- Email: [email protected]
Decreases in Systemic Arterial and Hindquarters Perfusion Pressure in Response to Nociceptin Are Not Inhibited by Naloxone in the Rat
Peptides, Vol. 18, No. 8, pp. 1197–1200, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/97 $17.00 1 .00
PII S0196-9781(97)00178-2
Decreases in Systemic Arterial and Hindquarters Perfusion Pressure in Response to Nociceptin Are Not Inhibited by Naloxone in the Rat M. A. CZAPLA, H. C. CHAMPION AND P. J. KADOWITZ1 Departments of Pharmacology and Physiology, Tulane University School of Medicine, New Orleans, LA 70112 Received 18 February 1997; Accepted 20 May 1997 CZAPLA, M. A., H. C. CHAMPION AND P. J. KADOWITZ. Decreases in systemic arterial and hindquarters perfusion pressure in response to nociceptin are not inhibited by naloxone in the rat. PEPTIDES 18(8) 1197–1200, 1997.—Nociceptin, the endogenous ligand for the ORL1 receptor, has been shown to decrease systemic arterial and hindquarters perfusion pressures in the rat. The present study was undertaken to determine if decreases in systemic arterial and hindquarters perfusion pressures, in response to nociceptin, are mediated by a naloxone-sensitive mechanism. Injections of nociceptin decreased systemic arterial and hindquarters perfusion pressures in a dose-related manner. The decreases in systemic arterial and hindquarters perfusion pressure in response to nociceptin were not altered by the administration of naloxone in a dose of 2 mg/kg IV. Met-enkephalin decreased systemic arterial and hindquarters perfusion pressures and responses to the opioid receptor agonist were significantly reduced by naloxone, whereas decreases in systemic arterial pressure in response to the nitric oxide donor, DEA/NO, were not altered. The results of the present study show that decreases in systemic arterial and hindquarters perfusion pressure in response to nociceptin are not mediated by a naloxone-sensitive mechanism in the rat. © 1997 Elsevier Science Inc. Nociceptin (orphanin FQ)
Naloxone
Systemic vascular bed
THE heptadecapeptide nociceptin, also known as orphanin FQ, is a newly discovered endogenous ligand that exhibits marked homology to the enkephalin, dynorphan, and endorphan opioid peptides and is an endogenous ligand for the ORL1 receptor (1,4,7–9,12,14 –20). The ORL1 receptor shares sequence homology with the m-, d-, or k-opioid receptors, but does not bind the m-, d-, or k-opioid agonists with high affinity (12,19). Nociceptin shares sequence homology with the m-, d-, or kopioid receptor agonists and has been shown to reverse stressinduced antinociception in the rat, suggesting that it is an opioid antagonist peptide (13). Nociceptin has been shown to bind to a single site on the ORL1 receptor with high affinity in a saturable manner, but does not bind to m-, d-, or k-opioid receptors (12,19). It has been reported that nociceptin binds to the ORL1 receptor, increasing potassium currents and inhibiting voltage-gated calcium channels in Xenopus oocytes and neuroblastoma cell lines (5,11). These effects of nociceptin, however, were not inhibited by the opioid antagonist, naloxone, suggesting a non-m-opioid receptor mechanism (5,11). Although studies have shown that nociceptin decreases systemic arterial and hindquarters perfusion pressure in the rat, little, if anything is known about the receptor-mediating responses to the peptide (2,3,6). The present study was therefore undertaken to investigate the effects of naloxone on decreases in 1
Hindquarters vascular bed
Hypotensive response
systemic arterial and hindquarters perfusion pressure in response to nociceptin in the rat. METHOD
Sprague–Dawley rats of either sex weighing 370 – 435 g were anesthetized with pentobarbital sodium (50 mg/kg, IP). Supplemental doses of pentobarbital were given as needed to maintain a uniform level of anesthesia. The trachea was cannulated and the rats were ventilated with a Harvard model 683 rodent ventilator at a tidal volume of 2.5 ml and at a rate of 30 breaths/min with room air enriched with 95% O2:5% CO2. Catheters were inserted into an external jugular vein for the IV administration of drugs and into the carotid artery for the measurement of systemic arterial pressure. Systemic arterial pressure was measured with a Viggo– Spectramed transducer (Viggo–Spectramed, Oxnard, CA) and was recorded on a Grass model 7 polygraph (Grass Instrument Co., Quincy, MA). Mean pressures were derived by electronic averaging. For constant-flow perfusion of the hindquarters vascular bed, a 1.0- to 1.5-cm segment of the distal aorta was exposed through a ventral midline incision and cleared of surrounding connective tissue. After administration of heparin sodium (1000U/kg, IV), the aorta was ligated, and catheters were inserted into the aorta both proximal and distal to the ligature. Blood was withdrawn from the
Requests for reprints should be addressed to Philip J. Kadowitz, Ph.D., Department of Pharmacology, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112.
