- Email: [email protected]
μ- and κ-opioids inhibit K+ evoked glutamate release from rat cerebrocortical slices
NIUROSCIINCI ELSEVIER
Neuroscience Letters 218 (1996) 79-82
l[ll[lS
and K-Opioids inhibit K + evoked glutamate release from rat cerebrocortical slices B. N i c o l , D . J . R o w b o t h a m ,
D.G. Lambert*
University Department of Anaesthesia, Leicester Royal Infirmary, Leicester, LEI SWW, UK
Received 2 August 1996; revised version received 20 September 1996; accepted 20 September 1996
Abstract
We have examined the effects of a range of opioid receptor subtype selective agonists on K ÷ evoked glutamate release from perfused rat cerebrocortical slices. Dual application (S~ and $2) of K ÷ (46 raM) evoked dual monophasic glutamate release profiles. When areas under the release curves were calculated an S2/S1 ratio for control slices of 1.07 + 0.08 (n = 75) was obtained, this was reduced by 80% with EGTA (0.1 mM) treatment confirming the presence of a Ca 2÷ regulated release process. Morphine produced a dose-dependent inhibition.of the S2/St ratio. At 1 tzM this amounted to 78 + 12% (mean + SEM; n = 6). (D-Ala2,MePhe4,gly(ol)5)enkephalin (DAMGO; 60 + 12%, n = 6 at 1 /~M), and spiradoline (53 + 14% at 1 and 71 + 11% at 100 #M, both n = 6) also inhibited glutamate release in a cyprodime (10/zM) and norbinaltorphimine (10/~M) reversible manner. (D-PenZ'S)enkephalin (DPDPE; 1 /zM) was ineffective. All agents tested did not affect basal glutamate release. Collectively these data implicate a role for ~ and r opioids in the control of evoked glutamate release and their potential for neuroprotective therapy. Keywords: Rat cerebrocortical slices; Opioids; Glutamate release
Opioids represent all important class of therapeutic agents used in the pre-, intra- and postoperative periods for analgesia [18], where their presumed mechanism of action is via a reduction in neurotransmission [12]. Opioids inhibit neurotransmitter release primarily by closing voltage sensitive Ce~2÷ channels and by hyperpolarising the cell membrane,, via an increased outward K ÷ conductance [17]. Opiates also inhibit adenylyl cyclase thereby reducing the formation of cAMP, however a role for cAMP in the acute control of neurotransmitter release remains to be established [4]. The effects of opiates on the release of glutamate is controversial. In rat synaptosomes morphine did not affect K+-evoked [3H]glutamate release [2]. However, Ueda et al. [20] reported that both (D-Ala2, MePhe4,gly(ol)5)enkephalin (DAMGO; 0 . 3 - 1 0 /~M) and morphine (1-30/~M) produced a concentration dependent reduction in capsaicin-evoked glutamate release from rat spinal dorsal horn slices. In this study the r-opioid agonists U-50,488H and U-69,593 were ineffective. At variance * Correspondingauthor. Tel.: +44 116 2585291; fax: +44 116 2854487; e-mail: [email protected]
with this general ineffectiveness of r-agonists, the r-agonist enadoline has been shown to inhibit glutamate release from ischemic brain [5] and to decrease 4-AP stimulated release of glutamate from rodent and primate striatum [6] and endogenous dynorphins block the induction of hippocampal long-term potentiation [21]. In this study we have examined the effects of a range of opioid receptor sub-type selective agonists on K + evoked glutamate release from rat cerebrocortical slices. Preliminary accounts of this work have been presented [14,15]. Female Wistar rats (200-250 g) were killed by cervical dislocation and decapitation. The brain was rapidly removed and placed in ice-cold oxygenated (95 02, 5% CO2) Krebs buffer, (pH 7.4) of the following composition (mM): NaC1 115, KCI 4.7, CaCI2 2.0, MgCI2 1.2, NaHCO3 25 mM and glucose 8.8. The outer cortex was removed from its internal structures, 350 × 350/zm slices were cut and suspended in Krebs buffer. The slices were washed three times in fresh Krebs buffer then agitated in a shaking water bath set at 37°C for 40 rain. Gravity (1 ml) packed slices were pipetted into a perfusion chamber constructed from a 2 ml syringe barrel, as described previously [1].
