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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Centrally Administered Nociceptin/Orphanin FQ (N/OFQ) Evokes Bradycardia, Hypotension, and Diuresis in Mice via Activation of Central N/OFQ Peptide Receptors

Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana

Received for publication January 23, 2007
Accepted April 19, 2007.

	Abstract

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The present studies examined the cardiovascular and renal responses produced by activation of central nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors in conscious mice. To assess this, we examined changes in heart rate (HR), mean arterial pressure (MAP), urine output (V), urinary sodium excretion (UNaV), and free water clearance (CH₂O) produced by acute i.c.v. injection of N/OFQ (0.03, 0.3, 1, or 3 nmol) or isotonic saline vehicle (2 µl) in conscious telemetered ICR-CD1 mice. After i.c.v. injection, N/OFQ, but not vehicle, dose dependently decreased HR and MAP and increased V. At 3 nmol, N/OFQ reduced HR [control (C), 672 ± 23 beats/min; 20 min, 411 ± 30 beats/min] and MAP (C, 108 ± 4 mm Hg; 20 min, 62 ± 6 mm Hg). In the same telemetered mice, i.c.v. N/OFQ significantly elevated V (0.65 ± 0.03 cc/2 h) compared with levels for the vehicle-treated group (0.15 ± 0.09 cc/2 h). Central N/OFQ/vehicle did not alter UNaV or CH₂O. In separate studies, 2-h i.c.v. pretreatment with the NOP receptor antagonist UFP-101 ([Nphe¹,Arg¹⁴,Lys¹⁵]N/OFQ-NH₂) (10 or 30 nmol) markedly, but transiently, reduced HR but not MAP, V, UNaV, or CH₂O. After 2-h UFP-101 (10 or 30 nmol) pretreatment, subsequent i.c.v. injection of N/OFQ (1 or 3 nmol) failed to alter cardiovascular or renal function. In contrast, in separate mice, 2-h pretreatment with N/OFQ (1 or 3 nmol) or vehicle failed to prevent the cardiodepressor and diuretic responses to a subsequent i.c.v. injection of the same dose of N/OFQ. Together, these findings demonstrate that in conscious mice, the central administration of N/OFQ evokes marked bradycardia, hypotension, and diuresis by selective activation of central NOP receptors.

There is a moderate to high density of prepro-N/OFQ (the polypeptide precursor of N/OFQ), N/OFQ, and NOP receptor mRNA and protein throughout the brain in rats (Anton et al., 1996; Neal et al., 1999a,b, 2001) and mice (Ikeda et al., 1998; Houtani et al., 2000), including regions known to participate in the central control of blood pressure and fluid/electrolyte balance. Accordingly, N/OFQ evokes novel cardiovascular and renal excretory responses following its injection into the brain (for review, see Kapusta, 2000; Salis et al., 2000). The i.c.v. injection of N/OFQ, for instance, concurrently decreases heart rate (HR) and mean arterial pressure (MAP) in conscious (Kapusta et al., 1997; Kapusta and Kenigs, 1999; Zhang et al., 1999) and anesthetized (Chen et al., 2002) rats by a mechanism that involves inhibition of central sympathetic outflow (Kapusta and Kenigs, 1999; Shirasaka et al., 1999). These findings are of particular interest because the central N/OFQ-evoked reduction in MAP is associated with a concurrent reduction in HR rather than a baroreflex-induced tachycardia (Kapusta and Kenigs, 1999; Shirasaka et al., 1999). In addition to cardiovascular depressor responses, central N/OFQ has also been shown to produce unique changes in renal excretory function and renal sympathetic nerve activity (for review, see Kapusta, 2000). In conscious rats, i.c.v. N/OFQ concomitantly increases urine output and decreases both urinary sodium excretion (Kapusta et al., 1997; Kapusta and Kenigs, 1999) and renal sympathetic nerve activity (Kapusta and Kenigs, 1999; Shirasaka et al., 1999). These diuretic and antinatriuretic responses to i.c.v. N/OFQ occur independently of a renal nerve mechanism because chronic bilateral renal denervation does not alter the renal responses produced by central N/OFQ (Kapusta and Kenigs, 1999).

