- Email: [email protected]
Evidence for functionally distinct μ receptors modulating acetylcholine release
Neuropeptides 5: 109-112, 1984
~JIDENCE
FOR
FUNCTIONALLY
DISTINCT 1-1 RECEPTORS MODULATING ACETYLCHOLINE
RELEASE. C Ennis and M G Wyllie, Department of Pharmacology, Wyeth Research (UK) Ltd, Huntercombe Lane South, Taplow, Nr Maidenhead, Berkshire. U.K. (reprint request to M G Wyllie) A comparison+of the potency of selective opioid agonists and antagonists to inhibit K -evoked ['HI-Ach release from slices of rat occipitoparietal cortex and rat striatum suggests that 1~opioid receptors are involved in the modulation of Ach release in both regions. However, only the receptor in the striatum was sensitive to blockade by pretreatment with naloxazone which is consistent with there being two types of ).I receptors. Such a subdivision is also supported by data from radioligand binding studies and the finding that meptazinol is an agonist at the naloxazone-sensitive site and an antagonist at the other. Introduction The results of recent biochemical and isolated tissue studies have suggested that the u class of opioid receptors may be divided into ul and )J subtypes (1,Z) but there is little unequivocal evidence of a functzonal separation between these subtypes in the CNS. Wood, Richard and Thakur (3) have suggested however, that acetylcholine (Ach) turnover in rat parietal cortex in vivo may be regulated by u1 receptors. The present experiments were performed to investigate the opioid modulation of Ach release in a more simple in vitro system using slices of rat parietal cortex. For comparison, the regulation of Ach release from slices of rat striatum was also examined since Ach turnover in this region is thought noi to involve opioid receptors. Methods The occipitoparietal cortex from one rat or the striata from two rats was chopped into 0.25 x 0.25 x 2mm slices using a McIlwain tissue chopper. The slices were incubated at 37OC in Krebs-Henseleit solution containing I'H(-choline (0.3um) for 30 min, washed with 3 x 5ml aliquots of Krebs-Henseleit solution and transferred to each of 10 superfusionchambers maintained at 37OC, approximately 25 mg tissue per chamber. The slices were superfused at 0.4 ml mine1 with Krebs-Henseleit solution containing hemicholinium - 3 (10nM) and physostigmine (10uM) for 120 min. Fractions of the superfusate were collected every 4 min. Two pulses of 4 min duration, of 40 mM K obtained by iso-osmotic replacement of NaCl by KCl, were given at t = 6B(S1) and t = 92(S2) min after the start of the superfusion. Drugs were added to the superfusing medium 109
immediately after Sl. The efflux of tritium was calculated as fractional release Tnd the amount of tritium released above basal in response to the The S2/sl ratio raised K concentration was expressed as the ratio S2/Sl. for drug-treated sites was expressed as a percentage of a paired control Four concentrations of agonist compounds were used in S2/Sl ratio. The pAl0 determinations were performed by duplicate in each experiment. the method of Schild (5). ('HI-choline chloride, specific acitivty 15 Ci mmol-1 was obtained from Amersham International. Statistical
analysis was performed
usingthe
Mann-Whitney
U test, 2 tailed.
Results The spontaneous release of tritium from slices of rat occipitoparietal cortex and rat striatum became steady after160 min of superfusion at a The addition rate of 0.57 + 0.04 % and 0.43 k 0.02% min respectively. of 40 mM K+ produced an increase in release of 2.8 +0.7% and 8.0 & 1.1%. The control s2,Sl ratios were 0.84 t 0.05, n=27 (occipitoparietal cortex) and 0.81 + 0.04, n=30 (striatum). M?rphine (0.01 to 10uM) produced a concentration-related inhibition of K -evoked tritium release in both brain areas. The concentration producing 30% inhibition (IC50) was 159 (49-275)nM for the cortex and 126 (6-630)nM Meptazinol (0.01 to 100uM) had no significant effect on in the striatum. K+-evoked tritium release from slices of occipitoparietal cortex but produced a concentration-related inhibi?ion of release in the striatum (IC30 = 501 (126-1,60O)nM), (Fig.1) .
1
1
Figure 1. Concentration-effect curves for morphine (circles) and meptazinol (squares) for the inhibition of ACh release in the occipitoparietal cortex (closed symbols) and striatum (open symbols). n 2 6.
80
4
USO, 488 (< 10uM). naloxone (< 0.1 PM), ICI 154129 (~10 uM) and ICI 174864 (11.1M) had no significant effect when tested alone on tritium release in either the cortex or striatum. None of the compounds used affected the spontaneous release of tritium.
