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Am J Physiol Regul Integr Comp Physiol 293: R931-R937, 2007. First published May 23, 2007; doi:10.1152/ajpregu.00196.2007
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WATER AND ELECTROLYTE HOMEOSTASIS

Tachykinin NK3 receptor contribution to systemic release of vasopressin and oxytocin in response to osmotic and hypotensive challenge

Gwendolen E. Haley and Francis W. Flynn

Graduate Neuroscience Program, University of Wyoming, Laramie, Wyoming

Submitted 20 March 2007 ; accepted in final form 16 May 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Activation of the neurokinin 3 receptor (NK3R) by a receptor agonist, hypotension, and hyperosmolarity results in the internalization of NK3R expressed by magnocellular neurons and the release of vasopressin (VP) and oxytocin (OT) into the circulation. The contribution of NK3R activation to the release of VP and OT in response to hyperosmolarity and hypotension was evaluated by measuring the release of both hormones following pretreatment with a selective NK3R antagonist, SB-222200. Freely behaving male rats were given an intraventricular injection of either 0.15 M NaCl or 250, 500, or 1,000 pmol SB-222200, and then were administered an intravenous infusion of 2 M NaCl or 0.15 M NaCl (experiment 1), or a bolus intra injection of 0.15 M NaCl or hydralazine (HDZ), a hypotension-inducing drug (experiment 2). Blood samples were taken from indwelling arterial catheters at various time points for 1–2 h, both before and after treatments. Plasma VP and OT levels were determined by ELISA. Blockade of NK3R did not affect the baseline levels of either hormone. In contrast, pretreatment with SB-222200 significantly reduced (~60%) or abolished the release of VP and OT, respectively, to 2 M NaCl infusion. HDZ-induced VP and OT release was eliminated by pretreatment with 500 pmol SB-222200. Therefore, NK3R activation contributes significantly to the systemic release of both VP and OT in response to osmotic and hypotensive challenges.

hydralazine; hyperosmolarity; hypotension

SYSTEMIC VASOPRESSIN (VP) and oxytocin (OT) are synthesized predominantly in the magnocellular neurons of the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Hyperosmolarity, hypovolemia, and hypotension elicit a simultaneous VP and OT release into the circulation (29, 33, 47, 53). Osmotic and volumetric information is relayed to the PVN via different pathways. The circumventricular organs, including the organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (SFO), contain central osmoreceptors (7, 8, 36) and project monosynaptically to the PVN and synapse on both VP and OT neurons (11, 50). Both the OVLT and the SFO project to the median preoptic area (MnPO), an area with osmosensitive neurons (52), which also directly projects to the magnocellular neurons (8). When these areas are ablated, hyperosmolarity no longer elicits VP and OT release into the circulation (9, 21, 37). Conversely, the A1 and A2 cell groups from the hindbrain project monosynaptically to the PVN and convey information about blood volume and pressure. Afferent projections from the A1 group synapse predominantly on VP neurons and sparsely on OT neurons (43), whereas neurons from the A2 cell group synapse on both VP and OT neurons (13, 14, 40, 43). When the A1 and A2 cell groups are ablated, VP response to moderate hemorrhage is almost completely abolished (10, 27). The pathways just described that relay blood osmolarity and volume information utilize a variety of transmitters, including atrial natriuretic peptide, ANG II, norephinephrine, ATP, glutamate, and substance P (25, 26, 28, 35, 38, 41, 49). Magnocellular VP and OT neurons express the receptors for the aforementioned peptides but also express other receptors as well (28, 46).