Although the cardiovascular and renal responses to i.c.v. N/OFQ have been well characterized in the rat, those evoked by this peptide in conscious mice remain largely unknown. In related but separate studies, we observed that N/OFQ produces a dose-dependent increase in urine output when injected centrally to conscious mice (D. Rizzi, G. Calo, R. Guerrini, and D. R. Kapusta, personal observation). Thus, the purpose of the present investigations was to extend these initial observations and more critically examine the cardiovascular and renal responses produced by the i.c.v. injection of N/OFQ in conscious mice. Having a basic understanding of central N/OFQ's effects in mice (wild type) also provides a foundation for comparison of results for future pharmacological and physiological studies that will explore the role of this peptide receptor system using transgenic NOP receptor (or N/OFQ) knockout mice. Therefore, the present studies examined the cardiovascular and renal excretory responses to increasing i.c.v. doses of N/OFQ in conscious mice. For these investigations, cardiovascular and renal excretory function was assessed simultaneously by using radiotelemetric and metabolic function methods, respectively. Additional studies were performed to determine whether the cardiovascular and renal responses to i.c.v. N/OFQ were mediated by selective activation of central NOP receptors. For these studies, the cardiovascular and renal responses to i.c.v. N/OFQ were examined in conscious mice pretreated centrally with the NOP receptor antagonist, UFP-101. UFP-101 has nanomolar affinity and high selectivity for NOP receptors and antagonizes the biological effects of N/OFQ in several in vivo and in vitro assays (Calo et al., 2002). Here, we report that activation of central NOP receptors via centrally administered N/OFQ markedly reduces HR and MAP in conscious mice at doses that also produce diuresis.

	Materials and Methods

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Subjects
Age-matched ICR-CD1 (Harlan Sprague-Dawley, Inc., Indianapolis, IN) weighing 20 to 25 g were used in these studies. Animals were housed and tested in a climate-controlled room on a 12-h dark/light cycle. All animals were fed a standard rodent chow diet and allowed food and tap water ad libitum. All experimental procedures were performed in accordance with Louisiana State University Health Sciences Center and National Institutes of Health Guidelines for the Care and Use of Experimental Animals.

Surgical Procedures
Mice were instrumented with a stainless steel cannula into the right lateral cerebral ventricle under anesthesia (200 mg/kg i.p. ketamine in combination with 10 mg/kg i.p. xylazine). The coordinates used for cannula implantation were derived from the atlas of the mouse brain by Paxinos and Franklin (2003): 0.3 mm posterior to the bregma, 1.0 mm lateral to the midline, and 3.1 mm below the skull surface. Custom-cut and fabricated guide, dummy (obturator), and internal cannula were purchased from Plastics One (Roanoke, VA). The guide cannula was fixed into position by jeweler's screws and cranioplastic cement. Verification of cannula position in the lateral cerebroventricle was made by observing spontaneous flow of cerebrospinal fluid from the tip of the cannula after removal of the obturator (after cannula implantation and before experimentation) and after the completion of the experiment by i.c.v. dye injection and subsequent postmortem brain section verification of dye placement.

After a 5-day recovery period, mice were anesthetized (200 mg/kg i.p. ketamine in combination with 10 mg/kg i.p. xylazine) and instrumented with a TA11PC20 radiotelemetry probe (Data Sciences International, Arden Hills, MN), which was implanted into and secured to the left common carotid artery. Proper catheter placement was verified by the quality of the audio output signal on an FM radio. The body of the probe was placed in a s.c. pocket created in the right flank, and the wound was closed an sutured. Body temperature was maintained at 37°C using a heat lamp until sternal recumbency had been recovered. After surgery, penicillin (60,000 U/kg i.m.) and yohimbine (0.1 mg/kg) were administered to prevent infection and facilitate recovery from surgery, respectively. Mice remained undisturbed in their home cages for 7 days before the initiation of cardiovascular and renal excretory function studies. Before testing, basal cardiovascular function was observed in all mice and shown to have characteristic circadian rhythm indicative of recovery from surgery.

