Departments of Medicine and Physiology, Wayne State University School of Medicine, and John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan 48201
Submitted 29 July 2003 ; accepted in final form 25 November 2003
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ABSTRACT |
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anteroventral third ventricle; posterior pituitary; supraoptic nucleus
The effects of ET in this system are complex, depending on the isoform, the locus of action, and the receptor subtype involved, and remain to be characterized fully. The ETA receptor has higher affinity for ET-1, whereas the ETB receptor has roughly equal affinities for ET-1 and ET-3 (5). After the initial report that ET induced AVP secretion by rat hypothalami in vitro (26), it was shown in compartmentalized hypothalamo-neurohypophysial explants that a nanomolar concentration of ET-3 stimulated AVP release at the posterior pituitary rather than at a hypothalamic site (21). Electrophysiological studies demonstrated that ET-1 inhibits phasically firing magnocellular neurons (a characteristic of AVP rather than oxytocin cells) within the supraoptic nuclei but exerts excitatory actions on neurons within the anteroventral third ventricular (Av3V) region (29). Neurons within the Av3V area project to the supraoptic nuclei. Thus it is likely that at least one synapse intervenes between the neurons stimulated by ET-1 in the Av3V region and the vasopressinergic neurons.
We hypothesize that release of AVP by the neurohypophysis will be increased by hypothalamic ETB receptor activation via a mechanism that involves at least one intervening synapse and will be directly stimulated by ETA receptor activation at the posterior pituitary. We further propose that somatodendritic AVP secretion at the hypothalamic level will differ from that observed from the neural lobe in response to the distinct subtype of ET receptor activated. The present studies were carried out in compartmentalized hypothalamo-neurohypophysial explants, thereby permitting evaluation of AVP release at both sites independent of potential systemic influences, such as baroreflex modulation (4, 23) or ET-induced actions on cerebral microvasculature (25).
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METHODS |
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Dissection and Culture of Explants
Explants of the hypothalamo-neurohypophysial complex (HNC) were dissected as described earlier (27). The explants contained both supraoptic nuclei with intact axonal projections to the neural lobe with the intermediate lobe attached. The organum vasculosum of the lamina terminalis and the arcuate, suprachiasmatic, preoptic, and ventromedial nuclei were present. The subfornical organ and paraventricular nuclei are not included.
Explants in standard culture. Explants cultured by the standard method were placed ventral side down onto Teflon mesh into separate incubation wells containing culture medium composed of Ham's F-12 nutrient mixture supplemented with 5.5 mM dextrose, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Grand Island, NY) fortified with 20% fetal bovine serum (Hyclone, Logan, UT) with a final osmolality of 297 ± 1 mosmol/kgH2O. Medium was changed every 24 h or after an experimental protocol. Both hypothalamic (HT) and posterior pituitary (PP) components were in the one culture well and exposed to the same medium.
Explants in compartmentalized cultures. After dissection, compartmentalized explants were immediately placed into custom-fabricated sterile incubation chambers that have a two-piece barrier separating the hypothalamus from the posterior pituitary (7, 21, 22). The explants were positioned onto Teflon mesh with the intact infundibular stalk lying in a 0.33-mm-wide by 0.2-mm-deep notch in the lower half of the barrier. The upper half of the barrier was then slid into place. The barrier interfaces and gaps were sealed with sterile silicone grease to prevent leaking from one chamber to the other. Each compartment of the chamber was filled with culture medium. The medium in each compartment was changed every 24 h. When properly positioned and sealed, the only communication between the two compartments was neuronal. Diffusion via the infundibular recess could not be entirely eliminated; however, the extent of leaking between compartments was assessed at the end of the experiment for each explant by adding 3H-labeled water (as a 150 mM NaCl solution) to one compartment and measuring its radioactivity in the opposite compartment. Explants displaying >0.05%/h leak were discarded from analysis. At the end of the experiments, the upper barrier was removed, and each explant was scrutinized under a stereomicroscope (magnification x25) for trauma to the infundibular stalk. Explants were classified as damaged if there was visual evidence of attenuation or lengthening of the stalk (commonly seen with excessive traction), nicks or cuts in the fibers of the stalk or any part of the explant (usually incurred during positioning of the explant within the chamber), and complete separation of the hypothalamus and pituitary. Tissues exhibiting damage to the stalk were discarded prospectively from further analysis. All explants were kept at 37°C under a humidified atmosphere of 95% O2-5% CO2 at pH 7.4.
