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The receptors activated by exogenous and endogenous opioids in the superior cervical ganglion of the cat
211
Brain Research, 622 (1993) 211-214 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 19199
The receptors activated by exogenous and endogenous opioids in the superior cervical ganglion of the cat C. Zhang, M. B a c h o o a n d C. P o l o s a Department of Physiology, McGill University, Montreal, Que. (Canada) (Accepted 20 April 1993)
Key words: Opiate receptor; Sympathetic ganglion; Synapse; Endogenous opioid
Nicotinic transmission in the superior cervical ganglion of the cat was studied in vivo and in vitro by recording the postganglionic compound action potential evoked, under partial block with hexamethonium, by supramaximal 0.2 Hz stimulation of the cervical sympathetic trunk. The compound action potential was depressed following a stimulus train (5 Hz, 40 s) applied to the same set of axons. The inhibition was antagonized by naloxone, suggesting mediation by endogenous opioids. Agonists selective for #-, 6- and K-opioid receptors, injected into the arterial supply of the in situ ganglion or added to the perfusion fluid in the in vitro ganglion, also produced a naloxone-sensitive inhibition of the compound action potential. The synaptic inhibition was antagonized by the 6-selective antagonist ICI 174,864 and by the/z-selective antagonist CTAP, but not by the K-selective antagonist NoroBNI. These results suggest that all three main types of opioid receptors are present in the ganglion but only/zand ~5-receptors are involved in the inhibition of ganglionic transmission mediated by the endogenous opioids.
INTRODUCTION
Opioid peptides have been demonstrated with immunohistochemical techniques in sympathetic ganglia22. Preganglionic nerve terminals 8 and the soma of sympathetic preganglionic neurons 11 contain these peptides. A physiological role of opioid peptides as neurotransmitters a n d / o r neuromodulators has been suggested by many studies (for reviews, see refs. 1 and 13). At most synapses opioids produce inhibitory effects on neuronal activity and synaptic transmission 19. In the autonomic nervous system, an inhibitory effect attributed to endogenous opioids was first demonstrated by Konishi ]2 in the inferior mesenteric ganglion. Similar effects were reported in colonic ganglial°, in bladder ganglia6 and in the superior cervical ganglion26. Three main types of opioid receptors (/z, ~ and K) have been characterized pharmacologically7'16'15. The types of opioid receptors mediating inhibition in autonomic ganglia have not been thoroughly investigated. There are studies indicating the involvement of 8opioid receptors in colonic ganglia1° and in bladder ganglia6. In the present study we demonstrate that
nicotinic transmission in the superior cervical ganglion of the cat can be depressed by exogenously applied/z-, 8- and K-opioid receptor agonists. We also present data suggesting that /z- and /~- but not K-receptors mediate the inhibitory effect of the endogenous opioid peptide(s) in the same ganglion. MATERIALS AND METHODS Cats of either sex, weighing 2.5-4.0 kg, were used under anesthesia with pentobarbital (40 mg/kg, i.p. followed by 5 mg/kg every 2 h). After paralysis with pancuronium (0.2 mg/kg, followed by supplements of 0.1 mg/kg every 2 h) and intubation of the trachea, the animals were artificially ventilated. In the in vivo experiments, the lingual artery was cannulated for injection of drugs into the arterial supply of the superior cervical ganglion. The thyroid and occipital arteries were ligated. A snare was placed around the external carotid artery to allow temporary closure of the artery during injections. Injections had a volume of 0.25 ml and took 2 s. An interval of 10 min was allowed between successive injections of the same or of different drugs except for the case of U 50,488 at high doses, in which an interval of 20 min was used. After exposure of the superior cervical ganglion, one of its postganglionic nerves (internal or external carotid nerve) was dissected and placed on bipolar hook electrodes for recording the postganglionic compound action potential. The segment of nerve between the electrodes was crushed. The cervical sympathetic trunk was dissected from the common carotid artery and cervical vagus nerve for a length of 1-2 cm and placed on
Correspondence: C. Polosa, Department of Physiology, McGill University, 3655 Drummond St., Montr6al, Qu6bec, Canada, H3G 1Y6. Fax: (1) (514) 398-7452.