1197
1198
CZAPLA, CHAMPION AND KADOWITZ
FIG. 1. Bar graphs showing the influence of naloxone on decreases in systemic arterial pressure in response to IV injections of nociceptin (1–30 nmol/kg), Met-enkephalin (100 nmol/kg), and DEA/NO (10 mg/kg). Responses were determined before and after administration of the opioid receptor antagonist in a dose of 2 mg/kg IV. n indicates the number of animals. The asterisk indicates the response is significantly different from control.
proximal catheter and pumped at a constant flow rate with a Masterflex Pump (Cole–Parmer, Chicago, IL) into the distal aortic catheter. Perfusion pressure was monitored from a lateral tap in the perfusion circuit located between the pump and the distal catheter. Agonists were injected directly into the hindquarters perfusion circuit distal to the pump in small volumes (30 –100 ml) in a random sequence. Hindquarters perfusion and systemic arterial pressures were measured with Viggo–Spectramed transducers and were recorded on a Grass model 7 polygraph. Mean pressures were derived from the pulsatile signals by electronic averaging. Hindquarters blood flow was set to achieve a baseline perfusion pressure of approximately 125 mmHg and was not changed during the remainder of an experiment. The flow rate was determined by timed collection and ranged from 3 to 6 ml/min. The hindquarters vascular bed was denervated by ligating and cutting the lumbar sympathetic chain ganglia between L2 and L4. Blood gases were measured periodically and were within the normal physiological range. Nociceptin (orphanin FQ) and Met-enkephalin (Phoenix Pharmaceuticals, Mountain View, CA) were dissolved in 0.9% NaCl and the solutions were divided into aliquots and stored in 1-ml plastic tubes in a freezer. The aliquots were thawed and used on the day of the experiment. Diethylamine/nitric oxide complex sodium (DEA/NO) (Research Biochemical International, Natick, MA), naloxone hydrochloride (DuPont Pharmaceuticals, Wilmington, DE), and acetylcholine chloride (Sigma Chemical Co., St. Louis, MO) were dissolved in 0.9% NaCl. During an experiment, the
agonist solutions were kept on crushed ice. The agonists were administered IV in small volumes (30 –100 ml) over a period of 10 –15 s in a random sequence. Control experiments with the saline vehicle had no effect on systemic arterial or hindquarters perfusion pressure or on responses to the vasoactive agonists. Responses were expressed as change from baseline in mmHg and were analyzed using a paired t-test. A p value of less than 0.05 was used as the criterion for statistical significance. RESULTS
The effects of naloxone on decreases in systemic arterial pressure and on decreases in hindquarters perfusion pressure in response to nociceptin were investigated and these data are summarized in Figs. 1 and 2. Injections of nociceptin in doses of 1–30 nmol/kg IV decreased systemic arterial pressure in a dose-related manner and injections of Met-enkephalin (100 nmol/kg, IV) and DEA/NO (10 mg/kg, IV) decreased systemic arterial pressure. Following administration of naloxone in a dose of 2 mg/kg IV, decreases in systemic arterial pressure in response to Met-enkephalin were reduced significantly, whereas decreases in systemic arterial pressure in response to nociceptin and to DEA/NO were not changed (Fig. 1). Nociceptin decreased hindquarters perfusion pressure when injected in doses of 1–30 nmol into the perfusion circuit and the decreases in perfusion pressure in response to nociceptin were not altered after administration of naloxone (2 mg/kg, IV) (Fig. 2). The decrease in hindquarters perfusion pres-
NALOXONE-INSENSITIVE RESPONSES TO NOCICEPTIN
1199
FIG. 2. Bar graphs showing decreases in hindquarters perfusion pressure in response to injections of nociceptin (1–30 nmol), Met-enkephalin (100 nmol), and acetylcholine (10 ng) into the hindquarters perfusion circuit. Responses were determined before and after administration of the opioid receptor antagonist, naloxone, in a dose of 2 mg/kg IV. n indicates the number of experiments. The asterisk indicate that the response is significantly different from control.
