NeuropharmacologyVol. 30, No. 4, pp. 323-327, 1991 Printed in Great Britain. All rights reserved
0028-3908/91 $3.00+ 0.00 Copyright © 1991 PergamonPress plc
FENTANYL AND SUFENTANIL INHIBIT AGONIST BINDING TO 5-HTIA RECEPTORS IN MEMBRANES FROM THE RAT BRAIN D. C. MARTIN,j'* R. P. INTRONA1 and R. S. ARONSTAM1'2 Departments of ~Anesthesiology and 2Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912-2700, U.S.A.
(Accepted 14 September 1990) Summary--The influence of several opioid narcotics and related drugs, on the binding of [3H]8-hydroxyN,N-dipropyl-2-aminotetralin ([3H]8-OH-DPAT), a serotonergic agonist, to 5-HTI^ receptors was determined in membranes from the brain of the rat. Sufentanil and fentanyl inhibited binding of [3H]8-OH-DPAT to hippocampal membranes, with IC5o values of 5.5 and 3.4/zM, respectively. In contrast, IC50values for meperidine, alfentanil and naloxone exceeded 100 # M. The inhibition of binding by sufentanil appeared to be competitive insofar as 10/zM sufentanil increased the apparent Kn from 1.0+ 0.1 to 3.9 + 0.3 nM, without affecting the number of binding sites and the inhibition was easily reversed. The binding of [3H]8-OH-DPAT to hippocampal membranes was inhibited by 5'-guanylylimidodiphosphate, a stable analogue of GTP, in a concentration-dependent manner. None of the opioid drugs examined altered the sensitivity of binding of [3H]8-OH-DPAT to guanine nucleotides. These results suggest that certain opioid narcotics, disrupt serotonergic neurotransmission as a result of direct interactions with 5-HTzAreceptors. No effects of opioid narcotics on 5-HTIA receptor-G protein coupling were noted.
Key words---opioids, serotonergic receptors (5-HTtA receptors), G proteins, sufentanil, 8-hydroxy-DPAT.
Numerous in vivo interactions between opioid narcotics and serotonergic systems in brain have been described: morphine-dependent rats possess fewer serotonergic receptors in the brain stem (Mennini, Poggesi, Cotecchia, De Blasi and Samanin, 1981). Moreover, serotonergic neuronal systems modulate the pharmacological activity of certain opioid narcotics. For example, Althaus, Miller, Moscicki, Hecker and DiFazio (1985) determined that the reduction of the minimum aveolar concentration of anesthetic, required to produce immobility in 50% of subjects exposed to a noxious stimulus for halothane, by sufentanil, required functional serotonergic pathways. Von Voigtlander, Lewis and Neff (1984) demonstrated that the full manifestation of analgesia induced by x opioids required serotonergic neuronal mediation. Central interactions between opiates and serotonin have also been implicated in the regulation of coronary baroreflexes (Moore, Porges, Gazibarich and White, 1985). Several subclasses of 5-HT receptors have been identified (5-HTtA.D, 5-HT2, 5-HT3; Watson and Abbott, 1990). The 5-HTIA receptors are members of the superfamily of G protein-coupled receptors and regulate adenylate cyclase and K ÷ channel activity. 8-Hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) and isapirone are selective agonists; selective antagonists have not been identified. How*To whom correspondence should be addressed.
ever, 5-HT1A receptors can be specifically labelled with the agonist probe [3H]8-OH-DPAT (Hall, E! Mestikawy, Emerit, Pichat, Hamon and Gozlan, 1985). The 5-HTIA receptor is present throughout the central nervous system of the rat but has the highest density of sites in the hippocampus, striatum and cortex. The 5-HT~A receptors have complex effects on nociceptive thresholds and cardiovascular function. These receptors are involved in opioid analgesia, insofar as the administration of 5-HT1A receptor agonists attenuate analgesia produced by administration of morphine in mice (Berge, Fasmer, Ogren and Mole, 1985) and administration of sufentanil and morphine in rats (Millan and Colpaert, 1990). A role of these receptors in the regulation of cardiovascular function is indicated by the fact that the administration of 8-OH-DPAT to rats causes a centrally-mediated sympathetic inhibition, resulting in hypotension and bradycardia (Mir and Fozard, 1987). Thus, pharmacological interference with 5-HTIA receptor function would have important implications regarding the regulation of nociceptive thresholds and cardiovascular dynamics. In order to increase understanding of the mechanism by which opioids alter serotonergic neuronal activity, the influence of opioids and other intravenous anesthetics on the binding of [3H]8-OH-DPAT to 5-HTtA receptors in membranes from the brain of the rat was examined.
