- Email: [email protected]
Spinal administration of selective opioid antagonists in amphibians: Evidence for an opioid unireceptor
Life Sciences,Vol. 64, No. 10,PP.PL 12S130,1999 CopyTight 0 1999 Elscvier science Inc. Printed in the USA. AI1 lights resewed otm-32asp9/ssce front matter
PI1 SOO24.3205(99)00013-2
ELSEVIER
PhWbU4COLOGYLElTERS Accelemted Communication
SPINAL ADMINISTRATION OF SELECTIVE OPIOID ANTAGONISTS EVIDENCE FOR AN OPIOID UNIRECEPTOR
IN AMPHIBIANS:
Craig W. Stevens and Leslie C. Newman Department
of Pharmacology and Physiology, Oklahoma State University, College of Osteopathic Medicine, Tulsa, OK USA (Submitted August X1,1998; accepted November 24,198; received in final form November 25, 1998)
Abstract. In mammals, opioids act by interactions with three distinct types of receptors: p, 6, or K opioid receptors. Using a novel assay of antinociception in the Northern grass frog, Rana pipiens, previous work demonstrated that selective p, 8, or K opioids produced a potent antinociception when administered by the spinal route. The relative potency of this effect was highly correlated to that found in mammals. Present studies employing selective opioid antagonists, p-FNA, NTI, or nor-BNI demonstrated that, in general, these antagonists were not selective in the amphibian model. These data have implications for the functional evolution of opioid receptors in vertebrates and suggest that the tested p, 6, and K opioids mediate antinociception via a single type of opioid receptor in amphibians, termed the unireceptor. 0 1999Else&r Science Inc. KV
Words:
opioid antinociception, morphine, opioid receptor, amphibian, unireceptor
Introduction Previous studies using Northern grass frogs, Rana pipiens, in a behavioral assay for nociception demonstrated a potent and reversible antinociception following systemic, spinal, and intracerebroventricular administration of morphine and other opioids (l-6). Surprisingly, the relative aniinociceptive potency of selective p, 6, and K opioid agonists in amphibians and rodents was highly correlated, even with regard to the actual dose of opioids when given by the spinal route (7). This was unexpected as opioid binding sites in amphibian and mammalian CNS tissue showed great differences in ligand recognition, type, and density. Tissue homogenate binding and receptor isolation studies using amphibian brain tissue demonstrated a high predominance of K-like opioid receptors with little detection of binding sites selective for p or 6 opioids (8,9). The main difference was a greaier affinity of p and &selective opioids and a lesser affinity of K-selective opioids at the main amphibian K-like opioid sites compared to mammalian K opioid sites (IO, 11). In the present study, we further characterized the antinociceptive action of p, 6, and K opioids following zspinal administration in frogs by using selective antagonists for p, 6, and K opioid receptors characterized in mammals; p-tialtrexamine; P-FNA (12), naltrindole; NT1 (13) and norbinaltorphi:mine; nor-BNI (13), respectively. In general, these selective antagonists were not selective o:pioid antagonists in amphibians. The present results suggest that selective p, 6, or K opioid antinociception in amphibians may be mediated by a single type of receptor termed the opioid unireceptor. Corresponding author: Craig W. Stevens, Ph.D., OSU-COM, Department. of Pharmacology Physiology, 1111 W. 17th Street, Tulsa, OK 74107 USA. email: [email protected]
and
PL-126
Selective Opioid Antagonists in Amphibians
Vol. 64, No. 10, 1999
Methods Animals. Male or female Northern grass frogs, Ranapipiens, snout-vent length of 5-7 cm (25-35 g) were used. Upon arrival, frogs are kept in stainless steel group holding aquaria, provided with flowing water and fed crickets and/or mealworms three to four times weekly. At least two days before the start of an experiment, frogs are transferred to the laboratory and randomly assigned to individual plastic cages with adequate water for acclimatization to laboratory conditions. Drugs and experimental design. Opioid agonists used were: u: morphine, fentanyl, DAMGO; 6: DADLE, DPDPE, [D-Ala2]-deltorphin, DSLET; K: U50488, CI-977 (enadoline) GR89696, obtained from commercial sources (RBI, Natick, MA). For studies of the selective opioid antagonists, the general strategy was to use, P-FNA, NTI, and nor-BNI to test for selective antagonism of p, 6, and K opioid antinociception after spinal administration. Antagonist doses for each agent were chosen based on the lowest dose conferring antagonism against a respective opioid agonist. Thus, intraspinal administration of P-FNA at 20 mnol was the lowest dose which blocked the antinociception