sure in response to Met-enkephalin (100 nmol) was reduced significantly by naloxone, whereas the opioid receptor antagonist had no significant effect on the decrease in perfusion pressure in response to acetylcholine (Fig. 2). DISCUSSION
The results of the present study demonstrate that decreases in systemic arterial pressure and hindquarters perfusion pressure in response to nociceptin are not altered by the administration of naloxone in a dose of 2 mg/kg IV. These results suggest that responses to nociceptin are not mediated by the activation of a m-, d-, or k-opioid receptor in the systemic or hindquarters vascular bed of the rat. Met-enkephalin activates the d-opioid receptor and produces significant decreases in systemic arterial and hindquarters perfusion pressure in the rat. The observation that decreases in systemic arterial pressure in response to Met-enkephalin are attenuated by naloxone, whereas the response to acetylcholine were not altered, suggests that vasodepressor responses to Met-enkephalin are mediated by a naloxone-sensitive mechanism. Moreover, because blood flow was maintained constant in experiments in the hindquarters vascular bed, the decreases in perfusion pressure in response to the opioid receptor agonist reflect decreases in hind-
quarters vascular resistance. The observation that responses to Met-enkephalin, but not to acetylcholine, are inhibited by naloxone suggests that vasodilator responses to Met-enkephalin are mediated by an opioid receptor and that the inhibitory effects of naloxone are selective in nature. It has been reported that nociceptin inhibits the opening of voltage-dependent calcium channels in neuroblastoma and hippocampal cell lines and that this effect is insensitive to the opioid receptor antagonist, naloxone (5,10). The results with naloxone in neuroblastoma and hippocampal cell lines are consistent with the present results in the systemic and hindquarters vascular bed of the rat and provide support for the hypothesis that responses to nociceptin are not mediated by the activation of classical m-, d-, or k-opioid receptors. In summary, the results of the present study demonstrate that nociceptin, also known as orphanin FQ, decreases systemic arterial and hindquarters perfusion pressure in the rat and that responses to nociceptin are not attenuated by the opioid receptor antagonist, naloxone. Naloxone attenuated decreases in systemic arterial and hindquarters perfusion pressure in response to the opioid receptor agonist, Met-enkephalin, but did not alter responses to the nitric oxide donor, DEA/NO, or to acetylcholine. These data indicate
1200
CZAPLA, CHAMPION AND KADOWITZ
that decreases in systemic arterial and hindquarters perfusion pressure in response to nociceptin are not mediated by the activation of a naloxone-sensitive receptor in the rat.
ACKNOWLEDGMENTS
These studies were supported, in part, by NIH Grant HL15580 and a grant from the American Heart Association-Louisiana, Inc.
