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Magnetic fields differentially inhibit mu, delta, kappa and sigma opiate-induced analgesia in mice
Peptides, Vol. 7. pp. 449-453, 1986. ©Ankho InternationalInc. Printed in the U.S.A.
0196-9781/86 $3.00 + .00
Magnetic Fields Differentially Inhibit Mu, Delta, Kappa and Sigma Opiate-Induced Analgesia in Mice MARTIN KAVALIERS*~ AND KLAUS-PETER
OSSENKOPPt
*Department o f Psychology, University o f Alberta, Edmonton, Alberta, Canada T6G 2E9 and tDepartment o f Psychology, University o f Western Ontario, London, Ontario, Canada N6A 5C2 R e c e i v e d 5 D e c e m b e r 1985 KAVALIERS, M. AND K.-P. OSSENKOPP. Magnetic fields differentially inhibit mu, delta, kappa and sigma opiateinduced analgesia in mice. PEPTIDES 7(3) 449--453, 1986.--An exposure for 60 min to a 0.5 Hz rotating magnetic field (1.5-90 G) significantly attenuated the daytime analgesic effects of the mu and kappa opiate agonists, morphine and U50,488H, respectively, and significantly inhibited the analgesic actions of the delta agonist, D-Ala2-D-Leu~-enkephalin, in mice. The magnetic stimuli had no significant effects on the analgesic effects of the prototypic sigma opiate agonist (_+) SKF-10,047. These results show that exposure to relatively weak magnetic stimuli has significant and differential inhibitory influences on various opioid systems. Mu, delta, kappa, and sigma opiates
Analgesia
Magnetic fields are altered in a manner similar to that observed with morphine. Accordingly, in view of the existence of multiple opioid systems and receptor types [27], it is of importance to determine whether exposure to magnetic fields has inhibitory influences on other opiate agonists and opioid systems. We report here that an exposure for 60 min to a 0.5 Hz rotating magnetic field significantly reduces the analgesic effects of mu (morphine sulfate); delta (DADLE; D-AlaZ-D-LeuS-enkephalin [27]) and kappa (U-50,488H; trans-3,4,-dichloroN-methyl-2-(l-pyrrolidinyl-cyclohexyl-benzeneacetaide methanesulfonate hydrate [27,37]) directed opiates in mice, while not having any significant actions on sigma ((-+) SKF-10-047; N-allynormetazocine [24-27]) induced analgesia.
T H E possibility that magnetic fields can significantly influence biological systems in a wide range of animals, including that of humans, has gained considerable acceptance [ 1, 3-6, 10, 12, 34]. Among the more dramatic effects of magnetic stimuli is the suppression of the analgesic and locomotory effects of morphine. Exposure of mice to earth-strength, 0-1.5 gauss, 60 Hz magnetic fields results in a reversible dosedependent inhibition of the nocturnal peak in the day-night rhythm of morphine-induced analgesia [17,18]. Natural geomagnetic disturbances arising from intense solar activity also reduce night-time morphine-induced analgesia [28]. Furthermore, relatively weak rotating magnetic fields have been demonstrated to reduce the day- and night-time analgesic effects of morphine [18, 20, 22], attenuate the locomotory effects of morphine [18,20], modify the rate of tolerance development to morphine-induced analgesia [19] and reduce stress-induced opioid analgesia in mice [2l]. Recently, it was shown that exposure to magnetic resonance imaging (MRI) procedures also suppressed the day- and night-time analgesic responses of mice receiving morphine injections [29, 31, 32]. Although the mechanisms for these inhibitory effects of magnetic fields on morphine-induced responses are poorly understood, results of our initial studies suggest that changes in the central distribution and levels of calcium and other divalent ions are likely to be involved [20]. These inhibitory effects of magnetic fields, and in particular those associated with MRI, have a number of practical and clinical implications. They raise the possibility that magnetic field components of MRI [32] may have inhibitory effects on responses to analgesics such as morphine in humans [31]. In addition, these observations also raise the question as to whether or not the effects of other exogenous opiates
METHOD
Animals Sexually mature male CF-1 mice (Charles River, Quebec) 3-4 months old and 30-35 g were housed in groups of 5 in polyethylene cages with stainless steel wire tops at 23_+ I°C under a 12 hr light:12 hr dark cycle. Food and water were available ad lib.
Experimental Apparatus At mid-photophase, two cages of mice with the stainless steel wire tops replaced by plastic covers, were placed on 5 cm form rubber sheets and positioned between the magnets of 0.5 Hz magnetic field apparatus [22]. The apparatus consisted of two horseshoe magnets that were rotated in opposite directions along their major axes at 29 rpm. Field intensities (with the magnets stationary and aligned along opposite
tRequests for reprints should be addressed to Martin Kavaliers, Division of Oral Biology, Faculty of Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.
449
450
KAVALIERS AND OSSENKOPP
50
50
A. mu
B. kappa
30
30
P
I
I lO!
