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Endocrinology Vol. 148, No. 2 479-489
Copyright © 2007 by The Endocrine Society

Vasopressin Autoreceptors and Nitric Oxide-Dependent Glutamate Release Are Required for Somatodendritic Vasopressin Release from Rat Magnocellular Neuroendocrine Cells Responding to Osmotic Stimuli

E. R. Gillard, C. G. Coburn, A. de Leon, E. P. Snissarenko, L. G. Bauce, Q. J. Pittman, B. Hou and M. C. Currás-Collazo

Department of Cell Biology and Neuroscience (E.R.G., A.d.L., E.P.S., B.H., M.C.C-C.), and Environmental Toxicology Program (C.G.C., M.C.C.-C.), University of California at Riverside, Riverside, California 92521; and Hotchkiss Brain Institute and Department of Physiology and Biophysics (L.G.B., Q.J.P.), University of Calgary, Calgary, Alberta, Canada T2N 4N1

Address all correspondence and requests for reprints to: E. R. Gillard, Department of Cell Biology and Neuroscience, 2110 Biological Sciences Building, University of California at Riverside, Riverside, California 92521. E-mail: erachelgillard{at}yahoo.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Magnocellular neuroendocrine cells of the supraoptic nucleus (SON) release vasopressin (VP) systemically and locally during osmotic challenge. Although both central VP and nitric oxide (NO) release appear to reduce osmotically stimulated systemic VP release, it is unknown whether they interact locally in the SON to enhance somatodendritic release of VP, a phenomenon believed to regulate systemic VP release. In this study, we examined the contribution of VP receptor subtypes and NO to local VP release from the rat SON elicited by systemic injection of 3.5 M saline. Treatment of SON punches with VP receptor antagonists decreased osmotically stimulated intranuclear VP release. Similarly, blockade of NO production, or addition of NO scavengers, reduced stimulated VP, glutamate, and aspartate release, suggesting that local NO production and activity are critical for osmotically induced intranuclear VP and excitatory amino acid release. An increase in endogenous NO release from SON punches in response to hyperosmolality was confirmed by enzymatic NO assay. Consistent with enhanced glutamate and VP release from stimulated rat SON punches, the ionotropic glutamate receptor blocker kynurenate decreased stimulated local VP release without affecting NO release. These data suggest that NO enhances local VP release in part by facilitating local release of glutamate/aspartate and that glutamate receptor activity is required for the stimulation of local VP release by osmotic challenge. Collectively, these results suggest that local VP receptors, NO, and glutamatergic signaling mediate the amplification of intranuclear VP release during hyperosmolality and may contribute to efficient, but not exhaustive, systemic release of VP during osmoregulatory challenge.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MAGNOCELLULAR NEUROENDOCRINE cells (MNCs) in the supraoptic nucleus (SON) and paraventricular nucleus of the hypothalamus synthesize either vasopressin (VP) or oxytocin and release these hormones into the circulatory system in response to elevated osmotic pressure in the local microenvironment and in the plasma (1). Although this osmotically stimulated release occurs with short latency and duration from MNC axon terminals in the pituitary (1, 2), these peptides are also released locally from cell bodies and dendrites in the SON with a longer delay [peaking 2–5 h later (3, 4)] and duration (3, 4) in response to systemic osmotic challenge. Somatodendritic VP release participates in autoregulation of MNC activity (5) and appears to inhibit further systemic VP output (6).

Local VP release in response to direct hypertonic stimulation of the SON via microdialysis is blocked by V2/V1 vasopressin receptor antagonism (7), suggesting that VP, like oxytocin (8), can facilitate its own intranuclear release by acting at MNC autoreceptors. However, the importance of autofacilitation to local SON VP release in response to systemic osmotic challenge and the relative contribution of VP autoreceptor subtypes to this phenomenon are unknown.

The second messenger, nitric oxide (NO), is also produced in MNCs of the SON, but its potential role in somatodendritic release of VP, especially during osmotic challenge, is unexplored. Like central VP, central NO appears to exert a largely inhibitory influence on MNC activity (9) and systemic VP release (10, 11). However, the site(s) at which central NO acts to reduce systemic VP output have not been precisely resolved due to the use of widespread central or systemic administration of NO synthase (NOS) inhibitors. Both neuronal NOS immunoreactivity and NOS activity are enhanced in MNCs during salt loading and dehydration (12, 13, 14), prompting us to ask whether NO signaling within the SON itself plays a role in the stimulation of local VP release during osmotic challenge.

The similarities between the effects of central VP and NO on MNC activity and systemic VP release raise the possibility that these two modulators might act in series and/or in parallel at the level of the SON to amplify somatodendritic VP release in the SON during strong physiological demand. Peripheral V2 receptors couple to NO production (15), raising the possibility that V2-like receptors linked to cAMP/PKA signaling in the SON (16) might enhance somatodendritic VP release in MNCs by coupling to NO signaling indirectly as has been shown for other cAMP-coupled receptors (17). In addition to other potential actions, both local VP receptor activity and NO might exert effects on local release of excitatory and/or inhibitory amino acids, levels of which are enhanced during direct osmotic stimulation of the SON (4). In turn, local release of excitatory amino acid neurotransmitters might contribute to local VP release within the SON. Because glutamate receptor activity influences both vasopressinergic MNC firing (18) and hormone output (19), modulation of glutamate release could be expected to be a target of endogenous VP and/or NO produced during osmotic activation. Although previous reports suggest such a relationship between NO and glutamatergic signaling in the SON under normosmotic conditions (20), the production and release of NO within the SON during systemic osmotic challenge, and its potential impact on local VP and excitatory amino acid release, have not been examined.

