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)P2 to generate PI(3, 4, 5)P3. We used common and PIK-specific inhibitors to evaluate the strength of calcium-secretion coupling in rat lactotrophs. Gene expression was analyzed by single-cell RNA sequencing and qRT-PCR analysis; intracellular and released hormones were assessed by radioimmunoassay and ELISA; and single-cell calcium signaling was recorded by Fura 2 imaging. Single-cell RNA sequencing revealed the expression of Pi4ka, Pi4kb, Pi4k2a, Pi4k2b, Pip5k1a, Pip5k1c, and Pik3ca, as well as Pikfyve and Pip4k2c, in lactotrophs. Wortmannin, a PI3K and PI4K inhibitor, but not LY294002, a PI3K inhibitor, blocked spontaneous action potential driven PRL release with a half-time of ~20 min when applied in 10 µM concentration, leading to accumulation of intracellular PRL content. Wortmannin also inhibited increase in PRL release by high potassium, the calcium channel agonist Bay K8644, and calcium mobilizing thyrotropin-releasing hormone without affecting accompanying calcium signaling. GSK-A1, a specific inhibitor of PI4KA, also inhibited calcium-driven PRL secretion without affecting calcium signaling and Prl expression. In contrast, PIK93, a specific inhibitor of PI4KB, and ISA2011B and UNC3230, specific inhibitors of PIP5K1A and PIP5K1C, respectively, did not affect PRL release. These experiments revealed a key role of PI4KA in calcium-secretion coupling in pituitary lactotrophs downstream of voltage-gated and PI(4, 5)P2-dependent calcium signaling.

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. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.

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 LßT2 cells. The blebs appear within ~2 min at a turnover rate of ~2-3 blebs/min and last for at least 90 min. Formation of the blebs requires active ERK1/2 and RhoA-ROCK but not active c-Src. Although the following ligands stimulate ERK1/2 in LßT2 cells: EGF > GnRH > PMA > cyclic adenosine monophosphate (cAMP), they produced little or no effect on bleb formation as compared to the robust effect of GnRH (GnRH > PMA > cAMP > EGF), indicating that ERK1/2 is required but not sufficient for bleb formation possibly due to compartmentalization. Members of the above mentioned signalosome are recruited to the blebs, some during bleb formation (GnRHR, c-Src, ERK1/2, focal adhesion kinase, paxillin, and tubulin), and some during bleb retraction (vinculin), while F-actin decorates the blebs during retraction. Fluorescence intensity measurements for the above proteins across the cells showed higher intensity in the blebs vs. intracellular area. Moreover, GnRH induces blebs in primary cultures of rat pituitary cells and isolated mouse gonadotropes in an ERK1/2-dependent manner. The novel signalosome-bleb pathway suggests that as with the signalosome, the blebs are apparently involved in cell migration. Hence, we have extended the potential candidates which are involved in the blebs life cycle in general and for the GnRHR in particular.

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. In pituitary cells from adult animals cultured in the absence of GnRH and steroid hormones, the Gnrhr expression was progressively reduced but not completely abolished. The basal Gnrhr expression was also operative in LßT2 immortalized gonadotrophs never exposed to GnRH. In both cell types, basal transcription was sufficient for the expression of functional GnRHRs. Continuous application of GnRH transiently elevated the Gnrhr expression in cultured pituitary cells followed by a sustained fall without affecting basal transcription. Both basal and regulated Gnrhr transcriptions were dependent on the protein kinase C signaling pathway. The GnRH-regulated Gnrhr expression was not operative in embryonal pituitary and LßT2 cells and was established neonatally, the sex-specific response patterns were formed at the juvenile-peripubertal stage and there was a strong correlation between basal and regulated gene expression during development. Thus, the age-dependent basal and regulated Gnrhr transcription could account for the initial blockade and subsequent activation of the reproductive system during development.