'E 10! U SI
C 0 Q. ffl
50
i
C RF Sh Morphine
U SI
i
C. delta
i
C RF Sh U-50,488H i
D. sigma
>, U C
30
McCormick [14], other mice (n=10, in all cases) received intracerebroventricular (ICV) injections of D A D L E (1.0/~g; Peninsula, Belmont, CA) in 2.0 ~1 saline (1.0 txl in each side of the brain) or ICV saline (2.0 p.i) alone which served as a control. In all cases the ICV procedure interrupted the activity of the animals for approximately 2-3 rain, after which they resumed apparently normal behaviors and activity. In order to gain an indication of the regions of the ventricular system reached by the solutions, dilute India ink (2.0 ~1) was injected into anesthetized mice. The brains were dissected, and the ink was observed to have penetrated the ventricular system. The dosages of the agonists were chosen to induce significant analgesia 30 rain after injection. After IP or ICV injections mice were returned to their previous exposure conditions (RF, sham R F or control). Thirty minutes later the latency of a foot-lifting response to an aversive thermal stimulus (47.5_+0.5°C, hot-plate, OmniTech, Columbus, OH) was determined from the individual mice. All thermal response latency determinations were carried out by an observer blind to the experimental procedures. Data were analyzed by analysis of variance procedures and post-hoc Neuman-Keuls tests. RESULTS
10
1E
U Sl
,C RFSh, DADLE
U Sl
1
,C RF Sh, .*SKF 10,047
FIG. I. Mean latencies of response to an aversive thermal stimulus of mice intraperitoneally (IP) injected with either A: the mu opiate agonist morphine sulfate (10 mg/kg); B: the kappa opiate agonist, U-50, 488H (1.0 mg/kg); C: intracerebroventricularly (1CV) injected with the delta opiate agonist DADLE (1.0 p,g); D: injected IP with the sigma opiate agonist SKF-10,047 (l.0 mg/kg) or the saline vehicle (SI ; 10 ml/kg, IP; 2.0/xl, ICV) and exposed to either the rotating magnetic field (RF), the sham rotating magnetic field (Sh) or control (C) conditions. Uninjected (U) mice were used as an additional control for the injection procedures. N= 10, in all cases. The vertical lines denote the standard error of the mean.
poles) as measured by a Bell Incremental Gauss Meter (Model 640) ranged from 1.5 to 90 gauss. The spatial distribution of this field is illustrated in Kavaliers et al. [23]. In sham exposure conditions, mice were exposed to the noise and vibrations of the motors with the magnets replaced by dummy lead weights of equivalent mass. In an additional control procedure different groups of mice received handling without any accompanying sham or magnetic field exposures. Experimental Procedures
Thirty minutes after either the magnetic field exposure (RF), sham exposures (Sham RF) or control handling procedures mice (n= 10, in all cases) received intraperitoneal (IP) injections of either morphine sulfate (10 rag/10 m/kg; B.D.H. Canada), U-50,488H (1.0 rag/10 ml/kg; The Upjohn Co., Kalamazoo, MI), (-+) SKF-10,047 (1.0 rag/10 ml/kg; N I D A , Rockville, MD) or the isotonic saline vehicle (10 mg/10 ml/kg). An additional group of 10 mice received handling only. Using the procedures described by Haley and
Control and sham RF-exposed mice treated with morphine, U-50,488H, D A D L E and SKF-10,047 displayed marked analgesic responses (Figs. IA-D). Their thermal response latencies were significantly, F(I,47)--8.9, p<0.01 for D A D L E , (Fig. 1C) greater than those of the saline (IP or ICV) treated, uninjected control, on sham RF-exposed animals. The mice IP treated with morphine, U-50,488H and SKF-10,047 all displayed equivalent analgesic responses that were significantly, F(1,47)=6.08, p<0.05, greater than those of mice injected with D A D L E . Results of previous [35] and control studies (not shown) using IP and ICV injections of morphine and U-50,488H (1.0 tzg in 2.0 l) indicated that the time-course and relative extent of the analgesic response was not significantly dependent on the mode of administration of the opiate agonist. Exposure to the R F significantly, F(1,47)= 10.7, p<0.01. reduced the levels of analgesia induced by morphine and U-50,488H, though the response latencies were still significantly, F(1,47)--5.8, p<0.05, greater than those of the saline-treated mice (Figs. IA,B). The rotating magnetic field significantly F(1,47)=6.4, p<0.05, inhibited the analgesic effects of D A D L E (Fig. 1C), while having no significant effect on the degree of analgesia induced by SKF-10,047 (Fig. I D). Results of additional control studies showed that the effects of the magnetic field exposure on thermal response latency were not affected by whether the opiate agonists were IP or ICV administered. In addition, in agreement with previous observations, the response latencies of saline treated animals were not affected by exposure to either the RF or Sham R F conditions [16-21]. DISCUSSION
The present results show that exposure to weak rotating 0.5 Hz magnetic fields significantly reduces mu, delta and kappa opiate-induced analgesia in mice, while not significantly affecting sigma opiate-directed analgesic responses. These results confirm and extend previous observations of significant reductions of morphine-induced analgesia and hyperactivity by rotating magnetic fields and magnetic field
OPIATES A N D M A G N E T I C F I E L D S components of MRI [17, 29, 31, 32] to delta and kappa opiate-mediated responses. This also complements earlier observations of significant nocturnal attenuations of morphine-induced analgesia by 60 Hz earth-strength magnetic fields [17,18] and magnetic field fluctuations associated with geomagnetic storms [28]. In addition, the present results are consistent with the significant inhibitory influences that exposure to magnetic fields have on stress-induced opioid analgesia and hyperactivity in CF-I and C57BL strains of mice, respectively [21]. As restraint stress-induced analgesia is associated with the activation of endogenous opioid systems [2,21], the aforementioned observations provide additional support for the present findings that magnetic stimuli have significant inhibitory influences on delta, kappa and mu opiate directed responses. The present observations are further confirmed by the results of our more recent studies which showed