produced by fentanyl administered 24 hours later. Unlike in mammals (12,14), P-FNA did not produce any acute antinociceptive effect (data not shown), so both a pretreatment and concurrent design was used. Likewise, co-administration of naltrindole at 0.I nmol was the lowest dose that blocked DPDPE antinociception and nor-BNI at 0.1 mnol was the minimal dose that significantly blocked antinociceptive effects of the K opioid, GR89696 (data not shown). Intraspinal (is.) administration. The technique of intraspinal administration in frogs was described fully elsewhere (1). Frogs are gently hand-held and injections made using a tapered 26-gauge needle fitted to a microsyringe. The needle is introduced into the spinal cord slightly lateral to the neural spine at the articulation between the seventh and eighth vertebrae. Penetration of the needle often produces a mild hindlimb extension or trembling which discontinues after a 2-3 second delivery of the drug. All drugs and doses were delivered in a volume of 5 pi/frog and expressed as nmol/animal. The acetic acid test. The nociceptive threshold (NT) in frogs is obtained by testing with 11 concentrations of acetic acid serially diluted from glacial acetic acid. The concentrations are given a code number from 0 to 10 with the lowest code number equal to the lowest concentration of acetic acid and range from 0.26 to 15M. Nociceptive testing is done by placing, with a Pasteur pipette, a single drop of acid on the dorsal surface of the frog’s thigh. The NT is defined as the code number of the lowest concentration of acid which causes the frog to vigorously wipe the treated leg with either hindlimb. To prevent tissue damage, the acetic acid is immediately wiped off with a gentle stream of distilled water once the animal responds or after four seconds if the animal fails to respond. An animal which fails to respond to the highest concentration (10) is assigned the cut-off valueof 11. Data collection and analysis. For quantification of antinociceptive effects, the NT was determined in animals before the administration of the test agent (baseline NT) and at 1, 3, 5h after administration. Raw data collected as the individual animal’s NT is entered on a spreadsheet and maximum percent effect (MPE) calculated by the following formula: M.P.E. = (post-treatment NT - baseline NT) x 100 (cut-off (11) - baseline NT) M.P.E. data was combined for individual animals receiving the same treatment and maximal effect over the time course averaged and plotted. Pharmacological software (Pharm PCS, MicroComputer Specialists, Philadelphia, PA) was used for statistical testing of treatment groups.
Vol. 64, No. 110,1999
Selective Opioid Antagonists in Amphibians
PL-127
Results All agonist does used produced significant antinociception compared to saline or selective antagonist alone administration (data not shown). In single dose studies, concurrent or 24h pretreatment of P-FNA significantly blocked the antinociceptive effects of morphine, fentanyl, DAMGO, DPDPE, C1977, and DADLE. U50488 was blocked by concurrent p-FNA but not by 24h pretreatment of P-FNA (Figure 1, below). Co-administration of naltrindole (0.1 nmol/fiog, i.s.) significantly attenuated the antinociception produced by DPDPE, DSLET and morphine, potentiated the effects of DADLE and fentanyl, and had no effect on the antinociception produced by DAMGO and GR89696 (Fig. 2, next page). As shown in Figure 3 (next page), coadministration of the K-selective opioid antagonist, nor-BNI (0.1 nmol/fiog, i.s.) significantly blocked the antinociceptive effect of the K agonists, U50488 and GR89696, as well as the p opioids, morphine and fentanyl, and the 6 opioids, DPDPE and deltorphin. The 6 agent, DSLET, was not blocked and the mixed p-6 agonist, DADLE, was significantly potentiated.
-
Drug Alone
0
Pre f3-FNA
SAL
MOR 30 nmol
FENT 30 nmol
t
DAMGO DADLE 3 nmol 100 nmol
DPDPE
U50488
Cl977
10 nmol 100 nmol 30 nmol
Fig. 1 Opioid antagonism of p, 6, and K opioid agonists by P-FNA (20 nmol/frog, i.s.) following co-administration or 24h pretreatment. N=6-12 animals/treatment group. First bar of each set is agonist alone, second bar is coadministration and third bar is 24h pretreatment with P-FNA. Dose of agonists given under each agent along the x-axis (nmol/fiog). Data presented as the maximum MPE obtained for groups of animals over the 5h testing time course (mean + s.e.m.). Asterisks (*) denote significant decrease compared to agonist alone effect, (PcO.05, one-tailed t-test). MOR = morphine, FENT = fentanyl, and the rest of the acronyms are abbreviations for opioid peptide analogs or drug company designations (see Methods section for selectivity of these opioid agonists).