212 hook electrodes for stimulation. After dissection, the ganglion and its nerves were covered with paraffin oil in a pool made by the skin flaps. Stimulus pulses (0.5 ms, supramaximal) were obtained from a Haer stimulator. A test shock was given at the frequency of 0.2 Hz. To evoke synaptic inhibition, a conditioning 5 Hz 40 s train was used. The postganglionic compound action potential was displayed on a storage oscilloscope and recorded on magnetic tape for later analysis. A femoral artery and vein were cannulated for monitoring systemic arterial pressure and for administering drugs, respectively. Hexamethonium (C6) was infused through the femoral vein at a rate of 0.05 + 0.003 mg-kg- 1. min- i to produce a steady partial block (compound action potential amplitude reduced by 40-50%) of nicotinic ganglionic transmission. The purpose of the block was to reduce the safety factor of nicotinic ganglionic transmission which is high in the superior cervical ganglion17'21, therefore making the compound action potential a more sensitive measure of effects of drugs or of conditioning stimulation. In the in vitro experiments, the common carotid artery was cannulated for perfusion. The superior cervical ganglion was dissected together with the segments of the common, internal and external carotid arteries which form the carotid bifurcation, with the CST and with the postganglionic nerves and placed in a perfusion chamber of 3 ml volume. The perfusion pressure, provided by a pump, was continuously monitored through a cannula placed in the lingual artery and connected to a pressure transducer. The ganglion was perfused with Krebs solution containing C6 (30-50/zM) of the following composition (in raM): NaCl 130.3, KCI 4.0, MgCl 2 1.0, KH2PO 4 1.0, NaHCO 3 12.0, CaC12 2.0 and D-glucose 11.0. The perfusion rate was kept at 0.2-0.3 ml/min, which was obtained with a perfusion pressure of 70-100 mmHg. The Krebs solution was equilibrated with a 95% 0 2 / 5 % CO 2 gas mixture and kept at room temperature (25°C). Drugs were dissolved in the Krebs solution and applied by switching from normal to drug-containing Krebs solution for 5-10 min. After application of a drug the ganglion was perfused with drug-free Krebs solution for 20-30 min before performing another test. The CST and internal carotid nerve were pulled into suction electrodes for stimulation and recording, respectively. Stimulus parameters were similar to those used in the in vivo experiment. When the effect of a conditioning train on ganglionic transmission was tested, the conditioning 5 Hz 40 s train and the 0.2 Hz test were given through the same electrode as in the in vivo preparations. Changes in compound action potential amplitude produced by the conditioning train or by application of drugs were expressed as percent of control compound action potential amplitude. Differences between means were tested with the t-test or the one-way ANOVA test. A difference was regarded as statistically significant if the P-value was less than 0.05. Numerical results reported in the text are expressed as mean ± S.E.M. Drugs used: Pentobarbital (M.T.C. Parmaceuticals), hexamethonium, naloxone, morphiceptin, [o-Pen2,o-PenS]enkephalin (DPDPE), dynorphin, trans-( ± )-3,4-dichloro-N-methyl-N-(2-[l-pyrrolidinyl]cyclohexyl)benzeneacetamide (U50,488, all from Sigma), Nor-Binaltorphimine (Nor-BNI, Research Biochemicals INC) and ICI 174,864 (Cambridge Research Biochemicals). o-Phe-Cys-Tyr-D-TrpArg-Thr-Pen-Thr-NH 2 (CTAP) was a gift from Dr. Victor J. Hruby, University of Arizona.
RESULTS
Inhibitory effect of exogenous opioid agonists on ganglionic nicotinic transmission In vivo experiments. As described earlier 26, the opioid peptide Leu-enkephalin, injected into the arterial supply of the ganglion, inhibits nicotinic transmission in the superior cervical ganglion. The inhibition is dosedependent. This inhibition was antagonized by the non-selective opioid receptor antagonist naloxone in a reversible manner. In the present study we tested the
A +
B
~
0.b~ 0.io 1.bolo:0o,o6.00
C
Concentration of agonist (~M)
_J Fig. 1. Inhibitory effect of opioid agonists on nicotinic ganglionic transmission. In vitro experiments. A: inhibition produced by morphiceptin (a), DPDPE (b) and U50,488 (c). Perfusion for 10 rain with the agonist-containing Krebs solution was started at the time indicated by the arrow. Each record shows the onset and peak of the inhibition (left) and the recovery (right). The break in a, b and c is 16, 17 and 20.5 min, respectively. Concentration of agonists: 1 /zM. Calibrations: vertical: 0.1 mV, horizontal: 50 s. B: examples of concentration-response relation. Ordinate is the percent depression of the compound action potential by the concentration of agonist indicated on abscissa. Open circle: morphiceptin. Open triangle: DPDPE. Filled triangle: U50,488. Data for morphiceptin and DPDPE are from the same ganglion.
