Nociceptin Induced Inhibition of K+ Evoked Glutamate Release from Rat Cerebrocortical Slices Beverley Nicol, 'David G

Home , Amastatin

SPECIAL REPORT Nociceptin induced inhibition of K+ evoked glutamate release from rat cerebrocortical slices Beverley Nicol, 'David G. Lambert, David J. Rowbotham, *Darren Smart & *Alexander T. McKnight

University Department of Anaesthesia, Leicester Royal Infirmary, Leicester LEI 5WW and *Parke-Davis Neuroscience Research Centre, Robinson Way, Cambridge, CB2 2QB Nociceptin, an endogenous ligand for the orphan receptor ORLI, has recently been described. In this study we have shown that nociception inhibits 46 mM K+-stimulated glutamate release from rat perfused cerebrocortical slices with an IC50 of 51 nM. At 100 nM the inhibition amounted to 68+ 14% and was naloxone (10 uM)-insensitive excluding an activation of i, 5 and K opioid receptors. These data demonstrate the functional coupling of ORLI in glutamatergic neurones and implicates a role for nociceptin in glutamatergic neurotransmission. Keywords: Nociceptin; orphanin FQ; orphan receptor; glutamate release; rat cerebrocortical slices

Introduction The structure, distribution and pharmacology of 1000 nm in the presence of 30 gM bestatin, amastatin, capto- an 'orphan' G-protein-coupled receptor with some homology pril, phosphoramidon and 0.1% BSA to prevent breakdown) to opioid receptors has been described (Bunzow et al., 1994; was applied immediately after SI until the end of the experi- Mollereau et al., 1994). In 1995 Meunier et al., and Reinscheid ment (also present during S2). In some experiments, naloxone et al., reported the isolation of a heptadecapeptide (10 /1M) was also included. Perfusate glutamate concentrations (H-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala- Arg- Lys- were measured fluorimetrically based on the conversion of Leu-Ala-Asn-Gln-OH) named nociceptin or orphanin FQ NADP to NADPH by glutamate dehydrogenase; 480 pl of which displayed nanomolar binding affinity for ORL, in perfusate was incubated with NADP (1.0 mM) and glutamate radioligand binding studies and was hyperalgesic when in- dehydrogenase (30 u). The resulting production of NADPH jected intracerebroventicularly (icv) in the mouse. In addition was detected fluorimetrically at excitation and emission wa- to inhibiting cyclic AMP formation (Meunier et al., 1995; velengths of 366 nm and 430 nm respectively in a Perkin-El- Reinscheid et al., 1995) nociceptin has recently been shown to mer LS50B spectrofluorimeter, and compared with a known increase inwardly rectifying K+ currents in dorsal raphe nu- set of glutamate standards. Glutamate release was expressed cleus neurones (Vaughan & Christie, 1996), inhibit N-type relative to the mean of the first three basal samples collected voltage-sensitive Ca2" channels and enhance the carbachol- and S2/Sj ratios calculated. All data are presented as mean+ stimulated increase in [Ca2"]i in SH-SY5Y cells (Connor et al., s.e.mean. IC50 value for nociceptin was obtained by computer- 1996) and inhibit tachykinin release from peripheral sensory assisted curve fitting of the composite curve using GRAPH- neurones (Giuliani & Maggi, 1996). High levels of expression PAD-PRIZM. Statistical comparisons of paired samples were of ORL1 have been reported in rat cortex (Bunzow et al., 1994) made using Wilcoxon Rank sum test and considered sig- and the post receptor coupling is similar to that seen with nificant when P < 0.05. opioid receptors (Resisine, 1995). As we have shown that opioid receptor occupation inhibits glutamate release from K+ Results Depolarization of rat cerebrocortical slices with depolarized rat cerebrocortical slices (Nicol et al., 1996) we 46 mM K+ produced a monophasic release of glutamate for have examined the effects of nociceptin on glutamate release. both S1 and S2 (Figure la) with a mean S2/Sj ratio from 26 We report that nociceptin produced a dose-dependent nalox- control experiments of 0.97+0.06. Nociceptin produced a one-insensitive release of glutamate. dose-dependent reduction in the S2/S ratio with an estimated IC50 of 51 nM (Figure la,b). At 100 nm, the inhibition Methods Female Wistar rats (200-250 g) were killed by amounted to 64+8% (Figure la). Naloxone (10 gM) did not cervical dislocation and decapitation. The cerebral cortex was reverse nociceptin (100 nM) inhibition of glutamate release rapidly dissected and placed into ice-cold oxygenated (95% 02, (Figure Ic). 5% CO2) Krebs buffer, pH 7.4 of the following composition (mM): NaCl 115, KCl 4.7, CaCl2 2.0, MgCl2 1.2, NaHCO3 25 Discussion We demonstrate that nociceptin inhibits K+- and glucose 8.8 Slices (350 x 350 Mm) were cut and washed evoked glutamate release from rat cerebrocortical slices, in a three times in fresh Krebs buffer prior to agitation in a shaking concentration-dependent manner, with an IC5o of 51 nM. IC/ water bath set at 37°C for 40 min; 1 ml of gravity packed slices ECm values of 42 nM and 60 nm were reported by Connor et were pipetted into a perfusion chamber constructed from the al. (1996) for N-type Ca2" channel current inhibition and the barrel of a 2 ml syringe with a custom-made flow diffuser as a enhancement of carbachol-stimulated increase in [Ca2+]i in modified plunger. Slices were perfused at 37°C for 60 min at SH-SY5Y human neuroblastoma. In addition, nociceptin en- I ml min- prior to collection of 2 min fractions for the esti- hances an outward K+ conductance in dorsal raphe neurones mation of glutamate concentration. A 2 min pulse of 46 mM with an ECm of 12 nM (Vaughan & Christie, 1996). A com- K+ (Na+ adjusted) was applied following 6 min of perfusion bination of Ca2" channel inhibition and hyperpolarization, (SI). Slices were perfused for a further 30 min, prior to the resulting from enhanced outward K+ fluxes, is likely to be the second application of a 2 min pulse of 46 mM K+ (S2). Frac- underlying mechanism of glutamate inhibition observed in the tions were collected for 8 min after S2. Nociception (10- present study. In these reports and the present study, naloxone was ineffective, confirming an action at non-p, 6, or K opioid receptors. The physiological significance of inhibition of glutamate release in cerebral cortex is unclear but if inhibition of 1 Author for correspondence. glutamatergic transmission is mirrored in the hippocampus 1082 B. Nicol et al Nociceptin and glutamate release

