Neurokinin 3 receptor (NK3R) signaling has an integral role in the stimulated oxytocin (OT) and vasopressin (VP) release in response to hyperosmolarity and hypotension. Peripheral injections of cholecystokinin (CCK) receptor agonists for the CCK-A (sulfated CCK-8) and CCK-B (nonsulfated CCK-8) receptors elicit an OT release in rat. It is unknown whether NK3R contributes to this endocrine response. Freely behaving male rats were administered an intraventricular pretreatment of 250 or 500 pmol of SB-222200, a specific NK3R antagonist, or 0.15 M NaCl before an intraperitoneal or intravenous injection of CCK-8 (nonsulfated or sulfated) or 0.15 M NaCl. Blood samples were taken before intraventricular treatment and 15 min after intraperitoneal or intravenous injection, and plasma samples were assayed for OT and VP concentration. Intraperitoneal injection of both nonsulfated and sulfated CCK-8 significantly increased plasma OT levels and had no effect on plasma VP levels. Intravenous injection of sulfated CCK-8 stimulated an increase in plasma OT levels and did not alter plasma VP levels. However, intravenous injection of nonsulfated CCK-8 stimulated a significant increase in plasma levels of both OT and VP. No other studies have demonstrated CCK-8-stimulated release of VP in rat. NK3R antagonist did not alter baseline levels of either hormone. However, pretreatment of NK3R antagonist significantly blocked the CCK-stimulated release of OT in all CCK treatment groups and blocked VP release in response to intravenous injection of nonsulfated CCK-8. Therefore, central NK3R signaling is required for OT and VP release in response to CCK administration.
the tachykinin system, specifically the neurokinin 3 receptor (NK3R), has a role in regulating the systemic release of oxytocin (OT) and vasopressin (VP) from the magnocellular neurons of the paraventricular (PVN) and supraoptic (SON) nuclei. NK3R is found in high concentrations in multiple brain areas that innervate magnocellular neurons, such as the nucleus of the solitary tract (NST) and median preoptic nucleus, as well as the magnocellular neurons themselves (20, 43, 61). The distribution of neurokinin B (NKB), the endogenous ligand for NK3R, fibers corresponds to the receptor distribution (37, 49). Intraventricular injection of the specific NK3R agonist, senktide, induces c-Fos expression in VP and OT magnocellular neurons (21, 62) and elicits a systemic release of both hormones (5, 27, 53). NK3R expressed on magnocellular neurons are activated by senktide and are internalized to the cytoplasm of magnocellular neurons following senktide injection (27). Furthermore, senktide administration activates neurons that innervate the magnocellular neurons. Specifically, central injection of senktide induces c-Fos in the median preoptic nucleus, caudal NTS (cNST; Ref. 62), an area that projects directly to OT neurons (49) and indirectly to VP neurons via projections to noradrenergic cell groups (54), and as well as magnocellular neurons (20, 62).
Physiological challenges, including hyperosmolarity and hypotension, induce the same NK3R internalization and systemic hormone release as demonstrated with senktide administration (27, 31). NK3R are required and must be activated for these two challenges to stimulate the release of OT and VP. This conclusion is based on findings that blockade of central NK3R using a specific NK3R antagonist, SB-222200, reduces the release of VP by 60% in response to a hypertonic saline injection. More striking was the observation that pretreatment with SB-222200 abolished the systemic release of OT in response to hypertonic saline and the release of both OT and VP in response to hypotension (28). In rats, plasma levels of the hormones following pretreatment with SB-222200 remained similar to baseline levels (27, 28). The magnitude of this effect suggests that the NK3R has an integral role in a central pathway that stimulates OT and VP release, at least in response to these two physiological challenges. The question remains whether NK3R has an integral role in other central pathways that stimulate OT and VP release. If this hypothesis is true, then NK3R could be a common path of activation to stimulate the release of OT and VP from the magnocellular neurons.
