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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana
Received for publication
August 19, 2005
Accepted
January 9, 2006.
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Abstract |
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; Reinscheid et al., 1995; Cox et al., 2000). Although structurally similar to the classic opioid family of peptides and receptors (e.g., the endorphins), the N/OFQ-NOP peptide system has been established as a distinct neurochemical entity. Anatomical distribution of N/OFQ and NOP receptor immunoreactivity and mRNA expression in rodent and human brain regions, including those involved in the regulation of cardiovascular function and fluid/electrolyte balance is well characterized (Isgor et al., 2003; Witta et al., 2004).
After i.c.v. administration in conscious rats, N/OFQ produces a significant decrease in mean arterial pressure, heart rate, and renal sympathetic nerve activity (RSNA) (Kapusta et al., 1997; Kapusta and Kenigs, 1999; Shirasaka et al., 1999). In addition, i.c.v. N/OFQ evokes a dose-dependent diuretic and antinatriuretic response (Kapusta et al., 1997; Kapusta and Kenigs, 1999) and a decrease in the circulating plasma levels of [Arg8]vasopressin (AVP) (Kakiya et al., 2000). Despite these findings, the specific brain sites and mechanisms by which N/OFQ affects systemic cardiovascular function and the renal handling of water and electrolytes in conscious rats are not completely known.
The hypothalamic paraventricular nucleus (PVN) is a brain site involved in the regulation of neuroendocrine function and activity of the autonomic nervous systems. The magnocellular neurons of the PVN synthesize the neurohypophysial hormones AVP and oxytocin and project to the posterior pituitary. The parvocellular PVN neurons project to the anterior pituitary to modulate activity of the hypothalamic-pituitary-adrenal axis and to other autonomic centers involved in the central nervous system regulation of sympathetic activity (Kenney et al., 2003). Functional studies also implicate the PVN in the regulation of cardiovascular function and the control of central sympathetic outflow (Coote, 2005). NOP receptors and N/OFQ-positive fibers have been identified in the mammalian PVN (Isgor et al., 2003), and N/OFQ has been shown to modulate the activity of magnocellular and parvocellular PVN neurons in vitro through activation of an inwardly rectifying K+ channel (Shirasaka et al., 2001). However, whether N/OFQ acts within the PVN to contribute to the central actions of N/OFQ on cardiovascular and renal function in vivo has not been examined previously.
Therefore, the present studies were performed to investigate the cardiovascular and renal responses produced by microinjection of the opioid-like peptide, N/OFQ, into the PVN of conscious rats. Studies were then performed to examine the role of the renal nerves and vasopressin in mediating the cardiovascular and renal responses to N/OFQ administration into this brain site. Participation of the renal sympathetic nerves in mediating N/OFQ-induced changes in renal excretory function was examined by directly measuring RSNA and by repeating the experimental protocol in rats that had the influence of the renal nerves on kidney function removed by chronic bilateral renal denervation. Separate studies were also performed in rats continuously infused i.v. with AVP to establish whether N/OFQ might act in the PVN to affect renal excretory function via a pathway that involves alterations in the circulating levels of this hormone. Finally, studies were performed in which rats were microinjected into the PVN with the selective NOP receptor antagonist UFP-101 (Calo et al., 2005) alone or as a pretreatment before N/OFQ injection. These studies examined whether native N/OFQ-NOP receptor systems in the PVN might elicit a tonic influence on cardiovascular and/or renal function and tested the NOP receptor selectivity of the action of N/OFQ in this brain region, respectively.
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Materials and Methods |
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Surgery. Aseptic technique was used for all operations. For microinjection of drugs (N/OFQ alone or after UFP-101 pretreatment), a chronic cannula was placed in the right PVN 5 to 8 days before the experimental day. For studies involving bilateral UFP-101 microinjection, a chronic cannula was also implanted into the right PVN. In brief, rats were anesthetized with an i.m. injection of ketamine and xylazine mixture (50 mg/kg ketamine plus 5 mg/kg xylazine, 1 ml/kg) and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). A 2-cm incision was made to expose the scalp, and connective tissue was removed to expose the cranial sutures. A 26-gauge stainless steel cannula was directed at the PVN at a 10° angle from the vertical plane through a burr hole located stereotaxically 1.6 to 1.7 mm posterior to the bregma, 1.8 to 1.9 mm laterally to the midline, and 7.8 mm ventral from the skull surface. Inner stylets were inserted into the guide cannulae to prevent obstruction. The guide cannulae were secured to the skull with stainless steel machine screws and dental acrylic. To prevent postsurgery infection, all rats received ampicillin (50 mg/kg s.c.). The animals were then placed in individual plastic cages and allowed at least 5 days to recover from the stereotaxic surgery.