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PI1 S0304-3940(96) 13104-9
80
B. Nicol et al. / Neuroscience Letters 218 (1996) 79-82
Slices were perfused at 37°C at lml/min for 60 min prior to collection of 2 rain fractions for the estimation of glutamate concentrations. Following 6 min of perfusion, 46 mM K + (Na + adjusted) was applied for 2 min ($1). Slices were perfused for a further 30 min prior to the second application of a 2 min pulse of 46 mM K + ($2). Fractions were collected for 8 min after $2. EGTA (0.1 mM in Ca 2+ free Krebs buffer), morphine (0.01-10 txM), D A M G O (1 /~M,/~-selective agonist), (D-Pen2'5)enkephalin (DPDPE; 1 ~tM, 6-selective agonist) and spiradoline (1 and 100 ~tM, Kselective agonist) were applied immediately after S~ until the end of the experiment (also present during $2). In some experiments cyprodime (/z-specific antagonist) and norbinaltorphimine (d-selective antagonist) were used. Perfusate glutamate concentrations were determined fluorimetrically using a modification of the method of Nicholls et al.[16]. Perfusate (480 tzl) was incubated with nicotinamide adenine dinucloeotide phosphate (NADP; 100 mM) and glutamate dehydrogenase (30 U). The resulting production of N A D P H was detected fluorimetrically at excitation and emission wavelengths of 366 and 430 nm, respectively in a Perkin-Elmer LS50B spectroflurimeter, and compared with a known set of glutamate standards. $2/S1 ratios were calculated from the area under the curves for control and drug treated samples. All data are presented as mean + SEM (n). Statistical comparisons of paired samples were made using Wilcoxon rank sum test and considered significant when P < 0.05. Depolarisation of rat cerebrocortical slices with 46 mM K ÷ produced a monophasic release of glutamate for both $1 and $2 (Fig. 1). There was considerable variation in the levels of stimulation between rats. The mean $2/S~ ratio from 75 control experiments was 1.07 + 0.08. Glutamate release was Ca 2+ dependent in that EGTA (0 1 raM) treat-
ment reduced the SJS1 ratio from 1.35 + 0 . 4 8 to 0.25 + 0.12 (80%, n = 5) (Fig. 1). Morphine produced a dose dependent inhibition of glutamate release (Fig. 2). At 1 tzM, D A M G O produced a 60% inhibition of glutamate release that was reversed by cyprodime (10 tzM) (Fig. 2). Spiradoline at 1 and 100 /xM also inhibited K ÷ evoked glutamate release by 52 and 71%, respectively (Fig. 2), with the response to 100 /~M spiradoline being reversed by nor-binaltorphimine (10/zM). DPDPE at 1/zM did not inhibit glutamate release (Fig. 2). D A M G O (1 /~M), DPDPE (1 /~M) and spiradoline (1 and 30 /~M) did not affect basal glutamate release (Fig. 3 and data not shown). In this preparation we have demonstrated Ca 2+ dependent glutamate release in that EGTA pre-treatment essentially abolished K ÷ evoked release. The measurement of endogenous glutamate has several advantages over measurements of [3H]glutamate overflow, the most important being the elimination of 3H-'pooling'. In addition perfused tissue enables the dynamics of release to be quantified./z, 6, K opioid receptors are found in frontal, piriform and entorhinal regions of the rat cerebral cortex in roughly equal proportions although in the frontal cortex the tz receptor density exceeds that of x with 6 being intermediate [10]. There have been a number of reports of the coupling of these subytpes to inhibition of various neurotransmitters (see [9]) including acetylcholine, where both stimulatory [13] and inhibitory [8] effects have been observed. In the present study we have shown that morphine, D A M G O (/x agonists), and spiradoline (K agonist) inhibit K + evoked glutamate release. These data confirm and extend the observations of Bradford et al. [3] who showed that/~ and ~ opioids inhibited veratrine-stimulated release of glutamate, the inhibition with morphine (10 #M) amounting to 116%. In our study D A M G O and 4-
Control
3
,, EGTA(0.traM)
2
ct..c~,t>..o
~"D"D/l
T
K+
K+
K÷ 16
26
3b
Time (rains)
46
50
0
'
20
30
40
50
Time(rains)
Fig. 1. K+ (46 mM) evokes glutamate release from perfused rat cerebrocortical slices. K + was applied twice (S1 and $2) for 2 min at the arrows. The effects of drugs were assessed by calculating the $2/S~ratios from the area under the release curves. In the right panel the effects of EGTA (0.1 mM) added between S~ and $2 and continuing in the presence of K+ stimulus during $2 is shown. EGTA reduced the magnitude of $2 and hence the SjS~ ratio. Data are expressed relative to the mean of the first three fractions collected and as mean + SEM from n = 75 control experiments in left panel and n = 5 in right panel.