Cholecystokinin (CCK) is an endogenous satiety signal released from the intestine to decrease food intake and stimulate the release of OT (26, 39, 46). The CCK-stimulated release of OT is involved in the inhibition of gastric motility and food intake (23). There are two different CCK receptors, CCK-A and CCK-B, and intraperitoneal and intravenous injections of agonists for both receptors stimulate the release of OT (39, 63, 67). Sulfated CCK-8 binds with high affinity to the CCK-A receptor, whereas nonsulfated CCK-8 binds with high affinity to CCK-B receptor but will also bind with a low affinity to CCK-A (47). CCK-A receptors are predominantly found in the gut but also within the area postrema (AP) and cNST (41, 47), whereas CCK-B receptors are predominantly found within the brain where they are distributed throughout the neuraxis (29, 42, 44, 58, 64). Intraperitoneal and intravenous injections of CCK receptor agonists may activate different central pathways to stimulate the release of OT; however, the neuronal pathways involved in intravenous injection of CCK receptor agonists have not been as thoroughly investigated as the neuronal pathways involved in intraperitoneal injections. It is thought that intraperitoneal injections of CCK receptor agonists stimulate CCK-A receptors on vagal afferents that terminate in the caudal medial NTS (cmNTS) (45, 56). Reports indicate that intraperitoneal injection of sulfated CCK-8 induces c-Fos expression in the AP/cmNST area and A2 cell group (18, 35, 45, 57) and that gastric vagotomy as well as lesions of the AP and underlying cmNST diminishes OT release in response to intraperitoneal injection of CCK-8 (14, 65). Intravenous injections of sulfated CCK-8 similarly induces c-Fos expression within the AP and cmNST (29). Fos expression in the AP may reflect a direct action of blood-borne sulfated CCK-8 on CCK-A receptors in this area (38, 47). From the hindbrain, ascending CCK information may be relayed directly to magnocellular OT neurons via the A2/C2 projections (11, 60). Functional evidence links A2 noradrenergic afferents, but not A1 projections, to the magnocellular neurons with sulfated CCK-8 and OT release (51, 63). Clearly, the noradrenergic cell groups convey ascending CCK information to the magnocellular neurons; however, it is important to note that other neurotransmitters, including tachykinins, are colocalized with norepinephrine (6, 22). Furthermore, norepinephrine interacts with glutamate in the regulation of magnocellular neuron function (8, 9), but at least OT release in response to sulfated CCK-8 does not appear to involve glutamatergic transmission (52). On the other hand, the role of CCK-B receptors in the control of neuroendocrine function is less clear. In vivo studies have tested the effects of sulfated CCK-8 but not nonsulfated CCK-8. In vitro, nonsulfated CCK-8 stimulates the release of both OT and VP from the neural lobe (7).
The objective of this study was to determine whether NK3R signaling has a stimulatory role in the release of neurohormones in response to CCK. As noted, NK3R plays a stimulatory role in both OT and VP release under several physiological conditions. Furthermore, NK3R and NKB fibers are distributed along the pathway that is activated in response to CCK-8 treatment (13, 35, 37, 49, 57). Thus NKB and NK3R may promote the release of OT following CCK injection. To evaluate this, rats were pretreated with a selective antagonist for the NK3R before an intraperitoneal or intravenous injection of one of two CCK receptor agonists, nonsulfated CCK-8 and sulfated CCK-8, which preferentially bind the CCK-B and CCK-A receptors. Blood samples were taken and assayed for OT and VP. Results from this study demonstrate that NK3R signaling has a critical stimulatory role in the release of OT in response to CCK-A receptor agonist and both OT and VP in response to CCK-B receptor agonist injection.
All procedures were approved by the University of Wyoming Animal Care and Use Committee. Rats (male, 300–350 g, n = 82; Charles River Laboratory) 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.
Rats (n = 80) were anesthetized with a mixture of ketamine and acepromazine (0.07 mg/kg ip) and mounted in a stereotaxic device. To position a cannula into the lateral ventricle, a hole was drilled in the skull 0.9 mm posterior to bregma and 1.8 mm lateral to the midline, and the cannula (26 g; Plastics One, Roanoke, VA) was then 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 re-anesthetized, 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 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 filled with heparinized saline and heat sealed until testing. Rats were returned to their home cages with ad libitum food and water for 24–48 h.