After recovery, rats were anesthetized with methohexital sodium (Brevital; 20 mg/kg i.p., supplemented with 10 mg/kg as needed; Lilly, Indianapolis, IN), and polyethylene catheters (PE-50; Becton Dickinson, Sparks, MD) were implanted into the left femoral artery and vein for recording of arterial pressure and infusion of isotonic saline or drugs, respectively. Through a suprapubic incision, a flanged polyethylene cannula (PE-240; Becton Dickinson) was inserted into the urinary bladder for collection of urine. The bladder catheter was then exteriorized and secured by suturing to adjacent muscle, tissue, and skin.
Rats (still anesthetized with methohexital sodium) were then implanted with a recording electrode on a renal nerve bundle for direct measurement of multifiber RSNA using techniques described previously (Kapusta and Kenigs, 1999). In brief, a left renal nerve bundle was dissected carefully via a retroperitoneal approach and freed from the surrounding tissue under a dissecting microscope (25x). The nerve was placed on a bipolar electrode made of Teflon-coated platinum wire (Cooner Wire, Chatsworth, CA). Spike potentials were amplified (10,000–50,000 times) and filtered (low, 30 Hz; high, 3000 Hz) with a Grass P511 bandpass amplifier (Grass Instruments, Quincy, MA). The amplified and filtered signal was channeled to a Grass model 7DA polygraph for visual evaluation, to an audio amplifier-loudspeaker (Grass model AM 8 Audio Monitor) for auditory evaluation, and to a rectifying voltage integrator (Grass model 7P10) and was continuously recorded on the Grass polygraph and a commercially available data acquisition system (model MP100 and AcqKnowledge version 3.7.2 software; BIOPAC Systems, Inc., Santa Barbara, CA). The quality of the renal sympathetic nerve signal was assessed by its pulse synchronous rhythm. When an optimal RSNA signal was observed, the recording electrode was fixed to the renal nerve branch with and covered with a silicone-based impression material (Coltene President, Alstatten, Switzerland). The electrode cable was then secured in position by suturing it to the abdominal trunk muscles. Finally, the electrode cable was exteriorized, and the flank incision was closed in layers.
In separate studies, the role of intact renal nerves in mediating the cardiovascular and renal excretory responses produced by the injection of N/OFQ into the PVN was examined by repeating the above protocol in rats (n = 6) having undergone chronic bilateral renal denervation 10 to 13 days before experimentation. Renal denervation was performed via bilateral flank incisions and stripping the renal arteries and veins of adventitia, cutting the renal nerve bundles, and coating the vessels with a solution of 10% phenol in absolute ethanol as previously described (DiBona and Sawin, 1983). This renal denervation procedure prevents the renal vasoconstrictor response to suprarenal lumbar sympathetic nerve stimulation, prevents the antinatriuretic response to environmental stress, reduces renal catecholamine histofluorescence to undetectable levels, and reduces the renal tissue noradrenaline concentration to <5% of control for up to 15 days postdenervation (DiBona and Sawin, 1983). Because our laboratory has previously and repeatedly verified that this renal denervation procedure completely removes the influence of the renal nerves on kidney function (Kapusta and Kenigs, 1999), renal denervation was not verified in these studies. Six to 7 days later, the same rats were anesthetized with ketamine-xylazine and implanted with a chronic PVN cannula as described previously.
After surgical preparation and recovery from anesthesia, the rat was placed in a rat holder (a chamber of stainless steel rods connected by Plexiglas ends; the metal rods formed an inverted U shape and a flat base in which the rat would sit) to minimize movement and damage to the renal nerve electrode preparation and to permit steady-state urine collection. An infusion (50 µl/min i.v.) of isotonic saline was then started and continued for the duration of the experiment. After stabilization of renal excretory parameters (4–6 h after recovery), the arterial catheter was flushed and attached to a pressure transducer (model P23XL; Viggo Spectramed, Oxnard, CA), and the urinary bladder catheter was led to a collection vial. Heart rate was derived from the pulse pressure by a tachograph (Grass model 7 P4H), and mean arterial pressure, heart rate, and RSNA were recorded on a Grass model 7 polygraph and the BIOPAC data acquisition system (MP100 and AcqKnowledge 3.7.2 software).
