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Nociceptin, an Endogenous Ligand for the ORL1 Receptor, Decreases Cardiac Output and Total Peripheral Resistance in the Rat
Peptides, Vol. 18, No. 5, pp. 729–732, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/97 $17.00 / .00
PII S0196-9781(97)00003-X
Nociceptin, an Endogenous Ligand for the ORL1 Receptor, Decreases Cardiac Output and Total Peripheral Resistance in the Rat H. C. CHAMPION, M. A. CZAPLA AND P. J. KADOWITZ1 Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana 70112 Received 12 December 1996; Accepted 24 January 1997 CHAMPION, H. C., M. A. CZAPLA AND P. J. KADOWITZ. Nociceptin, an endogenous ligand for the ORL1 receptor, decreases cardiac output and total peripheral resistance in the rat. PEPTIDES 18(5) 729–732, 1997.—The heptadecapeptide nociceptin, also known as Orphanin FQ, is a newly discovered endogenous ligand for the opioid-like G-protein coupled receptor, ORL1. In the present study, responses to intravenous injections of nociceptin were investigated in the systemic vascular bed of the rat. Nociceptin induced dose-related decreases in systemic arterial pressure and total peripheral resistance when injected in doses of 1–30 nmol/kg IV. Nociceptin decreased heart rate and in doses of 10 and 30 nmol/kg IV, significantly decreased cardiac output. In terms of relative vasodilator activity, nociceptin was approximately 10-fold less potent than the beta-adrenergic receptor agonist isoproterenol. These data show that nociceptin has novel vasodilator activity in the systemic vascular bed of the rat. q 1997 Elsevier Science Inc. Nociceptin
Orphanin FQ
ORL1 receptor
Vasodilator responses
Systemic vascular bed
Cardiac output
cells, which is a similar mechanism of action observed with traditional opioid receptors (9,16). However, unlike the traditional opioid agonists, nociceptin induces hyperalgesia when injected into the cerebral ventricles in the rat (9,16). In addition, administration of nociceptin into the cerebral ventricles of the rat decreases spontaneous motor activity, muscular tone, causes ataxia, and induces the loss of the righting reflex (9,16). While preliminary studies have shown that nociceptin decreases systemic arterial pressure in the rat (2), little if anything is known about the effects of nociceptin on cardiac output, heart rate, and systemic vascular resistance. Therefore, the present study was undertaken to investigate the effects of nociceptin on systemic arterial pressure, cardiac output, heart rate, and systemic vascular resistance in the rat and to compare responses with the beta-receptor agonist, isoproterenol, and adrenomedullin.
cDNA CLONING studies have led to the characterization of a novel G-protein coupled receptor (ORL1) that shares sequence homology to the m, d, and k opioid receptor subfamily (1,3– 5,8,10–14,17–19). Although the ORL1 receptor is negatively coupled to adenylyl cyclase, known opioid receptor agonists do not exhibit high affinity binding to the ORL1 receptor (1,3– 5,8,10–14,17–19). The ORL1 receptor transcripts are widely distributed in the central nervous system (CNS) of the rat and mouse, as well as in peripheral organs, such as the spleen, kidney, vas deferens, and intestine (1,3,4,8,10,11,13,14,17,18). In the CNS, ORL1 receptor transcripts are most abundant in the hypothalamus, pons, and the spinal cord and may have a role in regulating memory, attention and emotions, and sensory perception (1,8,10,11). Until recently, however, no known endogenous ligand was found to bind to the ORL1 receptor with high affinity (9,16). Nociceptin (H2N-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-LysSer-Ala-Arg-Lys-Leu-Ala-Asn-Gln-COOH), also known as Orphanin FQ, is an endogenous ligand for the ‘‘orphan’’ opioid receptor (ORL1) (9,16). Nociceptin is a 17 amino acid peptide that shares structural homology with the dynorphin family of peptides (9,16). Nociceptin is different from other opioid receptor agonists in that it does not possess the N-terminal tyrosine residue that is required for agonist activity at the m, d, and k opioid receptors (9,15,16). The isolation of nociceptin was based on its ability to inhibit adenylyl cyclase in ORL1 transfected
METHOD
Seventeen Sprague–Dawley rats of either sex weighing 340– 540 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 breathed room air or were ventilated with a Harvard model 683 rodent ventilator at a tidal volume of 2.4– 2.6 ml at a rate of 30–35 breaths/min. Catheters were inserted into the left external jugular vein for the iv administration of
1 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.