REFERENCES 1. Bunzow, J. R.; Saez, C.; Mortrud, M.; Bouvier, C.; Williams, J. T.; Low, M.; Grandy, D. K. Molecular cloning and tissue distribution of a putative member of the rat opiod receptor gene family that is not a mu, delta, or kappa opioid receptor type. FEBS Lett. 347:284 –288; 1994. 2. Champion, H. C.; Czapla, M. A.; Kadowitz, P. J. Nociceptin, an endogenous ligand for the ORL1 receptor, decreases cardiac output and total peripheral resistance in the rat. Peptides 18:729 –732; 1997. 3. Champion, H. C.; Kadowitz, P. J. Nociceptin, an endogenous ligand for the ORL1 receptor, has novel hypotensive activity in the rat. Life Sci. 60:PL241–PL245; 1997. 4. Chen, Y.; Fan, Y.; Liu, J.; Mestek, A.; Tian, M.; Kozak, C. A.; Yu, L. Molecular cloning, tissue distribution and chomasomal localization of a novel member of the opioid receptor gene family. FEBS Lett. 347:279 –283; 1994. 5. Connor, M.; Yeo, A.; Henderson, G. The effect of nociceptin on Ca21 channel current and intracellular Ca21 in the SH-SY5Y human neuroblastoma cell line. Br. J. Pharmacol. 118:205–207; 1996. 6. Czapla, M. A.; Champion, H. C.; Kadowitz, P. J. Nociceptin, an endogenous ligand for the ORL1 receptor, has vasodilator activity in the hindquarters vascular bed of the rat. Peptides 18:793–795; 1997. 7. Fukuda, K.; Kato, S.; Mori, K.; Nishi, M.; Takeshima, H.; Iwabe, N.; Miyata, T.; Houtani, T.; Sugimoto, T. cDNA cloning and regional distribution of a novel member of the opioid receptor family. FEBS Lett. 343:42– 46; 1994. 8. Halford, W. P.; Gebhardt, B. M.; Carr, D. J. J. Functional role and sequence analysis of a lymphocyte orphan opioid receptor. J. Neuroimmunol. 59:91–101; 1995. 9. Houtani, T.; Nishi, M.; Takeshima, H.; Nukada, T.; Sugimoto, T. Structure and regional distribution of nociceptin/orphanin FQ precursor. Biochem. Biophys. Res. Commun. 219:714 –719; 1996. 10. Knoflach, F.; Reinscheid, R. K.; Civelli, O.; Kemp, J. A. Modulation of voltage-gated calcium channels by orphanin FQ in freshly dissociated hippocampal neurons. J. Neurosci. 16(21):6657– 6664; 1996. 11. Matthes, H.; Seward, E. P.; Kieffer, B.; North, R. A. Functional selectivity of orphanin FQ for its receptor coexpressed with potassium
12.
13. 14.
15.
16.
17. 18. 19.
20.
channel subunits in Xenopus laevis oocytes. Mol. Pharmacol. 50:447– 450; 1996. Meunier, J.; Mollereau, C.; Toll, L.; Suaudeau, C.; Moisand, C.; Alvinerie, P.; Butour, J.; Guillemot, J.; Ferrara, P.; Monsarrat, B.; Mazaguil, H.; Vassart, G.; Parmentier, M.; Constetin, J. Isolation and structure of the endogenous agonist of opiod receptor-like ORL1 receptor. Nature 377:532–535; 1995. Mogil, J.; Grisel, J.; Reinscheid, R.; Civelli, O.; Belknap, J.; Grandy, D. Orphanin FQ is a functional anti-opioid peptide. Neuroscience 75(2):333–337; 1996. Mollereau, C.; Parmentier, M.; Mailleaux, P.; Butour, J. L.; Moisand, C.; Chalon, P.; Caput, D.; Vassart, G.; Meunier, J. C. ORL1, a novel member of the opioid receptor family. Cloning functional expression and localization. FEBS Lett. 341:33–38; 1994. Mollereau, C.; Simons, M. J.; Soularue, P.; Liners, F.; Vassart, G.; Meunier, J. C.; Paramentier, M. Structure, tissue distribution, and chromasomal localization of the prenociceptin gene. Proc. Natl. Acad. Sci. USA 93:8666 – 8670; 1996. Nothacker, H. P.; Reinscheid, R. K.; Mansour, A.; Henningsen, R. A.; Ardati, A.; Monsma, F. J.; Watson, S. J.; Civelli, O. Primary structure and tissue distribution of the orphanin FQ precursor. Proc. Natl. Acad. Sci. USA 93:8677– 8682; 1996. Pan, Y. X.; Xu, J.; Pasternak, G. W. Cloning and expression of a cDNA encoding a mouse brain orphanin FQ/nociceptin precursor. Biochem. J. 315:11–13; 1996. Reinscheid, R. K.; Ardati, A.; Monsma, F. J.; Civelli, O. Structure– activity relationship studies on the novel neuropeptide orphanin FQ. J. Biol. Chem. 271:14163–14168; 1996. Reinscheid, R. K.; Nothacker, H.; Bourson, A.; Ardati, A.; Henningsen, R. A.; Bunzow, J. R.; Grandy, D. K.; Langen, H.; Monsma, F. J.; Civelli, O. Orphanin FQ: A neuropeptide that activates an opioidlike G protein-coupled receptor. Science 270:792–794; 1995. Wang, J. B.; Johnson, P. S.; Imai, Y.; Persico, A. M.; Ozenberger, B. A.; Eppler, C. M.; Uhl, G. R. cDNA cloning of an orphan opioid receptor gene family member and its slice variant. FEBS Lett. 348: 75–79; 1994.