Selective Opioid Antagonists in Amphibians
PL.-128
80
0
Agonist Alone
m
Agonist
Vol. 64, No. 10, 1999
(is.)
+ Naltrindole
(0.1 nmolhg)
70 5 E y
60 50
5 Lu 40 is I$ 30 2 5
20 10 0
NTI DPDPE 0.1 nmol 10 nmol
DSLET 3 nmol
DADLE M R FENT 100 nmol 30 nmol 30 nmol
AMGO GR89898 1 nmol 10 nmol
Fig. 2 Opioid antagonism by the 6 antagonist, naltrindole (NTI) following coadministration (0.1 nmol/frog, i.s.) with ~.t,6, or K opioid agonists (agents and doses given on x-axis). N=6-12 animals/treatment group. Asterisks (*) denote significant decrease, plus (+) significant increase to agonist alone group (P~0.05, one-tailed t-test). 80 70 ; .
60
2 z
20
0 m
Agonist Agonist
Alone (i.s.) + nor-BNI (0.1 nmolhog)
T
10 0
nor-BNI MORPH FENT DPDPE DELT DADLE DSLET GR898 U50488 0.1 nmol30 nmol 30 nmol IO nmol 30 nmoliO0 nmol 3 nmol 10 nmollO0 nmol
Fig. 3 Opioid antagonism by the K antagonist, nor-BNI, following co-administration (0.1 nmol/fiog, i.s.) with l.~,6, or K opioid agonists (agents and doses given on axis). N=6-12 animals/bar. Asterisks (*) denote significant decrease, plus (+) significant increase to agonist alone group (PcO.05, one-tailed t-test).
Vol. 64, No. 10, 1999
Selective Opioid Antagonists in Amphibians
PC129
Discussion The present results are the first data of selective opioid antagonists used in a non-mammalian vertebrate model of opioid antinociception. It has been shown by a number of investigators that spinal administration of u, 6, or K opioids produces a dose-dependent elevation of nociceptive thresholds in common rodent models of antinociception (15-17). The strongest pharmacological evidence of antinociception mediated by distinct opioid receptor types in mammals comes from studies using selective opioid agonists in conjunction with selective opioid antagonists. Spinal opioid antircociception was shown to be mediated by p opioid receptors, as intrathecal (i.t.) administration of the p-selective antagonist, p-FNA, blocked the antinociceptive effects of i.t. p agonists, but not 6 or K agonists in mice (14) or rats (18). A 6 type of opioid receptor mediating spinal antinociception was shown by systemic pretreatment (19) or i.t. coadministration (20)of the &selective antagonist, NTI, and blockade of the antinociceptive effects of i.t. DPDPE or DSLET, but not p or K opioids. Antinociception mediated by K opioid receptors was demonstrated by i.t. pretreatment with the K selective antagonist, nor-BNI, as shown by subsequent failure of i.t. U50488H to produce antinociceptive effects which were still present after i.t. administration of u and 6 opioidls (21). The three main types of opioid receptors in mammals have been further divided in subtypes; however this issue is beyond the scope of the present study. The present results using amphibians suggest a quite different story from mammals. The general finding that selective opioid antagonists did not uniformly block their respective selective opioids lends credence to the parsimonious conclusion that an opioid unireceptor may be mediating the antinociceptive effect of all types of opioid agonists in amphibians. These data fit interpretation of the “negative determinant” hypothesis of receptor evolution which states that earlier-evolved vertebrates may contain ancestral and promiscuous receptors which through gene duplication and modification become selective receptor types (22,23). Should this be the case in amphibians, it is also possible that conservation of the “molecular efftcacy” of evolved opioid receptors would produce the surprising finding of correlated relative antinociceptive potency of p, 6, and K opioids in amphibians and mammals (7), despite that only one predominant opioid binding site is identified in competition binding studies using amphibian tissues. In contrast, p-,6- and K-like opioid receptors were recently cloned in fish (24) and binding studies suggest that invertebrates contain p and b-like opioid binding sites (25). That the presence of opioid binding sites has been detected in many invertebrate and vertebrates species forms the basis of the “conformation matching” proposal by Stefano (26). However, previous studies in non-mammalian vertebrates have not assessed opioid antinociception with regard to opioid receptor type using selective antagonists. The present findings confirm the broad conservation of opioid ligand and receptor conformation and provide novel data on the functional evolution of p, 6, and K opioid receptor types. These findings have implications for the evolution of opioid receptors mediating antinociception throughout the vertebrate p:hylogeny. Acknowledgements We gratefully acknowledge the past and hopefully future support of the National Institutes of Health (DAO7326) for these studies. Much appreciation also for institutional support provided by departmental funds. Thanks also to Drs. George Brenner, Dave Wallace, Patricia Claude, and Srinavasa Nagalla for stimulating discussions on the possibility of the opioid “unireceptor” in amphibians.