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FIG. 1. Bar graphs showing the influence of intravenous injection of nociceptin on (A) systemic arterial pressure, (B) heart rate, (C) cardiac output, and (D) total peripheral resistance expressed as percent change from baseline. Baseline values for systemic arterial pressure, heart rate, cardiac output, and total peripheral resistance were 121 { 4 mm Hg, 394 { 8 beats·min 01 , 134 { 12 ml·min 01 , and 1.1 { 0.14 mm Hg·ml·min 01 . n indicates number of experiments; *indicates that the response is significant from baseline value.
drugs and into the femoral artery for the measurement of systemic arterial (aortic) pressure. Systemic arterial pressure was measured with a Statham P23 pressure transducer and was recorded on a Grass model 7 polygraph. Mean pressure was derived by electronic averaging. Cardiac output was measured by the thermodilution technique using a Cardiotherm 500 cardiac output computer equipped with small animal interfaces (Columbus Instruments, Columbus, OH). The thermister microprobe (Columbus Instruments, Fr-1) was inserted into the right carotid artery and advanced to the aortic arch. A catheter placed in the left jugular vein was advanced to the right atrium for rapid bolus injection of 100 ml (plus catheter dead space) of 10–157C saline with a Hamilton constant-rate syringe to ensure rapid and repeatable injection of the saline indicator solution. Catheter placement was verified by
postmortem examination. Mean arterial pressure and heart rate were monitored continuously. Cardiac output was measured in the control period; and after administration of nociceptin when systemic arterial pressure was decreased to a steady level, and again after systemic arterial pressure had returned to control level. Nociceptin (Orphanin FQ; Phoenix Pharmaceuticals, Mountain View, CA), human synthetic adrenomedullin (Peptide Research Labs, Tulane Medical School, New Orleans, LA), and isoproterenol (Sigma Chemical Co., St. Louis, MO) were dissolved in 0.9% NaCl and the solutions were divided into aliquots and stored in 1 ml plastic tubes. The aliquots were stored frozen and thawed on the day of an experiment. During an experiment the agonist solutions were kept on crushed ice. The agonists were administered iv in small volumes (30–150 ml), and injections
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VASODILATOR RESPONSES TO NOCICEPTIN
731 approximately 10-fold less potent than isoproterenol in decreasing total peripheral resistance (Fig. 3A,B). DISCUSSION
FIG. 2. Records from an experiment illustrating a typical response to injection of nociceptin (10 nmol/kg IV) on systemic arterial blood pressure (SAP), cardiac output (ml/min), and heart rate (bts/min) in the rat. Cardiac output and heart rate were measured before injection of the peptide, at the plateau phase of the hypotensive response to nociceptin, and after systemic arterial pressure had returned to baseline value.
were made over a period of 10–15 s in a random sequence. In control experiments, injection of equal volumes of the saline vehicle had no effect on systemic arterial pressure, heart rate, or cardiac output. Responses are expressed as percent change from baseline and were analyzed using a one-way analysis of variance (ANOVA) and Scheffe’s F test or a paired t-test with a Bonferroni/Dunn procedure. Baseline values for systemic arterial pressure, heart rate, cardiac output, and total peripheral resistance were 121 { 4 mm Hg, 395 { 8 beats/min, 134 { 12 ml/min, and 1.1 { 0.14 mm Hg.ml.min 01 , respectively. A p value of less than 0.05 was used as the criterion for statistical significance.