To begin to address the potential interactions of VP and NO within the SON, the in vitro release of VP, amino acids, and NO from SON tissue punches was measured using competitive enzyme immunoassay, HPLC, and the Griess reaction, respectively. Both peptide and NO release from the isolated SON maintained in vitro should reflect mainly somatodendritic release, whereas amino acid release is likely to occur from presynaptic terminals (21), perinuclear interneurons, and/or from MNCs (22). We first tested the contribution of VP autoreceptors (7) to osmotically stimulated local VP and amino acid release in the SON using V2/V1, V1a, and V2-selective VP receptor antagonists applied to the SON of rats subjected to systemic osmotic challenge in vivo. The involvement of NO in osmotically stimulated local release of VP and amino acids was also tested by application of an NOS inhibitor (NG-monomethyl-L-arginine, L-NMMA) and two different extracellular NO scavengers to SON punches. To confirm the effect of L-NMMA and determine whether NO release is elevated in the SON in response to hyperosmolality, the release of endogenous NO was measured in the presence or absence of L-NMMA. We also examined the contribution of local ionotropic glutamate receptors to VP release and NO production in the SON in response to systemic hyperosmolality. Collectively, our findings suggest that osmotic stimulation enhances VP autoreceptor stimulation and local NO signaling and enhanced local glutamatergic signaling downstream of NO production contributes to the amplification of somatodendritic VP release in the SON during osmoregulatory challenge.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male Sprague Dawley Holtzmann rats (400–530 g, n = 147) were used in this study. Rats were housed in a vivarium with a 12-h light/12-h dark photoperiod and maintained with ad libitum access to standard rat chow pellets and water until the beginning of the experiment on the day of tissue harvest. For experiments measuring VP, amino acid, and NO release from brain tissue punches, each rat contributed two separate unilateral tissue samples from each brain site tested (left and right SON or hippocampus). All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with approval of the Institutional Animal Care and Use Committee (University of California Riverside) under the Animal Care and Use Protocol No. A-0506003-1.

In vivo osmotic challenge
For each experiment, animals were injected ip (0.6 cc/100 g body weight) with either 3.5 M NaCl (pH 7.4) or 0.9% NaCl (control physiological saline, 0.15 M, pH 7.4) as described previously (23), and water was withheld until the animals were decapitated. Hypertonic saline injection was used because it produces elevation of plasma osmolality (24) without appreciably altering blood volume (25). Tail blood (not exceeding 200 µl) was collected from each rat into chilled tubes just before decapitation, spun at 6000 rpm at 4 C for 10 min, and the osmolality of the plasma fraction was measured using a vapor pressure osmometer to confirm the effectiveness of the 3.5-M saline injections and to match the osmolality of the artificial cerebrospinal fluid (Locke’s solution; for details, see below) used for in vitro tissue incubation to the plasma osmolality for each rat. Because control animals in the physiological saline group were shown to have plasma osmolalities in the range of normal, uninjected rats just before they were decapitated (286–301 mOsm/kg water), they are referred to subsequently as "normosmotic."

In vitro tissue preparation
Animals were decapitated 4.5–6 h after injection and the brain was rapidly removed for dissection. The time delay used here was chosen because: 1) intra-SON VP release in response to 3.5 M saline measured both in vitro (23, 26) and in vivo (4) is robust at this time; and 2) it corresponds well with the latency for structural changes such as increased somal size that occur within the SON in response to a single hypertonic saline injection (5 h) (27).

For dissection and incubation of tissue punches, the plasma osmolality of each rat was measured and a modified Locke’s solution (pH = 7.4, described below) matching the plasma osmolality (within 0–5 mOsm) was used for that rat. Plasma-matched Locke’s solution was also used for in vitro maintenance of the punches (unless otherwise noted) to mimic the in vivo extracellular environment and to maintain changes in the SON induced in vivo by the hypertonic saline injection as has been done successfully with hypothalamic slices (28). The base Locke’s solution (290 mOsm) was adapted from that described previously (29) and is a glucose-containing, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Locke’s solution consisting of (in mM): NaCl (132), KCl (5), CaCl2 (2), MgCl2 (2), KH2PO4 (1.2), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (10), and glucose (10) with ascorbate (35 mg/liter), thiourea (15 mg/liter), and bacitracin (400 mg/liter) added to retard peptide degradation (however, for experiments requiring NO measurement, ascorbate and thiourea were omitted from the mixture). Locke’s solution of 300–360 mOsm was prepared in 10 mOsm increments by addition of NaCl to the base solution yielding a hypertonic, hyperosmotic solution. After decapitation, the SON was dissected out bilaterally as previously described (23) and the two sides were segregated so that each subject yielded two separate tissue punches. In some experiments, a pair of unilateral hippocampal samples was also removed from the same brain from which the SON punches were taken.

After dissection, each tissue punch was immediately transferred to a transwell containing 500 µl of Locke’s solution (pH 7.4) at 37 C as previously described (23), and oxygenation was provided by directing a gentle stream of 95%O2/5%CO2 onto the liquid/air interface. The initial incubation period for all punches was 30 min and served as an equilibration period during which neurochemicals released as a result of the dissection trauma were allowed to accumulate in the incubation solution. Drugs such as receptor antagonists, NO scavengers, and NOS inhibitor (or their vehicles) were also present throughout this period to allow time for diffusion and access to MNCs before the experimental period. All drugs were prepared as concentrated stock solutions and frozen at –20 C until just before use, at which time they were diluted to the appropriate concentrations in the Locke’s incubation solution. On completion of the equilibration period, the equilibration solution was discarded and replaced with 500 µl of fresh osmolality-matched (unless otherwise noted) Locke’s solution, in which punches were incubated for a 10-min experimental period in the presence of the same receptor antagonists, enzyme inhibitors, or their vehicles, as previously reported (23).

At the conclusion of the experimental period, the transwell containing the tissue punch was removed and individual aliquots of the Locke’s solution (150 µl for VP; 200 µl for NO and amino acids) were collected and immediately frozen for subsequent NO determination (–80 C) and VP, amino acid, and protein analysis (–20 C). Tissue punches were individually homogenized in protease inhibitor cocktail (for composition, see Ref. 23) and total protein was determined for each punch using the bicinchoninic acid method (BCA kit; Pierce, Rockford, IL) and neurochemical release was standardized for punch protein (reflective of punch size) as previously described (23). Typically, individual SON punches yielded total protein measurements of 65–110 µg in these experiments.