81
B. Nicol et al. / Neuroscience Letters 218 (1996) 79-82 |00
"l
100.
+ 75.
75.
t~
¢~
I
~e
I
T
_=
=
50.
J
25.
2s
0
0
'SI
DI
LoglMorphinel(M)
Fig. 2. Morphine produced a dose-dependent inhibition of K + evoked glutamate release from rat cerebrocortical slices (left panel). In the right panel DAMGO (D, 1 #M) and spiradoline (S, 1 and 100 #M) inhibited glutamate release in a cyprodime (C, 10/zM) and nor-binaltorphimine (N, 10 #M) reversible fashion. DPDPE (DP, 1 /~M) did not significantly affect glutamate release. Data are mean + SEM from n = 5-13. P < 0.05 reduced compared to K + control.
spiradoline (used as # and K selective agonists) inhibition was likely due to/z and x receptor activation as the selective antagonists cyprodime and nor-binaltorphimine (at high concentrations) reversed the observed inhibition. It is difficult to explain why DPDPE failed to inhibit glutamate release since the cerebral cortex expresses significant numbers of 6 receptors. If these 6 receptors are not found on glutamatergic neurones then 6 agonists would be ineffective. However, it is possible that 6 receptor occupation could produce a small inhibition of release that is below the sensitivity of our assay system. Bradford reported in veratrine stimulated cerebrocortical slices #, 6 and r agonist inhibition of 150, 45 and 98% with morphine, DPDPE and tifluadom at 100/~tvl [3]. Clearly the lowest degree of inhibition was observed with DPDPE used at very high 4
3.
G
Control
= DAMGO (lttM)
z
concentrations. It could also be argued that the concentration of DPDPE used was submaximal and that a higher dose may prove more effective as seen by Bradford et al. [3]. However, in a limited number of experiments with 10 #M DPDPE we could not observe a statistically significant inhibition of release. Furthermore, in a series of experiments examining the effects of DPDPE on cAMP and Ins(1,4,5)P3 formation in CHO cells expressing recombinant 6 receptors 1 /~M DPDPE produced a maximum response [7,19]. The clinical significance of these data are quite clear in that they support a role for/z and r agonists as neuroprotectants. However, the far superior side-effect profile of agonists (i.e. no respiratory depression) [11], may enable their use where cerebral ischaemia is anticipated. Indeed, Hayward et al. [5], have shown that the Kagonist enadoline is effective in reducing cerebral damage due to transient ischemia episodes in the cerebral cortex of rats. Clearly further studies are required to determine the most appropriate clinical uses for r agonists in neuroprotection. This
work
was
funded
by
Trent
Regional
Health
Authority.
I. .~..~
~
t
0
0
K+
]o
DAMGO 20
30
4o
5'0
Time (rains)
Fig. 3. Effect of DAMGO (1 /zM) on basal glutamate release from rat cerebrocortical slices. DAMGO addition is noted by the arrow. Data are mean _+ SEM for n = 3 and are expressed relative to the mean of the first three fractions collected. Similar results were obtained with DPDPE (1 #M) and spiradoline (1 and 3{) #M), (data not shown).
