Hypothalamo-neurohypophyseal System Explants Research Articles

Thermal stimulation of vasopressin and oxytocin (VP/OT) release from explants of the hypothalamo‐neurohypophyseal system (HNS)

Maintenance of a constant body temperature is essential in homeotherms. Since evaporative heat loss is necessary for preventing hyperthermia, integration of the mechanisms regulating water and thermal homeostasis is required. We used HNS explants from adult rats to study the impact of increases in temperature on stimulation of VP/OT release. Increasing the temperature from 37 to 39.5°C over 1 hr caused a dramatic increase in VP (850%) and OT (405% of basal) release (both p<0.001). Simultaneously exposing explants to a ramp increase in osmolality (25mOsm/2.6 hrs with mannitol) doubled the response to temperature alone (VP‐ 1780%; OT‐860% of basal). Thermal stimulation of VP/OT release was significantly attenuated by combined exposure to gadolinium (100μM), a non‐specific blocker of mechanosensitive channels, and capsazepine (10μM), a selective antagonist of TRPV1 channels, but not by capsazepine alone. This is consistent with other evidence that modified and capsazepine‐insensitive TRPV1 and TRPV4 channels participate in thermal regulation of VP/OT release. The gadolinium/capsazepine resistant response may reflect non‐specific thermal enhancement of metabolic processes. This is further supported by the thermal stimulus causing an increase in VP/OT release from isolated neural lobes similar to that retained by HNS explants in the presence of gadolinium/capsazepine. Supported by NIH RO1 NS‐27975

Role of purinergic P2Y1 receptors in regulation of vasopressin and oxytocin secretion

Pharmacological studies demonstrated that ATP elevates intracellular calcium ([Ca(2+)](i)) in supraoptic nucleus (SON) neurons primarily by activation of P2X2 and P2Y1 purinergic receptors [P2Y1R]. The current studies provide evidence for the presence of P2Y1R protein in SON neurons, evidence that activation of these P2Y1Rs induces an increase in [Ca(2+)](i) from both intracellular stores and Ca(2+) influx, and functional evidence that activation of P2Y1Rs induces vasopressin (VP) and oxytocin (OT) hormone release. Pretreatment of Fura-2 AM-loaded explants of the hypothalamo-neurohypophyseal system (HNS) with thapsigargin (TG) significantly (approximately 80%) reduced the increase in [Ca(2+)](i) induced by the P2Y1R-specific agonist, 2-methylthio-ADP (2-MeSADP). In contrast, the increase in [Ca(2+)](i) was slightly (approximately 20%) decreased in calcium-free medium. The calcium response to 2-MeSADP was completely blocked by the P2Y1R-specific antagonist, MRS2179 or by a combination of TG pretreatment and calcium-free medium. It was absent in P2Y1R knockout mice (P2Y1R(-/-)). 2-MeSADP significantly increased VP and OT release from perifused rat and wild-type mouse HNS explants compared with control. MRS2179 prevented this response in wild-type mouse, but it did not prevent ATP-induced hormone release from rat explants. 2-MeSADP did not induce hormone release from P2Y1R(-/-) explants. These findings support a potential role for P2Y1Rs in regulation of VP and OT release. The finding that P2Y1R activation induces a small Ca(2+) influx suggests that P2Y1Rs may regulate VP release by modifying ion channels such as stretch-inactivated cation channels.

P2X2 purinergic receptors (P2X2R) are responsible for ATP stimulated calcium influx in supraoptic nucleus neurons (SON)

