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Vol. 289, Issue 1, 173-180, April 1999
(CH2-NH)Gly2]Nociceptin(1-13)-NH2
and Orphanin FQ/Nociceptin (OFQ/N) Produce Similar Cardiovascular and
Renal Responses in Conscious Rats1
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Medical Center, New Orleans, Louisiana (D.R.K., V.A.K.); and Phoenix Pharmaceuticals Inc., Mountain View, California (J.-K.C.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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In vitro studies have shown that
[Phe1
(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)-NH2
produced 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.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Native
and synthetic opioid agonists exert their pharmacological and
physiological effects through binding to µ-, 
-, and/or 
-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-, -, and -opioid
receptors (Mollereau et al., 1994). Despite this similarity, native
opioid peptides or synthetic opioid agonists selective for µ-, -,
or -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 -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-, -, or -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-, -, and/or -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
[Phe1(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)-NH2
as 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.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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 by
Paxinos 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).
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
[Phe1(CH2-NH)Gly2]OFQ/N(1-13)-NH2
from 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)-NH2 from 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)-NH2
obtained from Phoenix Pharmaceuticals, Inc.
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).
|
). 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 -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 [Phe1(CH2-NH)Gly2]OFQ/N(1-13)-NH2.
[Phe1(CH2-NH)Gly2]OFQ/N(1-13)-NH2
was synthesized by solid-phase method using
p-methylbenzhydrylamine as the solid support. Formation of the
Phe1(CH2-NH)Gly2
bond 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
[Phe1(CH2-NH)Gly2]OFQ/N(1-13)-NH2
was 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.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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)-NH2
did 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 naïve 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).
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-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
agonist) but not OFQ/N (Kapusta et al., 1997).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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[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)-NH2 to 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)-NH2
pretreatment 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 
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 by
Guerrini 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)-NH2
from 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 |
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Accepted for publication November 4, 1998.
Received for publication July 1, 1998.
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.).
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
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Abbreviations |
|---|
CNS, central nervous system, OFQ/N, orphanin
FQ/nociceptin, [FG]OFQ/N(1-13)-NH2,
[Phe1(CH2-NH)Gly2]OFQ/N(1-13)-NH2,
ORL1, opioid receptor-like 1.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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, or opioid receptor type.
FEBS Lett
347:
284-288[Medline].-opioid and ORL1 receptors in isolated tissues from rat and guinea-pig.
Br J Pharmacol
119:
275P.-, -Opioid receptors and their subtypes: A critical review with emphasis on radioligand binding experiments.
Neurochem Int
24:
401-426[Medline]., and opioid receptors.
Mol Brain Res
27:
37-44[Medline].
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,
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,c p < .05, significant change from control within the 0.1-, 1-, and 10-µg group,
respectively.