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. In quiescent cells, CRH depolarized the membrane, leading to initial single spiking and sustained bursting; in active cells, CRH further facilitated or inhibited electrical activity and calcium spiking, depending on the initial activity pattern and CRH concentration. The stimulatory but not inhibitory action of CRH on electrical activity was mimicked by cAMP independently of the presence or absence of arachidonic acid. Removal of bath sodium silenced spiking and hyperpolarized the majority of cells; in contrast, the removal of bath calcium did not affect RMP but reduced CRH-induced depolarization, which abolished bursting electrical activity and decreased the spiking frequency but not the amplitude of single spikes. Corticotrophs with inhibited voltage-gated sodium channels fired calcium-dependent action potentials, whereas cells with inhibited L-type calcium channels fired sodium-dependent spikes; blockade of both channels abolished spiking without affecting the RMP. These results indicate that the background voltage-insensitive sodium conductance influences RMP, the CRH-depolarization current is driven by a cationic conductance, and the interplay between voltage-gated sodium and calcium channels plays a critical role in determining the status and pattern of electrical activity and calcium signaling.

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. Spontaneous electrical activity is coupled to small amplitude fluctuations in intracellular calcium, whereas TRH stimulates both calcium mobilization from intracellular pools and calcium influx. Non-receptor-mediated depletion of intracellular pool also leads to a prominent facilitation of calcium influx. Both receptor and non-receptor stimulated calcium influx is substantially attenuated but not completely abolished by inhibition of voltage-gated calcium channels, suggesting that depletion of intracellular calcium pool in these cells provides a signal for both voltage-independent and -dependent calcium influx, the latter by facilitating the pacemaking activity. These cells also express purinergic P2Y1 receptors and their activation by extracellular ATP mimics TRH action on calcium mobilization and influx. The thyroid hormone triiodothyronine prolongs duration of TRH-induced calcium spikes during 30-min exposure. These data indicate that TαT1 cells are capable of responding to natively feed-forward TRH signaling and intrapituitary ATP signaling with acute calcium mobilization and sustained calcium influx. Amplification of TRH-induced calcium signaling by triiodothyronine further suggests the existence of a pathway for positive feedback effects of thyroid hormones probably in a non-genomic manner.

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 LßT2 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. To rule out that the inhibitors acted upon PI(4,5)P2 levels, we followed the [Ca(2+)]i response to GnRH and found that wortmannin has no significant effect on GnRH-induced [Ca(2+)]i responses. Surprisingly, GnRH and PMA reduced, while IGF-1 increased AKT phosphorylation. We suggest that PI3K inhibits GnRH-stimulated αGSU activity, has no effect upon GnRH-stimulated LHß activity and enhanced the GnRH-stimulated FSHß transcription. Hence, PI4K and PI3K-AKT play a role in GnRH to ERK1/2 signaling, while PI3K may regulate also GnRH-induced gonadotropin gene expression.

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. When CRH perifusion started after 2 hours of glucocorticoid-free medium, corticosterone levels in the stress range (1 µM) caused a delayed (25 min) and prolonged inhibition of CRH-stimulated ACTH secretion, up to 60 minutes after corticosterone withdrawal. In contrast, after 6 hours of glucocorticoid-free medium, basal corticosterone levels inhibited CRH-stimulated ACTH within 5 minutes, after rapid recovery 5 minutes after corticosterone withdrawal. The latter effect was insensitive to actinomycin D but was prevented by the glucocorticoid receptor antagonist, RU486, suggesting nongenomic effects of the classical glucocorticoid receptor. In hypothalamic-derived 4B cells, 10 nM corticosterone increased immunoreactive glucocorticoid receptor content in membrane fractions, with association and clearance rates paralleling the effects on ACTH secretion from corticotrophs. Corticosterone did not affect CRH-stimulated calcium influx, but in AtT-20 cells, it had biphasic effects on CRH-stimulated Src phosphorylation, with early inhibition and late stimulation, suggesting a role for Src phosphorylation on the rapid glucocorticoid feedback. The data suggest that the nongenomic/membrane effects of classical GR mediate rapid and reversible glucocorticoid feedback inhibition at the pituitary corticotrophs downstream of calcium influx. The sensitivity and kinetics of these effects is consistent with the hypothesis that pituitary glucocorticoid feedback is part of the mechanism for adrenocortical ultradian pulse generation.