a b * 4 80-

CD ECD ~~~~~~~~~~CD-~~~~~~~~~~~~~~~~~~~~~ CDTm 4- 2 t~~ ~40

0.0 3 ~ ~ Tm -mn .0 a)[occpn 60M

-~~~~~~~~~~~~~~ ~~~

0- 0- 1/)~~~~~~~~~~~~.0 10 20 30 40 50 -8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5 Time (min) Iog[Nociceptin] m

E~~~~~~~~

C 1.5-

1.0 0 CU

0.5-

0.0 Control Noci Noci (100 nm) + (lO0 nm) NalI(10).Im) Figure 1 Nociceptin inhibits glutamate release from rat cerebrocortical slices. In panel (a) the effect of 100 nM nociceptin (U) applied between two 2min challenges (arrows) of 46mM K+, SI and S, is shown; (D) control. Data are presented relative to the mean of the first three fractions collected. In panel (b) a dose-dependent inhibition of the S2,/S1 ratio is presented and in panel (c) the effects of naloxone (Nal, 10yM) on the nociceptin (Noci, 100nM) inhibition of S2,'S1 ratio is shown. All data are mean+s.e.mean from at least 5 independent determinations. *P<0.05 significantly reduced compared with paired control.

where high levels of ORL1 expression are found (Bunzow et al., i.c.v. nociceptin produced ataxia and loss of righting reflex in 1994; Mollereau et al., 1994) then an involvement in learning mice (Reinscheid et al., 1995). Clearly further studies of ORL, and memory must be suspected. ORL1 is also located in the receptor-mediated inhibition of glutamate release are required cerebellum, albeit at low levels (Mollereau et al., 1994) and in these areas.

References

BUNZOW, J.R., SEAZ, C., MORTRUD, M., BOUVIER, C., WILLIAMS, GIULIANI, S. & MAGGI, C.A. (1996). Inhibition of tachykinin release J.T., LOW, M. & GRANDY, D.K. (1994). Molecular cloning and from peripheral endings of sensory nerves by nociceptin, a novel tissue distribution of a putative member of the rat opioid receptor opioid peptide. Br. J. Pharmacol., 118, 1567 - 1569. gene family that is not a i, 6 or K opioid receptor type. FEBS MEUNIER, J.C., MOLLEREAU, C., TOLL, L., SUAUDEAU, C., Lett., 347, 284-288. MOISAND, C., ALVINERIE, P., BUTOUR, J.L., GUILLEMOT, J.C., CONNOR, M., YEO, A. & HENDERSON. G. (1995). The effect of FERRARA, P., MONSARRAT, B., MAZARGUIL, H., VASSART, G., nociceptin on Ca2 + channel current and intracellular Ca2 tin the PARMENTIER, M. & COSTENTIN, J. (1995). Isolation and SH-SY5Y human neuroblastoma cell line. Br. J. Pharmacol., 118, structure of the endogenous agonist of opioid receptor-like 205 - 207. ORL1 receptor. Nature, 377, 532-535. B. Nicol et al Nociceptin and glutamate release 1083

MOLLEREAU, C., PARMENTIER, M., MAILLEUX, P., BUTOUR, J.L., REISINE, T. (1995). Opiate receptors. Neuropharmacology, 34, 463- MOISAND, C., CHALON, P., CAPUT, D., VASSART, G. & 472. MEUNIER, J.C. (1994). ORLI, a novel member of the opioid VAUGHAN, C.W. & CHRISTIE, M.J. (1996). Increase by the ORLI receptor family. FEBS Lett., 341, 33 - 38. receptor (opioid receptor-like,) ligand, nociceptin, of inwardly NICOL, B., ROWBOTHAM, D.J. & LAMBERT, D.G. (1996). Is rectifying K conductance in dorsal raphe nucleus neurones. Br. J. glutamate release from rat cerebrocortical slices opioid receptor Pharmacol., 117, 1609- 1611. subtype selective? Br. J. Anaesthesia., 77, 282P. REISCHEID, R.K., NOTHACKER, H.P., BOURSON, A., ARDATI, A., (Received August 20, 1996 HENNINGSEN, R.A., BUNZOW, J.R., GRANDY, D.K., LANGEN, Accepted September 9, 1996) H., MONSMA, Jr, F.J. & CIVELLI, 0. (1995). Orphanin FQ: A neuropeptide that activates an opioid like G protein-coupled receptor. Science, 270, 792-794.