opioid agonists morphiceptin, selective for /,-receptors 2, dynorphin, a possible endogenous ligand for Kreceptors 3, U50,488, a synthetic non-peptide agonist selective for K-receptors 24 and DPDPE selective for &receptors 5'1s. All agonists tested depressed the compound action potential in a dose-dependent manner. At the lowest dose used (1 /~g for morphiceptin and DPDPE; 10/~g for dynorphin and U50,488) the duration of the depression was between 45 and 63 s for morphiceptin, DPDPE and dynorphin, but was longer for U50,488 (120-300 s). The duration of the depression was dose-dependent. At the highest dose (100-200 /~g) the duration of the depression ranged from 4 to 19 min, again with the effect of U50,488 showing the slowest decay (P < 0.05). The inhibitory effect of these agonists was antagonised by i.v. naloxone. In vitro experiments. In order to determine the concentration at which /~-, 3- and K-agonists depress the compound action potential, experiments were performed in vitro. The agonists tested were morphiceptin, DPDPE and U50,488. Sample records of the effect produced at the 1 /~M concentration are shown in Fig. 1A. Concentration-response relations were obtained for the three agonists. Examples are shown in Fig. lB. The EDs0s for U50,488, morphiceptin and DPDPE were 234 + 30 (n = 5), 224 + 17 (n = 4) and 186 + 36 nM (n = 3), respectively. These values were not significantly different from each other. The selectivity of these agonists was tested with receptor-selective antagonists. These results are presented in table I.
213 As can be seen from the table, the 6-selective antagonist ICI 174,8644 blocked the DPDPE-induced inhibition but had no effect on the U50,488- and morphiceptin-induced inhibition. The /z-selective antagonist CTAP 14 blocked the inhibition produced by morphiceptin without effect on that produced by DPDPE and U50,488. The K-selective antagonist Nor-BN123 only blocked the inhibition produced by U50,488. These results suggest that at the concentration used morphiceptin, DPDPE and U50,488 act as selective agonists at/z-, 8- and K-opioid receptors.
A
co ..ci E
lOO 80 60
40
~0
2o 0
Antagonism of the naloxone-sensitive synaptic inhibition by opioid receptor-selective antagonists We have previously reported that a 5 Hz 40 s preganglionic train inhibits the postganglionic compound action potential and that the inhibition was antagonized by the non-selective opioid receptor antagonist naloxone 26. In in vitro experiments we tested the effects of the selective 6-antagonist ICI174,864, of the selective /z-antagonist CTAP and of the selective K-antagonist nor-BNI to determine the receptor type which mediates the inhibition. At a concentration of 1 /zM ICI 174,864, CTAP and Nor-BNI had no effect on the amplitude of the compound action potential. At the same concentration (1 /zM) ICI 174,864 depressed the inhibition by 53 _+5% (n = 4, P < 0.001). CTAP blocked the inhibition by 32 _+3% (n = 4, P < 0.01). When CTAP and ICI174,864, each at the 1 /zM concentration, were perfused simultaneously, the synaptic inhibition was depressed by 84 _+3% (n = 4, P < 0.001). The combined effect of these two antagonists was similar to the maximum antagonism produced by naloxone (10 /zM, 82 _+4%, n = 3, P < 0.001). The K-selective antagonist nor-BNI only produced a small blockade (8 +_ 2%, n = 4, P > 0.05). Fig. 2A shows the records obtained in one such experiment. The average results are shown in Fig. 2B. These results suggest that 8- and /z-receptors mediate the inhibition evoked by the preganglionic train.
Fig. 2. T h e synaptic inhibition is antagonized by naloxone, ICI 174,864 and CTAP, but not by nor-BNI. In vitro experiment. Antagonist concentration 1 ~ M . A: inhibition of the c o m p o u n d action potential by a standard 5 Hz 40 s train to the CST (a). Nor-BNI had little effect on the synaptic inhibition (b). T h e inhibition was antagonized by C T A P (c), ICI 174,864 (d) and naloxone (e). B: average data. Blank bar: C T A P (n = 4). Filled bar: ICI 174,864 (n = 4). Crosshatched bar: C T A P + I C I 1 7 4 , 8 6 4 ( n = 4 ) . Left diagonal bar: Nor-BNI (n = 4). * P < 0.01, * * P < 0.001.
cal ganglion is endowed with all three types of opioid receptors. The ED50 values for the three selective agonists were in the nM range, which is comparable to the concentrations of opioids found to be effective on central and peripheral neurons (e.g., refs. 10, 9, 20, 25). The synaptic inhibition was antagonized by the 6antagonist ICI 174,864 and the /z-antagonist CTAP, but not by the K-antagonist nor-BNI. As described earlier 26, the sensitivity of the synaptic inhibition to naloxone varies considerably from animal to animal, suggesting that the proportion each type of receptor contributes in different animals might be different. Since naloxone has affinity about 10 times higher for /z-receptors than for 6- and K-receptors (for a review,
TABLE I
DISCUSSION In the present study agonists selective for/z-, 8- and K-receptors were tested for their effect on nicotinic transmission in the superior cervical ganglion of the cat in vitro and in vivo. All agonists produced inhibition of transmission in a dose-dependent manner. The three selective agonists morphiceptin, DPDPE and U50,488 produced inhibition of similar magnitude at similar concentrations in the in vitro studies. Testing with specific antagonists revealed selectivity of the agonists. Thus, these results suggest that the cat superior cervi-
Effects of selective opioid receptor antagonists on the inhibition of ganglionic transmission produced by morphiceptin, U50,488 and DPDPE Values shown are peak inhibitory effect of agonist expressed as% decrease of c o m p o u n d action potential amplitude. Superscripts are n u m b e r of experiments. Concentration of agonists" 10/xM. Concentration of C T A P , ICI174,864 and Nor-BNI is 10/~M. Control is the inhibition produced by the agonist in absence of antagonist. Asterisk indicates significant difference from control ( P < 0.05). In vitro experiments.
