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OFQ in the hindquarters vascular bed of the rat
Peptides, Vol. 19, No. 9, pp. 1595–1602, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/98 $19.00 1 .00
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Nitric Oxide Release Mediates Vasodilator Responses to Endomorphin 1 But Not Nociceptin/ OFQ in the Hindquarters Vascular Bed of the Rat H. C. CHAMPION,* T. J. BIVALACQUA,* D. E. FRIEDMAN,* J. E. ZADINA,†‡ A. J. KASTIN†‡ AND P. J. KADOWITZ*1 Departments of *Pharmacology and †Medicine, Tulane University School of Medicine and ‡The Veterans Affairs Medical Center, New Orleans, LA Received 27 March 1998; Accepted 22 June 1998 CHAMPION, H. C., T. J. BIVALACQUA, D. E. FRIEDMAN, J. E. ZADINA, A. J. KASTIN AND P. J. KADOWITZ. Nitric oxide release mediates vasodilator responses to endomorphin 1 but not nociceptin/OFQ in the hindquarters vascular bed of the rat. PEPTIDES 19(9) 1595–1602, 1998 —We have recently shown that endomorphin1, an endogenous ligand for the m-opioid receptor, and nociceptin (Orphanin FQ; OFQ), an endogenous ligand for the ORL1 receptor, have substantial vasodilator activity in the hindquarters vascular bed of the rat. In the present study, the role of nitric oxide, vasodilator prostaglandins, and the opening of K1ATP channels in mediating vasodilator responses to endomorphin 1, PL017, and DAMGO was investigated in the regional vascular bed in the rat. Under constant-flow conditions, injections of the m-selective agonists endomorphin 1, PL017 ([N-MePhe3,D-Pro4]-morphiceptin), and DAMGO, and the ORL1 receptor agonist nociceptin/ OFQ produced dose-dependent decreases in hindquarters perfusion pressure. Vasodilator responses to endomorphin 1, PL017, and DAMGO, and the endothelium-dependent vasodilators acetylcholine and adrenomedullin were attenuated by the nitric oxide synthase inhibitor L-NAME (50 mg/kg IV) at a time when vasodilator responses to nociceptin/OFQ were not altered. Vasodilator responses to isoproterenol and prostaglandin E1, agents known to increase cAMP levels, and the nitric oxide donor DEA/NO were not altered by the nitric oxide synthase inhibitor. Responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ were not altered by sodium meclofenamate at a time when vasodilator responses to arachidonic acid were reduced significantly or after administration of U-37883A at a time when vasodilator responses to levcromakalim were reduced significantly. The results of these studies indicate that responses to endomorphin 1, PL017, and DAMGO are mediated in large part by the release of nitric oxide, while responses to nociceptin/OFQ are mediated by an L-NAME-insensitive mechanism. Moreover, these results demonstrate that responses to these peptides are not mediated by the release of vasodilator prostaglandins or the opening of K1ATP channels the hindquarters vascular bed. © 1998 Elsevier Science Inc. Nitric oxide-dependent vasodilator Endothelium-dependent vasodilator
Endomorphin 1
ENDOMORPHIN 1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin 2 (Tyr-Pro-Phe-Phe-NH2) are recently discovered endogenous peptides that display high affinity and selectivity for the m-opioid receptor (27). These tetrapeptides were isolated from bovine (27) and human (13) brain and shown to be differentially distributed in the nervous system by RIA
Hindquarters vascular bed
Nociceptin
(27) and ICC (19). Based on the potent and selective activity of the peptides at the m-opioid receptor, it has been suggested that the discovery of endomorphin 1 and 2 may lead to development of newer analgesics with less addictive properties than the traditional opioid agonists. Nociceptin (H2N-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-
1 Requests for reprints should be addressed to Philip J. Kadowitz, Ph.D., Department of Pharmacology SL83, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, Louisiana 70112 USA. E-Mail: [email protected]
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Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln-COOH), also known as orphanin FQ, is an endogenous ligand for the “orphan” opioid receptor (ORL1) (1,8,16,20 –22,24,25). Nociceptin/OFQ differs from the traditional opioid receptor agonists in that it does not possess the N-terminal tyrosine residue that is essential for activity at m, d, and k opioid receptor (20,25). The isolation of nociceptin/OFQ was based on its ability to inhibit adenylyl cyclase in ORL1 transfected cells (20,25). Although this signaling mechanism is similar to other opioid agonists when their receptors are activated, nociceptin has been shown to induce hyperalgesia when injected into the cerebral ventricles (ICV) in the rat (20). In addition, ICV administration of nociceptin/ OFQ decreases spontaneous activity and muscular tone and causes ataxia and loss of the righting reflex (20). Morphine and endogenous opioid peptides have vasodepressor (3,4,11,15) and vasodilator activity (3,15,26,69), and it has recently been shown that endomorphin 1 and 2 decrease systemic arterial pressure in the rabbit (5) and rat (7). In addition, it has recently been shown that endomorphin 1 and 2 have very similar vasodilator activity in the hindquarters vascular bed of the rat (6). However, the mechanism mediating vasodilator responses to the opioid peptides is not well understood (9 –11,14,15,23,26). Human arterial and rat microvascular endothelial cells contain a high affinity, saturable opioid receptor binding site that is antagonized in a stereo-selective manner by naloxone (26). This population of m-opioid receptors has been reported to mediate the release of nitric oxide in response to morphine but not DAMGO (26). In the aorta morphineinduced vasorelaxation is mediated by the release of nitric oxide, and it has been suggested that this release of nitric oxide is due to receptor activation by alkaloids, but not by peptides (26). Another study has shown concentrationdependent contractile responses to this m-selective synthetic agonist that is not dependent upon the presence of vascular endothelium (23,26). Although the explanation for these differences in the effects of morphine and DAMGO on isolated arteries is uncertain, these results are not consistent with the hypothesis that activation of m-opioid receptors release nitric oxide from the endothelium (23,26). Whereas previous studies have shown that the m-opioid agonists endomorphin 1 and 2 and the ORL1 receptor agonist nociceptin/OFQ decrease systemic arterial pressure and have vasodilator activity in the hindquarters vascular bed (3–7), little if anything is known about the role of nitric oxide in mediating vasodilator responses to these endogenous opioid-related peptides in the hindquarters vascular bed of the rat. The present study was, therefore, undertaken to investigate the effects of the nitric oxide synthase inhibitor Nv-nitro-L-arginine methyl ester (L-NAME) on vasodilator responses to the newly discovered m-opioid receptor selective peptide endomorphin 1 and on responses to PL017 and DAMGO in the hindquarters vascular bed of the rat. In