[1] Atcheson, R., Lambert, D.G., Hirst, R.A. and Rowbotharn, D.J., Studies on the mechanism of [3H]-noradrenaline release from SH-SY5Y ceils: the role of Ca 2+ and cyclic AMP, Br. J. Pharmacol., 111 (1994) 787-792. [2] Bartlett, S.E. and Smith, M.T., Effects of morphine-3-glucuronide and morphine on K+-evoked release of [H-3] glutamic acid and [C14] gamma aminobutyric-acid from rat brain synaptosomes, Life Sci., 58 (1995) 447-454. [3l Bradford, H.F., Crowder, J.M. and White, E.J., Inhibitory actions of opioid compounds on calcium fluxes and neurotransmitter release
82
[4]
[5]
[6]
[7]
[8]
[9] [10]
[11] [12]
B. Nicol et al. / Neuroscience Letters 218 (1996) 79-82
from mammalian cerebral cortical slices, Br. J. Pharmacol., 88 (1986) 87-93. Childers, S.R., Opioid receptor-coupled second messenger systems. In A. Herz (Ed.), Opioids 1, Springer-Verlag, Berlin, 1993, pp. 189-216. Hayward, N.J., Hitchcott, P.K., Woodruff, G.N. and McKnight, A.T., Inhibition of release of glutamate from ischaemic brain by the x-agonist enadoline (C1-977), Analgesia, l (1995) 453-456. Hill, M.P. and Brotchie, J.M., Modulation of glutamate release by a ~-opioid receptor agonist in rodent and primate striatum, Eur. J. Pharmacol., 281 (1995) R1-R2. Hirst, R.A., Devi, L.A. and Lambert, D.G., Is the C-terminus of the recombinant 6-opioid receptor important for adenylyl cyclase coupling?, Br. J. Pharmacol., 188 (1996) 27P. Lapchak, P.A., Araujo, D.M. and Collier, B., Regulation of endogenous acetylcholine release from mammalian brain slices by opiate receptors: hippocampus, striatum and cerebral cortex of guineapig and rat, Neuroscience, 31 (1989) 313-325. Leslie, F.M., Methods used lbr the study of opioid receptors, Pharmacol. Rev., 39 (1987) 197-249. Mansour, A., Khachaturian, H., Lewis, M.E., Akil, H. and Watson, S.J., Anatomy of CNS opioid receptors, Trends Neurol. Sci., 11 (1988) 308-314. Millan, M.J., ~-Opioid receptors and analgesia, Trends Pharmacol. Sci., 11 (1990)70-76. Mulder, A.M. and Schoffelmeer, A.N.M., Multiple opioid receptors and presynaptic modulation of neurotransmitter release in the brain. In A. Herz (Ed.), Opioids 1, Springer-Verlag, Berlin, 1993, pp. 125-144.
[13] Neal, M.J., Paterson, S.J. and Cunningham, J.R., Enhancement of retinal acetylcholine release by DAMGO: possibly a direct opioid receptor-mediated excitatory effect, Br. J. Pharmacol., 113 (1994) 789-794. [14] Nicol, B., Rowbotham, D.J. and Lambert, D.G., Morphine inhibits glutamate release from rat cerebrocortical slices, Br. J. Pharmacol., 117 (1996) 293P. [15] Nicol, B., Rowbotham, D.J. and Lambert, D.G., Is glutamate release opioid receptor sub-type selective?, Br. J. Anaesth., 77 (1996) 282P. [16] Nicholls, D.G., Shirr, T.S. and Sanchez-Prieto, J., Calcium-dependent and independent release of glutamate from synaptosomes monitored by continuous fluorimetry, J. Neurochem., 49 (1987) 50-57. [17] North, R.A., Opioid actions on membrane ion channels. In A. Herz (Ed.), Opioids 1, Springer-Verlag, Berlin, 1993, pp. 773-797. [18] Pasternak, G.W., Pharmacological mechanism of opioid analgesics, Clin. Neuropharmacol., 16 (1993) 1-18. [19] Smart, D., Devi, L.A. and Lambert, D.G., Is the C-terminus of the recombinant 6-opioid receptor important for phospholipase C coupling?, Br. J. Pharmacol., 188 (1996) 28P. [20] Ueda, M., Sugimoto, K., Oyama, T., Kuraishi, Y. and Satoh, M., Opioidergic inhibition of capsaicin-evoked glutamate release from rat spinal dorsal horn slices, Neuropharmacology, 34 (1995) 303308. [21] Wagner, J.J., Terman, G.W. and Chavkin, C., Endogenous dynorphins inhibit excitatory neurotransmission and block LTP induction in the hippocampus, Nature, 363 (1993) 451-454.