Agonist
Control
CTAP
ICI174, 864
Nor-BNI
Morphiceptin U50,488 DPDPE
55+6 49+6 54+4
5 + 2 5, 47_+3 3 47_+3 3
51+5 3 47+6 3 1 1 + 6 3,
50+3 3 1 7 + 6 3, 51 + 4 3
214 see ref. 19), it is possible that in the cases in which the inhibition was more sensitive to naloxone /x-receptors were predominant, while in the cases less sensitive to naloxone g-receptors were predominant. The fact that both ~- and g-antagonists attenuate the synaptic inhibition, at concentrations within the range in which they show selectivity when tested against selective opioid receptor agonists (Table I), suggests that both /x- and g-receptors are activated by the endogenous opioid. Acknowledgements. This work was supported by the Medical Research Council of Canada and the Quebec Heart Foundation. C.Z. is recipient of an MRC fellowship. We thank Dr. Victor J. Hruby for providing CTAP, Mr. Joe Petrella for technical assistance and Mrs. Christine Pamplin for preparing the manuscript.
REFERENCES 1 Akil, H., Watson, S.J., Young, E., Lewis, M.E., Khachaturian, H. and Walker, J.M., Endogenous opioids: biology and function, Annu. Reu. Neurosci., 7 (1984) 223-255. 2 Chang, K., Killian, A. and Cuatrecasas, P., Morphiceptin (NH 4Tyr-Pro-Phe-CONH2): a potent specific agonist for morphine (/z) receptors, Science, 212 (1981) 75-77. 3 Chavkin, C., James, I.F. and Goldstein, A., Dynorphin as a specific endogenous ligand of the K opioid receptor, Science, 215 (1982) 413-415. 4 Cotton, R., Kosterlitz, H.W., Paterson, S.J., Rance, M.J. and Traynor, J.R., The use of [3H]-[D-Pen2, D-PenS]enkephalin as a highly selective ligand for the g-binding site, Br. J. Pharmacol., 84 (1985) 927-932. 5 Cotton, R., Giles, M.G., Miller, L., Shaw, J.S. and Timms, D., ICI 174864: a highly selective antagonist for the opioid g-receptor, Eur. J. PharmacoL, 97 (1984) 331-332. 6 De Groat, W.C. and Kawatani, M., Enkephalinergic inhibition in parasympathetic ganglia of the urinary bladder of the cat, J. Physiol., 413 (1989) 13-29. 7 Gilbert, P.E. and Martin, W.R., The effects of morphine- and nalorphine-like drugs in the nondependent, morphine-dependent and cyclazocine-dependent chronic spinal dog, J. Pharmacol. Exp. Then, 198 (1976) 66-82. 8 Kanagawa-Terayama, Y., Wanaka, A., Yamasaki, H., Matsuyama, T., Matsumoto, M., Kamada, C. and Tohyama, M., Immunocytochemical analysis of [MetS]enkephalin-arg6-glyT-Leu s immunoreactive structures in the rat superior cervical ganglion, Brain Res., 494 (1989) 75-84. 9 Katayama, Y. and Nishi, S., Sites and mechanisms of actions of enkephalin in the feline parasympathetic ganglion, J. PhysioL, 251 (1984) 111-121. 10 Kennedy, C. and Krier, J., g-Opioid receptors mediate inhibition
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of fast excitatory postsynaptic potentials in cat parasympathetic colonic ganglia, Br. J. PharmacoL, 92 (1987)437-443. Kondo, H., Kuramoto, H., Wainer, B.H. and Yanaihara, N., Evidence for the coexistence of acetylcholine and enkephalin in the sympathetic preganglionic neurons of rats, Brain Res., 335 (1985) 309-314. Konishi, S., Tsunoo, A. and Otsuka, M., Enkephalin as a transmitter for presynaptic inhibition in sympathetic ganglia, Nature, 294 (1981) 80-82. Kow, L.