Quantification of VP by enzyme immunoassay
As described previously, VP content in individual aliquots of incubation solution (100 µl) was measured without extraction using a highly specific, commercially available competitive enzyme immunoassay kit (arg8-vasopressin Correlate-Enzyme-Immunoassay kit, Assay Designs; for details, see Ref. 23) with a sensitivity of 3.52 pg/ml and intra- and interassay coefficients of variation of 7.9% and 8.5%, respectively. In separate experiments using receptor antagonists incubated without brain tissue, this assay showed minimal (<0.0014%) cross-reactivity with SR49059 or adamantane-VP, the noncyclic VP receptor antagonist (doses of which were tested up to 7 µM and 1 µM, respectively). Vasopressin concentrations (pg/ml) in the sample aliquots were calculated using a four-parameter curve-fitting computer program (STATLIA; Brendan Technologies Inc., Carlsbad, CA). Vasopressin values for each punch were then standardized to the protein measured in the tissue punch and expressed as pg/ml/µg protein.

Quantification of NO by the Griess reaction
Aliquots removed from the incubation solution of each SON (or hippocampal) tissue punch were assayed for NO by the Griess method (Oxford Biomedical Research, Oxford, MI; enzymatic kit) for detection of the oxygenation products of NO (nitrate and nitrite) after enzymatic conversion of nitrate to nitrite. The concentration of NO (µM) was analyzed in 85 µl of incubation solution and values were used to calculate the total NO produced by each punch (in pmol) in the original 500-µl incubation volume. Total NO values from each punch were then standardized to the amount of protein in the punch and expressed as pmol/µg protein. The efficacy of the reaction was verified by running aliquots of Locke’s solution to which the NO liberator S-nitrosoglutathione had been added, in the absence of tissue, as a positive control for NO detection.

Quantification of amino acids by HPLC
In some experiments, amino acid levels in aliquots of incubation solution (200 µl) were determined by HPLC after derivitization with OPA reagent (o-phthaldialdehyde) as previously described (23). Sensitivity was 4–8 pmol/aliquot for each amino acid. Results for the analyzed aliquot were used to calculate the amount of each amino acid (nmol) released by each tissue punch, and this release was standardized to total punch protein and expressed as nmol/mg protein.

Experimental design
Each individual experiment typically consisted of multiple trials of an identical experimental design conducted on multiple days and in close temporal proximity. The number of samples required for meaningful statistical analyses necessitated the distribution of subjects in this manner to preserve consistent timing for all phases of the experiments. Data were pooled only within each individual experiment to minimize the impact of variable VP release among different rat cohorts, and n values are given in the figure legends.

Experiment 1: involvement of VP receptors in local VP and amino acid release elicited by systemic osmotic challenge.
To examine whether the autofacilitatory effect of endogenous VP release within the SON (7) might contribute to stimulated somatodendritic VP release, SON tissue punches prepared from hyperosmotic rats were maintained in vitro in the presence or absence of the V2/V1 receptor antagonist 1-adamantane-acetyl1) D-tyr (Et2), Val4, Aln6, Arg8, 9-vasopressin (adamantane-vasopressin, 1 nM or 1 µM; Bachem, Torrance, CA) (30) (n = 13 rats). Glutamate, aspartate, serine, and glycine release from these punches was also measured to examine whether amino acid release within the SON in response to osmotic challenge might be subject to modulation by VP and/or its receptors. Aspartate was studied in addition to glutamate because it is a selective agonist of NMDA receptors (31) and serine in the SON is an endogenous ligand for the glycine coagonist site on the NMDA receptor (32). Although we were able to detect basal levels of GABA, its levels were not statistically sensitive to the systemic osmotic challenge in initial experiments, and results for this amino acid are not reported for further experiments.

Hippocampal punches were also prepared from the same animals and received the same drug treatments. Aliquots of incubation solution were analyzed for VP simultaneously with the SON samples and served as negative controls for osmotically stimulated VP release. Under the experimental conditions examined here (sampling several hours after intraperitoneal injection), hippocampal punches do not display elevated VP release in response to hyperosmolality.

Punches containing the SON were also prepared from a separate group of control (normosmotic) rats and were maintained with or without drug treatment (adamantane-VP, 1 nM) to determine whether this antagonist alters basal release of VP (n = 5 rats).

Experiment 2: role of V1a receptors in somatodendritic VP release elicited by systemic osmotic challenge.
SON tissue punches were prepared from a separate group of normosmotic and hyperosmotic rats. Tissue punches prepared from hyperosmotic rats were tested in the presence or absence of the selective V1a receptor antagonist SR49059 (7 nM, 70 nM, and 700 nM, a gift from Sanofi-Aventis, Paris, France). Normosmotic SON punches were maintained without drug treatment with the exception of a small subset of punches that received 700 nM SR49059 to examine whether this dose of antagonist alters basal VP release in the SON. Hippocampal punches were also removed from the same rats and served as a negative control for osmotically stimulated VP release (n = 33 rats).

Experiment 3: role of V2 receptors in local VP release elicited by systemic osmotic challenge.
To test the potential contribution of V2 (or pharmacologicallyV2-like) receptors in osmotically stimulated local VP release, the selective V2 antagonist SR121463B (10 and 100 nM, a gift from Sanofi-Aventis) was tested in SON punches obtained from normosmotic and hyperosmotic rats (n = 12 rats).

Experiment 4: role of NO in local VP release during direct osmotic stimulation of the SON.
Somatodendritic VP release in vivo can occur in response to direct osmotic stimulation of the SON by retrodialysis (7). To examine whether the isolated SON in vitro also responds to direct osmotic stimulation (without prior in vivo challenge), and whether NO production might contribute to VP release elicited only by local hyperosmolality, SON punches were prepared from normosmotic control rats and were maintained under normosmotic conditions or received in vitro osmotic stimulation in the presence or absence of L-NMMA (Sigma-Aldrich, St. Louis, MO; 7 µM), an inhibitor of NOS activity (33). Control, normosmotic rat SON punches were equilibrated and incubated in solution matching the plasma osmolality for each rat just before sacrifice; punches receiving in vitro osmotic stimulation incubated in the same solution during equilibration but received NaCl-hypertonic Locke’s (350 mOsm) during the experimental period only. A group of SON punches prepared from 3.5 M saline-injected animals was also incubated in plasma-matched solution in the absence of drugs for purposes of comparison (n = 15 rats).

