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CHARACTERISATION OF [3H]-CI-977 LABELLED Kc OPIOID BINDING SITES IN RODENT CNS
S.J. Boyle, K.G. Keecham, J.C. Hunter and J. Hughes
Parke-Davis Pesearch Unit, Addenbrookes Hospital Site, Hills Road,
ambridge,
CB2 2QB.
The rent develcpwnt of the kappa-selective arcylatamides (Lahti et al, 1985; Clark et al, 1988) has allowed a detaied instigaticn of the ctaracteristics and distributicn of kappa cpioid sites in the CIS. s is CI-977, a highly kapa-secive pioid oist The mstrnt aition to this series of c acteristics of JH]-CI-977 birdir arnd (Hunter et al, 1990). In the present study we have examined the distribution in the forebrains of the rat and guinea-pig. The mthodology for ligard birdig experiments anxd for autorad iy was as previonsly described (Clark et al, 1988, Smith et al, 1989). H]-CI-977 bcurd with high affinity to an apparent sirgle pcpulation of nKn-interactir sites in both rodent forebrain preparations. The equilibrium dissociation oMstant ( btaine for ;HJ-CI-977 in the guinea-pig was 0.10 + 0.lrf (90% specific birdie) with a maximl bir ained was 0.24 + 0.02rt (70% value capacity (maia) of 94.3 + 5.8 frvl/ng protein. In the rat the specific bixdirg) with a lower Blax value of 19.3 + 1.8 ftol/gprotein. 3 In ccmpetition sttdies against [HJ-CI-977 (0.01r the high affinity of unlabelled C1-977, U69593 and PD117302 ocmbined with the low affinity of the mu an delta selective ligards A0L ad DPD wee consistent with a kappa receptor profile (Table 1).
Ki (rm) (Man + Stardard
Table 1.
Ermr for n experiments) PD117302 DPDPE
= NAWXCME RNAZ0CIE Guinea-Pig 0.19+0.02 (7) 1.65+0.44 (6) 280+53 (6) >30000 (6) 1.03+0.20 (5) 0.14+0.04 (7) 5.00+1.00 (5) 0.17+0.02 (7) 4.39+1.20 (4) 307+73 (5) >30000 (4) 1.43+0.09 (4) 0.09+0.01 (4) 4.48+1.19 (4) Rat H] -CI-977 birdir to kappa raceptors The autoradiograpthic pattern of distribution was also consistent wi in both rat aid guinea-pig forebrain. Thus the highest levels of HJ-CI-977 bdiq in the guiapig were to be fond in the interpeduncmlar nuclas (ON), stantia nigra (SN) and cortical layers V aid VI, whilst levels h t absolute h in the rat the highest levels were present in the IK, SN and nucleus a-cohene althc
CI-977
U69593
DAK0L
were raah lower than in the guinea-pig. re B., Sharif N.A., Hunter J.C., Hill R.G. & Huhes J. (1988) Br. J. E~amco1. 93, Clark C.R., Bir 618-626 Hunter J.C., Leighton G.E., Horwell D.C., Rees D.C. & Hughes J. (1990) Br. J. Eba l. 24P. Iahti R.A., Micklson M.M., 1Cial1 J.M. & Von Voigtlander P.F. (1985) Eur. J. Flamool. 109, 281-284. Smith J.A.M., Hunter J.C., Hill R.G. &Hhes J. (1989) J. Neurochem. 53, 27-36.
CHARACTERIZATION OF THE PHARMACOLOGY OF SIGMA OPIOID SITES IN GUINEA-PIG BRAIN AND NCB20 CELL MEMBRANES A.R. Knight, J. Gillard and E.H.F. Wong. Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre,
304P
Terlings Park, Eastwick Road, Harlow, Essex, CM20 2QR. Sigma binding sites, which have been postulated to mediate the psychotomimetic actions of benzomorphan drugs (Zukin et al 1981), have been characterised in the CNS (Weber et al, 1986). In order to determine whether NCB20 cells can be used as a model system to study mammalian sigma sites, the pharmacology of the sigma binding site in membranes prepared from these cells and guinea pig whole brain P2 membranes has been compared, using the potent and specific ligand [3H]-Di-tolylguanidine (DTG). Sigma binding was performed by incubating aliquots of membranes with 5nM [3H]-DTG for 90 minutes at 230C. Non specific binding was defined with 1AM haloperidol. Scatchard analysis of [3H]-DTG binding in NCB20 cells revealed the site density to be 4.37 ± 0.13pmol/mg protein (n = 3) and the Kd 90.2nM (+ 98.5, - 81.9nM) (geometric mean, + sem, - sem). A range of sigma ligands were tested in both tissues (see Table). All of the compounds were 1.7 to 0.2 log units less potent in NCB 20 cell membranes. The (-) isomers of SKF 10,047 and pentazocine were more potent than the (+)-isomers in NCB20 cells whereas the reverse was true in guinea pig brain. (+) SKF 10,047, (+) pentazocine and (+)3PPP displaced sigma binding in guinea pig brain membranes with low Hill coefficients, suggesting heterogeneity in [3H]-DTG binding sites. In NCB20 membranes no heterogeneity was apparent with any of the compounds tested.