Experimental Protocols. Studies were performed to examine cardiovascular and renal excretory responses to microinjection of N/OFQ into the PVN of conscious animals. After stabilization of cardiovascular, renal excretory, and RSNA parameters, urine was collected during a 20-min control period. After that, a 33-gauge injection needle connected to 10-µl microsyringe was inserted into the guide cannula so that the tip of the injection needle extended beyond the guide cannula by 0.5 to 0.75 mm and reached the PVN. Vehicle or N/OFQ (10 or 100 pmol, n = 7–8 per group) was injected into the PVN in a volume of 60 nl over 10 s. Artificial cerebrospinal fluid (aCSF) of the following composition was used as vehicle: 140 mM NaCl, 3.35 mM KCl, 1.15 mM MgCl2, 1.26 mM CaCl2, 1.2 mM Na2HPO, and 0.3 mM NaH2PO4 pH 7.4. A 30-s period was allowed for diffusion. Then, the injection cannula was withdrawn. Immediately after PVN administration, urine was collected during eight consecutive 10-min experimental urine samples. The doses of N/OFQ used in the present study were derived from our previous dose-response study performed in chloralose-anesthetized rats (Krowicki et al., 1997). In this study, microinjection of N/OFQ into the dorsal vagal complex produced consistent reductions in heart rate and arterial blood pressure.
Because our initial experiments demonstrated that free-water clearance increased in response to N/OFQ in the PVN (Fig. 3), we investigated whether the renal excretory responses produced by the peptide were caused by decreases in the systemic circulating levels of AVP. For these experiments, the microinjection study described above was repeated with the exception that the cardiovascular and renal responses produced by the injection of N/OFQ into the PVN were examined in rats that were continuously infused with isotonic saline containing AVP [85 pg (60 fmol)/kg/min i.v.]. Because AVP has a relatively short half-life of approximately 2 min in rats, steady-state plasma concentration of AVP should be achieved within a 15-min period. Indeed, antidiuretic and natriuretic responses to continuous i.v. infusion of AVP (63 fmol/kg/min) have been shown to require 20 min to reach a plateau and persist for the duration of AVP infusion (Smith and Balment, 1993). Therefore, in our previous (Kapusta and Dzialowski, 1995) and present studies, AVP was allowed to infuse for 30 min and then consecutive urine samples (10 min each) were collected before (control) and after the microinjection of N/OFQ (100 pmol) into the PVN.
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) was microinjected into the PVN 10 min before vehicle or N/OFQ (100 pmol). Cardiovascular and renal functions were measured during consecutive 10-min periods before (control) and after drug injection (experimental) for 80 min.
At the end of all microinjection experiments, the site of drug injection in the brain was examined functionally and histologically. The microinjection of L-glutamate (15 nmol) was used to confirm localization of the tip of an injection needle in the PVN. Typical responses to L-glutamate in the PVN are increases in heart rate and mean arterial pressure (Martin and Haywood, 1992). In addition, at the end of the experiments, rats received injections of 60 nl of pontamine sky blue dye (1%) to mark the injection site(s).
Histology. The animals were euthanized with an overdose of methohexital sodium and perfused transcardially with saline and 4% paraformaldehyde. Their brains were removed, postfixed in the same solution, sectioned at 50 µm, and counterstained with neutral red dye for histological confirmation of injection sites. Injections were considered acceptable if the cannula tip was shown to be within a distance of 0.5 mm of the PVN boundaries (Lessard and Bachelard, 2002). Several sites around the PVN served as anatomical controls for the N/OFQ injections.
Data Analysis. Urine volume was determined gravimetrically. Urine sodium and potassium concentration was measured by flame photometry (model 943; Instrumentation Laboratories, Lexington, MA) and expressed as urinary sodium (UNaV) and urinary potassium (UKV) excretion. Urine osmolality was measured by vapor pressure osmometer (model 5500; WESCOR, Logan, UT). Free water clearance (CH2O) was calculated as a difference between the rate of urine volume (microliters) per minute and the osmolar clearance. Cardiovascular function and RSNA were measured through a computer-driven data acquisition software (AcqKnowledge 3.7.2). Integrated RSNA was expressed as microvolt-seconds per 1-s intervals. For each 10-min control and experimental period, the values for integrated RSNA were sampled over the entire collection period, and the numbers were averaged. The data for RSNA were expressed as the percentage of the baseline value obtained during the control period with this being expressed as 100% for each animal. After completion of each experiment, the level of postmortem background noise was measured, and the value was then subtracted from all control and experimental values of RSNA. Because of the limitations of comparing values for multifiber RSNA between animals, the data are expressed as percentage of control with the control values for each animal taken as 100%.