Experimental Protocols
Concurrent Cardiovascular and Renal Function Studies. In the first set of experiments, cardiovascular and renal excretory function studies were performed simultaneously. This was achieved by placing the telemetered mice individually in metabolic cages that sat directly on top of the telemetry receiver plates, which allowed for the simultaneous measurement of MAP and HR as well as collection of spontaneously voided urine. The metabolic cages were constructed of Plexiglas (walls) and stainless steel (row of bars positioned horizontally that the animal sat upon) and were designed to allow spontaneously voided urine to be collected onto a large plastic weigh boat placed underneath the animal. On the day before experimentation, each mouse was removed from its home cage and trained by being placed individually in a metabolic cage for 20 min. The animals were then removed, and the lower abdominal region was gently messaged to void any urine already present in the bladder. Each animal was then touched in the region of the i.c.v. cannulae and then returned to the metabolic cages for 2.5 hours. On the day of the experiment, mice were again placed in metabolic cages that were positioned on top of the telemetry plates. The telemetry probes were then turned on with the least possible disturbance to the animals for the measurement of baseline MAP and HR (sampling frequency, 500 Hz; sampling interval, 2 s). After baseline sampling, the mice were removed from the metabolic cages, and urine was voided from the bladder of each animal by massaging the abdominal area as described. An i.c.v. injector was then securely placed into each animal's indwelling i.c.v. guide cannula. The i.c.v. injector was attached via polyethylene-10 tubing to a Hamilton syringe for injection of drug/vehicle. Animals were then returned to the metabolic cages. The polyethylene tubing was extended outside of the cages to keep disturbance to the animals to a minimum and to prevent it from being gnawed and damaged. After stabilization and additional baseline sampling (i.e., two 10-min predrug control periods), N/OFQ (0.03, 0.3, 1, and 3 nmol) or isotonic saline vehicle (2 µl) was delivered i.c.v. via the Hamilton syringe injector assembly. Then, HR and MAP were measured continuously during a 120-min experimental period (12 consecutive 10-min periods). Parameters analyzed were the MAP and HR responses to N/OFQ and isotonic saline control. Renal excretory studies involved the hourly collection of spontaneous urine voided immediately following vehicle/drug administration for two hours. Parameters analyzed were urine flow rate, urinary excretion of sodium and potassium, urine osmolality, and free water clearance.

Cardiovascular Antagonist Studies. Studies were performed to determine the cardiovascular responses to i.c.v. N/OFQ in conscious mice that were pretreated centrally for 2 h with either UFP-101 or N/OFQ. In these studies, mice that were chronically instrumented with both radiotelemeters and indwelling i.c.v. cannulae received a 2-h i.c.v. pretreatment with either UFP-101 (10 or 30 nmol) or N/OFQ (1 or 3 nmol), and drug-evoked changes in HR and MAP were assessed using the experimental protocol described above. Renal function was not measured. After 2-h drug pretreatment, animals then received a second i.c.v. injection, this time with N/OFQ (1 or 3 nmol). HR and MAP were then measured for an additional 2 h. After all testing, animals were disconnected from their injectors and returned to their home cages.

Renal Antagonist Studies. Studies were performed to determine the renal excretory responses to i.c.v. N/OFQ in conscious mice that were pretreated centrally for 2 h with either vehicle or UFP-101. For these studies, mice that were instrumented with chronic i.c.v. cannulae (i.e., no radiotelemeters) received a 2-h i.c.v. pretreatment with either isotonic saline (2 µl) or UFP-101 (10 or 30 nmol). Drug-evoked changes in urine output were then assessed during the pretreatment period using the experimental protocol described above. After the 2-h pretreatment, animals then received a second i.c.v. injection, this time of N/OFQ (1 or 3 nmol). Urine output was then measured hourly during a 2-h experimental period. In other experiments, urine output was also assessed in additional groups of mice that received simultaneous administration (i.e., cotreatment) of N/OFQ (1 or 3 nmol) and UFP-101 (10 or 30 nmol, respectively). After all testing, animals were disconnected from their injectors and returned to their home cages.

Analytical Techniques
Urine volume was determined gravimetrically. Urine sodium and potassium concentrations were measured by flame photometry (model 943; Instrumentation Laboratories, Lexington, MA). Urine osmolality was analyzed using a vapor pressure osmometer (model 5500; WESCOR, Inc., Logan, UT). Telemetry data were compiled and analyzed using Dataquest A.R.T. Software (version 2.3; Data Sciences International, St. Paul, MN) and Microsoft Excel (Microsoft, Redmond, WA). The output from the radiotelemetry probes was recorded (500 Hz) using receiver plates placed beneath the home or metabolic cages. Data were sent to a consolidation matrix before being stored on a personal computer. Results were plotted and graphed using GraphPad Prism software (version 4.00; GraphPad Software Inc., San Diego, CA).