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. In the presence of dopamine, paliperidone rescued cAMP/calcium signaling and prolactin release in a concentration-dependent manner, whereas aripiprazole was only partially effective. In the absence of dopamine, paliperidone stimulated cAMP/calcium signaling and prolactin release, whereas aripiprazole inhibited signaling and secretion more potently but less effectively than dopamine. Forskolin-stimulated cAMP production was facilitated by paliperidone and inhibited by aripiprazole, although the latter was not as effective as dopamine. None of the compounds affected prolactin transcript activity, intracellular prolactin accumulation, or growth hormone secretion. These data indicate that paliperidone has dual hyperprolactinemic actions in lactotrophs i) by preserving the coupling of spontaneous electrical activity and prolactin secretion in the presence of dopamine and ii) by inhibiting intrinsic dopamine receptor activity in the absence of dopamine, leading to enhanced calcium signaling and secretion. In contrast, aripiprazole acts on prolactin secretion by attenuating, but not abolishing, calcium-secretion coupling.

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 ß-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 expression of Tshb could not be reversed via pulsatile or continuous TRH application in variable concentrations and treatment duration or by the removal of thyroid and steroid hormones from the sera. In parallel, the expression of CGA and TSHB proteins declined progressively in pituitary cells from both sexes. The lack of the effect of TRH on Tshb expression was not related to the age of pituitary cultures and the presence of functional TRH receptors. In cultured pituitary fragments, there was also a rapid decline in expression of these genes, but TRH was able to induce transient Tshb expression. In vivo, thyrotrophs were often in close proximity to each other and to somatotroph and folliculostellate cell networks and especially to the lactotroph cell network; such an organization pattern was lost in vitro. These observations suggest that the lack of influence of anterior pituitary architecture and/or intrapituitary factors probably accounts for the loss of basal and TRH-stimulated Tshb expression in dispersed pituitary cells.

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. However, elevated intracellular cAMP levels enhance the excitability of lactotrophs by stimulating the depolarizing non-selective cationic hyperpolarization-activated cyclic nucleotide-regulated and background channels, whereas cGMP inhibits it by activating Ca(2+)-controlled K(+) channels. Elevated cAMP also modulates prolactin release downstream of Ca(2+) influx by changing the kinetic of secretory pores: stimulate at low and inhibit at high concentrations. Induction of prolactin gene and lactotroph proliferation is also stimulated by elevated cAMP through protein kinase A. Together, these observations suggest that in lactotrophs cAMP exhibits complex regulatory effects on voltage-gated Ca(2+) influx and Ca(2+)-dependent cellular processes.

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. Activation of naïve (not previously stimulated) receptors by low agonist concentrations caused monophasic slow desensitizing currents and internalization of receptors without other changes in the cellular morphology, much like other P2XRs. In contrast, saturating agonist concentrations induced high-amplitude biphasic currents, reflecting pore dilation and causing rapid cell swelling and lysis. The existence of these two signaling patterns was accounted for using a revised Markov-state model that included, in addition to naïve and sensitized states, desensitized states. Occupancy of one or two ATP-binding sites of naïve receptors favored a slow transition to desensitized states, whereas occupancy of the third binding site favored a transition to sensitized/dilated states. Consistent with model predictions, nondilating P2X7R mutants always generated desensitizing currents. These results suggest that the level of saturation of the ligand binding sites determines the nature of the P2X7R gating and cellular actions.

Multiple cholinergic signaling pathways in pituitary gonadotrophs.