323
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Fig. 1. Inhibition of the binding of [3H]8-OH-DPAT to 5-HTtA receptors in hippocampal membranes by sufentanil and fentanyl. The specific binding of 1 nM [3H]8-OH-DPAT to 5-HT~Areceptors was measured in hippocampal membranes (100/zg protein), in the presence of the indicated concentrations of sufentanil (A) and fentanyl (B). Binding is expressed as the percentage of specific binding, measured in the absence of the opioid. Binding was measured in the absence (open symbols) and presence (closed symbols) of 10/zM Gpp(NH)p. (At this concentration, Gpp(NH)p depressed the binding of [3H]8-OH-DPAT by approximately 40%, from 124 to 73 fmol/mg protein.) Data represent mean values + SEM, N = 5-6. METHODS Preparation o f membranes
Adult, male Sprague-Dawley rats were decapitated and their brains removed. Hippocampus, corpus striatum and cerebral cortex were dissected and suspended in 15ml of 50raM Tris-HCl buffer (2-4°C), pH 7.4, containing 5 mM MgCI 2 and 1 mM dithiothreitol. The tissues were homogenized using a Teflon-glass tissue grinder and then centrifuged at 17,000 g for 20 rain. The supernatants were discarded and the pellets resuspended in Tris-HC1 buffer and recentrifuged under identical conditions. Following an additional wash, the pellets were suspended in buffer at a concentration of 1 mg protein/ml, protein being determined by the method described by Smith, Krohn, Hermanson, Mallia, Gartner, Provenzano, Fujimoto, Goeke, Olsen and Klenk (1985), using bicinchoninic acid as reagent and bovine serum albumin as standard. Binding assays
Membranes were stored at 2-4°C and were incubated at 23°C for 5 min prior to all assays. Assays were initiated by addition of the membranes to a binding media containing [3H]8-OH-DPAT (142.9Ci/mmol; Dupont/NEN) and 50mM TrisHCI, pH 7.4, in a final volume of 1 ml. Some assays included 5'-guanylylimidodiphosphate (Gpp(NH)p; Sigma Chemicals Co.), a non-hydrolyzable analogue of GTP. Experimental compounds were added to the binding assay as aliquots from concentrated stock solutions. The following compounds were used: sufentanil, fentanyl and alfentanil (Janssen Pharmaceutica); meperidine, naloxone, morphine and methadone (Sigma); and ketamine (Parke Davis). Assays were carried out at 23°C, until the binding reached equilibrium (approximately 40 min). Nonspecific binding was determined in the presence of 100#M 5-hydroxytryptamine (Sigma). Membranes were collected by vacuum filtration on glass fiber
filters (Schleicher and Schueil, No. 32) and the filters were washed twice with 2.5 ml of buffer. The radioactivity content of the filters was determined by liquid scintillation spectrometry. Binding parameters ( K n and Bmax)were determined in saturation experiments, using 7 concentrations of [3H]8-OH-DPAT from 0.1 to 10nM. Binding data were fitted to a single site binding model by nonlinear regression analyses, using custom software based on the Marquardt-Levenberg algorithm. RESULTS The binding of [3H]8-OH-DPAT reached equilibrium by 40 min at 23°C and was linear with respect to protein concentration between 5 and 300/~g. Consistent with previous results (Hall et al., 1985), serotonin inhibited the binding of [3H]8-OH-DPAT with a nanomolar affinity, while methysergide inhibited binding with a micromolar affinity. The binding of [3H]8-OH-DPAT to 5-HT~A receptors in the hippocampus was inhibited in a concentration-dependent manner, by both sufentanil and fentanyl (Fig. 1; Table I); Gpp(NH)p (10/~M) did not affect the inhibition of agonist binding by either drug but Gpp(NH)p itself inhibited the binding of [3H]8-OH-DPAT in a concentration-dependent Table 1. Influence of opioid drugs on the binding of [3H]8-OHDPAT to 5-HT]Areceptors ICs0 (/~M)a Drug Hippocampus Striatum Cortex Sufentanil 5.5 + 0.5 2.2 + 0.8 3.4 + 1.5 Fentanyl 3.4 _+0.6 NTb 2.3 _+0.5 Alfentanil > 100 > 100 NT aThe concentrations of drugs, which inhibited the binding of 1 nM [3H]8-OH-DPAT to 5-HTIA receptors by 50%. Means + SEM, N = 5-8. bNT = not tested. The following compounds did not inhibit the binding of 1 nM [3H]8-OH-DPAT to 5-HTIA receptors in the hippocampus, with an ICs0 below 100/~M: meperidine, naloxone, morphine, ketamine, pentobarbital.
