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Research ArticleArticle

Central Administration of [Phe1Ψ(CH2-NH)Gly2]Nociceptin(1-13)-NH2and Orphanin FQ/Nociceptin (OFQ/N) Produce Similar Cardiovascular and Renal Responses in Conscious Rats

Daniel R. Kapusta, Jaw-Kang Chang and Velga A. Kenigs
Journal of Pharmacology and Experimental Therapeutics April 1999, 289 (1) 173-180;

Abstract

In vitro studies have shown that [Phe1Ψ(CH2-NH)Gly2]OFQ/N(1–13)-NH2(referred to as [FG]OFQ/N(1–13)-NH2) is the first selective antagonist to prevent the binding of the endogenous ligand orphanin FQ/Nociceptin (OFQ/N) at the orphan opioid-like receptor. In the present study, we examined the potential changes in cardiovascular and renal function produced by the i.c.v. injection of [FG]OFQ/N(1–13)-NH2 in conscious Sprague-Dawley rats. In conscious rats, i.c.v. injection of [FG]OFQ/N(1–13)-NH2produced a marked and sustained decrease in heart rate, mean arterial pressure, and urinary sodium excretion and a profound increase in urine flow rate (i.e., a water diuresis). The cardiovascular and renal excretory responses produced by i.c.v. [FG]OFQ/N(1–13)-NH2 were dose dependent and were similar in pattern but of longer duration than responses evoked by i.c.v. OFQ/N. In other animals, the i.c.v. injection of OFQ/N(1–13)-NH2, a potential metabolite of [FG]OFQ/N(1–13)-NH2, produced changes in cardiovascular and renal function that were comparable to those evoked by i.c.v. [FG]OFQ/N(1–13)-NH2. In contrast, OFQ/N(2–17), a fragment of OFQ/N [OFQ/N(1–17)], was inactive when administered centrally. Finally, studies were performed to determine whether [FG]OFQ/N(1–13)-NH2 may be an antagonist at the orphan opioid-like receptor receptor when administered centrally at a dose that alone was inactive. In these studies, i.c.v. pretreatment of animals with low-dose [FG]OFQ/N(1–13)-NH2 failed to prevent the cardiovascular and renal excretory response to i.c.v. OFQ/N. Although [FG]OFQ/N(1–13)-NH2 is reported to be an antagonist of the OFQ/N receptor in vitro, these findings indicate that this compound has agonist activity similar to that of the endogenous ligand OFQ/N when administered centrally in vivo.

Native and synthetic opioid agonists exert their pharmacological and physiological effects through binding to μ-, δ-, and/or κ-opioid receptors in the central nervous system (CNS) and periphery (Fowler and Fraser, 1994). In addition to these classic opioid receptors, cDNA expression cloning techniques have been used to isolate and identify a fourth opioid receptor subtype referred to here as opioid receptor-like 1 (ORL1) (Mollereau et al., 1994) but also called ROR-C (Fukuda et al., 1994), oprl (Chen et al., 1994), LC132 (Bunzow et al., 1994), XOR1 (Wang et al., 1994), Hyp 8–1 (Wick et al., 1994), C3 (Lachowicz et al., 1995), and KOR-3 (Pan et al., 1994). ORL1 is a G protein-coupled receptor that shares a high degree of nucleotide sequence homology in the transmembrane domains with the cloned μ-, δ-, and κ-opioid receptors (Mollereau et al., 1994). Despite this similarity, native opioid peptides or synthetic opioid agonists selective for μ-, δ-, or κ-opioid receptors do not show specific binding to ORL1 (Bunzow et al., 1994; Chen et al., 1994; Mollereau et al., 1994; Wang et al., 1994; Wick et al., 1994; Lachowicz et al., 1995).

An endogenous peptide has now been isolated from brain tissue and shown to be the endogenous ligand of the orphan ORL1 (Meunier et al., 1995;Reinscheid et al., 1995). This novel peptide named orphanin FQ (Reinscheid et al., 1995) and nociceptin (Meunier et al., 1995), but referred to here as OFQ/N, is a heptadecapeptide with an amino acid sequence most similar to that of dynorphin A(1–17), a proposed endogenous ligand of the κ-opioid receptor. OFQ/N binds in a saturable manner and with high affinity to ORL1 (Reinscheid et al., 1995). Moreover, OFQ/N potently inhibits forskolin-stimulated cAMP accumulation (Meunier et al., 1995; Reinscheid et al., 1995), a property shared by interaction of native opioids with all three classic opioid receptor subtypes. Despite these features, OFQ/N appears to be pharmacologically distinct from other opioids because in binding and biological assays, it does not interact with μ-, δ-, or κ-opioid receptors (Meunier et al., 1995; Reinscheid et al., 1995).

The association of OFQ/N to the endogenous opioid peptide systems and the functional importance of this new peptide in vivo are not fully known. In previous investigations, we demonstrated that in conscious rats, the i.c.v. injection of OFQ/N evoked a significant decrease in heart rate, mean arterial pressure, and urinary sodium excretion and an increase in urine flow rate (Kapusta et al., 1997). These findings are of interest because the central administration of native or synthetic opioid agonists produces marked changes in cardiovascular and renal function through the activation of μ-, δ-, and/or κ-opioid receptors (Kapusta, 1995). Despite these similar effects, our previous studies revealed that the changes in cardiovascular and renal function produced by i.c.v. OFQ/N were mediated via a pathway independent of classic opioid receptors (Kapusta et al., 1997). These findings suggest that the endogenous OFQ/N system may participate as a novel pathway in the central regulation of cardiovascular and renal function (Kapusta et al., 1997).