Displacement of [3H]-DTG (5nM) radioligand binding in guinea pig whole brain and NCB20 cell membranes NCB20 Cells G.P. brain nH nl pICso pICN 1.19 ±0.07 7.49 ± 0.04 1.04 ± 0.03 6.93 ± 0.06 DTG 5.69 ± 0.10 0.48 ± 0.05 4.13 ± 0.10 0.9 ± 0.08 (+)-SKF 10,047 4.42 ± 0.06 0.72 ± 0.07 5.0 ± 0.13 0.82 ± 0.09 (-)SF 10,047 6.92 ± 0.18 0.49 ± 0.06 5.18 ± 0.33 1.08 ± 0.20 (+)-Pentazocine
6.84 ± 0.02 (W)-Pentazocine Figures represent mean ± SEM, n = 3
0.83 ± 0.07
6.65 ± 0.14
1.15 ± 0.13
Thus although haloperidol sensitive [3H]-DTG binding is present in NCB20 cells, there are differences in the pharmacology seen in these membranes and in guinea pig brain, notably differing affinity, reverse stereoselectivity and Hill coefficients close to unity. Weber E., Sanders M.S., Quarum M., McLean S., Pou S. and Keana J.F.S. (1986) Proc. Natl. Acad. Sci. USA 83,8784-8788. Zukin R.S. and Zukin S.R. (1981) Mol. Pharmacol. 20,246-255.
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SHORT-TERM LITHIUM INCREASES THE RELEASE OF HIPPOCAMPAL 5-HT EVOKED BY ELECTRICAL STIMULATION OF THE DORSAL RAPHt NUCLEUS IN THE RAT IN VIVO T. Sharp S.R. Bramwell & D.G. Grahame-Smith, M.R.C. Clinical Pharmacology Unit, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE. The addition of lithium to other antidepressant drugs is reported to have a rapid therapeutic effect in otherwise drug resistant depression (De Montigny et al, 1983). This rapid antidepressant effect of lithium might relate to an increased release of S-HT in the brain which is proposed to occur in rats on the basis of earlier biochemical and behavioural measurements of presynaptic 5-HT function (Green & Grahame-Smith, 1974) and more recent electrophysiological studies of serotoninergic neurotransmission (Blier & De Montigny, 1985). Previously, we have shown using the microdialysis technique that electrical stimulation of the dorsal raphe nucleus (DRN) evokes a release of 5-HT in the rat hippocampus in vivo (Sharp et al, 1989). Here we have examined whether electrically-evoked release of hippocampal 5-HT is altered in rats receiving short-term treatment with lithium. Microdialysis was carried out as previously described (Sharp et al, 1989) in control or lithium chloridetreated (3 mmol/kg s.c. twice daily for 3 days), chloral hydrate-anaesthetized rats. Perfusates were collected every 20 min and analysed for 5-HT by HPLC-EC. Following a baseline period of up to 3 h, the DRN was electrically stimulated (300 iAi .0 msec) for 20 min at 1 h intervals. The current frequency was increased successively with each period of stimulation (2, 3, 5 and 10 Hz). In control rats, electrical stimulation of the DRN evoked a frequency-dependent, short-lasting release of 5-HT in hippocampus. The effect of electrical stimulation was enhanced in rats treated with lithium particularly when low frequency stimulation was used (table 1). Basal 5-HT output was not significantly different between the lithium-treated and control groups.
Effect of short-term lithium treatment on electrically-evoked release of hippocampal 5-HT in vivo
Table 1
2 3 5 10 0.076 ± 0.025 (4) 0.045 ± 0.01 (8) 0.030 ± 0.009 (9) 0.016 ± 0.003 (9) Control 0.071 ± 0.005 (6) 0.060 ± 0.003 (6)* 0.106 ± 0.013 (4) 0.049 ± 0.004 (6)** Lithium (values are absolute amounts of 5-HT (pmol) released during 20 min electrical stimulation at the pulse frequency indicated; mean ± S.E.M. (n), **p
CHARACTERISATION OF [3H]-CI-977 LABELLED Kc OPIOID BINDING SITES IN RODENT CNS
S.J. Boyle, K.G. Keecham, J.C. Hunter and J. Hughes
Parke-Davis Pesearch Unit, Addenbrookes Hospital Site, Hills Road,
ambridge,
CB2 2QB.