Opioids and 5-HTt^ receptors
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Fig. 2. The influence of sufentanil on inhibition by Gpp(NH) of the binding of [3H]8-OH-DPAT to 5-HTIA receptors in hippocampal membranes. The specific binding of l nM [3H]8-OH-DPAT to 5-HT~A receptors was measured in hippocampal membranes (100 #g), in the presence and absence of the indicated concentrations of Gpp(NH)p. Binding was determined in the absence (open symbols) and presence (closed symbols) of 1 #M sufentanil and is expressed as the percentage of total specific binding, measured in the absence of Gpp(NH)p. Data represent mean values -t- SEM, N = 4. manner; 50% inhibition was observed with 100/~M Gpp(NH)p (Fig. 2). Sufentanil had little effect on the inhibition of [3H]8-OH-DPAT binding by Gpp(NH)p (Fig, 2). The potency of both sufentanil and fentanyl as displacers of the binding of [3H]8OH-DPAT to 5-HTtA receptors in striatum and cortex was similar to their potency in the hippocampus (Fig. 3; Table 1). Saturation analyses of the binding of [3H]8-OH-DPAT, performed in the absence and presence of sufentanil, revealed that 10#M sufentanil decreased the apparent affinity ( K D = I . 0 + 0 . 1 n M , control; 3 . 9 + 0 . 3 n M , sufentanil; N = 3), without affecting the number of binding sites (Bmax = 266 + 33 fmol/mg, control; 211 _ 45 fmol/mg, sufentanil; N = 3), suggesting a competitive interaction. Qualitatively similar results were obtained with fentanyl. Inhibition by sufentanil was also readily reversed: after incubation with 10#M 1.2
'~
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Fig. 4. The reversibility of the inhibition of binding of [3H]8-OH-DPAT by sufentanil. Hippocampal membranes (200#g) were incubated in the presence (sufentanil) and absence (control) of I0/~M sufentanil for 5 rain. The membranes were either left at room temperature (unwashed) or washed by centrifugation at 12,000g for 5 min, followed by aspiration of the supernatant and resuspension of the pellet in 800/d buffer. The specific binding of l nM [3H]8-OHDPAT to 5-HTIA receptors was then measured. Data represent mean total binding values _+SEM, N = 3. sufentanil, the binding of [3H]8-OH-DPAT returned to control values after a single wash of the membranes (Fig. 4). Inhibition by fentanyl was similarly easily reversed. Alfentanil, naloxone and meperidine were considerably less potent as inhibitors of the binding of [3H]8-OH-DPAT (Fig. 5; Table 1); morphine, ketamine and pentobarbital had ICs0 values greater than 100/~M (Table 1). DISCUSSION The present results indicate that sufentanil and fentanyl interacted directly with 5-HTtA receptors, thereby inhibiting agonist binding. Other related opioid drugs were considerably less potent inhibitors of 5-HTIAreceptor binding. Sufentanil and fentanyl inhibited the binding of [3H]8-OH-DPAT in hippocampus, striatum and cortex with nearly identical potencies, indicating the similar nature of the receptor in these three regions. Saturation analyses revealed the inhibition by sufentanil and fentanyl =:~ 1.2 n (:3 I 1.0 -10 I 0.8 O0 0.6
.,~ 0.4 L_I ~
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Fig. 3. Inhibition of the binding of [3H]8-OH-DPAT to LoO concentration (M) 5-HTtA receptors in striatal and cortical membranes by sufentanil. The specific binding of 1 nM [3H]8-OH-DPAT Fig. 5. The effects of meperidine and naloxone on the was measured in membranes (100#g) from cortex (closed binding of [3H]8-OH-DPAT to 5-HT~Areceptors in hipposymbols) and striatum (open symbols), in the presence and campal membranes. The specific binding of [3H]8-OHabsence of the indicated concentrations of sufentanil. Data DPAT to 5-HTtA receptors was measured in hippocampal are mean values of a representative experiment, performed 'membranes (100#g), in the presence and absence of the in triplicate, that was repeated twice with essentially similar indicated concentrations of meperidine (©) or naloxone (0). Data represent mean values _+SEM, N = 4. results.
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D.C. MARTINet al.
to be competitive and reversible. None of the drugs examined affected 5-HT~A receptor-G protein coupling, as indicated by the sensitivity of agonist binding to a guanine nucleotide (Gpp(NH)p). The 5-HTIA receptor was the subject of the present investigation because of its documented roles in the integration of nociceptive mechanisms and cardiovascular function. Though serotonergic neuronal modulation of opioid analgesia (Berge et al., 1985) and cardiovascular function (Moore et al., 1985) has been demonstrated, the molecular mechanisms subserving these actions have not been defined. The 5-HT~A receptors in hippocampus are strongly coupled to guanine nucleotide-dependent transducer proteins (G proteins) that transduce receptorgenerated signals to postsynaptic intracellular effector mechanisms (Hall et al., 1985; Schlegel and Peroutka, 1986). Receptors coupled to G proteins are sensitive to exogenous guanine nucleotides, which usually decrease receptor affinity for agonists by converting high affinity, G protein-coupled receptors to a low affinity, uncoupled state (Schlegel and Peroutka, 1986). Accordingly, the application of Gpp(NH)p (a non-hydrolyzable analogue of GTP) resulted in decreased binding of the agonist probe, [3H]8-OH-DPAT, as receptors were converted to a low affinity, G protein-uncoupled state (low affinity [3H]8-OH-DPAT binding to uncoupled receptors would not be detected in this filtration assay due to the rapid dissociation of [3H]8-OH-DPAT from these receptors). Several volatile general anesthetics have been demonstrated to disrupt receptor-G protein