Full characterization of the cardiovascular and renal responses produced by OFQ/N in different physiological and/or pathophysiological conditions requires the use of an antagonist selective for ORL1 receptors. Moreover, a selective antagonist for the OFQ/N receptor is necessary to investigate the role of the endogenous OFQ/N system in the tonic regulation of cardiovascular and renal function. Such an antagonist would provide a means to interrupt an ongoing tonic influence of the endogenous OFQ/N system on each cardiovascular and/or renal regulatory process.

Recently, the structure and in vitro pharmacology of a new selective antagonist of the OFQ/N receptor were described (Guerrini et al., 1998). In these studies, the compound [Phe1Ψ(CH2-NH)Gly2]OFQ/N(1–13)-NH2([FG]OFQ/N(1–13)-NH2) was shown to displace to the right the concentration-response curves of nociceptin in the guinea pig ileum and the mouse vas deferens (Guerrini et al., 1998), two preparations in which ORL1 has been demonstrated and characterized with use of a number of agonists and OFQ/N analogs (Guerrini et al., 1997). Based on these findings, it was suggested that [FG]OFQ/N(1–13)-NH2) is a potent antagonist of the OFQ/N receptor.

The present investigation was performed to further characterize the pharmacological action of [FG]OFQ/N(1–13)-NH2as an antagonist of the effects of OFQ/N at the ORL1 receptor in vivo. For this purpose, we examined the potential changes in systemic cardiovascular and renal excretory functions produced by the i.c.v. administration of [FG]OFQ/N(1–13)-NH2 in conscious Sprague-Dawley rats. Additional studies were performed to determine whether i.c.v. pretreatment of animals with [FG]OFQ/N(1–13)-NH2 prevents the cardiovascular and renal responses produced by i.c.v. injection of OFQ/N [OFQ/N(1–17)], the endogenous ligand of the ORL1 receptor. For comparative purposes, the cardiovascular and renal responses produced by central administration of OFQ/N(1–13)-NH2, a potential metabolite of [FG]OFQ/N(1–13)-NH2, and OFQ/N(2–17), a fragment of OFQ/N proclaimed to be inactive in other biological systems (Matthes et al., 1996; Reinscheid et al., 1996; Champion and Kadowitz, 1997), were also studied.

Materials and Methods

Subjects.

Male Sprague-Dawley rats (Harlan Inc., Indianapolis, IN) weighing between 275 and 300 g were used in these studies. Rats were housed in groups of five or fewer under a 12-h light/dark cycle until the day of the experiments. Rats that had undergone prior surgical procedures were housed in individual cages during the period of recovery. All rats were fed with normal sodium diets (sodium content, 163 mEq/kg) and were allowed tap water ad libitum. All experimental procedures were conducted in accordance with the Louisiana State University Medical Center and the National Institutes of Health guidelines for the care and use of animals.

Surgery.

At 5 to 7 days before experimentation, certain rats were implanted with a stainless steel cannula into the right lateral cerebral ventricle while under anesthesia (30 mg/kg i.m. ketamine in combination with 3 mg/kg i.m. xylazine). The coordinates used for cannula implantation were derived from the atlas of the rat brain byPaxinos and Watson (1986): 0.3 mm posterior to the bregma, 1.3 mm lateral to the midline, and 4.5 mm below the skull surface. Custom-cut and -fabricated guide, dummy (obturator), and internal cannula were purchased from Plastics One, Inc. (Roanoke, VA). The guide cannula was fixed into position with the use of 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 stereotaxic cannula implantation and before experimentation), and after completion of the experimental protocol by injection of dye through the cannula with subsequent postmortem brain section (Koepke and DiBona, 1986; Kapusta et al., 1989, 1997;Kapusta and Obih, 1993).

On the experimental day, rats were anesthetized with methohexital sodium (Brevital, 20 mg/kg i.p. supplemented with 10 mg/kg i.v. as needed; Eli Lilly, Indianapolis, IN). Polyethylene catheters (PE-10 tubing attached to PE-50; Becton Dickinson and Company, Sparks, MD) were then implanted into the left femoral artery and vein for recording of arterial pressure and infusion of isotonic saline, respectively. Through a suprapubic incision, a flanged polyethylene cannula (PE-240, Becton Dickinson and Company) was inserted into the urinary bladder. The bladder catheter was then exteriorized and secured by suturing to adjacent muscle, tissue, and skin. After surgical preparation, rats were placed in rat holders to minimize movement after recovery from anesthesia and to permit steady-state urine collections. An i.v. infusion of isotonic saline (50 μl/min) was then started and continued for the duration of the experiment. At 4 to 6 h after recovery and the start of isotonic saline infusion, the arterial catheter was flushed and attached to a pressure transducer (model P23Db; Statham, Oxnard, CA), and a collection beaker was placed under the bladder catheter. Heart rate was derived from the pulse pressure with a tachograph (model 7 P4H; Grass Instruments, Quincy, MA), and arterial pressure and heart rate were recorded on a Grass model 7 polygraph.