Acetylcholine (ACh) has been established as a paracrine factor in the anterior pituitary gland, but the receptors mediating ACh action and the cell types bearing these receptors have not been identified. Our results showed that the expression of the nicotinic subunits mRNAs followed the order ß2 > ß1 = α9 > α4 in cultured rat pituitary cells. The expression of the subunits in immortalized LßT2 mouse gonadotrophs followed the order ß2 > α4 = α1. M4 > M3 muscarinic receptor mRNA were also identified in pituitary and LßT2 cells. The treatment of cultured pituitary cells with GnRH down-regulated the expression of α9 and α4 mRNAs, without affecting the expression of M3 and M4 receptor mRNAs, and ACh did not alter the expression of GnRH receptor mRNA. We also performed double immunostaining to show the expression of ß2-subunit and M4 receptor proteins in gonadotrophs. Functional nicotinic channels capable of generating an inward current, facilitation of electrical activity, and Ca(2+) influx were identified in single gonadotrophs and LßT2 cells. In both cell types, the M3 receptor-mediated, phospholipase C-dependent Ca(2+) mobilization activated an outward apamin-sensitive K(+) current and caused hyperpolarization. The activation of M4 receptors by ACh inhibited cAMP production and GnRH-induced LH release in a pertussis toxin-sensitive manner. We concluded that multiple cholinergic receptors are expressed in gonadotrophs and that the main secretory action of ACh is inhibitory through M4 receptor-mediated down-regulation of cAMP production. The expression of nicotinic receptors in vitro compensates for the lack of regular GnRH stimulation of gonadotrophs.

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. However, pore dilation was accompanied with a decrease rather than an increase in the total conductance, which temporally coincided with rapid and partial desensitization. During sustained agonist application, receptors continued to desensitize in calcium-independent and calcium-dependent modes. Calcium-independent desensitization was more pronounced in P2X2bR, and calcium-dependent desensitization was more pronounced in P2X2aR. In whole cell recording, we also observed use-dependent facilitation of desensitization of both receptors. Such behavior was accounted for by a 16-state Markov kinetic model describing ATP binding/unbinding and activation/desensitization. The model assumes that naive receptors open when two to three ATP molecules bind and undergo calcium-independent desensitization, causing a decrease in the total conductance, or pore dilation, causing a shift in the reversal potential. In calcium-containing media, receptor desensitization is facilitated and the use-dependent desensitization can be modeled by a calcium-dependent toggle switch. The experiments and the model together provide a rationale for the lack of sustained current growth in dilating P2X2Rs and show that receptors in the dilated state can also desensitize in the presence of calcium.

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. Panx1c is lacking the Phe108-Gln180 sequence and P2X1d is missing the Val307-Cys426 C-terminal end sequence. Confocal microscopy and biotin labeling revealed that Panx1a is expressed in the plasma membrane, whereas Panx1c and Panx1d show the cytoplasmic localization when expressed as homomeric proteins. The three Panx1 isoforms and Panx2 form homomeric and heteromeric complexes in any combination. These splice forms can also physically associate with ATP-gated P2X2, P2X3, P2X4, and P2X7 receptor channels. The Panx1a-mediated ATP release in AtT-20 immortalized pituitary cells is attenuated when co-expressed with Panx1c or Panx1d. These results suggest that Panx1c and Panx1d may serve as dominant-negative effectors to modulate the functions of Panx1a through formation of heteromeric channels. The complex patterns of Panx1 expression and association could also define the P2X-dependent roles of these channels in cell types co-expressing both proteins.

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. Spontaneous and forskolin-stimulated calcium influx was present in cells with inhibited voltage-gated sodium and hyperpolarization-activated cation channels but not in cells bathed in medium, in which sodium was replaced with organic cations. Consistent with the role of sodium-conducting nonselective cation channels in PKA-stimulated Ca(2+) influx, cAMP induced a slowly developing current with a reversal potential of about 0 mV. Two TRP (transient receptor potential) channel blockers, SKF96365 and 2-APB, as well as flufenamic acid, an inhibitor of nonselective cation channels, also inhibited spontaneous and forskolin-stimulated electrical activity and calcium influx. Quantitative RT-PCR analysis indicated the expression of mRNA transcripts for TRPC1 >> TRPC6 > TRPC4 > TRPC5 > TRPC3 in rat pituitary cells. These experiments suggest that in pituitary cells constitutively active cation channels are stimulated further by PKA and contribute to calcium signaling indirectly by controlling the pacemaking depolarization in a sodium-dependent manner and directly by conducting calcium.

