Differential distribution and potential regulatory roles of estrogen receptor 2a and 2b in the pituitary of ricefield eel Monopterus albus.

Estrogens play important regulatory roles in the pituitary of vertebrates. Two forms of estrogen receptor 2 (Esr2), namely Esr2a and Esr2b, are identified in teleosts, but their differential roles remain to be fully elucidated. In the present study, expression and potential functional roles of Esr2a and Esr2b were characterized in ricefield eels. esr2a and esr2b mRNA were broadly distributed in tissues, with high levels observed in the brain, pituitary, and gonads. In order to examine the cellular localization of Esr2a and Esr2b in the pituitary, specific antisera against ricefield eel Esr2a and Esr2b were generated, respectively. Interestingly, immunohistochemistry and Western blot analysis revealed that Esr2a and Esr2b were differentially distributed in the pituitary, with the former localized to the adenohypophysis while the latter to the neurohypophysis. Dual fluorescent immunostaining showed that immunoreactive Esr2a was present in Gh and Prl cells, but not in Lh and Fsh cells. Estradiol (E2) stimulated lhb and prl gene expression in dispersed pituitary cells of intersexual ricefield eels, but had no effects on gh, fshb, and gnrhr2 gene expression and Gh release. Results of the present study are helpful for further understanding the roles and mechanisms of estrogen signals in the pituitary.

Critical role of rabphilin-3A in the pathophysiology of experimental lymphocytic neurohypophysitis.

Autoimmune hypophysitis (AH) is thought to be an autoimmune disease characterized by lymphocytic infiltration of the pituitary gland. Among AH pathologies, lymphocytic infundibulo-neurohypophysitis (LINH) involves infiltration of the neurohypophysis and/or the hypothalamic infundibulum, causing central diabetes insipidus resulting from insufficiency of arginine vasopressin secretion. The pathophysiological and pathogenetic mechanisms underlying LINH are largely unknown. Clinically, differentiating LINH from other pituitary diseases accompanied by mass lesions, including tumours, has often been difficult, because of similar clinical manifestations. We recently reported that rabphilin-3A is an autoantigen and that anti-rabphilin-3A antibodies constitute a possible diagnostic marker for LINH. However, the involvement of rabphilin-3A in the pathogenesis of LINH remains to be elucidated. This study was undertaken to explore the role of rabphilin-3A in lymphocytic neurohypophysitis and to investigate the mechanism. We found that immunization of mice with rabphilin-3A led to neurohypophysitis. Lymphocytic infiltration was observed in the neurohypophysis and supraoptic nucleus 1 month after the first immunization. Mice immunized with rabphilin-3A showed an increase in the volume of urine that was hypotonic as compared with control mice. Administration of a cocktail of monoclonal anti-rabphilin-3A antibodies did not induce neurohypophysitis. However, abatacept, which is a chimeric protein that suppresses T-cell activation, decreased the number of T cells specific for rabphilin-3A in peripheral blood mononuclear cells (PBMCs). It ameliorated lymphocytic infiltration of CD3+ T cells in the neurohypophysis of mice that had been immunized with rabphilin-3A. Additionally, there was a linear association between the number of T cells specific for rabphilin-3A in PBMCs and the number of CD3+ T cells infiltrating the neurohypophysis. In conclusion, we suggest that rabphilin-3A is a pathogenic antigen, and that T cells specific for rabphilin-3A are involved in the pathogenesis of neurohypophysitis in mice. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Osmoregulation requires brain expression of the renal Na-K-2Cl cotransporter NKCC2.