Experimental Protocols.

After stabilization of urine flow rate and urinary sodium excretion, urine was collected during a 20-min control period. After this, the putative OFQ/N receptor antagonist [FG]OFQ/N(1–13)-NH2 was injected i.c.v. (0.1,n = 7; 1, n = 6; or 10 μg total in isotonic saline vehicle, n = 9). Immediately after central administration, urine was collected during seven consecutive 10-min experimental [FG]OFQ/N(1–13)-NH2 urine samples. For studies involving the central administration of [FG]OFQ/N(1−13)-NH2 at a dose of 10 μg, two sources of the drug were tested: Phoenix Pharmaceuticals Inc. (Mountain View, CA) and Tocris Cookson Inc. (Ballwin, MO). HPLC analysis (coinjection of samples from each source) and mass spectra data (molecular weight, 1367.6) demonstrated that samples of [Phe1Ψ(CH2-NH)Gly2]OFQ/N(1–13)-NH2from these two sources were identical (personal communication, J.-K. Chang, Phoenix Pharmaceuticals). Because in these studies, the i.c.v. injection of 10 μg of [FG]OFQ/N(1–13)-NH2from Phoenix Pharmaceuticals and Tocris Cookson Inc. (adjusted for variation in peptide content) produced indistinguishable effects on cardiovascular and renal function, all data collected for 10 μg of [FG]OFQ/N(1–13)-NH2 in these dose-response studies were pooled. Subsequent experiments described below, however, were performed with [FG]OFQ/N(1–13)-NH2obtained from Phoenix Pharmaceuticals, Inc.

Additional studies were also performed in which urine samples were collected during the control period (20 min) and immediately after the i.v. bolus administration of 10 μg of [FG]OFQ/N(1–13)-NH2, the highest dose of this peptide used in the i.c.v. studies. Consecutive experimental urine samples were collected for 80 min (consecutive 10-min periods).

Studies were also performed to determine the ability of [FG]OFQ/N(1−13)-NH2 to prevent the cardiovascular and renal responses evoked by central administration of OFQ/N, the endogenous ligand for the ORL1 receptor. This was performed by examining, in separate group of rats, the changes produced by i.c.v. OFQ/N after i.c.v. pretreatment (20 min) with [FG]OFQ/N(1–13)-NH2. The i.c.v. dose of [FG]OFQ/N(1–13)-NH2 used in these studies (0.1 μg) was determined from the results of the dose-response experiments described above and was shown to have no effects itself on heart rate, mean arterial pressure, or urine flow rate (Fig.1). After the control urine period (20 min), [FG]OFQ/N(1–13)-NH2 (0.1 μg) was injected i.c.v. and allowed 10 min to distribute. A 10-min experimental [FG]OFQ/N(1–13)-NH2 urine sample was then collected. After this urine collection (a total of 20 min of [FG]OFQ/N(1–13)-NH2 pretreatment time), OFQ/N (10 μg total) was injected i.c.v. Consecutive 10-min experimental OFQ/N urine samples then were collected for 80 min. For comparative purposes, in other studies, the experimental protocol was repeated, and the cardiovascular and renal responses produced by the i.c.v. injection of OFQ/N (10 μg) were examined in rats pretreated i.c.v. with isotonic saline vehicle (5 μl).

Figure 1
Figure 1

Dose-response study of the cardiovascular and renal responses produced by the i.c.v. injection of [FG]OFQ/N(1–13)-NH2 in conscious Sprague-Dawley rats. The values are mean ± S.E.M. and illustrate the systemic cardiovascular and renal excretory responses produced by i.c.v. injection of 0.1 μg (●, n = 6), 1 μg (▾,n = 6), or 10 μg (▪, n = 9) per rat of the OFQ/N receptor antagonist [FG]OFQ/N(1–13)-NH2. Urine samples were collected during control (C, 20 min) and immediately after [FG]OFQ/N(1–13)-NH2 injection for 70 min, denoted as time periods 10 to 70 min (consecutive 10-min samples). HR, heart rate; MAP, mean arterial pressure; V, urine flow rate; UNaV, urinary sodium excretion. a,b,cp < .05, significant change from control within the 0.1-, 1-, and 10-μg group, respectively.

Previous studies in our laboratory have shown that in conscious Sprague-Dawley rats under similar experimental conditions, the i.c.v. administration of OFQ/N (1, 10, or 30 μg) produced profound reductions in heart rate, mean arterial pressure, and urinary sodium excretion (all responses were rapid in onset) and a concurrent increase in urine flow rate (delayed response) (Kapusta et al., 1997). In these previous studies, the 10-μg dose of OFQ/N administered centrally produced the diuretic response of the greatest magnitude (Kapusta et al., 1997).