coupling in muscarinic and cq-adrenergic receptor systems (Aronstam and Dennison, 1989; Dennison, Anthony, Narayanan and Aronstam, 1987; Baumgartner, Dennison, Narayanan and Aronstam, 1990). A similar action by opioids would have resulted in an attenuation of the inhibition of [3H]8-OH-DPAT binding by Gpp(NH)p. The failure of sufentanil and fentanyl to substantially affect the sensitivity of agonist binding to a guanine nucleotide indicates that these drugs do not affect 5-HT~A receptor-G protein coupling. Apparently, disruption of receptorG protein coupling is not a property common to all classes of general anesthetics. The potency of opioids as displacers of 5-HT~A receptor ligand binding roughly paralleled their lipophilicity. Fentanyl and sufentanil have octanolwater partition coefficients of 813 and 1778, respectively, while meperidine and alfentanil have coefficients of 39 and 145, respectively (Corssen, Reves and Stanley, 1988). Hug and Murphy (1981) observed that peak concentrations of fentanyl in plasma in dogs after a 50/zg/kg bolus injection were approximately 0.2/zM and concentrations in brain tissue were approximately 2-3 times greater. In the present study, analogous concentrations of fentanyl inhibited the binding of [~H]8-OH-DPAT by approximately 20%. Thus, while the extrapolation of concentrations of
drugs, used in crude membrane preparations, to the clinical situation is problematic, fentanyl appears to disrupt 5-HT~A receptor function at clinicallyrelevant concentrations. The involvement of serotonergic neuronal mechanisms in the pharmacology of opioid narcotics is incompletely understood. The present results demonstrate an action by the opioids at 5-HT~A receptors in three regions of the brain. Serotonergic systems have been implicated in opiate-induced neuromuscular rigidity and in analgesic mechanisms (Weinger, Cline, Smith and Koob, 1987; Von Voigtlander et al., 1984). One of the most clearly established functions of 5-HTIA receptors is the regulation of sympathetic outflow; stimulation of 5-HT~A receptors decreases heart rate and blood pressure in rats (Fozard and McDermott, 1985). The present observations suggest a mechanism whereby opioid narcotics could alter cardiovascular function during clinical anesthesia: antagonism at the 5-HT1A receptor could attenuate decreases in sympathetic outflow. Although the role of 5-HTIA receptors in the spinal cord is not known (Huang and Peroutka, 1987), disruption of ascending serotonergic neurotransmission by intrathecal or peridural administration of these potent narcotics could influence nociceptive thresholds. In summary, the binding of [3H]8-OH-DPAT to 5-HT~A receptors was disrupted by the opioid drugs sufentanil, fentanyl, meperidine and alfentanil. The potency of these drugs at inhibiting the binding of [3H]8-OH-DPAT paralleled their lipophilicity. The inhibition of the binding of [3H]8-OH-DPAT by sufentanil was characterized as competitive and reversible. None of the drugs tested had any effects on the sensitivity of 5-HTIA receptors to guanine nucleotides. The findings suggest that sufentanil and fentanyl disrupt neurotransmission at 5-HT~A receptors during clinical anesthesia and that this disruption could contribute to the clinical pharmacology of these drugs. by a grant-in-aid from the Georgia Affiliate of the American Heart Association, PHS grants GM-37948 and AA-07698, and a research starter grant from the Foundation for Anesthesia Education and Research. Acknowledgements--Supported
REFERENCES
Althaus J. S., Miller E. D., Moscicki J. C., Hecker B. R. and DiFazio C. A. (1985) Analgetic contribution of sufentanil during halothane anesthesia: A mechanism involving serotonin. Anesth. Analg. 64: 857-863. Aronstam R. S. and Dennison R. L. (1989) Anesthetic effects on muscarinic signal transduction. Int. Anesth. Clin. 27: 265-272. Baumgartner M. K., Dennison R. L., Narayanan T. K. and Aronstam R. S. (1990) Halothane disruption of ~2-adrenergic receptor-mediated inhibition of adenylate cyclase and receptor-G protein coupling in rat brain. Biochem. Pharmac. 39: 223-225.
Opioids and 5-HTIA receptors Berge O. G., Fasmer O. B., Ogren S. O. and Mole K. (1985) The putative serotonin receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin antagonizes the antinociceptive effect of morphine. Neurosci. Lett. 54: 71-75. Corssen G., Reves J. G. and Stanley T. H. (1988) Intravenous Anesthesia and Analgesia, chap. 4, Pharmacokinetics of narcotic compounds, pp. 31-38. Lea and Febiger, Philadelphia. Dennison R. L., Anthony B. L., Narayanan T. K. and Aronstam R. S. (1987) Effects of halothane on high affinity agonist binding and guanine nucleotide sensitivity of muscarinic acetylcholine receptors from rat brainstem. Neuropharrnacology 16: 1201-1205. Fozard J. R. and McDermott I. (1985) The cardiovascular response to 8-hydroxy-2-(di-n-propylamino) tetralin (8OH-DPAT) in the rat. Br. J. Pharmac. 84: 69P. Hall M. D., E1 Mestikawy S., Emerit M. B., Pichat L., Hamon M. and Gozlan H. (1985) [3H]8-Hydroxy-2(di-n-propylamino)tetralin binding to pre- and postsynaptic 5-hydroxytryptamine sites in various regions of the rat brain. J. Neurochem. 44: 1685-1696. Huang J. C. and Peroutka S. J. (1987) Identification of 5-hydroxytryptamine 1 binding site subtypes in rat spinal cord. Brain Res. 436: 173-176. Hug C. C. and Murphy M. R. (1981) Tissue redistribution of fentanyl and termination of its effects in rats. Anesthesiology 55: 369-375. Mennini T., Poggesi E., Cotecchia S., De Blasi A. and Samanin R. (1981) Changes in serotonin, but not catecholamine, receptor binding in the brain of morphinedependent rats. Molec. Pharmac. 20: 237-239.