PII S0196-9781(97)00178-2
Decreases in Systemic Arterial and Hindquarters Perfusion Pressure in Response to Nociceptin Are Not Inhibited by Naloxone in the Rat M. A. CZAPLA, H. C. CHAMPION AND P. J. KADOWITZ1 Departments of Pharmacology and Physiology, Tulane University School of Medicine, New Orleans, LA 70112 Received 18 February 1997; Accepted 20 May 1997 CZAPLA, M. A., H. C. CHAMPION AND P. J. KADOWITZ. Decreases in systemic arterial and hindquarters perfusion pressure in response to nociceptin are not inhibited by naloxone in the rat. PEPTIDES 18(8) 1197–1200, 1997.—Nociceptin, the endogenous ligand for the ORL1 receptor, has been shown to decrease systemic arterial and hindquarters perfusion pressures in the rat. The present study was undertaken to determine if decreases in systemic arterial and hindquarters perfusion pressures, in response to nociceptin, are mediated by a naloxone-sensitive mechanism. Injections of nociceptin decreased systemic arterial and hindquarters perfusion pressures in a dose-related manner. The decreases in systemic arterial and hindquarters perfusion pressure in response to nociceptin were not altered by the administration of naloxone in a dose of 2 mg/kg IV. Met-enkephalin decreased systemic arterial and hindquarters perfusion pressures and responses to the opioid receptor agonist were significantly reduced by naloxone, whereas decreases in systemic arterial pressure in response to the nitric oxide donor, DEA/NO, were not altered. The results of the present study show that decreases in systemic arterial and hindquarters perfusion pressure in response to nociceptin are not mediated by a naloxone-sensitive mechanism in the rat. © 1997 Elsevier Science Inc. Nociceptin (orphanin FQ)
Naloxone
Systemic vascular bed
THE heptadecapeptide nociceptin, also known as orphanin FQ, is a newly discovered endogenous ligand that exhibits marked homology to the enkephalin, dynorphan, and endorphan opioid peptides and is an endogenous ligand for the ORL1 receptor (1,4,7–9,12,14 –20). The ORL1 receptor shares sequence homology with the m-, d-, or k-opioid receptors, but does not bind the m-, d-, or k-opioid agonists with high affinity (12,19). Nociceptin shares sequence homology with the m-, d-, or kopioid receptor agonists and has been shown to reverse stressinduced antinociception in the rat, suggesting that it is an opioid antagonist peptide (13). Nociceptin has been shown to bind to a single site on the ORL1 receptor with high affinity in a saturable manner, but does not bind to m-, d-, or k-opioid receptors (12,19). It has been reported that nociceptin binds to the ORL1 receptor, increasing potassium currents and inhibiting voltage-gated calcium channels in Xenopus oocytes and neuroblastoma cell lines (5,11). These effects of nociceptin, however, were not inhibited by the opioid antagonist, naloxone, suggesting a non-m-opioid receptor mechanism (5,11). Although studies have shown that nociceptin decreases systemic arterial and hindquarters perfusion pressure in the rat, little, if anything is known about the receptor-mediating responses to the peptide (2,3,6). The present study was therefore undertaken to investigate the effects of naloxone on decreases in 1
Hindquarters vascular bed
Hypotensive response
systemic arterial and hindquarters perfusion pressure in response to nociceptin in the rat. METHOD
Sprague–Dawley rats of either sex weighing 370 – 435 g were anesthetized with pentobarbital sodium (50 mg/kg, IP). Supplemental doses of pentobarbital were given as needed to maintain a uniform level of anesthesia. The trachea was cannulated and the rats were ventilated with a Harvard model 683 rodent ventilator at a tidal volume of 2.5 ml and at a rate of 30 breaths/min with room air enriched with 95% O2:5% CO2. Catheters were inserted into an external jugular vein for the IV administration of drugs and into the carotid artery for the measurement of systemic arterial pressure. Systemic arterial pressure was measured with a Viggo– Spectramed transducer (Viggo–Spectramed, Oxnard, CA) and was recorded on a Grass model 7 polygraph (Grass Instrument Co., Quincy, MA). Mean pressures were derived by electronic averaging. For constant-flow perfusion of the hindquarters vascular bed, a 1.0- to 1.5-cm segment of the distal aorta was exposed through a ventral midline incision and cleared of surrounding connective tissue. After administration of heparin sodium (1000U/kg, IV), the aorta was ligated, and catheters were inserted into the aorta both proximal and distal to the ligature. Blood was withdrawn from the
Requests for reprints should be addressed to Philip J. Kadowitz, Ph.D., Department of Pharmacology, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112.