Sampling Procedures
The sampling techniques have been reported earlier (7, 27). All protocols were performed with 0.05 mg/ml bacitracin (Sigma, St. Louis, MO) to prevent peptide degradation (27). Each compartment was treated identically in all respects. During the basal period, AVP release rate was ascertained after 1 h of exposure to standard medium administered to both sides of the barrier. A 1-h test period followed immediately, and the test agent(s) dissolved directly in medium was delivered only to the specified compartment. Unless otherwise stated, standard medium was administered to the other compartment. Samples were obtained for determination of AVP degradation during basal and test periods from both chambers (7, 27). Explants in standard cultures were treated as a single compartment. Samples for AVP radioimmunoassay were frozen and stored at -70°C. Osmolality was determined on the remaining medium.
Standard Explant Protocols
In all protocols (standard or compartmentalized), each explant was used for only one basal and test period.
Dose response to ET agonists. ET-1 (ETA selective), IRL-1620 (ETB selective), or ET-3 (ETA/ETB nonselective) was added in increasing log concentrations from 0.01 to 10 nM during the test period.
ETA and ETB antagonism. During the test period, the selective ETA antagonist BQ-123 was added to explants either alone or at 2, 20, or 200 nM together with a concentration of each agonist that stimulated AVP to roughly equivalent levels when given alone (350-400%·HNC-1·h-1): 1 nM ET-1, 1 nM ET-3, or 0.01 nM IRL 1620. Similarly, explants were tested with the agonists in the presence of the selective ETB antagonist IRL-1038 at 6, 60, or 600 nM.
Compartmentalized Protocols
Dose response to ET agonists. ET-1, ET-3, or IRL-1620 was added to the hypothalamic side of the barrier at 0.1, 1, or 10 nM. Standard medium was added to the posterior pituitary side. In separate experiments, the agonist was added to the posterior pituitary compartment and medium to the hypothalamus. At the end of the test hour, samples for release were obtained from each compartment regardless of the site of application of agonist.
ETA and ETB antagonism of hypothalamic sites. Either 20 nM BQ-123 or 60 nM IRL-1038 was added to the hypothalamus alone or with 10 nM ET-1, 10 nM ET-3, or 0.1 nM IRL-1620. The choice of concentrations was based on the following: 1) 10 nM ET-3 produced a maximal stimulation of hypothalamic AVP release and no change at any dose on pituitary AVP release; 2) 0.1 nM IRL-1620 resulted in a maximal stimulation of pituitary AVP release and also resulted in significant hypothalamic release; and 3) none of the doses of ET-1 evoked a change in AVP from basal release, so 10 nM was picked arbitrarily.
ETA and ETB antagonism of posterior pituitary sites. Either 20 nM BQ-123 or 600 nM IRL-1038 was added to the posterior pituitary alone or with 10 nM ET-1, 1 nM ET-3, or 1 nM IRL-1620. The choice of concentrations was based on the following: 10 nM ET-1 maximally stimulated both hypothalamic and pituitary AVP release. Likewise, 1 nM ET-3 was maximally stimulating for release from both sites. One nanomolar IRL-1620 resulted in maximal release from the hypothalamus and had no significant effect on posterior pituitary AVP release, but neither did the lower or higher concentrations.
Tetrodotoxin studies. The maximally stimulating dose of 0.1 nM IRL-1620 alone or in the presence of 10 µM tetrodotoxin was added to the hypothalamus. Control medium was added to the pituitary compartment. AVP release was measured in medium from the pituitary compartment. In separate explants, 10 nM ET-1 either with or without 10 µM tetrodotoxin was added to the pituitary, and the hypothalamus received standard medium. AVP release was assessed from the pituitary.
Analytical Methods
AVP content of the medium was measured by radioimmunoassay with methods previously reported from our laboratory (20-24). Samples of medium were diluted 1:50 and directly assayed in duplicate. Media from one set of experiments were assayed together to avoid interassay variability. Standards were prepared with purified AVP (Ferring, Malmo, Sweden). [125I]iodotyrosyl-AVP was used as the tracer (Amersham Pharmacia Biotech, Piscataway, NJ). Anti-AVP serum no. 2849 (prepared by J. Durr, Veterans Affairs Medical Center, Tampa, FL) was used at a final dilution of 1:3.6 x 105.