Statistics
Results are expressed as the mean ± S.E.M. The magnitude of the changes in cardiovascular and renal excretory parameters at different time points after i.c.v. injection of drugs were compared with respective group control values by a one-way repeated-measures analysis of variance (ANOVA) with subsequent Dunnett's test. Differences occurring between treatment groups (e.g., multiple doses of drug) were assessed by two-way repeated measures ANOVA with treatment being one fixed effect and time the other, with the interaction included. The time (minutes) was the repeated factor. Post hoc analysis was performed using Bonferroni's test. Where appropriate, a Student's t test was also used to compare means between two groups. In each case, statistical significance was defined as p < 0.05.

Drugs
N/OFQ (FGGFTGARKSARKLANQ) and UFP-101 ([Nphe¹,Arg¹⁴, Lys¹⁵]N/OFQ-NH₂) were synthesized and graciously provided by Drs. Remo Guerrini and Severo Salvadori (University of Ferrara, Ferrara, Italy). Stock solutions of peptides were prepared fresh and stored frozen. All drugs/vehicle were administered i.c.v. in a volume of 2 µl.

	Results

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The cardiovascular responses produced by the i.c.v. administration of N/OFQ in conscious ICR-CD1 mice are presented in Fig. 1a (typical tracing), Fig. 1b (time course), and Fig. 1c (peak delta). In Fig. 1b, mean values for each parameter are depicted during control (C; 10 min) and consecutive 10-min experimental periods (time, 10–120 min) beginning immediately after the i.c.v. injection of N/OFQ (0.03, 0.3, 1, or 3 nmol) or isotonic saline vehicle. As shown (Fig. 1b), the i.c.v. administration of isotonic saline did not alter HR or MAP at any point throughout the study. After i.c.v. injection of N/OFQ at 1 or 3 nmol, however, HR and MAP began to decrease within 30 s to 1 min (Fig. 1, a and b). At 3 nmol, the highest dose tested, N/OFQ decreased HR from 672 ± 23 (C) to 411 ± 30 beats/min (20 min) and MAP from 108 ± 4 (C) to 62 ± 6 mm Hg (20 min) (Fig. 1b). The peak reductions in each parameter occurred 20 min after central drug administration, with each response returning to baseline by 50 and 40 min, respectively, postinjection (Fig. 1b). Moreover, the peak cardiovascular depressor responses elicited by central N/OFQ in conscious mice were dose-dependent (Fig. 1c), with responses in HR being significantly different from the vehicle-treated group at 1 nmol and maximal at 3 nmol. Likewise, the central N/OFQ-evoked reduction in MAP was rapid in onset (Fig. 1, a and b) and significantly different from control values at 0.3-, 1-, and 3-nmol doses (Fig. 1, b and c); maximal reduction in MAP was produced by 1 nmol of N/OFQ.