that exposure to these rotating magnetic stimuli also inhibited the analgesic effects of other specific mu and delta directed opiates such as Fentanyl, DAGO and D-Pen 5 (2,5)-enkephalin, respectively ([36], in preparation). The results of earlier studies have shown that daytime exposure to the rotating 0.5 Hz field does not have any significant effects on basal levels of activity or aversive thermal responses [ 18,20]. The inhibitory effects of the magnetic stimuli may arise from the increased levels of the magnetic field as compared to earth strength and/or fluctuations in field strength. Although data have been presented to suggest that both of these components can influence biological systems [1], evidence is accumulating to indicate that the inhibitory effects of the magnetic fields on biological responses are primarily mediated by fluctuations in field strength. Results of studies with MRI procedures have shown that the time-varying and radio-frequency components significantly reduce, while the strong static field (1500 G) has no evident effects on morphine-induced analgesia in mice [32]. However, as earlier reported for magnetophosphenes [7] this effect seems to be also related to the waveform (frequency and amplitude) of the time-varying magnetic fields. Blackman et al. [5,6] have hypothesized that the extent of the biological effects of weak magnetic fields is dependent on the relative intensity and orientations of both the steady-state (local geomagnetic field) and oscillating field components. They showed that exposure to different and specific combinations of time varying and local geomagnetic fields cause significant changes in the effiux of calcium ions from in vitro preparations of chick brain tissue. The ability of magnetic fields to inhibit the in vivo firing rate of neural cells and nocturnal metabolic activity of the pineal gland of several species of animals has also been shown to be dependent on the orientation of the head with respect to the horizontal component of the earth's magnetic field [38]. However, the mechanisms whereby these effects are exerted are not well understood [I]. Magnetic fields have been shown to influence the release and levels of neurotransmitters, as well as to affect a number of behavioral, biochemical, developmental and molecular processes that are opioid mediated [I, 6, 10]. These effects of exposure to magnetic fields have been suggested to involve alterations in the levels of intra- and extra-cellular calcium (Ca++), rates of cellular Ca ++ effiux, as well as the stability of Ca ++ binding to neuronal membranes [2-6, 10, 20]. Results of recent investigations with morphine-induced analgesia have
451 shown that at least some of the inhibitory effects of magnetic stimuli on this response are compatible with actions on Ca ++ and possibly other divalent ions [20]. It was found that the calcium chelator, EGTA, could in part reverse the inhibitory actions of rotating magnetic fields on morphine-induced analgesia in mice, while the calcium ionophore, A21387 potentiated the reduction in the antinociceptive effects of morphine. Moreover, these findings are consistent with the available data on adverse relations between calcium as well as that of other divalent ions and the effects of mu, delta and kappa opiates [8, 13, 15, 20, 33]. The locus of the magnetic field may incorporate direct adverse effects between calcium and other divalent ions and/or a variety of indirect actions. Speculation as to the latter can range from modifications in activity of metallo enzyme systems (calmodulin-dependent phosphates, i.e., calcineurin), to alterations in the release and activity endopeptidases or putative endogenous opioid antagonists, such as FMRFamide-like immunoreactive neuropeptides [20]. Recently, evidence has appeared to indicate that mu, delta and kappa opioid agonists depress calcium spikes and alter calcium dependent channels in neuronal membranes [11, 39-42]. Furthermore, it is also likely that the various opioid system agonists may be variably affected by Ca ++ efflux and levels [42]. Thus, it is likely that any alterations in Ca ++ levels and efflux induced by the rotating magnetic fields would differentially, and either directly or indirectly, alter mu, delta and kappa opiate directed effects. In order to more fully establish whether or not these magnetic stimuli exert similar degrees of inhibition on the analgesic effects of mu, delta and kappa opiates, examinations of the effects of multiple doses of the different agonists, coupled with exposure to various durations and/or intensities of magnetic fields are required. The absence of any significant inhibitory influences of exposure to the magnetic fields on SKF-10,047 induced analgesia suggests that sigma opioid systems may be relatively insensitive to the presently applied magnetic stimuli. The analgesic effects of SKF-10,047 in CF-1 mice can be partially blocked by naloxone, suggesting that some opiate sensitive mechanism is present [24,27]. However, it is also worthy of note the analgesic effects of U-50,488H, which are inhibited by the magnetic stimuli, are also only partially blocked by naloxone in this strain of mice [23,27]. Whether SKF-10,047 and sigma opiates exert their actions through Ca ++ dependent or mediated processes in a similar manner as mu, delta and kappa opiates is not well established. SKF10,047 is also reported to have non-opiate, psychotomimetic effects which may confound interpretations of analgesic actions [25,26]. Whether or not psychotomimetic responses, as well as the effects of non-opiate analgesics, are influenced by magnetic stimuli is under investigation. It should be noted that a variety of other behavioral, physiological and psychological responses have been indicated to be unaffected by exposure to magnetic stimuli [9, 16, 30]. However, in view of the broad range of functions in which opioid systems are implicated [2], slight alterations in opioid activity could initiate a wide spectrum of subtle biological and possibly clinically relevant effects [31]. Further investigations of the modes of action of magnetic stimuli, especially those associated with MRI procedures, on opioid systems in both humans and laboratory animals are essential.