The Na-K-2Cl cotransporter 2 (NKCC2) was thought to be kidney specific. Here we show expression in the brain hypothalamo-neurohypophyseal system (HNS), wherein upregulation follows osmotic stress. The HNS controls osmotic stability through the synthesis and release of the neuropeptide hormone, arginine vasopressin (AVP). AVP travels through the bloodstream to the kidney, where it promotes water conservation. Knockdown of HNS NKCC2 elicited profound effects on fluid balance following ingestion of a high-salt solution-rats produced significantly more urine, concomitant with increases in fluid intake and plasma osmolality. Since NKCC2 is the molecular target of the loop diuretics bumetanide and furosemide, we asked about their effects on HNS function following disturbed water balance. Dehydration-evoked GABA-mediated excitation of AVP neurons was reversed by bumetanide, and furosemide blocked AVP release, both in vivo and in hypothalamic explants. Thus, NKCC2-dependent brain mechanisms that regulate osmotic stability are disrupted by loop diuretics in rats.

µ-Opioid inhibition of Ca2+ currents and secretion in isolated terminals of the neurohypophysis occurs via ryanodine-sensitive Ca2+ stores.

µ-Opioid agonists have no effect on calcium currents (I(Ca)) in neurohypophysial terminals when recorded using the classic whole-cell patch-clamp configuration. However, µ-opioid receptor (MOR)-mediated inhibition of I(Ca) is reliably demonstrated using the perforated-patch configuration. This suggests that the MOR-signaling pathway is sensitive to intraterminal dialysis and is therefore mediated by a readily diffusible second messenger. Using the perforated patch-clamp technique and ratio-calcium-imaging methods, we describe a diffusible second messenger pathway stimulated by the MOR that inhibits voltage-gated calcium channels in isolated terminals from the rat neurohypophysis (NH). Our results show a rise in basal intracellular calcium ([Ca(2+)]i) in response to application of [D-Ala(2)-N-Me-Phe(4),Gly5-ol]-Enkephalin (DAMGO), a MOR agonist, that is blocked by D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a MOR antagonist. Buffering DAMGO-induced changes in [Ca(2+)]i with BAPTA-AM completely blocked the inhibition of both I(Ca) and high-K(+)-induced rises in [Ca(2+)]i due to MOR activation, but had no effect on κ-opioid receptor (KOR)-mediated inhibition. Given the presence of ryanodine-sensitive stores in isolated terminals, we tested 8-bromo-cyclic adenosine diphosphate ribose (8Br-cADPr), a competitive inhibitor of cyclic ADP-ribose (cADPr) signaling that partially relieves DAMGO inhibition of I(Ca) and completely relieves MOR-mediated inhibition of high-K(+)-induced and DAMGO-induced rises in [Ca(2+)]i. Furthermore, antagonist concentrations of ryanodine completely blocked MOR-induced increases in [Ca(2+)]i and inhibition of I(Ca) and high-K(+)-induced rises in [Ca(2+)]i while not affecting KOR-mediated inhibition. Antagonist concentrations of ryanodine also blocked MOR-mediated inhibition of electrically-evoked increases in capacitance. These results strongly suggest that a key diffusible second messenger mediating the MOR-signaling pathway in NH terminals is [Ca(2+)]i released by cADPr from ryanodine-sensitive stores.

P2X7 receptors in neurohypophysial terminals: evidence for their role in arginine-vasopressin secretion.

Arginine-vasopressin (AVP) plays a major role in maintaining cardiovascular function and related pathologies. The mechanism involved in its release into the circulation is complex and highly regulated. Recent work has implicated the purinergic receptor, P2X7R, in a role for catecholamine-enhanced AVP release in the rat hypothalamic-neurohypophysial (NH) system. However, the site of P2X7R action in this endocrine system, and whether or not it directly mediates release in secretory neurons have not been determined. We hypothesized that the P2X7R is expressed and mediates AVP release in NH terminals. P2X7R function was first examined by patch-clamp recordings in isolated NH terminals. Results revealed that subpopulations of isolated terminals displayed either high ATP-sensitivity or low ATP-sensitivity, the latter of which was characteristic of the rat P2X7R. Additional recordings showed that terminals showing sensitivity to the P2X7R-selective agonist, BzATP, were further inhibited by P2X7R selective antagonists, AZ10606120 and brilliant blue-G. In confocal micrographs from tissue sections and isolated terminals of the NH P2X7R-immunoreactivity was found to be localized in plasma membranes. Lastly, the role of P2X7R on AVP release was tested. Our results showed that BzATP evoked sustained AVP release in NH terminals, which was inhibited by AZ10606120. Taken together, our data lead us to conclude that the P2X7R is expressed in NH terminals and corroborates its role in AVP secretion.

