.

The role of PI4K and PI3K-AKT in ERK1/2 activation by GnRH was examined. A relatively long preincubation (60 min) with wortmannin (10 nM and 10 μM), and LY294002 (10 μM and 100 μM) (doses known to inhibit PI3K and PI4K, respectively), were required to inhibit GnRH-and PMA-stimulated ERK1/2 activity in αT3-1 and LβT2 gonadotrope cells. A similar preincubation protocol was required to demonstrate inhibition of IGF-1-stimulated AKT activation lending support for the need of prolonged incubation (60 min) with wortmannin in contrast to other cellular systems.

Rapid Glucocorticoid Feedback Inhibition of ACTH Secretion Involves Ligand-Dependent Membrane Association of Glucocorticoid Receptors.

The hypothesis that rapid glucocorticoid inhibition of pituitary ACTH secretion mediates a feedforward/feedback mechanism responsible for the hourly glucocorticoid pulsatility was tested in cultured pituitary cells. Perifusion with 30 pM CRH caused sustained the elevation of ACTH secretion. Superimposed corticosterone pulses inhibited CRH-stimulated ACTH release, depending on prior glucocorticoid clearance.

Paliperidone and aripiprazole differentially affect the strength of calcium-secretion coupling in female pituitary lactotrophs.

Hyperprolactinemia is a common adverse in vivo effect of antipsychotic medications that are used in the treatment of patients with schizophrenia. Here, we compared the effects of two atypical antipsychotics, paliperidone and aripiprazole, on cAMP/calcium signaling and prolactin release in female rat pituitary lactotrophs in vitro. Dopamine inhibited spontaneous cAMP/calcium signaling and prolactin release.

Loss of basal and TRH-stimulated Tshb expression in dispersed pituitary cells.

This study addresses the in vivo and in vitro expression pattern of three genes that are operative in the thyrotroph subpopulation of anterior pituitary cells: glycoprotein α-chain (Cga), thyroid-stimulating hormone β-chain (Tshb), and TRH receptor (Trhr). In vivo, the expression of Cga and Tshb was robust, whereas the expression of Trhr was low. In cultured pituitary cells, there was a progressive decline in the expression of Cga, Tshb, and Trhr.

The role of cyclic nucleotides in pituitary lactotroph functions.

Lactotrophs are one of the five secretory anterior pituitary cell types specialized to synthesize and release prolactin. In vitro, these cells fire action potentials (APs) spontaneously and the accompanied Ca(2+) transients are of sufficient amplitude to keep the exocytotic pathway, the transcription of prolactin gene, and de novo hormone synthesis continuously active. Basal cyclic nucleotide production is also substantial in cultured cells but not critical for the APs secretion/transcription coupling in lactotrophs.

Dual gating mechanism and function of P2X7 receptor channels.

The ATP-gated P2X7 receptor channel (P2X7R) operates as a cytolytic and apoptotic receptor but also controls sustained cellular responses, including cell growth and proliferation. However, it has not been clarified how the same receptor mediates such opposing effects. To address this question, we have combined electrophysiological, imaging, and mathematical studies using wild-type and mutant rat P2X7Rs.

Gating properties of the P2X2a and P2X2b receptor channels: experiments and mathematical modeling.

Adenosine triphosphate (ATP)-gated P2X2 receptors exhibit two opposite activation-dependent changes, pore dilation and pore closing (desensitization), through a process that is incompletely understood. To address this issue and to clarify the roles of calcium and the C-terminal domain in gating, we combined biophysical and mathematical approaches using two splice forms of receptors: the full-size form (P2X2aR) and the shorter form missing 69 residues in the C-terminal domain (P2X2bR). Both receptors developed conductivity for N-methyl-D-glucamine within 2-6 s of ATP application.

Characterization of novel Pannexin 1 isoforms from rat pituitary cells and their association with ATP-gated P2X channels.

Our previous studies have showed that Pannexin 1 (Panx1), a member of a recently discovered family of gap junction proteins, is expressed in the pituitary gland. Here we investigated the presence and expression pattern of Panx1 isoforms in pituitary cells, their roles in ATP release, and their association with purinergic P2X receptor subtypes that are native to pituitary cells. In addition to the full-size Panx1, termed Panx1a, pituitary cells also express two novel shorter isoforms, termed Panx1c and Panx1d, which formation reflects the existence of alternative splicing sites in exons 2 and 4, respectively.

Role of nonselective cation channels in spontaneous and protein kinase A-stimulated calcium signaling in pituitary cells.

Several receptors linked to the adenylyl cyclase signaling pathway stimulate electrical activity and calcium influx in endocrine pituitary cells, and a role for an unidentified sodium-conducting channel in this process has been proposed. Here we show that forskolin dose-dependently increases cAMP production and facilitates calcium influx in about 30% of rat and mouse pituitary cells at its maximal concentration. The stimulatory effect of forskolin on calcium influx was lost in cells with inhibited PKA (cAMP-dependent protein kinase) and in cells that were haploinsufficient for the main PKA regulatory subunit but was preserved in cells that were also haploinsufficient for the main PKA catalytic subunit.