ATP and phenylephrine (PE, α1 adrenergic receptor agonist) co‐applied to explants of the hypothalamo‐neurohypophyseal system (HNS) act synergistically to stimulate vasopressin and oxytocin release and generate an extended elevation in intracellular calcium ([Ca++]i) in SON (AJP:Regul 291:R31,'06). The latter may be the driving force for synergism in hormone release. ATP activated both P2X and P2YRs to increase [Ca++]i in SON (AJP:Regul 292:R423, ‘07). Our current studies evaluated the relative contribution of different P2XR subtypes to ATP stimulated Ca++ influx. HNS explants were loaded with the Ca++‐sensitive dye, fura 2AM (100 uM for 10 min., then 20 uM for 50 min in 0.02% pluronic acid), and [Ca++]i in SON was monitored by ratiometric analysis of fluorescence emitted by excitation at 340 and 380 nm. Neither A317491 (300 nM, P2X3 and P2X2/3R antag.) nor BBG (1 uM, P2X7R antag.) attenuated the increase in [Ca++]i induced by ATP or ATP+PE. α,β‐MethyleneATP (100 uM, a P2X3 and P2X2/3R agonist) did not increase [Ca++]i. BzATP increased [Ca++]i at 100 and 200 uM (activates P2X2Rs), but not at 20 uM (activates only P2X7Rs). Moreover, ATP γ S (100 uM, P2X2 and P2X4R agonist) also induced an increase in [Ca++]i. Since P2X4Rs are not sensitive to PPADS, and PPADS (10uM) blocked ATP stimulated Ca++ influx, together these findings support a primary role for P2X2Rs in stimulating Ca++ influx in SON. Since the non‐desensitizing P2X2/3 and P2X7Rs are important for sustained VP release induced by ATP+PE (Gomes et al, EB'08), but do not participate in Ca++ signal generation, ATP+PE synergism in hormone release may require a shift in P2XR subtypes. Supported by NIH R01‐NS27975.

Simultaneous exposure to ATP and phenylephrine induces a sustained elevation in the intracellular calcium concentration in supraoptic neurons

Vasopressin (VP) release from the hypothalamo-neurohypophyseal system (HNS) is stimulated by ATP activation of P2X purinergic receptors and by activation of 1-adrenergic receptors by phenylephrine (PE). These responses are potentiated by simultaneous exposure to ATP+PE. Potentiation was blocked by depleting intracellular calcium stores with thapsigargin. To test the hypothesis that the synergistic response to ATP+PE reflects alterations in the intracellular calcium concentration ([Ca2+]i), [Ca2+]i was monitored in supraoptic neurons in HNS explants loaded with fura 2-AM. Both ATP and PE induced rapid, but transient, elevations in [Ca2+]i. In 0.3 mM Ca2+, the peak response to ATP was greater than to PE but did not differ from the peak response to ATP+PE. A sustained elevation in [Ca2+]i was induced by ATP+PE, that was greater than ATP or PE alone. In 2 mM Ca2+, the peak response to ATP+PE was significantly greater than to either ATP or PE alone, and the sustained response to ATP+PE was greater than to either agent alone. Responses were comparable in the presence of TTX. The sustained elevation in [Ca2+]i was also observed when ATP+PE was removed after 1 min, but it was eliminated by either thapsigargin or removing external calcium, indicating that both calcium influx and calcium release from internal stores are required. Some cells were vasopressinergic based on a VP-induced increase in [Ca2+]i. These observations support the hypothesis that simultaneous exposure to ATP+PE induces a different pattern of [Ca2+]i than either agent alone that may initiate events leading to synergistic stimulation of VP release.

Does conversion of ATP to adenosine terminate ATP-stimulated vasopressin release from hypothalamo-neurohypophyseal explants?