Neuroscience Letters 218 (1996) 79-82
l[ll[lS
and K-Opioids inhibit K + evoked glutamate release from rat cerebrocortical slices B. N i c o l , D . J . R o w b o t h a m ,
D.G. Lambert*
University Department of Anaesthesia, Leicester Royal Infirmary, Leicester, LEI SWW, UK
Received 2 August 1996; revised version received 20 September 1996; accepted 20 September 1996
Abstract
We have examined the effects of a range of opioid receptor subtype selective agonists on K ÷ evoked glutamate release from perfused rat cerebrocortical slices. Dual application (S~ and $2) of K ÷ (46 raM) evoked dual monophasic glutamate release profiles. When areas under the release curves were calculated an S2/S1 ratio for control slices of 1.07 + 0.08 (n = 75) was obtained, this was reduced by 80% with EGTA (0.1 mM) treatment confirming the presence of a Ca 2÷ regulated release process. Morphine produced a dose-dependent inhibition.of the S2/St ratio. At 1 tzM this amounted to 78 + 12% (mean + SEM; n = 6). (D-Ala2,MePhe4,gly(ol)5)enkephalin (DAMGO; 60 + 12%, n = 6 at 1 /~M), and spiradoline (53 + 14% at 1 and 71 + 11% at 100 #M, both n = 6) also inhibited glutamate release in a cyprodime (10/zM) and norbinaltorphimine (10/~M) reversible manner. (D-PenZ'S)enkephalin (DPDPE; 1 /zM) was ineffective. All agents tested did not affect basal glutamate release. Collectively these data implicate a role for ~ and r opioids in the control of evoked glutamate release and their potential for neuroprotective therapy. Keywords: Rat cerebrocortical slices; Opioids; Glutamate release
Opioids represent all important class of therapeutic agents used in the pre-, intra- and postoperative periods for analgesia [18], where their presumed mechanism of action is via a reduction in neurotransmission [12]. Opioids inhibit neurotransmitter release primarily by closing voltage sensitive Ce~2÷ channels and by hyperpolarising the cell membrane,, via an increased outward K ÷ conductance [17]. Opiates also inhibit adenylyl cyclase thereby reducing the formation of cAMP, however a role for cAMP in the acute control of neurotransmitter release remains to be established [4]. The effects of opiates on the release of glutamate is controversial. In rat synaptosomes morphine did not affect K+-evoked [3H]glutamate release [2]. However, Ueda et al. [20] reported that both (D-Ala2, MePhe4,gly(ol)5)enkephalin (DAMGO; 0 . 3 - 1 0 /~M) and morphine (1-30/~M) produced a concentration dependent reduction in capsaicin-evoked glutamate release from rat spinal dorsal horn slices. In this study the r-opioid agonists U-50,488H and U-69,593 were ineffective. At variance * Correspondingauthor. Tel.: +44 116 2585291; fax: +44 116 2854487; e-mail: [email protected]
with this general ineffectiveness of r-agonists, the r-agonist enadoline has been shown to inhibit glutamate release from ischemic brain [5] and to decrease 4-AP stimulated release of glutamate from rodent and primate striatum [6] and endogenous dynorphins block the induction of hippocampal long-term potentiation [21]. In this study we have examined the effects of a range of opioid receptor sub-type selective agonists on K + evoked glutamate release from rat cerebrocortical slices. Preliminary accounts of this work have been presented [14,15]. Female Wistar rats (200-250 g) were killed by cervical dislocation and decapitation. The brain was rapidly removed and placed in ice-cold oxygenated (95 02, 5% CO2) Krebs buffer, (pH 7.4) of the following composition (mM): NaC1 115, KCI 4.7, CaCI2 2.0, MgCI2 1.2, NaHCO3 25 mM and glucose 8.8. The outer cortex was removed from its internal structures, 350 × 350/zm slices were cut and suspended in Krebs buffer. The slices were washed three times in fresh Krebs buffer then agitated in a shaking water bath set at 37°C for 40 rain. Gravity (1 ml) packed slices were pipetted into a perfusion chamber constructed from a 2 ml syringe barrel, as described previously [1].