PL-130
Selective Opioid Antagonists in Amphibians
Vol. 64, No. 10, 1999
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 2 1. 22. 23. 24. 25. 26.
C.W. STEVENS and P.D. PEZALLA, Life Sci. 33 2097-2103 (1983). C.W. STEVENS and P.D. PEZALLA, Brain Res. 301 171-174 (1984). C.W. STEVENS, P.D. PEZALLA and T.L. YAKSH, Brain Res. 402 201-203 (1987). C.W. STEVENS and K. KIRKENDALL, Life Sci. 52 PLll l-l 16 (1993). C.W. STEVENS, A.J. KLOPP and J.A. FACELLO, J.Pharmacol.Exp.Ther. 269 1086-1093 (1994). C.W. STEVENS and K. ROTHE-SKINNER, Eur.J.Pharmacol. 331 15-21 (1997). C.W. STEVENS, J.Pharmacol.Exp.Ther. 276 440-448 (1996). E.J. SIMON, J.M. HILLER, J. GROTH, Y. ITZHAK, M.J. HOLLAND and S.G. BECK, Life Sci. 31 1367-1370 (1982). M. WOLLEMANN, S. BENYHE and J. SIMON, Life Sci. 52 599-611 (1993). C. MOLLEREAU, A. PASCAUD, G. BAILLAT, H. MAZARGUIL, A. PUGET and J.C. MEUNIER, Eur.J.Pharmacol. 150 75-84 (1988). S. BENYHE, E. VARGA, J. HEPP, A. MAGYAR, A. BORSODI and M. WOLLEMANN, Neurochem.Res. 15 899-904 (1990). A.E. TAKEMORI, Arm.Rev.Pharmacol.Toxicol. 25 193-223 (1985). A.E. TAKEMORI and P.S. PORTOGHESE, Atm.Rev.Pharmacol.Toxicol. 32 239-269 (1992). Q.I. JIANG, J.S. HEYMAN, R.J. SHELDON, R.J. KOSLO and F. PORRECA, J.Pharmacol.Exp.Ther. 252 1006-1011 (1990). F. PORRECA, H.I. MOSBERG, J.R. OMNAAS, T.F. BURKS and A. COWAN, J.Pharmacol.Exp.Ther. 240 890-894 (1987). C.W. STEVENS and T.L. YAKSH, J.Pharmacol.Exp.Ther. 238 833-838 (1986). J.C. HUNTER, G.E. LEIGHTON, K.G. MEECHAM, S.J. BOYLE, D.C. HORWELL, D.C. REES and J. HUGHES, Br.J.Pharmacol. 101 183-189 (1990). E. MJANGER and T.L. YAKSH, J.Pharmacol.Exp.Ther. 258 544-550 (1991). M. SOFUOGLU, P.S. PORTOGHESE and A.E. TAKEMORI, J.Pharmacol.Exp.Ther. 257 676-680 (1991). P.J. TISEO and T.L. YAKSH, Eur.J.Pharmacol. 236 89-96 (1993). A.E. TAKEMORI, B.Y. HO, J.S. NAESETH and P.S. PORTOGHESE, J.Pharmacol.Exp.Ther. 246 255-258 (1988). W.R. MOYLE, R.K. CAMPBELL, R.V. MYERS, M.P. BERNARD, Y. HAN and X. WANG, Nature 368 251-254 (1994). R.K. CAMPBELL, E.R. BERGERT, Y. WANG, J.C. MORRIS and W.R. MOYLE, nature biotechnology 15 439-443 (1997). M.G. DARLISON, F.R. GRETEN, R.J. HARVEY, H. KREIENKAMP, T. STUHMER, H. ZWIERS, K. LEDERIS and D. RICHTER, Proc.Natl.Acad.Sci.USA 94 8214-8219 (1997). STEFANO, G.B., Comp. Biochem. Physiol. C 90 287-294 (1988). SALZET, M. and G.B. STEFANO, Brain Res. 768 224-232 (1997).