Naloxone produced
a concentration-related
110
antagonism
of the response to
,morphinein both brain regionswith pAl0 values of 7.9 + 0.19 (cortex) and 8.1 -+0.26 (striatum). The inhibition of tritium release produced by meptazinol in the striatum was also antagonised bynaloxone with a pAlO value of 8.2 f 0.27. The response to morphine in the cortex was significantly reduced in the presence of meptazinol in concentrations above 1pM. ICI 154129, up to lOuM, had no effect on the response to morphine in the cortex but antagonised the response to morphine in the striatum (pAlO = 6.2 _+0.33). ICI 174864 (1uM) had no effect on the response to morphine in the striatum. -1 Pretreatmentwithnaloxazone (30 mg kg , S.C. )24h prior to sacrifice, had no effect per se on tritium release but abolished the responses to both morphine and meptazinol in the striatum. The response to morphine in the cortex was not significantly changed, IC = 224 (50-603) cf 159 (49-275). 30 Discussion In recent years the concept of multiple opioid receptors has received much attention and the identification of multiple classes of binding sites has led to considerable effort being expended to correlate these sites with particular physiological effects in vitro and in vivo. As yet the results of such investigations remain equivocal. The data from the present study are consistent with the suggestion that Ach release in vitro may be influenced by different opioid receptors in different brain regions. The potency of naloxone (pAlo z 8.0) suggests that it may act a p receptor in both regions (6)_ However, the finding that only one of these sites was sensitive to blockade by naloxazone is consistent with a subdivision of these receptors. Naloxazone, a selective ul antagonist (7) has been shown to antagonise morphine-induced analgesia in the same dose as that used in the present study (Hartley, unpublished). Kappa opioid receptors do not appear to be involved in the modulation of Ach release in vitro since the K agonist US0488 was ineffective. It has been reported (8) that 6 receptors may be involved in the modulation of striatal Ach release in vitro. This view was not supported by the findings of the present study in which the antagonist ICI 154129 reduced the effect of morphine whereas another more selective 6 antagonist ICI 174864 was without effect. The discrepancies between the results of the previous (8) and the present studies may be due to methodological differences and to the difference in agonists used ie DADLE and morphine. The relative naloxazone sensitivity of cortical and striatal responses to morphine would favour a subdivision of 1-1 sites (cortex p - striatum, ~1). This subdivision is further supported by comparing the pres&t data with that from radioligand binding studies (Table 1).
111
Table 1:
IC values (nM) for the inhibition of Ach release compared with K.3!nd Na+ ratios for the displacement of I'HI naloxone binding O!ZnM (~1) and 2.OnM (u2).
u1 Agonist
IC30
striatum
Ki(nM)*
Morphine 126(6.3-630) 2.6kO.50 Meptazinol 500(126-1000) 57.5klO.7 *Data from
p2 Na+ ratio
IC30cortex
27.6 9.6
159(49-275) 7.08k1.41 69.8 ~10000 363+34 1.4
Ki(nM)*
Na+ ratio
Wood, M.D., unpublished
Thus, morphine with agonist (high Na+ratio) activity at both u and u 2 sites depressed Ach release in both brain regions. Meptazinollwith agonist activity at 1-1 (Na+ratio 9.6) inhibited Ach release in the striatum whereas in tie cortex (p,, Na+ratio 2.1.0) it had no effect alone but antagonised the response to morphine. Overall, therefore the data supports the concept of a subdivision of u sites, both of which are involved in the modulation of Ach release in vitro. 1.
Nishimura, S.L., Recht, L.D and Pasternak, G.W. (1984) Biochemical characterization of high-affinity 'H-opioid binding. Mol.Pharmac. 25: 29-37
2.
Sayre, L.M., Portoghese, P.S. and Takemori, A.E. (1983) Difference between mu-receptors in the guinea-pig ileum and the mouse vas deferens. Eur. J. Pharmacol. 90: 159-160.
3.
Wood, P-L., Richard, J.W. and Thakur, M. (1982). Mu opiate isoreceptors: differentiation with kappa agonists. Life Sci. 31:2313-2317
4.
Wood, P.L. and Stotland, L.M. (1980) Actions of enkephalin, mu and partial agonist analgesics on acetylcholine turnover in rat brain.
5.
Schild, H.O. (1947). A new scale for measurement of drug antagonism Br. J. Pharmac. 2: 189-206.
6.
Lord, J.A.H., Waterfield, A.A. Hughes, J and Kosterlitz, H.W. (1977). Endogenous opioid peptides: multiple agonists and receptors. Nature 267: 495-499.
7.
Childers, S.R. and Pasternak, G.W. (1982). Naloxazone, a novel opiate antagonist: irreversible blockade of rat brain opiate receptors in vitro Bell and Mol. Neurobiol. 2: 93-103
8.
Mulder, A.H., Wardeh, G. Hogenboom, F. and Frankhuyzen A.L. (1984) K and 6 -opioid receptor agonists differentially inhibit striatal dopamine and acetylcholine release. Nature 308: 278-280.