The results of the present investigation show that nociceptin produced dose-related decreases in systemic arterial pressure and total peripheral resistance and are the first to demonstrate that nociceptin induces vasodilation in the systemic vascular bed of the rat. The decreases in systemic arterial pressure were associated with decreases in heart rate and, at the highest doses studied, decreases in cardiac output. However, decreases in systemic arterial pressure were greater than decreases in cardiac output at the 10 and 30 nmol/kg doses, so that total peripheral resistance was reduced. In terms of its relative hypotensive activity, nociceptin was approximately 10-fold less potent than the vasodilator peptide adrenomedullin or the beta-receptor agonist isoproterenol. In terms of its relative vasodilator activity in the systemic vascular bed, nociceptin was approximately 10-fold less potent than isoproterenol. The mechanism by which nociceptin decreases total peripheral resistance, cardiac output, or heart rate is unknown at the present time. The decreases in heart rate and cardiac output observed along with the decrease in systemic ar-
RESULTS
The effects of IV injection of nociceptin on systemic arterial pressure, heart rate, cardiac output, and total peripheral resistance in the anesthetized rat are illustrated in Figs. 1 and 2. Intravenous injections of nociceptin in doses of 1–30 nmol/kg caused doserelated decreases in systemic arterial pressure (Fig. 1A). Heart rate was decreased significantly in response to all doses of nociceptin studied (Fig. 1B), whereas cardiac output was decreased when the peptide was injected in doses of 10 and 30 nmol/kg IV (Fig. 1C). Total peripheral resistance was decreased in a significant dose-related manner when nociceptin was injected in doses of 1–30 nmol/kg IV (Fig. 1D). The time-course of the decrease in systemic arterial pressure in response to injection of nociceptin in a dose of 10 nmol/kg IV is shown in Fig. 2. The decrease in systemic arterial pressure in response to iv injection of nociceptin was rapid in onset, and pressure returned to control value over a 6-min period (Fig. 2). The cardiac output decreased from a control value of 128 ml/min to a value of 108 ml/min when measured during the plateau phase of the hypotensive response and returned to control value (127 ml/min) 6 min after the injection of nociceptin (Fig. 1). Heart rate decreased from a value of 380 beats/min during the control period to 310 beats/min during the plateau phase of the hypotensive response when cardiac output was measured and heart rate returned to control value (390 beats/ min) 6 min after the injection of the peptide (Fig. 2). The decreases in systemic arterial pressure in response to nociceptin, adrenomedullin, and isoproterenol are compared in Fig. 3A, and the decreases in total peripheral resistance in response to nociceptin and isoproterenol are compared in Fig. 3B. Nociceptin was approximately 10-fold less potent than adrenomedullin or isoproterenol in decreasing systemic arterial pressure and
FIG. 3. (A) Line graphs comparing the percent decrease in systemic arterial pressure in response to intravenous injections of nociceptin, adrenomedullin, and the b-adrenergic agonist isoproterenol in the rat. (B) Line graphs comparing the percent decrease in total peripheral resistance (TPR) in response to intravenous injections of nociceptin and isoproterenol in the rat. Doses of the agonists are expressed on a nmol basis to take molecular weight into account. n indicates number of experiments.
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terial pressure in response to nociceptin are unexpected, since decreases in systemic arterial pressure are usually associated with reflex increases in heart rate and, if stroke volume is not diminished, increases in cardiac output. In contrast, injections of the beta-receptor agonist isoproterenol resulted in a decrease in systemic arterial pressure and an increase in heart rate and cardiac output. The present data may be interpreted to suggest that nociceptin may interfere with the baroreceptor reflex in the rat. Preliminary studies in the rat have shown that nociceptin has novel hypotensive activity (2). The results of the present study extend this finding by showing that this novel vasodepressor activity is associated with a decrease in heart rate and total peripheral resistance and, at the higher doses, a decrease in cardiac output. These data indicate that nociceptin has vasodilator activity in the systemic vascular bed of the rat. The reason for the decrease in cardiac output associated with the injection of nociceptin is uncertain, but may be due in part to the decrease in heart rate. This decrease in cardiac output and heart rate may be due to a central effect of the peptide or to a direct action of the peptide on the heart. Nociceptin, also known as Orphanin FQ, is a 17 amino acid peptide recently isolated from the rat brain (9,16). The peptide was named nociceptin because of its ability to induce enhanced reactivity to noxious thermal stimuli (9). Nociceptin shares marked sequence homology with dynorphin A, one of five endogenous ligands for the opioid receptor subfamily that is classified into three main receptor subtypes ( m, d, and k ) (9,16).