After the i.c.v. injection of [FG]OFQ/N(1–13)-NH2 in conscious rats, it is possible that this peptide is metabolized in the CNS to OFQ/N(1–13)-NH2, a reported agonist at the ORL1 receptor (Calo et al., 1996; Guerrini et al., 1997). Further studies were therefore performed to examine the changes in cardiovascular and renal excretory functions produced by this modified OFQ/N fragment. These studies used the same experimental protocol as that described above for studying the cardiovascular and renal responses produced by i.c.v. injection of [FG]OFQ/N(1–13)-NH2 alone, with the exception that OFQ/N(1–13)-NH2 was administered centrally (10 μg i.c.v.). For comparative purposes, in separate rats, the cardiovascular and renal responses produced by the i.c.v. administration of OFQ/N(2–13) (10 μg) were also examined. OFQ/N(2–17) is a fragment of the authentic OFQ/N peptide [OFQ/N(1–17)] and is reported to be devoid of pharmacological activity in other physiological systems (Matthes et al., 1996;Reinscheid et al., 1996; Champion and Kadowitz, 1997).

Analytical Techniques.

Urine volume was determined gravimetrically. Urine sodium concentration was measured by flame photometry (model 943; Instrumentation Laboratories, Lexington, MA).

Drugs.

OFQ/N(1–13)-NH2, OFQ/N(1–17), and OFQ/N(2–17) were from Phoenix Pharmaceuticals, Inc. The i.c.v. stock solutions of each drug were prepared fresh in isotonic saline vehicle and stored frozen. Injection of drugs in isotonic saline vehicle (5 μl volume) was made with a 10-μl Hamilton syringe. The i.c.v. doses of [FG]OFQ/N(1–13)-NH2 (0.1, 1, or 10 μg total) were derived from previous investigations in which we demonstrated that the i.c.v. administration of OFQ/N produced a maximal diuresis in conscious rats when administered at a dose of 10 μg total (5.528 nmol; molecular weight, 1809) (Kapusta et al., 1997). In these studies, the magnitudes of increase in urine flow rate and decrease in urinary sodium excretion produced by OFQ/N were shown to be similar to those produced by the i.c.v. injection of 10 μg of dynorphin A, an endogenous ligand of the κ-opioid receptor (Kapusta et al., 1997). The physiological effects produced by the in vivo administration of OFQ/N have been studied in other investigations in mice and rats using a similar i.c.v. dose range (0.1–10 μg = 0.0553–5.527 nmol) as those used in the present investigation (Reinscheid et al., 1995; Mogil et al., 1996; Rossi et al., 1997; Mathis et al., 1998).

Synthesis of [Phe1Ψ(CH2-NH)Gly2]OFQ/N(1–13)-NH2.

[Phe1Ψ(CH2-NH)Gly2]OFQ/N(1–13)-NH2was synthesized by solid-phase method usingp-methylbenzhydrylamine as the solid support. Formation of the Phe1Ψ(CH2-NH)Gly2bond was carried out by complexing Boc-phe-CHO to Gly2 of the protected OFQ/N(2–13)-p-methylbenzhydrylamine resin and reduction by NaBH4. After HF cleavage of the protected peptide resin, the crude [Phe1Ψ(CH2-NH)Gly2]OFQ/N(1–13)-NH2was purified by preparative HPLC using 0.1% trifluoroacetic acid and 60% CH3CN in 0.1% trifluoroacetic acid as initial and final buffer, respectively. The final sample purity was more than 95%, and mass spectra molecular weight was 1367.6.

Data Analysis.

The data were analyzed statistically by using repeated measures ANOVA for main effects and interactions. Bonferroni’s test was used for pairwise comparisons between mean values. Statistical significance was defined as P < .05.

Results

Effects of i.c.v. [FG]OFQ/N(1–13)-NH2.

In the present investigation, the i.c.v. injection of [FG]OFQ/N(1–13)-NH2 (0.1–10 μg) or OFQ/N (10 μg) did not produce any overt sedative or behavioral effects (e.g., excitation, enhanced exploratory activity, convulsions) in conscious rats. Although changes in locomotor activity were not apparent in these animals, this may have been concealed by the fact that before drug administration, the rats remained calm and were without locomotor activity due to their placement in rat holders. After the i.c.v. injection of drugs, the rats continued to remain calm and conscious throughout the duration of the protocol. It should be noted, however, that if rats treated i.c.v. with [FG]OFQ/N(1–13)-NH2 or OFQ/N were presented with a food pellet (or nonfood item, such as a wooden applicator), these animals would demonstrate excessive gnawing (personal observation), demonstrating biological activity of the compound.