Rats 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). Rats were left undisturbed in a quiet room for 15–20 min before testing. The infusion pump was turned on to deliver the intraventricular injection of 5 μl (1 μl/min for 5 min) of 0.15 M NaCl (n = 12), 250 pmol (n = 12), or 500 pmol (n = 12) NK3R-specific antagonist SB-222200 (courtesy of GlaxoSmithKlein). The pharmacokinetic profile shows that SB-222200 is a selective NK3R antagonist (59), and pharmacological results show that it blocks NK3R agonist-induced effects both in vitro (33, 59) and in vivo (27, 59) without affecting other tachykinin actions (31). Immediately following the end of the intraventricular infusion, an intraperitoneal injection of nonsulfated CCK-8 (Sigma-Aldrich, 50 μg/kg, n = 12), sulfated CCK-8 (Phoenix Pharmaceuticals, 50 μg/kg, n = 12), or equal amount of 0.15 M NaCl (n = 12) was administered. One-third of the rats from each pretreatment group was randomly assigned to one of the three intraperitoneal groups and used only once. Blood samples (2 ml each) were taken before the start of the intraventricular infusion and 15 min after the intraperitoneal injection. With each blood sample, an equal volume (2 ml) of whole blood was simultaneously infused. Donor blood was collected from a cardiac draw 1–2 h prior from an ether-anesthetized rat and kept at 37.6°C until used (2, 3, 16). Blood samples were spun in a refrigerated centrifuge (2,000 revolutions/min for 20 min), and plasma was aspirated into a clean Eppendorf tube and stored at −80°C until extraction.
Rats were removed from their home cages and transferred to a clean, transparent cage in a quiet room. Femoral artery and venous catheters were attached to heparinized saline-filled tubes attached to a length of PE-50 tubing, and the intraventricular injector was attached to a length of PE-50 tubing connected to a microliter syringe positioned on an infusion pump (as stated above). The infusion pump was turned on to deliver an intraventricular injection of 5 μl (1 μl/min) of 0.15 M NaCl (n = 12), 250 pmol (n = 12), or 500 pmol (n = 12) of SB-222200. Immediately following the intraventricular infusion, rats were administered a bolus intravenous injection of non-sulfated CCK-8 (Sigma-Aldrich, 50 μg/kg, n = 12), sulfated CCK-8 (Phoenix Pharmaceuticals, 50 μg/kg, n = 12), or equal amounts of 0.15 M NaCl (n = 12). One-third of the rats from each pretreatment group was randomly assigned to one of the three intravenous groups and used only once. Blood samples with replacement were taken as previously described.
VP and OT assay.
Hormones were extracted from plasma samples using C-18 column extraction method. Briefly, a 1% trifluoracetic acid (TFA) solution was used to acidify 1 ml of plasma for each blood 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% dH20, 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 OT and VP ELISA kits (Assay Designs, Ann Arbor, MI).
Sensitivity of the OT ELISA kit was 11.7 pg/ml, whereas the VP ELISA kit was 3.39 pg/ml (performance characteristics for Assay Design kits). Specificity of the ELISA kits was determined by applying OT standards to VP kits and vice versa. Neither the OT nor the VP kit recognized the standards from the other kit. Intra-assay and interassay precision were determined with calculated variants, and in both assays the values were <6%. Extraction efficiency was ∼90% for both OT and VP extractions.
Plasma Data Analysis
Plasma OT and VP levels were analyzed using SPSS 13.0. Plasma levels were analyzed using separate, three-factor ANOVA with repeated measures [intraventricular treatment × intravenous treatment × time (repeated measure)]. Significant main effects and interactions were further analyzed with one-way ANOVA, 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.