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PI1 S0304-3940(96) 13104-9
80
B. Nicol et al. / Neuroscience Letters 218 (1996) 79-82
Slices were perfused at 37°C at lml/min for 60 min prior to collection of 2 rain fractions for the estimation of glutamate concentrations. Following 6 min of perfusion, 46 mM K + (Na + adjusted) was applied for 2 min ($1). Slices were perfused for a further 30 min prior to the second application of a 2 min pulse of 46 mM K + ($2). Fractions were collected for 8 min after $2. EGTA (0.1 mM in Ca 2+ free Krebs buffer), morphine (0.01-10 txM), D A M G O (1 /~M,/~-selective agonist), (D-Pen2'5)enkephalin (DPDPE; 1 ~tM, 6-selective agonist) and spiradoline (1 and 100 ~tM, Kselective agonist) were applied immediately after S~ until the end of the experiment (also present during $2). In some experiments cyprodime (/z-specific antagonist) and norbinaltorphimine (d-selective antagonist) were used. Perfusate glutamate concentrations were determined fluorimetrically using a modification of the method of Nicholls et al.[16]. Perfusate (480 tzl) was incubated with nicotinamide adenine dinucloeotide phosphate (NADP; 100 mM) and glutamate dehydrogenase (30 U). The resulting production of N A D P H was detected fluorimetrically at excitation and emission wavelengths of 366 and 430 nm, respectively in a Perkin-Elmer LS50B spectroflurimeter, and compared with a known set of glutamate standards. $2/S1 ratios were calculated from the area under the curves for control and drug treated samples. All data are presented as mean + SEM (n). Statistical comparisons of paired samples were made using Wilcoxon rank sum test and considered significant when P < 0.05. Depolarisation of rat cerebrocortical slices with 46 mM K ÷ produced a monophasic release of glutamate for both $1 and $2 (Fig. 1). There was considerable variation in the levels of stimulation between rats. The mean $2/S~ ratio from 75 control experiments was 1.07 + 0.08. Glutamate release was Ca 2+ dependent in that EGTA (0 1 raM) treat-
ment reduced the SJS1 ratio from 1.35 + 0 . 4 8 to 0.25 + 0.12 (80%, n = 5) (Fig. 1). Morphine produced a dose dependent inhibition of glutamate release (Fig. 2). At 1 tzM, D A M G O produced a 60% inhibition of glutamate release that was reversed by cyprodime (10 tzM) (Fig. 2). Spiradoline at 1 and 100 /xM also inhibited K ÷ evoked glutamate release by 52 and 71%, respectively (Fig. 2), with the response to 100 /~M spiradoline being reversed by nor-binaltorphimine (10/zM). DPDPE at 1/zM did not inhibit glutamate release (Fig. 2). D A M G O (1 /~M), DPDPE (1 /~M) and spiradoline (1 and 30 /~M) did not affect basal glutamate release (Fig. 3 and data not shown). In this preparation we have demonstrated Ca 2+ dependent glutamate release in that EGTA pre-treatment essentially abolished K ÷ evoked release. The measurement of endogenous glutamate has several advantages over measurements of [3H]glutamate overflow, the most important being the elimination of 3H-'pooling'. In addition perfused tissue enables the dynamics of release to be quantified./z, 6, K opioid receptors are found in frontal, piriform and entorhinal regions of the rat cerebral cortex in roughly equal proportions although in the frontal cortex the tz receptor density exceeds that of x with 6 being intermediate [10]. There have been a number of reports of the coupling of these subytpes to inhibition of various neurotransmitters (see [9]) including acetylcholine, where both stimulatory [13] and inhibitory [8] effects have been observed. In the present study we have shown that morphine, D A M G O (/x agonists), and spiradoline (K agonist) inhibit K + evoked glutamate release. These data confirm and extend the observations of Bradford et al. [3] who showed that/~ and ~ opioids inhibited veratrine-stimulated release of glutamate, the inhibition with morphine (10 #M) amounting to 116%. In our study D A M G O and 4-
Control
3
,, EGTA(0.traM)
2
ct..c~,t>..o
~"D"D/l
T
K+
K+
K÷ 16
26
3b
Time (rains)
46
50
0
'
20
30
40
50
Time(rains)
Fig. 1. K+ (46 mM) evokes glutamate release from perfused rat cerebrocortical slices. K + was applied twice (S1 and $2) for 2 min at the arrows. The effects of drugs were assessed by calculating the $2/S~ratios from the area under the release curves. In the right panel the effects of EGTA (0.1 mM) added between S~ and $2 and continuing in the presence of K+ stimulus during $2 is shown. EGTA reduced the magnitude of $2 and hence the SjS~ ratio. Data are expressed relative to the mean of the first three fractions collected and as mean + SEM from n = 75 control experiments in left panel and n = 5 in right panel.
