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Morphine and methadone have different effects on calcium channel currents in neuroblastoma cells
Brain Research 870 (2000) 199–203 www.elsevier.com / locate / bres
Short communication
Morphine and methadone have different effects on calcium channel currents in neuroblastoma cells a, b a b J.C. Yang M.D. *, J. Shan Ph.D. , K.F. Ng MBChB , P. Pang Ph.D. a
Department of Anaesthesiology, The University of Hong Kong, Room 424, Block K, Queen Mary Hospital, Pokfulam Road, Hong Kong, Hong Kong b C.V. Technologies, Edmonton, Canada Accepted 4 April 2000
Abstract Using patch-clamp technique, cellular calcium channel currents were studied on nine mouse neuroblastoma N1E115 cells. Both morphine and methadone decreased the T-type calcium currents in dose-dependent fashion. These effects were partially blocked by naloxane. On L-type calcium currents, morphine showed no effect. However, methadone inhibited the L-calcium currents in dosedependent fashion. This effect was not antagonised by naloxone. Hence, the action of methadone on L-calcium channel is probably not associated with m receptors. 2000 Elsevier Science B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Calcium channel physiology, pharmacology, and modulation Keywords: Morphine; Methadone; Calcium channel current; Neuroblastoma cell
It is well known that clinically one strong opioid could provide adequate analgesia when another one failed to do so. Animal study has shown that d-methadone attenuates the development of tolerance and prevent the increase of tail-flick ED50 by morphine [2]. These facts indicate that, in spite of their similar action on receptors, opioids may have different effects on other mechanisms. Morphine has been shown to change intracellular calcium [5,8]. The involvement of T-type [1] and L-type [3] calcium channels with opioids have also been reported. Increase intracellular Ca 21 will lead to depolarization of neurons [6]. This suggests the link between calcium channel currents and analgesic effect. The present study is to investigate the effects of morphine and methadone which are m, d and k agonists, and their combine effect with naloxone, a pure m antagonist, on the cloned neuroblastoma cells (strain N1E115). Nine mouse neuroblastoma cells (N1E115) were cultured in Dubecco’s modified Eagle’s medium (GIBCO) containing 10% fetal bovine serum at 378C in a humidified atmosphere of 5% CO 2 in air. The medium was changed *Corresponding author. Tel.: 1852-2855-33-03; fax: 1852-2855-1654.
every 3–4 days. After mechanical agitation, 3310 4 cells were replanted in 35-mm tissue culture dishes containing 4 ml of bath solution. After cell attachment, the dish was mounted on the stage of an inverted phase-contrast microscope (Nikon) for Ca 21 channel current recording. These cells expressed predominately T channel currents. In experiments where L channels were specifically sought, the cells were grown and maintained at confluence for 3–4 weeks under the same culture conditions with the addition of 2% (v / v) dimethylsulfoxide [7]. Three to 5 days before use, the cells were replanted with the same medium. These cells expressed predominately L channel currents. A small number of these cells also expressed T channel currents. Hence, cells were selected so that at a holding potential of 240 mV, the T channel component, was very small and the inward current measured was conducted predominantly by L channels. The whole-cell variation of the patch-clamp technique was used. The pipettes had resistance of 2–15 MV. Membrane current recordings were made with an Axopatch-1C (Axon Instruments) patch-clamp amplifier. All signals were filtered at 1 kHz and stored in the computer. Since the peak currents measured with 20 mM Ba 21 as the charge carrier were usually small (about 200
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02369-6
200
J.C. Yang et al. / Brain Research 870 (2000) 199 – 203
Fig. 1. The effects of morphine on T-calcium currents in N1E115 cells (n59). Data are expressed as percentage of the control which is counted as 100%. One and 10 mM morphine significantly inhibited the currents and different from each other.