View larger version (30K): [in this window] [in a new window]   Fig. 1. a, representative tracing of the cardiovascular responses produced by the i.c.v. injection of N/OFQ in a conscious telemetered ICR-CD1 mouse. The tracing illustrates the typical systemic cardiovascular depressor responses produced by the i.c.v. injection of 3 nmol of N/OFQ in mice. Heart rate and mean arterial pressure were measured via telemetry during C (5 min) and immediately after i.c.v. N/OFQ injection for 120 min (500 Hz, consecutive 2-s sampling interval). b, time course of the cardiovascular responses produced by the i.c.v. injection of N/OFQ in conscious, telemetered ICR-CD1 mice. The values are mean ± S.E.M. and illustrate the systemic cardiovascular responses produced by the i.c.v. injection of 0.03 nmol of N/OFQ (, n = 7), 0.3 nmol of N/OFQ (, n = 6), 1 nmol of N/OFQ (, n = 9), 3 nmol of N/OFQ (, n = 7), or isotonic saline vehicle (2 µl, , n = 8). Heart rate and mean arterial pressure were measured during C (10 min) and immediately after i.c.v. drug/vehicle injection for 120 min denoted as time periods 10 to 120 min (consecutive 10-min periods). *, p < 0.05 compared with respective predrug control (time C). c, peak cardiovascular responses produced by the i.c.v. injection of N/OFQ in conscious, telemetered ICR-CD1 mice. The values are mean ± S.E.M. and illustrate the peak systemic cardiovascular responses produced by the i.c.v. injection of 0.03 nmol of N/OFQ (n = 7), 0.3 nmol of N/OFQ (n = 6), 1 nmol of N/OFQ (n = 9), 3 nmol of N/OFQ (n = 7), or isotonic saline vehicle (2 µl; n = 8) measured within the first 20 min (0–20 min) following vehicle/drug administration. Note that data depicted in this figure are from the same groups of telemetered mice for which cardiovascular data are presented in b. *, p < 0.05 compared with the saline-treated group.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Changes in urine output produced by the i.c.v. injection of N/OFQ in conscious, telemetered ICR-CD1 mice. The values are mean ± S.E.M. and illustrate the changes in cumulative urine output produced by the i.c.v. injection of 0.03 nmol of N/OFQ (n = 8), 0.3 nmol of N/OFQ (n = 7), 1 nmol of N/OFQ (n = 14), 3 nmol of N/OFQ (n = 12), or isotonic saline vehicle (2 µl; n = 13). Note that data depicted in this figure are from the same groups of telemetered mice in which cardiovascular data are presented in Fig. 1, b and c. Data shown in this figure represent mean values for cumulative urine collected throughout the entire 120-min experimental protocol beginning immediately after vehicle/drug administration. V, urine output. **, p < 0.01 compared with isotonic saline vehicle-treated mice.