Pituitary Extraction and Western Blot Analysis
Two naive rats were deeply anesthetized with ketamine/acepromazine mixture, and once deeply anesthetized, they were decapitated. Their brains were immediately removed from the skull, and the pituitary glands were isolated and homogenized using 1 ml of RIPA buffer (Pierce Pharmaceuticals, Rockford, IL) with Halt protease inhibitor (Pierce Pharmaceuticals). Cerebellum and brain stem were removed, and the forebrain and midbrain were homogenized in 5 ml of RIPA buffer with Halt protease inhibitor. Forebrain-midbrain tissue served as a positive control since CCK-B receptors are found throughout the forebrain and midbrain (24, 64). Homogenized tissue was spun at 12,000 g for 15 min, and supernatant was kept. Protein concentrations were determined using Coomassie protein assay reagent (Pierce Pharmaceuticals). For each sample, 40 μg of protein were run in each lane. Proteins were denatured for 15 min at 99°C in a solution of Laemmli's buffer (95 μl; Bio-Rad, Hercules CA) and 2-mercaptoethanol (5 μl; Bio-Rad). Pre-prepared gels (Bio-Rad Ready Gels, 4–15% Tris·HCl) were placed in the electrophoresis apparatus with one lane of TriChromeRange Marker (Pierce Pharmaceuticals), one lane of forebrain-midbrain homogenate, and one lane of isolated pituitary preparation. Gels were run with a Bio-Rad Power Pac (Bio-Rad) for 35 min at 200 V. Proteins were transferred to membranes for 55 min at 100 V.
Membranes were placed in 5% dry-milk TBS-Tween blocking buffer for 1 h. Membranes were washed in TBS-Tween buffer (4 × 5 min) and CCK-B receptor antibody (Santa Cruz Biotechnology, raised in goat, 1 μg/ml) in the dry-milk buffer for 1 h at room temperature. Membranes were washed in the TBS-Tween buffer (4 × 5 min) and were incubated in the secondary antibody in the dry-milk buffer for 1 h (Santa Cruz, donkey anti-goat-HRP, 0.6 μg/ml). Membranes were incubated in SuperSignal West Pico Chemiluminescent Substrate (Pierce Pharmaceuticals) for 5 min and apposed to Kodak film. Kodak film was scanned to a digital image file using HP ScanJet 8270. Pixel densities were determined using UN-SCAN-IT gel 6.1 (Silk Scientific). Background levels were automatically determined by the software using upper- and lower-edge interpolation. Semiquantification with densitometry of the CCK-B bands was measured between forebrain-midbrain homogenate and pituitary samples. Also, densitometry measured the CCK-B antibody bands and the preadsorbed CCK-B antibody bands.
To determine whether VP release from nonsulfated CCK-8 was due to a drop in blood pressure, rats (n = 8) fitted with arterial and venous catheters were allowed to acclimate in a quiet room. 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-filled syringe. Mean arterial blood pressure (MAP) was continually measured 5 min before nonsulfated CCK-8 intraperitoneal or intravenous injection until 20 min after the CCK-8 injection. No blood samples were taken during this time, and blood pressure was measured without interruption. Blood pressure was analyzed using repeated-measures and one-way ANOVA analyses using Bonferroni table for critical values in multiple comparisons. MAP is presented as means ± SE.
Plasma OT levels.
Plasma OT levels before intraventricular infusion (baseline) were not significantly different between treatment groups [F(5,18) = 2.1; P > 0.10; Fig. 1]. Intraventricular injection of 0.15 M NaCl had no effect on plasma OT levels compared with baseline hormone levels (P = 1.0). However, intraperitoneal injection of nonsulfated CCK-8 significantly increases plasma OT levels from 12.9 ± 0.9 pg/ml at time 0 to 67.5 ± 7.3 pg/ml at time 15 (P < 0.001). The stimulated release of OT was blocked by 95% (P < 0.001) with pretreatment of both concentrations of the NK3R antagonist, and the plasma OT levels were not significantly different from baseline levels (P > 0.6).