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AND OSSENKOPP
ACKNOWLEDGEMENTS We thank Dr. P. VonVoigtlander and the Upjohn company for the gift of the U-50,488H and the National Institute of Drug Abuse for supplying the SKF-10,047. Linda Scharr provided technical assistance. This work was supported by Natural Sciences and Engineering Research Council of Canada grants to M.K. ($222A9) and K.-P.O. (UOI51).
REFERENCES 1. Adey, W. R. Tissue interactions with non-ionizing electromagnetic fields. Physiol Rev 61: 435-513, 1981. 2. Akil, H., S. J. Watson, E. Young, M. E. Lewis and J. M. Walker. Endogenous opioids: biology and function. Annu Rev Neurosci 7: 223-255, 1984. 3. Bawin, S. M. and W. R. Adey. Sensitivity of calcium binding in cerebral tissue to weak environmental electrical fields oscillating at low frequency. Proc Natl Acad Sci USA 73: 1999-2003, 1976. 4. Bawin, S. M., W. R. Adey and I. M. Sabbot. Ionic factors in the release of Ca ++ from chicken cerebral tissue by electromagnetic fields. Proc Nat! Acad Sci USA 75: 6314-6318, 1978. 5. Blackman, C. F., S. G. Benane, D. E. House and W. T. Joines. Effects of E L F (1-1120 Hz) and modulated (50 Hz) RF fields on the efflux of calcium ions from brain tissue in vitro. Bioelectromagnetics 6:1-11, 1985. 6. Blackman, C. F., S. G. Benane, J. R. Rabinowitz, D. E. House and W. T. Joines. A role for the magnetic field in the radiationinduced effiux of calcium ions from brain tissue in vitro. Bioelectromagnetics 6: 327-337, 1985. 7. Budinger, T. F. Thresholds for physiological effects due to RF and magnetic fields used in NMR imaging. IEEE Trans Nucl Sci NS-26: 2821-2825, 1979. 8. Chapman, D. B. and E. L. Way. Modification of endorphin/enkephalin analgesia and stress-induced analgesia by divalent ions, a cation chelator and an ionophore. Br J Pharmacol 75: 38%396, 1982. 9. Davis, H. P., S. J. Y. Nizumori, H. Allen, A. R. Rosenzweig, E. L. Bennet and T. S. Tenforde. Behavioral studies with mice exposed to DC and 60-Hz magnetic fields. Bioelectromagnetics 5: 147-164, 1985. 10. Dixey, D. R. and G. Rein. H3-noradrenaline release potentiated in a clonal cell line by low intensity pulsed magnetic fields. Nature 206: 253-256, 1982. 11. Frank, G. B. Stereospecific opioid drug receptors on excitable cell membranes. Can J PhysioIPharmaco163: 1023-1032, 1985. 12. Golding, G. P., L. Newboult, J. M. H. Rees and B. R. Varlow. The effects of 50 Hz magnetic fields on opioid peptide mediated inhibition of guinea pig ileum. Neuropeptides 5: 357-358, 1985. 13. Guerrero-Monoz, F., C. Adames, Z. Fearon and E. L. Way. Calcium-opiate antagonism in the periaqueductal grey (PAG) region. Eur J Pharmacol 76: 417-419, 1981. 14. Haley, T. J. and W. G. McCormick. Pharmacological effects produced by intracerebral injections of drugs in the conscious mouse. Br J Pharmacol Chem 12: 12-15, 1957. 15. Harris, R. A., H. H. Loh and E. L. Way. Effect of divalent cations, cation chelators and an ionophore on morphine analgesia and tolerance. J Pharmacol Exp Ther 195: 488-498, 1975. 16. Innis, N. K., K.-P. Ossenkopp, F. S. Prato and E. Sestini. Behavioral effects of exposure to nuclear magnetic resonance imaging. If. Spatial memory tasks. Mag Res lmag, in press, 1986. 17. Kavaliers, M. and K.-P. Ossenkopp. Effects of 0.5 Hz and 60 Hz magnetic fields on morphine-induced behavioral changes in mice. Soc Neurosci Abstr 10:1105, 1984. 18. Kavaliers, M. and K.-P. Ossnenkopp. Exposure to rotating magnetic fields alters morphine-induced behavioral responses in two strains of mice. Neuropharmacology 4: 337-340, 1985.