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addition to the release of nitric oxide, vasodilator responses have been shown to be mediated by the release of cyclooxygenase products, as well as the opening of K1ATP channels. We, therefore, tested the role of these mechanisms in mediating vasodilator responses to the opioid peptides in the hindquarters vascular bed of the rat. METHOD Sprague–Dawley rats (Harlan Sprague–Dawley, Indianapolis, IN) weighing 270 – 420 g were anesthetized with pentobarbital sodium (50 mg/kg IV). Supplemental doses of pentobarbital were given during the course of the experiment to ensure a uniform level of anesthesia. The trachea was cannulated, and the rats breathed spontaneously or were ventilated with a Harvard model 683 rodent ventilator (Harvard Apparatus, South Natick, MA) 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 the external jugular vein for the IV administration of drugs and into the carotid artery for the measurement of systemic arterial pressure. 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 (1000 U/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 proximal catheter and pumped at a constant-flow rate with a Masterflex Pump (Cole Parmer Instrument, 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 hindquarter 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 (Oxnard, CA) transducers and were recorded on a Grass model 7 polygraph (Grass Instruments, Quincy, MA). Mean pressures were derived from the pulsatile signals by electronic averaging. Hindquarters blood flow was set to achieve a baseline perfusion pressure of about 125 mm Hg and was not changed during the remainder of an experiment. The flow rate was determined by timed collection and ranged from 5 to 7 ml/min. In order to denervate the hindquarters vascular bed, the lumbar sympathetic chain ganglia were ligated between L2 and L4. Blood gases were measured periodically and were within the physiological range. These procedures have been previously described (2,6). In the first set of experiments responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ were investigated in the hindquarters vascular bed under constant-flow conditions and compared on a nmol basis to take molecular weight into account. In the second set of experiments the
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role of nitric oxide release in mediating hindquarters vasodilator responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ were determined. Responses were compared before and after administration of L-NAME in a dose of 50 mg/kg IV. In the third series of experiments the role of prostaglandin release in mediating vasodilator responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ was investigated in the hindquarters vascular bed of the rat. Vasodilator responses to endomorphin 1, PL017, DAMGO, nociceptin/OFQ, and arachidonic acid were compared before and beginning 15 min after administration of the cyclooxygenase inhibitor sodium meclofenamate in a dose of 1.5 mg/kg IV. In the fourth series of experiments the role of K1ATP channel activation in mediating vasodilator responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ was investigated in the hindquarters vascular bed of the rat. Vasodilator responses to endomorphin 1, PL017, DAMGO, nociceptin/OFQ, and the K1ATP channel agonist levcromakalim were compared before and beginning 15 min after administration of the K1ATP channel antagonist U-37883A in a dose of 5 mg/kg IV. Endomorphin 1 (L. Hackler, VA Medical Center, New Orleans, LA; Tulane University School of Medicine, New Orleans, LA), diethylamine/NO complex (DEA/NO; Research Biochemicals Inc., Natick, MA), and human synthetic adrenomedullin (hADM; Peptide Research Labs, Tulane University School of Medicine, New Orleans, LA) were dissolved in 0.9% NaCl. Nociceptin (Orphanin FQ), PL017 ([N-MePhe3,D-Pro4]-morphiceptin) and DAMGO (Phoenix Pharmaceuticals, Mountain View, CA) were dissolved in 0.9% NaCl. Acetylcholine chloride, isoproterenol hydrochloride, and arachidonic acid (Sigma Chemical Co., St. Louis, MO) were dissolved in 0.9% NaCl. Prostaglandin E1 (PGE1; Upjohn, Kalamazoo, MI) was dissolved in 100% ethanol and diluted with 0.9% NaCl. Levcromakalim (SmithKline Beecham, Sussex, UK) was dissolved in 10% ethanol-saline solution at a concentration of 10 mg/ml and was diluted with 0.9% NaCl. Drug solutions were stored frozen in amber bottles. During an experiment the agonist solutions were kept on crushed ice. The agonists were administered IA in small volumes (30 –100 ml) over a period of 10 –15 s in a random sequence. Sodium meclofenamate, Nv-nitro-L-arginine methyl ester hydrochloride (LNAME; Sigma Chemical Co.) and naloxone hydrochloride (DuPont Pharmaceuticals, Wilmington, DE) were dissolved in 0.9% NaCl. U-37883A (Upjohn) was dissolved in 0.9% NaCl with sonication. The antagonists used in these studies were prepared immediately before use and injected IV. Control injections of the vehicles for the agents used in this study had no significant effect on baseline systemic arterial or hindquarters perfusion pressure or on responses to the vasoactive agonists. Responses were analyzed with a oneway analysis of variance and Scheffe’s F-test or a paired t-test. L-NAME produced a significant increase in hindquar-
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FIG. 1. Dose–response curves comparing decreases in hindquarters perfusion pressure in response to injections of endomorphin 1, PL017, DAMGO, and nociceptin. Doses of the peptides are expressed on a nmol basis to take molecular weight into account. n indicates number of experiments.
ters perfusion pressure, and responses were expressed as percent decrease from baseline in order to take the increase in baseline perfusion pressure into account. A p value of less than 0.05 was used as the criterion for statistical significance. RESULTS Responses to Endomorphin 1, PL017, DAMGO, and Nociceptin in the Hindquarters Vascular Bed of the Rat Hindquarters vasodilator responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ were compared under constant-flow conditions, and these data are shown in Figure 1. Injections of endomorphin 1, PL017, DAMGO, and nociceptin/OFQ into the perfusion circuit in doses of 3–1000 nmol caused dose-related decreases in hindquarters perfusion pressure (Fig. 1). In terms of relative vasodilator activity, the dose–response curve for endomorphin 1 was about one log-unit to the right of the dose–response curve for nociceptin/OFQ when doses of the peptides were compared on a nmol basis to take molecular weight into account (Fig. 1). The dose–response curves for PL017 and DAMGO were about one log unit to the right of the dose–response curve for endomorphin 1 (Fig. 1). Influence of L-NAME The role of nitric oxide release in mediating vasodilator responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ was investigated, and these results are summarized in Figure 2. After administration of L-NAME in a dose
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FIG. 2. Influence of the nitric oxide synthesis inhibitor L-NAME on vasodilator responses to endomorphin 1, PL017, DAMGO, and nociceptin in the hindquarters vascular bed. Responses were compared before and after administration of L-NAME in a dose of 50 mg/kg IV. Vasodilator responses to the vasoactive peptides are expressed as percent decrease in perfusion pressure to take the L-NAME-induced increase in baseline perfusion pressure into account. n indicates number of experiments and the asterisk indicates that the response is significantly different than control (p , 0.05).