-M. and Pfaff, D.W., Neuromodulatory action of peptides, Annu. Rev. Pharmacol. Toxicol., 28 (1988) 163-188. Kramer, T.H., Shook, J.E., Kazmierski, W., Ayres, E.A., Wire, W.S., Hruby, V.J. and Burks, T.F., Novel peptidic mu opioid antagonists: pharmacologic characterization in vitro and in vivo, J. PharmacoL Exp. Then, 249 (1989) 544-551. Lord, J.A.H., Waterfield, A.A., Hughes, J. and Kosterlitz, H.W., Endogenous opioid peptides: multiple agonists and receptors, Nature, 267 (1977) 495-499. Martin, W.R., Eades, C.G., Thompson, J.A., Huppler, R.E. and Gilbert, P.E., The effects of morphine- and nalorphine-like drugs in the nondependent and morphine-dependent chronic spinal dog, J. Pharmacol. Exp. Then, 197 (1976) 517-532. McCandless, D.L., Zablocka-Esplin, B. and Esplin, D.W., Rates of transmitter turnover in the cat superior cervical ganglion estimated by electrophysiological techniques, J. Neurophysiol., 34 (1971) 817-830. Mosberg, H.I., Hurst, R., Hruby, V.J., Gee, K., Yamamura, H.I., Galligan, J.J. and Burks, T.F., Bis-penicillamine enkephalins possess highly improved specificity toward g opioid receptors, Proc. Natl. Acad. Sci. USA, 80 (1983) 5871-5874. North, R.A., Opioid receptor types and membrane ion channels, TINS (March 1986) 114-117. North, R.A., Williams, J.T., Surprenant, A. and Christie, M.J.,/z and g-receptors belong to a family of receptors that are coupled to potassium channels, Proc. Natl. Acad. Sci. USA, 84 (1987) 5487-5491 Perri, V., Sacchi, O. and Casella, C., Synaptically mediated potentials elicited by the stimulation of post-ganglionic trunks in the guinea pig superior cervical ganglion, Pfliiger's Arch., 314 (1970) 57-67. Schultzberg, M., H6kfelt, T., Elfvin, T.L.G., Lundberg, J.M., Brandt, J., Elde, R.P. and Goldstein, M., Enkephalin immunoreactive nerve fibers and cell bodies in sympathetic ganglia of the guinea-pig and rat, Neuroscience, 4 (1979) 249-270. Takemori, A.E., Ho, B.Y., Naeseth, J.S. and Portoghese, P.S., Nor-binaltorphimine, a highly selective kappa-opioid antagonist in analgesic and receptor binding assays, J. PharmacoL Exp. Ther., 246 (1988) 255-258. Vonvoigtlander, P.F., Lahti, R.A. and Ludens, J.H., U-50,488: a selective and structurally novel non-mu (kappa) opioid agonist, J. Pharmacol. Exp. Ther., 224 (1983) 7-12. Williams, J.T., North, R.A. and Tokimasa, T., Inward rectification of resting and opiate-activated potassium currents in rat locus coeruleus neurons, J. Neurosci., 8 (1988) 4299-4306. Zhang, C., Bachoo, M. and Polosa, C., Naloxone-sensitive inhibition of nicotinic transmission in the superior cervical ganglion of the cat, Brain Res., 548 (1991) 29-34.
Brain Research, 622 (1993) 211-214 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 19199
The receptors activated by exogenous and endogenous opioids in the superior cervical ganglion of the cat C. Zhang, M. B a c h o o a n d C. P o l o s a Department of Physiology, McGill University, Montreal, Que. (Canada) (Accepted 20 April 1993)
Key words: Opiate receptor; Sympathetic ganglion; Synapse; Endogenous opioid
Nicotinic transmission in the superior cervical ganglion of the cat was studied in vivo and in vitro by recording the postganglionic compound action potential evoked, under partial block with hexamethonium, by supramaximal 0.2 Hz stimulation of the cervical sympathetic trunk. The compound action potential was depressed following a stimulus train (5 Hz, 40 s) applied to the same set of axons. The inhibition was antagonized by naloxone, suggesting mediation by endogenous opioids. Agonists selective for #-, 6- and K-opioid receptors, injected into the arterial supply of the in situ ganglion or added to the perfusion fluid in the in vitro ganglion, also produced a naloxone-sensitive inhibition of the compound action potential. The synaptic inhibition was antagonized by the 6-selective antagonist ICI 174,864 and by the/z-selective antagonist CTAP, but not by the K-selective antagonist NoroBNI. These results suggest that all three main types of opioid receptors are present in the ganglion but only/zand ~5-receptors are involved in the inhibition of ganglionic transmission mediated by the endogenous opioids.