19. Kavaliers, M. and K.-P. Ossenkopp. Tolerance to morphineinduced analgesia in mice: magnetic fields function as environmental specific cues and reduce tolerance development. Lift, Sci 37: 1125-1135, 1985. 20. Kavaliers, M. and K.-P. Ossenkopp. Magnetic field inhibition of morphine-induced analgesia and behavioral activity in two strains of mice: evidence for involvement of calcium ions. Brain Res, in press, 1986. 21. Kavaliers, M. and K.-P. Ossenkopp. Stress-induced opioid analgesia and activity in mice: inhibitory influences of exposure to magnetic fields. Psychopharmacology (Berlin). in press, 1986. 22. Kavaliers, M., K.-P. Ossenkopp and M. Hirst. Magnetic fields abolish the enhanced nocturnal analgesic response to morphine in mice. Physiol Behav 32: 261-264, 1984. 23. Kavaliers, M., G. C. Teskey and M. Hirst. Effects of aging on day-night rhythms of kappa opioid mediated feeding in the mouse. Psychopharmacology (Berlin) 87: 286-291, 1985. 24. Kavaliers, M. and M. Hirst. Aging and day-night rhythms in feeding: Effects of the putative sigma opiate agonist N-allynormetazocine (SKF-10,047). Neurobiol Aging 7: 17%183, 1986. 25. Khazan, N., G. A. Young, E. E. EI-Takany, O. Hong and D. Calligaro. Sigma receptors mediate the psychotomimetic effects of N-allynormetazocine (SKF-10,047) but not its opioid agonist-antagonist properties. Neuropharmacology 23: 983-987. 1984. 26. Largent, B. L., A. L. Gundlach and S. H. Snyder. Psychotomimetic receptors labeled and visualized with (_+)-[H]3-(3-hydroxphenyl)-N-tl-propyl)-piperidine. Proc Nat/ Acad Sei USA 81: 4983--4987, 1984. 27. Martin, W. R. Pharmacology of opioids. Pharmacol Rev 35: 282-323, 1984. 28. Ossenkopp, K.-P., M. Kavaliers and M. Hirst. Reduced nocturnal morphine analgesia in mice following a geomagnetic disturbance. Neurosci Lett 40: 321-325, 1983. 29. Ossenkopp, K.-P., M. Kavaliers, F. S. Prato, G. C. Teskey, E. Sestini and M. Hirst. Exposure to nuclear magnetic resonance imaging procedures attenuates morphine-induced analgesia in mice. Lift, Sci 37: 1507-1514, 1985. 30. Ossenkopp, K.-P., N. K. lnnis, F. S. Prato and E. Sestini. Behavioral effects of exposure to nuclear magnetic resonance imaging. I. Open field behavior passive avoidance learning in rats. Mag Res hnag, in press, 1986. 31. Ossenkopp, K.-P. and M. Kavaliers. Clinical and applied aspects of magnetic field exposure: a possible role for the endogenous opioid systems. J Bioelectricity', in press. 1986. 32. Prato, F. S., F.-P. Ossenkopp and M. Kavaliers. Nuclear magnetic resonance inhibition of morphine-induced analgesia in mice: differential effects of the static, radio-frequency and time-varying magnetic field components. Mug Res hnag. submitted. 33. Sadee, W., A. Pfeiffer and A. Herz. Opiate receptors: multiple effects of metal ions. J Neurochem 39: 65%667, 1982. 34. Seegal, R. F. Exposure to 60 Hz electric and magnetic fields alters biogenic amine metabolite concentration in non-human primate cerebrospinal fluid. Soc Neurosci Abstr 11: 443, 1985. 35. Teskey, G. C., M. Kavaliers and M. Hirst. Social conflict activates opioid analgesic and ingestive behaviors in male mice. Ltifi" Sci 35: 303-315, 1984.
OPIATES AND MAGNETIC
FIELDS
36. Teskey, G. C., K.-P. Ossenkopp, F. S. Prato and M. Kavaliers. Exposure to magnetic resonance imaging attenuates fentanylinduced analgesia in mice. Soc Neurosci Abstr 12: in press, 1986. 37. VonVoigtlander, P. F., R. A. Lahti and J. H. Ladens. U50,488H: A selective and structurally novel non-mu (kappa) opioid agonist. J Pharmacol Exp Ther 224: 7-12, 1983. 38. Welker, H. A., P. Semm, R. P. Willig, J. C. Commentz, W. Wiltschko and L Vollrath. Effects of an artificial magnetic field on serotonin N-acetyltransferase activity and melatonin content of the rat pineal gland. Exp Brain Re 50: 426--432, 1985. 39. Werz, M. A. and R. L. Macdonald. Opioid peptides decrease calcium-dependent action potential duration of mouse dorsal root ganglion neurons in cell culture. Brain Res 239: 315-321, 1982.
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40. Werz, M. A. and R. L. Macdonald. Opioid peptides with differential affinity for mu and delta receptors decrease sensory neuron calcium-dependent action potentials. J Pharmacol Exp Ther 227: 394-402, 1983. 41. Werz, M. A. and R. L. Macdonald. Opioid peptides selective for mu- and delta-opiate receptors reduce calcium-dependent action potential duration by increasing potassium conductance. Neurosci Lett 42: 173-178, 1983. 42. Werz, M. A. and R. L. Macdonald. Dynorphin reduced calcium-dependent action potential duration by decreasing voltage-dependent calcium conductance. Neurosci Lett 46: 185-190, 1985.