Experiment 5: role of endogenous NO production and activity in VP and amino acid release elicited by systemic osmotic challenge.
To test potential contributions of NO signaling to neurochemical release within the SON in response to hyperosmolality, SON punches prepared from both normosmotic rats and rats subjected to systemic osmotic challenge were maintained in vitro in the presence or absence of the NO scavenger vitamin E (vitamin E, 100 µM) or the NOS inhibitor L-NMMA (700 nM, 7 µM). The release of VP and amino acids by the same SON punches was measured (n = 21 rats).

In a separate experiment, another NO scavenger of the nitronyl nitroxide class, 4-(carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO, 100 µM) was tested in the same manner on SON tissue punches prepared from osmotically stimulated and control rats (n = 24 rats). This NO scavenger is not an antioxidant and has been shown to be effective in reducing rat duodenal relaxation elicited by NO (10 µM) in vitro by roughly 75% at 100 µM (34).

Experiment 6: measurement of NO release from SON tissue during systemic osmotic challenge.
To verify a role for NO production in the SON during systemic hyperosmolality and to confirm the effects of L-NMMA on NO production, endogenous NO release from SON punches was measured using the Griess method. Parallel aliquots were removed for NO and VP analysis after completion of the experimental incubation. Tissue punches containing the SON MNCs were removed from osmotically stimulated rats and maintained in vitro in the presence or absence of the NOS inhibitor L-NMMA (7 µM); SON punches removed from normosmotic rats were also tested (n = 25 rats).

Experiment 7: importance of excitatory amino acids for local VP release in response to systemic osmotic challenge.
To investigate the contribution of excitatory transmission and ionotropic glutamate receptors to local VP release, the general ionotropic glutamate receptor blocker kynurenic acid (1 mM; Sigma-Aldrich) was tested for ability to block osmotically stimulated VP release from SON punches prepared from hyperosmotic rats. Punches prepared from both normosmotic and hyperosmotic rats were also tested in the absence of drug (n = 24 rats distributed among all groups). Endogenous NO release under these conditions was also measured by the Griess reaction in a subset (n = 18) of the same subjects.

Statistical analysis
For each individual experiment, VP, amino acid, and/or NO release values were averaged for punches in each treatment group and analyzed for main effects of treatment by one-way analysis of variance (ANOVA) with the exception of experiment 4 (vitamin E, L-NMMA) using Sigma Stat software. Two-way ANOVA was used in this case because the experimental design was completely crossed (each level of in vivo treatment received each level of in vitro treatment).

For all analyses, General linear model ANOVA was used where data met normal distribution/equal variance assumptions; otherwise, the Kruskal-Wallis ANOVA on ranks was used ({alpha} = 0.05 for both types). Multiple comparisons ({alpha} = 0.05) were used to detect specific differences, with Student-Newman-Keuls test applied following General linear model ANOVA and Dunn’s test applied following Kruskal-Wallis ANOVA on ranks unless otherwise noted. The same statistical testing was applied to plasma osmolalities for all experiments, and for all experiments reported here, osmotically stimulated rats had significantly elevated plasma osmolalities relative to normosmotic rats.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
For all results reported here, normosmotic rats within each experiment had measured plasma osmolalities that ranged between 290 ± 1 mOsm and 297 ± 4 mOsm among separate experiments. Within each experiment (the data for which different in vitro treatments of the tissue were exclusively pooled), plasma osmolality did not differ statistically between any normosmotic rat groups whose tissue punches subsequently received different in vitro drug treatments after decapitation; likewise, within each experiment, osmolality values did not differ significantly between hyperosmotic rat groups whose tissue punches subsequently received different in vitro drug treatments (P greater than or equal to 0.5036). In hypertonic saline-injected rats, plasma osmolalities were between 334 ± 3 mOsm and 347 ± 5 mOsm and in each experiment were statistically greater than that of the normosmotic rats.

Involvement of VP receptors in local VP and amino acid release elicited by systemic osmotic challenge
The contribution of VP receptors and autofacilitatory (7) somatodendritic VP release in response to systemic hyperosmolality was tested by examining SON VP release in the presence of adamantane-VP using SON punches prepared from osmotically stimulated rats. As shown in Fig. 1AGo, VP release from stimulated rat punches was significantly greater than basal VP release from normosmotic rat SON punches (ANOVA df = 3,17, F = 10.4, P < 0.001 followed by Dunn’s test) in the absence of the V2/V1 receptor antagonist. In contrast, inclusion of adamantane-VP in the incubation medium eliminated dehydration-elicited VP release (Fig. 1AGo) at doses as low as 1 nM, suggesting that VP receptors are critical for normal expression of osmotically induced somatodendritic VP release.


Figure 1
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  		FIG. 1. A, Release of VP (shaded bars) in the SON in response to systemic osmotic challenge is blocked by antagonism of V2/V1 vasopressin receptors. Mean (±SEM) release of VP, glutamate (glu), aspartate (asp), and serine (ser) from SON punches prepared from normal rats injected ip with 0.9% saline (normosmotic controls) or from rats injected with 3.5 M saline and incubated in the presence or absence of the V2/V1 VP receptor antagonist 1-adamantaneacetyl1, D-tyr(Et)2, Val4, Aln6, Arg8,9-vasopressin (adamantane-VP, 1 nM or 1 µM). Vasopressin was measured by enzyme immunoassay, and amino acids by HPLC, in aliquots of the incubation solution in which SON tissue punches were incubated for a 10-min experimental period. Corresponding bars with different letters are significantly different from each other (P < 0.05). Downward karats indicate significantly lower glutamate release relative to that from dehydrated SON in the absence of adamantane-VP. Sample sizes are n = 4, 4, 9, and 4 from left to right. B, The V2/V1 vasopressin receptor antagonist 1-adamantaneacetyl1, D-tyr(Et)2, Val4, Aln6, Arg8,9)-vasopressin (adamantane-VP, 1 nM) does not reduce basal VP release in SON punches. Mean (±SEM) VP release is reported for tissue punches prepared from normosmotic rats and incubated in matching Locke’s solution in the absence (control) and presence of adamantane-VP. Sample sizes are n = 4 and 4 for bars read from left to right.