A similar pattern was observed with an intraperitoneal injection of sulfated CCK-8. Baseline plasma OT levels were not different with any of the treatment groups [F(5,18) = 3.0; P > 0.6; Fig. 1], nor did the intraventricular injection of the antagonist alter the plasma OT levels from baseline levels (P = 1.0). However, intraperitoneal injection of sulfated CCK-8 significantly increased plasma OT levels from 12.9 ± 1.0 pg/ml at time 0 to 56.7 ± 2.4 pg/ml at time 15 (P < 0.001). Intraventricular pretreatment of 500 pmol SB-222200 blocked over 96% (P < 0.001) of the sulfated CCK-8-stimulated release of OT, and plasma levels were not significantly different from baseline levels (P > 0.6). Therefore, both nonsulfated and sulfated CCK-8 stimulates the release of OT with an intraperitoneal injection, and this release is blocked by pretreatment with SB-222200.
Plasma VP levels.
In contrast to plasma OT levels, intraperitoneal injections of nonsulfated CCK-8 did not alter plasma VP levels compared with baseline levels (P > 0.7; Fig. 1). In fact, no significant difference in plasma VP levels was found between the different treatment groups at time 15 (P > 0.4). Likewise, plasma VP levels following intraperitoneal injections of sulfated CCK-8 were not significantly different from baseline levels (P = 1.0). Therefore, intraperitoneal injections of CCK-8 have no effect on plasma VP levels.
Plasma OT levels.
Baseline plasma OT levels were not significantly different in any of the treatment groups [F(5,18) = 2.4; P > 0.3; Fig. 2]. Intraventricular treatment had no effect on baseline plasma OT levels following intravenous injection of 0.15 M NaCl (P = 1.0). Intravenous injection of nonsulfated CCK-8 significantly increased plasma OT levels from 12.6 ± 1.3 pg/ml at time 0 to 52.9 ± 8.1 pg/ml at time 15 (P < 0.001). Pretreatment of both doses of the NK3R antagonist blocked over 92% of the stimulated OT release, and plasma OT levels were not significantly different from baseline plasma OT levels (P = 1.0).
Baseline plasma OT levels were not different in any of the treatment groups [F(5,18) = 1.3; P > 0.28; Fig. 2]. Intraventricular injection of the antagonist did not change plasma OT levels from baseline (P = 1.0). However, intravenous injection of sulfated CCK-8 significantly increased plasma OT levels from 12.9 ± 0.9 pg/ml at time 0 to 35.8 ± 1.8 pg/ml at time 15 (P < 0.001). Pretreatment of SB-222200 at both concentrations blocked over 90% of the sulfated CCK-8-stimulated OT release as plasma OT levels remained close to baseline levels (P > 0.9).
Plasma VP levels.
Baseline plasma VP levels were not significantly different between the groups [F(5,18) = 2.7; P > 0.14; Fig. 2]. Intravenous injection of nonsulfated CCK-8 significantly increased plasma VP levels from 2.7 ± 0.3 pg/ml at time 0 (range: 2.0–3.6 pg/ml) to 16.4 ± 1.2 pg/ml at time 15 (P < 0.001). This novel finding was consistent in all four rats 15 min after the CCK-8 injection (individual data: 17.1, 19.0, 13.2, and 16.6 pg/ml at time 15). Pretreatment with both 250 and 500 pmol of SB-222200 significantly blocked the release of VP following intravenous injection of nonsulfated CCK-8 by over 91% (P < 0.001). However, intravenous injection of sulfated CCK-8 did not alter VP levels compared with baseline hormone levels (P > 0.17; Fig. 2).
Western Blot Analysis
An equal amount of total protein (40 μg) from forebrain-midbrain sample and pituitary sample were loaded in the Western blot. CCK-B receptor protein was detected in both samples at a weight of ∼50 kDa (Fig. 3). Densitometry revealed that roughly equal amounts of CCK-B receptor protein were detected in the 40-μg protein samples from the forebrain-midbrain and pituitary; pituitary CCK-B receptor protein band was 98% of the density that was the forebrain-midbrain CCK-B protein band. Preadsorbing the CCK-8 antibody virtually eliminated the detection of CCK-8 protein in both tissue samples.