Drugs. N/OFQ (1–17) was obtained from Phoenix Pharmaceuticals (Mountain View, CA), and AVP was purchased from Bachem California, Inc. (Torrance, CA). UFP-101 was synthesized and graciously provided by Dr. Remo Guerrini (University of Ferrara, Ferrara, Italy). Stock solutions of peptides were prepared fresh and stored frozen.
Statistics. Results are expressed as means ± S.E.M. The magnitude of the changes in cardiovascular and renal parameters at different time points after injection of drugs in the PVN were compared with respective group control values by a one-way repeated-measures analysis of variance (ANOVA) with a subsequent Dunnett's test. Differences occurring between treatment groups (e.g., intact versus renal denervated; infusion with vehicle versus AVP) were assessed by a two-way repeated measures ANOVA with treatment being one fixed effect and time as another, with the interaction included. The time (in minutes) was the repeated factor, and post hoc analysis was performed using the Holm-Sidak or Bonferroni test. Data were verified for normality of distribution and equality of variances. If needed, data were normalized using a log10 transformation for the purpose of statistical analyses; however, results are reported in standard units. Statistical testing was carried out using Prism and SigmaStat programs. In each case, statistical significance was defined as p < 0.05.
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Figure 2 depicts the changes in free water clearance produced by the microinjection of N/OFQ (100 pmol) and aCSF into the PVN for the same rats depicted in Fig. 1. As shown, N/OFQ evoked a significant increase in free water clearance over time points 20 to 50 min with the peak response observed 40 min after injection (C, –52 ± 7 ml/min; 40 min, 49 ± 21 ml/min; p < 0.001). Microinjection of aCSF did not significantly alter free water clearance over the course of the study.
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). Several sites around the PVN served as anatomical controls for the N/OFQ injections. No significant responses were elicited from these microinjections (data not shown). Cardiovascular and Renal Effects of N/OFQ Microinjected into the PVN of Rats Infused with AVP. The cardiovascular and renal responses produced by the microinjection of N/OFQ into the PVN of conscious rats continuously infused i.v. with isotonic saline containing AVP (85 pg/kg/min) are shown in Fig. 4. For comparison, superimposed are data from Fig. 1 that illustrate the cardiovascular and renal responses to microinjection of N/OFQ into the PVN of rats infused with isotonic saline vehicle alone.
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Although not depicted, continuous infusion of AVP in rats during the first 40 min (stabilization period) evoked a decrease in urinary flow rate, heart rate, and RSNA accompanied with an increase in mean arterial pressure and no apparent change in UNaV. In separate animals, it was shown that all of these changes tended to reach plateau within the first 30 min and remained relatively unchanged until the AVP infusion was stopped at the end of the experiment (i.e., 80 min after aCSF injection). As illustrated in Fig. 4, the i.v. infusion of AVP completely prevented the diuretic response produced by the injection of N/OFQ into the PVN of conscious rats. Likewise, in contrast to rats infused with vehicle alone, the injection of N/OFQ into the PVN of AVP-infused animals did not significantly alter urinary sodium or potassium excretion. Although administration of N/OFQ into the PVN produced similar patterns of cardiovascular and RSNA responses between groups, N/OFQ did evoke a slight, but statistically significant, reduction in mean arterial blood pressure in the AVP-treated group.
Cardiovascular and Renal Effects of N/OFQ Microinjected into the PVN of Renal-Denervated Rats. Microinjection of N/OFQ (100 pmol) into the PVN of renal-denervated rats (n = 6) caused a transient increase in urine flow rate with a peak at 30 min after injection (C, 55 ± 6 µl/min; 30 min, 98 ± 4 µl/min; p < 0.05; Fig. 5). However, in comparison to the responses observed in rats with intact kidneys, the diuresis produced by this dose of N/OFQ into the PVN was significantly (p < 0.001) blunted. In renal-denervated rats, N/OFQ also decreased urinary sodium and potassium excretion with nadirs attained 30 min after injection (C, 8.7 ± 0.3 µEq/min, 30 min, 5.4 ± 0.8 µEq/min; p < 0.01 and C, 3.0 ± 0.1 µEq/min; 30 min, 1.8 ± 0.2 µEq/min; p < 0.05; Fig. 5), respectively.
