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Inhibition by morphine of the N-like calcium current in the human neuroblastoma cell line SH-SY5Y
ltnduly alter the analgesic properties and 6-phenyl derivatives were weak antinociceptive agents similarly to 6-methyl, 6-ethyl or 6-butyl analogs, the ;.mportance of the 3.8 A° long linear azido-group which has a firmly fixed conformation and its steric (fl) orientation is well documented by the fact, that dihydroisomorphine proved tO be much weaker analgesic than AM (hot plate EDso > 20 mg/kg and 0.023 mg/kg resp.). Also, 3-O-methyl and 3-O-ethyl substituted members of azidogroup proved to be 40 times and 50 times more active analgesics, than the parent compounds, and ethylmorphine, resp. Our experiments strongly suggested, that azidomorphine interacted with mu receptor, since its agonist effects are readily antagonized by naloxone, both in vivo (analgesia, sedation, catalepsy, etc.) and in vitro (longitudinal muscle strip of guinea pig ileum, mouse vas deferens) and also it was supported by receptor binding study of Horvath and Wollemann (1986). Moreover, AM appeared to us a pure agonist, since we were not able to detect any antagonist activity. Furthermore, among the N-allyl-substituted azidomorphines we have found strong antagonists, as N-allyl-norazidomorphine and its 14-OH derivative, estimating their antagonist activity (ADs0:0.031 and 0.033 mg/kg s.c. resp., against morphine) in oxymorphine fighting test. When N-cyclopropylmethyl was the substituent, we have observed a kappa agonist and mu antagonist activity as well, while its 14-OH-derivative failed to show antagonist activity, neither in vivo nor in vitro tests besides its s~ong mu antagonist efficacy. Furthermore, AM prolonged significantly the latency to first myoclonus or convulsion induced by pentetrazol, similarly to morphine, while this time was observed to be shortened by administration of antagonist type azidomorphines, which also showed procon,~dsive effects, as well.
References Bognkr, R., Maldcit, S., 1968, Acta China. Acad. Sci. Hung. 58, 203. Horvfith, K. and Wollemann, M. 1986, Neurochem. Res. 1565. Knoll. J., F"urst,S. and Kelemen, K., 1973, J. Pharm. Pharmacol., 25, 929. P.fr.232 [
Inlfib|tion by morphine of the N-fike calcium current in the human neuroblastoma cell line SH-SYSY Kennedy, C. and Henderson, G. Department of Pharmacology, Universityof Cambridge, Tennis Court RtZ, Cambridge CB2 I QJ, U.K.
The SH-SY5Y cell line is a human neuroblastoma cell line which displays a voltage-sensitive, N-like Ca 2"~ current, but not a T- or L-like Ca 2+ current, following retinoic acid-induced differentiation (Henderson and Seward, 1989). This current is inhibited following activation of both p and 8 opioid receptors present on these cells (Seward et al., 1989). Thus, this cell line may be a useful model in which to study opioid-induced tolerance and dependence. The aim of this study was to characterise the effects of acute and chronic administration of morphine on the N-like Ca 2+ current present in these cells. Ca 2+ currents were recorded using the whole cell patch clamp technique, with Ba 2+ as the charge carrier. The recording electrode contained (mM): CsCI 100, EGTA 10, MgCI 2 5, ATP 2, cAMP 0.25, HEPES 40, pH 7.2. The superfusate contained (ruM): NaCI 140, CsCI 5.4, BaCI 2 10.8, MgCI 2 1, D-glucose 10, HEPES 10 and TTX 0.5/~M, pH 7.2. Drugs were applied in known concentrations in the superfusate. To study tolerance and dependence cells were incubated for 3-6 days with incubation medium containing morphine (1 pM). Subsequently, cells were bathed with morphine-free superfusate when studying tolerance and with superfusate containing 1 / t M morphine when studying dependence. Ca 2+ currents were evoked from a holding potential of - 9 0 mV by step depolarisation to + 10 mV for 500 ms at 0.05 Hz. Morphine (1 pM) depressed both the peak and the sustained components of the Ca 2+ current. The peak current was inhibited to a greater extent than the sustained current and both effects were antagonised by naloxone (1 pM). These results are similar to those reported by Henderson and Seward (1989) for the p-selective agonist [D-AIa2, NMe-Phe 4, Gly-ol] enkephalin (DAGO). Neither comvonent of the Ca 2+ current was increased by BAY K 8644 (3 ~M) applied for up to 5 rain.