Neurohypophyseal response to fluid resuscitation with hypertonic saline during septic shock in rats.

Septic shock is a serious condition with a consequent drop in blood pressure and inadequate tissue perfusion. Small-volume resuscitation with hypertonic saline (HS) has been proposed to restore physiological haemodynamics during haemorrhagic and endotoxic shock. In the present study, we sought to determine the effects produced by an HS infusion in rats subjected to caecal ligation and perforation (CLP). Male Wistar rats were randomly grouped and submitted to either CLP or sham surgery. Either HS (7.5% NaCl, 4 ml kg(-1) i.v.) or isotonic saline (IS; 0.9% NaCl, 4 ml kg(-1) i.v.) was administered 6 h after CLP. Recordings of mean arterial pressure and heart rate were made during this protocol. Moreover, measurements of electrolyte, vasopressin and oxytocin secretion were analysed after either the HS or the IS treatment. Six hours after CLP, we observed a characteristic decrease in mean arterial pressure that occurs after CLP. The HS infusion in these rats produced a transient elevation of the plasma sodium concentration and osmolality and increased plasma vasopressin and oxytocin levels. Moreover, the HS infusion could restore the mean arterial pressure after CLP, which was completely blunted by the previous injection of the vasopressin but not the oxytocin antagonist. The present study demonstrated that rats subjected to CLP and an infusion of hypertonic saline respond with secretion of neurohypophyseal hormones and a transient increase in blood pressure mediated by the V(1) receptor.

Molecular tolerance of voltage-gated calcium channels is evident after short exposures to alcohol in vasopressin-releasing nerve terminals.

BACKGROUND: Voltage-gated calcium channels (VGCCs) in rat neurohypophysial terminals exhibit molecular tolerance to alcohol, including desensitization to the drug and increased current density, after 3 weeks of alcohol drinking. Moreover, after this time, terminals from drinking rats exhibit diminished alcohol inhibition of vasopressin (AVP) release. METHODS: We took advantage of organotypic cultures (explants) of the hypothalamo-neurohypophysial system (HNS) to extend our analysis of molecular tolerance to 2 classes of the VGCC. The isolated HNS explant allows much finer temporal resolution of molecular tolerance than do voluntary drinking paradigms. After exposure of the HNS explant to alcohol, terminals are isolated by mechanical treatment and plated in a dish. Patch clamp recording techniques are used to obtain VGCC currents, and immunohistochemistry is used to determine VGCC distribution. A release assay is used to provide functional readout of AVP release. RESULTS: We show that even a brief, 1-hour exposure to a clinically relevant concentration of alcohol is sufficient to evoke similar changes to those observed after several weeks of exposure. Acute ethanol (EtOH) exposure inhibits high K(+) -induced AVP release from naïve terminals. However, terminals pre-exposed to 20 mM EtOH for 1 hour become tolerant to EtOH, and subsequent exposure has significantly less effect on high K(+) -induced AVP release. Electrophysiological recordings indicate that among different types of VGCCs present in the neuronal terminal, the L-type is the most affected by alcohol. The current density of L-type current is significantly increased (approximately 50%), while its responsiveness to alcohol is significantly diminished (approximately 50%), after brief alcohol exposure. Fluorescent imaging results were consistent with the electrophysiology and suggest that the increased current density of VGCCs after brief exposure is attributable to combined synthesis of 1.2 and 1.3 subtypes of the L-type VGCC and redistribution of channel protein into terminal plasma membrane. CONCLUSIONS: These data indicate that a brief alcohol exposure affects subsequent alcohol sensitivity of VGCCs and neuropeptide release from presynaptic terminals.