327
Millan M. J. and Colpaert F. C. (1990) Attenuation of opioid induced antinociception by 5-HT,A partial agonists in the rat. Neuropharmacology 29:315-318. Mir A. K. and Fozard J. R. (1987) Cardiovascular Effects of 8-Hydroxy-2-(di-n-Propylamino)Tetralin. Brain 5HT~A receptors. (Dourish C. T., Ahlenius S. and Hutson P. H., Eds), pp. 120-134. Horwood, Chichester. Mir A. K., Hibert M., Tricklebank M. D., Middlemiss D. N., Kidd E. J. and Fozard J. R. (1988) MDL 72832; a potent and stereoselective ligand at central and peripheral 5-HTIA receptors. Eur. J. Pharmac. 149: 107-120. Moore P. G., Porges W. L., Gazibarich G. J. and White S. W. (1985) Central nervous system opiate and 5hydroxytryptamine influences on baroreflex control of the coronary circulation. J. Aut. Ner. Sys. 12: 185-194. Schlegel J. R. and Peroutka S. J. (1986) Nucleotide interactions with 5-HTtA binding sites directly labeled by [3H]-8-hydroxy-2-(di-n-propylamino)tetralin ([3H]-8-OHDPAT). Biochem. Pharmac. 35: 1943-1949. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olsen B. J. and Klenk D. C. (1985) Measurement of protein using bicinchoninic acid. Analyt. Bioehem. 150: 76-85. Von Voigtlander P, F., Lewis R. A. and Neff G. L. (1984) Kappa opioid analgesia is dependent on serotonergic mechanisms. J. Pharmac. exp. Ther. 231: 270-274. Watson S. and Abbott A. (1990) Receptor nomenclature supplement. Trends Pharmac. Sci. IlS. Weinger M. B., Cline E. J., Smith N. T. and Koob J. F. (1987) Ketanserin pretreatment reverses alfentanilinduced muscle rigidity. Anesthesiology 67: 348-354.
0028-3908/91 $3.00+ 0.00 Copyright © 1991 PergamonPress plc
FENTANYL AND SUFENTANIL INHIBIT AGONIST BINDING TO 5-HTIA RECEPTORS IN MEMBRANES FROM THE RAT BRAIN D. C. MARTIN,j'* R. P. INTRONA1 and R. S. ARONSTAM1'2 Departments of ~Anesthesiology and 2Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912-2700, U.S.A.
(Accepted 14 September 1990) Summary--The influence of several opioid narcotics and related drugs, on the binding of [3H]8-hydroxyN,N-dipropyl-2-aminotetralin ([3H]8-OH-DPAT), a serotonergic agonist, to 5-HTI^ receptors was determined in membranes from the brain of the rat. Sufentanil and fentanyl inhibited binding of [3H]8-OH-DPAT to hippocampal membranes, with IC5o values of 5.5 and 3.4/zM, respectively. In contrast, IC50values for meperidine, alfentanil and naloxone exceeded 100 # M. The inhibition of binding by sufentanil appeared to be competitive insofar as 10/zM sufentanil increased the apparent Kn from 1.0+ 0.1 to 3.9 + 0.3 nM, without affecting the number of binding sites and the inhibition was easily reversed. The binding of [3H]8-OH-DPAT to hippocampal membranes was inhibited by 5'-guanylylimidodiphosphate, a stable analogue of GTP, in a concentration-dependent manner. None of the opioid drugs examined altered the sensitivity of binding of [3H]8-OH-DPAT to guanine nucleotides. These results suggest that certain opioid narcotics, disrupt serotonergic neurotransmission as a result of direct interactions with 5-HTzAreceptors. No effects of opioid narcotics on 5-HTIA receptor-G protein coupling were noted.
Key words---opioids, serotonergic receptors (5-HTtA receptors), G proteins, sufentanil, 8-hydroxy-DPAT.
Numerous in vivo interactions between opioid narcotics and serotonergic systems in brain have been described: morphine-dependent rats possess fewer serotonergic receptors in the brain stem (Mennini, Poggesi, Cotecchia, De Blasi and Samanin, 1981). Moreover, serotonergic neuronal systems modulate the pharmacological activity of certain opioid narcotics. For example, Althaus, Miller, Moscicki, Hecker and DiFazio (1985) determined that the reduction of the minimum aveolar concentration of anesthetic, required to produce immobility in 50% of subjects exposed to a noxious stimulus for halothane, by sufentanil, required functional serotonergic pathways. Von Voigtlander, Lewis and Neff (1984) demonstrated that the full manifestation of analgesia induced by x opioids required serotonergic neuronal mediation. Central interactions between opiates and serotonin have also been implicated in the regulation of coronary baroreflexes (Moore, Porges, Gazibarich and White, 1985). Several subclasses of 5-HT receptors have been identified (5-HTtA.D, 5-HT2, 5-HT3; Watson and Abbott, 1990). The 5-HTIA receptors are members of the superfamily of G protein-coupled receptors and regulate adenylate cyclase and K ÷ channel activity. 8-Hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) and isapirone are selective agonists; selective antagonists have not been identified. How*To whom correspondence should be addressed.
ever, 5-HT1A receptors can be specifically labelled with the agonist probe [3H]8-OH-DPAT (Hall, E! Mestikawy, Emerit, Pichat, Hamon and Gozlan, 1985). The 5-HTIA receptor is present throughout the central nervous system of the rat but has the highest density of sites in the hippocampus, striatum and cortex. The 5-HT~A receptors have complex effects on nociceptive thresholds and cardiovascular function. These receptors are involved in opioid analgesia, insofar as the administration of 5-HT1A receptor agonists attenuate analgesia produced by administration of morphine in mice (Berge, Fasmer, Ogren and Mole, 1985) and administration of sufentanil and morphine in rats (Millan and Colpaert, 1990). A role of these receptors in the regulation of cardiovascular function is indicated by the fact that the administration of 8-OH-DPAT to rats causes a centrally-mediated sympathetic inhibition, resulting in hypotension and bradycardia (Mir and Fozard, 1987). Thus, pharmacological interference with 5-HTIA receptor function would have important implications regarding the regulation of nociceptive thresholds and cardiovascular dynamics. In order to increase understanding of the mechanism by which opioids alter serotonergic neuronal activity, the influence of opioids and other intravenous anesthetics on the binding of [3H]8-OH-DPAT to 5-HTtA receptors in membranes from the brain of the rat was examined.