Medium osmolality was measured by freezing point depression (Precision Systems, Sudbury, MA).
Statistical Analysis
Release rates for basal and test periods were calculated and corrected for volume and degradation as reported previously (27). All comparisons of AVP release between basal period and test period release rates were performed on the absolute release rates after correction for degradation. Each explant acted as its own control. Because basal secretory rates varied from explant to explant, the release of AVP during the test hour was normalized as the percentage of AVP released during the preceding basal hour for the same compartment. Comparisons among test periods from different groups of explants were then performed on the normalized release rates.
Differences between basal and test period release of AVP in the same explants were compared using the paired t-test. Comparisons among test periods from separate groups of explants were assessed by analysis of variance and the Tukey-Kramer test for multiple comparisons. All data are expressed as means ± SE. P values < 0.05 were considered significant.
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RESULTS |
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BQ-123 dose-dependently inhibited the stimulatory effect of 1 nM ET-1 or ET-3 but did not change AVP release by 0.01 nM IRL-1620 (Fig. 2). Average basal AVP secretion was 69 ± 22 pg·HNC-1·h-1 (n = 85). IRL-1038 significantly antagonized AVP release to IRL-1620 but did not inhibit the response to ET-1 (Fig. 3). Notably, the ETB blocker did not block the effect of the nonselective agonist ET-3, but at the highest dose IRL-1038 significantly potentiated the AVP secretory rate to 1 nM ET-3. Basal AVP secretion for this group of experiments averaged 65 ± 21 pg·HNC-1·h-1 (n = 74). When administered alone, neither BQ-123 nor IRL-1038 at the highest doses tested changed AVP release: 100 ± 20 (basal) vs. 103 ± 40%·HNC-1·h-1 (200 nM BQ-123, n = 6); 100 ± 28 (basal) vs. 108 ± 31%·HNC-1·h-1 (600 nM IRL-1038, n = 7).
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When medium was added to both sides of the barrier in compartmentalized explants during both basal and test periods, AVP secretory rates were unchanged from hypothalamus (100 ± 27 vs. 99 ± 40%·HT-1·h-1, n = 4) and from posterior pituitary (100 ± 31 vs. 66 ± 39%·PP-1·h-1, n = 4). Figure 4 shows the AVP release when the ET agonists were added to the hypothalamic compartment. Figure 4A depicts release of AVP by the hypothalamus, and Fig. 4B shows secretion by the posterior pituitary during the test period. Basal AVP release averaged 23 ± 5 pg·HT-1·h-1 by the hypothalamus and 37 ± 6 pg·PP-1·h-1 by the pituitary (n = 71). Maximal stimulation with IRL-1620 was observed at a concentration of 0.01 nM IRL-1620 (414 ± 89%·PP-1·h-1, P < 0.01 vs, basal, n = 6).
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Figure 5 shows the effect of BQ-123 or IRL-1038 on AVP secretion by the hypothalamus and the neurohypophysis when the ET agonists and antagonists are applied to the hypothalamic side. Hypothalamic AVP release induced by the nonselective ETB agonist ET-3 was significantly lower in the presence of IRL-1038. Although pituitary release of AVP in these explants was lower (53 ± 20% basal·PP-1·h-1) when ET-3 was added in the presence of IRL-1038 than with ET-3 alone (81 ± 43% basal·PP-1·h-1), this did not achieve statistical significance. This is due, in part, to the difficulty in demonstrating inhibition when the release rate was already very low. ETA antagonism with BQ-123 potentiated AVP release by the neural lobe in response to ET-1 or ET-3 but did not alter AVP released into the hypothalamic compartment. Pituitary AVP release in response to 1 nM IRL-1620 was also higher in the presence of the ETA inhibitor. Significantly, BQ-123 reversed the decline in hormone release with the highest dose of IRL-1620 tested: 93 ± 18%·PP-1·h-1 (10 nM IRL-1620, n = 10) vs. 340 ± 75%·PP-1·h-1 (10 nM IRL-1620 plus 20 nM BQ-123, n = 6, P < 0.05 vs. IRL-1620 alone); but it had no effect on pituitary AVP release to 0.01 nM IRL-1620 (323 ± 101%·PP-1·h-1, n = 5). In contrast, IRL-1038 blocked the maximal stimulation by 0.01 nM IRL-1620 (88 ± 54%·PP-1·h-1, n = 6, P < 0.025). Overall, absolute basal AVP release in this group was 27 ± 7 pg·HT-1·h-1 by the hypothalamus and 34 ± 10 pg·PP-1·h-1 by the posterior pituitary (n = 66).