INTRODUCTION
Opioid peptides have been demonstrated with immunohistochemical techniques in sympathetic ganglia22. Preganglionic nerve terminals 8 and the soma of sympathetic preganglionic neurons 11 contain these peptides. A physiological role of opioid peptides as neurotransmitters a n d / o r neuromodulators has been suggested by many studies (for reviews, see refs. 1 and 13). At most synapses opioids produce inhibitory effects on neuronal activity and synaptic transmission 19. In the autonomic nervous system, an inhibitory effect attributed to endogenous opioids was first demonstrated by Konishi ]2 in the inferior mesenteric ganglion. Similar effects were reported in colonic ganglial°, in bladder ganglia6 and in the superior cervical ganglion26. Three main types of opioid receptors (/z, ~ and K) have been characterized pharmacologically7'16'15. The types of opioid receptors mediating inhibition in autonomic ganglia have not been thoroughly investigated. There are studies indicating the involvement of 8opioid receptors in colonic ganglia1° and in bladder ganglia6. In the present study we demonstrate that
nicotinic transmission in the superior cervical ganglion of the cat can be depressed by exogenously applied/z-, 8- and K-opioid receptor agonists. We also present data suggesting that /z- and /~- but not K-receptors mediate the inhibitory effect of the endogenous opioid peptide(s) in the same ganglion. MATERIALS AND METHODS Cats of either sex, weighing 2.5-4.0 kg, were used under anesthesia with pentobarbital (40 mg/kg, i.p. followed by 5 mg/kg every 2 h). After paralysis with pancuronium (0.2 mg/kg, followed by supplements of 0.1 mg/kg every 2 h) and intubation of the trachea, the animals were artificially ventilated. In the in vivo experiments, the lingual artery was cannulated for injection of drugs into the arterial supply of the superior cervical ganglion. The thyroid and occipital arteries were ligated. A snare was placed around the external carotid artery to allow temporary closure of the artery during injections. Injections had a volume of 0.25 ml and took 2 s. An interval of 10 min was allowed between successive injections of the same or of different drugs except for the case of U 50,488 at high doses, in which an interval of 20 min was used. After exposure of the superior cervical ganglion, one of its postganglionic nerves (internal or external carotid nerve) was dissected and placed on bipolar hook electrodes for recording the postganglionic compound action potential. The segment of nerve between the electrodes was crushed. The cervical sympathetic trunk was dissected from the common carotid artery and cervical vagus nerve for a length of 1-2 cm and placed on
Correspondence: C. Polosa, Department of Physiology, McGill University, 3655 Drummond St., Montr6al, Qu6bec, Canada, H3G 1Y6. Fax: (1) (514) 398-7452.
212 hook electrodes for stimulation. After dissection, the ganglion and its nerves were covered with paraffin oil in a pool made by the skin flaps. Stimulus pulses (0.5 ms, supramaximal) were obtained from a Haer stimulator. A test shock was given at the frequency of 0.2 Hz. To evoke synaptic inhibition, a conditioning 5 Hz 40 s train was used. The postganglionic compound action potential was displayed on a storage oscilloscope and recorded on magnetic tape for later analysis. A femoral artery and vein were cannulated for monitoring systemic arterial pressure and for administering drugs, respectively. Hexamethonium (C6) was infused through the femoral vein at a rate of 0.05 + 0.003 mg-kg- 1. min- i to produce a steady partial block (compound action potential amplitude reduced by 40-50%) of nicotinic ganglionic transmission. The purpose of the block was to reduce the safety factor of nicotinic ganglionic transmission which is high in the superior cervical ganglion17'21, therefore making the compound action potential a more sensitive measure of effects of drugs or of conditioning stimulation. In the in vitro experiments, the common carotid artery was cannulated for perfusion. The superior cervical ganglion was dissected together with the segments of the common, internal and external carotid arteries which form the carotid bifurcation, with the CST and with the postganglionic nerves and placed in a perfusion chamber of 3 ml volume. The perfusion pressure, provided by a pump, was continuously monitored through a cannula placed in the lingual artery and connected to a pressure transducer. The ganglion was perfused with Krebs solution containing C6 (30-50/zM) of the following composition (in raM): NaCl 130.3, KCI 4.0, MgCl 2 1.0, KH2PO 4 1.0, NaHCO 3 12.0, CaC12 2.0 and D-glucose 11.0. The perfusion rate was kept at 0.2-0.3 ml/min, which was obtained with a perfusion pressure of 70-100 mmHg. The Krebs solution was equilibrated with a 95% 0 2 / 5 % CO 2 gas mixture and kept at room temperature (25°C). Drugs were dissolved in the Krebs solution and applied by switching from normal to drug-containing Krebs solution for 5-10 min. After application of a drug the ganglion was perfused with drug-free Krebs solution for 20-30 min before performing another test. The CST and internal carotid nerve were pulled into suction electrodes for stimulation and recording, respectively. Stimulus parameters were similar to those used in the in vivo experiment. When the effect of a conditioning train on ganglionic transmission was tested, the conditioning 5 Hz 40 s train and the 0.2 Hz test were given through the same electrode as in the in vivo preparations. Changes in compound action potential amplitude produced by the conditioning train or by application of drugs were expressed as percent of control compound action potential amplitude. Differences between means were tested with the t-test or the one-way ANOVA test. A difference was regarded as statistically significant if the P-value was less than 0.05. Numerical results reported in the text are expressed as mean ± S.E.M. Drugs used: Pentobarbital (M.T.C. Parmaceuticals), hexamethonium, naloxone, morphiceptin, [o-Pen2,o-PenS]enkephalin (DPDPE), dynorphin, trans-( ± )-3,4-dichloro-N-methyl-N-(2-[l-pyrrolidinyl]cyclohexyl)benzeneacetamide (U50,488, all from Sigma), Nor-Binaltorphimine (Nor-BNI, Research Biochemicals INC) and ICI 174,864 (Cambridge Research Biochemicals). o-Phe-Cys-Tyr-D-TrpArg-Thr-Pen-Thr-NH 2 (CTAP) was a gift from Dr. Victor J. Hruby, University of Arizona.