ATP stimulates vasopressin (VP) release from explants of the hypothalamo-neurohypophyseal system (HNS), but the response is not sustained for the duration of exposure to ATP. Since adenosine, a metabolite of ATP, inhibits VP release from neurohypophysial terminals and adenosine receptors (AR) are expressed in supraoptic nucleus (SON) neurons, we postulated that conversion of ATP to adenosine contributed to termination of ATP-stimulated VP release from HNS explants. This was tested using a non-selective AR antagonist, 5-amino-9-chloro-2-(2-furyl)-1, 2, 4-triazolo [1, 5- c] quinazoline (CGS-15943). CGS-15943 did not affect basal VP release and did not alter the initial response to ATP. A selective A 1R antagonist, 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX), increased basal VP release at 1 μM, without altering the response to ATP. However, at a higher concentration of DPCPX (10 μM), VP release was enhanced by ATP for an extended period of time. Inhibition of the enzymatic conversion of ATP to adenosine using a combination of a potent ecto-5′-nucleotidase inhibitor, α,β-methylene adenosine 5′-diphosphate (AMP-CP), and a competitive substrate for ecto-5′-nucleotidase (guanosine monophosphate, GMP) did not affect basal VP release. Enzymatic inhibition did slightly prolong the response to ATP, but it was not sustained for the duration of exposure to ATP. We conclude that an endogenous inhibitory influence of adenosine decreases basal VP release from HNS explants and that conversion of exogenously applied ATP to adenosine contributes to termination of ATP-induced stimulation of VP release, but additional mechanisms such as receptor desensitization also limit the response to extended exposure to ATP.

Role of Neurokinin 3 Receptors in Supraoptic Vasopressin and Oxytocin Neurons

Neurokinin 3 receptors (NK3-Rs) are expressed in the supraoptic nucleus (SON), and SON is innervated by substance P (SP)-expressing A1 neurons in the medulla. Because SP stimulates vasopressin (VP) and oxytocin release from explants of the hypothalamo-neurohypophyseal system (HNS), two hypotheses were tested: (1) SP-stimulated VP release is mediated by NK3-Rs, and (2) stimulation of the A1 pathway by hypotension activates SON NK3-Rs. Senktide, an NK3-R agonist, stimulated VP release from HNS explants, but neither a neurokinin 1 receptor antagonist [L732,138 (N-acetyl-L-tryptophan 3,5-bis(tri-fluoromethyl)benzyl ester)] nor two NK3-R antagonists (SB222200 and SB235375) prevented SP-stimulated VP release. Because the affinity of these antagonists for rat NK-Rs may limit their efficacy, NK3-R internalization was used to assess the ability of SP to activate SON NK3-Rs. Senktide, SP, or vehicle was microinjected above SON. The brain was perfused 5 min after injection and stained for NK3-R immunoreactivity. Using confocal microscopy, the number of NK3-R-immunoreactive (-IR) endosomes was counted in a 5.6(2) mu region of cytoplasm in SON neurons. Senktide, but not SP or vehicle, significantly increased the number of NK3-R-IR endosomes in the cytoplasm. When hypotension was induced with hydralazine, NK3-R internalization was observed within 5 min (p < 0.005). A decrease in cytoplasmic NK3-R immunoreactivity was observed within 15 min of hypotension. Unexpectedly, both senktide and hypotension resulted in translocation of NK3-R-IR immunoreactivity to the nucleus. Thus, although these studies do not identify SP as the NK3-R ligand, they do provide evidence for hypotension-induced release of an endogenous tachykinin in SON and evidence suggesting a role for NK3-Rs in transcription regulation.

Role of estrogen receptor-beta in regulation of vasopressin and oxytocin release in vitro.

In rats, the magnocellular neurons that produce vasopressin (VP) and oxytocin (OT) express estrogen receptor-beta (ER-beta). Physiological concentrations of estrogen (E2) inhibit N-methyl-D-aspartate (NMDA)-stimulated VP and OT release from explants of the hypothalamo-neurohypophyseal system (HNS). To determine whether ER-beta mediates inhibition by E2, HNS explants were perifused with and without NMDA (50 microM) in the presence of E2 (50 pg/ml), E2 coupled to BSA (E2:BSA), genistein (100 nM, a phytoestrogen with affinity for ER-beta), or tetrahydrochrysene-R,R,-enantiomer (R,R-THC, a ligand that acts as an agonist on ER-alpha but an antagonist on ER-beta). VP and OT released into the perifusate were measured by RIA. E2 and genistein inhibited NMDA-stimulated VP release, but E2:BSA and R,R,THC were not effective inhibitors. However, R,R,THC blocked E2 inhibition of NMDA-stimulated VP release. The inability of E2:BSA to mimic the effect of E2 indicates that E2 inhibition is not mediated by membrane receptors. The ability of genistein to mimic the effect of E2 suggests that the effect is mediated by ERbeta. This interpretation is supported by the ability of R,R,THC to block but not to mimic the effect of E2. Thus, E2 inhibition of NMDA-stimulated VP and OT release may be mediated by ER-beta.