PI1 SOO24.3205(99)00013-2
ELSEVIER
PhWbU4COLOGYLElTERS Accelemted Communication
SPINAL ADMINISTRATION OF SELECTIVE OPIOID ANTAGONISTS EVIDENCE FOR AN OPIOID UNIRECEPTOR
IN AMPHIBIANS:
Craig W. Stevens and Leslie C. Newman Department
of Pharmacology and Physiology, Oklahoma State University, College of Osteopathic Medicine, Tulsa, OK USA (Submitted August X1,1998; accepted November 24,198; received in final form November 25, 1998)
Abstract. In mammals, opioids act by interactions with three distinct types of receptors: p, 6, or K opioid receptors. Using a novel assay of antinociception in the Northern grass frog, Rana pipiens, previous work demonstrated that selective p, 8, or K opioids produced a potent antinociception when administered by the spinal route. The relative potency of this effect was highly correlated to that found in mammals. Present studies employing selective opioid antagonists, p-FNA, NTI, or nor-BNI demonstrated that, in general, these antagonists were not selective in the amphibian model. These data have implications for the functional evolution of opioid receptors in vertebrates and suggest that the tested p, 6, and K opioids mediate antinociception via a single type of opioid receptor in amphibians, termed the unireceptor. 0 1999Else&r Science Inc. KV
Words:
opioid antinociception, morphine, opioid receptor, amphibian, unireceptor
Introduction Previous studies using Northern grass frogs, Rana pipiens, in a behavioral assay for nociception demonstrated a potent and reversible antinociception following systemic, spinal, and intracerebroventricular administration of morphine and other opioids (l-6). Surprisingly, the relative aniinociceptive potency of selective p, 6, and K opioid agonists in amphibians and rodents was highly correlated, even with regard to the actual dose of opioids when given by the spinal route (7). This was unexpected as opioid binding sites in amphibian and mammalian CNS tissue showed great differences in ligand recognition, type, and density. Tissue homogenate binding and receptor isolation studies using amphibian brain tissue demonstrated a high predominance of K-like opioid receptors with little detection of binding sites selective for p or 6 opioids (8,9). The main difference was a greaier affinity of p and &selective opioids and a lesser affinity of K-selective opioids at the main amphibian K-like opioid sites compared to mammalian K opioid sites (IO, 11). In the present study, we further characterized the antinociceptive action of p, 6, and K opioids following zspinal administration in frogs by using selective antagonists for p, 6, and K opioid receptors characterized in mammals; p-tialtrexamine; P-FNA (12), naltrindole; NT1 (13) and norbinaltorphi:mine; nor-BNI (13), respectively. In general, these selective antagonists were not selective o:pioid antagonists in amphibians. The present results suggest that selective p, 6, or K opioid antinociception in amphibians may be mediated by a single type of receptor termed the opioid unireceptor. Corresponding author: Craig W. Stevens, Ph.D., OSU-COM, Department. of Pharmacology Physiology, 1111 W. 17th Street, Tulsa, OK 74107 USA. email: [email protected]
and
PL-126
Selective Opioid Antagonists in Amphibians
Vol. 64, No. 10, 1999
Methods Animals. Male or female Northern grass frogs, Ranapipiens, snout-vent length of 5-7 cm (25-35 g) were used. Upon arrival, frogs are kept in stainless steel group holding aquaria, provided with flowing water and fed crickets and/or mealworms three to four times weekly. At least two days before the start of an experiment, frogs are transferred to the laboratory and randomly assigned to individual plastic cages with adequate water for acclimatization to laboratory conditions. Drugs and experimental design. Opioid agonists used were: u: morphine, fentanyl, DAMGO; 6: DADLE, DPDPE, [D-Ala2]-deltorphin, DSLET; K: U50488, CI-977 (enadoline) GR89696, obtained from commercial sources (RBI, Natick, MA). For studies of the selective opioid antagonists, the general strategy was to use, P-FNA, NTI, and nor-BNI to test for selective antagonism of p, 6, and K opioid antinociception after spinal administration. Antagonist doses for each agent were chosen based on the lowest dose conferring antagonism against a respective opioid agonist. Thus, intraspinal administration of P-FNA at 20 mnol was the lowest dose which blocked the antinociception produced by fentanyl administered 24 hours later. Unlike in mammals (12,14), P-FNA did not produce any acute antinociceptive effect (data not