The tachykinin, neurokinin 3 receptor (NK3R) is expressed in multiple nuclei, including the median preoptic nucleus, preoptic nucleus, and the nucleus of the solitary tract, which relay osmotic and volumetric afferent information to magnocellular neurons (42). Also, NK3Rs are highly expressed by the magnocellular neurons of the PVN and SON (16, 34, 45). These receptors are colocalized with vasopressinergic (15, 22) and oxytocinergic magnocellular neurons (20). Exogenous activation of the NK3R, by intraventricular injection of the selective NK3R agonist senktide, causes VP (18, 22, 39) and OT (6, 24) release into the circulation, as well as antidiuresis in water-loaded rats (18). As for a site of action, intraventricular injections of senktide elicit c-Fos expression in all of the aforementioned nuclei (48). As such, the intraventricular injection of NK3R agonists could target one or more populations of NK3R. Also, the intraventricular injection of senktide may directly affect NK3R expressed by magnocellular neurons because intraventricular injections of senktide cause the activation and internalization of these receptors (22), and injections of NK3R agonists directly into the PVN are highly potent in stimulating VP release (32). Indeed, on the basis of receptor internalization, NK3Rs expressed by magnocellular neurons are activated in response to senktide injection. Upon binding a ligand, NK3R, like other G-protein coupled receptors, are internalized to the cytoplasm (3, 23, 31), and this translocation can be visualized with immunohistochemical methods. NK3Rs are largely membrane-bound following intraventricular injections of saline, but following senktide injection, NK3Rs appear in endosomes in the cytoplasm of VP magnocellular neurons (22, 24). Furthermore, NK3R internalization is a specific pharmacological marker of receptor activation by an agonist and is prevented by pretreatment with a specific NK3R antagonist, SB-222200 (22). Also, pretreatment with the NK3R antagonist blocks senktide-induced release of VP into circulation (22). The results of the antagonist blocking the actions of the specific agonist for the receptor lead to the conclusion that the SB-222200 is binding the NK3R, thus blocking its activation.

NK3Rs expressed by magnocellular neurons are internalized in response to physiological challenges. Recent studies show that hyperosmolarity and hypotension, induced by hydralazine (HDZ), cause the translocation of NK3R from membrane to the cytoplasm of magnocellular neurons in the PVN and SON (22, 24). Neurons adjacent to the VP magnocellular neurons in the PVN also expressed NK3Rs, which were internalized in response to hyperosmolarity as well (22). The size and proximity of the neurons to VP immunoreactive magnocellular neurons led to the suggestion that the unidentified neurons were oxytocinergic (22). This NK3R internalization by magnocellular neurons requires binding of an endogenous ligand, neurokinin B (NKB), which is presumed to have been locally released in response to hyperosmolarity and hypotension-induced by HDZ (30, 42).

NK3R are activated by two potent physiological challenges, hyperosmolarity and hypotension, that elicit both VP and OT release. As pointed out above, multiple receptors and transmitter systems are involved in the release of VP and OT, and the significance of NK3R signaling to the release of VP and OT under these physiological conditions is not known. The objective of this study was to determine whether NK3R signaling contributes to the systemic release of VP and OT during two physiological challenges. The effects of blockade of NK3R on VP and OT release in response to osmotic (2 M NaCl, experiment 1) and hypotensive challenges (HDZ, experiment 2) were determined. We show that blockade of NK3R by intraventricular pretreatment with a selective NK3R antagonist, SB-222200, blunts or abolishes the systemic release of VP and OT in response to these two challenges.


   METHODS
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 METHODS
RESULTS
 DISCUSSION
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Animals

All procedures were approved by the University of Wyoming Animal Care and Use Committee. Rats (male, 300–350 g, n = 62; Charles River Laboratory, Wilmington, MA) were individually housed in standard wire cages in a temperature-controlled room on a 12:12-h light-dark cycle. Rats had free access to Purina Rat chow and water.

Surgery

Rats were anesthetized with a mixture of ketamine and acepromazine (0.07 mg/kg ip) and mounted in a stereotaxic device. A hole was drilled in the skull 0.9 mm posterior to bregma, 1.8 mm lateral to the midline, and a cannula (26 G; Plastics One, Roanoke, VA) was lowered 4.5 mm ventral to the dura. The cannula was secured in place with four jewelry screws and dental acrylic. Rats were allowed to recover for 24 h. Then, rats were reanesthetized and a small incision was made in the right hind leg. The femoral artery and vein were exposed and catheters were inserted 3 cm into the vessels. The femoral vein catheter was constructed of 15 cm of polyethylene (PE)-50 tubing and the femoral artery catheter was constructed from 3 cm of PE-10 tubing attached to 15 cm of PE-50 tubing. Once the tubing was set in place, it was sutured to the vessel and then subcutaneously drawn to an exit wound between the two scapulae. The catheters were then filled with heparinized saline and heat sealed until testing. Rats were then returned to their home cages with ad libitum food and water for 24–48 h.