Role of Estrogen Receptor- in Regulation of Vasopressin and Oxytocin Release in Vitro

In rats, the magnocellular neurons that produce vasopressin (VP) and oxytocin (OT) express estrogen receptor-β (ER-β). Physiological concentrations of estrogen (E2) inhibit N-methyl-d-aspartate (NMDA)-stimulated VP and OT release from explants of the hypothalamo-neurohypophyseal system (HNS). To determine whether ER-β mediates inhibition by E2, HNS explants were perifused with and without NMDA (50 μm) in the presence of E2 (50 pg/ml), E2 coupled to BSA (E2:BSA), genistein (100 nm, a phytoestrogen with affinity for ER-β), or tetrahydrochrysene-R,R,-enantiomer (R,R-THC, a ligand that acts as an agonist on ER-α but an antagonist on ER-β). VP and OT released into the perifusate were measured by RIA. E2 and genistein inhibited NMDA-stimulated VP release, but E2:BSA and R,R,THC were not effective inhibitors. However, R,R,THC blocked E2 inhibition of NMDA-stimulated VP release. The inability of E2:BSA to mimic the effect of E2 indicates that E2 inhibition is not mediated by membrane receptors. The ability of genistein to mimic the effect of E2 suggests that the effect is mediated by ERβ. This interpretation is supported by the ability of R,R,THC to block but not to mimic the effect of E2. Thus, E2 inhibition of NMDA-stimulated VP and OT release may be mediated by ER-β.

Role of non-NMDA receptors in osmotic and glutamate stimulation of vasopressin release: effect of rapid receptor desensitization.

Previous studies demonstrated that the increase in vasopressin (VP) release and induction of VPmRNA content by osmotic stimulation was blocked by kynurenic acid, a non-specific antagonist of excitatory amino acid (EAA) receptors. In order to identify the type of EAA receptor involved, perifused explants of the hypothalamo-neurohypophyseal system (HNS) were exposed to a ramp increase in osmolality (40 mOsm over 6 h achieved by increasing NaCl) in the presence and absence of 10 microM 6,7-dinitroquinoxaline-2,3-dione (DNQX), an antagonist of non-n-methyl-d-aspartate (NMDA) excitatory amino acid receptors. Vasopressin release and VP mRNA content were significantly increased by exposure to the osmotic stimulus. 6,7-dinitroquinoxaline-2,3-dione inhibited osmotically stimulated VP release (F=16.65, P=0.0008) without significantly reducing basal release. It also prevented the osmotically stimulated increase in VP mRNA content (P <0.05). Although these results implicated glutamate, the primary endogenous ligand for EAA receptors, in the regulation of VP, exogenous glutamate was ineffective in stimulating VP release from HNS explants in either low-Mg2+ or Mg2+-replete medium. However, blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor desensitization with cyclothiazide (100 microM) caused a marked increase in VP release in response to 100 microM glutamate, and blockade of kainate receptor desensitization with concanavalin A resulted in a small, but significant increase in VP release in response to 1 mM glutamate. These results support a role for non-NMDA receptor activation in osmotic regulation of VP release.

N-methyl-D-aspartic acid stimulation of vasopressin release: role in osmotic regulation and modulation by gonadal steroids.