The rent develcpwnt of the kappa-selective arcylatamides (Lahti et al, 1985; Clark et al, 1988) has allowed a detaied instigaticn of the ctaracteristics and distributicn of kappa cpioid sites in the CIS. s is CI-977, a highly kapa-secive pioid oist The mstrnt aition to this series of c acteristics of JH]-CI-977 birdir arnd (Hunter et al, 1990). In the present study we have examined the distribution in the forebrains of the rat and guinea-pig. The mthodology for ligard birdig experiments anxd for autorad iy was as previonsly described (Clark et al, 1988, Smith et al, 1989). H]-CI-977 bcurd with high affinity to an apparent sirgle pcpulation of nKn-interactir sites in both rodent forebrain preparations. The equilibrium dissociation oMstant ( btaine for ;HJ-CI-977 in the guinea-pig was 0.10 + 0.lrf (90% specific birdie) with a maximl bir ained was 0.24 + 0.02rt (70% value capacity (maia) of 94.3 + 5.8 frvl/ng protein. In the rat the specific bixdirg) with a lower Blax value of 19.3 + 1.8 ftol/gprotein. 3 In ccmpetition sttdies against [HJ-CI-977 (0.01r the high affinity of unlabelled C1-977, U69593 and PD117302 ocmbined with the low affinity of the mu an delta selective ligards A0L ad DPD wee consistent with a kappa receptor profile (Table 1).
Ki (rm) (Man + Stardard
Table 1.
Ermr for n experiments) PD117302 DPDPE
= NAWXCME RNAZ0CIE Guinea-Pig 0.19+0.02 (7) 1.65+0.44 (6) 280+53 (6) >30000 (6) 1.03+0.20 (5) 0.14+0.04 (7) 5.00+1.00 (5) 0.17+0.02 (7) 4.39+1.20 (4) 307+73 (5) >30000 (4) 1.43+0.09 (4) 0.09+0.01 (4) 4.48+1.19 (4) Rat H] -CI-977 birdir to kappa raceptors The autoradiograpthic pattern of distribution was also consistent wi in both rat aid guinea-pig forebrain. Thus the highest levels of HJ-CI-977 bdiq in the guiapig were to be fond in the interpeduncmlar nuclas (ON), stantia nigra (SN) and cortical layers V aid VI, whilst levels h t absolute h in the rat the highest levels were present in the IK, SN and nucleus a-cohene althc
CI-977
U69593
DAK0L
were raah lower than in the guinea-pig. re B., Sharif N.A., Hunter J.C., Hill R.G. & Huhes J. (1988) Br. J. E~amco1. 93, Clark C.R., Bir 618-626 Hunter J.C., Leighton G.E., Horwell D.C., Rees D.C. & Hughes J. (1990) Br. J. Eba l. 24P. Iahti R.A., Micklson M.M., 1Cial1 J.M. & Von Voigtlander P.F. (1985) Eur. J. Flamool. 109, 281-284. Smith J.A.M., Hunter J.C., Hill R.G. &Hhes J. (1989) J. Neurochem. 53, 27-36.
CHARACTERIZATION OF THE PHARMACOLOGY OF SIGMA OPIOID SITES IN GUINEA-PIG BRAIN AND NCB20 CELL MEMBRANES A.R. Knight, J. Gillard and E.H.F. Wong. Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre,
304P
Terlings Park, Eastwick Road, Harlow, Essex, CM20 2QR. Sigma binding sites, which have been postulated to mediate the psychotomimetic actions of benzomorphan drugs (Zukin et al 1981), have been characterised in the CNS (Weber et al, 1986). In order to determine whether NCB20 cells can be used as a model system to study mammalian sigma sites, the pharmacology of the sigma binding site in membranes prepared from these cells and guinea pig whole brain P2 membranes has been compared, using the potent and specific ligand [3H]-Di-tolylguanidine (DTG). Sigma binding was performed by incubating aliquots of membranes with 5nM [3H]-DTG for 90 minutes at 230C. Non specific binding was defined with 1AM haloperidol. Scatchard analysis of [3H]-DTG binding in NCB20 cells revealed the site density to be 4.37 ± 0.13pmol/mg protein (n = 3) and the Kd 90.2nM (+ 98.5, - 81.9nM) (geometric mean, + sem, - sem). A range of sigma ligands were tested in both tissues (see Table). All of the compounds were 1.7 to 0.2 log units less potent in NCB 20 cell membranes. The (-) isomers of SKF 10,047 and pentazocine were more potent than the (+)-isomers in NCB20 cells whereas the reverse was true in guinea pig brain. (+) SKF 10,047, (+) pentazocine and (+)3PPP displaced sigma binding in guinea pig brain membranes with low Hill coefficients, suggesting heterogeneity in [3H]-DTG binding sites. In NCB20 membranes no heterogeneity was apparent with any of the compounds tested.