Intravenous injection of nonsulfated CCK-8 did not alter MAP from baseline levels (P > 0.2; Fig. 4). In fact, the MAP from rats treated with intraperitoneal injection and intravenous injection were not statistically different (P > 0.3).
In this experiment, we find that intraperitoneal and intravenous injections of both nonsulfated and sulfated CCK-8 stimulated an OT release (39, 63, 65, 66). A novel finding in this experiment is that intraventricular injection of SB-222200 blocked over 95% of OT release in response to intraperitoneal injections of nonsulfated CCK-8 and sulfated CCK-8. Furthermore, pretreatment of SB-222200 blocked over 90% of the OT release in response to intravenous injection of nonsulfated and sulfated CCK-8. Intraventricular injection of SB-222200 did not alter baseline OT levels. Therefore, NK3R signaling is important for the CCK-stimulated OT release. Earlier experiments demonstrate that pretreatment with 500 pmol SB-222200 before an osmotic or hypotensive challenge blocks the release of OT by 95% (28). Collectively, these experiments indicate that NK3R signaling plays a critical role in the release of OT under multiple physiological conditions.
A number of studies report that administration of sulfated CCK-8 by intravenous (29, 39, 63) or intraperitoneal injection (14, 67) elicits the release of OT but not VP in rat (63). The pattern of c-Fos expression following intraperitoneal injection of sulfated CCK-8 mimics the hormone profile in that c-Fos protein is induced in OT neurons and not VP immunoreactive neurons (67). We similarly show that intraperitoneal and intravenous injection of sulfated CCK-8 stimulates selectively OT release.
Nonsulfated CCK-8, administered via intraperitoneal or intravenous injection, stimulated the systemic release of OT. Interestingly, the route of administration influenced whether or not nonsulfated CCK stimulated VP release. Intraperitoneal administration of nonsulfated CCK-8 had no effect on VP release, yet intravenous injection of nonsulfated CCK-8 stimulated a significant increase in plasma VP levels. MAP was measured to determine whether the intraperitoneal and intravenous routes of nonsulfated CCK injection differentially affected MAP, which could account for the release of VP. Neither route of nonsulfated CCK administration affected MAP (see also Ref. 68); thus VP release not was due to baroreceptor activation.
We are not aware of other in vivo experiments that evaluated the effects of intravenous injection of nonsulfated CCK-8, a CCK-B receptor agonist, on VP release in rats. However, in humans, intravenous injections of CCK-B receptor agonists stimulate the release of VP (1). Furthermore, in vitro experiments demonstrate that perfusion of rat pituitary with nonsulfated CCK-8 and sulfated CCK-8 stimulates the release of both OT and VP (7). Autoradiography results show a high concentration of CCK-8 binding sites in rat neural lobe (7), and our present Western analyses demonstrate that the pituitary CCK-8 binding sites include CCK-B receptors. The intravenous injection of nonsulfated CCK-8 may directly stimulate pituitary CCK-B receptors to release VP. Our observation that intraperitoneal injections of nonsulfated CCK-8 were ineffective in stimulating VP release may imply that nonsulfated CCK-8 administered in this manner did not readily access pituitary receptors or not at a significant concentration to stimulate VP release. The presence of CCK-B receptors and their activation by an intravenous CCK-8 injection may possibly shed light on how intravenous, but not intraperitoneal, CCK-8 injections stimulate VP release. This putative target of intravenous injections of CCK-8 does not lend itself to explaining how the intraventricular injection of NK3R blocks the release of VP. Alternatively, as just suggested, CCK-8 delivered by an intravenous route may better access brain receptors. VP as well as OT magnocellular neurons express the CCK-B receptor (40), and intravenous injections of the nonsulfated CCK-8 may access CCK-B receptors that are otherwise not accessible.