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. Pannexin 1 was more abundantly expressed in the anterior lobe, whereas pannexin 2 was more abundantly expressed in the intermediate and posterior pituitary. Pannexin 1 was identified in corticotrophs and a fraction of somatotrophs, the S100-positive pituicytes of the posterior pituitary and AtT-20 (mouse pituitary adrenocorticotropin-secreting cells) and rat immortalized pituitary cells secreting prolactin, whereas pannexin 2 was detected in the S100-positive folliculostellate cells of the anterior pituitary, melanotrophs of the intermediate lobe, and vasopressin-containing axons and nerve endings in the posterior lobe. Overexpression of pannexins 1 and 2 in AtT-20 pituitary cells enhanced the release of ATP in the extracellular medium, which was blocked by the gap junction inhibitor carbenoxolone. Basal ATP release in At-T20 cells was also suppressed by down-regulating the expression of endogenous pannexin 1 but not pannexin 2 with their short interfering RNAs. These results indicate that pannexins may provide a pathway for delivery of ATP, which is a native agonist for numerous P2X cationic channels and G protein-coupled P2Y receptors endogenously expressed in the pituitary gland.

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. The slow secondary growth of current in the biphasic response coincided temporally with pore dilation. Repetitive stimulation with the same agonist concentration caused sensitization of receptors, which manifested as a progressive increase in the current amplitude, accompanied by a slower deactivation rate. Once a steady level of the secondary current was reached, responses at high agonist concentrations were no longer biphasic but monophasic. Sensitization of receptors was independent of Na(+) and Ca(2+) influx and ∼30 min washout was needed to reestablish the initial gating properties. T15E- and T15K-P2X7 mutants showed increased sensitivity for agonists, responded with monophasic currents at all agonist concentrations, activated immediately with dilated pores, and deactivated slowly. The complex pattern of gating exhibited by wild-type channels can be accounted for by a Markov state model that includes negative cooperativity of agonist binding to unsensitized receptors caused by the occupancy of one or two binding sites, opening of the channel pore to a low conductance state when two sites are bound, and sensitization with pore dilation to a high conductance state when three sites are occupied.

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). AA release was followed by a marked induction of cyclooxygenase (COX)-1 and COX-2 by GnRH via the protein kinase C/c-Src/phosphatidylinositol 3-kinase/MAPK pathway. COX-2 transcription by GnRH is mediated by the two nuclear factor-kappaB sites and the CCAAT/enhancer-binding protein site within its promoter. Indeed, GnRH stimulates p65/RelA phosphorylation (22-fold) in LbetaT2 cells and the two nuclear factor-kappaB sites apparently act as a composite response element. Although GnRH stimulates cAMP formation in LbetaT2 cells, we found no role for cAMP acting via the cAMP response element site in the COX-2 promoter. PGF(2alpha), PGI(2), or PGE(2) had no effect on GnRH-stimulated ERK, c-Jun N-terminal kinase, and p38MAPK activation or on GnRH- and high K(+)-stimulated intracellular Ca(2+) elevation in LbetaT2 and gonadotropes in primary culture. Although, PGF(2alpha), PGI(2), and PGE(2) reduced GnRH-stimulated cAMP formation, we could not correlate it to the inhibition of GnRH receptor expression, which is exerted only by PGF(2alpha) and PGI(2.) Hence, the inhibition by PGF(2alpha) and PGI(2) of the autoregulation of GnRH receptor expression is most likely mediated via inhibition of GnRH-stimulated phosphoinositide turnover and not by inhibition of Ca(2+) elevation and MAPK activation.

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 channels were activated and desensitized in a dose-dependent manner and deactivated rapidly. Activation of these channels led to stimulation of electrical activity and promotion of voltage-gated and voltage-insensitive Ca(2+) influx. In the presence of ivermectin, a specific allosteric modulator of P2X(4)Rs, there was an approximately fourfold increase in the maximum amplitude of the ATP-induced inward current, accompanied by an increase in the sensitivity of receptors for ATP, slowed deactivation of receptors, and enhanced ATP-induced prolactin release. These results indicate that thyrotropin-releasing hormone-responsive cells, including lactotrophs, express homomeric and/or heteromeric P2X(4)Rs, which facilitate Ca(2+) influx and hormone secretion.