These receptors belong to a closely related subfamily of G-protein coupled receptors; and, using homology-based screening procedures, a novel receptor of the family that does not bind any of the known opiate ligands with high affinity was identified (1,3–5,8,10–12,14,17,19). It has been shown that nociceptin is an endogenous ligand that binds to this orphan receptor with high affinity and induces hyperalgesia when injected into the cerebral ventricle of the rat (9,16). The observation that nociceptin may induce hyperalgesia by opposing the actions of other opiate agonists is consistent with the hypothesis of Kastin and co-workers that MIF-I can antagonize opiate actions in the mouse (7). In summary, the results of the present study show that nociceptin, a novel opioid receptor agonist which binds to the ORL1 receptor and induces hyperalgesia, decreases systemic arterial pressure, cardiac output, heart rate, and total peripheral resistance in the rat. Although the mechanism of action or the properties of the receptor with which nociceptin interact are unknown, the results of the present study show for the first time that this novel opioid peptide has significant vasodilator activity in the systemic vascular bed in the rat, and suggest that nociceptin could play a role in regulating arterial pressure in the rat. ACKNOWLEDGEMENTS
The authors wish to thank Ms. Janice Ignarro for editorial assistance. The studies were supported by NIH grant HL15580 and a grant from the American Heart Association-Louisiana, Inc. Hunter C. Champion was supported by NIH grant HL09474.
REFERENCES 1. Bunzow, J. R.; Saez, C.; Mortrud, M.; Bouvier, C.; William, J. T.; Low, M.; Grandy, D. K. Molecular cloning and tissue distribution of a putative member of the rat opioid receptor gene family that is not a mu, delta, or kappa opioid receptor type. FEBS Lett. 347:284– 288; 1994. 2. Champion, H. C.; Kadowitz, P. J. Nociceptin, an endogenous ligand for the ORL1 receptor, has novel hypotensive activity in the rat. (abstract) FASEB J. 1996. 3. Chen, Y.; Fan, Y.; Liu, J.; Mestek, A.; Tian, M.; Kozak, C. A.; Yu, L. Molecular cloning, tissue distribution and chromosomal localization of a novel member of the opioid receptor gene family. FEBS Lett. 347:279–283; 1994. 4. 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. 5. 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. 6. 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. 7. Kastin, A. J.; Olson, R. D.; Enrensing, R. H.; Berzas, M. C.; Schally, A. V.; Coy, D. H. MIF-I’s differential actions as an opiate antagonist. Pharmacol. Biochem. Behav. 11:721–723; 1979. 8. Lachowicz, J. E.; Shen, Y.; Monsma, F. J.; Sibley, D. R. Molecular cloning of a novel G-protein coupled receptor related to the opioid receptor family. J. Neurochem. 64:34–40; 1995. 9. Meunier, J.; Molereau, 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 opioid receptor-like ORL1 receptor. Nature 377:532–535; 1995. 10. 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.
11. Mollereau, C.; Simons, M. J.; Soularue, P.; Liners, F.; Vassart, G.; Meunier, J. C.; Paramentier, M. Structure, tissue distribution, and chromosomal localization of the prenociceptin gene. Proc. Natl. Acad. Sci. USA 93:8666–8670; 1996. 12. Nishi, M.; Takeshima, H.; Mori, M.; Nakagawara, K.; Takeuchi, T. Structure and chromosomal mapping of genes for the mouse kappaopioid receptor and an opioid receptor homologue (MOR-C). Biochem. Biophys. Res. Commun. 205:1353–1357; 1994. 13. 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. 14. 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. 15. Reinscheid, R. K.; Ardati, A.; Monsma, F. J.; Civelli, O. Structureactivity relationship studies on the novel neuropeptide orphanin FQ. J. Biol. Chem. 271:14163–14168; 1996. 16. 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 opioid-like G protein-coupled receptor. Science 270:792–794; 1995. 17. 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. 18. Wick, M. J.; Minnerath, S. R.; Lin, X.; Elde, R.; Law, P. Y.; Loh, H. H. Isolation of a novel cDNA encoding a putative membrane receptor with high homology to the cloned mu, delta, and kappa opioid receptors. Molec. Brain Res. 27:37 – 44; 1994. 19. Wick, M. J.; Minnerath, S. R.; Roy, S.; Ramakrishnan, S.; Loh, H. H. Expression of alternate forms of brain opioid ‘‘orphan’’ receptor mRNA in activated human peripheral blood lymphocytes and lymphocytic cell lines. Molec. Brain Res. 32:342 – 347; 1995.