Previous experiments demonstrated that excitatory amino acids participate in the osmotic regulation of vasopressin secretion, but the specific involvement of N-methyl-D-aspartic acid (NMDA) receptors was not evaluated. This was demonstrated in the present studies. NMDA stimulated vasopressin release from perifused explants of the hypothalamo-neurohypophyseal system (HNS), and osmotic stimulation of vasopressin release was inhibited by MK-801 (10 microM) and AP5 (100 microM) NMDA receptor antagonists. The effective concentration of NMDA was dependent upon the Mg2+ concentration of the perifusate with stimulation observed at 1 microM NMDA in Mg2+-replete compared with 5 microM in low-Mg2+ medium. Previous experiments also demonstrated that estradiol and dihydrotestosterone (DHT) inhibited osmotically stimulated vasopressin secretion, and a nongenomic mechanism of action was suggested by the ability of steroids conjugated to bovine serum albumin to replicate the effect. Experiments were performed to explore the potential role of NMDA receptors in this mechanism. Estradiol (50 pg/ml) and DHT (3 ng/ml) inhibited NMDA stimulated vasopressin release in perifused HNS explants. These results suggest a role of NMDA receptors in the mediation of vasopressin secretion in osmotically stimulated release. Furthermore, estradiol and DHT may exert their inhibitory effect on osmotically stimulated vasopressin release via the NMDA receptor.

Gonadal steroid modulation of vasopressin secretion in response to osmotic stimulation.

As deficiencies in osmotic stimulation of vasopressin (VP) messenger RNA (mRNA) content in castrated rats have been reported, experiments were performed to determine whether castration altered osmotically stimulated VP release in vitro. Perifused explants of the hypothalamo-neurohypophyseal system were obtained from sham and gonadectomized male rats. There were no significant differences in VP release stimulated by a ramp increase in the osmolality of the culture medium between the two groups. As testosterone was undetectable in the perifusion medium, the effect of addition of testosterone on osmotically stimulated VP release was evaluated. Testosterone (3 ng/ml) and its metabolites, estradiol (50 pg/ml) and dihydrotestosterone (DHT; 3 ng/ml), inhibited osmotically stimulated VP release in hypothalamo-neurohypophyseal system explants. The osmotically induced increase in VP mRNA content was also inhibited by testosterone and estradiol, but not by DHT. Neither estradiol nor DHT affected stimulus-secretion coupling of hormone secretion, because they did not inhibit KCl (25 mM)-stimulated VP release. BSA conjugates of estradiol (200 nM) and DHT (10 mM) also inhibited osmotically stimulated VP release, and VP mRNA content was inhibited by BSA-estradiol, but not by BSA-DHT, suggesting nongenomic actions of the steroids. The differential effects of estradiol and DHT on VP mRNA imply distinct actions for these steroids, and the DHT mechanism uncouples regulation of VP release from VP mRNA content.

Gonadal Steroid Modulation of Vasopressin Secretion in Response to Osmotic Stimulation

As deficiencies in osmotic stimulation of vasopressin (VP) messenger RNA (mRNA) content in castrated rats have been reported, experiments were performed to determine whether castration altered osmotically stimulated VP release in vitro. Perifused explants of the hypothalamo-neurohypophyseal system were obtained from sham and gonadectomized male rats. There were no significant differences in VP release stimulated by a ramp increase in the osmolality of the culture medium between the two groups. As testosterone was undetectable in the perifusion medium, the effect of addition of testosterone on osmotically stimulated VP release was evaluated. Testosterone (3 ng/ml) and its metabolites, estradiol (50 pg/ml) and dihydrotestosterone (DHT; 3 ng/ml), inhibited osmotically stimulated VP release in hypothalamo-neurohypophyseal system explants. The osmotically induced increase in VP mRNA content was also inhibited by testosterone and estradiol, but not by DHT. Neither estradiol nor DHT affected stimulus-secretion coupling of hormone secretion, because they did not inhibit KCl (25 mm)-stimulated VP release. BSA conjugates of estradiol (200 nm) and DHT (10 mm) also inhibited osmotically stimulated VP release, and VP mRNA content was inhibited by BSA-estradiol, but not by BSA-DHT, suggesting nongenomic actions of the steroids. The differential effects of estradiol and DHT on VP mRNA imply distinct actions for these steroids, and the DHT mechanism uncouples regulation of VP release from VP mRNA content.