shown), so both a pretreatment and concurrent design was used. Likewise, co-administration of naltrindole at 0.I nmol was the lowest dose that blocked DPDPE antinociception and nor-BNI at 0.1 mnol was the minimal dose that significantly blocked antinociceptive effects of the K opioid, GR89696 (data not shown). Intraspinal (is.) administration. The technique of intraspinal administration in frogs was described fully elsewhere (1). Frogs are gently hand-held and injections made using a tapered 26-gauge needle fitted to a microsyringe. The needle is introduced into the spinal cord slightly lateral to the neural spine at the articulation between the seventh and eighth vertebrae. Penetration of the needle often produces a mild hindlimb extension or trembling which discontinues after a 2-3 second delivery of the drug. All drugs and doses were delivered in a volume of 5 pi/frog and expressed as nmol/animal. The acetic acid test. The nociceptive threshold (NT) in frogs is obtained by testing with 11 concentrations of acetic acid serially diluted from glacial acetic acid. The concentrations are given a code number from 0 to 10 with the lowest code number equal to the lowest concentration of acetic acid and range from 0.26 to 15M. Nociceptive testing is done by placing, with a Pasteur pipette, a single drop of acid on the dorsal surface of the frog’s thigh. The NT is defined as the code number of the lowest concentration of acid which causes the frog to vigorously wipe the treated leg with either hindlimb. To prevent tissue damage, the acetic acid is immediately wiped off with a gentle stream of distilled water once the animal responds or after four seconds if the animal fails to respond. An animal which fails to respond to the highest concentration (10) is assigned the cut-off valueof 11. Data collection and analysis. For quantification of antinociceptive effects, the NT was determined in animals before the administration of the test agent (baseline NT) and at 1, 3, 5h after administration. Raw data collected as the individual animal’s NT is entered on a spreadsheet and maximum percent effect (MPE) calculated by the following formula: M.P.E. = (post-treatment NT - baseline NT) x 100 (cut-off (11) - baseline NT) M.P.E. data was combined for individual animals receiving the same treatment and maximal effect over the time course averaged and plotted. Pharmacological software (Pharm PCS, MicroComputer Specialists, Philadelphia, PA) was used for statistical testing of treatment groups.
Vol. 64, No. 110,1999
Selective Opioid Antagonists in Amphibians
PL-127
Results All agonist does used produced significant antinociception compared to saline or selective antagonist alone administration (data not shown). In single dose studies, concurrent or 24h pretreatment of P-FNA significantly blocked the antinociceptive effects of morphine, fentanyl, DAMGO, DPDPE, C1977, and DADLE. U50488 was blocked by concurrent p-FNA but not by 24h pretreatment of P-FNA (Figure 1, below). Co-administration of naltrindole (0.1 nmol/fiog, i.s.) significantly attenuated the antinociception produced by DPDPE, DSLET and morphine, potentiated the effects of DADLE and fentanyl, and had no effect on the antinociception produced by DAMGO and GR89696 (Fig. 2, next page). As shown in Figure 3 (next page), coadministration of the K-selective opioid antagonist, nor-BNI (0.1 nmol/fiog, i.s.) significantly blocked the antinociceptive effect of the K agonists, U50488 and GR89696, as well as the p opioids, morphine and fentanyl, and the 6 opioids, DPDPE and deltorphin. The 6 agent, DSLET, was not blocked and the mixed p-6 agonist, DADLE, was significantly potentiated.
-
Drug Alone
0
Pre f3-FNA
SAL
MOR 30 nmol
FENT 30 nmol
t
DAMGO DADLE 3 nmol 100 nmol
DPDPE
U50488
Cl977
10 nmol 100 nmol 30 nmol
Fig. 1 Opioid antagonism of p, 6, and K opioid agonists by P-FNA (20 nmol/frog, i.s.) following co-administration or 24h pretreatment. N=6-12 animals/treatment group. First bar of each set is agonist alone, second bar is coadministration and third bar is 24h pretreatment with P-FNA. Dose of agonists given under each agent along the x-axis (nmol/fiog). Data presented as the maximum MPE obtained for groups of animals over the 5h testing time course (mean + s.e.m.). Asterisks (*) denote significant decrease compared to agonist alone effect, (PcO.05, one-tailed t-test). MOR = morphine, FENT = fentanyl, and the rest of the acronyms are abbreviations for opioid peptide analogs or drug company designations (see Methods section for selectivity of these opioid agonists).