323
324
D.C. MARTINet al. ),-
c~ I
c~ i LJ
A. 1.0
~
1.Ol
Control
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o.81
0.6
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0.4l
0.2 0 -10
B,
o.21
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-6
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-2
Log [Sufentanil], M
.lO
A i
-8
-6
-4
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Log [Fentanyl], M
Fig. 1. Inhibition of the binding of [3H]8-OH-DPAT to 5-HTtA receptors in hippocampal membranes by sufentanil and fentanyl. The specific binding of 1 nM [3H]8-OH-DPAT to 5-HT~Areceptors was measured in hippocampal membranes (100/zg protein), in the presence of the indicated concentrations of sufentanil (A) and fentanyl (B). Binding is expressed as the percentage of specific binding, measured in the absence of the opioid. Binding was measured in the absence (open symbols) and presence (closed symbols) of 10/zM Gpp(NH)p. (At this concentration, Gpp(NH)p depressed the binding of [3H]8-OH-DPAT by approximately 40%, from 124 to 73 fmol/mg protein.) Data represent mean values + SEM, N = 5-6. METHODS Preparation o f membranes
Adult, male Sprague-Dawley rats were decapitated and their brains removed. Hippocampus, corpus striatum and cerebral cortex were dissected and suspended in 15ml of 50raM Tris-HCl buffer (2-4°C), pH 7.4, containing 5 mM MgCI 2 and 1 mM dithiothreitol. The tissues were homogenized using a Teflon-glass tissue grinder and then centrifuged at 17,000 g for 20 rain. The supernatants were discarded and the pellets resuspended in Tris-HC1 buffer and recentrifuged under identical conditions. Following an additional wash, the pellets were suspended in buffer at a concentration of 1 mg protein/ml, protein being determined by the method described by Smith, Krohn, Hermanson, Mallia, Gartner, Provenzano, Fujimoto, Goeke, Olsen and Klenk (1985), using bicinchoninic acid as reagent and bovine serum albumin as standard. Binding assays
Membranes were stored at 2-4°C and were incubated at 23°C for 5 min prior to all assays. Assays were initiated by addition of the membranes to a binding media containing [3H]8-OH-DPAT (142.9Ci/mmol; Dupont/NEN) and 50mM TrisHCI, pH 7.4, in a final volume of 1 ml. Some assays included 5'-guanylylimidodiphosphate (Gpp(NH)p; Sigma Chemicals Co.), a non-hydrolyzable analogue of GTP. Experimental compounds were added to the binding assay as aliquots from concentrated stock solutions. The following compounds were used: sufentanil, fentanyl and alfentanil (Janssen Pharmaceutica); meperidine, naloxone, morphine and methadone (Sigma); and ketamine (Parke Davis). Assays were carried out at 23°C, until the binding reached equilibrium (approximately 40 min). Nonspecific binding was determined in the presence of 100#M 5-hydroxytryptamine (Sigma). Membranes were collected by vacuum filtration on glass fiber
filters (Schleicher and Schueil, No. 32) and the filters were washed twice with 2.5 ml of buffer. The radioactivity content of the filters was determined by liquid scintillation spectrometry. Binding parameters ( K n and Bmax)were determined in saturation experiments, using 7 concentrations of [3H]8-OH-DPAT from 0.1 to 10nM. Binding data were fitted to a single site binding model by nonlinear regression analyses, using custom software based on the Marquardt-Levenberg algorithm. RESULTS The binding of [3H]8-OH-DPAT reached equilibrium by 40 min at 23°C and was linear with respect to protein concentration between 5 and 300/~g. Consistent with previous results (Hall et al., 1985), serotonin inhibited the binding of [3H]8-OH-DPAT with a nanomolar affinity, while methysergide inhibited binding with a micromolar affinity. The binding of [3H]8-OH-DPAT to 5-HT~A receptors in the hippocampus was inhibited in a concentration-dependent manner, by both sufentanil and fentanyl (Fig. 1; Table I); Gpp(NH)p (10/~M) did not affect the inhibition of agonist binding by either drug but Gpp(NH)p itself inhibited the binding of [3H]8-OH-DPAT in a concentration-dependent Table 1. Influence of opioid drugs on the binding of [3H]8-OHDPAT to 5-HT]Areceptors ICs0 (/~M)a Drug Hippocampus Striatum Cortex Sufentanil 5.5 + 0.5 2.2 + 0.8 3.4 + 1.5 Fentanyl 3.4 _+0.6 NTb 2.3 _+0.5 Alfentanil > 100 > 100 NT aThe concentrations of drugs, which inhibited the binding of 1 nM [3H]8-OH-DPAT to 5-HTIA receptors by 50%. Means + SEM, N = 5-8. bNT = not tested. The following compounds did not inhibit the binding of 1 nM [3H]8-OH-DPAT to 5-HTIA receptors in the hippocampus, with an ICs0 below 100/~M: meperidine, naloxone, morphine, ketamine, pentobarbital.