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Cardiovascular and Renal Effects of UFP-101 Microinjected into the PVN of Conscious Rats. Figure 6 illustrates the cardiovascular and renal responses produced by the unilateral microinjection of aCSF or N/OFQ into the PVN of rats pretreated in the same brain site with the selective NOP receptor antagonist UFP-101. When microinjected into the PVN at a dose of 300 pmol with subsequent aCSF (n = 6), UFP-101 caused a significant increase in RSNA with a peak 30 min after injection (C, 100%; 30 min, 129 ± 12%; p < 0.05). In this same group of animals, UFP-101 did not alter mean arterial pressure, but heart rate was significantly elevated (time periods 40–60 min) compared with the respective group control value. In regard to renal excretory responses, microinjection of UFP-101 into the PVN reduced urine flow rate and urinary sodium excretion, although only the reduction in urine output reached statistical significance (C, 46 ± 6 µl/min; 30 min, 25 ± 4 µl/min; p < 0.05). As also shown in Fig. 6, pretreatment (10 min) with UFP-101 (300 pmol, left PVN) completely prevented the cardiovascular and renal excretory responses produced by the subsequent microinjection of N/OFQ (100 pmol) into the PVN (compare with N/OFQ responses in Fig. 1).
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As shown in Fig. 7, the bilateral microinjection of UFP-101 also produced significant increases in heart rate and RSNA and a reduction in urine flow rate, but the magnitudes of the sympathoexcitatory and antidiuretic responses were greater than those produced by unilateral UFP-101 injection (compare with responses depicted in Fig. 6).
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Discussion |
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), the present findings suggest that the PVN of the hypothalamus is a brain site where N/OFQ acts to produce a selective water diuresis (e.g., aquaresis) after central administration (e.g., i.c.v. injection) or endogenous release of the peptide.
In addition to altering renal excretory function, the microinjection of N/OFQ into the PVN of conscious rats reduced heart rate and RSNA but did not significantly change mean arterial pressure. Likewise, i.c.v. administration of N/OFQ evokes slight hypotension (10–15 mm Hg) and a pronounced bradycardia and renal sympathoinhibitory response. However, the reduction in RSNA was delayed (30 min) until mean arterial pressure recovered toward predrug baseline levels (Kapusta and Kenigs, 1999; Shirasaka et al., 1999). Thus, although the N/OFQ-induced activation of NOP receptors in the PVN causes a reduction in central sympathetic outflow to the kidneys (renal sympathoinhibition) and heart (bradycardia), it appears that mean arterial pressure is either not significantly affected by drug administration into this site or that other compensatory mechanisms (e.g., involving the baroreflex) contributed to maintain mean arterial blood pressure constant. It should be noted that, in the experiments of the present investigations, basal mean arterial pressure was observed to be slightly elevated in N/OFQ and vehicle-treated rats, with all groups having essentially the same starting mean arterial pressure. However, we strongly believe that the neurohumoral status of these animals was not altered to an extent that would have compromised cardiovascular and/or renal function from being studied, particularly because the hypotensive effect of drugs in animals with elevated blood pressure are typically greater (Irvine et al., 1997).