Selective Opioid Antagonists in Amphibians
PL.-128
80
0
Agonist Alone
m
Agonist
Vol. 64, No. 10, 1999
(is.)
+ Naltrindole
(0.1 nmolhg)
70 5 E y
60 50
5 Lu 40 is I$ 30 2 5
20 10 0
NTI DPDPE 0.1 nmol 10 nmol
DSLET 3 nmol
DADLE M R FENT 100 nmol 30 nmol 30 nmol
AMGO GR89898 1 nmol 10 nmol
Fig. 2 Opioid antagonism by the 6 antagonist, naltrindole (NTI) following coadministration (0.1 nmol/frog, i.s.) with ~.t,6, or K opioid agonists (agents and doses given on x-axis). N=6-12 animals/treatment group. Asterisks (*) denote significant decrease, plus (+) significant increase to agonist alone group (P~0.05, one-tailed t-test). 80 70 ; .
60
2 z
20
0 m
Agonist Agonist
Alone (i.s.) + nor-BNI (0.1 nmolhog)
T
10 0
nor-BNI MORPH FENT DPDPE DELT DADLE DSLET GR898 U50488 0.1 nmol30 nmol 30 nmol IO nmol 30 nmoliO0 nmol 3 nmol 10 nmollO0 nmol
Fig. 3 Opioid antagonism by the K antagonist, nor-BNI, following co-administration (0.1 nmol/fiog, i.s.) with l.~,6, or K opioid agonists (agents and doses given on axis). N=6-12 animals/bar. Asterisks (*) denote significant decrease, plus (+) significant increase to agonist alone group (PcO.05, one-tailed t-test).
Vol. 64, No. 10, 1999
Selective Opioid Antagonists in Amphibians
PC129
Discussion The present results are the first data of selective opioid antagonists used in a non-mammalian vertebrate model of opioid antinociception. It has been shown by a number of investigators that spinal administration of u, 6, or K opioids produces a dose-dependent elevation of nociceptive thresholds in common rodent models of antinociception (15-17). The strongest pharmacological evidence of antinociception mediated by distinct opioid receptor types in mammals comes from studies using selective opioid agonists in conjunction with selective opioid antagonists. Spinal opioid antircociception was shown to be mediated by p opioid receptors, as intrathecal (i.t.) administration of the p-selective antagonist, p-FNA, blocked the antinociceptive effects of i.t. p agonists, but not 6 or K agonists in mice (14) or rats (18). A 6 type of opioid receptor mediating spinal antinociception was shown by systemic pretreatment (19) or i.t. coadministration (20)of the &selective antagonist, NTI, and blockade of the antinociceptive effects of i.t. DPDPE or DSLET, but not p or K opioids. Antinociception mediated by K opioid receptors was demonstrated by i.t. pretreatment with the K selective antagonist, nor-BNI, as shown by subsequent failure of i.t. U50488H to produce antinociceptive effects which were still present after i.t. administration of u and 6 opioidls (21). The three main types of opioid receptors in mammals have been further divided in subtypes; however this issue is beyond the scope of the present study. The present results using amphibians suggest a quite different story from mammals. The general finding that selective opioid antagonists did not uniformly block their respective selective opioids lends credence to the parsimonious conclusion that an opioid unireceptor may be mediating the antinociceptive effect of all types of opioid agonists in amphibians. These data fit interpretation of the “negative determinant” hypothesis of receptor evolution which states that earlier-evolved vertebrates may contain ancestral and promiscuous receptors which through gene duplication and modification become selective receptor types (22,23). Should this be the case in amphibians, it is also possible that conservation of the “molecular efftcacy” of evolved opioid receptors would produce the surprising finding of correlated relative antinociceptive potency of p, 6, and K opioids in amphibians and mammals (7), despite that only one predominant opioid binding site is identified in competition binding studies using amphibian tissues. In contrast, p-,6- and K-like opioid receptors were recently cloned in fish (24) and binding studies suggest that invertebrates contain p and b-like opioid binding sites (25). That the presence of opioid binding sites has been detected in many invertebrate and vertebrates species forms the basis of the “conformation matching” proposal by Stefano (26). However, previous studies in non-mammalian vertebrates have not assessed opioid antinociception with regard to opioid receptor type using selective antagonists. The present findings confirm the broad conservation of opioid ligand and receptor conformation and provide novel data on the functional evolution of p, 6, and K opioid receptor types. These findings have implications for the evolution of opioid receptors mediating antinociception throughout the vertebrate p:hylogeny. Acknowledgements We gratefully acknowledge the past and hopefully future support of the National Institutes of Health (DAO7326) for these studies. Much appreciation also for institutional support provided by departmental funds. Thanks also to Drs. George Brenner, Dave Wallace, Patricia Claude, and Srinavasa Nagalla for stimulating discussions on the possibility of the opioid “unireceptor” in amphibians.