Opioids and 5-HTt^ receptors
325
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Fig. 2. The influence of sufentanil on inhibition by Gpp(NH) of the binding of [3H]8-OH-DPAT to 5-HTIA receptors in hippocampal membranes. The specific binding of l nM [3H]8-OH-DPAT to 5-HT~A receptors was measured in hippocampal membranes (100 #g), in the presence and absence of the indicated concentrations of Gpp(NH)p. Binding was determined in the absence (open symbols) and presence (closed symbols) of 1 #M sufentanil and is expressed as the percentage of total specific binding, measured in the absence of Gpp(NH)p. Data represent mean values -t- SEM, N = 4. manner; 50% inhibition was observed with 100/~M Gpp(NH)p (Fig. 2). Sufentanil had little effect on the inhibition of [3H]8-OH-DPAT binding by Gpp(NH)p (Fig, 2). The potency of both sufentanil and fentanyl as displacers of the binding of [3H]8OH-DPAT to 5-HTtA receptors in striatum and cortex was similar to their potency in the hippocampus (Fig. 3; Table 1). Saturation analyses of the binding of [3H]8-OH-DPAT, performed in the absence and presence of sufentanil, revealed that 10#M sufentanil decreased the apparent affinity ( K D = I . 0 + 0 . 1 n M , control; 3 . 9 + 0 . 3 n M , sufentanil; N = 3), without affecting the number of binding sites (Bmax = 266 + 33 fmol/mg, control; 211 _ 45 fmol/mg, sufentanil; N = 3), suggesting a competitive interaction. Qualitatively similar results were obtained with fentanyl. Inhibition by sufentanil was also readily reversed: after incubation with 10#M 1.2
'~
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Fig. 4. The reversibility of the inhibition of binding of [3H]8-OH-DPAT by sufentanil. Hippocampal membranes (200#g) were incubated in the presence (sufentanil) and absence (control) of I0/~M sufentanil for 5 rain. The membranes were either left at room temperature (unwashed) or washed by centrifugation at 12,000g for 5 min, followed by aspiration of the supernatant and resuspension of the pellet in 800/d buffer. The specific binding of l nM [3H]8-OHDPAT to 5-HTIA receptors was then measured. Data represent mean total binding values _+SEM, N = 3. sufentanil, the binding of [3H]8-OH-DPAT returned to control values after a single wash of the membranes (Fig. 4). Inhibition by fentanyl was similarly easily reversed. Alfentanil, naloxone and meperidine were considerably less potent as inhibitors of the binding of [3H]8-OH-DPAT (Fig. 5; Table 1); morphine, ketamine and pentobarbital had ICs0 values greater than 100/~M (Table 1). DISCUSSION The present results indicate that sufentanil and fentanyl interacted directly with 5-HTtA receptors, thereby inhibiting agonist binding. Other related opioid drugs were considerably less potent inhibitors of 5-HTIAreceptor binding. Sufentanil and fentanyl inhibited the binding of [3H]8-OH-DPAT in hippocampus, striatum and cortex with nearly identical potencies, indicating the similar nature of the receptor in these three regions. Saturation analyses revealed the inhibition by sufentanil and fentanyl =:~ 1.2 n (:3 I 1.0 -10 I 0.8 O0 0.6
.,~ 0.4 L_I ~
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Fig. 3. Inhibition of the binding of [3H]8-OH-DPAT to LoO concentration (M) 5-HTtA receptors in striatal and cortical membranes by sufentanil. The specific binding of 1 nM [3H]8-OH-DPAT Fig. 5. The effects of meperidine and naloxone on the was measured in membranes (100#g) from cortex (closed binding of [3H]8-OH-DPAT to 5-HT~Areceptors in hipposymbols) and striatum (open symbols), in the presence and campal membranes. The specific binding of [3H]8-OHabsence of the indicated concentrations of sufentanil. Data DPAT to 5-HTtA receptors was measured in hippocampal are mean values of a representative experiment, performed 'membranes (100#g), in the presence and absence of the in triplicate, that was repeated twice with essentially similar indicated concentrations of meperidine (©) or naloxone (0). Data represent mean values _+SEM, N = 4. results.
326
D.C. MARTINet al.