In separate studies, changes in HR and MAP were examined in telemetered mice that were pretreated centrally with the purported NOP receptor antagonist UFP-101 (2-h pretreatment period) and then challenged with a subsequent i.c.v. injection of N/OFQ. As shown in Fig. 3a (data for the pretreatment period), i.c.v. injection of 10 or 30 nmol of UFP-101 alone significantly decreased HR in conscious mice (C, 697 ± 59 and 628 ± 22 beats/min, respectively, versus 30 min, 508 ± 73 and 383 ± 28 bpm, respectively). At the lower dose, the bradycardia persisted for 90 min, whereas at the 30-nmol dose, HR did not fully recover over the 2-h pretreatment period. In contrast, UFP-101 did not appreciably alter MAP. These two doses of UFP-101 were selected so as to maintain a 10:1 antagonist to agonist ratio that other investigators have reported is optimal for the antagonist action of UFP-101 at NOP receptors in vivo (Calo et al., 2002; Hashiba et al., 2003; Vitale et al., 2006; Nazzaro et al., 2007). Figure 3b (left) illustrates the peak changes in HR and MAP produced by the subsequent i.c.v. injection of N/OFQ (1 or 3 nmol) observed in these same groups of mice that were pretreated centrally with UFP-101. As shown (Fig. 3b, left), 2-h i.c.v. pretreatment with 10 nmol of UFP-101 completely abolished the bradycardic and hypotensive responses to i.c.v. injection of 1 nmol of N/OFQ (C, 600 ± 45 beats/min versus N/OFQ 20 min, 628 ± 33 beats/min and C, 122 ± 6 mm Hg versus N/OFQ 20 min, 121 ± 5 mm Hg, respectively). Likewise, the bradycardia and hypotensive effects typically produced by i.c.v. injection of 3 nmol of N/OFQ were completely prevented in mice that received 2-h i.c.v. pretreatment with 30 nmol of UFP-101 (Fig. 3b; C, 605 ± 31 beats/min versus N/OFQ 20 min, 600 ± 29 beats/min and C, 108 ± 3 mm Hg versus N/OFQ 20 min, 104 ± 4 mm Hg, respectively). Note that this antagonist action of UFP-101 (30 nmol) occurred despite the fact that in this group the control level for HR measured before N/OFQ injection had not fully recovered by the end of the 2-h pretreatment period (see Fig. 3a). In contrast to these findings, Fig. 3b (right) shows that i.c.v. N/OFQ produced marked reductions in HR and MAP in conscious mice that had instead been pretreated centrally for 2 h with N/OFQ (1 or 3 nmol). The magnitudes of these depressor responses with the 1-nmol (C, 674 ± 31 bpm and 112 ± 6 mm Hg; N/OFQ 20 min, 515 ± 51 bpm and 89 ± 11 mm Hg) and 3-nmol dose (C, 699 ± 31 bpm and 110 ± 6 mm Hg; N/OFQ 20 min, 516 ± 31 bpm and 81 ± 2 mm Hg) were similar to those observed when N/OFQ was initially given as a pretreatment (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 3. a, time course of the cardiovascular responses produced by i.c.v. pretreatment of conscious, telemetered ICR-CD1 mice with the purported NOP receptor antagonist, UFP-101. The values are mean ± S.E.M. and illustrate the systemic cardiovascular responses produced by the i.c.v. injection of 10 nmol of UFP-101 (, n = 5), 30 nmol of UFP-101 (, n = 5), or isotonic saline vehicle (2 µl, , n = 8). Heart rate and mean arterial pressure were measured during C (10 min) and immediately after i.c.v. drug/vehicle injection during a 120-min "pretreatment" period denoted as time periods 10 to 120 min (consecutive 10-min samples). *, p < 0.05 compared with respective predrug control (time C). b, peak cardiovascular responses produced by i.c.v. N/OFQ in conscious ICR-CD1 mice that were pretreated centrally for 2-h with either UFP-101 or N/OFQ. The values are mean ± S.E.M. and illustrate the peak systemic cardiovascular responses produced by the i.c.v. injection of N/OFQ (1 or 3 nmol) following a 2-h i.c.v. pretreatment with either UFP-101 (left; 10 nmol, n = 6; 30 nmol; n = 5) or N/OFQ (right; 1 nmol, n = 5; 3 nmol, n = 4). An antagonist:agonist ratio of 10:1 was used for studies with UFP-101 and N/OFQ. Data shown are the peak changes in cardiovascular function that occurred within the first 20 min (0–20 min) following i.c.v. drug/vehicle administration. *, p < 0.05 compared with baseline value immediately before N/OFQ.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Changes in urine output produced by the i.c.v. injection of N/OFQ in conscious mice pretreated i.c.v. for 2 h with either isotonic saline vehicle or UFP-101. The values are mean ± S.E.M. and illustrate the changes in cumulative urine output produced by i.c.v. injection of N/OFQ (1 or 3 nmol) in mice that received a 2-h pretreatment (pt) with isotonic saline vehicle (2 µl; n = 8/group) or UFP-101 (10 nmol, n = 8; 30 nmol, n = 8). An antagonist:agonist ratio of 10:1 was used for studies with UFP-101 and N/OFQ. Data shown are mean values for N/OFQ-evoked changes in cumulative urine output measured during a 2-h experimental period, which began after completion of the 2-h pretreatment. Values (mean ± S.E.M.) are also illustrated (hatched bars) for the changes in 2-h cumulative urine output produced by the cotreatment of UFP-101 and N/OFQ (1 and 10 nmol, respectively, n = 8; 3 and 30 nmol, respectively, n = 8). V, urine output. *, p < 0.05 compared with isotonic saline vehicle pretreatment; , p < 0.05 compared with N/OFQ in animals pretreated with isotonic saline.

	Discussion

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In comparison with findings observed in conscious rats (Kapusta and Kenigs, 1999; Kapusta, 2000), the pattern of the changes in cardiovascular function produced by i.c.v. N/OFQ in conscious mice was similar in direction and duration but remarkably different in magnitude. More specifically, at maximally effective doses, N/OFQ lowers HR and MAP in both conscious male ICR-CD1 mice and Sprague-Dawley rats, with peak responses in each species occurring 20 min following the peptide's i.c.v. administration. However, compared with respective predrug control values, the 10-min peak HR response to i.c.v. N/OFQ (3 nmol) was considerably greater in mice (=-261 ± 27 bpm; nadir, 20 min; Fig. 1c of present study) than that produced by N/OFQ (5.5 nmol) in rats (=-82 ± 11 bpm; nadir, 20 min; Kapusta and Kenigs, 1999). The 10-min peak MAP response to i.c.v. N/OFQ was also greater in mice (=-46 ± 5 mm Hg, 3 nmol, Fig. 1c) than in rats (=-16 ± 5 mm Hg, 5.5 nmol; Kapusta and Kenigs, 1999). Note that the i.c.v. doses for this comparison are based on dose-response studies that established the lowest i.c.v. dose of N/OFQ that produced maximal cardiovascular and renal responses in each species; 3 nmol for mouse (current study) and 5.5 nmol for rat (Kapusta et al., 1997). The observation that i.c.v. N/OFQ evoked greater bradycardic and hypotensive responses in conscious mice is probably due to the increased contribution of the sympathetic nervous system to basal autonomic tone in mice versus rats. In this setting, in mice, the N/OFQ-mediated inhibition of central sympathetic outflow (and possibly activation of parasympathetic outflow) would, therefore, be anticipated to elicit greater magnitudes of cardiodepressive responses.