There has been considerable focus on the role of two excitatory neurotransmitters, norepinephrine and glutamate, in magnocellular neuron function. Both norepinephrine and glutamate stimulate magnocellular neurons to release OT and VP (17, 34, 52). Conversely, injections of adrenergic receptor antagonists may decrease the systemic release of OT and VP (4, 10, 17, 19, 55). Specifically, CCK injection increases norepinephrine release in the PVN as well as the firing rate of OT neurons (63). Pretreatment of phentolamine blocked the effects of sulfated CCK-8 on firing rate (63), suggesting an adrenergic receptor role in the CCK-stimulated release of OT. In contrast, NMDA receptor antagonists decrease the release of OT and VP following hypertonic saline, but not following CCK injection (52). On the other hand, we have previously reported that central blockade of NK3R blocks the release of OT and VP in response to both hyperosmolarity and hypotension (28). In the present study, we show that blockade of NK3R blocks the release of both OT and VP in response to nonsulfated and sulfated CCK. Collectively, these studies highlight that NK3R signaling is fundamental in the systemic release of both OT and VP under a variety of conditions and has a broad role in magnocellular neuron function.
Afferent signals regarding hyperosmolarity, hypotension, and peripheral CCK injections converge on magnocellular neurons by several pathways. Osmotic information is conveyed to the magnocellular neurons via axons arising in the anterior ventral third ventricle (AV3V) region, including the organum vasculosum of the lamina terminalis and the median preoptic area (50, 69). On the other hand, blood pressure and volume information is relayed to the magnocellular neurons via the A1 and A2 cell groups in the brain stem (12, 29). The stimulatory effect of sulfated CCK-8 on OT release is similarly conveyed to magnocellular neurons by afferents arising from A1 and caudal NST neurons (11, 63). NK3R are distributed in each of these pathways; specifically, NK3R are expressed in the nuclei comprising the AV3V region (61) and the cNST (13). Acting at these sites, blockade of NK3R function by intraventricular injections of SB-222200 could prevent the release of OT and VP in response to these different challenges. In addition, NK3R are heavily expressed by the final path, magnocellular neurosecretory neurons (20, 43, 61). Previous studies show that NK3R expressed by magnocellular neurons in the PVN and SON are activated, based on receptor internalization, in response to hyperosmolarity and hypotension (27, 31). Thus central injections of an NK3R antagonist may block tachykinin excitatory input to magnocellular neurons and thereby prevent the release of OT and VP in response to hyperosmolarity, hypotension (28), and CCK.
Perspectives and Significance
A novel finding in this study is that CCK-B agonists stimulate a systemic VP release, which may relate to one of two physiological processes. First, CCK is a putative satiety signal released rapidly following a meal (15, 26), and the subsequent VP release and resulting fluid retention could serve to correct meal-induced alterations in osmolality. Second, intravenous injection of nonsulfated CCK-8 may be sufficient to activate CCK-B receptors in emetic pathways that stimulate VP release (36, 48, 66). Nevertheless, the NK3R antagonist blocked OT and VP release in response to peripheral administration of CCK-A and CCK-B receptor agonists. The current findings, taken with our previous report that SB-222200 blocks both OT and VP release in response to hyperosmolarity and hypotension (28), identify a fundamental and previously unsuspected role of NK3R signaling in OT and VP release under a variety of conditions. The extent of the involvement of NK3R signaling in OT and VP release will need to be expanded by testing the effects of NK3R antagonists on neurohormone release in response to additional challenges, such as suckling (32) and stress (25, 30). Results show that multiple stimuli activate NK3R, and this activation is not only required for the systemic OT and VP release but may directly affect genomics of the cells. Several reports show that the NK3R translocates to the nuclei of magnocellular neurons, presumably through interactions with importins, following activation (Refs. 27, 31; Jensen D, Zhang Z, and Flynn FW, unpublished observations). Current studies are aimed at identifying the genomic actions of nuclear NK3R and the resulting long-term changes in the responsivity of magnocellular neurons to physiological challenges.
This publication was made possible by National Institutes of Health Grants DK-50586, NS-57823, and P20 RR-15640.
We thank GlaxoSmithKline for their generous gift of SB-222200. Also, we thank Shawna McBride, Dane Jensen, and Donald Pratt for technical assistance.
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.
- Copyright © 2008 the American Physiological Society