to be competitive and reversible. None of the drugs examined affected 5-HT~A receptor-G protein coupling, as indicated by the sensitivity of agonist binding to a guanine nucleotide (Gpp(NH)p). The 5-HTIA receptor was the subject of the present investigation because of its documented roles in the integration of nociceptive mechanisms and cardiovascular function. Though serotonergic neuronal modulation of opioid analgesia (Berge et al., 1985) and cardiovascular function (Moore et al., 1985) has been demonstrated, the molecular mechanisms subserving these actions have not been defined. The 5-HT~A receptors in hippocampus are strongly coupled to guanine nucleotide-dependent transducer proteins (G proteins) that transduce receptorgenerated signals to postsynaptic intracellular effector mechanisms (Hall et al., 1985; Schlegel and Peroutka, 1986). Receptors coupled to G proteins are sensitive to exogenous guanine nucleotides, which usually decrease receptor affinity for agonists by converting high affinity, G protein-coupled receptors to a low affinity, uncoupled state (Schlegel and Peroutka, 1986). Accordingly, the application of Gpp(NH)p (a non-hydrolyzable analogue of GTP) resulted in decreased binding of the agonist probe, [3H]8-OH-DPAT, as receptors were converted to a low affinity, G protein-uncoupled state (low affinity [3H]8-OH-DPAT binding to uncoupled receptors would not be detected in this filtration assay due to the rapid dissociation of [3H]8-OH-DPAT from these receptors). Several volatile general anesthetics have been demonstrated to disrupt receptor-G protein coupling in muscarinic and cq-adrenergic receptor systems (Aronstam and Dennison, 1989; Dennison, Anthony, Narayanan and Aronstam, 1987; Baumgartner, Dennison, Narayanan and Aronstam, 1990). A similar action by opioids would have resulted in an attenuation of the inhibition of [3H]8-OH-DPAT binding by Gpp(NH)p. The failure of sufentanil and fentanyl to substantially affect the sensitivity of agonist binding to a guanine nucleotide indicates that these drugs do not affect 5-HT~A receptor-G protein coupling. Apparently, disruption of receptorG protein coupling is not a property common to all classes of general anesthetics. The potency of opioids as displacers of 5-HT~A receptor ligand binding roughly paralleled their lipophilicity. Fentanyl and sufentanil have octanolwater partition coefficients of 813 and 1778, respectively, while meperidine and alfentanil have coefficients of 39 and 145, respectively (Corssen, Reves and Stanley, 1988). Hug and Murphy (1981) observed that peak concentrations of fentanyl in plasma in dogs after a 50/zg/kg bolus injection were approximately 0.2/zM and concentrations in brain tissue were approximately 2-3 times greater. In the present study, analogous concentrations of fentanyl inhibited the binding of [~H]8-OH-DPAT by approximately 20%. Thus, while the extrapolation of concentrations of
drugs, used in crude membrane preparations, to the clinical situation is problematic, fentanyl appears to disrupt 5-HT~A receptor function at clinicallyrelevant concentrations. The involvement of serotonergic neuronal mechanisms in the pharmacology of opioid narcotics is incompletely understood. The present results demonstrate an action by the opioids at 5-HT~A receptors in three regions of the brain. Serotonergic systems have been implicated in opiate-induced neuromuscular rigidity and in analgesic mechanisms (Weinger, Cline, Smith and Koob, 1987; Von Voigtlander et al., 1984). One of the most clearly established functions of 5-HTIA receptors is the regulation of sympathetic outflow; stimulation of 5-HT~A receptors decreases heart rate and blood pressure in rats (Fozard and McDermott, 1985). The present observations suggest a mechanism whereby opioid narcotics could alter cardiovascular function during clinical anesthesia: antagonism at the 5-HT1A receptor could attenuate decreases in sympathetic outflow. Although the role of 5-HTIA receptors in the spinal cord is not known (Huang and Peroutka, 1987), disruption of ascending serotonergic neurotransmission by intrathecal or peridural administration of these potent narcotics could influence nociceptive thresholds. In summary, the binding of [3H]8-OH-DPAT to 5-HT~A receptors was disrupted by the opioid drugs sufentanil, fentanyl, meperidine and alfentanil. The potency of these drugs at inhibiting the binding of [3H]8-OH-DPAT paralleled their lipophilicity. The inhibition of the binding of [3H]8-OH-DPAT by sufentanil was characterized as competitive and reversible. None of the drugs tested had any effects on the sensitivity of 5-HTIA receptors to guanine nucleotides. The findings suggest that sufentanil and fentanyl disrupt neurotransmission at 5-HT~A receptors during clinical anesthesia and that this disruption could contribute to the clinical pharmacology of these drugs. by a grant-in-aid from the Georgia Affiliate of the American Heart Association, PHS grants GM-37948 and AA-07698, and a research starter grant from the Foundation for Anesthesia Education and Research. Acknowledgements--Supported
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Opioids and 5-HTIA receptors Berge O. G., Fasmer O. B., Ogren S. O. and Mole K. (1985) The putative serotonin receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin antagonizes the antinociceptive effect of morphine. Neurosci. Lett. 54: 71-75. Corssen G., Reves J. G. and Stanley T. H. (1988) Intravenous Anesthesia and Analgesia, chap. 4, Pharmacokinetics of narcotic compounds, pp. 31-38. Lea and Febiger, Philadelphia. Dennison R. L., Anthony B. L., Narayanan T. K. and Aronstam R. S. (1987) Effects of halothane on high affinity agonist binding and guanine nucleotide sensitivity of muscarinic acetylcholine receptors from rat brainstem. Neuropharrnacology 16: 1201-1205. Fozard J. R. and McDermott I. (1985) The cardiovascular response to 8-hydroxy-2-(di-n-propylamino) tetralin (8OH-DPAT) in the rat. Br. J. Pharmac. 84: 69P. Hall M. D., E1 Mestikawy S., Emerit M. B., Pichat L., Hamon M. and Gozlan H. (1985) [3H]8-Hydroxy-2(di-n-propylamino)tetralin binding to pre- and postsynaptic 5-hydroxytryptamine sites in various regions of the rat brain. J. Neurochem. 44: 1685-1696. Huang J. C. and Peroutka S. J. (1987) Identification of 5-hydroxytryptamine 1 binding site subtypes in rat spinal cord. Brain Res. 436: 173-176. Hug C. C. and Murphy M. R. (1981) Tissue redistribution of fentanyl and termination of its effects in rats. Anesthesiology 55: 369-375. Mennini T., Poggesi E., Cotecchia S., De Blasi A. and Samanin R. (1981) Changes in serotonin, but not catecholamine, receptor binding in the brain of morphinedependent rats. Molec. Pharmac. 20: 237-239.
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