The cardiovascular and renal responses produced by the i.c.v. administration of the putative OFQ/N receptor antagonist [FG]OFQ/N(1–13)-NH2 in conscious Sprague-Dawley rats are shown in Fig. 1. Mean values for each parameter are depicted during control (C, 20 min) and during consecutive 10-min experimental urine collections (time periods, 10–70 min) beginning immediately after the i.c.v. administration of [FG]OFQ/N(1–13)-NH2, (0.1, 1, or 10 μg total/5 μl isotonic saline vehicle). In previous investigations, we have demonstrated that i.c.v. isotonic saline vehicle does not produce a change in any cardiovascular or renal parameter measured throughout the duration of study, thus demonstrating the stability of the parameters measured under these experimental conditions (Kapusta et al., 1993). Compared with respective group control values for each parameter (Fig. 1), the i.c.v. injection of [FG]OFQ/N(1–13)-NH2 produced dose-related changes in cardiovascular and renal excretory function. The i.c.v. administration of 10 μg of [FG]OFQ/N(1–13)-NH2, the highest dose tested, produced a significant decrease in heart rate (369 ± 10 versus 333 ± 18 beats/min, C versus 30-min time point, respectively) and mean arterial pressure (116 ± 3 versus 100 ± 3 mm Hg, C versus 30-min time point, respectively). The bradycardia and hypotension evoked by 10 μg of [FG]OFQ/N(1–13)-NH2 were sustained over the course of the study (time periods, 20–70 min) and did not return to control levels for an additional 90 to 120 min (i.e., approximately 160–190 min after i.c.v. injection; data not shown). In contrast to these responses, the i.c.v. injection of 1 or 0.1 μg of [FG]OFQ/N(1–13)-NH2 did not produce a significant change in heart rate or mean arterial pressure in conscious rats. Note that 1 μg of [FG]OFQ/N(1–13)-NH2did produce a decrease in mean arterial pressure (116 ± 2 versus 108 ± 4 mm Hg, C versus 20-min time period), although this reduction did not attain statistical significance. Concurrent with these cardiovascular responses, the central administration of [FG]OFQ/N(1–13)-NH2 also produced a profound and dose-dependent diuretic response. A group ANOVA confirmed that the peak diuresis produced by i.c.v. injection of 10 μg of [FG]OFQ/N(1–13)-NH2 (180 ± 15 μl/min, 40 min) was significantly greater (p < .05) than that produced by the 1-μg dose (124 ± 5 μl/min, 30 min). In contrast to these higher doses, the i.c.v. injection of 0.1 μg of [FG]OFQ/N(1–13)-NH2 did not alter urine output at any time. Despite the failure of this low i.c.v. dose to affect urine flow rate, 0.1 μg of [FG]OFQ/N(1–13)-NH2 did produce a significant decrease in urinary sodium excretion 20 min after administration. Similarly, compared with each group control, higher i.c.v. doses of [FG]OFQ/N(1–13)-NH2 (1 and 10 μg) also produced significant antinatriuretic responses. In contrast to these responses, in other animals the i.v. bolus injection of 10 μg of [FG]OFQ/N(1–13)-NH2 (the highest i.c.v. dose tested) did not significantly alter any cardiovascular or renal excretory parameter (data not shown), thereby excluding the possibility that centrally administered [FG]OFQ/N(1–13)-NH2 affected cardiovascular or renal function subsequent to its leakage into the periphery.

Additional studies were performed to determine whether pretreatment of rats with a low i.c.v. dose of [FG]OFQ/N(1–13)-NH2 (0.1 μg) prevents the cardiovascular (bradycardia and hypotension) and/or renal excretory (diuretic and antinatriuretic) responses produced by OFQ/N [OFQ/N(1–17)], the endogenous ligand of ORL1 receptors. For these studies, and as depicted in Fig. 2, a 10-min urine sample was collected in conscious rats starting 10 min after i.c.v. pretreatment with 0.1 μg of [FG]OFQ/N(1–13)-NH2 [pretreatment period (PT), 10 min]. After completion of this PT urine sample, OFQ/N was administered i.c.v. (10 μg total), and consecutive experimental urine samples were collected immediately for 80 min. For comparative purposes, Fig. 2 also shows the cardiovascular and renal responses produced by the i.c.v. injection of 10 μg of OFQ/N in rats pretreated with isotonic saline vehicle. Compared with respective control values, the i.c.v. pretreatment of 0.1 μg of [FG]OFQ/N(1–13)-NH2 itself (pretreatment period, ●) did not affect any cardiovascular or renal excretory function. These findings are in agreement with data presented in Fig.1, which indicates that this low i.c.v. dose of [FG]OFQ/N(1–13)-NH2 does not alter heart rate, mean arterial pressure, or urine flow rate (Fig. 1). After central [FG]OFQ/N(1–13)-NH2 pretreatment (Fig. 2, PT), the i.c.v. injection of 10 μg of OFQ/N produced a significant reduction in heart rate (fast onset and large magnitude change), mean arterial pressure, and urinary sodium excretion and an increase in urine flow rate (delayed onset). Thus, pretreatment of rats with a low i.c.v. dose of [FG]OFQ/N(1–13)-NH2 failed to prevent the cardiovascular and renal excretory responses to central OFQ/N. Note that the pattern and magnitude changes in cardiovascular and renal excretory function produced by OFQ/N in rats pretreated with [FG]OFQ/N(1–13)-NH2 (Fig. 2) are similar to those that occur when the same dose of this drug is administered centrally to rats pretreated i.c.v. with isotonic saline vehicle (Fig.2, ○) or to naı̈ve rats (Kapusta et al., 1997). Two important exceptions, however, are that in rats pretreated with [FG]OFQ/N(1–13)-NH2 (Fig. 2), the bradycardia and hypotension evoked by i.c.v. OFQ/N persisted for a longer duration than those observed in rats pretreated with isotonic saline vehicle (in which heart rate and mean arterial pressure returned to control levels by 40 min after injection) (Fig. 2 and Kapusta et al., 1997). In addition, although i.c.v. OFQ/N typically produces a peak diuretic response in naive rats 40 min after drug injection (Fig. 2 and Kapusta et al., 1997), the pretreatment of animals with low-dose [FG]OFQ/N(1–13)-NH2 delayed the onset and time of peak diuresis (60 min) produced by i.c.v. OFQ/N. It is likely that this delayed diuretic response was related to the prolonged and greater-magnitude hypotensive response produced by OFQ/N in [FG]OFQ/N(1–13)-NH2-pretreated rats (Fig. 2).