 
Analysis of amino acid release measured in the same punches revealed significant effects of the treatment conditions on glutamate release (ANOVA, F3,17 = 3.85, P < 0.04; multiple comparisons by Student-Newman-Keuls). There was a significant increase in glutamate release from hyperosmotic rat SON (Fig. 1Go, P < 0.05) relative to normosmotic rat SON, an apparent increase in aspartate levels that did not reach statistical significance (P = 0.054) (Fig. 1AGo) and no significant change in serine, glycine, or GABA release (for the latter three amino acids, n identical to that for glutamate). Inclusion of the V2/V1 receptor antagonist in the incubation medium eliminated the increase in glutamate release in response to osmotic stimulation (P < 0.05), but the apparent reduction in aspartate did not reach significance (Fig. 1AGo). There was no significant effect of drug treatment on serine (P > 0.12; n identical to that for glutamate) or glycine (P > 0.4; data not shown; n identical to that for glutamate). Under the conditions tested here, neither osmotically stimulated VP release nor the effects of V2/V1 receptor antagonism generalized to hippocampal tissue (data not shown).

In a separate experiment, adamantane-VP (1 nM) was tested on normosmotic rat SON. As shown in Fig. 1BGo, there was no significant effect of this dose on basal VP release from SON punches (ANOVA, F1,6 = 0.0335, P = 0.8608).

Role of V1a receptors in somatodendritic VP release elicited by systemic osmotic challenge
To begin to examine which subtypes of VP receptor might be important in mediating endogenous VP release in the SON in response to systemic hyperosmolality, the selective V1a antagonist SR49059 was tested on SON punches prepared from a separate group of normosmotic and hyperosmotic rats. There was a significant effect of treatment condition on VP release (ANOVA on ranks, H = 17, 5 df, P < 0.005). Multiple comparisons revealed that systemic hyperosmolality significantly stimulated SON VP release relative to basal levels (P < 0.05 by Dunn’s test) in the absence of the V1a antagonist, as expected. Inclusion of the V1a antagonist in the incubation medium significantly reduced osmotically stimulated VP release (Fig. 2AGo) by nearly 50% at the 700-nM dose (P < 0.05 by Dunn’s test). The apparent reduction in VP release at 70 nM did not reach statistical significance. Basal vasopressin release from normosmotic rat SON punches was not affected by SR49059 (700 nM; data not shown).


Figure 2
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  		FIG. 2. A, Release of VP in the SON in response to systemic osmotic challenge is reduced by antagonism of VP receptors. Release of VP (mean ± SEM) from SON punches prepared from control and osmotically stimulated rats is shown in the presence or absence of the V1a-selective VP receptor antagonist SR49059 (more information on the preparation can be obtained from the legend for Fig. 1Go). Significant reduction in VP release occurs only at a dose that also blocks V2 receptors. Corresponding bars with different letters are significantly different from each other (P < 0.05). Sample sizes are n = 18, 18, 6, 10, and 8 for bars shown from left to right. B, In contrast, the V2 antagonist SR121463B significantly blocks stimulated local VP release at selectively low doses (10–100 nM). Bars with different letters are significantly different from each other (P < 0.05). Sample sizes are 6, 5, and 8 for bars read from left to right.

 
Role of V2 receptors in local VP release elicited by systemic osmotic challenge
The selective V2 antagonist SR121463B (10–100 nM) significantly reduced local SON VP release in response to hyperosmolality (F2,16 = 4.05, P < 0.04; multiple comparisons by Student-Newman-Keuls with P < 0.05), as shown in Fig. 2BGo. The blockade was effective at doses as low as 10 nM; because there was no significant difference in VP release between the 10 nM and 100-nM doses, these data were pooled. In normosmotic rats SON, SR121463B (100 nM) did not significantly impact local VP release (mean VP release was 4.1 ± 1.7 pg/ml/µg; data not shown).

Role of NO in local VP release during direct osmotic stimulation of the SON
As shown in Fig. 3Go, hyperosmotic stimulation in vitro resulted in robust release of VP from the isolated SON (ANOVA, H = 6.75 with 2 df, P = 0.0342; followed by Dunn’s test, P < 0.05). This stimulus resulted in VP release values that were more than sevenfold that measured in normosmotic incubation solution and which were more than twice that observed in tissue punches removed from rats subjected to systemic osmotic challenge (21.8 ± 8.6 pg/ml/µg; data not shown). Inclusion of the NOS inhibitor L-NMMA (7 µM) in the incubation significantly reduced VP release from osmotically stimulated punches (Dunn’s test, P < 0.05).


Figure 3
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  		FIG. 3. Release of VP within the SON in response to direct osmotic stimulation is dependent on NO production. Release of VP (mean ± SEM) from SON punches prepared from normosmotic rats and maintained in either normosmotic incubation solution (control) or hypertonic (350 mOsm) incubation solution in the presence or absence of the NOS inhibitor L-NMMA. The asterisk indicates significantly greater VP release relative to both control scores and relative to VP release from L-NMMA-treated SON punches subjected to in vitro osmotic stimulation (P < 0.05). Sample sizes are n = 6, 8, and 4 for bars read from left to right.

 
Role of endogenous NO production and activity in VP and amino acid release elicited by systemic osmotic challenge
Neurochemical release was measured in SON punches prepared from control (normosmotic) or hyperosmotic rats and incubated in the presence or absence of drugs that interfere with NO production or diffusion. Analysis of VP release values in these punches showed a significant interaction between the effects of systemic saline injection and drug treatment (two-way ANOVA; F = 3.46, P = 0.0274). Application of vitamin E and L-NMMA did not appreciably affect VP or amino acid release from SON punches prepared from normosmotic control rats (Fig. 4AGo). Multiple comparisons showed that SON punches removed from hyperosmotic rats released significantly more VP (6.6 ± 0.9 pg/ml/µg, Student-Newman-Keuls test, P < 0.05) than did SON from normosmotic control rats in the absence of vitamin E and L-NMMA (Fig. 4BGo). Similarly, release of both glutamate and aspartate in the various incubation conditions was dependent on the type of systemic injection as well as drug treatment (significant interaction effects in two-way ANOVA were F = 3.46, P = 0.0207 for glutamate; F = 7.80, P = 0.0001 for aspartate) with both glutamate and aspartate release from hyperosmotic rat SON being significantly higher relative to their release from normosmotic rat SON in the absence of drugs (Fig. 4BGo; Student-Newman-Keuls test, P < 0.05).