pA) the series resistance compensation was not usually employed. If the capacitive transient overlapped with the onset of the inward current, or if the spatial voltage control was inadequate (i.e., N1E-115 cells with long neural outgrowths), the experimental data were rejected. Unless otherwise specified the current–voltage plots were constructed by using the peak values (corrected for leakage) from the original records for both T and L channel currents. The holding membrane potential was fixed at 280 mV when the L channels were studied. Ba 21 currents through Ca 21 channels were elicited by 200 ms depolariza-
tion at intervals of 5 s. For every single-cell recording, stable readings were first obtained for 5 min. The drugs, naloxone (Merck) morphine sulphate (Faulding), methadone (Glaxo Wellcome) and their mixtures (e.g., morphine1naxolone) were then added to the bath solution. Experiments were performed at room temperature (21– 228C) to prolong cell survival and channel recording time. The bath solution contained 110 mM Tris, 5 mM CsCl, 20 mM Hepes, 30 mM glucose, 20 mM BaCl 2 , and 0.5 mM tetrodotoxin. The pipette (internal) solution contained 70 mM Cs 2 -
Fig. 2. The effects of methadone on T-calcium currents in N1E115 cells (n59). Data are expressed as percentage of control.
J.C. Yang et al. / Brain Research 870 (2000) 199 – 203
201
Fig. 3. The antagonism of naloxone on morphine in T-calcium currents in N1E115 cells (n59). Naloxane with morphine showed significant less inhibition than morphine alone.
aspartame, 10 mM EGTA 10, 2 mM ATP-Na 2 , 5 mM K-pyruvate, 5 mM K-succinate, 5 mM phosphocreatineNa 2 , 15 units / ml creatine kinase, 20 mM Hepes and 5 mM glucose. The osmolality of all solutions was adjusted to 310–320 mOsm and pH to 7.4 using HCl or CsOH as required. T-type calcium channels: morphine inhibited calcium currents. These effects were significant at 1 and 10 mM. The inhibition was dosage dependent because 10 mM morphine showed significantly more inhibition than 1 mM (Fig. 1). The inhibition with methadone was significant at a lower dose (0.1 mM) (Fig. 2). Naloxone 10 mM showed
no effect on its own. However, it antagonised both the effect of morphine and methadone in calcium current (Figs. 3 and 4). L-type calcium channels: up to 10 mM, morphine showed no effect by itself, nor any effect when it was given together with naloxone (10 mM). However, methadone inhibited the calcium currents in a dose-dependent fashion (Fig. 5). The inhibition was not antagonised by 10 mM naloxone (Fig. 6). The effect of opioids on calcium channels have been controversial. Both inhibition [8] and activation [5] have been reported. In addition, there is still no general agree-
Fig. 4. The antagonism of naloxane on methadone in T-calcium currents in N1E115 cells (n59). Naloxane with methadone is significantly different from methadone alone.
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J.C. Yang et al. / Brain Research 870 (2000) 199 – 203
Fig. 5. The effects of methadone on L-calcium currents in N1E115 cells (n59). 0.1, 1 and 10 mM are significantly different from control. Ten mM dose is different from 0.1 mM.
ment whether the effect of morphine on calcium channel is via m-, d- or k-type receptors [9]. Since m receptors are the most important one for analgesia, we used the pure m antagonist, naloxone, as a marker. The specific relationship between m receptors, morphine, methadone and calcium channels could then be studied. Our findings suggest that these two opioids were similar in their action on T-type calcium channel currents. Since they were both antagonised by naloxone, these effects are at least partially mediated by m receptors. However, these two opioids showed their differences on the L-channels. Morphine neither alone nor when combined with naloxone showed any effect. On the other hand, methadone decreased L-type
calcium currents in a dose-dependent manner. This effect was not antagonised by naloxone. The results suggest that the action of methadone on L-channel calcium currents is not associated with m receptor. The drugs used in the study were not the pure powder. But all the inactive gradients in these opioid preparations (sodium hydroxide, sodium chloride and hydrochloric acid), have not been shown to have any effect on calcium currents. The commercial preparation of methadone is a racemic mixture of d–l form. The affinity of l-methadone for m-opioid receptor is 10 times higher than that of d-methadone and twice of the racemic mixture [4]. Hence the analgesia by l-methadone is likely via m receptors. On
Fig. 6. The antagonism of naloxane on methadone in L-calcium currents in N1E115 cells (n59). Although the inhibitory effect of methadone is significant, there is no different between methadone and naloxone with methadone.