Figure 2
Figure 2

Effects of i.c.v. [FG]OFQ/N(1–13)-NH2or isotonic saline vehicle pretreatment on OFQ/N-induced cardiovascular and renal responses in Sprague-Dawley rats. The values are mean ± S.E.M. and illustrate the systemic cardiovascular and renal excretory responses produced by i.c.v. OFQ/N administration (10 μg) in conscious rats pretreated with the putative OFQ/N receptor antagonist [FG]OFQ/N(1–13)NH2 (●, 0.1 μg, i.c.v.,n = 6) or isotonic saline vehicle (○, 5 μl,n = 6). Urine samples were collected during control (C, 20 min) and 10 min after i.c.v. injection of [FG]OFQ/N(1–13)-NH2 or vehicle (pretreatment period, PT). After the PT urine collection, OFQ/N was injected i.c.v. Consecutive 10-min urine samples were then collected immediately after OFQ/N injection, denoted as time periods 10 to 80 min. HR, heart rate; MAP, mean arterial pressure; V, urine flow rate; UNaV, urinary sodium excretion. *P < .05, significantly different from corresponding PT value for each group.

Figure 3 illustrates the cardiovascular and renal responses produced by the central administration of OFQ/N(1–13)-NH2 (a potential metabolite of [FG]OFQ/N(1–13)-NH2) and OFQ/N(2–17) [a fragment of the endogenous ligand OFQ/N(1–17) in conscious rats]. As shown in Fig. 3, the i.c.v. injection of 10 μg of OFQ/N(1–13)-NH2 produced a similar pattern and magnitude decrease in heart rate, mean arterial pressure, and urinary sodium excretion and increase in urine flow rate as that produced by the i.c.v. administration of 10 μg of [FG]OFQ/N(1–13)-NH2 (Fig. 1). The bradycardia and hypotension produced by central OFQ/N(1–13)-NH2 were, however, transient and returned to control levels 30 and 50 min after injection, respectively In contrast to these changes, in other animals the i.c.v. injection of 10 μg of OFQ/N(2–17) did not alter any cardiovascular or renal excretory parameter (Fig. 3).

Figure 3
Figure 3

Effects of i.c.v. administration of OFQ/N(1–13)-NH2 and OFQ/N(2–17) on cardiovascular and renal functions in Sprague-Dawley rats. The values are mean ± S.E.M. and illustrate the systemic cardiovascular and renal excretory responses produced by i.c.v. injection of OFQ/N(1–13)-NH2(●, 10 μg, n = 5) or OFQ/N(2–17) (○, 10 μg, n = 5) in conscious Sprague-Dawley rats. Urine samples were collected during control (C, 20 min) and immediately after i.c.v. drug injection, denoted as time periods 10 to 80 min (consecutive 10-min periods). HR, heart rate; MAP, mean arterial pressure; V, urine flow rate; UNaV, urinary sodium excretion. *P < .05, significantly different from corresponding control.

In other studies, it was observed that the i.c.v. pretreatment of rats with the selective κ-opioid receptor antagonist, norbinaltorphimine (1 μg total), did not prevent the cardiovascular or renal responses produced by [FG]OFQ/N(1–13)-NH2 (10 μg). We have previously shown that this i.c.v. dose of norbinaltorphimine completely prevents the cardiovascular and renal responses produced by the i.c.v. injection of 10 μg of dynorphin A(1–17) (an endogenous κagonist) but not OFQ/N (Kapusta et al., 1997).

Discussion

[FG]OFQ/N(1–13)-NH2 has recently been reported to be the first selective antagonist of the receptor for the endogenous opioid-like peptide OFQ/N (Guerrini et al., 1998). As an extension of these in vitro investigations, the present study examined the potential physiological effects of this putative OFQ/N receptor antagonist in vivo. In conscious Sprague-Dawley rats, the i.c.v. injection of [FG]OFQ/N(1–13)-NH2 produced marked changes in cardiovascular and renal excretory functions. The cardiovascular and renal excretory responses were dose dependent, with 0.1 μg of [FG]OFQ/N(1–13)-NH2 affecting only urinary sodium excretion, 1 μg of [FG]OFQ/N(1–13)-NH2 producing a concurrent diuretic and antinatriuretic response and transient decrease in mean arterial pressure (but not heart rate), and 10 μg of [FG]OFQ/N(1–13)-NH2 (the highest dose tested) producing profound and sustained reductions in heart rate, mean arterial pressure, and urinary sodium excretion and an increase in urine flow rate. The changes in cardiovascular and renal function produced by the i.c.v. injection of 10 μg of [FG]OFQ/N(1–13)-NH2 were shown to be mediated via an action of the drug in the CNS because the i.v. bolus injection of this same dose did not elicit a change in either physiological parameter. The ability of [FG]OFQ/N(1–13)-NH2to produce significant centrally mediated changes in cardiovascular and renal function in conscious rats demonstrates that this proposed ORL1 antagonist is active physiologically.