Figure 4
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  		FIG. 4. Inhibition of NO synthesis or activity reduces osmotically stimulated VP and amino acid release in the SON. Mean (±SEM) release of VP, glutamate (glu), and aspartate (asp) from SON punches prepared from normosmotic control rats (A) or osmotically stimulated rats (B) in the presence or absence of the NO scavenger vitamin E (100 µM) or the NOS inhibitor L-NMMA (more information on the preparation can be obtained from Fig. 1Go legend). Asterisks indicate significantly greater release of the corresponding neurochemical relative to both control scores and release measured from osmotically stimulated rat SON punches treated with vitamin E or L-NMMA (P < 0.05). Sample sizes for bars read from left to right are n = 8, 6, 4, and 7 in A and n = 5, 4, 4, and 4 in B. C, Inclusion of the NO scavenger carboxy-PTIO (100 µM) in the medium reduces stimulated VP release (means ± SEM) from SON punches (more information on the preparation can be obtained from Fig. 1Go legend). Bars with different letters are significantly different from each other. Sample sizes for bars read from left to right are n = 14, 14, and 16.

 
As shown in Fig. 4Go, incubation of hyperosmotic rat SON in the presence of either the NO scavenger vitamin E [100 µM, an effective dose typically used for NO scavenging activity (35)] or the NOS inhibitor L-NMMA (700 nM, 7 µM) significantly reduced osmotically stimulated VP release to the level found in analysates of control, normosmotic SON tissue (Student-Newman-Keuls test, P < 0.05). Concomitantly, release of both glutamate and aspartate from hyperosmotic rat SON was also reduced to approximately basal levels (Student-Newman-Keuls test, P < 0.05). Differences in neurochemical release among the groups shown are unlikely to be a result of nonrandom distribution of plasma osmolalities; analysis of plasma osmolality values showed that although animals injected with physiological saline had significantly different plasma osmolality than those receiving 3.5 M saline (ANOVA, F = 66.29, P < 0.0001), there was no significant difference in plasma osmolality between osmotically stimulated rats whose tissues received different drug treatments after decapitation (F = 1.65, P = 0.2451).

The NO scavenger carboxy-PTIO (100 µM) was similarly tested on SON punches removed from osmotically challenged rats. Addition of the scavenger reduced stimulated local release of VP to levels that were not significantly different from control punch VP release (ANOVA F3,44 = 3.23, P = 0.0312; comparisons by Student-Newman-Keuls test, P < 0.05), as shown in Fig. 4CGo. In punches removed from normosmotic rats, CPTIO treatment did not alter VP release from the SON.

Measurement of NO release from SON tissue after systemic osmotic challenge
Nitric oxide release from SON punches was measured to determine whether systemic hyperosmolality stimulates NO release from the SON and to confirm the efficacy of L-NMMA in inhibiting NO production in SON punches.

There was a significant effect of treatment condition on VP (ANOVA on ranks, H = 15.5 with 2 df, P < 0.0005) and NO (ANOVA F2,46 = 7.02, P < 0.003) release from SON punches. Concomitant with a more than twofold increase in VP release from punches prepared from dehydrated rats relative to control rats (P < 0.05, Dunn’s test), there was a more modest increase in the NO measured in the incubation solution after the 10-min experimental period (P < 0.05, Student-Newman-Keuls test) (Fig. 5Go). However, incubation in the presence of L-NMMA (7 µM) virtually eliminated both osmotically induced VP and NO release (P < 0.05), as shown in Fig. 5Go. The efficacy of L-NMMA (7 µM) in reducing NO levels in the analysate suggests that this pharmacologic inhibitor reduces VP release by inhibiting NO production and subsequent release in this preparation.


Figure 5
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  		FIG. 5. Both VP and NO release (presented as means ± SEM) are elevated in SON punches prepared from osmotically challenged rats relative to control values. Incubation of SON punches with the NOS inhibitor L-NMMA eliminated the intranuclear VP response to osmotic challenge. Vasopressin was measured by enzyme immunoassay, and NO was measured using the Griess method in aliquots of punch incubation solution. Corresponding bars with different letters are significantly different from each other (P < 0.05). Sample sizes are n = 12, 18, and 19 for bars read from left to right.

 
Importance of excitatory amino acids for local VP release in response to systemic osmotic challenge
As shown in Fig. 6Go, incubation of hyperosmotic rat SON in the presence of kynurenic acid reduced osmotically stimulated local VP release. The treatments resulted in mean group values that were significantly different (ANOVA, F2,44 = 8.87, P < 0.0007) with osmotically stimulated VP release being significantly higher than basal levels in the absence of drugs, but not in the presence of kynurenic acid (P < 0.05, Student-Newman-Keuls). In the absence of kynurenic acid, endogenous NO release from osmotically stimulated rat SON punches was also significantly elevated over basal levels (ANOVA, F2,32 = 3.90, P < 0.031, followed by Dunnett’s test at P < 0.05), but addition of kynurenate did not significantly alter the osmotically induced increase in NO release.


Figure 6
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  		FIG. 6. Local VP release in response to systemic osmotic stimulation is dependent on stimulation of ionotropic glutamate receptors. Release of VP (mean ± SEM) from SON punches prepared from normosmotic control rats or from 3.5 M-saline-injected, hyperosmotic rats and maintained in the presence or absence or absence of kynurenic acid (kynurenate; 1 mM). Corresponding bars with different letters are significantly different from each other. Sample sizes are 14, 14, and 19 for bars read from left to right for VP measurements; and 10, 10, and 16 for NO measurements.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The activity and function of hypothalamic MNCs responsible for body hydromineral balance are shaped by a variety of intrinsic and extrinsic factors. Local somatodendritic release of peptides is also emerging as a potential modulator of MNC activity and systemic hormone output, particularly during conditions of physiological challenge (8). We report here, for the first time, that stimulation of local VP release within the SON in response to osmotic challenge is dependent on stimulation of MNC VP autoreceptors by locally released VP, signaling mediated by NO, and ionotropic glutamate receptor activity.