Socially modulated cell proliferation is independent of gonadal steroid hormones in the brain of the adult green treefrog (Hyla cinerea).

Gonadal steroid hormones have been shown to influence adult neurogenesis in addition to their well-defined role in regulating social behavior. Adult neurogenesis consists of several processes including cell proliferation, which can be studied via 5-bromo-2'-deoxyuridine (BrdU) labeling. In a previous study we found that social stimulation altered both cell proliferation and levels of circulating gonadal steroids, leaving the issue of cause/effect unclear. In this study, we sought to determine whether socially modulated BrdU-labeling depends on gonadal hormone changes. We investigated this using a gonadectomy-implant paradigm and by exposing male and female green treefrogs (Hyla cinerea) to their conspecific chorus or control stimuli (i.e. random tones). Our results indicate that socially modulated cell proliferation occurred independently of gonadal hormone levels; furthermore, neither androgens in males nor estrogen in females increased cell proliferation in the preoptic area (POA) and infundibular hypothalamus, brain regions involved in endocrine regulation and acoustic communication. In fact, elevated estrogen levels decreased cell proliferation in those brain regions in the implanted female. In male frogs, evoked calling behavior was positively correlated with BrdU-labeling in the POA; however, statistical analysis showed that this behavior did not mediate socially induced cell proliferation. These results show that the social modulation of cell proliferation can occur without gonadal hormone involvement in either male or female adult anuran amphibians, and confirms that it is independent of a behavioral response in males.

Effects of sodium diclofenac on the concentration function in animals with different neurohypophyseal status.

The characteristics of the osmotic concentration system under conditions of sodium diclofenac treatment were studied in Wistar rats with normally functioning vasopressin gene and homozygotic Brattleboro rats completely lacking endogenous vasopressin. Blockade of prostaglandin synthesis in rats with different neurohypophyseal status stimulated urinary osmolality to a different degree. Different contribution of sodium cations and urea to osmotic concentration was revealed.

Changes in angiotensin II receptor bindings in the hen neurohypophysis before and after oviposition.

The present study was performed to elucidate whether the angiotensin II (ANG II) receptor exists in the plasma membrane fraction of the neurohypophysis in hens, to estimate the time of action of ANG II on the neurohypophysis before and after oviposition, and to examine relationships between the action of ANG II on the neurohypophysis and those of estrogen and prostaglandin F(2α) (PGF(2α)) in relation to arginine vasotocin (AVT) release. The specific binding had a binding specificity to chicken ANG II (cANG II), reversibility, and saturation in the [(125)I]cANG II binding assay. Scatchard analysis revealed that the binding sites are of a single class. The equilibrium dissociation constant (K(d)) obtained by kinetic analysis and Scatchard analysis suggested a high affinity, and the maximum binding capacity (B(max)) obtained by Scatchard analysis suggested a limited capacity. These results suggest that an ANG II receptor exists in the neurohypophysis of hens. The K(d) and the B(max) value was significantly smaller in laying hens than in nonlaying hens, which suggests that bindings of the cANG II receptor change, depending on the difference in laying condition. Values of the K(d) and the B(max) decreased approximately 15 min before oviposition in laying hens, and decreased 1 h after an intramuscular injection of estradiol-17ß and 5 min after an intravenous injection of cANG II in nonlaying hens. The amount of specific binding of PGF(2α) receptor in the neurohypophysis also decreased and AVT concentration in blood increased after the cANG II injection. It seems likely that the action of cANG II in the neurohypophysis increases due to the effect of estrogen approximately 15 min before oviposition, and the cANG II action stimulates AVT release through the increase in the PGF(2α) action in this tissue.

Ionic conditions modulate stimulus-induced capacitance changes in isolated neurohypophysial terminals of the rat.

