The electrical activity pattern of endocrine pituitary cells regulates their basal secretion level. Rat somatotrophs and lactotrophs exhibit spontaneous bursting and have high basal levels of hormone secretion, while gonadotrophs exhibit spontaneous spiking and have low basal hormone secretion. It has been proposed that the difference in electrical activity between bursting somatotrophs and spiking gonadotrophs is due to the presence of large conductance potassium (BK) channels on somatotrophs but not on gonadotrophs. This is one example where the role of an ion channel type may be clearly established. We demonstrate here that BK channels indeed promote bursting activity in pituitary cells. Blocking BK channels in bursting lacto-somatotroph GH4C1 cells changes their firing activity to spiking, while further adding an artificial BK conductance via dynamic clamp restores bursting. Importantly, this burst-promoting effect requires a relatively fast BK activation/deactivation, as predicted by computational models. We also show that adding a fast activating BK conductance to spiking gonadotrophs converts the activity of these cells to bursting. Together, our results suggest that differences in BK channel expression may underlie the differences in electrical activity and basal hormone secretion levels among pituitary cell types and that the rapid rate of BK channel activation is key to its role in burst promotion.

Pituitary cells fire action potentials independently of external stimuli, and such spontaneous electrical activity is modulated by a large variety of hypothalamic and intrapituitary agonists. Here, we focused on the potential role of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels in electrical activity of cultured rat anterior pituitary cells. Quantitative RT-PCR analysis showed higher level of expression of mRNA transcripts for HCN2 and HCN3 subunits and lower expression of HCN1 and HCN4 subunits in these cells. Western immunoblot analysis of lysates from normal and GH(3) immortalized pituitary cells showed bands with appropriate molecular weights for HCN2, HCN3, and HCN4. Electrophysiological experiments showed the presence of a slowly developing hyperpolarization-activated inward current, which was blocked by Cs(+) and ZD7288, in gonadotrophs, thyrotrophs, somatotrophs, and a fraction of lactotrophs, as well as in other unidentified pituitary cell types. Stimulation of adenylyl cyclase and addition of 8-Br-cAMP enhanced this current and depolarized the cell membrane, whereas 8-Br-cGMP did not alter the current and hyperpolarized the cell membrane. Both inhibition of basal adenylyl cyclase activity and stimulation of phospholipase C signaling pathway inhibited this current. Inhibition of HCN channels affected the frequency of firing but did not abolish spontaneous electrical activity. These experiments indicate that cAMP and cGMP have opposite effects on the excitability of endocrine pituitary cells, that basal cAMP production in cultured cells is sufficient to integrate the majority of HCN channels in electrical activity, and that depletion of phosphatidylinositol 4,5-bisphosphate caused by activation of phospholipase C silences them.