The SON punch preparation has been used successfully in our laboratory (23, 26) and others (36) to examine somatodendritic VP release and depolarization-induced amino acid release (21). The values obtained for VP release in the current studies are on the order of those reported previously (23, 36) and are likely to reflect mainly MNC peptide release at the somatodendritic level due to the scarcity of MNC axon collaterals terminating within the SON (37). Similar to the SON in situ (7), normosmotic rat SON punches exhibited VP release in response to direct local osmotic stimulation. More importantly, VP release from the SON of hyperosmotic rats (350 mOsm mean plasma osmolality) was only 50% of that released from the normosmotic rat SON responding to direct osmotic (350 mOsm) stimulation in vitro (experiment 4), consistent with the diminished response to direct osmotic stimulation of the SON in salt-loaded rats observed in vivo (38). The similar responses of local VP release in the isolated SON and the SON in situ along with the ability of partially deafferented SON preparations to maintain dehydration-elicited changes for hours in vitro (28) suggest that VP release from SON punches, although certainly not identical to that in vivo, is likely to reflect ongoing physiological conditions at the time of decapitation to a reasonable degree.

In these studies, both adamantane-VP [a V2/V1 receptor antagonist (30)] and SR49059 (a V1a-selective antagonist) reduced osmotically stimulated local VP release in SON tissue punches. Based on the Ki values for adamantane-VP at V2 and V1a receptors in other preparations (39, 40, 41), it is likely that both receptor subtypes were blocked to some extent at 1 nM (to our knowledge, in vitro data are not available for this drug on rat V1b receptors). The V1a-selective antagonist SR49059 (7–70 nM) did not significantly block SON VP release in response to a systemic osmotic stimulus, although binding studies have shown that it inhibits labeled VP binding to native rat V1a receptors with a Ki of 1.65 nM (42). This suggests that the V1a receptor is not required for local VP release during osmotic challenge. The effectiveness of SR49059 at 700 nM might be explained by an additional action at V2 (or V2-like; see Ref. 43) or V1b (43) receptors, where it has Kis of 280 and 220 nM, respectively (42). Consistent with this, the highly potent and selective V2 antagonist SR121463B blocked stimulated local VP release at doses selective for the V2 receptor (44). Collectively, these results suggest that although V1a receptors may play a permissive role, V2 or V2-like autoreceptors are required for osmotically stimulated VP release in the SON, consistent with previous results demonstrating V2 receptor agonist-induced VP release from the normosmotic rat SON (45). Local SON VP release elicited by both V1a and V2 agonists is dependent on extracellular Ca2+ (45) gated by high voltage activated (HVA) Ca2+ channels in MNCs (46). In addition, both V1a and V2 agonists stimulate local SON VP release by recruiting multiple signaling pathways, including liberation of intracellular Ca2+ stores (45). Although a role for V1b receptors in osmotically elicited VP release is not excluded by our findings, it has been reported that a selective V1b agonist does not elicit VP release from normosmotic SON (45).

Consistent with the importance of excitatory amino acid transmission as a modulator of MNC function (47, 48), local release of the excitatory amino acids glutamate and aspartate was significantly stimulated in SON punches removed from osmotically stimulated rats, and overall levels correspond well with values previously observed in SON punches (21). Similar enhancement of glutamate release has been reported in response to direct osmotic stimulation of the SON in vivo (4) and is consistent with the reported increase in glutamatergic transmission in the SON of dehydrated rats (47). In the current study, the reduction of osmotically elicited VP release by kynurenic acid suggests that glutamatergic signaling contributes to local VP release under hyperosmotic conditions. Glutamate application can elicit VP release from isolated normosmotic SON in an extracellular Ca2+-dependent manner (49), suggesting that Ca2+ gated by ionotropic glutamate receptors might stimulate local VP release by acting on the exocytotic machinery controlling VP secretion (Fig. 7Go). Glutamate and aspartate release in osmotically stimulated rat SON is likely to be attributable to increased release from presynaptic terminals and/or MNCs themselves, because the other major source of amino acids (glial cells), release them in response to hypotonic, not hypertonic, extracellular conditions (50).


Figure 7
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  		FIG. 7. Hypothetical model of interactions between VP autoreceptors, nitric oxide and amino acid release, and somatodendritic VP release from MNCs during osmotic stimulation. Local increases in osmolality alter cell volume and stimulate somatodendritic VP release independently of action potential generation in the SON (8 ). In addition, local hyperosmolality elicits Ca2+ influx into MNCs via stretch-inactivated cation channels (59 ). Direct osmotic stimulation of the SON also enhances local release of glutamate (4 ); actions on presynaptic release are consistent with evidence of enhanced glutamatergic transmission in the SON of dehydrated rats (47 ) and with the presynaptic effects of local osmolality as reported in SON slices (62 ). Other potential sources of amino acids not shown in the diagram include release from MNCs and/or from perinuclear interneurons present in the punch. Vasopressin receptor stimulation triggers Ca2+ influx via L-, N-, and T-type channels (46 ) and elicits extracellular Ca2+-dependent (51 ) liberation of intracellular Ca2+ stores (45 ). Both V1a and V2 receptors couple either directly or indirectly to cAMP/PKA and PLC/PKC/IP3 signaling in SON MNCs (45 ). Potential interactions between receptor-initiated signaling pathways (such as cAMP/PKA signaling and associated Ca2+ influx) and NO production, as well as potential actions of NO on synaptic release, are based on previous evidence of functional links as reported in other neurons (17 ) (see Discussion). VPr, VP receptor; NMDAr, N-methyl-D-aspartate receptor; AMPA/KAr, {alpha}-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainic acid receptor; SIC, stretch-inactivated cation channel; IP3, inositol triphosphate; AC, adenylate cyclase; PKA, protein kinase A; NOS, nitric oxide synthase; SNARE, N-ethylmaleimide sensitive factor attachment protein receptors (synaptobrevin, syntaxin, SNAP-25) forming a core complex whose exocytotic activity is Ca2+-stimulable with the addition of synaptotagmin.

 
An unexpected result was that V2/V1 receptor antagonism resulted in a decrease in local glutamate release stimulated by hyperosmolality. Application of VP/oxytocin has been shown to decrease depolarization-stimulated excitatory amino acid release from normosmotic rat SON punches (21). It is conceivable that adamantane-VP might reduce osmotically stimulated glutamate release by blocking VP receptor-mediated Ca2+ responses (51) in MNCs, which express vesicular glutamate transporters (22) and might, therefore, be a source of Ca2+-dependent glutamate release along with presynaptic terminals present in SON punches (52, 53). Alternatively, blockade of VP autoreceptors may have caused a reduction in a downstream mediator (i.e. NO; see below) that promotes local glutamate release.