Studies were conducted to determine the roles of AVP and the renal sympathetic nerves in mediating the renal excretory responses evoked by administration of N/OFQ into the PVN of conscious rats. Although plasma levels of AVP were not measured, the results of the present study suggest that the N/OFQ-induced diuresis (UFP-101-sensitive) was secondary to suppression of circulating AVP levels. Support for this premise is demonstrated by the observation that a continuous low-dose infusion of AVP abolished the diuretic response to injection of N/OFQ in the PVN. This finding is in accord with previous reports demonstrating that N/OFQ acts in the PVN to inhibit firing of AVP neurons (Olszewski et al., 2000). Furthermore, although changes in renal function were not measured, other studies have shown that i.c.v administration of N/OFQ suppresses circulating plasma levels of AVP in dehydrated rats and blunts the increase in plasma AVP to either hyperosmolality or hypovolemia (Kakiya et al., 2000). These observations strongly imply that NOP receptor activation in the PVN by N/OFQ produces diuresis by a pathway (e.g., reduction in plasma AVP levels) in which the hydroosmotic effect of AVP in the collecting duct is reduced. However, the present data also suggests that, in addition to AVP, the renal sympathetic nerves contribute in mediating the N/OFQ-evoked diuresis. This possibility is first supported by the observation that the microinjection of N/OFQ into the PVN significantly reduced RSNA over the course of the diuresis. Moreover, compared with the pattern observed in intact rats, the diuretic (but not antinatriuretic or antikaliuretic) response produced by N/OFQ injection into the PVN of chronic bilateral renal denervated animals was markedly decreased in magnitude and time course. Together, these findings indicate that stimulation of NOP receptors in the PVN produces a selective water diuresis by suppressing AVP secretion/release and RSNA. In accord with these observations, the activation of PVN neurons (e.g., via bicuculline microinjection and disinhibition) has been shown to produce antidiuresis by pathways involving augmentation of AVP and renal sympathetic nerve pathways (Haselton and Vari, 1998).
As demonstrated by the findings discussed above and from previous investigations (Coote, 2005), the PVN can influence renal function via the renal nerves. However, in the present study, it is clear that the renal nerves did not contribute to the PVN-mediated affects of N/OFQ on urinary sodium excretion. This is supported by the fact that the microinjection of N/OFQ into the PVN decreased urinary sodium excretion instead of producing natriuresis, the latter of which would have been expected if the drug's effect on urinary sodium excretion had been due to renal sympathoinhibition (DiBona, 2001). In addition, chronic bilateral renal denervation did not alter the antinatriuresis produced by N/OFQ injection into the PVN. Therefore, although the mechanisms have yet to be determined, it appears that N/OFQ acts in the PVN via pathways, other than the renal nerves, to influence the renal handling of sodium and potassium. Although yet to be tested, there is the possibility that N/OFQ may act from the PVN to evoke antinatriuresis and antikaliuresis by decreasing circulating levels of oxytocin, a hypothalamic hormone that produces natriuresis and kaliuresis (Sjoquist et al., 1999). Oxytocin has been suggested to have an important role in renal sodium homeostasis under basal conditions (Conrad et al., 1993). Moreover, N/OFQ inhibits rat oxytocin and vasopressin supraoptic nucleus neurons in vitro (Doi et al., 1998).
UFP-101 is a peptide antagonist with nanomolar affinity and high selectivity for NOP receptors (Calo et al., 2005). In the present studies, we demonstrated that the pretreatment of conscious rats in the PVN with UFP-101 completely blocked the cardiovascular and renal excretory responses produced by the subsequent injection of N/OFQ into the same brain site. Therefore, these studies, which were performed with unilateral drug (UFP-101 and N/OFQ) administration, clearly demonstrate the selectivity of NOP receptors in mediating N/OFQ responses. Of additional interest, it was observed that the unilateral administration of UFP-101 alone produced an increase in heart rate and RSNA and a slight but statistically significant reduction in urine flow rate. These UFP-101-mediated responses are of significance because they suggest that endogenous N/OFQ may play a tonic role in the PVN in the regulation of these physiological parameters. To further examine this possibility, additional cardiovascular and renal function studies were performed in which UFP-101 was microinjected bilaterally into the PVN of conscious rats. The results of these experiments confirmed this premise and in fact revealed that bilateral injection of UFP-101 into the PVN produced tachycardia, renal sympathoexcitatory responses, and antidiuretic responses of even greater magnitude. Thus, considering the fact that the microinjection of the native ligand N/OFQ into the PVN produced bradycardia, renal sympathoinhibition, and diuresis, these findings with bilateral UFP-101 provide strong evidence that the N/OFQ-NOP receptor system in the rat PVN provides tonic inhibitory influences on central sympathetic outflow to the heart (e.g., to produce bradycardia) and on the secretion/release of AVP and RSNA, which together affect the renal handling of water (e.g., to produce diuresis). These observations are of interest because the existence of a population of neurons in the PVN that can inhibit RSNA has been demonstrated (Deering and Coote, 2000). Moreover, Badoer et al. (2002) provided evidence that renal sympathoinhibitory neurons in the PVN are tonically active.