of 50 mg/kg IV, decreases in hindquarters perfusion pressure in response to endomorphin 1, PL017, and DAMGO were significantly reduced under conditions in which vasodilator responses to nociceptin/OFQ were not altered (Fig. 2). Hindquarters vasodilator responses to acetylcholine and adrenomedullin were significantly reduced after administration of L-NAME (Fig. 3). In contrast to the effects of L-NAME on responses to endomorphin 1, PL017, DAMGO, acetylcholine, and adrenomedullin, vasodilator responses to isoproterenol, DEA/NO, and PGE1 were not altered after administration of the nitric oxide synthase inhibitor (Fig. 3). The nitric oxide synthase inhibitor increased systemic arterial and hindquarters perfusion pressures from 127 6 9 to 191 6 8 and from 124 6 12 to 198 6 14, respectively. Effects of Sodium Meclofenamate and U-37883A The effects of the cyclooxygenase inhibitor sodium meclofenamate and the K1ATP channel antagonist U-37883A on responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ were investigated, and these results are summarized in Figures 4 and 5. Hindquarters vasodilator responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ were not altered by sodium meclofenamate (2.5 mg/kg IV) that attenuated vasodilator responses to the prostaglandin precursor arachidonic acid (Fig. 5). Administration of sodium meclofenamate in a dose of 2.5 mg/kg IV did not alter systemic arterial or hindquarters perfusion pressure. Hindquarters vasodilator responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ were not altered by U-37883A in a dose of 5 mg/kg IV (Fig.
4). Vasodilator responses to the K1ATP channel opener levcromakalim were significantly attenuated by the K1ATP channel antagonist U-37883A (Fig. 5). Administration of U-37883A in a dose of 5 mg/kg IV did not alter systemic arterial or hindquarters perfusion pressure. DISCUSSION While it has long been recognized that morphine has vasodepressor activity in man and in experimental animals, the mechanisms mediating the vasodilator actions of m-selective opioid receptor agonists are not well understood (9 –11,14,15,23,26). It has been reported that the synthetic m-opioid receptor agonist DAMGO induces vasodilation of rat pial arteries (9). It has also been reported, however, that DAMGO does not relax vascular smooth muscle and that this peptide induced concentration-dependent contractile responses in rat aortic strips (23,26). The effects of the m-selective opioid agonists endomorphin 1, PL017, and DAMGO on regional vascular resistance were studied in the denervated hindquarters vascular bed of the rat under constant-flow conditions. Injections of endomorphin 1, PL017, and DAMGO into the perfusion circuit induced dose-related decreases in hindquarters perfusion pressure. Inasmuch as blood flow was maintained constant, the decreases in perfusion pressure in response to the peptides reflect decreases in hindquarters vascular resistance. The vasodilator response to these peptides was independent of an action on the adrenergic nervous system, since the hindquarters vascular bed was surgically denervated. The observation that endomorphin 1, PL017, and DAMGO have
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FIG. 3. Influence of L-NAME on vasodilator responses to acetylcholine (ACh), adrenomedullin (ADM), DEA/NO, PGE1, and isoproterenol in the hindquarters vascular bed. Responses were compared before and after administration of L-NAME in a dose of 50 mg/kg IV. Responses were compared on a percent decrease basis to take the L-NAME-induced increase in baseline perfusion pressure into account. n indicates number of experiments, and the asterisk indicates that the response is significantly different than control (p , 0.05).
similar activity suggest that m-opioid receptor activation induces vasodilation in the hindquarters vascular bed of the rat. When the dose–response curves for endomorphin 1, PL017, and DAMGO were compared on a nmol basis to take molecular weight into account, endomorphin 1 was about tenfold more potent than PL017 and DAMGO in decreasing hindquarters vascular resistance. The present data may be interpreted to suggest that endomorphin 1 has greater affinity for the m-opioid receptor than do PL017 or DAMGO. The dose–response curve for endomorphin 1 was similar to the dose–response curve for nociceptin, the endogenous ligand for the ORL1 receptor. These results suggest that activation of m-opioid receptors and ORL1 receptors induces vasodilation, although these peptides have been reported to produce opposite effects in other experimental models. Mu-opioid receptors are present on endothelial cells, and morphine has vasorelaxant activity mediated by the release of nitric oxide from the endothelium (26). The synthetic m-opioid receptor agonist DAMGO induces vasodilation of
rat pial arteries mediated by the release of nitric oxide (9). It was, however, reported that DAMGO does not relax vascular smooth muscle or release nitric oxide and that this peptide induces contractile responses in rat aortic strips (23,26). The difference in response to DAMGO may involve differences in the vascular preparation studied; and the results of the present study in the rat hindquarters vascular bed show that endomorphin 1, PL017, and DAMGO induced dose-related decreases in perfusion pressure that were attenuated by L-NAME in a dose that reduced responses to the endothelium-dependent vasodilator agents acetylcholine and adrenomedullin. The observation that vasodilator responses to endomorphin 1, PL017, DAMGO, acetylcholine, and adrenomedullin were attenuated by LNAME whereas vasodilator responses to the nitric oxide donor DEA/NO, to isoproterenol, PGE1, and to nociceptin/ OFQ were not altered, provides support for the hypothesis that vasodilator responses to m-opioid receptor activation are mediated at least in part by the release of nitric oxide
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FIG. 4. Influence of the cyclooxygenase inhibitor sodium meclofenamate in vasodilator responses to endomorphin 1, PL017, DAMGO, nociceptin, and arachidonic acid (AA) in the hindquarters vascular bed of the rat. n indicates number of experiments, and the asterisk indicates that the response is significantly different than control (p , 0.05).