Effect of Extended Exposure to Hypertonicity on Vasopressin Messenger Ribonucleic Acid Content in Hypothalamo-Neurohypophyseal Explants*

The feasibility of using organ-cultured explants of the rat hypothalamo-neurohypophyseal system (HNS) to study the mechanisms regulating the vasopressin (VP) mRNA content of the HNS was examined by evaluating the effect of exposure to hypertonicity on the VP mRNA content of these explants. Different effects were observed after a step increase in osmolality and a gradual increase in the same amount over 24 h. The VP mRNA content of control HNS explants determined from a RNA protection assay was 22 +/- 6 pg. It gradually decreased to 23% and 9% of the control value during 24 and 48 h in culture, respectively. Northern blot analysis revealed a single band of VP mRNA approximately 700 bases long in explants cultured for 36 h. Explants exposed to the step increase in osmolality were maintained in static culture. The control explants were placed directly into isotonic medium (299 mosmol/kg H2O). The explants exposed to the step increase were placed directly into hypertonic medium (greater than 304 mosmol/kg H2O). After 24 h in culture, basal VP release was measured, and all explants were then exposed to a further acute 15 mosm/kg H2O increase in osmolality. The highest basal release of VP was observed in the explants maintained under isotonic conditions (299 mosm/kg H2O). These explants significantly increased VP release in response to the acute increase in osmolality. Basal VP release was lower in explants maintained in hypertonic medium (greater than 304 mosmol/kg H2O), and these explants did not respond to the acute hypertonic pulse. VP mRNA content was significantly decreased in explants maintained for 24 or 48 h in hypertonic medium compared to that in explants maintained in isotonic medium (47 +/- 10% and 57 +/- 6%, respectively; P less than 0.01). No significant difference existed in the VP content of the posterior pituitary between the groups. To achieve a slow increase in osmolality, explants were perifused in individual chambers with medium at 2.1 ml/h. A gradual increase in osmolality (16 mosmol/kg H2O medium) was achieved by increasing the NaCl concentration in the perifusion medium. In response to this stimulus there was a significant increase in VP release, which was sustained for 9 h. VP mRNA content in the hypertonic group was 165 +/- 19% of that in control explants (P less than 0.001), but no difference existed in VP content in the posterior pituitary compared to that in time control explants.(ABSTRACT TRUNCATED AT 400 WORDS)

γ-Aminobutyric Acid Antagonists Stimulate Vasopressin Release from Organ-Cultured Hypothalamo-Neurohypophyseal Explants*

gamma-Aminobutyric acid (GABA) has been identified in axon terminals innervating neurons of the supraoptic nucleus and has been shown to inhibit the electrical activity of supraoptic neurons when applied iontophoretically. This study examines the effects of GABA and GABA antagonists on vasopressin (VP) release from organ-cultured explants of the hypothalamo-neurohypophyseal system (HNS). The GABA antagonists bicuculline and picrotoxin stimulated VP release in a concentration-dependent manner. These observations suggest that VP release by HNS explants is tonically inhibited by GABA. Exposure of HNS explants to GABA (10(-8)-10(-3) M) did not consistently alter basal VP release. This was true even when penicillin, which can block GABA-activated chloride channels, was omitted from the medium. Similarly, nipecotic acid, an agent that potentiates GABA activity by inhibiting GABA uptake, did not alter basal VP release; stimulation of VP release by acetylcholine and increases in osmolality was not diminished by the addition of 10(-5) M GABA. The failure of exogenous GABA to modify basal and stimulated VP release suggests that GABAergic inhibition of VP release is maximally activated by endogenously released GABA in cultured HNS explants. This is consistent with evidence for a local source of GABA in the supraoptic nucleus and suggests that one role of GABA in the regulation of VP release is that of a potent local inhibitory neurotransmitter.

Cholinergic stimulation of vasopressin release in spontaneously hypertensive rats.