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Likewise, Fig. 6 illustrates the AVP secretory response by the neurohypophysis when the ET agonists were added to the posterior pituitary side of the barrier. In these explants, the average basal AVP secretory rate was 39 ± 12 pg·PP-1·h-1 (n = 52). The effects of ET agonists and antagonists added to the posterior pituitary on AVP secretion are displayed in Fig. 7. Average basal neural lobe AVP release was 34 ± 8 pg·PP-1·h-1 in this group (n = 53).
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The effects of tetrodotoxin on AVP release into the pituitary compartment by stimulation of the pituitary with ET-1 or the hypothalamus with IRL-1620 are depicted in Table 1. When IRL-1620 was added together with tetrodotoxin to the hypothalamus, pituitary release of AVP was blocked. Hypothalamic release of AVP in response to IRL-1620 was also prevented by tetrodotoxin. Tetrodotoxin did not block the effect of ET-1 when both were coapplied to the pituitary. Normalized basal hypothalamic release was 100 ± 26%·HT-1·h-1, and test period release in the presence of both IRL-1620 and tetrodotoxin was 103 ± 26%·HT-1·h-1 (n = 6).
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Medium osmolality at the end of the experiments averaged 301 ± 1 mosmol/kgH2O overall and never exceeded 304 mosmol/kgH2O.
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DISCUSSION |
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The present studies highlight the complex interactions that occur when multiple isoforms of a peptide, such as ET, act via distinct receptor subtypes located on different neuronal elements of the same or synaptically related neurons. The hypothalamo-neurohypophysial explant in standard culture provides a useful experimental model for the study of AVP release. Even so, these explants possess more than one site for ET peptide action. The AVP released into the medium will be the net hormone secreted in response to both inhibitory and excitatory inputs from both somatodendritic and neural lobe (axonal) elements. Our data with explants in such standard cultures confirmed earlier results showing a progressive net increase in AVP release with higher concentrations of ET-1 by perifused hypothalami (26) or ET-3 by hypothalamo-neurohypophysial explants in standard culture (21). Because ET-3, which has roughly equal affinities for ETA and ETB receptors (5), potently stimulates AVP secretion from the neural lobe (26) and neurons in the Av3V region that possess mostly ETB receptors were excited by ET-1 (29), the dose-dependent decrease in AVP secretion with the selective ETB agonist was somewhat unexpected. However, this finding was further corroborated by the observation that, once the ETB receptors were maximally blocked with IRL-1038, then unopposed ETA agonism resulted in potentiation of the neurohormone secretory response by ET-3. Overall, these results supported a stimulatory role for ETA receptor activation on AVP release. That there are at least two sites of stimulatory action, one via ETA receptors and another via ETB receptors within the structures included in the explants is suggested by the observation that picomolar concentrations of the ETB agonist also stimulated AVP and could be selectively and dose-dependently inhibited by IRL-1038. In addition, any working model should be able to reconcile the secretory data with electrophysiological studies that have shown that ET-1 inhibits phasically firing magnocellular neurons (29) whose somata are primarily endowed with ETA receptors (30) but excites neurons in the Av3V region (29), which encompasses the organum vasculosum of the lamina terminalis where ETB receptors are located (30).
The experiments using compartmentalized hypothalamo-neurohypophysial explants provide new insights into the interplay of ET receptor subtypes and sites of action that result in AVP secretion. Application of the lowest concentration of IRL-1620 exclusively to the hypothalamus clearly resulted in AVP release by the neural lobe. The lack of stimulation by 10 nM IRL-1620 suggests that, at higher doses the ETB agonist begins to bind to inhibitory hypothalamic ETA receptors similar to binding characteristics of ET ligands reported in rat anterior pituitary (8). This interpretation is also consistent with electrophysiological findings that ET-1, an ETA agonist, inhibits presumptive vasopressinergic neurons within the supraoptic nucleus (29) and by the present observation that concurrent blockade of hypothalamic ETA receptors with BQ-123 resulted in significant neurohypophysial AVP secretion by ET-1 or ET-3.