from the endothelium and are consistent with results showing that vasodilator responses to DAMGO are attenuated by inhibitors of nitric oxide synthesis in rat pial arteries (9). Moreover, these results provide evidence in support for the hypothesis that vasodilator responses to nociceptin/OFQ are not mediated by the release of nitric oxide. The effects of the cyclooxygenase inhibitor sodium meclofenamate and of the K1ATP channel antagonist U-37883A on vasodilator responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ were investigated, and these results show that responses were not altered by sodium meclofenamate in a dose that attenuated vasodilator responses to the prostaglandin precursor arachidonic acid or to U-37883A in a dose that reduced vasodilator responses to the K1ATP channel opener levcromakalim. These results suggest that the release of vasodilator prostaglandins or the opening of K1ATP channels is not involved in mediating responses to endomorphin 1, PL017, DAMGO, or nociceptin/OFQ in the hindquarters vascular bed of the rat. In summary, the results of the present study show that, under constant-flow conditions, endomorphin 1 produced
dose-dependent decreases in hindquarters perfusion pressure that were similar to responses to the endogenous ligand for the ORL1 receptor nociceptin/OFQ, and that endomorphin 1 was more potent than PL017 and DAMGO in decreasing hindquarters vascular resistance. Vasodilator responses to endomorphin 1, PL017, and DAMGO in the hindquarters vascular bed were attenuated by the nitric oxide synthase inhibitor L-NAME at a time when vasodilator responses were not altered by the K1ATP channel antagonist U-37883A or the cyclooxygenase inhibitor sodium meclofenamate. These results suggest that vasodilator responses to endomorphin 1, PL017, and DAMGO are mediated by a nitric oxide-dependent mechanism within the hindquarters vascular bed of the rat and that endomorphin 1 may act as an endothelium-dependent vasodilator agent. ACKNOWLEDGEMENTS These studies were supported in part by NIH Grant HL15580, the VA, and a grant from the American Heart Association–Louisiana, Inc. Hunter C. Champion was supported by NIH Grant HL9474.
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FIG. 5. Effect of the nonsulfonylurea K1ATP channel antagonist U-37883A on vasodilator responses to endomorphin 1, PL017, DAMGO, nociceptin, and levcromkalim (LK) in the hindquarters vascular bed of the rat. Responses were compared before and after administration of U-37883A in a dose of 5 mg/kg IV. n indicates number of experiments, and the asterisk indicates that the response is significantly different than control (p , 0.05)
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 opioid receptor gene family that is not a mu, delta, or kappa opioid receptor type. FEBS Lett. 34:284 –288; 1994. 2. Champion, H. C.; Czapla, M. A.; Friedman, D. E.; Lambert, D. G.; Murphy, W. A.; Coy, D. H.; Kadowitz, P. J. Tonedependent vasodilator responses to proadrenomedullin NH2terminal 20 peptide in the rat. Peptides. 18:513–518; 1997. 3. 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. 4. 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. 5. Champion, H. C.; Zadina, J. E.; Kastin, A. J.; Hackler, L.; Ge, L-J.; Kadowitz, P. J. Endomorphin 1 and 2, the endogenous m-opioid agonists, have novel hypotensive activity in the rabbit. Biochem. Biophys. Res. Commun. 235:567–570; 1997. 6. Champion, H. C.; Zadina, J. E.; Kastin, A. J; Kadowitz, P. J.
7.
8.
9.
10.
11.
The endogenous m-opioid agonists, endomorphin 1 and 2, have vasodilator activity in the hindquarters vascular bed of the rat. Life Sci. 61:PL409 –PL415; 1997. Czapla, M. A.; Champion, H. C.; Zadina, J. E.; Kastin, A. J.; Hackler, L.; Ge, L.; Kadowitz, P. J. Endomorphin 1 and 2, endogenous m-opioid agonists, decrease systemic arterial pressure in the rat. Life Sci. 62:PL175–PL179; 1998. 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. Devine, J. O.; Armstead, W. M. The role of nitric oxide in opioid-induced pial artery vasodilation. Brain Res. 675:257– 263; 1995. Ferreira, S. H.; Duarte, I. D.; Lorenzetti, B. B. The molecular mechanism of action of peripheral morphine analgesia: stimulation of the cGMP system via nitric oxide release. Eur. J. Pharmacol. 201:121–122; 1991. Feuerstein, G.; Siren, L. E. The opioid system in cardiac and vascular regulation of normal and hypertensive states. Circulation. 75:I125–I129; 1987.
1602 12. Gyires, K. The role of endogenous nitric oxide in the gastroprotective action of morphine. Eur. J. Pharmacol. 255:33–37; 1994. 13. Hackler, L.; Zadina, J. E.; Ge, L.; Kastin, A. J. Isolation of relatively large amounts of endomorphin 1 and endomorphin 2 from human brain cortex. Peptides. 18:1635–1639; 1997. 14. Holaday, J. W. Cardiovascular effects of endogenous opiate systems. Ann. Rev. Pharmacol. 23:541–594; 1983. 15. Johnson, M. W.; Mitch, W. E.; Wilcox, C. S. The cardiovascular actions of morphine and the endogenous opioid peptides. Prog. Cardiovasc. Dis. 27:435– 450; 1985. 16. Lachowicz, J. E.; Shen, Y.; Monsma, F. J.; Sibley, D. R. Molecular cloning of novel G-protein coupled receptor related to the opioid receptor family. J. Neurochem. 64:34 – 40; 1995. 17. Liu, Y.; Shenouda, D.; Bilfinger, T. V.; Stefano, M. L.; Magazine, H. I.; Stefano, G. B. Morphine stimulates nitric oxide release from invertebrate microglia. Brain Res. 722:125–131; 1996. 18. Majeed, N. H.; Przewlocka B.; Machelska, H.; Przewloki, R. Inhibition of nitric oxide attenuates the development of morphine tolerance and dependence in mice. Neuropharmacology. 33:189 –192; 1994. 19. Martin-Schild, S.; Zadina, J. E.; Gerall, A. A.; Vigh, S.; Kastin, A. J. Localization of endomorphin 2-like immunoreactivity in the rat medulla and spinal cord. Peptides. 18:1641– 1649; 1997. 20. 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 opioid receptor-like ORL1 receptor. Nature (London). 377: 532–535; 1995.
CHAMPION ET AL. 21. 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. 22. 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. 23. Parra, L.; Perez–Vizcaino, F.; Alsasua, A.; Martin, M. I.; Tamargo, J. m-.; and d-opioid receptor-mediated contractile effects of rat aorta vascular smooth muscle. Eur. J. Pharmacol. 277:99 –105; 1995. 24. 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. 25. 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 (Washington DC). 270:792–794; 1995. 26. Stefano, G. B.; Hartman, A.; Bilfinger, T. V.; Magazine, H. I.; Liu, Y.; Casares, F.; Goligorsky, M. S. Presence of the m opiate receptor in endothelial cells: coupling to nitric oxide production and vasodilation. J. Biol. Chem. 270:30290 – 30293; 1995. 27. Zadina, J. E.; Hackler, L.; Ge, L-J.; Kastin, A. J. A potent and selective endogenous agonist for the m-opiate receptor. Nature (London). 386:499 –502; 1997.