Peptidergic nerve terminals of the neurohypophysis (NH) secrete both oxytocin and vasopressin upon stimulation with peptide-specific bursts of action potentials from magnocellular neurons. These bursts vary in both frequency and action potential duration and also induce in situ ionic changes both inside and outside the terminals in the NH. These temporary effects include the increase of external potassium and decrease of external calcium, as well as the increase in internal sodium and chloride concentrations. In order to determine any mechanism of action that these ionic changes might have on secretion, stimulus-induced capacitance recordings were performed on isolated terminals of the NH using action potential burst patterns of varying frequency and action potential width. The results indicate that in NH terminals: (1) increased internal chloride concentration improves the efficiency of action potential-induced capacitance changes, (2) increasing external potassium increases stimulus-induced capacitance changes, (3) decreasing external calcium decreases the capacitance induced by low frequency broadened action potentials, while no capacitance change is observed with high frequency un-broadened action potentials, and (4) increasing internal sodium increases the capacitance change induced by low frequency bursts of broadened action potentials, more than for high frequency bursts of narrow action potentials. These results are consistent with previous models of stimulus-induced secretion, where optimal secretory efficacy is determined by particular characteristics of action potentials within a burst. Our results suggest that positive effects of increased internal sodium and external potassium during a burst may serve as a compensatory mechanism for secretion, counterbalancing the negative effects of reduced external calcium. In this view, high frequency un-broadened action potentials (initial burst phase) would condition the terminals by increasing internal sodium for optimal secretion by the physiological later phase of broadened action potentials. Thus, ionic changes occurring during a burst may help to make such stimulation more efficient at inducing secretion. Furthermore, these effects are thought to occur within the initial few seconds of incoming burst activity at both oxytocin and vasopressin types of NH nerve terminals.

Multiple alpha1-adrenergic receptor subtypes support synergistic stimulation of vasopressin and oxytocin release by ATP and phenylephrine.

Simultaneous exposure of explants of the hypothalamo-neurohypophyseal system (HNS) to ATP and the α(1)-adrenergic receptor (α(1)-R) agonist, phenylephrine (ATP+PE) induces a synergistic stimulation of vasopressin and oxytocin (VP/OT) release that is sustained for hours. The current studies confirm that the synergism is dependent upon activation of α(1)-R by demonstrating that an α(1)-R antagonist prevents the response. The role of the α(1)A, B, and D-adrenergic receptor subtypes in the synergistic effect of ATP+PE on intracellular calcium ([Ca(2+)](i)) in supraoptic nucleus (SON) neurons and VP/OT release from neural lobe was evaluated. The increase in [Ca(2+)](i) induced by PE in SON predominantly reflects release from intracellular stores and is mediated by activation of the α(1)A adrenergic receptor subtype. The α(1)A subtype is also required for the sustained elevation in [Ca(2+)](i) induced by ATP+PE. In contrast, although synergistic stimulation of VP/OT release was eliminated by removal of PE and was blunted by benoxathian, an α(1)-R antagonist that is not subtype selective, no single α(1)-R subtype selective antagonist prevented sustained stimulation of VP/OT release by ATP+PE. Thus, sustained activation of α(1)-R is essential for the synergistic VP and OT response to ATP+PE, but multiple α(1)-R subtypes can support the response. Redundancy amongst the α(1)-R subunits in supporting this response is consistent with the predicted importance of the response for sustaining the elevated VP release required to prevent cardiovascular collapse during hemorrhage and sepsis.

Daily pattern of pituitary glutamine, glutamate, and aspartate content disrupted by cadmium exposure.

Cadmium is a neurotoxic heavy metal and is considered endocrine disruptor. In this work, we investigate the effects of cadmium on the 24 h changes of aspartate, glutamate, and glutamine content in the pituitary. Adult male Sprague-Dawley rats were treated with 25 or 50 mg/l of cadmium chloride (CdCl(2)) in the drinking water for 30 days. Metal exposure with the lowest dose induced the disappearance of the nocturnal peak of anterior pituitary amino acid content, and the appearance of a peak of glutamine concentration during the resting phase of the photoperiod. After exposure to 50 mg/l of CdCl(2), the peaks of anterior pituitary amino acid content at 12:00 and 00:00 h disappeared, and two minimal values at these same hours and a peak at 08:00 h appeared. In the posterior pituitary, cadmium treatment with the lowest dose induced the appearance of a peak of aspartate and glutamate concentration at 12:00 h, and the disappearance of the peak of glutamine content at 16:00 h. After exposure to 50 mg/l of CdCl(2) aspartate and glutamate daily pattern presented two maximal values between 00:00 and 04:00 h, and the metal abolished glutamine daily pattern. These results suggest that cadmium disrupted aspartate, glutamate, and glutamine daily pattern in the pituitary.