1197
1198
CZAPLA, CHAMPION AND KADOWITZ
FIG. 1. Bar graphs showing the influence of naloxone on decreases in systemic arterial pressure in response to IV injections of nociceptin (1–30 nmol/kg), Met-enkephalin (100 nmol/kg), and DEA/NO (10 mg/kg). Responses were determined before and after administration of the opioid receptor antagonist in a dose of 2 mg/kg IV. n indicates the number of animals. The asterisk indicates the response is significantly different from control.
proximal catheter and pumped at a constant flow rate with a Masterflex Pump (Cole–Parmer, Chicago, IL) into the distal aortic catheter. Perfusion pressure was monitored from a lateral tap in the perfusion circuit located between the pump and the distal catheter. Agonists were injected directly into the hindquarters perfusion circuit distal to the pump in small volumes (30 –100 ml) in a random sequence. Hindquarters perfusion and systemic arterial pressures were measured with Viggo–Spectramed transducers and were recorded on a Grass model 7 polygraph. Mean pressures were derived from the pulsatile signals by electronic averaging. Hindquarters blood flow was set to achieve a baseline perfusion pressure of approximately 125 mmHg and was not changed during the remainder of an experiment. The flow rate was determined by timed collection and ranged from 3 to 6 ml/min. The hindquarters vascular bed was denervated by ligating and cutting the lumbar sympathetic chain ganglia between L2 and L4. Blood gases were measured periodically and were within the normal physiological range. Nociceptin (orphanin FQ) and Met-enkephalin (Phoenix Pharmaceuticals, Mountain View, CA) were dissolved in 0.9% NaCl and the solutions were divided into aliquots and stored in 1-ml plastic tubes in a freezer. The aliquots were thawed and used on the day of the experiment. Diethylamine/nitric oxide complex sodium (DEA/NO) (Research Biochemical International, Natick, MA), naloxone hydrochloride (DuPont Pharmaceuticals, Wilmington, DE), and acetylcholine chloride (Sigma Chemical Co., St. Louis, MO) were dissolved in 0.9% NaCl. During an experiment, the
agonist solutions were kept on crushed ice. The agonists were administered IV in small volumes (30 –100 ml) over a period of 10 –15 s in a random sequence. Control experiments with the saline vehicle had no effect on systemic arterial or hindquarters perfusion pressure or on responses to the vasoactive agonists. Responses were expressed as change from baseline in mmHg and were analyzed using a paired t-test. A p value of less than 0.05 was used as the criterion for statistical significance. RESULTS
The effects of naloxone on decreases in systemic arterial pressure and on decreases in hindquarters perfusion pressure in response to nociceptin were investigated and these data are summarized in Figs. 1 and 2. Injections of nociceptin in doses of 1–30 nmol/kg IV decreased systemic arterial pressure in a dose-related manner and injections of Met-enkephalin (100 nmol/kg, IV) and DEA/NO (10 mg/kg, IV) decreased systemic arterial pressure. Following administration of naloxone in a dose of 2 mg/kg IV, decreases in systemic arterial pressure in response to Met-enkephalin were reduced significantly, whereas decreases in systemic arterial pressure in response to nociceptin and to DEA/NO were not changed (Fig. 1). Nociceptin decreased hindquarters perfusion pressure when injected in doses of 1–30 nmol into the perfusion circuit and the decreases in perfusion pressure in response to nociceptin were not altered after administration of naloxone (2 mg/kg, IV) (Fig. 2). The decrease in hindquarters perfusion pres-
NALOXONE-INSENSITIVE RESPONSES TO NOCICEPTIN
1199
FIG. 2. Bar graphs showing decreases in hindquarters perfusion pressure in response to injections of nociceptin (1–30 nmol), Met-enkephalin (100 nmol), and acetylcholine (10 ng) into the hindquarters perfusion circuit. Responses were determined before and after administration of the opioid receptor antagonist, naloxone, in a dose of 2 mg/kg IV. n indicates the number of experiments. The asterisk indicate that the response is significantly different from control.