Tetrodotoxin blockade of the hypothalamic IRL-1620-induced AVP release by the posterior pituitary further indicates that the stimulatory effect is not located on the magnocellular neuron itself but resides on neurons that project to the AVP secretory cell. Previous studies from our laboratory (22) implicate an N-methyl-D-aspartate receptor, but other receptors may be involved as well. Although the present data do not directly address the locus of these ET-responsive neurons or whether the projection is monosynaptic or polysynaptic, the Av3V region, which contains neurons excited by ET peptides (29) and includes the organum vasculosum of the lamina terminalis richly endowed with ETB receptors (30), is a candidate locus (Fig. 8).
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That tetrodotoxin applied with ET-1 to the posterior pituitary did not block AVP release is not surprising. Nevertheless, receptor-binding studies in this brain region have not discriminated definitively between binding at the neural lobe and binding at the intermediate lobe of the posterior pituitary (12). The present data show that ET-1 indeed acts on receptors located directly on nerve terminals within the neural lobe rather than on the intermediate lobe, which is also present in the explant preparation (27).
The importance of somatodendritic release of AVP in auto-control of magnocellular neurons and optimizing the efficiency of hormone release has become increasingly apparent (1, 3, 6, 11). AVP secreted into the hypothalamic compartment largely reflects somatodendritic release, although hormone release from axon projections that terminate in the proximate median eminence may also contribute. The pattern of AVP release into this compartment differed from simultaneously assessed neural lobe secretion. Selective ETA agonism failed to elicit AVP release into either compartment. In contrast, however, higher doses of ET-3 resulted in progressively greater AVP release by the hypothalamic components as did IRL-1620. This stimulatory effect was due to ETB receptor activation and involved at least one synapse. However, blockade of ETA receptors did not alter somatodendritic release as it did axonal AVP secretion into the pituitary compartment. Taken together with the observation that the mixed agonist increased hypothalamic AVP secretion, it appears that stimulation of the ETA receptor can inhibit the vasopressinergic neurons in the hypothalamus and modulate neurohypophysial AVP release without altering ETB receptor-induced somatodendritic secretion. Independent modulation of the vasopressinergic neuron itself by ETA receptors would permit even more plasticity in the net response to ET peptides.
Immunocytochemical studies have shown that AVP receptors exist on neurons within the supraoptic nucleus (2). Either direct AVP modulation of the AVP neuron (1, 11) or indirect action by depolarization of neighboring interneurons (3) can influence the firing pattern of the vasopressinergic cell so as to optimize AVP secretory activity (6). Therefore, under pathophysiological conditions such as septic or hemorrhagic shock, where circulating and/or brain ET levels are high (9, 14, 17, 18), activation of hypothalamic ETB receptors would result in a coordinated signal to the AVP neuron by somatodendritic release of AVP that would amplify hormone secretion by the neural lobe and result in sustained circulating levels of AVP.
Besides hypothalamic actions, ET peptides also exerted a stimulatory effect on the posterior pituitary. Specifically, ET-1 dose-dependently increased AVP release, which was inhibited by ETA antagonism. Analogous to the hypothalamus, ETA blockade prevented stimulation by the nonselective agonist ET-3 and uncovered the stimulatory effect of IRL-1620, presumably by displacing the ETB agonist to ETA sites. A direct inhibitory action was not definitively evident but may be suggested by the decrease in AVP secretion with the highest dose of ET-3. These findings are noteworthy in view of the colocalization of ET peptides and AVP in neurosecretory vesicles within the axon terminals in the neural lobe (16, 31). This would permit coreleased ET, yet another site for amplification (or modulation) of AVP secretion. Moreover, both the Av3V region and the posterior pituitary are devoid of the blood-brain barrier and may be responsive to circulating ET peptides as well as ET generated within the central nervous system.
In summary, the response of whole explants exposed to ET agonists and antagonists reflects the net result of both stimulatory and inhibitory inputs. Accordingly, even within the relatively simplified slice model of the whole hypothalamo-neurohypophysial explant, ET may exert complex actions on AVP release depending on the isoform, receptor subtype, and locus of the action. The integration of these excitatory and inhibitory inputs endows the vasopressinergic system with a greater plasticity in its response to physiological and pathophysiological states. In this context, the compartmentalized paradigm provides an approach for systematic analysis of these influences on AVP secretion from both somatodendritic as well as neural lobe sources.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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