Despite the findings noted above, the patterns of cardiovascular and renal excretory responses produced by i.c.v. [FG]OFQ/N(1–13)-NH2 are in opposition to those that would have been predicted based on the presumed antagonist effect of this compound at ORL1 receptors. For instance, under similar experimental conditions, we have previously shown that activation of central ORL1 receptor systems evoked by the i.c.v. injection of OFQ/N produces a significant reduction in heart rate, mean arterial pressure, and urinary sodium excretion and a marked increase in urine flow rate (Kapusta et al., 1997). Based on these findings in which ORL1 receptors are activated, it would be predicted that the i.c.v. administration of a selective OFQ/N receptor antagonist alone (i.e., in the absence of exogenous OFQ/N) may evoke a change in any of these cardiovascular or renal excretory parameters but in a direction opposite that produced by i.c.v. OFQ/N. This would specifically be the case if [FG]OFQ/N(1–13)-NH2 were to interrupt an ongoing tonic influence of endogenous central OFQ/N systems in the control of cardiovascular and/or renal function. Alternatively, if the endogenous OFQ/N and ORL1 systems do not participate in the tonic regulation of these physiological processes (in conscious rats under our experimental conditions), then blockade of ORL1 with an antagonist selective for this receptor would not evoke a change in any cardiovascular or renal excretory parameter. In contrast to these possibilities, the i.c.v. injection of [FG]OFQ/N(1–13)-NH2 evoked marked cardiovascular and renal excretory responses (dose dependent; Fig. 1) that were similar in direction and pattern as those produced by the agonist OFQ/N (Kapusta et al., 1997). Therefore, these findings indicate that when administered centrally in vivo, [FG]OFQ/N(1–13)-NH2 has agonist properties similar to those elicited by OFQ/N.

Additional studies were performed in the present investigation to determine whether [FG]OFQ/N(1–13)-NH2 is an antagonist at the ORL1 receptor when administered centrally at a dose that is inactive physiologically. This premise was tested because other compounds (e.g., naloxone benzoylhydrazone) have been reported to antagonize the effects of OFQ/N at ORL1 receptors in vitro when administered at a concentration that is without agonist action (Dunnill et al., 1996). In our studies, the i.c.v. pretreatment of rats with 0.1 μg of [FG]OFQ/N(1–13)-NH2, a dose that does not effect heart rate, mean arterial pressure, or urine flow rate (Fig.1), failed to prevent or attenuate the cardiovascular (rapid-onset bradycardia and hypotension) or renal excretory (diuresis and antinatriuresis) responses produced by i.c.v. OFQ/N (Fig. 2). However, compared with our previous findings (Kapusta et al., 1997), low-dose [FG]OFQ/N(1–13)-NH2 pretreatment prolonged the time course of the bradycardia and hypotensive response evoked by i.c.v. OFQ/N but did not alter the time of onset or the peak change (Fig. 2). In addition, [FG]OFQ/N(1–13)-NH2pretreatment delayed the onset, time of peak magnitude change, and total duration of the diuretic response produced by OFQ/N (10 μg; Fig. 2). Although not studied in this investigation, the altered time course of the diuresis may be related to the prolonged hypotensive response produced by i.c.v. OFQ/N in [FG]OFQ/N(1–13)-NH2-pretreated rats. Despite these variances, these findings provide further physiological evidence to indicate that [FG]OFQ/N(1–13)-NH2 is devoid of antagonist action at the OFQ/N receptor (presumably ORL1) when administered centrally in vivo.

At present, it is not known why the results we obtain from the i.c.v. administration of [FG]OFQ/N(1–13)-NH2 in the intact rat, which show agonist actions, differ from those of Guerrini et al. (1998), which showed an apparent competitive antagonism of the actions of OFQ/N on the electrically stimulated guinea pig isolated ileum and mouse isolated vas deferens. One possibility is that [FG]OFQ/N(1–13)-NH2 is a much lower efficacy agonist than OFQ/N. If the receptor density and/or coupling of the ORL1 receptor is much lower in the ileum and vas deferens than in the CNS, then [FG]OFQ/N(1–13)-NH2, as a low efficacy agonist, could exhibit an apparent competitive antagonism of OFQ/N. Such has been shown to be the case for the low-efficacy β agonist prenalterol (Kenakin and Beek, 1980, 1984). Other possibilities are that there are multiple subtypes of the ORL1 receptor (Bunzow et al., 1994; Mollereau et al., 1994; Pan et al., 1994, 1995; Wang et al., 1994; Wick et al., 1994; Mathis et al., 1997) and that [FG]OFQ/N(1–13)-NH2 and OFQ/N differ in their selectivity and/or efficacies at these sites. It is possible that OFQ/N shows only agonist actions at each of the ORL1 receptor subtypes, whereas [FG]OFQ/N(1–13)-NH2 may show agonist as well as antagonist actions. Agents that exhibit agonist actions at one subtype and antagonist actions at another subtype of the same receptor are well known (Gergen et al., 1996; Caudle et al., 1997). Further studies are clearly required to understand the mechanisms by which [FG]OFQ/N(1–13)-NH2 interacts with the OFQ/N receptor in different tissues.