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Nitric Oxide Release Mediates Vasodilator Responses to Endomorphin 1 But Not Nociceptin/ OFQ in the Hindquarters Vascular Bed of the Rat H. C. CHAMPION,* T. J. BIVALACQUA,* D. E. FRIEDMAN,* J. E. ZADINA,†‡ A. J. KASTIN†‡ AND P. J. KADOWITZ*1 Departments of *Pharmacology and †Medicine, Tulane University School of Medicine and ‡The Veterans Affairs Medical Center, New Orleans, LA Received 27 March 1998; Accepted 22 June 1998 CHAMPION, H. C., T. J. BIVALACQUA, D. E. FRIEDMAN, J. E. ZADINA, A. J. KASTIN AND P. J. KADOWITZ. Nitric oxide release mediates vasodilator responses to endomorphin 1 but not nociceptin/OFQ in the hindquarters vascular bed of the rat. PEPTIDES 19(9) 1595–1602, 1998 —We have recently shown that endomorphin1, an endogenous ligand for the m-opioid receptor, and nociceptin (Orphanin FQ; OFQ), an endogenous ligand for the ORL1 receptor, have substantial vasodilator activity in the hindquarters vascular bed of the rat. In the present study, the role of nitric oxide, vasodilator prostaglandins, and the opening of K1ATP channels in mediating vasodilator responses to endomorphin 1, PL017, and DAMGO was investigated in the regional vascular bed in the rat. Under constant-flow conditions, injections of the m-selective agonists endomorphin 1, PL017 ([N-MePhe3,D-Pro4]-morphiceptin), and DAMGO, and the ORL1 receptor agonist nociceptin/ OFQ produced dose-dependent decreases in hindquarters perfusion pressure. Vasodilator responses to endomorphin 1, PL017, and DAMGO, and the endothelium-dependent vasodilators acetylcholine and adrenomedullin were attenuated by the nitric oxide synthase inhibitor L-NAME (50 mg/kg IV) at a time when vasodilator responses to nociceptin/OFQ were not altered. Vasodilator responses to isoproterenol and prostaglandin E1, agents known to increase cAMP levels, and the nitric oxide donor DEA/NO were not altered by the nitric oxide synthase inhibitor. Responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ were not altered by sodium meclofenamate at a time when vasodilator responses to arachidonic acid were reduced significantly or after administration of U-37883A at a time when vasodilator responses to levcromakalim were reduced significantly. The results of these studies indicate that responses to endomorphin 1, PL017, and DAMGO are mediated in large part by the release of nitric oxide, while responses to nociceptin/OFQ are mediated by an L-NAME-insensitive mechanism. Moreover, these results demonstrate that responses to these peptides are not mediated by the release of vasodilator prostaglandins or the opening of K1ATP channels the hindquarters vascular bed. © 1998 Elsevier Science Inc. Nitric oxide-dependent vasodilator Endothelium-dependent vasodilator
Endomorphin 1
ENDOMORPHIN 1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin 2 (Tyr-Pro-Phe-Phe-NH2) are recently discovered endogenous peptides that display high affinity and selectivity for the m-opioid receptor (27). These tetrapeptides were isolated from bovine (27) and human (13) brain and shown to be differentially distributed in the nervous system by RIA
Hindquarters vascular bed
Nociceptin
(27) and ICC (19). Based on the potent and selective activity of the peptides at the m-opioid receptor, it has been suggested that the discovery of endomorphin 1 and 2 may lead to development of newer analgesics with less addictive properties than the traditional opioid agonists. Nociceptin (H2N-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-
1 Requests for reprints should be addressed to Philip J. Kadowitz, Ph.D., Department of Pharmacology SL83, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, Louisiana 70112 USA. E-Mail: [email protected]
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Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln-COOH), also known as orphanin FQ, is an endogenous ligand for the “orphan” opioid receptor (ORL1) (1,8,16,20 –22,24,25). Nociceptin/OFQ differs from the traditional opioid receptor agonists in that it does not possess the N-terminal tyrosine residue that is essential for activity at m, d, and k opioid receptor (20,25). The isolation of nociceptin/OFQ was based on its ability to inhibit adenylyl cyclase in ORL1 transfected cells (20,25). Although this signaling mechanism is similar to other opioid agonists when their receptors are activated, nociceptin has been shown to induce hyperalgesia when injected into the cerebral ventricles (ICV) in the rat (20). In addition, ICV administration of nociceptin/ OFQ decreases spontaneous activity and muscular tone and causes ataxia and loss of the righting reflex (20). Morphine and endogenous opioid peptides have vasodepressor (3,4,11,15) and vasodilator activity (3,15,26,69), and it has recently been shown that endomorphin 1 and 2 decrease systemic arterial pressure in the rabbit (5) and rat (7). In addition, it has recently been shown that endomorphin 1 and 2 have very similar vasodilator activity in the hindquarters vascular bed of the rat (6). However, the mechanism mediating vasodilator responses to the opioid peptides is not well understood (9 –11,14,15,23,26). Human arterial and rat microvascular endothelial cells contain a high affinity, saturable opioid receptor binding site that is antagonized in a stereo-selective manner by naloxone (26). This population of m-opioid receptors has been reported to mediate the release of nitric oxide in response to morphine but not DAMGO (26). In the aorta morphineinduced vasorelaxation is mediated by the release of nitric oxide, and it has been suggested that this release of nitric oxide is due to receptor activation by alkaloids, but not by peptides (26). Another study has shown concentrationdependent contractile responses to this m-selective synthetic agonist that is not dependent upon the presence of vascular endothelium (23,26). Although the explanation for these differences in the effects of morphine and DAMGO on isolated arteries is uncertain, these results are not consistent with the hypothesis that activation of m-opioid receptors release nitric oxide from the endothelium (23,26). Whereas previous studies have shown that the m-opioid agonists endomorphin 1 and 2 and the ORL1 receptor agonist nociceptin/OFQ decrease systemic arterial pressure and have vasodilator activity in the hindquarters vascular bed (3–7), little if anything is known about the role of nitric oxide in mediating vasodilator responses to these endogenous opioid-related peptides in the hindquarters vascular bed of the rat. The present study was, therefore, undertaken to investigate the effects of the nitric oxide synthase inhibitor Nv-nitro-L-arginine methyl ester (L-NAME) on vasodilator responses to the newly discovered m-opioid receptor selective peptide endomorphin 1 and on responses to PL017 and DAMGO in the hindquarters vascular bed of the rat. In