0196-9781/86 $3.00 + .00
Magnetic Fields Differentially Inhibit Mu, Delta, Kappa and Sigma Opiate-Induced Analgesia in Mice MARTIN KAVALIERS*~ AND KLAUS-PETER
OSSENKOPPt
*Department o f Psychology, University o f Alberta, Edmonton, Alberta, Canada T6G 2E9 and tDepartment o f Psychology, University o f Western Ontario, London, Ontario, Canada N6A 5C2 R e c e i v e d 5 D e c e m b e r 1985 KAVALIERS, M. AND K.-P. OSSENKOPP. Magnetic fields differentially inhibit mu, delta, kappa and sigma opiateinduced analgesia in mice. PEPTIDES 7(3) 449--453, 1986.--An exposure for 60 min to a 0.5 Hz rotating magnetic field (1.5-90 G) significantly attenuated the daytime analgesic effects of the mu and kappa opiate agonists, morphine and U50,488H, respectively, and significantly inhibited the analgesic actions of the delta agonist, D-Ala2-D-Leu~-enkephalin, in mice. The magnetic stimuli had no significant effects on the analgesic effects of the prototypic sigma opiate agonist (_+) SKF-10,047. These results show that exposure to relatively weak magnetic stimuli has significant and differential inhibitory influences on various opioid systems. Mu, delta, kappa, and sigma opiates
Analgesia
Magnetic fields are altered in a manner similar to that observed with morphine. Accordingly, in view of the existence of multiple opioid systems and receptor types [27], it is of importance to determine whether exposure to magnetic fields has inhibitory influences on other opiate agonists and opioid systems. We report here that an exposure for 60 min to a 0.5 Hz rotating magnetic field significantly reduces the analgesic effects of mu (morphine sulfate); delta (DADLE; D-AlaZ-D-LeuS-enkephalin [27]) and kappa (U-50,488H; trans-3,4,-dichloroN-methyl-2-(l-pyrrolidinyl-cyclohexyl-benzeneacetaide methanesulfonate hydrate [27,37]) directed opiates in mice, while not having any significant actions on sigma ((-+) SKF-10-047; N-allynormetazocine [24-27]) induced analgesia.
T H E possibility that magnetic fields can significantly influence biological systems in a wide range of animals, including that of humans, has gained considerable acceptance [ 1, 3-6, 10, 12, 34]. Among the more dramatic effects of magnetic stimuli is the suppression of the analgesic and locomotory effects of morphine. Exposure of mice to earth-strength, 0-1.5 gauss, 60 Hz magnetic fields results in a reversible dosedependent inhibition of the nocturnal peak in the day-night rhythm of morphine-induced analgesia [17,18]. Natural geomagnetic disturbances arising from intense solar activity also reduce night-time morphine-induced analgesia [28]. Furthermore, relatively weak rotating magnetic fields have been demonstrated to reduce the day- and night-time analgesic effects of morphine [18, 20, 22], attenuate the locomotory effects of morphine [18,20], modify the rate of tolerance development to morphine-induced analgesia [19] and reduce stress-induced opioid analgesia in mice [2l]. Recently, it was shown that exposure to magnetic resonance imaging (MRI) procedures also suppressed the day- and night-time analgesic responses of mice receiving morphine injections [29, 31, 32]. Although the mechanisms for these inhibitory effects of magnetic fields on morphine-induced responses are poorly understood, results of our initial studies suggest that changes in the central distribution and levels of calcium and other divalent ions are likely to be involved [20]. These inhibitory effects of magnetic fields, and in particular those associated with MRI, have a number of practical and clinical implications. They raise the possibility that magnetic field components of MRI [32] may have inhibitory effects on responses to analgesics such as morphine in humans [31]. In addition, these observations also raise the question as to whether or not the effects of other exogenous opiates
METHOD
Animals Sexually mature male CF-1 mice (Charles River, Quebec) 3-4 months old and 30-35 g were housed in groups of 5 in polyethylene cages with stainless steel wire tops at 23_+ I°C under a 12 hr light:12 hr dark cycle. Food and water were available ad lib.
Experimental Apparatus At mid-photophase, two cages of mice with the stainless steel wire tops replaced by plastic covers, were placed on 5 cm form rubber sheets and positioned between the magnets of 0.5 Hz magnetic field apparatus [22]. The apparatus consisted of two horseshoe magnets that were rotated in opposite directions along their major axes at 29 rpm. Field intensities (with the magnets stationary and aligned along opposite
tRequests for reprints should be addressed to Martin Kavaliers, Division of Oral Biology, Faculty of Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.
449
450
KAVALIERS AND OSSENKOPP
50
50
A. mu
B. kappa
30
30
P
I
I lO!