RESULTS
Inhibitory effect of exogenous opioid agonists on ganglionic nicotinic transmission In vivo experiments. As described earlier 26, the opioid peptide Leu-enkephalin, injected into the arterial supply of the ganglion, inhibits nicotinic transmission in the superior cervical ganglion. The inhibition is dosedependent. This inhibition was antagonized by the non-selective opioid receptor antagonist naloxone in a reversible manner. In the present study we tested the
A +
B
~
0.b~ 0.io 1.bolo:0o,o6.00
C
Concentration of agonist (~M)
_J Fig. 1. Inhibitory effect of opioid agonists on nicotinic ganglionic transmission. In vitro experiments. A: inhibition produced by morphiceptin (a), DPDPE (b) and U50,488 (c). Perfusion for 10 rain with the agonist-containing Krebs solution was started at the time indicated by the arrow. Each record shows the onset and peak of the inhibition (left) and the recovery (right). The break in a, b and c is 16, 17 and 20.5 min, respectively. Concentration of agonists: 1 /zM. Calibrations: vertical: 0.1 mV, horizontal: 50 s. B: examples of concentration-response relation. Ordinate is the percent depression of the compound action potential by the concentration of agonist indicated on abscissa. Open circle: morphiceptin. Open triangle: DPDPE. Filled triangle: U50,488. Data for morphiceptin and DPDPE are from the same ganglion.
opioid agonists morphiceptin, selective for /,-receptors 2, dynorphin, a possible endogenous ligand for Kreceptors 3, U50,488, a synthetic non-peptide agonist selective for K-receptors 24 and DPDPE selective for &receptors 5'1s. All agonists tested depressed the compound action potential in a dose-dependent manner. At the lowest dose used (1 /~g for morphiceptin and DPDPE; 10/~g for dynorphin and U50,488) the duration of the depression was between 45 and 63 s for morphiceptin, DPDPE and dynorphin, but was longer for U50,488 (120-300 s). The duration of the depression was dose-dependent. At the highest dose (100-200 /~g) the duration of the depression ranged from 4 to 19 min, again with the effect of U50,488 showing the slowest decay (P < 0.05). The inhibitory effect of these agonists was antagonised by i.v. naloxone. In vitro experiments. In order to determine the concentration at which /~-, 3- and K-agonists depress the compound action potential, experiments were performed in vitro. The agonists tested were morphiceptin, DPDPE and U50,488. Sample records of the effect produced at the 1 /~M concentration are shown in Fig. 1A. Concentration-response relations were obtained for the three agonists. Examples are shown in Fig. lB. The EDs0s for U50,488, morphiceptin and DPDPE were 234 + 30 (n = 5), 224 + 17 (n = 4) and 186 + 36 nM (n = 3), respectively. These values were not significantly different from each other. The selectivity of these agonists was tested with receptor-selective antagonists. These results are presented in table I.
213 As can be seen from the table, the 6-selective antagonist ICI 174,8644 blocked the DPDPE-induced inhibition but had no effect on the U50,488- and morphiceptin-induced inhibition. The /z-selective antagonist CTAP 14 blocked the inhibition produced by morphiceptin without effect on that produced by DPDPE and U50,488. The K-selective antagonist Nor-BN123 only blocked the inhibition produced by U50,488. These results suggest that at the concentration used morphiceptin, DPDPE and U50,488 act as selective agonists at/z-, 8- and K-opioid receptors.