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addition to the release of nitric oxide, vasodilator responses have been shown to be mediated by the release of cyclooxygenase products, as well as the opening of K1ATP channels. We, therefore, tested the role of these mechanisms in mediating vasodilator responses to the opioid peptides in the hindquarters vascular bed of the rat. METHOD Sprague–Dawley rats (Harlan Sprague–Dawley, Indianapolis, IN) weighing 270 – 420 g were anesthetized with pentobarbital sodium (50 mg/kg IV). Supplemental doses of pentobarbital were given during the course of the experiment to ensure a uniform level of anesthesia. The trachea was cannulated, and the rats breathed spontaneously or were ventilated with a Harvard model 683 rodent ventilator (Harvard Apparatus, South Natick, MA) 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 the external jugular vein for the IV administration of drugs and into the carotid artery for the measurement of systemic arterial pressure. 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 (1000 U/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 proximal catheter and pumped at a constant-flow rate with a Masterflex Pump (Cole Parmer Instrument, 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 hindquarter 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 (Oxnard, CA) transducers and were recorded on a Grass model 7 polygraph (Grass Instruments, Quincy, MA). Mean pressures were derived from the pulsatile signals by electronic averaging. Hindquarters blood flow was set to achieve a baseline perfusion pressure of about 125 mm Hg and was not changed during the remainder of an experiment. The flow rate was determined by timed collection and ranged from 5 to 7 ml/min. In order to denervate the hindquarters vascular bed, the lumbar sympathetic chain ganglia were ligated between L2 and L4. Blood gases were measured periodically and were within the physiological range. These procedures have been previously described (2,6). In the first set of experiments responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ were investigated in the hindquarters vascular bed under constant-flow conditions and compared on a nmol basis to take molecular weight into account. In the second set of experiments the
NO-MEDIATED RESPONSES TO ENDOMORPHIN-1
role of nitric oxide release in mediating hindquarters vasodilator responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ were determined. Responses were compared before and after administration of L-NAME in a dose of 50 mg/kg IV. In the third series of experiments the role of prostaglandin release in mediating vasodilator responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ was investigated in the hindquarters vascular bed of the rat. Vasodilator responses to endomorphin 1, PL017, DAMGO, nociceptin/OFQ, and arachidonic acid were compared before and beginning 15 min after administration of the cyclooxygenase inhibitor sodium meclofenamate in a dose of 1.5 mg/kg IV. In the fourth series of experiments the role of K1ATP channel activation in mediating vasodilator responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ was investigated in the hindquarters vascular bed of the rat. Vasodilator responses to endomorphin 1, PL017, DAMGO, nociceptin/OFQ, and the K1ATP channel agonist levcromakalim were compared before and beginning 15 min after administration of the K1ATP channel antagonist U-37883A in a dose of 5 mg/kg IV. Endomorphin 1 (L. Hackler, VA Medical Center, New Orleans, LA; Tulane University School of Medicine, New Orleans, LA), diethylamine/NO complex (DEA/NO; Research Biochemicals Inc., Natick, MA), and human synthetic adrenomedullin (hADM; Peptide Research Labs, Tulane University School of Medicine, New Orleans, LA) were dissolved in 0.9% NaCl. Nociceptin (Orphanin FQ), PL017 ([N-MePhe3,D-Pro4]-morphiceptin) and DAMGO (Phoenix Pharmaceuticals, Mountain View, CA) were dissolved in 0.9% NaCl. Acetylcholine chloride, isoproterenol hydrochloride, and arachidonic acid (Sigma Chemical Co., St. Louis, MO) were dissolved in 0.9% NaCl. Prostaglandin E1 (PGE1; Upjohn, Kalamazoo, MI) was dissolved in 100% ethanol and diluted with 0.9% NaCl. Levcromakalim (SmithKline Beecham, Sussex, UK) was dissolved in 10% ethanol-saline solution at a concentration of 10 mg/ml and was diluted with 0.9% NaCl. Drug solutions were stored frozen in amber bottles. During an experiment the agonist solutions were kept on crushed ice. The agonists were administered IA in small volumes (30 –100 ml) over a period of 10 –15 s in a random sequence. Sodium meclofenamate, Nv-nitro-L-arginine methyl ester hydrochloride (LNAME; Sigma Chemical Co.) and naloxone hydrochloride (DuPont Pharmaceuticals, Wilmington, DE) were dissolved in 0.9% NaCl. U-37883A (Upjohn) was dissolved in 0.9% NaCl with sonication. The antagonists used in these studies were prepared immediately before use and injected IV. Control injections of the vehicles for the agents used in this study had no significant effect on baseline systemic arterial or hindquarters perfusion pressure or on responses to the vasoactive agonists. Responses were analyzed with a oneway analysis of variance and Scheffe’s F-test or a paired t-test. L-NAME produced a significant increase in hindquar-
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FIG. 1. Dose–response curves comparing decreases in hindquarters perfusion pressure in response to injections of endomorphin 1, PL017, DAMGO, and nociceptin. Doses of the peptides are expressed on a nmol basis to take molecular weight into account. n indicates number of experiments.
ters perfusion pressure, and responses were expressed as percent decrease from baseline in order to take the increase in baseline perfusion pressure into account. A p value of less than 0.05 was used as the criterion for statistical significance. RESULTS Responses to Endomorphin 1, PL017, DAMGO, and Nociceptin in the Hindquarters Vascular Bed of the Rat Hindquarters vasodilator responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ were compared under constant-flow conditions, and these data are shown in Figure 1. Injections of endomorphin 1, PL017, DAMGO, and nociceptin/OFQ into the perfusion circuit in doses of 3–1000 nmol caused dose-related decreases in hindquarters perfusion pressure (Fig. 1). In terms of relative vasodilator activity, the dose–response curve for endomorphin 1 was about one log-unit to the right of the dose–response curve for nociceptin/OFQ when doses of the peptides were compared on a nmol basis to take molecular weight into account (Fig. 1). The dose–response curves for PL017 and DAMGO were about one log unit to the right of the dose–response curve for endomorphin 1 (Fig. 1). Influence of L-NAME The role of nitric oxide release in mediating vasodilator responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ was investigated, and these results are summarized in Figure 2. After administration of L-NAME in a dose
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FIG. 2. Influence of the nitric oxide synthesis inhibitor L-NAME on vasodilator responses to endomorphin 1, PL017, DAMGO, and nociceptin in the hindquarters vascular bed. Responses were compared before and after administration of L-NAME in a dose of 50 mg/kg IV. Vasodilator responses to the vasoactive peptides are expressed as percent decrease in perfusion pressure to take the L-NAME-induced increase in baseline perfusion pressure into account. n indicates number of experiments and the asterisk indicates that the response is significantly different than control (p , 0.05).