VP and OT Assay

Hormones were extracted from plasma samples using C-18 column extraction method. Briefly, a 1% trifluoroacetic acid (TFA) solution was used to acidify the sample, which was then filtered through an equilibrated C-18-OH column. Columns were washed with the 1% TFA solution, and proteins were eluted with a 60% acetonitrile, 39% dH2O, and 1% TFA solution. Samples were spun in a vaccufuge until only a small pellet remained and stored at –80°C. The pellet was reconstituted in assay buffer 24 h before assay. Plasma samples were analyzed using VP and OT ELISA kits (Assay Designs, Ann Arbor, MI). Sensitivity of the VP ELISA kit was 3.39 pg/ml, while the OT ELISA kit was 11.7 pg/ml. Specificity of the ELISA kits were determined by applying VP standards to OT kits and vice versa. Neither the VP nor the OT kit recognized the standards from the other kit. Intra-assay and inter-assay precision were determined with calculated variants, and in both assays, the values were less than 6%. Extraction efficiency was ~90% for both VP and OT extractions.

Data Analysis

Plasma VP and OT levels were analyzed using SPSS 13.0. Plasma levels were analyzed using separate, three-factor ANOVA with repeated-measures (intraventricular treatment x intravenous treatment x time [repeated measures]). Significant main effects and interactions were further analyzed with one-way ANOVAs, and Student's t-test was used for specific comparisons with critical values from Bonferroni table for multiple comparisons. Results are presented as group means ± SE.

Experiment 1: Osmotic Challenge

Procedure. Rats (n = 32) were removed from their home cages and transferred to a clean, transparent plastic cage. Femoral artery and venous catheters were attached to heparinized saline-filled tubes attached to a length of PE-50 tubing. Intraventricular injectors were attached to a length of PE-50 tubing connected to a microliter syringe positioned on an infusion pump (model 200; KD Scientific, Holliston, MA). Rats were then left undisturbed in a quiet room for 15–20 min before testing. The infusion pump was then turned on to deliver intraventricular infusion of 5 µl (1 µl/min for 5 min) of 0.15 M NaCl (n = 8) or 250 pmol (n = 8), 500 pmol (n = 8), 1,000 pmol (n = 8) of the NK3R specific antagonist, SB-222200 (Courtesy of GlaxoSmithKline, Durham, NC), into the lateral ventricle. Immediately following the end of the intraventricular infusion, a syringe filled with either 0.15 M (n = 16) or 2 M (n = 16) NaCl was positioned on the infusion pump, and 2 ml of the NaCl solution was infused intravenously (0.1 ml/min for 20 min). Half of the rats from each of the intraventricular treatment groups was randomly assigned to one of the two intravenous treatment groups, and rats were only treated once. Blood samples were taken before the start of the intraventricular infusion, 25 and 40 (15 min after the intravenous infusion ended) min after the intraventricular infusion began. With each blood sample, an equal volume of donor blood was simultaneously infused. Donor blood was collected from a cardiac draw 1–2 h prior from a ether-anesthetized rat and kept at 37.6°C until used (1, 2, 12). Blood samples were spun in a refrigerated centrifuge (2,000 rpm for 20 min), and plasma was aspirated into a clean Eppendorf tube and stored at –80°C until extraction.

The effect of the intravenous infusions on plasma osmolality was determined in a separate group of male rats (n = 4). Blood samples (1 ml) were collected from the femoral artery before and immediately after intravenous infusions of 0.15 M NaCl or 2 M NaCl. Blood samples were spun in a refrigerated centrifuge (2,000 rpm for 20 min). Plasma was aspirated and osmolality was measured with an osmometer (Multi-Osmette; Precision Services, Shelby Township, Michigan).

To verify the reported (5) increase of blood pressure following intravenous infusions of 2 M NaCl, a rat (n = 1) was fitted with arterial and venous catheters and allowed to acclimate in a quiet room for 15 min. The arterial catheter was connected to a blood pressure transducer, which interfaced a computer with blood pressure monitoring software (Power Laboratory; AD Instruments, Castle Hill, New South Wales, Australia), and the venous catheter was attached to a 2 M NaCl syringe. The syringe was then attached to the infusion pump. Once the blood pressure had stabilized, the infusion pump was turned on. Blood pressure was measured continuously for 120 min.