sure in response to Met-enkephalin (100 nmol) was reduced significantly by naloxone, whereas the opioid receptor antagonist had no significant effect on the decrease in perfusion pressure in response to acetylcholine (Fig. 2). DISCUSSION
The results of the present study demonstrate that decreases in systemic arterial pressure and hindquarters perfusion pressure in response to nociceptin are not altered by the administration of naloxone in a dose of 2 mg/kg IV. These results suggest that responses to nociceptin are not mediated by the activation of a m-, d-, or k-opioid receptor in the systemic or hindquarters vascular bed of the rat. Met-enkephalin activates the d-opioid receptor and produces significant decreases in systemic arterial and hindquarters perfusion pressure in the rat. The observation that decreases in systemic arterial pressure in response to Met-enkephalin are attenuated by naloxone, whereas the response to acetylcholine were not altered, suggests that vasodepressor responses to Met-enkephalin are mediated by a naloxone-sensitive mechanism. Moreover, because blood flow was maintained constant in experiments in the hindquarters vascular bed, the decreases in perfusion pressure in response to the opioid receptor agonist reflect decreases in hind-
quarters vascular resistance. The observation that responses to Met-enkephalin, but not to acetylcholine, are inhibited by naloxone suggests that vasodilator responses to Met-enkephalin are mediated by an opioid receptor and that the inhibitory effects of naloxone are selective in nature. It has been reported that nociceptin inhibits the opening of voltage-dependent calcium channels in neuroblastoma and hippocampal cell lines and that this effect is insensitive to the opioid receptor antagonist, naloxone (5,10). The results with naloxone in neuroblastoma and hippocampal cell lines are consistent with the present results in the systemic and hindquarters vascular bed of the rat and provide support for the hypothesis that responses to nociceptin are not mediated by the activation of classical m-, d-, or k-opioid receptors. In summary, the results of the present study demonstrate that nociceptin, also known as orphanin FQ, decreases systemic arterial and hindquarters perfusion pressure in the rat and that responses to nociceptin are not attenuated by the opioid receptor antagonist, naloxone. Naloxone attenuated decreases in systemic arterial and hindquarters perfusion pressure in response to the opioid receptor agonist, Met-enkephalin, but did not alter responses to the nitric oxide donor, DEA/NO, or to acetylcholine. These data indicate
1200
CZAPLA, CHAMPION AND KADOWITZ
that decreases in systemic arterial and hindquarters perfusion pressure in response to nociceptin are not mediated by the activation of a naloxone-sensitive receptor in the rat.
ACKNOWLEDGMENTS
These studies were supported, in part, by NIH Grant HL15580 and a grant from the American Heart Association-Louisiana, Inc.
REFERENCES 1. Bunzow, J. R.; Saez, C.; Mortrud, M.; Bouvier, C.; Williams, J. T.; Low, M.; Grandy, D. K. Molecular cloning and tissue distribution of a putative member of the rat opiod receptor gene family that is not a mu, delta, or kappa opioid receptor type. FEBS Lett. 347:284 –288; 1994. 2. Champion, H. C.; Czapla, M. A.; Kadowitz, P. J. Nociceptin, an endogenous ligand for the ORL1 receptor, decreases cardiac output and total peripheral resistance in the rat. Peptides 18:729 –732; 1997. 3. Champion, H. C.; Kadowitz, P. J. Nociceptin, an endogenous ligand for the ORL1 receptor, has novel hypotensive activity in the rat. Life Sci. 60:PL241–PL245; 1997. 