J.C. Yang et al. / Brain Research 870 (2000) 199 – 203
the other hand, the anti-nociceptive action by d-methadone has been shown not affected by naloxone and appears to be the result of NMDA activity [10]. NMDA receptor activation increases cytoplasmic calcium and enhances action potential in spinal cord neuron [6]. Thus our data not only underscore the hypothesis that opioids induce hyperpolarization after combination with receptors, but also provide additional evidence concerning the effect of d-methadone on NMDA [2]. Our observation that naloxone was inactive on the effect of methadone on L-channel suggests further the possible linkage between L-channel and NMDA. The different effects of morphine and methadone on intracellular L-type calcium channels offer a possible explanation for their variable efficacy in producing analgesia in human.
Acknowledgements The work is supported in part by a grant from Hong Kong Research Council (HKU240 / 95M).
References [1] F.A. Abulla, P.A. Smith, Nociceptin inhibits T-type Ca 21 channel current in rat sensory neuron by a G-protein-independent mechanism, J. Neurosci. 17 (1997) 8721–8728.
203
[2] A.M. Davis, C.E. Inturrissi, d-methadone blocks morphine tolerance and N-methyl-D-aspartate-induced hyperalgesia, J. Pharmacol. Exp. Ther. 289 (1999) 1048–1053. [3] A.E. Hasegawa, J.P. Zancy, The influence of three L-type calcium channel blockers on morphine effects in healthy volunteers, Anesth. Analg. 85 (1997) 633–638. [4] N.A. Inqoglia, V.P. Dole, Localization of d-and l-methadone after intraventricular injection into rat brains, J. Pharmacol. Exp. Ther. 175 (1970) 84–87. [5] M. Lorentz, B. Hendlund, P. Arhem, Morphine activates calcium channels in cloned mouse neuroblastoma cell lines, Brain Res. 445 (1988) 157–159. [6] A.B. MacDermott, A.B. Mayer, M.L. Mayer, G.I. Westbrook, S.J. Smith, J.L. Barker, NMDA-receptor activation increase cytoplasmic calcium concentrations in cultured spinal cord neurons, Nature 321 (1986) 519–522. [7] T. Narahash, A. Tsunoo, M. Yoshii, Characterisation of two types of calcium channels in mouse neuroblastoma cells, J. Physiol. 383 (1987) 231–249. [8] A. Neil, Affinities of some opioid analgesics towards four binding sites in mouse brain, Naunyn-schmiedeberg’s, Arch. Pharmacol. 328 (1984) 24–29. [9] R.A. North, Opioid receptors types and membrane ion channels, Trends Neurosci. 11 (1986) 114–117. [10] N. Shimoyama, M. Shimoyama, K.J. Eilloitt, C.E. Inturrissi, dMethadone is antinociceptive in rat Formalin tests, J. Pharmacol. Exp. Ther. 283 (1997) 648–652.
Short communication
Morphine and methadone have different effects on calcium channel currents in neuroblastoma cells a, b a b J.C. Yang M.D. *, J. Shan Ph.D. , K.F. Ng MBChB , P. Pang Ph.D. a
Department of Anaesthesiology, The University of Hong Kong, Room 424, Block K, Queen Mary Hospital, Pokfulam Road, Hong Kong, Hong Kong b C.V. Technologies, Edmonton, Canada Accepted 4 April 2000
Abstract Using patch-clamp technique, cellular calcium channel currents were studied on nine mouse neuroblastoma N1E115 cells. Both morphine and methadone decreased the T-type calcium currents in dose-dependent fashion. These effects were partially blocked by naloxane. On L-type calcium currents, morphine showed no effect. However, methadone inhibited the L-calcium currents in dosedependent fashion. This effect was not antagonised by naloxone. Hence, the action of methadone on L-calcium channel is probably not associated with m receptors. 2000 Elsevier Science B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Calcium channel physiology, pharmacology, and modulation Keywords: Morphine; Methadone; Calcium channel current; Neuroblastoma cell
It is well known that clinically one strong opioid could provide adequate analgesia when another one failed to do so. Animal study has shown that d-methadone attenuates the development of tolerance and prevent the increase of tail-flick ED50 by morphine [2]. These facts indicate that, in spite of their similar action on receptors, opioids may have different effects on other mechanisms. Morphine has been shown to change intracellular calcium [5,8]. The involvement of T-type [1] and L-type [3] calcium channels with opioids have also been reported. Increase intracellular Ca 21 will lead to depolarization of neurons [6]. This suggests the link between calcium channel currents and analgesic effect. The present study is to investigate the effects of morphine and methadone which are m, d and k agonists, and their combine effect with naloxone, a pure m antagonist, on the cloned neuroblastoma cells (strain N1E115). Nine mouse neuroblastoma cells (N1E115) were cultured in Dubecco’s modified Eagle’s medium (GIBCO) containing 10% fetal bovine serum at 378C in a humidified atmosphere of 5% CO 2 in air. The medium was changed *Corresponding author. Tel.: 1852-2855-33-03; fax: 1852-2855-1654.