2313 Incubation with morphine (1/~M) for 3-6 days induced tolerance to opioids as the inhibitory action of DAGO on the Ca 2+ current was substantially reduced compared with that seen in untreated cells (Seward et al., 1989). In the continued presence of morphine (1 I~M) naloxone (1 pM) increased the amplitude of the Ca 2+ current and this was reversed on washout. Bathing in morphine-free superfusate also increased the amplitude of the Ca 2+ current and this too was reversed on returning to morphine-containing superfusate. BAY K 8644 (3 pM) did not increase the amplitude of the Ca 2+ current when added in morphine-containing superfusate. These results show that morphine acutely depresses the N-like Ca 2+ current in the SH-SY5Y human neuroblastoma cell line. This action appears to be mediated via p-opioid receptors. Chronic administration of morphine leads to a) tolerance to the inhibitory action of p-agonists and b) an increase in the amplitude of the Ca 2+ current following washout of morphine or administration of naloxone. It is not yet clear whether this represents an increase in available Ca 2+ conductance or whether it is simply due to reversal of a prolonged inhibitory action of morphine. However, the increased Ca 2+ current is unlikely to be due to the appearance of functional L-type Ca 2+ channels. References Henderson, G. and Seward, E.P. (1989) J. Physiol., in press. Seward, E.P., Henderson, G. and Sadee, W. (1989) Adv. in the Biosci., 75, 181-184. Pergamon.
P.fr.233 1
Effect of GTP-¥-S on the action of morphine in hippocampal slices Saito, K., Ohnishi *, T., M a t s u m o t o *, K., Sakuda *, M. and Inoki, R. Dept. of Pharmacology and * The Second Dept. of Oral and Maxillofacial Surgery, Faculty of Dentistry, Osaka University, Yamadaoka 1-8, Suita 565, Japan
Lines of evidence have been accumulated on the mechanism of action of morphine in which it inhibits the influx of calcium followed by the reduction of the release of transmitters. Recent report has more precisely demonstrated the processes of the calcium influx by morphine (Hescheler et al. 1987). In neuroblastoma × glioma hybrid cells, morphine inhibits calcium influx through a process in which the GTP binding proteins (Go) are involved. It has been shown that morphine and opioid peptides inhibit firing of basket cells and cause disinhibition of pyramidal cells in hippocampal slice preparations (Siggins and Zieglgansberger 1981). In the present study, the effect of GTP-y-S, a stable GTP analogue, on the action of morphine was examined employing the hippocampal slice preparations in the aim to characterize the GTP-binding proteins. SD rats were killed by decapitation and parasagittal slices containing hippocampus were cut at a thickness of 300 /~m. They were superfused with Krebs-Ringer solution in a recording chamber. Stimulation was applied through a bipolar stainless steel electrode to Schaffer collaterals and field potentials were recorded from pyramidal cells in cornu ammonis. Inositol 1,4,5-triphosphate (IP3) in hippocampal slices was measured by the radioimmunoassay procedure. In rat brain hippocampal slices, morphine enlarged the amplitude of the field potentials evoked in pyramidal neurons by Sehaffer collateral stimulation. It was found that D-AIa, D-Leu enke~phalip, a delta agonist, was more effective than morphine, whereas nalorphine, a kappa agonist, had no effect. The effect of morphine was reduced when the Ca concentration was elevated. In contrast, the reduction of the Ca concentrati~-a enhanced the effect of morphine. These findings suggested an inhibition of Ca dynamics by morphine through delta receptors. In rat brain hippocampal slices, GTP-~,-S reduced the morphine enhancement of the field potentials in a dose-dependent manner (Table 1). The effect of the GTP-y-S was irreversible° GDP-fl-S and ATP failed to iL4fibit the action of morphine. It was also observed that GDP-jS-S alone enhanced the amplitude of the field potentials. Stimulation of IP3 formation by GTP-y-S, but not by GDP-fl-S was observed in hippocampal slices. Incorporation of 35S-GTP-y-S into synaptosomal fraction was observed to some extent (30 fmol/mg protein). These results indicate that GTP-y-S permeates partly into nerve terminals in slice preparations and affect the action of morphine through GTP-binding proteins.
References Bognkr, R., Maldcit, S., 1968, Acta China. Acad. Sci. Hung. 58, 203. Horvfith, K. and Wollemann, M. 1986, Neurochem. Res. 1565. Knoll. J., F"urst,S. and Kelemen, K., 1973, J. Pharm. Pharmacol., 25, 929. P.fr.232 [
Inlfib|tion by morphine of the N-fike calcium current in the human neuroblastoma cell line SH-SYSY Kennedy, C. and Henderson, G. Department of Pharmacology, Universityof Cambridge, Tennis Court RtZ, Cambridge CB2 I QJ, U.K.
The SH-SY5Y cell line is a human neuroblastoma cell line which displays a voltage-sensitive, N-like Ca 2"~ current, but not a T- or L-like Ca 2+ current, following retinoic acid-induced differentiation (Henderson and Seward, 1989). This current is inhibited following activation of both p and 8 opioid receptors present on these cells (Seward et al., 1989). Thus, this cell line may be a useful model in which to study opioid-induced tolerance and dependence. The aim of this study was to characterise the effects of acute and chronic administration of morphine on the N-like Ca 2+ current present in these cells. Ca 2+ currents were recorded using the whole cell patch clamp technique, with Ba 2+ as the charge carrier. The recording electrode contained (mM): CsCI 100, EGTA 10, MgCI 2 5, ATP 2, cAMP 0.25, HEPES 40, pH 7.2. The superfusate contained (ruM): NaCI 140, CsCI 5.4, BaCI 2 10.8, MgCI 2 1, D-glucose 10, HEPES 10 and TTX 0.5/~M, pH 7.2. Drugs were applied in known concentrations in the superfusate. To study tolerance and dependence cells were incubated for 3-6 days with incubation medium containing morphine (1 pM). Subsequently, cells were bathed with morphine-free superfusate when studying tolerance and with superfusate containing 1 / t M morphine when studying dependence. Ca 2+ currents were evoked from a holding potential of - 9 0 mV by step depolarisation to + 10 mV for 500 ms at 0.05 Hz. Morphine (1 pM) depressed both the peak and the sustained components of the Ca 2+ current. The peak current was inhibited to a greater extent than the sustained current and both effects were antagonised by naloxone (1 pM). These results are similar to those reported by Henderson and Seward (1989) for the p-selective agonist [D-AIa2, NMe-Phe 4, Gly-ol] enkephalin (DAGO). Neither comvonent of the Ca 2+ current was increased by BAY K 8644 (3 ~M) applied for up to 5 rain.
