Taka-aki Koshimizu

The vasopressin V1b receptor critically regulates hypothalamic-pituitary-adrenal axis activity under both stress and resting conditions

The neurohypophyseal peptide [Arg(8)]-vasopressin (AVP) exerts major physiological actions through three distinct receptor isoforms designated V1a, V1b, and V2. Among these three subtypes, the vasopressin V1b receptor is specifically expressed in pituitary corticotrophs and mediates the stimulatory effect of vasopressin on ACTH release. To investigate the functional roles of V1b receptor subtypes in vivo, gene targeting was used to create a mouse model lacking the V1b receptor gene (V1bR-/-). Under resting conditions, circulating concentrations of ACTH and corticosterone were lower in V1bR-/- mice compared with WT mice (V1bR+/+). The normal increase in circulating ACTH levels in response to exogenous administration of AVP was impaired in V1bR-/- mice, while corticotropin-releasing hormone-stimulated ACTH release in the V1bR-/- mice was not significantly different from that in the V1bR+/+ mice. AVP-induced ACTH release from primary cultured pituitary cells in V1bR-/- mice was also blunted. Furthermore, the increase in ACTH after a forced swim stress was significantly suppressed in V1bR-/- mice. Our results clearly demonstrate that the V1b receptor plays a crucial role in regulating hypothalamic-pituitary-adrenal axis activity. It does this by maintaining ACTH and corticosterone levels, not only under stress but also under basal conditions.

Contributions of the C-terminal domain to the control of P2X receptor desensitization.

The P2X purinergic receptor channels (P2XRs) differ among themselves with respect to the rates of desensitization during prolonged agonist stimulation. Here we studied the desensitization of recombinant channels by monitoring the changes in intracellular free Ca2+ concentration in cells stimulated with ATP, the native and common agonist for all P2XRs. The focus in our investigations was on the relevance of the P2XR C terminus in controlling receptor desensitization. When expressed in GT1 cells, the P2XRs desensitized with rates characteristic to each receptor subtype: P2X1R = P2X3R > P2X2bR > P2X4R > P2X2aR > P2X7R. A slow desensitizing pattern of P2X2aR was mimicked partially by P2X3R and fully by P2X4R when the six-amino acid sequences of these channels located in the cytoplasmic C terminus were substituted with the corresponding arginine 371 to proline 376 sequence of P2X2aR. Changing the total net charge in the six amino acids of P2X4R to a more positive direction also slowed the receptor desensitization. On the other hand, substitution of arginine 371-proline 376 sequence of P2X2aR with the corresponding sequences of P2X1R, P2X3R, and P2X4R increased the rate of receptor desensitization. Furthermore, heterologous polymerization of wild-type P2X2aR and mutant P2X3R having the C-terminal six amino acids of P2X2aR at its analogous position resulted in a functional channel whose desensitization was significantly delayed. These results suggest that composition of the C-terminal six-amino acid sequence and its electrostatic force influence the rate of receptor desensitization.

IDENTIFICATION OF AMINO ACID RESIDUES CONTRIBUTING TO DESENSITIZATION OF THE P2X2 RECEPTOR CHANNEL

The P2X2 receptor (P2X2R) is a member of the ATP-gated ion channels that mediate Ca2+ entry in several tissues, including the brain, adrenal medulla, and pituitary. Alternative usage of cryptic splice sites in the primary P2X2R transcript accounts for the existence of several transcript types, one of which (P2X2–2R) encodes a functional channel. P2X2–2R lacks a stretch of cytoplasmic C-terminal amino acids (Val370-Gln438) and exhibits rapid and complete desensitization, whereas P2X2R desensitizes slowly and incompletely. The role of the C terminus in P2X2R desensitization was studied by generating several channel mutants and monitoring intracellular free Ca2+ changes in transfected single GT1–7 neurons. Deletion studies indicated that the Arg371-Ile391 segment of the P2X2R is required for sustained Ca2+ influx. To identify the important residues within this segment, three contiguous amino acids were sequentially changed to alanine. Only two of these replacement mutants, at Arg371-Thr372-Pro373and Lys374-His375-Pro376, had an enhanced rate of desensitization. Single amino acid deletions in the P2X2R C terminus and a series of insertions of wild-type sequences into the corresponding spliced site identified four residues, Pro373-Lys374-His375-Pro376, required for sustained Ca2+ influx through agonist-occupied wild-type channels. Thus, it is likely that the Pro373-Pro376 sequence of P2X2R represents a functional motif that is critical for the development of the slow desensitization profile observed in these channels. Consequently, deletion of this motif by alternative splicing provides an effective mechanism for generating a channel with controlled Ca2+ influx.

Characterization of a plasma membrane calcium oscillator in rat pituitary somatotrophs.