Experiment 2: HDZ-Induced Hypotension

Procedure. Rats (n = 24) were placed in cages, and catheters were attached to syringes in the same manner as experiment 1. Following a 15–20 min acclimation period, rats were administered intraventricular infusion (5 µl; 1 µl/min for 5 min) of 0.15 M NaCl (n = 8), 250 pmol (n = 8), or 500 pmol (n = 8) of SB-222200. Immediately after the completion of the intraventricular infusion, rats were given a bolus intravenous injection of HDZ (10 mg/kg, n = 12; Sigma-Aldrich, St. Louis, MO) or equal amount of 0.15 M NaCl (n = 12). As in the previous experiment, one-half of the rats from each intraventricular treatment group was randomly assigned to one of the two intravenous treatments and only treated once. Blood samples were taken before the start of the intraventricular infusion, and then 20, 40, and 60 min later. Replacement blood was given as previously described. Whole blood was spun in a refrigerated centrifuge. Plasma was aspirated into a clean Eppendorf tube and stored at –80°C until extraction.

To verify HDZ-induced hypotension, a rat (n = 1) fitted with arterial and venous catheters was allowed to acclimate in a quiet room 15–20 min before testing. The arterial catheter was connected to a blood pressure transducer, which interfaced a computer with blood pressure monitoring software, and the venous catheter was attached to a heparinized saline-filled syringe. A bolus intravenous injection of HDZ was administered once mean arterial pressure (MAP) was stable. MAP was continually measured 5 min before HDZ injection and 95 min after HDZ injection. Intravenous HDZ treatment decreased MAP from 122 mmHg to 64 mmHg within 10 min of injection, and MAP remained at 64 mmHg for more than 90 min after injection (data not shown).


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Experiment 1: Osmotic Challenge

Plasma osmolality (282 ± 2.0, 285 ± 1.4 mosmol/kgH2O) was not significantly different in the two groups before the start of the intravenous infusions. Infusions of 0.15 M NaCl had no effect on plasma osmolality, but 2 M NaCl caused a significant elevation in plasma osmolality, which peaked immediately following the end of the infusion (time 25; 334 ± 6.1 mosmol/kgH2O, P < 0.008). Plasma osmolality decreased once the infusion stopped and was significantly lower than the peak plasma osmolality at both time 40 (314 ± 5.7 mosmol/kgH2O, P < 0.02) and time 120 (295 ± 3.4 mosmol/kgH2O, P < 0.0001). Blood pressure followed a similar pattern as osmolality, and MAP increased from 115 mmHg to a peak of ~135 mmHg by the end of the infusion of 2 M NaCl. However, by 120 min, MAP returned to the baseline level, 115 mmHg (data not shown).

Plasma VP levels. Plasma VP levels before intraventricular infusions (basal levels, time 0) were not significantly different between the groups, [F(7,31) = 1.8, P > 0.1, (Fig. 1)], but thereafter, there was a significant interaction of pretreatment x intravenous injection x time [F(6,48) = 21.7, P < 0.0001]. Plasma VP levels in rats administered 0.15 M NaCl or NK3R antagonist pretreatment and then intravenous 0.15 M NaCl were not significantly different from each other at any time point (P = 1.0). Furthermore, in these rats, plasma VP levels during the test did not significantly vary. As such, intravenous infusion of 0.15 M NaCl had no effect on plasma VP levels, and furthermore, blockade of NK3R had no significant effect on baseline (intravenous infusion of 0.15 M NaCl) levels of VP.


Figure 1
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  		Fig. 1. SB-222200 blocks vasopressin (VP) release in response to 2 M NaCl. Plasma VP levels following 0.15 M NaCl or 2 M NaCl infusion. Pretreatment of SB-222200 had no effect on baseline VP levels. Intravenous infusion of 2 M NaCl elicited an increase in plasma VP levels. Pretreatment of 250 pmol of SB-222200 had no effect on stimulated VP release to 2 M NaCl, as the lines and symbols representing the 0.15 M NaCl and 250 pmol pretreatment overlap substantially. Conversely, pretreatment of 500 pmol and 1,000 pmol of SB-222200 significantly attenuated stimulated VP release (**P < 0.0001).