4. Chen, Y.; Fan, Y.; Liu, J.; Mestek, A.; Tian, M.; Kozak, C. A.; Yu, L. Molecular cloning, tissue distribution and chomasomal localization of a novel member of the opioid receptor gene family. FEBS Lett. 347:279 –283; 1994. 5. Connor, M.; Yeo, A.; Henderson, G. The effect of nociceptin on Ca21 channel current and intracellular Ca21 in the SH-SY5Y human neuroblastoma cell line. Br. J. Pharmacol. 118:205–207; 1996. 6. Czapla, M. A.; Champion, H. C.; Kadowitz, P. J. Nociceptin, an endogenous ligand for the ORL1 receptor, has vasodilator activity in the hindquarters vascular bed of the rat. Peptides 18:793–795; 1997. 7. Fukuda, K.; Kato, S.; Mori, K.; Nishi, M.; Takeshima, H.; Iwabe, N.; Miyata, T.; Houtani, T.; Sugimoto, T. cDNA cloning and regional distribution of a novel member of the opioid receptor family. FEBS Lett. 343:42– 46; 1994. 8. Halford, W. P.; Gebhardt, B. M.; Carr, D. J. J. Functional role and sequence analysis of a lymphocyte orphan opioid receptor. J. Neuroimmunol. 59:91–101; 1995. 9. Houtani, T.; Nishi, M.; Takeshima, H.; Nukada, T.; Sugimoto, T. Structure and regional distribution of nociceptin/orphanin FQ precursor. Biochem. Biophys. Res. Commun. 219:714 –719; 1996. 10. Knoflach, F.; Reinscheid, R. K.; Civelli, O.; Kemp, J. A. Modulation of voltage-gated calcium channels by orphanin FQ in freshly dissociated hippocampal neurons. J. Neurosci. 16(21):6657– 6664; 1996. 11. Matthes, H.; Seward, E. P.; Kieffer, B.; North, R. A. Functional selectivity of orphanin FQ for its receptor coexpressed with potassium
12.
13. 14.
15.
16.
17. 18. 19.
20.
channel subunits in Xenopus laevis oocytes. Mol. Pharmacol. 50:447– 450; 1996. Meunier, J.; Mollereau, C.; Toll, L.; Suaudeau, C.; Moisand, C.; Alvinerie, P.; Butour, J.; Guillemot, J.; Ferrara, P.; Monsarrat, B.; Mazaguil, H.; Vassart, G.; Parmentier, M.; Constetin, J. Isolation and structure of the endogenous agonist of opiod receptor-like ORL1 receptor. Nature 377:532–535; 1995. Mogil, J.; Grisel, J.; Reinscheid, R.; Civelli, O.; Belknap, J.; Grandy, D. Orphanin FQ is a functional anti-opioid peptide. Neuroscience 75(2):333–337; 1996. Mollereau, C.; Parmentier, M.; Mailleaux, P.; Butour, J. L.; Moisand, C.; Chalon, P.; Caput, D.; Vassart, G.; Meunier, J. C. ORL1, a novel member of the opioid receptor family. Cloning functional expression and localization. FEBS Lett. 341:33–38; 1994. Mollereau, C.; Simons, M. J.; Soularue, P.; Liners, F.; Vassart, G.; Meunier, J. C.; Paramentier, M. Structure, tissue distribution, and chromasomal localization of the prenociceptin gene. Proc. Natl. Acad. Sci. USA 93:8666 – 8670; 1996. Nothacker, H. P.; Reinscheid, R. K.; Mansour, A.; Henningsen, R. A.; Ardati, A.; Monsma, F. J.; Watson, S. J.; Civelli, O. Primary structure and tissue distribution of the orphanin FQ precursor. Proc. Natl. Acad. Sci. USA 93:8677– 8682; 1996. Pan, Y. X.; Xu, J.; Pasternak, G. W. Cloning and expression of a cDNA encoding a mouse brain orphanin FQ/nociceptin precursor. Biochem. J. 315:11–13; 1996. Reinscheid, R. K.; Ardati, A.; Monsma, F. J.; Civelli, O. Structure– activity relationship studies on the novel neuropeptide orphanin FQ. J. Biol. Chem. 271:14163–14168; 1996. Reinscheid, R. K.; Nothacker, H.; Bourson, A.; Ardati, A.; Henningsen, R. A.; Bunzow, J. R.; Grandy, D. K.; Langen, H.; Monsma, F. J.; Civelli, O. Orphanin FQ: A neuropeptide that activates an opioidlike G protein-coupled receptor. Science 270:792–794; 1995. Wang, J. B.; Johnson, P. S.; Imai, Y.; Persico, A. M.; Ozenberger, B. A.; Eppler, C. M.; Uhl, G. R. cDNA cloning of an orphan opioid receptor gene family member and its slice variant. FEBS Lett. 348: 75–79; 1994.