The nature of hypthalamo-neurohypophyseal neurosecretion in the rat. A study by light- and electron microscope autoradiography.

The nature of hypothalamo-neurohypophyseal neurosecretion was examined in the rat by means of intraventricular injections of tritiated amino acids. Quantitation of autoradiographs was used at the light microscope level to study the sites of synthesis of proteins and their time of arrival in the neural lobe. Electron microscope autoradiographs were used to study the labeling of neural lobe tissue. It was concluded that the great majority of the labeled material was translocated inside dense-cored granules and was probably composed mostly of neurophysins. The effect of ether anesthesia was also examined. It was found to remove the dense cores from about 20% of the granules in the neural lobe tissue, a process accompanied by the loss of most of their labeled material. The mechanism of the ether effect is discussed and compared to the normal secretion process.

Methionine and leucine enkephalin in rat neurohypophysis: different responses to osmotic stimuli and T2 toxin.

Specific radioimmunoassays were used to measure the effects of hypertonic saline (salt loading), water deprivation, and trichothecene mycotoxin (T2 toxin) on the content of methionine enkephalin (ME), leucine enkephalin (LE), alpha-neoendorphin, dynorphin A, dynorphin B, vasopressin, and oxytocin in the rat posterior pituitary. Concentrations of vasopressin and oxytocin decreased in response to both osmotic stimuli and treatment with T2 toxin, but the decrease was greater with osmotic stimulations. Similarly, concentrations of LE and dynorphin-related peptides declined after salt loading and water deprivation; LE concentrations also decreased after treatment with T2 toxin. The concentration of ME decreased after water deprivation, did not change after salt loading, and increased after T2 toxin treatment. The differentiating effects of these stimuli on the content of immunoreactive LE and ME are consistent with the hypothesis that LE and ME may be localized in separate populations of nerve endings with different roles in the posterior pituitary.

GABA-activated chloride channels in secretory nerve endings.

Neurotransmitters acting on presynaptic terminals regulate synaptic transmission and plasticity. Because of the difficulty of direct electrophysiological recording from small presynaptic terminals, little is known about the ion channels that mediate these actions or about the mechanisms by which transmitter secretion is altered. The patch-clamp technique is used to show that the predominant inhibitory presynaptic neurotransmitter, gamma-aminobutyric acid (GABA), activates a GABAA receptor and gates a chloride channel in the membranes of peptidergic nerve terminals of the posterior pituitary. The opening of a chloride channel by GABA weakly depolarizes the nerve terminal membrane and blocks action potentials. In this way, GABA limits secretion by retarding the spread of excitation into the terminal arborization.

Prostaglandin F(2alpha) receptor in the neurohypophysis of hens.

To elucidate whether the receptor for prostaglandin (PG) F(2alpha), one of PG, exists in the neurohypophysis in hens and whether the binding of receptor changes with relation to oviposition, the PGF(2alpha) binding component in the membrane fraction of the neurohypophysis of laying hens was analyzed by radioligand binding assay using [5,6,8,9,11,12,14,15(n)-(3)H]PGF(2alpha). The binding component had characteristics of a receptor such as binding specificity, high affinity, and limited capacity for PGF(2alpha). Scatchard analysis indicated that the binding site was of a single class. The binding capacity of the receptor was smaller in laying hens than in nonlaying hens, whereas the binding affinity was not significantly different between these hens. When non-laying hens received an i.m. injection of estradiol-17beta or progesterone (0.5 mg/hen), the specific binding of the PGF(2alpha) receptor in the neurohypophysis was decreased. In laying hens, the specific binding decreased and the blood arginine vasotocin (AVT) concentration increased just after oviposition but did not change during a 24-h day in nonlaying hens. An i.v. injection of PGF(2alpha) (2 microg/hen) induced oviposition and caused an increase in the blood AVT concentration with a decrease in the specific binding of PGF(2alpha) receptor. The present study suggests a possibility that PGF(2alpha) may directly cause the AVT release from the neurohypophysis at oviposition time in hens.