Plasma vasopressin (VP) concentration is elevated in spontaneously hypertensive rats (SHRs) relative to their normotensive Wistar-Kyoto (WKY) controls. The possibility that this reflects altered responsiveness of the hypothalamo-neurohypophyseal system (HNS) in SHRs was examined by comparing VP release in response to acetylcholine from organ cultured HNS explants obtained from SHR and WKY donors. Explants were prepared from 5-, 8-, and 18-week-old animals. Blood pressure was significantly elevated in the 8- and 18-week-old SHR donors relative to their age-matched WKY donors. VP release was assessed on the 4th day of culture during a control hour and during the subsequent hour in the presence of acetylcholine. Acetylcholine caused a concentration-dependent stimulation of VP release from both types of explants, but the response was significantly greater in the explants from 5- and 8-week-old SHRs than in explants from age-matched WKYs. The explants from 18-week-old SHRs and WKYs demonstrated comparable sensitivity to acetylcholine. Basal VP release was not significantly different in explants from age-matched SHRs and WKYs, but it did increase with donor age in both strains. These studies indicate potential hyperresponsiveness of the HNS to excitatory stimuli in SHRs during the developmental phase of hypertension. The hyperresponsiveness disappears in the chronically hypertensive phase. Thus, increased sensitivity of the HNS during the development of hypertension may contribute to the elevation of plasma VP concentration in SHRs.

Effect of anteroventral third ventricle lesions on vasopressin release by organ-cultured hypothalamo-neurohypophyseal explants.

Rats with electrolytic lesions of the tissue surrounding the third ventricle (AV3V) of the hypothalamus exhibit hypernatremia and chronic drinking deficits in response to hypertonic NaCl. These findings are suggestive of impaired osmoreception. The organ-cultured rat hypothalamo-neurohypophyseal system (HNS) previously has been shown to release vasopressin (VP) in response to osmotic stimuli. The ventral portion of the region damaged by AV3V lesions is included in the HNS explant. Thus, these studies were initiated to evaluate the ability of HNS explants which were obtained from rats previously prepared with AV3V lesions to respond to an increase in osmolality, acetylcholine, or angiotensin II with an increase in VP release. Following electrolytic ablation of the AV3V region or sham lesions and a 2-week recovery period, HNS explants were removed from rats with sham or AV3V lesions. The explants were maintained in organ culture for 4 days. On the third day in culture, increasing the osmolality of the culture medium from 295 to 315 mosm/kg H2O by the addition of NaCl resulted in a 2.5-fold increase in VP release from the explants with sham lesions, but did not significantly alter VP release from the explants with AV3V lesions. On the subsequent day in culture, acetylcholine (10(-5) M) stimulated VP release from the explants with AV3V lesions as well as the explants with sham lesions. Angiotensin II (10-5 M) also stimulated VP release from explants obtained from rats with both AV3V and sham lesions. These data suggest that the osmoreceptors which are involved in controlling VP release from the organ cultured HNS may be located in the region of the AV3V.

Angiotensin Stimulation of Vasopressin Release from the Rat Hypothalamo-Neurohypophyseal System in Organ Culture*

Angiotensin II (AII) stimulated vasopressin (VP) release from the rat hypothalamo-neurohypophyseal system (HNS) in organ culture in a concentration-dependent manner. Exposure to AII at 10(-8) M for 1 hr yielded a 1.8-fold increase in VP release over control release (P less than 0.01), while a 1-h exposure to 10(-5) M AII resulted in a 4-fold increment over control VP release by HNS explants maintained in organ culture for 3 days (P less than 0.01). Saralasin, an AII antagonist, blocked AII stimulation of VP release without significantly altering basal VP release by the HNS explants. Saralasin did not interfere with stimulation of VP release by acetylcholine or nicotine. Tetrodotoxin (10(-7) g/ml) also blocked AII stimulation of VP release. These findings suggest that action potentials are generated in response to AII stimulation of specific receptors in the HNS and are requisite for VP release in response to this stimulus.