2313 Incubation with morphine (1/~M) for 3-6 days induced tolerance to opioids as the inhibitory action of DAGO on the Ca 2+ current was substantially reduced compared with that seen in untreated cells (Seward et al., 1989). In the continued presence of morphine (1 I~M) naloxone (1 pM) increased the amplitude of the Ca 2+ current and this was reversed on washout. Bathing in morphine-free superfusate also increased the amplitude of the Ca 2+ current and this too was reversed on returning to morphine-containing superfusate. BAY K 8644 (3 pM) did not increase the amplitude of the Ca 2+ current when added in morphine-containing superfusate. These results show that morphine acutely depresses the N-like Ca 2+ current in the SH-SY5Y human neuroblastoma cell line. This action appears to be mediated via p-opioid receptors. Chronic administration of morphine leads to a) tolerance to the inhibitory action of p-agonists and b) an increase in the amplitude of the Ca 2+ current following washout of morphine or administration of naloxone. It is not yet clear whether this represents an increase in available Ca 2+ conductance or whether it is simply due to reversal of a prolonged inhibitory action of morphine. However, the increased Ca 2+ current is unlikely to be due to the appearance of functional L-type Ca 2+ channels. References Henderson, G. and Seward, E.P. (1989) J. Physiol., in press. Seward, E.P., Henderson, G. and Sadee, W. (1989) Adv. in the Biosci., 75, 181-184. Pergamon.
P.fr.233 1
Effect of GTP-¥-S on the action of morphine in hippocampal slices Saito, K., Ohnishi *, T., M a t s u m o t o *, K., Sakuda *, M. and Inoki, R. Dept. of Pharmacology and * The Second Dept. of Oral and Maxillofacial Surgery, Faculty of Dentistry, Osaka University, Yamadaoka 1-8, Suita 565, Japan
Lines of evidence have been accumulated on the mechanism of action of morphine in which it inhibits the influx of calcium followed by the reduction of the release of transmitters. Recent report has more precisely demonstrated the processes of the calcium influx by morphine (Hescheler et al. 1987). In neuroblastoma × glioma hybrid cells, morphine inhibits calcium influx through a process in which the GTP binding proteins (Go) are involved. It has been shown that morphine and opioid peptides inhibit firing of basket cells and cause disinhibition of pyramidal cells in hippocampal slice preparations (Siggins and Zieglgansberger 1981). In the present study, the effect of GTP-y-S, a stable GTP analogue, on the action of morphine was examined employing the hippocampal slice preparations in the aim to characterize the GTP-binding proteins. SD rats were killed by decapitation and parasagittal slices containing hippocampus were cut at a thickness of 300 /~m. They were superfused with Krebs-Ringer solution in a recording chamber. Stimulation was applied through a bipolar stainless steel electrode to Schaffer collaterals and field potentials were recorded from pyramidal cells in cornu ammonis. Inositol 1,4,5-triphosphate (IP3) in hippocampal slices was measured by the radioimmunoassay procedure. In rat brain hippocampal slices, morphine enlarged the amplitude of the field potentials evoked in pyramidal neurons by Sehaffer collateral stimulation. It was found that D-AIa, D-Leu enke~phalip, a delta agonist, was more effective than morphine, whereas nalorphine, a kappa agonist, had no effect. The effect of morphine was reduced when the Ca concentration was elevated. In contrast, the reduction of the Ca concentrati~-a enhanced the effect of morphine. These findings suggested an inhibition of Ca dynamics by morphine through delta receptors. In rat brain hippocampal slices, GTP-~,-S reduced the morphine enhancement of the field potentials in a dose-dependent manner (Table 1). The effect of the GTP-y-S was irreversible° GDP-fl-S and ATP failed to iL4fibit the action of morphine. It was also observed that GDP-jS-S alone enhanced the amplitude of the field potentials. Stimulation of IP3 formation by GTP-y-S, but not by GDP-fl-S was observed in hippocampal slices. Incorporation of 35S-GTP-y-S into synaptosomal fraction was observed to some extent (30 fmol/mg protein). These results indicate that GTP-y-S permeates partly into nerve terminals in slice preparations and affect the action of morphine through GTP-binding proteins.