'E 10! U SI
C 0 Q. ffl
50
i
C RF Sh Morphine
U SI
i
C. delta
i
C RF Sh U-50,488H i
D. sigma
>, U C
30
McCormick [14], other mice (n=10, in all cases) received intracerebroventricular (ICV) injections of D A D L E (1.0/~g; Peninsula, Belmont, CA) in 2.0 ~1 saline (1.0 txl in each side of the brain) or ICV saline (2.0 p.i) alone which served as a control. In all cases the ICV procedure interrupted the activity of the animals for approximately 2-3 rain, after which they resumed apparently normal behaviors and activity. In order to gain an indication of the regions of the ventricular system reached by the solutions, dilute India ink (2.0 ~1) was injected into anesthetized mice. The brains were dissected, and the ink was observed to have penetrated the ventricular system. The dosages of the agonists were chosen to induce significant analgesia 30 rain after injection. After IP or ICV injections mice were returned to their previous exposure conditions (RF, sham R F or control). Thirty minutes later the latency of a foot-lifting response to an aversive thermal stimulus (47.5_+0.5°C, hot-plate, OmniTech, Columbus, OH) was determined from the individual mice. All thermal response latency determinations were carried out by an observer blind to the experimental procedures. Data were analyzed by analysis of variance procedures and post-hoc Neuman-Keuls tests. RESULTS
10
1E
U Sl
,C RFSh, DADLE
U Sl
1
,C RF Sh, .*SKF 10,047
FIG. I. Mean latencies of response to an aversive thermal stimulus of mice intraperitoneally (IP) injected with either A: the mu opiate agonist morphine sulfate (10 mg/kg); B: the kappa opiate agonist, U-50, 488H (1.0 mg/kg); C: intracerebroventricularly (1CV) injected with the delta opiate agonist DADLE (1.0 p,g); D: injected IP with the sigma opiate agonist SKF-10,047 (l.0 mg/kg) or the saline vehicle (SI ; 10 ml/kg, IP; 2.0/xl, ICV) and exposed to either the rotating magnetic field (RF), the sham rotating magnetic field (Sh) or control (C) conditions. Uninjected (U) mice were used as an additional control for the injection procedures. N= 10, in all cases. The vertical lines denote the standard error of the mean.
poles) as measured by a Bell Incremental Gauss Meter (Model 640) ranged from 1.5 to 90 gauss. The spatial distribution of this field is illustrated in Kavaliers et al. [23]. In sham exposure conditions, mice were exposed to the noise and vibrations of the motors with the magnets replaced by dummy lead weights of equivalent mass. In an additional control procedure different groups of mice received handling without any accompanying sham or magnetic field exposures. Experimental Procedures
Thirty minutes after either the magnetic field exposure (RF), sham exposures (Sham RF) or control handling procedures mice (n= 10, in all cases) received intraperitoneal (IP) injections of either morphine sulfate (10 rag/10 m/kg; B.D.H. Canada), U-50,488H (1.0 rag/10 ml/kg; The Upjohn Co., Kalamazoo, MI), (-+) SKF-10,047 (1.0 rag/10 ml/kg; N I D A , Rockville, MD) or the isotonic saline vehicle (10 mg/10 ml/kg). An additional group of 10 mice received handling only. Using the procedures described by Haley and
Control and sham RF-exposed mice treated with morphine, U-50,488H, D A D L E and SKF-10,047 displayed marked analgesic responses (Figs. IA-D). Their thermal response latencies were significantly, F(I,47)--8.9, p<0.01 for D A D L E , (Fig. 1C) greater than those of the saline (IP or ICV) treated, uninjected control, on sham RF-exposed animals. The mice IP treated with morphine, U-50,488H and SKF-10,047 all displayed equivalent analgesic responses that were significantly, F(1,47)=6.08, p<0.05, greater than those of mice injected with D A D L E . Results of previous [35] and control studies (not shown) using IP and ICV injections of morphine and U-50,488H (1.0 tzg in 2.0 l) indicated that the time-course and relative extent of the analgesic response was not significantly dependent on the mode of administration of the opiate agonist. Exposure to the R F significantly, F(1,47)= 10.7, p<0.01. reduced the levels of analgesia induced by morphine and U-50,488H, though the response latencies were still significantly, F(1,47)--5.8, p<0.05, greater than those of the saline-treated mice (Figs. IA,B). The rotating magnetic field significantly F(1,47)=6.4, p<0.05, inhibited the analgesic effects of D A D L E (Fig. 1C), while having no significant effect on the degree of analgesia induced by SKF-10,047 (Fig. I D). Results of additional control studies showed that the effects of the magnetic field exposure on thermal response latency were not affected by whether the opiate agonists were IP or ICV administered. In addition, in agreement with previous observations, the response latencies of saline treated animals were not affected by exposure to either the RF or Sham R F conditions [16-21]. DISCUSSION
The present results show that exposure to weak rotating 0.5 Hz magnetic fields significantly reduces mu, delta and kappa opiate-induced analgesia in mice, while not significantly affecting sigma opiate-directed analgesic responses. These results confirm and extend previous observations of significant reductions of morphine-induced analgesia and hyperactivity by rotating magnetic fields and magnetic field
OPIATES A N D M A G N E T I C F I E L D S components of MRI [17, 29, 31, 32] to delta and kappa opiate-mediated responses. This also complements earlier observations of significant nocturnal attenuations of morphine-induced analgesia by 60 Hz earth-strength magnetic fields [17,18] and magnetic field fluctuations associated with geomagnetic storms [28]. In addition, the present results are consistent with the significant inhibitory influences that exposure to magnetic fields have on stress-induced opioid analgesia and hyperactivity in CF-I and C57BL strains of mice, respectively [21]. As restraint stress-induced analgesia is associated with the activation of endogenous opioid systems [2,21], the aforementioned observations provide additional support for the present findings that magnetic stimuli have significant inhibitory influences on delta, kappa and mu opiate directed responses. The present observations are further confirmed by the results of our more recent studies which showed that exposure to these rotating magnetic stimuli also inhibited the analgesic effects of other specific mu and delta directed opiates such as Fentanyl, DAGO and D-Pen 5 (2,5)-enkephalin, respectively ([36], in preparation). The