In excitable cells, oscillations in intracellular free calcium concentrations ([Ca2+] i ) can arise from action-potential-driven Ca2+ influx, and such signals can have either a localized or global form, depending on the coupling of voltage-gated Ca2+ influx to intracellular Ca2+ release pathway. Here we show that rat pituitary somatotrophs generate spontaneous [Ca2+] i oscillations, which rise from fluctuations in the influx of external Ca2+ and propagate within the cytoplasm and nucleus. The addition of caffeine and ryanodine, modulators of ryanodine-receptor channels, and the depletion of intracellular Ca2+ stores by thapsigargin and ionomycin did not affect the global nature of spontaneous [Ca2+] i signals. Bay K 8644, an L-type Ca2+ channel agonist, initiated [Ca2+] i signaling in quiescent cells, increased the amplitude of [Ca2+] i spikes in spontaneously active cells, and stimulated growth hormone secretion in perifused pituitary cells. Nifedipine, a blocker of L-type Ca2+channels, decreased the amplitude of spikes and basal growth hormone secretion, whereas Ni2+, a blocker of T-type Ca2+ channels, abolished spontaneous [Ca2+] i oscillations. Spiking was also abolished by the removal of extracellular Na+ and by the addition of 10 mm Ca2+, Mg2+, or Sr2+, the blockers of cyclic nucleotide-gated channels. Reverse transcriptase-polymerase chain reaction and Southern blot analyses indicated the expression of mRNAs for these channels in mixed pituitary cells and purified somatotrophs. Growth hormone-releasing hormone, an agonist that stimulated cAMP and cGMP productions in a dose-dependent manner, initiated spiking in quiescent cells and increased the frequency of spiking in spontaneously active cells. These results indicate that in somatotrophs a cyclic nucleotide-controlled plasma membrane Ca2+oscillator is capable of generating global Ca2+ signals spontaneously and in response to agonist stimulation. The Ca2+-signaling activity of this oscillator is dependent on voltage-gated Ca2+ influx but not on Ca2+ release from intracellular stores.

Signaling by extracellular nucleotides in anterior pituitary cells

Pituitary cells secrete ATP, which acts as an autocrine and/or paracrine extracellular messenger on two families of purinergic receptors: G-protein-coupled P2Y receptors (P2YRs) and ion-conducting P2X receptors (P2XRs). Lactotrophs and GH(3)-immortalized cells express the P2Y(2)R subtype. Several P2XR subtypes are expressed in pituitary cells. Gonadotrophs and somatotrophs express P2X(2a)R and P2X(2b)R, which occur as heteromeric channels. Lactotrophs and GH(3) cells express one or more ion-conducting subtypes from among P2X(3)R, P2X(4)R and P2X(7)R in homomeric form. Thyrotrophs and corticotrophs also express P2XRs, but their identification requires further study. Pituitary cells express purinergic P1 receptors, which are activated by adenosine. The A(1)R subtype of these receptors is expressed in melanotrophs and GH(3) cells. In this review, we briefly discuss the expression and coupling of A(1)R and P2Y(2)R, and focus on the expression and Ca(2+) signaling of P2XRs.

Dependence of Soluble Guanylyl Cyclase Activity on Calcium Signaling in Pituitary Cells

The role of nitric oxide (NO) in the stimulation of soluble guanylyl cyclase (sGC) is well established, but the mechanism by which the enzyme is inactivated during the prolonged NO stimulation has not been characterized. In this paper we studied the interactions between NO and intracellular Ca2+ in the control of sGC in rat anterior pituitary cells. Experiments were done in cultured cells, which expressed neuronal and endothelial NO synthases, and in cells with elevated NO levels induced by the expression of inducible NO synthase and by the addition of several NO donors. Basal sGC-dependent cGMP production was stimulated by the increase in NO levels in a time-dependent manner. In contrast, depolarization of cells by high K+ and Bay K 8644, an L-type Ca2+ channel agonist, inhibited sGC activity. Depolarization-induced down-regulation of sGC activity was also observed in cells with inhibited cGMP-dependent phosphodiesterases but not in cells bathed in Ca2+-deficient medium. This inhibition was independent from the pattern of Ca2+ signaling (oscillatoryversus nonoscillatory) and NO levels, and was determined by averaged concentration of intracellular Ca2+. These results indicate that inactivation of sGC by intracellular Ca2+serves as a negative feedback to break the stimulatory action of NO on enzyme activity in intact pituitary cells.

Signaling by purinergic receptors and channels in the pituitary gland

Adenosine 5-triphosphate is frequently released by cells and acts as an agonist for G protein-coupled P2Y receptors and ligand-gated P2X cationic channels in numerous tissues. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors. In the pituitary gland, adenosine 5-triphosphate is released from the endings of magnocellular hypothalamic neurons and by anterior pituitary cells through pathway(s) that are still not well characterized. This gland also expresses several members of each family of purinergic receptors. P2X and adenosine receptors are co-expressed in the somata and nerve terminals of vasopressin-releasing neurons as well as in some secretory pituitary cells. P2X receptors stimulate electrical activity and modulate InsP(3)-dependent calcium release from intracellular stores, whereas adenosine receptors terminate electrical activity. Calcium-mobilizing P2Y receptors are expressed in pituicytes, folliculo-stellate cells and some secretory cells of the anterior pituitary.