PL-130
Selective Opioid Antagonists in Amphibians
Vol. 64, No. 10, 1999
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 2 1. 22. 23. 24. 25. 26.
C.W. STEVENS and P.D. PEZALLA, Life Sci. 33 2097-2103 (1983). C.W. STEVENS and P.D. PEZALLA, Brain Res. 301 171-174 (1984). C.W. STEVENS, P.D. PEZALLA and T.L. YAKSH, Brain Res. 402 201-203 (1987). C.W. STEVENS and K. KIRKENDALL, Life Sci. 52 PLll l-l 16 (1993). C.W. STEVENS, A.J. KLOPP and J.A. FACELLO, J.Pharmacol.Exp.Ther. 269 1086-1093 (1994). C.W. STEVENS and K. ROTHE-SKINNER, Eur.J.Pharmacol. 331 15-21 (1997). C.W. STEVENS, J.Pharmacol.Exp.Ther. 276 440-448 (1996). E.J. SIMON, J.M. HILLER, J. GROTH, Y. ITZHAK, M.J. HOLLAND and S.G. BECK, Life Sci. 31 1367-1370 (1982). M. WOLLEMANN, S. BENYHE and J. SIMON, Life Sci. 52 599-611 (1993). C. MOLLEREAU, A. PASCAUD, G. BAILLAT, H. MAZARGUIL, A. PUGET and J.C. MEUNIER, Eur.J.Pharmacol. 150 75-84 (1988). S. BENYHE, E. VARGA, J. HEPP, A. MAGYAR, A. BORSODI and M. WOLLEMANN, Neurochem.Res. 15 899-904 (1990). A.E. TAKEMORI, Arm.Rev.Pharmacol.Toxicol. 25 193-223 (1985). A.E. TAKEMORI and P.S. PORTOGHESE, Atm.Rev.Pharmacol.Toxicol. 32 239-269 (1992). Q.I. JIANG, J.S. HEYMAN, R.J. SHELDON, R.J. KOSLO and F. PORRECA, J.Pharmacol.Exp.Ther. 252 1006-1011 (1990). F. PORRECA, H.I. MOSBERG, J.R. OMNAAS, T.F. BURKS and A. COWAN, J.Pharmacol.Exp.Ther. 240 890-894 (1987). C.W. STEVENS and T.L. YAKSH, J.Pharmacol.Exp.Ther. 238 833-838 (1986). J.C. HUNTER, G.E. LEIGHTON, K.G. MEECHAM, S.J. BOYLE, D.C. HORWELL, D.C. REES and J. HUGHES, Br.J.Pharmacol. 101 183-189 (1990). E. MJANGER and T.L. YAKSH, J.Pharmacol.Exp.Ther. 258 544-550 (1991). M. SOFUOGLU, P.S. PORTOGHESE and A.E. TAKEMORI, J.Pharmacol.Exp.Ther. 257 676-680 (1991). P.J. TISEO and T.L. YAKSH, Eur.J.Pharmacol. 236 89-96 (1993). A.E. TAKEMORI, B.Y. HO, J.S. NAESETH and P.S. PORTOGHESE, J.Pharmacol.Exp.Ther. 246 255-258 (1988). W.R. MOYLE, R.K. CAMPBELL, R.V. MYERS, M.P. BERNARD, Y. HAN and X. WANG, Nature 368 251-254 (1994). R.K. CAMPBELL, E.R. BERGERT, Y. WANG, J.C. MORRIS and W.R. MOYLE, nature biotechnology 15 439-443 (1997). M.G. DARLISON, F.R. GRETEN, R.J. HARVEY, H. KREIENKAMP, T. STUHMER, H. ZWIERS, K. LEDERIS and D. RICHTER, Proc.Natl.Acad.Sci.USA 94 8214-8219 (1997). STEFANO, G.B., Comp. Biochem. Physiol. C 90 287-294 (1988). SALZET, M. and G.B. STEFANO, Brain Res. 768 224-232 (1997).