In addition to marked cardiovascular depressor responses, the results of the present studies demonstrate that the central administration of N/OFQ to conscious mice significantly increased urine output. This finding is in agreement with those of a separate but related study (Rizzi et al., 2004) in which the renal excretory responses produced by the central and peripheral administration of N/OFQ and a novel NOP receptor ligand UFP-102 were examined in mice under conditions in which cardiovascular function was not measured. In the present study, compared with control levels in mice treated with vehicle, i.c.v. N/OFQ (3 nmol) increased cumulative urine output over the 2-h study by 333% (Fig. 2). Interestingly, the N/OFQ-evoked diuresis occurred in both the 1st and 2nd hour periods of the protocol; thus, the diuresis continued well after the N/OFQ-evoked bradycardia and hypotension had returned to baseline (approximately 40–50 min). As previously reported (Kapusta and Kenigs, 1999), the central administration of N/OFQ (5.5 nmol) to conscious rats also increased urine output by approximately 254%. In these rat studies, it was observed that the peak diuresis produced by central N/OFQ also occurred at a time (40 min postdrug injection) in which the cardiovascular depressor responses to central N/OFQ had fully recovered. In this and other rat studies, however, the duration of the central N/OFQ-evoked diuresis was shorter and lasted only for approximately 1 h (Kapusta et al., 1997; Kapusta and Kenigs, 1999) compared with persisting for 2 h in the present studies in mice. Another important difference is that in rats, i.c.v. N/OFQ has been shown to produce a marked decrease in urinary sodium excretion that occurs concurrent with the diuresis; thus, in rats, central N/OFQ produces diuresis, antinatriuresis, and an increase in free water clearance (i.e., a water diuresis) (Kapusta et al., 1997; Kapusta and Kenigs, 1999). Yet, in contrast, in the present studies, i.c.v. N/OFQ did not alter urinary sodium excretion or free water clearance in conscious telemetered mice. In addition to species differences, it is likely that variations in the experimental methods and protocols used in the rat versus mouse studies contributed to the differences in the renal excretory responses observed between these two species. Of merit, in the rat studies, urine was collected from chronically implanted bladder catheters from animals that were continuously infused i.v. with isotonic saline (55 µl/min) (Kapusta et al., 1997; Kapusta and Kenigs, 1999). Under these conditions (i.e., i.v. isotonic saline infusion), drug-induced changes (e.g., increases or decreases) in urine flow rate and/or urinary sodium excretion can readily be detected (Kapusta et al., 1997; Kapusta and Kenigs, 1999). This is in contrast to the present studies in which urine was spontaneously collected from euvolemic mice (i.e., no volume load) that were placed in individual metabolic cages. In this latter case, because basal levels for urine output and urinary sodium excretion were substantially low, it is likely that a bona fide antinatriuretic effect of central N/OFQ could not be detected in the present studies in mice. Regardless, it is clear from these investigations that in both species, the central administration of N/OFQ markedly enhanced the renal excretion of water. Although not tested, it is likely that in mice, i.c.v. N/OFQ evokes diuresis via inhibiting the secretion/release of vasopressin into the systemic circulation as this peptide has been shown to do in the rat (Kapusta et al., 1997; Kakiya et al., 2000).