every 3–4 days. After mechanical agitation, 3310 4 cells were replanted in 35-mm tissue culture dishes containing 4 ml of bath solution. After cell attachment, the dish was mounted on the stage of an inverted phase-contrast microscope (Nikon) for Ca 21 channel current recording. These cells expressed predominately T channel currents. In experiments where L channels were specifically sought, the cells were grown and maintained at confluence for 3–4 weeks under the same culture conditions with the addition of 2% (v / v) dimethylsulfoxide [7]. Three to 5 days before use, the cells were replanted with the same medium. These cells expressed predominately L channel currents. A small number of these cells also expressed T channel currents. Hence, cells were selected so that at a holding potential of 240 mV, the T channel component, was very small and the inward current measured was conducted predominantly by L channels. The whole-cell variation of the patch-clamp technique was used. The pipettes had resistance of 2–15 MV. Membrane current recordings were made with an Axopatch-1C (Axon Instruments) patch-clamp amplifier. All signals were filtered at 1 kHz and stored in the computer. Since the peak currents measured with 20 mM Ba 21 as the charge carrier were usually small (about 200
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02369-6
200
J.C. Yang et al. / Brain Research 870 (2000) 199 – 203
Fig. 1. The effects of morphine on T-calcium currents in N1E115 cells (n59). Data are expressed as percentage of the control which is counted as 100%. One and 10 mM morphine significantly inhibited the currents and different from each other.
pA) the series resistance compensation was not usually employed. If the capacitive transient overlapped with the onset of the inward current, or if the spatial voltage control was inadequate (i.e., N1E-115 cells with long neural outgrowths), the experimental data were rejected. Unless otherwise specified the current–voltage plots were constructed by using the peak values (corrected for leakage) from the original records for both T and L channel currents. The holding membrane potential was fixed at 280 mV when the L channels were studied. Ba 21 currents through Ca 21 channels were elicited by 200 ms depolariza-
tion at intervals of 5 s. For every single-cell recording, stable readings were first obtained for 5 min. The drugs, naloxone (Merck) morphine sulphate (Faulding), methadone (Glaxo Wellcome) and their mixtures (e.g., morphine1naxolone) were then added to the bath solution. Experiments were performed at room temperature (21– 228C) to prolong cell survival and channel recording time. The bath solution contained 110 mM Tris, 5 mM CsCl, 20 mM Hepes, 30 mM glucose, 20 mM BaCl 2 , and 0.5 mM tetrodotoxin. The pipette (internal) solution contained 70 mM Cs 2 -
Fig. 2. The effects of methadone on T-calcium currents in N1E115 cells (n59). Data are expressed as percentage of control.
J.C. Yang et al. / Brain Research 870 (2000) 199 – 203
201
Fig. 3. The antagonism of naloxone on morphine in T-calcium currents in N1E115 cells (n59). Naloxane with morphine showed significant less inhibition than morphine alone.
aspartame, 10 mM EGTA 10, 2 mM ATP-Na 2 , 5 mM K-pyruvate, 5 mM K-succinate, 5 mM phosphocreatineNa 2 , 15 units / ml creatine kinase, 20 mM Hepes and 5 mM glucose. The osmolality of all solutions was adjusted to 310–320 mOsm and pH to 7.4 using HCl or CsOH as required. T-type calcium channels: morphine inhibited calcium currents. These effects were significant at 1 and 10 mM. The inhibition was dosage dependent because 10 mM morphine showed significantly more inhibition than 1 mM (Fig. 1). The inhibition with methadone was significant at a lower dose (0.1 mM) (Fig. 2). Naloxone 10 mM showed
no effect on its own. However, it antagonised both the effect of morphine and methadone in calcium current (Figs. 3 and 4). L-type calcium channels: up to 10 mM, morphine showed no effect by itself, nor any effect when it was given together with naloxone (10 mM). However, methadone inhibited the calcium currents in a dose-dependent fashion (Fig. 5). The inhibition was not antagonised by 10 mM naloxone (Fig. 6). The effect of opioids on calcium channels have been controversial. Both inhibition [8] and activation [5] have been reported. In addition, there is still no general agree-
Fig. 4. The antagonism of naloxane on methadone in T-calcium currents in N1E115 cells (n59). Naloxane with methadone is significantly different from methadone alone.