112
~JIDENCE
FOR
FUNCTIONALLY
DISTINCT 1-1 RECEPTORS MODULATING ACETYLCHOLINE
RELEASE. C Ennis and M G Wyllie, Department of Pharmacology, Wyeth Research (UK) Ltd, Huntercombe Lane South, Taplow, Nr Maidenhead, Berkshire. U.K. (reprint request to M G Wyllie) A comparison+of the potency of selective opioid agonists and antagonists to inhibit K -evoked ['HI-Ach release from slices of rat occipitoparietal cortex and rat striatum suggests that 1~opioid receptors are involved in the modulation of Ach release in both regions. However, only the receptor in the striatum was sensitive to blockade by pretreatment with naloxazone which is consistent with there being two types of ).I receptors. Such a subdivision is also supported by data from radioligand binding studies and the finding that meptazinol is an agonist at the naloxazone-sensitive site and an antagonist at the other. Introduction The results of recent biochemical and isolated tissue studies have suggested that the u class of opioid receptors may be divided into ul and )J subtypes (1,Z) but there is little unequivocal evidence of a functzonal separation between these subtypes in the CNS. Wood, Richard and Thakur (3) have suggested however, that acetylcholine (Ach) turnover in rat parietal cortex in vivo may be regulated by u1 receptors. The present experiments were performed to investigate the opioid modulation of Ach release in a more simple in vitro system using slices of rat parietal cortex. For comparison, the regulation of Ach release from slices of rat striatum was also examined since Ach turnover in this region is thought noi to involve opioid receptors. Methods The occipitoparietal cortex from one rat or the striata from two rats was chopped into 0.25 x 0.25 x 2mm slices using a McIlwain tissue chopper. The slices were incubated at 37OC in Krebs-Henseleit solution containing I'H(-choline (0.3um) for 30 min, washed with 3 x 5ml aliquots of Krebs-Henseleit solution and transferred to each of 10 superfusionchambers maintained at 37OC, approximately 25 mg tissue per chamber. The slices were superfused at 0.4 ml mine1 with Krebs-Henseleit solution containing hemicholinium - 3 (10nM) and physostigmine (10uM) for 120 min. Fractions of the superfusate were collected every 4 min. Two pulses of 4 min duration, of 40 mM K obtained by iso-osmotic replacement of NaCl by KCl, were given at t = 6B(S1) and t = 92(S2) min after the start of the superfusion. Drugs were added to the superfusing medium 109
immediately after Sl. The efflux of tritium was calculated as fractional release Tnd the amount of tritium released above basal in response to the The S2/sl ratio raised K concentration was expressed as the ratio S2/Sl. for drug-treated sites was expressed as a percentage of a paired control Four concentrations of agonist compounds were used in S2/Sl ratio. The pAl0 determinations were performed by duplicate in each experiment. the method of Schild (5). ('HI-choline chloride, specific acitivty 15 Ci mmol-1 was obtained from Amersham International. Statistical
analysis was performed
usingthe
Mann-Whitney
U test, 2 tailed.
Results The spontaneous release of tritium from slices of rat occipitoparietal cortex and rat striatum became steady after160 min of superfusion at a The addition rate of 0.57 + 0.04 % and 0.43 k 0.02% min respectively. of 40 mM K+ produced an increase in release of 2.8 +0.7% and 8.0 & 1.1%. The control s2,Sl ratios were 0.84 t 0.05, n=27 (occipitoparietal cortex) and 0.81 + 0.04, n=30 (striatum). M?rphine (0.01 to 10uM) produced a concentration-related inhibition of K -evoked tritium release in both brain areas. The concentration producing 30% inhibition (IC50) was 159 (49-275)nM for the cortex and 126 (6-630)nM Meptazinol (0.01 to 100uM) had no significant effect on in the striatum. K+-evoked tritium release from slices of occipitoparietal cortex but produced a concentration-related inhibi?ion of release in the striatum (IC30 = 501 (126-1,60O)nM), (Fig.1) .
1
1
Figure 1. Concentration-effect curves for morphine (circles) and meptazinol (squares) for the inhibition of ACh release in the occipitoparietal cortex (closed symbols) and striatum (open symbols). n 2 6.
80
4
USO, 488 (< 10uM). naloxone (< 0.1 PM), ICI 154129 (~10 uM) and ICI 174864 (11.1M) had no significant effect when tested alone on tritium release in either the cortex or striatum. None of the compounds used affected the spontaneous release of tritium.