UFP-101 has been demonstrated to behave as a selective and pure NOP receptor antagonist in many species and biological systems (for review, see Calo et al., 2005). Furthermore, i.c.v. UFP-101 effectively blocks the diuretic response to i.c.v. N/OFQ in conscious mice when the two compounds are administered as a cotreatment (Rizzi et al., 2004). Here, we report that, in conscious telemetered mice, a 2-h pretreatment with i.c.v. UFP-101 effectively blocked the diuretic response to a subsequent i.c.v. N/OFQ injection. UFP-101 itself did not alter urine output, thereby confirming that UFP-101 is a selective NOP receptor antagonist in preventing N/OFQ's action on renal excretory function. However, when administered i.c.v. either alone or as a cotreatment with N/OFQ (data not presented), UFP-101 profoundly and selectively lowered HR. Despite this residual agonist-like activity of UFP-101, it was shown that once HR recovered to/toward baseline (2 h), the bradycardia and hypotensive responses typically elicited by i.c.v. N/OFQ injection were completely blocked (Fig. 3b). This antagonism was most probably due to a receptor antagonist action of UFP-101 at NOP receptors and not attributable to tachyphylaxis. This possibility is suggested since in other mice, a 2-h i.c.v. pretreatment with N/OFQ did not block the cardiovascular depressor responses to a subsequent (i.e., second) i.c.v. N/OFQ injection (Fig. 3b).

Thus, although the mechanism by which UFP-101 affects HR has yet to be determined, these findings strongly support the notion that in mice, i.c.v. N/OFQ produces cardiovascular depressor and diuretic responses via selective activation of central NOP receptors. This hypothesis is further supported by pilot data in which we have shown that the cardiovascular depressor (i.e., decreases in HR and MAP) responses to i.c.v. N/OFQ, but not the bradycardia to i.c.v. UFP-101, are abolished in transgenic NOP receptor knockout mice (M. A. Burmeister and D. R. Kapusta, personal observation).

In addition to characterizing the cardiovascular and renal responses produced by exogenous administration of N/OFQ, it is of physiological importance to understand how the endogenous N/OFQ-NOP receptor system participates in the central (and peripheral) control of cardiovascular and/or renal function. The findings of the current studies with UFP-101 indicate that the native N/OFQ-NOP receptor system in mice is not involved in the tonic regulation of MAP or the renal excretion of water/sodium. However, given that UFP-101 did affect HR (produced bradycardia) following acute i.c.v. administration, this conclusion must be carefully interpreted. Considering this point, it is likely that other investigations that incorporate transgenic NOP receptor (or N/OFQ ligand) knockout mice as well as small interfering RNA targeted against the NOP receptor may serve as additional approaches for investigating N/OFQ's endogenous physiological role(s) in cardiovascular and renal function during health and stress/pathology. With this likelihood, the findings of the present studies, which were performed in wild-type mice, will provide a foundation for comparison of results from investigations that will examine N/OFQ's role in the central control of cardiovascular and renal function using these transgenic or small interfering RNA model systems.

In summary, our results show that in conscious mice, the central administration of the opioid-like peptide, N/OFQ, produces marked cardiovascular depressor responses at doses that also elicit diuresis. These responses were completely prevented by i.c.v. pretreatment of mice with UFP-101, thus demonstrating that the central effects of N/OFQ on cardiovascular function and urine output are mediated by a NOP receptor pathway.

	Acknowledgements

 
We thank Drs. Severo Salvadori, and Remo Guerrini at the University of Ferrara (Ferrara, Italy) for graciously providing the N/OFQ and UFP-101 peptides used in the studies. We also thank Daniela Rizzi for technical assistance and discussion of data.

	Footnotes

 
This study was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (Grants DK-43337 and DK-02605 to D.R.K.) and by the Heart Blood and Lung Institute (Grant HL71212 to D.R.K.). Additional funding for this work was provided to the Cardiac and Vascular Core Facility in the Department of Pharmacology and Center of Excellence in Cardiovascular Research at Louisiana State University Health Sciences Center by COBRE Grant P20 RR018766 (to D.R.K.) from the National Center for Research Resources, a component of the National Institutes of Health.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.120394.

ABBREVIATIONS: N/OFQ, nociceptin/orphanin FQ; NOP, nociceptin/orphanin FQ peptide; HR, heart rate; MAP, mean arterial pressure; V, urine output; UFP-101, [Nphe¹,Arg¹⁴,Lys¹⁵]N/OFQ-NH₂; ANOVA, analysis of variance; C, control.

Address correspondence to: Daniel R. Kapusta, Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112. E-mail: dkapus{at}lsuhsc.edu

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