of 50 mg/kg IV, decreases in hindquarters perfusion pressure in response to endomorphin 1, PL017, and DAMGO were significantly reduced under conditions in which vasodilator responses to nociceptin/OFQ were not altered (Fig. 2). Hindquarters vasodilator responses to acetylcholine and adrenomedullin were significantly reduced after administration of L-NAME (Fig. 3). In contrast to the effects of L-NAME on responses to endomorphin 1, PL017, DAMGO, acetylcholine, and adrenomedullin, vasodilator responses to isoproterenol, DEA/NO, and PGE1 were not altered after administration of the nitric oxide synthase inhibitor (Fig. 3). The nitric oxide synthase inhibitor increased systemic arterial and hindquarters perfusion pressures from 127 6 9 to 191 6 8 and from 124 6 12 to 198 6 14, respectively. Effects of Sodium Meclofenamate and U-37883A The effects of the cyclooxygenase inhibitor sodium meclofenamate and the K1ATP channel antagonist U-37883A on responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ were investigated, and these results are summarized in Figures 4 and 5. Hindquarters vasodilator responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ were not altered by sodium meclofenamate (2.5 mg/kg IV) that attenuated vasodilator responses to the prostaglandin precursor arachidonic acid (Fig. 5). Administration of sodium meclofenamate in a dose of 2.5 mg/kg IV did not alter systemic arterial or hindquarters perfusion pressure. Hindquarters vasodilator responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ were not altered by U-37883A in a dose of 5 mg/kg IV (Fig.
4). Vasodilator responses to the K1ATP channel opener levcromakalim were significantly attenuated by the K1ATP channel antagonist U-37883A (Fig. 5). Administration of U-37883A in a dose of 5 mg/kg IV did not alter systemic arterial or hindquarters perfusion pressure. DISCUSSION While it has long been recognized that morphine has vasodepressor activity in man and in experimental animals, the mechanisms mediating the vasodilator actions of m-selective opioid receptor agonists are not well understood (9 –11,14,15,23,26). It has been reported that the synthetic m-opioid receptor agonist DAMGO induces vasodilation of rat pial arteries (9). It has also been reported, however, that DAMGO does not relax vascular smooth muscle and that this peptide induced concentration-dependent contractile responses in rat aortic strips (23,26). The effects of the m-selective opioid agonists endomorphin 1, PL017, and DAMGO on regional vascular resistance were studied in the denervated hindquarters vascular bed of the rat under constant-flow conditions. Injections of endomorphin 1, PL017, and DAMGO into the perfusion circuit induced dose-related decreases in hindquarters perfusion pressure. Inasmuch as blood flow was maintained constant, the decreases in perfusion pressure in response to the peptides reflect decreases in hindquarters vascular resistance. The vasodilator response to these peptides was independent of an action on the adrenergic nervous system, since the hindquarters vascular bed was surgically denervated. The observation that endomorphin 1, PL017, and DAMGO have
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FIG. 3. Influence of L-NAME on vasodilator responses to acetylcholine (ACh), adrenomedullin (ADM), DEA/NO, PGE1, and isoproterenol in the hindquarters vascular bed. Responses were compared before and after administration of L-NAME in a dose of 50 mg/kg IV. Responses were compared on a percent decrease basis to take the L-NAME-induced increase in baseline perfusion pressure into account. n indicates number of experiments, and the asterisk indicates that the response is significantly different than control (p , 0.05).
similar activity suggest that m-opioid receptor activation induces vasodilation in the hindquarters vascular bed of the rat. When the dose–response curves for endomorphin 1, PL017, and DAMGO were compared on a nmol basis to take molecular weight into account, endomorphin 1 was about tenfold more potent than PL017 and DAMGO in decreasing hindquarters vascular resistance. The present data may be interpreted to suggest that endomorphin 1 has greater affinity for the m-opioid receptor than do PL017 or DAMGO. The dose–response curve for endomorphin 1 was similar to the dose–response curve for nociceptin, the endogenous ligand for the ORL1 receptor. These results suggest that activation of m-opioid receptors and ORL1 receptors induces vasodilation, although these peptides have been reported to produce opposite effects in other experimental models. Mu-opioid receptors are present on endothelial cells, and morphine has vasorelaxant activity mediated by the release of nitric oxide from the endothelium (26). The synthetic m-opioid receptor agonist DAMGO induces vasodilation of
rat pial arteries mediated by the release of nitric oxide (9). It was, however, reported that DAMGO does not relax vascular smooth muscle or release nitric oxide and that this peptide induces contractile responses in rat aortic strips (23,26). The difference in response to DAMGO may involve differences in the vascular preparation studied; and the results of the present study in the rat hindquarters vascular bed show that endomorphin 1, PL017, and DAMGO induced dose-related decreases in perfusion pressure that were attenuated by L-NAME in a dose that reduced responses to the endothelium-dependent vasodilator agents acetylcholine and adrenomedullin. The observation that vasodilator responses to endomorphin 1, PL017, DAMGO, acetylcholine, and adrenomedullin were attenuated by LNAME whereas vasodilator responses to the nitric oxide donor DEA/NO, to isoproterenol, PGE1, and to nociceptin/ OFQ were not altered, provides support for the hypothesis that vasodilator responses to m-opioid receptor activation are mediated at least in part by the release of nitric oxide
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FIG. 4. Influence of the cyclooxygenase inhibitor sodium meclofenamate in vasodilator responses to endomorphin 1, PL017, DAMGO, nociceptin, and arachidonic acid (AA) in the hindquarters vascular bed of the rat. n indicates number of experiments, and the asterisk indicates that the response is significantly different than control (p , 0.05).