 
Intravenous infusions of 2 M NaCl elicited a significant rise in plasma VP concentrations, and there was a significant interaction with the intraventricular pretreatment [F(6,24) = 8.0, P < 0.0001]. Further comparisons revealed that compared with baseline (time 0), intravenous infusion of 2 M NaCl elicited a similar and significant rise in VP levels in rats pretreated with 0.15 M NaCl and 250 pmol SB-222200 (P < 0.0001). In rats pretreated with 0.15 M NaCl and 250 pmol SB-222200 prior to intravenous injection of 2 M NaCl plasma VP levels remained significantly above time 0 at time 40 (P < 0.002).

Intravenous infusion of 2 M NaCl elicited a significant rise in plasma VP levels in rats treated with 500 pmol and 100 pmol SB-222200 [F(2,6) = 21, P < 0.002; F(2,6) = 10.6, P < 0.01], but the increase in plasma VP levels at the conclusion of the intravenous infusion was significantly less than seen in rats pretreated with 0.15 M NaCl (P < 0.0001). At time 25, pretreatment with 500 and 1,000 pmol SB-222200 reduced VP release in response to 2 M NaCl infusion by ~60% compared with rats pretreated with 0.15 M NaCl. At time 40, plasma VP levels of rats pretreated with 500 or 1,000 pmol SB-222200 before the 2 M NaCl infusion had returned to baseline levels and were not significantly different from rats that were administered intravenous infusion of 0.15 M NaCl (P = 1.0).

Plasma OT levels. Plasma OT levels significantly varied as a function of pretreatment, intravenous infusion and time [F(6,54) = 10.9, P < 0.0001, (Fig. 2)]. At time 0, before the treatments, plasma OT levels were not significantly different between the groups (P > 0.05). Intravenous infusion of 0.15 M NaCl had no significant effect on plasma OT levels in rats pretreated with 0.15 M NaCl, 250 pmol, or 500 pmol SB-222200 (P > 0.2).


Figure 2
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  		Fig. 2. SB-222200 blocks OT release in response to 2 M NaCl. Plasma OT levels following 0.15 M NaCl or 2 M NaCl infusion. Intraventricular treatment of SB-222200 had no effect on baseline OT levels. Intravenous infusion of 2 M NaCl significantly increased plasma OT levels. However, pretreatment with both 250 pmol and 500 pmol concentrations of specific neurokinin 3 receptor (NK3R) antagonist blocked OT release after intravenous infusion of 2 M NaCl. (**P < 0.0001).

 
Intravenous infusion of 2 M NaCl elicited a significant rise in plasma OT concentrations in rats pretreated with either intraventricular injections of 0.15 M NaCl or 250 pmol SB-222200 (P < 0.0001), but not in rats pretreated with 500 pmol SB-222200 (P > 0.05). In fact, plasma OT levels of rats pretreated with 500 pmol SB-222200 before 2 M NaCl infusion were not significantly different from those of rats administered the 0.15 M NaCl infusion at any time point (P = 1.0). Intravenous 2 M NaCl infusion stimulated the greatest release of OT in rats pretreated with 0.15 M NaCl. In these rats, plasma OT levels increased to ~60 pg/ml with relatively little change in levels at 120 min. Intravenous infusion of 2 M NaCl caused a significant elevation in plasma OT levels in rats pretreated with 250 pmol SB-222200 [F(3,9) = 43, P < 0.0001]. However, OT levels in rats pretreated with 250 pmol SB-222200 were significantly less than those measured in osmotically challenged rats that were pretreated with 0.15 M NaCl (P < 0.05).

Experiment 2: HDZ-Induced Hypotension

Plasma VP levels. The overall three-way ANOVA revealed significant main effects and a significant time x pretreatment (intraventricular infusion) x intravenous injection interaction, [F(6,54) = 7.3, P < 0.001 (Fig. 3)]. At time 0, basal plasma VP levels were not significantly different between the groups (P = 0.3). In control rats pretreated with intraventricular infusion of 0.15 M NaCl, intravenous injection of 0.15 M NaCl had no reliable effect on plasma VP levels during the experiment. Furthermore, plasma VP levels of rats administered 250 or 500 pmol SB-222200 and the intravenous injection of 0.15 M NaCl were not different from those of control rats (P = 1.0).