Naloxone produced
a concentration-related
110
antagonism
of the response to
,morphinein both brain regionswith pAl0 values of 7.9 + 0.19 (cortex) and 8.1 -+0.26 (striatum). The inhibition of tritium release produced by meptazinol in the striatum was also antagonised bynaloxone with a pAlO value of 8.2 f 0.27. The response to morphine in the cortex was significantly reduced in the presence of meptazinol in concentrations above 1pM. ICI 154129, up to lOuM, had no effect on the response to morphine in the cortex but antagonised the response to morphine in the striatum (pAlO = 6.2 _+0.33). ICI 174864 (1uM) had no effect on the response to morphine in the striatum. -1 Pretreatmentwithnaloxazone (30 mg kg , S.C. )24h prior to sacrifice, had no effect per se on tritium release but abolished the responses to both morphine and meptazinol in the striatum. The response to morphine in the cortex was not significantly changed, IC = 224 (50-603) cf 159 (49-275). 30 Discussion In recent years the concept of multiple opioid receptors has received much attention and the identification of multiple classes of binding sites has led to considerable effort being expended to correlate these sites with particular physiological effects in vitro and in vivo. As yet the results of such investigations remain equivocal. The data from the present study are consistent with the suggestion that Ach release in vitro may be influenced by different opioid receptors in different brain regions. The potency of naloxone (pAlo z 8.0) suggests that it may act a p receptor in both regions (6)_ However, the finding that only one of these sites was sensitive to blockade by naloxazone is consistent with a subdivision of these receptors. Naloxazone, a selective ul antagonist (7) has been shown to antagonise morphine-induced analgesia in the same dose as that used in the present study (Hartley, unpublished). Kappa opioid receptors do not appear to be involved in the modulation of Ach release in vitro since the K agonist US0488 was ineffective. It has been reported (8) that 6 receptors may be involved in the modulation of striatal Ach release in vitro. This view was not supported by the findings of the present study in which the antagonist ICI 154129 reduced the effect of morphine whereas another more selective 6 antagonist ICI 174864 was without effect. The discrepancies between the results of the previous (8) and the present studies may be due to methodological differences and to the difference in agonists used ie DADLE and morphine. The relative naloxazone sensitivity of cortical and striatal responses to morphine would favour a subdivision of 1-1 sites (cortex p - striatum, ~1). This subdivision is further supported by comparing the pres&t data with that from radioligand binding studies (Table 1).
111
Table 1:
IC values (nM) for the inhibition of Ach release compared with K.3!nd Na+ ratios for the displacement of I'HI naloxone binding O!ZnM (~1) and 2.OnM (u2).
u1 Agonist
IC30
striatum
Ki(nM)*
Morphine 126(6.3-630) 2.6kO.50 Meptazinol 500(126-1000) 57.5klO.7 *Data from
p2 Na+ ratio
IC30cortex
27.6 9.6
159(49-275) 7.08k1.41 69.8 ~10000 363+34 1.4
Ki(nM)*
Na+ ratio
Wood, M.D., unpublished
Thus, morphine with agonist (high Na+ratio) activity at both u and u 2 sites depressed Ach release in both brain regions. Meptazinollwith agonist activity at 1-1 (Na+ratio 9.6) inhibited Ach release in the striatum whereas in tie cortex (p,, Na+ratio 2.1.0) it had no effect alone but antagonised the response to morphine. Overall, therefore the data supports the concept of a subdivision of u sites, both of which are involved in the modulation of Ach release in vitro. 1.
Nishimura, S.L., Recht, L.D and Pasternak, G.W. (1984) Biochemical characterization of high-affinity 'H-opioid binding. Mol.Pharmac. 25: 29-37
2.
Sayre, L.M., Portoghese, P.S. and Takemori, A.E. (1983) Difference between mu-receptors in the guinea-pig ileum and the mouse vas deferens. Eur. J. Pharmacol. 90: 159-160.
3.
Wood, P-L., Richard, J.W. and Thakur, M. (1982). Mu opiate isoreceptors: differentiation with kappa agonists. Life Sci. 31:2313-2317
4.
Wood, P.L. and Stotland, L.M. (1980) Actions of enkephalin, mu and partial agonist analgesics on acetylcholine turnover in rat brain.
5.
Schild, H.O. (1947). A new scale for measurement of drug antagonism Br. J. Pharmac. 2: 189-206.
6.
Lord, J.A.H., Waterfield, A.A. Hughes, J and Kosterlitz, H.W. (1977). Endogenous opioid peptides: multiple agonists and receptors. Nature 267: 495-499.
7.
Childers, S.R. and Pasternak, G.W. (1982). Naloxazone, a novel opiate antagonist: irreversible blockade of rat brain opiate receptors in vitro Bell and Mol. Neurobiol. 2: 93-103
8.
Mulder, A.H., Wardeh, G. Hogenboom, F. and Frankhuyzen A.L. (1984) K and 6 -opioid receptor agonists differentially inhibit striatal dopamine and acetylcholine release. Nature 308: 278-280.
112