A
co ..ci E
lOO 80 60
40
~0
2o 0
Antagonism of the naloxone-sensitive synaptic inhibition by opioid receptor-selective antagonists We have previously reported that a 5 Hz 40 s preganglionic train inhibits the postganglionic compound action potential and that the inhibition was antagonized by the non-selective opioid receptor antagonist naloxone 26. In in vitro experiments we tested the effects of the selective 6-antagonist ICI174,864, of the selective /z-antagonist CTAP and of the selective K-antagonist nor-BNI to determine the receptor type which mediates the inhibition. At a concentration of 1 /zM ICI 174,864, CTAP and Nor-BNI had no effect on the amplitude of the compound action potential. At the same concentration (1 /zM) ICI 174,864 depressed the inhibition by 53 _+5% (n = 4, P < 0.001). CTAP blocked the inhibition by 32 _+3% (n = 4, P < 0.01). When CTAP and ICI174,864, each at the 1 /zM concentration, were perfused simultaneously, the synaptic inhibition was depressed by 84 _+3% (n = 4, P < 0.001). The combined effect of these two antagonists was similar to the maximum antagonism produced by naloxone (10 /zM, 82 _+4%, n = 3, P < 0.001). The K-selective antagonist nor-BNI only produced a small blockade (8 +_ 2%, n = 4, P > 0.05). Fig. 2A shows the records obtained in one such experiment. The average results are shown in Fig. 2B. These results suggest that 8- and /z-receptors mediate the inhibition evoked by the preganglionic train.
Fig. 2. T h e synaptic inhibition is antagonized by naloxone, ICI 174,864 and CTAP, but not by nor-BNI. In vitro experiment. Antagonist concentration 1 ~ M . A: inhibition of the c o m p o u n d action potential by a standard 5 Hz 40 s train to the CST (a). Nor-BNI had little effect on the synaptic inhibition (b). T h e inhibition was antagonized by C T A P (c), ICI 174,864 (d) and naloxone (e). B: average data. Blank bar: C T A P (n = 4). Filled bar: ICI 174,864 (n = 4). Crosshatched bar: C T A P + I C I 1 7 4 , 8 6 4 ( n = 4 ) . Left diagonal bar: Nor-BNI (n = 4). * P < 0.01, * * P < 0.001.
cal ganglion is endowed with all three types of opioid receptors. The ED50 values for the three selective agonists were in the nM range, which is comparable to the concentrations of opioids found to be effective on central and peripheral neurons (e.g., refs. 10, 9, 20, 25). The synaptic inhibition was antagonized by the 6antagonist ICI 174,864 and the /z-antagonist CTAP, but not by the K-antagonist nor-BNI. As described earlier 26, the sensitivity of the synaptic inhibition to naloxone varies considerably from animal to animal, suggesting that the proportion each type of receptor contributes in different animals might be different. Since naloxone has affinity about 10 times higher for /z-receptors than for 6- and K-receptors (for a review,
TABLE I
DISCUSSION In the present study agonists selective for/z-, 8- and K-receptors were tested for their effect on nicotinic transmission in the superior cervical ganglion of the cat in vitro and in vivo. All agonists produced inhibition of transmission in a dose-dependent manner. The three selective agonists morphiceptin, DPDPE and U50,488 produced inhibition of similar magnitude at similar concentrations in the in vitro studies. Testing with specific antagonists revealed selectivity of the agonists. Thus, these results suggest that the cat superior cervi-
Effects of selective opioid receptor antagonists on the inhibition of ganglionic transmission produced by morphiceptin, U50,488 and DPDPE Values shown are peak inhibitory effect of agonist expressed as% decrease of c o m p o u n d action potential amplitude. Superscripts are n u m b e r of experiments. Concentration of agonists" 10/xM. Concentration of C T A P , ICI174,864 and Nor-BNI is 10/~M. Control is the inhibition produced by the agonist in absence of antagonist. Asterisk indicates significant difference from control ( P < 0.05). In vitro experiments.
Agonist
Control
CTAP
ICI174, 864
Nor-BNI
Morphiceptin U50,488 DPDPE
55+6 49+6 54+4
5 + 2 5, 47_+3 3 47_+3 3
51+5 3 47+6 3 1 1 + 6 3,
50+3 3 1 7 + 6 3, 51 + 4 3
214 see ref. 19), it is possible that in the cases in which the inhibition was more sensitive to naloxone /x-receptors were predominant, while in the cases less sensitive to naloxone g-receptors were predominant. The fact that both ~- and g-antagonists attenuate the synaptic inhibition, at concentrations within the range in which they show selectivity when tested against selective opioid receptor agonists (Table I), suggests that both /x- and g-receptors are activated by the endogenous opioid. Acknowledgements. This work was supported by the Medical Research Council of Canada and the Quebec Heart Foundation. C.Z. is recipient of an MRC fellowship. We thank Dr. Victor J. Hruby for providing CTAP, Mr. Joe Petrella for technical assistance and Mrs. Christine Pamplin for preparing the manuscript.
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