Figure 3
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  		Fig. 3. SB-222200 blocks VP release in response to hypotension. Plasma VP levels following bolus 0.15 M NaCl or 10 mg/kg hydralazine (HDZ) treatment. Pretreatment with SB-222200 had no effect on baseline VP levels. Intravenous injection of HDZ elicited a significant increase in plasma VP levels. However, following pretreatment with the NK3R antagonist at both the 250 pmol and 500 pmol concentrations, no increase in plasma VP was seen after HDZ injection compared with baseline plasma VP levels (**P < 0.0001).

 
HDZ elicited a significant increase in plasma VP levels compared with 0.15 M NaCl intravenous injection and a significant interaction was observed with intraventricular pretreatment [F(2,18) = 21.0, P < 0.0001]. In rats pretreated with 0.15 M NaCl before HDZ, plasma VP levels significantly increased from basal levels to 8.2 ± 1.3 pg/ml, 10.7 ± 2.0 pg/ml, and 11.7 ± 1.3 pg/ml at times 20, 40, and 60 min, respectively. At each of the time points, plasma VP levels following HDZ were significantly greater than those of rats treated with 0.15 M NaCl (P < 0.002). However, pretreatment with 250 and 500 pmol SB-222200 blocked VP release in response to HDZ. Plasma VP levels in rats administered HDZ following 250 or 500 pmol SB-222200 were not significantly different from those of rats administered intravenous injections of 0.15 M NaCl at any of the time points (P = 1.0).

Plasma OT levels. The overall analysis revealed a significant interaction of pretreatment, intravenous injection and time [F(6,54) = 4.1, P < 0.002 (Fig. 4)]. At time 0, prior to any treatments, plasma OT levels of the groups were not significantly different (P = 0.3). Plasma OT levels did not significantly change over time in rats administered intravenous injections of 0.15 M NaCl following pretreatment of 0.15 M NaCl or SB-222200 (P < 0.2). Intravenous injection of HDZ resulted in a significant increase in plasma OT levels in rats pretreated with 0.15 M NaCl [F(3,9) = 18.7, P < 0.0001]. Plasma OT levels increased from 10.7 ± 1.3 pg/ml at time 0 to 33.2 ± 2.7 pg/ml at time 60 in these rats. Pretreatment with 500 pmol SB-222200 blocked the systemic release of OT in response to HDZ. Plasma OT levels of rats in this group did not change significantly during the experiment (P = 0.3), and OT levels were significantly less than that of rats pretreated with 0.15 M NaCl before HDZ at 40 and 60 min (P < 0.0001). Plasma levels rose in rats pretreated with 250 pmol SB-222200 before HDZ, but this was not statistically reliable (P < 0.07). Moreover, there were no significant differences in plasma OT levels of rats pretreated with 250 pmol SB-222200 before HDZ and OT levels of rats given intravenous 0.15 M NaCl injection (P = 1.0).


Figure 4
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  		Fig. 4. SB-222200 blocks OT release in response to hypotension. Plasma OT levels following 0.15 M NaCl or 10 mg/kg HDZ treatment. Pretreatment of SB-222200 had no effect on baseline OT levels. Intravenous injection of HDZ elicited a significant increase in plasma OT levels. However, pretreatment of both 250 pmol and 500 pmol concentrations of the NK3R antagonist significantly blocked OT release after HDZ injection. At the 20-min time point, OT release was attenuated by both the 250 and 500 pmol antagonist 15% and 60%, respectively. However, at the 40 and 60 min time points, the OT release was virtually eliminated. (**P < 0.0001).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Hyperosmolarity and hypotension, induced by HDZ treatment, stimulate the systemic release of VP and OT (29, 33, 47, 53). The high concentration of NK3R on magnocellular neurons in the SON and PVN (16, 34, 45) suggests that this receptor system may play a significant role in the release of VP and OT. Previous studies show that exogenous injections of NK3R agonists stimulate the release of both hormones into the circulation (6, 18, 22). The link between NK3R and the release of OT and VP in response to physiological challenges was provided by studies showing that in response to hyperosmolarity and hypotension, induced by HDZ treatment, NK3R expressed by magnocellular neurons were trafficked from the membrane to the cytoplasm (22, 24). The NK3R trafficking requires that the physiological challenges cause the local release of an endogenous NK3R ligand, presumably NKB, which then binds and activates the receptor expressed by the magnocellular neurons (30, 42). Although the results establish that physiological challenges result in the activation of NK3R expressed by PVN and SON magnocellular neurons, magnocellular neurons express multiple receptors (28, 46), and the relative contribution of NK3R activation in the release of VP and OT was not established by receptor internalization.