from the endothelium and are consistent with results showing that vasodilator responses to DAMGO are attenuated by inhibitors of nitric oxide synthesis in rat pial arteries (9). Moreover, these results provide evidence in support for the hypothesis that vasodilator responses to nociceptin/OFQ are not mediated by the release of nitric oxide. The effects of the cyclooxygenase inhibitor sodium meclofenamate and of the K1ATP channel antagonist U-37883A on vasodilator responses to endomorphin 1, PL017, DAMGO, and nociceptin/OFQ were investigated, and these results show that responses were not altered by sodium meclofenamate in a dose that attenuated vasodilator responses to the prostaglandin precursor arachidonic acid or to U-37883A in a dose that reduced vasodilator responses to the K1ATP channel opener levcromakalim. These results suggest that the release of vasodilator prostaglandins or the opening of K1ATP channels is not involved in mediating responses to endomorphin 1, PL017, DAMGO, or nociceptin/OFQ in the hindquarters vascular bed of the rat. In summary, the results of the present study show that, under constant-flow conditions, endomorphin 1 produced
dose-dependent decreases in hindquarters perfusion pressure that were similar to responses to the endogenous ligand for the ORL1 receptor nociceptin/OFQ, and that endomorphin 1 was more potent than PL017 and DAMGO in decreasing hindquarters vascular resistance. Vasodilator responses to endomorphin 1, PL017, and DAMGO in the hindquarters vascular bed were attenuated by the nitric oxide synthase inhibitor L-NAME at a time when vasodilator responses were not altered by the K1ATP channel antagonist U-37883A or the cyclooxygenase inhibitor sodium meclofenamate. These results suggest that vasodilator responses to endomorphin 1, PL017, and DAMGO are mediated by a nitric oxide-dependent mechanism within the hindquarters vascular bed of the rat and that endomorphin 1 may act as an endothelium-dependent vasodilator agent. ACKNOWLEDGEMENTS These studies were supported in part by NIH Grant HL15580, the VA, and a grant from the American Heart Association–Louisiana, Inc. Hunter C. Champion was supported by NIH Grant HL9474.
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FIG. 5. Effect of the nonsulfonylurea K1ATP channel antagonist U-37883A on vasodilator responses to endomorphin 1, PL017, DAMGO, nociceptin, and levcromkalim (LK) in the hindquarters vascular bed of the rat. Responses were compared before and after administration of U-37883A in a dose of 5 mg/kg IV. n indicates number of experiments, and the asterisk indicates that the response is significantly different than control (p , 0.05)
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 opioid receptor gene family that is not a mu, delta, or kappa opioid receptor type. FEBS Lett. 34:284 –288; 1994. 2. Champion, H. C.; Czapla, M. A.; Friedman, D. E.; Lambert, D. G.; Murphy, W. A.; Coy, D. H.; Kadowitz, P. J. Tonedependent vasodilator responses to proadrenomedullin NH2terminal 20 peptide in the rat. Peptides. 18:513–518; 1997. 3. 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. 4. 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. 5. Champion, H. C.; Zadina, J. E.; Kastin, A. J.; Hackler, L.; Ge, L-J.; Kadowitz, P. J. Endomorphin 1 and 2, the endogenous m-opioid agonists, have novel hypotensive activity in the rabbit. Biochem. Biophys. Res. Commun. 235:567–570; 1997. 6. Champion, H. C.; Zadina, J. E.; Kastin, A. J; Kadowitz, P. J.
7.
8.
9.
10.
11.
The endogenous m-opioid agonists, endomorphin 1 and 2, have vasodilator activity in the hindquarters vascular bed of the rat. Life Sci. 61:PL409 –PL415; 1997. Czapla, M. A.; Champion, H. C.; Zadina, J. E.; Kastin, A. J.; Hackler, L.; Ge, L.; Kadowitz, P. J. Endomorphin 1 and 2, endogenous m-opioid agonists, decrease systemic arterial pressure in the rat. Life Sci. 62:PL175–PL179; 1998. 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. Devine, J. O.; Armstead, W. M. The role of nitric oxide in opioid-induced pial artery vasodilation. Brain Res. 675:257– 263; 1995. Ferreira, S. H.; Duarte, I. D.; Lorenzetti, B. B. The molecular mechanism of action of peripheral morphine analgesia: stimulation of the cGMP system via nitric oxide release. Eur. J. Pharmacol. 201:121–122; 1991. Feuerstein, G.; Siren, L. E. The opioid system in cardiac and vascular regulation of normal and hypertensive states. Circulation. 75:I125–I129; 1987.
1602 12. Gyires, K. The role of endogenous nitric oxide in the gastroprotective action of morphine. Eur. J. Pharmacol. 255:33–37; 1994. 13. Hackler, L.; Zadina, J. E.; Ge, L.; Kastin, A. J. Isolation of relatively large amounts of endomorphin 1 and endomorphin 2 from human brain cortex. Peptides. 18:1635–1639; 1997. 14. Holaday, J. W. Cardiovascular effects of endogenous opiate systems. Ann. Rev. Pharmacol. 23:541–594; 1983. 15. Johnson, M. W.; Mitch, W. E.; Wilcox, C. S. The cardiovascular actions of morphine and the endogenous opioid peptides. Prog. Cardiovasc. Dis. 27:435– 450; 1985. 16. Lachowicz, J. E.; Shen, Y.; Monsma, F. J.; Sibley, D. R. Molecular cloning of novel G-protein coupled receptor related to the opioid receptor family. J. Neurochem. 64:34 – 40; 1995. 17. Liu, Y.; Shenouda, D.; Bilfinger, T. V.; Stefano, M. L.; Magazine, H. I.; Stefano, G. B. Morphine stimulates nitric oxide release from invertebrate microglia. Brain Res. 722:125–131; 1996. 18. Majeed, N. H.; Przewlocka B.; Machelska, H.; Przewloki, R. Inhibition of nitric oxide attenuates the development of morphine tolerance and dependence in mice. Neuropharmacology. 33:189 –192; 1994. 19. Martin-Schild, S.; Zadina, J. E.; Gerall, A. A.; Vigh, S.; Kastin, A. J. Localization of endomorphin 2-like immunoreactivity in the rat medulla and spinal cord. Peptides. 18:1641– 1649; 1997. 20. 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 opioid receptor-like ORL1 receptor. Nature (London). 377: 532–535; 1995.
CHAMPION ET AL. 21. 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. 22. 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. 23. Parra, L.; Perez–Vizcaino, F.; Alsasua, A.; Martin, M. I.; Tamargo, J. m-.; and d-opioid receptor-mediated contractile effects of rat aorta vascular smooth muscle. Eur. J. Pharmacol. 277:99 –105; 1995. 24. 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. 25. 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 (Washington DC). 270:792–794; 1995. 26. Stefano, G. B.; Hartman, A.; Bilfinger, T. V.; Magazine, H. I.; Liu, Y.; Casares, F.; Goligorsky, M. S. Presence of the m opiate receptor in endothelial cells: coupling to nitric oxide production and vasodilation. J. Biol. Chem. 270:30290 – 30293; 1995. 27. Zadina, J. E.; Hackler, L.; Ge, L-J.; Kastin, A. J. A potent and selective endogenous agonist for the m-opiate receptor. Nature (London). 386:499 –502; 1997.