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Nociceptin, an Endogenous Ligand for the ORL1 Receptor, Decreases Cardiac Output and Total Peripheral Resistance in the Rat H. C. CHAMPION, M. A. CZAPLA AND P. J. KADOWITZ1 Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana 70112 Received 12 December 1996; Accepted 24 January 1997 CHAMPION, H. C., M. A. CZAPLA AND P. J. KADOWITZ. Nociceptin, an endogenous ligand for the ORL1 receptor, decreases cardiac output and total peripheral resistance in the rat. PEPTIDES 18(5) 729–732, 1997.—The heptadecapeptide nociceptin, also known as Orphanin FQ, is a newly discovered endogenous ligand for the opioid-like G-protein coupled receptor, ORL1. In the present study, responses to intravenous injections of nociceptin were investigated in the systemic vascular bed of the rat. Nociceptin induced dose-related decreases in systemic arterial pressure and total peripheral resistance when injected in doses of 1–30 nmol/kg IV. Nociceptin decreased heart rate and in doses of 10 and 30 nmol/kg IV, significantly decreased cardiac output. In terms of relative vasodilator activity, nociceptin was approximately 10-fold less potent than the beta-adrenergic receptor agonist isoproterenol. These data show that nociceptin has novel vasodilator activity in the systemic vascular bed of the rat. q 1997 Elsevier Science Inc. Nociceptin
Orphanin FQ
ORL1 receptor
Vasodilator responses
Systemic vascular bed
Cardiac output
cells, which is a similar mechanism of action observed with traditional opioid receptors (9,16). However, unlike the traditional opioid agonists, nociceptin induces hyperalgesia when injected into the cerebral ventricles in the rat (9,16). In addition, administration of nociceptin into the cerebral ventricles of the rat decreases spontaneous motor activity, muscular tone, causes ataxia, and induces the loss of the righting reflex (9,16). While preliminary studies have shown that nociceptin decreases systemic arterial pressure in the rat (2), little if anything is known about the effects of nociceptin on cardiac output, heart rate, and systemic vascular resistance. Therefore, the present study was undertaken to investigate the effects of nociceptin on systemic arterial pressure, cardiac output, heart rate, and systemic vascular resistance in the rat and to compare responses with the beta-receptor agonist, isoproterenol, and adrenomedullin.
cDNA CLONING studies have led to the characterization of a novel G-protein coupled receptor (ORL1) that shares sequence homology to the m, d, and k opioid receptor subfamily (1,3– 5,8,10–14,17–19). Although the ORL1 receptor is negatively coupled to adenylyl cyclase, known opioid receptor agonists do not exhibit high affinity binding to the ORL1 receptor (1,3– 5,8,10–14,17–19). The ORL1 receptor transcripts are widely distributed in the central nervous system (CNS) of the rat and mouse, as well as in peripheral organs, such as the spleen, kidney, vas deferens, and intestine (1,3,4,8,10,11,13,14,17,18). In the CNS, ORL1 receptor transcripts are most abundant in the hypothalamus, pons, and the spinal cord and may have a role in regulating memory, attention and emotions, and sensory perception (1,8,10,11). Until recently, however, no known endogenous ligand was found to bind to the ORL1 receptor with high affinity (9,16). Nociceptin (H2N-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-LysSer-Ala-Arg-Lys-Leu-Ala-Asn-Gln-COOH), also known as Orphanin FQ, is an endogenous ligand for the ‘‘orphan’’ opioid receptor (ORL1) (9,16). Nociceptin is a 17 amino acid peptide that shares structural homology with the dynorphin family of peptides (9,16). Nociceptin is different from other opioid receptor agonists in that it does not possess the N-terminal tyrosine residue that is required for agonist activity at the m, d, and k opioid receptors (9,15,16). The isolation of nociceptin was based on its ability to inhibit adenylyl cyclase in ORL1 transfected
METHOD
Seventeen Sprague–Dawley rats of either sex weighing 340– 540 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 breathed room air or were ventilated with a Harvard model 683 rodent ventilator at a tidal volume of 2.4– 2.6 ml at a rate of 30–35 breaths/min. Catheters were inserted into the left external jugular vein for the iv administration of
1 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.