The contribution of NK3R in the release of VP and OT in response to two physiological challenges was assessed by measuring the effects of blockade of NK3R on OT and VP levels. Injections of SB-222200 had no effect on basal or unstimulated (0.15 M NaCl infusion) conditions. As such, NK3R does not appear to be involved in the control of either neurohormone under these conditions. Intravenous infusions of 2 M NaCl led to a significant elevation in both VP and OT levels. Hypertonic saline-stimulated VP levels were reduced by 60% or more by pretreatment with SB-222200. Blockade of NK3R had a greater effect on OT than VP release in response to osmotic challenge. Pretreatment with 500 pmol SB-222200 virtually abolished OT release in response to intravenous infusions of 2 M NaCl. However, OT levels are present at higher levels for a longer period of time compared with VP levels (51). One possible explanation for the prolonged presence of OT in the plasma is the longer degradation time for the peptide (19, 51). Therefore, NK3R signaling is necessary for the VP and OT release into the circulation in response to hyperosmolarity and hypotension.

Overall, the hypotensive challenge was less potent than the osmotic challenge in stimulating VP and OT release. The dose of HDZ used in this experiment causes moderate hypotension and stimulates not only VP and OT release but also NK3R activation (24, 44). Nevertheless, intraventricular pretreatment with the NK3R antagonist virtually abolished the release of VP and OT in response to moderate hypotension. Taken together, the results show that intraventricular injections of SB-222200 are effective at blocking the endogenous activation of NK3R and release of OT and VP in response to two, potent physiological challenges.

Sensory information related to hyperosmolarity and hypotension is relayed to magnocellular neurons via separate pathways. The OVLT, SFO, and MnPO are essential for the systemic release of VP and OT in response to an osmotic challenge (8, 37). Conversely, neurons in the A1 and A2 cell groups project to magnocellular neurons in the hypothalamus and are essential for the systemic release of VP and OT in response to hypovolemia (17, 54). While these two stimuli activate two different pathways originating in different parts of the brain, NK3R signaling appears to be common to both pathways and is involved in the stimulated release of VP and OT. Intraventricular administration of the NK3R antagonist SB-222200 does not identify the location of NK3R affected by the antagonist, leaving the identity of the NK3R activation site unclear. Possible targets of the NK3R antagonist action are suggested by overlap of NK3R immunoreactivity with areas that are involved in both the osmotic and volume neural pathways. NK3R immunoreactivity is reported in the A2 cell group and the MnPO, but not in the A1 cell group, OVLT, or SFO (16, 34, 45). Out of these areas, one that could be the site of the NK3R antagonist action after both osmotic and hypotensive challenge is the MnPO. Not only is it sensitive to both osmotic and hemodynamic changes (4, 52), but lesions of the MnPO decrease the stimulated release of VP in both hyperosmotic and hypovolemic challenges (55). Alternatively, the magnocellular neurons could be the site of NK3R antagonist action. High amounts of NK3R immunoreactivity are found on the magnocellular neurons (16, 34, 45) and are activated on these neurons in response to both hyperosmolarity and hypovolemia (22, 24).

In conclusion, NK3R-specific antagonist SB-222200 blocks the systemic release of two hormones in response to two different stimuli. Both hyperosmolarity and hypotension are potent stimulators of systemic release of VP and OT, and the sensory information is relayed to the magnocellular neuron through different neural pathways. These results indicate that NK3R signaling is necessary for the stimulated release of VP and OT, which is located in an area common to both osmotic and volume neural pathways.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This publication was made possible by National Institutes of Health Grant DK-50586, and Grant P-20-RR15640 from the National Center of Research Resources, a component of the National Institutes of Health.


   ACKNOWLEDGMENTS
 
We thank GlaxoSmithKline for their generous gift of SB-222200. Also, we would like to thank Shawna McBride and Donald Pratt for their assistance in preparing the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Francis W. Flynn, Dept. of Zoology and Physiology, Univ. of Wyoming, 1000 E. Univ. Dept. 3166, Laramie, WY 82071 (e-mail: flynn{at}uwyo.edu)

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.


   REFERENCES
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 ABSTRACT
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