Two types of calcium channels coexist in peptide-releasing vertebrate nerve terminals.

The properties of the Ca2+ channels mediating transmitter release in vertebrate neurons have not yet been described with voltage-clamp techniques. Several types of voltage-dependent Ca2+ channels are known to exist on neuronal somata, but the small size and inaccessibility of most vertebrate nerve endings have precluded direct characterization of the presynaptic channels. However, large nerve endings, which release the peptides oxytocin and vasopressin in a Ca2(+)-dependent manner, can be dissociated from the rat neurohypophysis. Using both single-channel and whole-cell patch-clamp techniques, we have characterized two types of Ca2+ channels that coexist in these terminals. One is a large-conductance, high-threshold, dihydropyridine-sensitive channel that contributes a slowly inactivating current. The second is a smaller conductance channel, which is also activated at high thresholds, but underlies a rapidly inactivating, dihydropyridine-insensitive current. Both types of Ca2+ channels may participate in the peptide release process.

cGMP-mediated facilitation in nerve terminals by enhancement of the spike afterhyperpolarization.

cGMP has long been suspected to play a role in synaptic plasticity, but the inaccessibility of nerve terminals to electrical recording has impeded tests of this hypothesis. In posterior pituitary nerve terminals, nitric oxide enhanced Ca(2+)-activated K+ channel activity by activating guanylate cyclase and PKG. This enhancement occurred only at depolarized potentials, so the spike threshold remained unaltered but the afterhyperpolarization became larger. During spike trains, the enhanced afterhyperpolarization promoted Na+ channel recovery from inactivation, thus reducing action potential failures and allowing more Ca(2+) to enter. Activating guanylate cyclase, either with applied nitric oxide, or with physiological stimulation to activate nitric oxide synthase, increased action potential firing. Thus, the cGMP/nitric oxide cascade generates a short-term, use-dependent enhancement of release.

Endogenous ATP potentiates only vasopressin secretion from neurohypophysial terminals.

Exogenous ATP induces inward currents and causes the release of arginine-vasopressin (AVP) from isolated neurohypophysial terminals (NHT); both effects are inhibited by the P2X2 and P2X3 antagonists, suramin and PPADS. Here we examined the role of endogenous ATP in the neurohypophysis. Stimulation of NHT caused the release of both AVP and ATP. ATP induced a potentiation in the stimulated release of AVP, but not of oxytocin (OT), which was blocked by the presence of suramin. In loose-patch clamp recordings, from intact neurohypophyses, suramin or PPADS produces an inhibition of action potential currents in a static bath, that can be mimicked by a hyperpolarization of the resting membrane potential (RMP). Correspondingly, in a static versus perfused bath there is a depolarization of the RMP of NHT, which was reduced by either suramin or PPADS. We measured an accumulation of ATP (3.7 +/- 0.7 microM) released from NHT in a static bath. Applications of either suramin or PPADS to a static bath decreased burst-stimulated capacitance increases in NHT. Finally, only vasopressin release from electrically stimulated intact neurohypophyses was reduced in the presence of Suramin or PPADS. These data suggest that there was sufficient accumulation of ATP released from the neurohypophysis during stimulations to depolarize its nerve terminals. This would occur via the opening of P2X2 and P2X3 receptors, inducing an influx of Ca2+. The subsequent elevation in [Ca2+](i) would further increase the stimulated release of only vasopressin from NHT terminals. Such purinergic feedback mechanisms could be physiologically important at most CNS synapses.