Expression and roles of pannexins in ATP release in the pituitary gland.

Pannexins are a newly discovered three-member family of proteins expressed in the brain and peripheral tissues that belong to the superfamily of gap junction proteins. However, in mammals pannexins do not form gap junctions, and their expression and function in the pituitary gland have not been studied. Here we show that the rat pituitary gland expresses mRNA and protein transcripts of pannexins 1 and 2 but not pannexin 3.

Experimental characterization and mathematical modeling of P2X7 receptor channel gating.

The P2X7 receptor is a trimeric channel with three binding sites for ATP, but how the occupancy of these sites affects gating is still not understood. Here we show that naive receptors activated and deactivated monophasically at low and biphasically at higher agonist concentrations. Both phases of response were abolished by application of Az10606120, a P2X7R-specific antagonist.

Elucidation of mechanisms of the reciprocal cross talk between gonadotropin-releasing hormone and prostaglandin receptors.

We recently described a novel GnRH receptor signaling pathway mediated by the prostaglandins (PGs) F(2alpha) and PGI(2), which acts through an autocrine/paracrine modality to limit autoregulation of the GnRH receptor and inhibit LH but not FSH release. Here we further explore the cross talk between GnRH and the PG receptors. GnRH stimulates arachidonic acid (AA) release from LbetaT2 gonadotrope cells via the Ca(2+)-independent phospholipase A(2) (iPLA(2)) and not via the more common Ca(2+)-dependent cytosolic phospholipase A(2)alpha (cPLA(2)alpha).

Characterization of purinergic P2X4 receptor channels expressed in anterior pituitary cells.

Anterior pituitary cells express cation-conducting P2X receptor channels (P2XRs), but their molecular identity, electrophysiological properties, cell-specific expression pattern, and physiological roles have been only partially characterized. In this study, we show by quantitative RT-PCR that mRNA transcripts for the P2X(4) subunit are the most abundant in rat anterior pituitary tissue and confirm the P2X(4)R protein expression by Western blot analysis. Single-cell patch-clamp recordings show that extracellular ATP induced an inward depolarizing current in a majority of thyrotropin-releasing hormone-responsive pituitary cells, which resembled the current profile generated by recombinant P2X(4)R.

The P2X7 receptor channel pore dilates under physiological ion conditions.

Activation of the purinergic P2X(7) receptor leads to the rapid opening of an integral ion channel that is permeable to small cations. This is followed by a gradual increase in permeability to fluorescent dyes by integrating the actions of the pannexin-1 channel. Here, we show that during the prolonged agonist application a rapid current that peaked within 200 ms was accompanied with a slower current that required tens of seconds to reach its peak.

Molecular, pharmacological and functional properties of GABA(A) receptors in anterior pituitary cells.

Anterior pituitary cells express gamma-aminobutyric acid (GABA)-A receptor-channels, but their structure, distribution within the secretory cell types, and nature of action have not been clarified. Here we addressed these questions using cultured anterior pituitary cells from postpubertal female rats and immortalized alphaT3-1 and GH(3) cells. Our results show that mRNAs for all GABA(A) receptor subunits are expressed in pituitary cells and that alpha1/beta1 subunit proteins are present in all secretory cells.

Release and extracellular metabolism of ATP by ecto-nucleotidase eNTPDase 1-3 in hypothalamic and pituitary cells.

Hypothalamic and pituitary cells express G protein-coupled adenosine and P2Y receptors and cation-conducting P2X receptor-channels, suggesting that extracellular ATP and other nucleotides may function as autocrine and/or paracrine signaling factors in these cells. Consistent with this hypothesis, we show that cultured normal and immortalized pituitary and hypothalamic cells release ATP under resting conditions. RT-PCR analysis also revealed the presence of transcripts for ecto-nucleotidase eNTPDase 1-3 in these cells.

Dopamine inhibits basal prolactin release in pituitary lactotrophs through pertussis toxin-sensitive and -insensitive signaling pathways.

Dopamine D2 receptors signal through the pertussis toxin (PTX)-sensitive G(i/o) and PTX-insensitive G(z) proteins, as well as through a G protein-independent, beta-arrestin/glycogen synthase kinase-3-dependent pathway. Activation of these receptors in pituitary lactotrophs leads to inhibition of prolactin (PRL) release. It has been suggested that this inhibition occurs through the G(i/o)-alpha protein-mediated inhibition of cAMP production and/or G(i/o)-betagamma dimer-mediated activation of inward rectifier K(+) channels and inhibition of voltage-gated Ca(2+) channels.