81
B. Nicol et al. / Neuroscience Letters 218 (1996) 79-82 |00
"l
100.
+ 75.
75.
t~
¢~
I
~e
I
T
_=
=
50.
J
25.
2s
0
0
'SI
DI
LoglMorphinel(M)
Fig. 2. Morphine produced a dose-dependent inhibition of K + evoked glutamate release from rat cerebrocortical slices (left panel). In the right panel DAMGO (D, 1 #M) and spiradoline (S, 1 and 100 #M) inhibited glutamate release in a cyprodime (C, 10/zM) and nor-binaltorphimine (N, 10 #M) reversible fashion. DPDPE (DP, 1 /~M) did not significantly affect glutamate release. Data are mean + SEM from n = 5-13. P < 0.05 reduced compared to K + control.
spiradoline (used as # and K selective agonists) inhibition was likely due to/z and x receptor activation as the selective antagonists cyprodime and nor-binaltorphimine (at high concentrations) reversed the observed inhibition. It is difficult to explain why DPDPE failed to inhibit glutamate release since the cerebral cortex expresses significant numbers of 6 receptors. If these 6 receptors are not found on glutamatergic neurones then 6 agonists would be ineffective. However, it is possible that 6 receptor occupation could produce a small inhibition of release that is below the sensitivity of our assay system. Bradford reported in veratrine stimulated cerebrocortical slices #, 6 and r agonist inhibition of 150, 45 and 98% with morphine, DPDPE and tifluadom at 100/~tvl [3]. Clearly the lowest degree of inhibition was observed with DPDPE used at very high 4
3.
G
Control
= DAMGO (lttM)
z
concentrations. It could also be argued that the concentration of DPDPE used was submaximal and that a higher dose may prove more effective as seen by Bradford et al. [3]. However, in a limited number of experiments with 10 #M DPDPE we could not observe a statistically significant inhibition of release. Furthermore, in a series of experiments examining the effects of DPDPE on cAMP and Ins(1,4,5)P3 formation in CHO cells expressing recombinant 6 receptors 1 /~M DPDPE produced a maximum response [7,19]. The clinical significance of these data are quite clear in that they support a role for/z and r agonists as neuroprotectants. However, the far superior side-effect profile of agonists (i.e. no respiratory depression) [11], may enable their use where cerebral ischaemia is anticipated. Indeed, Hayward et al. [5], have shown that the Kagonist enadoline is effective in reducing cerebral damage due to transient ischemia episodes in the cerebral cortex of rats. Clearly further studies are required to determine the most appropriate clinical uses for r agonists in neuroprotection. This
work
was
funded
by
Trent
Regional
Health
Authority.
I. .~..~
~
t
0
0
K+
]o
DAMGO 20
30
4o
5'0
Time (rains)
Fig. 3. Effect of DAMGO (1 /zM) on basal glutamate release from rat cerebrocortical slices. DAMGO addition is noted by the arrow. Data are mean _+ SEM for n = 3 and are expressed relative to the mean of the first three fractions collected. Similar results were obtained with DPDPE (1 #M) and spiradoline (1 and 3{) #M), (data not shown).
[1] Atcheson, R., Lambert, D.G., Hirst, R.A. and Rowbotharn, D.J., Studies on the mechanism of [3H]-noradrenaline release from SH-SY5Y ceils: the role of Ca 2+ and cyclic AMP, Br. J. Pharmacol., 111 (1994) 787-792. [2] Bartlett, S.E. and Smith, M.T., Effects of morphine-3-glucuronide and morphine on K+-evoked release of [H-3] glutamic acid and [C14] gamma aminobutyric-acid from rat brain synaptosomes, Life Sci., 58 (1995) 447-454. [3l Bradford, H.F., Crowder, J.M. and White, E.J., Inhibitory actions of opioid compounds on calcium fluxes and neurotransmitter release
82
[4]
[5]
[6]
[7]
[8]
[9] [10]
[11] [12]
B. Nicol et al. / Neuroscience Letters 218 (1996) 79-82
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