results of earlier studies have shown that daytime exposure to the rotating 0.5 Hz field does not have any significant effects on basal levels of activity or aversive thermal responses [ 18,20]. The inhibitory effects of the magnetic stimuli may arise from the increased levels of the magnetic field as compared to earth strength and/or fluctuations in field strength. Although data have been presented to suggest that both of these components can influence biological systems [1], evidence is accumulating to indicate that the inhibitory effects of the magnetic fields on biological responses are primarily mediated by fluctuations in field strength. Results of studies with MRI procedures have shown that the time-varying and radio-frequency components significantly reduce, while the strong static field (1500 G) has no evident effects on morphine-induced analgesia in mice [32]. However, as earlier reported for magnetophosphenes [7] this effect seems to be also related to the waveform (frequency and amplitude) of the time-varying magnetic fields. Blackman et al. [5,6] have hypothesized that the extent of the biological effects of weak magnetic fields is dependent on the relative intensity and orientations of both the steady-state (local geomagnetic field) and oscillating field components. They showed that exposure to different and specific combinations of time varying and local geomagnetic fields cause significant changes in the effiux of calcium ions from in vitro preparations of chick brain tissue. The ability of magnetic fields to inhibit the in vivo firing rate of neural cells and nocturnal metabolic activity of the pineal gland of several species of animals has also been shown to be dependent on the orientation of the head with respect to the horizontal component of the earth's magnetic field [38]. However, the mechanisms whereby these effects are exerted are not well understood [I]. Magnetic fields have been shown to influence the release and levels of neurotransmitters, as well as to affect a number of behavioral, biochemical, developmental and molecular processes that are opioid mediated [I, 6, 10]. These effects of exposure to magnetic fields have been suggested to involve alterations in the levels of intra- and extra-cellular calcium (Ca++), rates of cellular Ca ++ effiux, as well as the stability of Ca ++ binding to neuronal membranes [2-6, 10, 20]. Results of recent investigations with morphine-induced analgesia have
451 shown that at least some of the inhibitory effects of magnetic stimuli on this response are compatible with actions on Ca ++ and possibly other divalent ions [20]. It was found that the calcium chelator, EGTA, could in part reverse the inhibitory actions of rotating magnetic fields on morphine-induced analgesia in mice, while the calcium ionophore, A21387 potentiated the reduction in the antinociceptive effects of morphine. Moreover, these findings are consistent with the available data on adverse relations between calcium as well as that of other divalent ions and the effects of mu, delta and kappa opiates [8, 13, 15, 20, 33]. The locus of the magnetic field may incorporate direct adverse effects between calcium and other divalent ions and/or a variety of indirect actions. Speculation as to the latter can range from modifications in activity of metallo enzyme systems (calmodulin-dependent phosphates, i.e., calcineurin), to alterations in the release and activity endopeptidases or putative endogenous opioid antagonists, such as FMRFamide-like immunoreactive neuropeptides [20]. Recently, evidence has appeared to indicate that mu, delta and kappa opioid agonists depress calcium spikes and alter calcium dependent channels in neuronal membranes [11, 39-42]. Furthermore, it is also likely that the various opioid system agonists may be variably affected by Ca ++ efflux and levels [42]. Thus, it is likely that any alterations in Ca ++ levels and efflux induced by the rotating magnetic fields would differentially, and either directly or indirectly, alter mu, delta and kappa opiate directed effects. In order to more fully establish whether or not these magnetic stimuli exert similar degrees of inhibition on the analgesic effects of mu, delta and kappa opiates, examinations of the effects of multiple doses of the different agonists, coupled with exposure to various durations and/or intensities of magnetic fields are required. The absence of any significant inhibitory influences of exposure to the magnetic fields on SKF-10,047 induced analgesia suggests that sigma opioid systems may be relatively insensitive to the presently applied magnetic stimuli. The analgesic effects of SKF-10,047 in CF-1 mice can be partially blocked by naloxone, suggesting that some opiate sensitive mechanism is present [24,27]. However, it is also worthy of note the analgesic effects of U-50,488H, which are inhibited by the magnetic stimuli, are also only partially blocked by naloxone in this strain of mice [23,27]. Whether SKF-10,047 and sigma opiates exert their actions through Ca ++ dependent or mediated processes in a similar manner as mu, delta and kappa opiates is not well established. SKF10,047 is also reported to have non-opiate, psychotomimetic effects which may confound interpretations of analgesic actions [25,26]. Whether or not psychotomimetic responses, as well as the effects of non-opiate analgesics, are influenced by magnetic stimuli is under investigation. It should be noted that a variety of other behavioral, physiological and psychological responses have been indicated to be unaffected by exposure to magnetic stimuli [9, 16, 30]. However, in view of the broad range of functions in which opioid systems are implicated [2], slight alterations in opioid activity could initiate a wide spectrum of subtle biological and possibly clinically relevant effects [31]. Further investigations of the modes of action of magnetic stimuli, especially those associated with MRI procedures, on opioid systems in both humans and laboratory animals are essential.
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ACKNOWLEDGEMENTS We thank Dr. P. VonVoigtlander and the Upjohn company for the gift of the U-50,488H and the National Institute of Drug Abuse for supplying the SKF-10,047. Linda Scharr provided technical assistance. This work was supported by Natural Sciences and Engineering Research Council of Canada grants to M.K. ($222A9) and K.-P.O. (UOI51).
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