Anterior pituitary cells express nucleotide-gated G protein-coupled P2 receptors (P2YRs) and cation-conducting channels (P2XRs). However, the identification of P2 receptors subtypes and their native ligands, and the distribution and function of these receptors within the secretory and non-secretory pituitary cells has been incompletely characterized. The focus in this study was on lactotroph subpopulation of cells. ATP and ADP, but not UTP and UDP, triggered calcium signaling in a majority (85%) of lactotrophs and prolactin release in mixed pituitary cells. Consistent with the role of P2 receptors in signaling and secretion, the actions of ATP and ADP were abolished in the presence of apyrase, an ectonucleotidase. Transcripts for G q -coupled calciummobilizing P2Y 1 R, P2Y 2 R, P2Y 4 R, and P2Y 6 R, as well as G i -coupled P2Y 12 R, were identified in mixed anterior pituitary cells. The ligand-selectivity profile of calcium mobilization-dependent signaling and prolactin secretion and the blockade of these responses by pyridoxal 5-phosphate 6-azophenyl-2,4-disulphonic acid indicated that P2Y 1 R mediates the stimulatory action of ATP and ADP. Within the channels expressed in anterior pituitary (P2X 2 R, P2X 3 R, P2X 4 R, and P2X 7 R), the P2X 4 R subtype provides a major pathway for calcium influx-dependent signaling and prolactin secretion. This conclusion was based on comparison of native to recombinant channels with respect to their ligand preference, sensitivity to pyridoxal 5-phosphate 6-azophenyl-2,4-disulphonic acid, and the rates of calcium signal desensitization.Purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) are not only important intracellular molecules, but also function as extracellular messengers by activating distinct plasma membrane receptors. These receptors were termed "purinergic" in the seventies and belong to two groups: P1 receptors (P1Rs) 1 that are activated by adenosine and P2 receptors (P2Rs) that recognize mainly ADP, ATP, UDP, and UTP. Four subtypes comprise the P1R family of G protein-coupled receptors, termed A 1 , A 2A , A 2B , and A 3 , whereas P2Rs are composed of two families: the ligand-gated channels (P2XR) and the G protein-coupled receptors (P2YR). To date, seven functional channels (P2X 1 to P2X 7 ) and six receptors (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , and P2Y 12 ) have been identified in mammals (1-3). P2Rs are expressed in a variety of non-excitable and excitable tissues. Three lines of previously published evidence also indicate that an extracellular nucleotide-controlled signaling system is expressed and operative in anterior pituitary cells. First, ATP is co-stored with hormones in secretory vesicles and co-secreted by anterior pituitary cells (4, 5). Second, P1Rs, P2YRs, and P2XRs are expressed in these cells and their activation leads to amplification of calcium signals and secretion triggered by hypothalamic neurohormones (6 -8). Third, the autocrine/paracrine actions of ATP are controlled by ectonucleotidases (4). These enzymes degrade extracel...

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 1Y3 in these cells. These enzymes were functional as documented by degradation of endogenously released and exogenously added ATP. Blocking the activity of eNTPDases by ARL67156 led to an increase in ATP release in perifused pituitary cells and inhibition of degradation of extracellularly added ATP. Furthermore, the addition of apyrase, a soluble ecto-nucleotidase, and the expression of recombinant mouse eNTPDase-2, enhanced degradation of both endogenously released and exogenously added ATP. The released ATP by resting hypothalamic cells was sufficient to activate and desensitize high-affinity recombinant P2X receptors, whereas facilitation of ATP metabolism by the addition of apyrase protected their desensitization. These results indicate that colocalization of ATP release sites and ecto-nucleotidase activity at the plasma membrane of hypothalamic and pituitary cells provides an effective mechanism for the operation of nucleotides as extracellular signaling molecules.Abbreviations: [Ca 2+ ] i -intracellular calcium concentration; eNTPDase -ecto-nucleoside triphosphate diphosphohydrolase;

Pituitary lactotrophs in vitro fire extracellular Ca2+-dependent action potentials spontaneously through still unidentified pacemaking channels, and the associated voltage-gated Ca2+influx (VGCI) is sufficient to maintain basal prolactin (PRL) secretion high and steady. Numerous plasma membrane channels have been characterized in these cells, but the mechanism underlying their pacemaking activity is still not known. Here we studied the relevance of cyclic nucleotide signaling pathways in control of pacemaking, VGCI, and PRL release. In mixed anterior pituitary cells, both VGCI-inhibitable and -insensitive adenylyl cyclase (AC) subtypes contributed to the basal cAMP production, and soluble guanylyl cyclase was exclusively responsible for basal cGMP production. Inhibition of basal AC activity, but not soluble guanylyl cyclase activity, reduced PRL release. In contrast, forskolin stimulated cAMP and cGMP production as well as pacemaking, VGCI, and PRL secretion. Elevation in cAMP and cGMP levels by inhibition of phosphodiesterase activity was also accompanied with increased PRL release. The AC inhibitors attenuated forskolin-stimulated cyclic nucleotide production, VGCI, and PRL release. The cell-permeable 8-bromo-cAMP stimulated firing of action potentials and PRL release and rescued hormone secretion in cells with inhibited ACs in an extracellular Ca2+-dependent manner, whereas 8-bromo-cGMP and 8-(4-chlorophenylthio)-2'-O-methyl-cAMP were ineffective. Protein kinase A inhibitors did not stop spontaneous and forskolin-stimulated pacemaking, VGCI, and PRL release. These results indicate that cAMP facilitates pacemaking, VGCI, and PRL release in lactotrophs predominantly in a protein kinase A- and Epac cAMP receptor-independent manner.