Displacement of [3H]-DTG (5nM) radioligand binding in guinea pig whole brain and NCB20 cell membranes NCB20 Cells G.P. brain nH nl pICso pICN 1.19 ±0.07 7.49 ± 0.04 1.04 ± 0.03 6.93 ± 0.06 DTG 5.69 ± 0.10 0.48 ± 0.05 4.13 ± 0.10 0.9 ± 0.08 (+)-SKF 10,047 4.42 ± 0.06 0.72 ± 0.07 5.0 ± 0.13 0.82 ± 0.09 (-)SF 10,047 6.92 ± 0.18 0.49 ± 0.06 5.18 ± 0.33 1.08 ± 0.20 (+)-Pentazocine
6.84 ± 0.02 (W)-Pentazocine Figures represent mean ± SEM, n = 3
0.83 ± 0.07
6.65 ± 0.14
1.15 ± 0.13
Thus although haloperidol sensitive [3H]-DTG binding is present in NCB20 cells, there are differences in the pharmacology seen in these membranes and in guinea pig brain, notably differing affinity, reverse stereoselectivity and Hill coefficients close to unity. Weber E., Sanders M.S., Quarum M., McLean S., Pou S. and Keana J.F.S. (1986) Proc. Natl. Acad. Sci. USA 83,8784-8788. Zukin R.S. and Zukin S.R. (1981) Mol. Pharmacol. 20,246-255.
305P
SHORT-TERM LITHIUM INCREASES THE RELEASE OF HIPPOCAMPAL 5-HT EVOKED BY ELECTRICAL STIMULATION OF THE DORSAL RAPHt NUCLEUS IN THE RAT IN VIVO T. Sharp S.R. Bramwell & D.G. Grahame-Smith, M.R.C. Clinical Pharmacology Unit, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE. The addition of lithium to other antidepressant drugs is reported to have a rapid therapeutic effect in otherwise drug resistant depression (De Montigny et al, 1983). This rapid antidepressant effect of lithium might relate to an increased release of S-HT in the brain which is proposed to occur in rats on the basis of earlier biochemical and behavioural measurements of presynaptic 5-HT function (Green & Grahame-Smith, 1974) and more recent electrophysiological studies of serotoninergic neurotransmission (Blier & De Montigny, 1985). Previously, we have shown using the microdialysis technique that electrical stimulation of the dorsal raphe nucleus (DRN) evokes a release of 5-HT in the rat hippocampus in vivo (Sharp et al, 1989). Here we have examined whether electrically-evoked release of hippocampal 5-HT is altered in rats receiving short-term treatment with lithium. Microdialysis was carried out as previously described (Sharp et al, 1989) in control or lithium chloridetreated (3 mmol/kg s.c. twice daily for 3 days), chloral hydrate-anaesthetized rats. Perfusates were collected every 20 min and analysed for 5-HT by HPLC-EC. Following a baseline period of up to 3 h, the DRN was electrically stimulated (300 iAi .0 msec) for 20 min at 1 h intervals. The current frequency was increased successively with each period of stimulation (2, 3, 5 and 10 Hz). In control rats, electrical stimulation of the DRN evoked a frequency-dependent, short-lasting release of 5-HT in hippocampus. The effect of electrical stimulation was enhanced in rats treated with lithium particularly when low frequency stimulation was used (table 1). Basal 5-HT output was not significantly different between the lithium-treated and control groups.
Effect of short-term lithium treatment on electrically-evoked release of hippocampal 5-HT in vivo
Table 1
2 3 5 10 0.076 ± 0.025 (4) 0.045 ± 0.01 (8) 0.030 ± 0.009 (9) 0.016 ± 0.003 (9) Control 0.071 ± 0.005 (6) 0.060 ± 0.003 (6)* 0.106 ± 0.013 (4) 0.049 ± 0.004 (6)** Lithium (values are absolute amounts of 5-HT (pmol) released during 20 min electrical stimulation at the pulse frequency indicated; mean ± S.E.M. (n), **p