One last consideration is that when administered centrally, [FG]OFQ/N(1–13)-NH2 may be metabolized to an analog of OFQ/N that is an agonist at the ORL1 receptor. As reported byGuerrini et al. (1997, 1998), [FG]OFQ/N(1–13)-NH2 was discovered in the frame of a structure-activity study on the OFQ/N fragment OFQ/N(1–13)-NH2. In these investigations, [FG]OFQ/N(1–13)-NH2 was designed in an attempt to protect the OFQ/N fragment OFQ/N(1–13)-NH2from aminopeptidase degradation. Although [FG]OFQ/N(1–13)-NH2 appears to be resistant to enzymatic cleavage in the guinea pig isolated ileum and mouse isolated vas deferens (Guerrini et al., 1998), this peptide may be metabolized to OFQ/N(1–13)-NH2 when administered centrally in vivo. This possibility is of concern because OFQ/N(1–13)-NH2 is a potent OFQ/N receptor agonist in different biological systems (Calo et al., 1996, 1997). Thus, in the present study, we also examined the cardiovascular and renal responses produced by the central administration of this potential metabolite. In these experiments (Fig. 3), i.c.v. injection of OFQ/N(1–13)-NH2 (10 μg) also produced marked changes in cardiovascular and renal function similar in direction, magnitude, and time course to those produced by [FG]OFQ/N(1–13)-NH2 (10 μg; Fig. 1). In contrast, the OFQ/N fragment OFQ/N(2-17) did not alter cardiovascular or renal function when injected i.c.v. in conscious rats (Fig. 3), thus confirming that this peptide fragment is inactive in different biological systems (Matthes et al., 1996; Reinscheid et al., 1996;Champion and Kadowitz, 1997). These findings suggest that the agonist effects of [FG]OFQ/N(1–13)-NH2 observed in our physiological studies may be mediated, at least in part, by the potential CNS metabolism of [FG]OFQ/N(1–13)-NH2 to OFQ/N(1–13)-NH2. Although our results demonstrate a central action of [FG]OFQ/N(1–13)-NH2 and OFQ/N, it remains to be established which brain sites (e.g., paraventricular nucleus of the hypothalamus, rostral ventrolateral medulla, anteroventral region of the third ventricle, and so on) are involved in mediating the changes in cardiovascular and renal function produced by these compounds and potential metabolites.

In conclusion, we examined the cardiovascular and renal responses produced by the central administration of the putative OFQ/N receptor antagonist [FG]OFQ/N(1–13)-NH2 in vivo. In conscious Sprague-Dawley rats, the i.c.v. injection of [FG]OFQ/N(1–13)-NH2 produced profound dose-dependent changes in cardiovascular (bradycardia and hypotension) and renal excretory (diuresis and antinatriuresis) function that were similar to those produced by OFQ/N, the endogenous ligand of ORL1 receptors. These findings suggest that the pattern of cardiovascular and renal responses produced by i.c.v. [FG]OFQ/N(1–13)-NH2 result from the agonist effects of this compound at the OFQ/N receptor (presumably the ORL1 receptor). In other studies, the i.c.v. pretreatment of animals with a physiologically inactive low dose of [FG]OFQ/N(1–13)-NH2 did not prevent the cardiovascular or renal excretory responses produced by central [FG]OFQ/N(1–13)-NH2 administration. Although [FG]OFQ/N(1–13)-NH2 is reported to be an antagonist of the OFQ/N receptor in vitro, these findings indicate that this compound has agonist activity similar to that of the endogenous ligand OFQ/N when administered centrally in vivo. Further studies designed to investigate the role of the central endogenous OFQ/N system in various physiological and pathological processes must await the development of a compound that retains selective antagonist effects at the OFQ/N receptor.

Footnotes

  • Send reprint requests to: Daniel R. Kapusta, Ph.D., Department of Pharmacology, Louisiana State University Medical Center, 1901 Perdido St., New Orleans, LA 70112. E-mail: dkapus{at}lsumc.edu

  • ↵1 This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-43337 and American Heart Association, Louisiana Affiliate, Grant 91-6-08B (to D.R.K.).

  • Abbreviations:
    CNS
    central nervous system, OFQ/N, orphanin FQ/nociceptin, [FG]OFQ/N(1–13)-NH2, [Phe1Ψ(CH2-NH)Gly2]OFQ/N(1–13)-NH2, ORL1, opioid receptor-like 1
    • Received July 1, 1998.
    • Accepted November 4, 1998.

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Central Administration of [Phe1Ψ(CH2-NH)Gly2]Nociceptin(1-13)-NH2and Orphanin FQ/Nociceptin (OFQ/N) Produce Similar Cardiovascular and Renal Responses in Conscious Rats

Daniel R. Kapusta, Jaw-Kang Chang and Velga A. Kenigs
Journal of Pharmacology and Experimental Therapeutics April 1, 1999, 289 (1) 173-180;
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