Central NO signaling has been reported to inhibit MNC firing (48) and systemic VP output, especially during strong activation of the hypothalamo-neurohypophysial system (11, 48). We have shown for the first time that both systemic osmotic activation and local osmotic stimuli significantly enhance the production and release of endogenous NO within the SON. Although NO synthase activity and/or immunoreactivity is found in the vasculature and in glia (48), the high percentage of SON MNCs expressing NOS activity (54), and the marked upregulation of nNOS in these cells (14) during osmoregulatory challenge, suggest that MNCs themselves contribute heavily to NO release under stimulated conditions.

The present results are the first to implicate NO production, release, and subsequent activity in osmotically stimulated somatodendritic VP release within the SON. Because vitamin E is lipophilic and inserts into cell membranes, it exerts its scavenging activity at the aqueous/lipid interface (55). Carboxy-PTIO is hydrophilic, does not permeate cell membranes and exerts its NO scavenging activity extracellularly (56). Because these NO scavengers should effectively limit the ability of NO to diffuse out of the cells of origin and/or persist extracellularly as a free radical, their effectiveness in these studies suggests that NO diffusion from the cells of origin is important for local SON VP release in response to hyperosmolality. The present results are consistent with previous findings showing that retrodialysis of NO into the normosmotic rat SON increases intranuclear release of VP in rats subjected to forced swim stress (57). Although basal (10) as well as osmotically stimulated (10, 11) systemic VP release can be inhibited by NO, the current results suggest that local SON NO production modulates somatodendritic VP release preferentially during strong physiological demand.

We report here that blockade of NO synthesis by L-NMMA reduced local glutamate release from the SON of osmotically challenged animals, suggesting that NO production enhances glutamate release. Enhancement of amino acid release by NO has been reported for other brain areas (58) and for the SON during forced swimming (20). Because glutamate stimulates VP release from normosmotic rat MNCs (49), it is possible that inhibition of NO synthesis reduced osmotically stimulated local VP release by attenuating glutamate release from afferent terminals in the SON (see Fig. 7Go). This interpretation is consistent with the efficacy of the extracellular NO scavenger CPTIO, which should effectively block such a retrograde action of NO at presynaptic sites. That glutamatergic signaling occurs downstream of NO production is supported by the ability of kynurenate to block osmotically stimulated VP release in the SON without affecting osmotically stimulated NO release. The failure of kynurenate to block NO release suggests that there are multiple mechanisms responsible for enhanced NO production under hyperosmotic conditions (Fig. 7Go). For example, hyperosmotically induced opening of stretch-inhibited cation channels and subsequent Ca2+ entry into MNCs (59) might be expected to stimulate Ca2+-dependent NOS isoforms (58) directly, and/or to stimulate cAMP/PKA signaling, which is positively linked to NOS activity (17).

The finding that either blocking the activity of VP autoreceptors or inhibiting NO signaling is sufficient to prevent amplified local VP release in the SON might reflect actions of VP and NO at a common target such as vesicle release machinery. Alternately, this might also suggest that VP autoreceptors are indirectly and positively linked to NO production, a phenomenon that has been demonstrated for renal V2 receptors (15). The finding that kynurenic acid reduced local VP release, but not NO release, in response to osmotic challenge suggests that NO signaling is necessary, but not sufficient, for osmotically stimulated somatodendritic VP release in the SON and that some degree of glutamate receptor activity is also required. A hypothetical model of the potential relationships suggested by our findings, within the framework of known NO signaling pathways, is presented in Fig. 7Go. Our results show that both glutamatergic signaling and NO signaling are enhanced within the SON in response to hyperosmolality and that both are required for maximal stimulation of somatodendritic VP release in these conditions. The effects of VP on MNC firing rate are activity-dependent, resulting in inhibition in the most active MNCs (60). Because strong physiological challenge (such as that produced by hyperosmolality) elicits intense firing activity in VPergic cells, locally released VP is likely to have largely inhibitory effects on firing rate and systemic VP release in this condition, consistent with the reported effects of central VP on systemic VP output in vivo (6, 61). At the same time, enhancement of NO release in the SON should have similar inhibitory effects on MNC firing (9) as well as systemic VP release (10, 11). Although NO-mediated enhancement of local glutamate release might oppose these inhibitory effects by its postsynaptic actions on MNCs, glutamatergic signaling also appears to enhance local VP release. It is possible that NO-mediated glutamate release occurs in close proximity to microdomains (i.e. dendrites) in MNCs that are physically distant from the point of action potential generation. Although this possibility has not been investigated, local Ca2+ influx in response to glutamate signaling in these microdomains might stimulate somatodendritic VP release while failing to elicit action potential firing and consequent systemic release. It is tempting to speculate that this outcome might be particularly likely during the hyperosmotic stimulus induced here when both local VP and NO signaling are enhanced. We propose that in addition to the more extensively studied effects of VP and NO on MNC synaptic transmission and electrical activity, convergent effects on the amplification of somatodendritic VP release might mediate the effects of central VP and NO on systemic hormone release, potentially contributing to efficient, but not exhaustive, release of VP during systemic osmotic challenges.


   Acknowledgments
 
We thank Sanofi-Aventis for their generous gift of SR49059 and SR121463B. We also thank Jennifer Shultz, Glenn Blanco, Mark Gaertner, and Anoush Shahidzadeh for valuable technical assistance and Dr. Glenn Hatton for sharing his StatLIA program.


   Footnotes
 
This work was supported by a National Science Foundation grant to M.C.C.-C. and a Canadian Institutes of Health Research grant to Q.P.

Author Disclosure Summary: All of the authors have nothing to disclose.

First Published Online November 2, 2006

Abbreviations: L-NMMA, NG-monomethyl-L-arginine; MNC, magnocellular neuroendocrine cell; NO, nitric oxide; NOS, NO synthase; SON, supraoptic nucleus; VP, vasopressin.

Received July 25, 2006.

Accepted for publication October 25, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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