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. Here we show that the dopamine agonist-induced inhibition of spontaneous Ca(2+) influx and release of prestored PRL was preserved when cAMP levels were elevated by forskolin treatment. We further observed that dopamine agonists inhibited both spontaneous and depolarization-induced Ca(2+) influx in untreated but not in PTX-treated cells. This inhibition was also observed in cells with blocked inward rectifier K(+) channels, suggesting that the dopamine effect on voltage-gated Ca(2+) channel gating is sufficient to inhibit spontaneous Ca(2+) influx. However, agonist-induced inhibition of PRL release was only partially relieved in PTX-treated cells, indicating that dopamine receptors also inhibit exocytosis downstream of voltage-gated Ca(2+) influx. The PTX-insensitive step in agonist-induced inhibition of PRL release was not affected by the addition of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and lithium, an inhibitor of glycogen synthase kinase-3, but was attenuated in the presence of phorbol 12-myristate 13-acetate, which inhibits G(z) signaling pathway in a protein kinase C-dependent manner. Thus, dopamine inhibits basal PRL release by blocking voltage-gated Ca(2+) influx through the PTX-sensitive signaling pathway and by desensitizing Ca(2+) secretion coupling through the PTX-insensitive and protein kinase C-sensitive signaling pathway.

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 P2X4 subunit are the most abundant in rat anterior pituitary tissue and confirm the P2X4R 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 P2X4R. 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 voltageinsensitive Ca 2ϩ influx. In the presence of ivermectin, a specific allosteric modulator of P2X4Rs, 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 P2X4Rs, which facilitate Ca 2ϩ influx and hormone secretion. adenosine 5=-triphosphatase; adenosine 5=-triphosphatase-gated receptor channels; calcium; lactotrophs; prolactin; ivermectin IN ADDITION TO ITS INTRACELLULAR FUNCTIONS, ATP can be released by excitable and nonexcitable cells and act as an extracellular messenger through the stimulation of P2Y receptors (P2YRs) and P2X receptors (P2XRs). To date, eight mammalian P2YRs have been identified and are denoted as

Prolactin and oxytocin are important reproductive hormones implicated in several common adaptive functions during pregnancy, pseudopregnancy and lactation. Recently, extracellular recordings of supraoptic neurons have shown that prolactin may modulate electrical activity of oxytocinergic neurons. However, no study has been conducted to establish whether prolactin directly influences this activity in oxytocinergic paraventricular neurons. Here we addressed this question by studying the effects of prolactin on the electrical activity and voltage-current relationship of identified paraventricular neurons in rat brain slices. Whole-cell recordings were obtained and neurons were classified on the basis of their morphological and electrophysiological fingerprint (magnocellular or parvicellular) and neuropeptide phenotype (oxytocinergic or non-oxytocinergic). We report that prolactin elicited a hyperpolarizating current in 37% of the neurons in this nucleus, of which the majority (67%) were identified as putative magnocellular oxytocin neurons and the reminder (33%) were regarded as oxytocin-negative, parvicellular neuroendocrine neurons. Our results suggest that, in addition to the well-established negative feedback loop between prolactin-secreting lactotrophs and dopaminergic neurons in the arcuate nucleus, an inhibitory feedback loop also exists between lactotrophs and oxytocinergic paraventricular neurons.