In summary, the results of the present investigations demonstrate that microinjection of N/OFQ into the PVN of the hypothalamus of conscious rats produces significant reductions in systemic cardiovascular function and RSNA and a water diuresis. These N/OFQ-evoked responses were shown to be mediated by activation of NOP receptors because they were completely blocked by PVN pretreatment with the selective NOP receptor antagonist UFP-101. In other studies, microinjection of the antagonist UFP-101 alone produced tachycardia, renal sympathoexcitation, and antidiuresis. Together, these findings provide evidence that the PVN is a brain site where centrally administered and endogenously released N/OFQ mediate cardiovascular depressor and renal sympathoinhibitory and water diuretic responses in conscious rats. Moreover, UFP-101 blocks a tonically active inhibitory influence of endogenous N/OFQ on central sympathetic outflow and AVP pathways that arise from the PVN to affect heart rate and urine output.
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Footnotes |
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ABBREVIATIONS: N/OFQ, nociceptin/orphanin FQ; NOP, nociceptin/orphanin FQ peptide; RSNA, renal sympathetic nerve activity; AVP, [Arg8]vasopressin; PVN, hypothalamic paraventricular nucleus; UFP-101, [Nphe1,Arg14,Lys15]N/OFQ-NH2; aCSF, artificial cerebrospinal fluid; ANOVA, analysis of variance; C, control.
Address correspondence to: Dr. Zbigniew K. Krowicki, MediProfile, Inc., 1825 Broadway St., New Orleans, LA 70118. E-mail: zkrowi{at}hotmail.com
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References |
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Badoer E, Ng C-W, and De Matteo R (2002) Tonic sympathoinhibition arising from the hypothalamic PVN in the conscious rabbit. Brain Res 947: 17–24.[CrossRef][Medline]
Calo G, Guerrini R, Rizzi A, Salvadori S, Burmeister M, Kapusta DR, Lambert DG, and Regoli D (2005) UFP-101, a peptide antagonist selective for the nociceptin/orphanin FQ receptor. CNS Drug Rev 11: 97–112.[Medline]
Conrad KP, Gellai M, North WG, and Valtin H (1993) Influence of oxytocin on renal hemodynamics and sodium excretion. Ann NY Acad Sci 689: 346–362.[Medline]
Coote JH (2005) A role for the paraventricular nucleus of the hypothalamus in the autonomic control of heart and kidney. Exp Physiol 90: 169–173.
Cox BM, Chavkin C, Christie MJ, Civelli O, Evans C, Hamon MD, Hoellt V, Kieffer B, Kitchen I, McKnight AT, et al. (2000) Opioid receptors, in The IUPHAR Compendium of Receptor Characterization and Classification (Girdlestone D ed) pp 321–333, IUPHAR Media, Ltd., London.
Deering J and Coote JH (2000) Paraventricular neurones elicit a volume expansion-like change of activity in sympathetic nerves to the heart and kidney in the rabbit. Exp Physiol 85: 177–186.[Abstract]
DiBona GF (2001) Peripheral and central interactions between the renin-angiotensin system and the renal sympathetic nerves in control of renal function. Ann NY Acad Sci 940: 395–406.[Medline]
DiBona GF and Sawin LL (1983) Renal nerves in renal adaptation to dietary sodium restriction. Am J Physiol 245: F322–F328.[Medline]
Doi N, Dutia MB, and Russell JA (1998) Inhibition of rat oxytocin and vasopressin supraoptic nucleus neurons by nociceptin in vitro. Neuroscience 84: 913–921.[CrossRef][Medline]
Haselton JR and Vari RC (1998) Neuronal cell bodies in paraventricular nucleus affect renal hemodynamics and excretion via the renal nerves. Am J Physiol 275: R1334–R1342.[Medline]
Irvine RJ, White J, and Chan R (1997) The influence of restraint on blood pressure in the rat. J Pharmacol Toxicol Methods 38: 157–162.[CrossRef][Medline]
Isgor C, Shieh KR, Akil H, and Watson SJ (2003) Colocalization of estrogen 
-receptor messenger RNA with orphanin FQ, vasopressin and oxytocin in the rat hypothalamic paraventricular and supraoptic nuclei. Anat Embryol 206: 461–469.[Medline]
Kakiya S, Murase T, Arima H, Yokoi H, Iwasaki Y, Miura Y, and Oiso Y (2000) Role of endogenous nociceptin in the regulation of arginine vasopressin release in conscious rats. Endocrinology 141: 4466–4471.