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FIG. 1. Bar graphs showing the influence of intravenous injection of nociceptin on (A) systemic arterial pressure, (B) heart rate, (C) cardiac output, and (D) total peripheral resistance expressed as percent change from baseline. Baseline values for systemic arterial pressure, heart rate, cardiac output, and total peripheral resistance were 121 { 4 mm Hg, 394 { 8 beats·min 01 , 134 { 12 ml·min 01 , and 1.1 { 0.14 mm Hg·ml·min 01 . n indicates number of experiments; *indicates that the response is significant from baseline value.
drugs and into the femoral artery for the measurement of systemic arterial (aortic) pressure. Systemic arterial pressure was measured with a Statham P23 pressure transducer and was recorded on a Grass model 7 polygraph. Mean pressure was derived by electronic averaging. Cardiac output was measured by the thermodilution technique using a Cardiotherm 500 cardiac output computer equipped with small animal interfaces (Columbus Instruments, Columbus, OH). The thermister microprobe (Columbus Instruments, Fr-1) was inserted into the right carotid artery and advanced to the aortic arch. A catheter placed in the left jugular vein was advanced to the right atrium for rapid bolus injection of 100 ml (plus catheter dead space) of 10–157C saline with a Hamilton constant-rate syringe to ensure rapid and repeatable injection of the saline indicator solution. Catheter placement was verified by
postmortem examination. Mean arterial pressure and heart rate were monitored continuously. Cardiac output was measured in the control period; and after administration of nociceptin when systemic arterial pressure was decreased to a steady level, and again after systemic arterial pressure had returned to control level. Nociceptin (Orphanin FQ; Phoenix Pharmaceuticals, Mountain View, CA), human synthetic adrenomedullin (Peptide Research Labs, Tulane Medical School, New Orleans, LA), and isoproterenol (Sigma Chemical Co., St. Louis, MO) were dissolved in 0.9% NaCl and the solutions were divided into aliquots and stored in 1 ml plastic tubes. The aliquots were stored frozen and thawed on the day of an experiment. During an experiment the agonist solutions were kept on crushed ice. The agonists were administered iv in small volumes (30–150 ml), and injections
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731 approximately 10-fold less potent than isoproterenol in decreasing total peripheral resistance (Fig. 3A,B). DISCUSSION
FIG. 2. Records from an experiment illustrating a typical response to injection of nociceptin (10 nmol/kg IV) on systemic arterial blood pressure (SAP), cardiac output (ml/min), and heart rate (bts/min) in the rat. Cardiac output and heart rate were measured before injection of the peptide, at the plateau phase of the hypotensive response to nociceptin, and after systemic arterial pressure had returned to baseline value.
were made over a period of 10–15 s in a random sequence. In control experiments, injection of equal volumes of the saline vehicle had no effect on systemic arterial pressure, heart rate, or cardiac output. Responses are expressed as percent change from baseline and were analyzed using a one-way analysis of variance (ANOVA) and Scheffe’s F test or a paired t-test with a Bonferroni/Dunn procedure. Baseline values for systemic arterial pressure, heart rate, cardiac output, and total peripheral resistance were 121 { 4 mm Hg, 395 { 8 beats/min, 134 { 12 ml/min, and 1.1 { 0.14 mm Hg.ml.min 01 , respectively. A p value of less than 0.05 was used as the criterion for statistical significance.
The results of the present investigation show that nociceptin produced dose-related decreases in systemic arterial pressure and total peripheral resistance and are the first to demonstrate that nociceptin induces vasodilation in the systemic vascular bed of the rat. The decreases in systemic arterial pressure were associated with decreases in heart rate and, at the highest doses studied, decreases in cardiac output. However, decreases in systemic arterial pressure were greater than decreases in cardiac output at the 10 and 30 nmol/kg doses, so that total peripheral resistance was reduced. In terms of its relative hypotensive activity, nociceptin was approximately 10-fold less potent than the vasodilator peptide adrenomedullin or the beta-receptor agonist isoproterenol. In terms of its relative vasodilator activity in the systemic vascular bed, nociceptin was approximately 10-fold less potent than isoproterenol. The mechanism by which nociceptin decreases total peripheral resistance, cardiac output, or heart rate is unknown at the present time. The decreases in heart rate and cardiac output observed along with the decrease in systemic ar-
RESULTS