Kapusta DR and Dzialowski EM (1995) Central mu opioids mediate differential control of urine flow rate and urinary sodium excretion in conscious rats. Life Sci 56: PL243–PL248.[CrossRef][Medline]
Kapusta DR and Kenigs VA (1999) Cardiovascular and renal responses produced by central orphanin FQ/nociceptin occur independent of renal nerves. Am J Physiol 277: R987–R995.[Medline]
Kapusta DR, Sezen SF, Chang JK, Lippton H, and Kenigs VA (1997) Diuretic and antinatriuretic responses produced by the endogenous opioid-like peptide, nociceptin (orphanin FQ). Life Sci 60: PL15–PL21.[CrossRef][Medline]
Kenney MJ, Weiss ML, and Haywood JR (2003) The paraventricular nucleus: an important component of the central neurocircuitry regulating sympathetic nerve outflow. Acta Physiol Scand 177: 7–15.[CrossRef][Medline]
Krowicki ZK, Kapusta DR, Nathan NA, and Hornby PJ (1997) Excitatory gastric motor and cardiovascular effects of nociceptin in the dorsal vagal complex of the rat. Soc Neurosci Abstr 23: 429.
Lessard A and Bachelard H (2002) Tonic inhibitory control exerted by opioid peptides in the paraventricular nuclei of the hypothalamus on regional hemodynamic activity in rats. Br J Pharmacol 136: 753–763.[CrossRef][Medline]
Martin DS and Haywood JR (1992) Sympathetic nervous system activation by glutamate injections into the paraventricular nucleus. Brain Res 577: 261–267.[CrossRef][Medline]
Meunier JC, Mollereau C, Toll L, Suaudeau C, Moisand C, Alvinerie P, Butour JL, Guillemot JC, Ferrara P, Monsarrat B, et al. (1995) Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature (Lond) 377: 532–535.[CrossRef][Medline]
Olszewski PK, Shi Q, Billington CJ, and Levine AS (2000) Opioids affect acquisition of LiCl-induced conditioned taste aversion: involvement of OT and VP systems. Am J Physiol 279: R1504–R1511.
Paxinos G and Watson C (1998) The Rat Brain in Stereotaxic Coordinates, 4th Ed, Academic Press, New York.
Reinscheid RK, Nothacker HP, Bourson A, Ardati A, Henningsen RA, Bunzow JR, Grandy DK, Langen H, Monsma FJ Jr, and Civelli O (1995) Orphanin FQ: a neuropeptide that activates an opioid-like G protein-coupled receptor. Science (Wash DC) 270: 792–794.
Shirasaka T, Kunitake T, Kato K, Takasaki M, and Kannan H (1999) Nociceptin modulates renal sympathetic nerve activity through a central action in conscious rats. Am J Physiol 277: R1025–R1032.[Medline]
Shirasaka T, Miyahara S, Takasaki M, and Kannan H (2001) Nociceptin/orphanin FQ and [Phe1
(CH2-NH)Gly2]nociceptin(1–13)NH2 modulates the activity of hypothalamic paraventricular nucleus neurons in vitro. Brain Res 890: 147–153.[CrossRef][Medline]
Sjoquist M, Huang W, Jacobsson E, Skott O, Stricker EM, and Sved AF (1999) Sodium excretion and renin secretion after continuous versus pulsatile infusion of oxytocin in rats. Endocrinology 140: 2814–2818.
Smith CP and Balment RJ (1993) Arginine vasopressin-induced natriuresis in the anaesthetized rat: involvement of V1 and V2 receptors. J Endocrinol 136: 283–288.
Witta J, Palkovits M, Rosenberger J, and Cox BM (2004) Distribution of nociceptin/orphanin FQ in adult human brain. Brain Res 997: 24–29.[CrossRef][Medline]
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, n = 7), 100 pmol of N/OFQ (
, n = 8), or aCSF vehicle (
, n = 7) into the PVN of rats. Urine samples were collected during control (C, 20 min) and, immediately after drug/vehicle microinjection for 80 min, denoted as time periods 10 to 80 min (consecutive 10-min samples). MAP, mean arterial pressure; HR, heart rate, UV, urinary flow rate; UNaV, urinary sodium excretion; UKV, urinary potassium excretion. *, p < 0.05; **, p < 0.01 compared with respective predrug control (time C).
, n = 5) compared with responses in rats infused with saline (