The effects of IV injection of nociceptin on systemic arterial pressure, heart rate, cardiac output, and total peripheral resistance in the anesthetized rat are illustrated in Figs. 1 and 2. Intravenous injections of nociceptin in doses of 1–30 nmol/kg caused doserelated decreases in systemic arterial pressure (Fig. 1A). Heart rate was decreased significantly in response to all doses of nociceptin studied (Fig. 1B), whereas cardiac output was decreased when the peptide was injected in doses of 10 and 30 nmol/kg IV (Fig. 1C). Total peripheral resistance was decreased in a significant dose-related manner when nociceptin was injected in doses of 1–30 nmol/kg IV (Fig. 1D). The time-course of the decrease in systemic arterial pressure in response to injection of nociceptin in a dose of 10 nmol/kg IV is shown in Fig. 2. The decrease in systemic arterial pressure in response to iv injection of nociceptin was rapid in onset, and pressure returned to control value over a 6-min period (Fig. 2). The cardiac output decreased from a control value of 128 ml/min to a value of 108 ml/min when measured during the plateau phase of the hypotensive response and returned to control value (127 ml/min) 6 min after the injection of nociceptin (Fig. 1). Heart rate decreased from a value of 380 beats/min during the control period to 310 beats/min during the plateau phase of the hypotensive response when cardiac output was measured and heart rate returned to control value (390 beats/ min) 6 min after the injection of the peptide (Fig. 2). The decreases in systemic arterial pressure in response to nociceptin, adrenomedullin, and isoproterenol are compared in Fig. 3A, and the decreases in total peripheral resistance in response to nociceptin and isoproterenol are compared in Fig. 3B. Nociceptin was approximately 10-fold less potent than adrenomedullin or isoproterenol in decreasing systemic arterial pressure and
FIG. 3. (A) Line graphs comparing the percent decrease in systemic arterial pressure in response to intravenous injections of nociceptin, adrenomedullin, and the b-adrenergic agonist isoproterenol in the rat. (B) Line graphs comparing the percent decrease in total peripheral resistance (TPR) in response to intravenous injections of nociceptin and isoproterenol in the rat. Doses of the agonists are expressed on a nmol basis to take molecular weight into account. n indicates number of experiments.
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terial pressure in response to nociceptin are unexpected, since decreases in systemic arterial pressure are usually associated with reflex increases in heart rate and, if stroke volume is not diminished, increases in cardiac output. In contrast, injections of the beta-receptor agonist isoproterenol resulted in a decrease in systemic arterial pressure and an increase in heart rate and cardiac output. The present data may be interpreted to suggest that nociceptin may interfere with the baroreceptor reflex in the rat. Preliminary studies in the rat have shown that nociceptin has novel hypotensive activity (2). The results of the present study extend this finding by showing that this novel vasodepressor activity is associated with a decrease in heart rate and total peripheral resistance and, at the higher doses, a decrease in cardiac output. These data indicate that nociceptin has vasodilator activity in the systemic vascular bed of the rat. The reason for the decrease in cardiac output associated with the injection of nociceptin is uncertain, but may be due in part to the decrease in heart rate. This decrease in cardiac output and heart rate may be due to a central effect of the peptide or to a direct action of the peptide on the heart. Nociceptin, also known as Orphanin FQ, is a 17 amino acid peptide recently isolated from the rat brain (9,16). The peptide was named nociceptin because of its ability to induce enhanced reactivity to noxious thermal stimuli (9). Nociceptin shares marked sequence homology with dynorphin A, one of five endogenous ligands for the opioid receptor subfamily that is classified into three main receptor subtypes ( m, d, and k ) (9,16).
These receptors belong to a closely related subfamily of G-protein coupled receptors; and, using homology-based screening procedures, a novel receptor of the family that does not bind any of the known opiate ligands with high affinity was identified (1,3–5,8,10–12,14,17,19). It has been shown that nociceptin is an endogenous ligand that binds to this orphan receptor with high affinity and induces hyperalgesia when injected into the cerebral ventricle of the rat (9,16). The observation that nociceptin may induce hyperalgesia by opposing the actions of other opiate agonists is consistent with the hypothesis of Kastin and co-workers that MIF-I can antagonize opiate actions in the mouse (7). In summary, the results of the present study show that nociceptin, a novel opioid receptor agonist which binds to the ORL1 receptor and induces hyperalgesia, decreases systemic arterial pressure, cardiac output, heart rate, and total peripheral resistance in the rat. Although the mechanism of action or the properties of the receptor with which nociceptin interact are unknown, the results of the present study show for the first time that this novel opioid peptide has significant vasodilator activity in the systemic vascular bed in the rat, and suggest that nociceptin could play a role in regulating arterial pressure in the rat. ACKNOWLEDGEMENTS
The authors wish to thank Ms. Janice Ignarro for editorial assistance. The studies were supported by NIH grant HL15580 and a grant from the American Heart Association-Louisiana, Inc. Hunter C. Champion was supported by NIH grant HL09474.
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