202
J.C. Yang et al. / Brain Research 870 (2000) 199 – 203
Fig. 5. The effects of methadone on L-calcium currents in N1E115 cells (n59). 0.1, 1 and 10 mM are significantly different from control. Ten mM dose is different from 0.1 mM.
ment whether the effect of morphine on calcium channel is via m-, d- or k-type receptors [9]. Since m receptors are the most important one for analgesia, we used the pure m antagonist, naloxone, as a marker. The specific relationship between m receptors, morphine, methadone and calcium channels could then be studied. Our findings suggest that these two opioids were similar in their action on T-type calcium channel currents. Since they were both antagonised by naloxone, these effects are at least partially mediated by m receptors. However, these two opioids showed their differences on the L-channels. Morphine neither alone nor when combined with naloxone showed any effect. On the other hand, methadone decreased L-type
calcium currents in a dose-dependent manner. This effect was not antagonised by naloxone. The results suggest that the action of methadone on L-channel calcium currents is not associated with m receptor. The drugs used in the study were not the pure powder. But all the inactive gradients in these opioid preparations (sodium hydroxide, sodium chloride and hydrochloric acid), have not been shown to have any effect on calcium currents. The commercial preparation of methadone is a racemic mixture of d–l form. The affinity of l-methadone for m-opioid receptor is 10 times higher than that of d-methadone and twice of the racemic mixture [4]. Hence the analgesia by l-methadone is likely via m receptors. On
Fig. 6. The antagonism of naloxane on methadone in L-calcium currents in N1E115 cells (n59). Although the inhibitory effect of methadone is significant, there is no different between methadone and naloxone with methadone.
J.C. Yang et al. / Brain Research 870 (2000) 199 – 203
the other hand, the anti-nociceptive action by d-methadone has been shown not affected by naloxone and appears to be the result of NMDA activity [10]. NMDA receptor activation increases cytoplasmic calcium and enhances action potential in spinal cord neuron [6]. Thus our data not only underscore the hypothesis that opioids induce hyperpolarization after combination with receptors, but also provide additional evidence concerning the effect of d-methadone on NMDA [2]. Our observation that naloxone was inactive on the effect of methadone on L-channel suggests further the possible linkage between L-channel and NMDA. The different effects of morphine and methadone on intracellular L-type calcium channels offer a possible explanation for their variable efficacy in producing analgesia in human.
Acknowledgements The work is supported in part by a grant from Hong Kong Research Council (HKU240 / 95M).
References [1] F.A. Abulla, P.A. Smith, Nociceptin inhibits T-type Ca 21 channel current in rat sensory neuron by a G-protein-independent mechanism, J. Neurosci. 17 (1997) 8721–8728.
203
[2] A.M. Davis, C.E. Inturrissi, d-methadone blocks morphine tolerance and N-methyl-D-aspartate-induced hyperalgesia, J. Pharmacol. Exp. Ther. 289 (1999) 1048–1053. [3] A.E. Hasegawa, J.P. Zancy, The influence of three L-type calcium channel blockers on morphine effects in healthy volunteers, Anesth. Analg. 85 (1997) 633–638. [4] N.A. Inqoglia, V.P. Dole, Localization of d-and l-methadone after intraventricular injection into rat brains, J. Pharmacol. Exp. Ther. 175 (1970) 84–87. [5] M. Lorentz, B. Hendlund, P. Arhem, Morphine activates calcium channels in cloned mouse neuroblastoma cell lines, Brain Res. 445 (1988) 157–159. [6] A.B. MacDermott, A.B. Mayer, M.L. Mayer, G.I. Westbrook, S.J. Smith, J.L. Barker, NMDA-receptor activation increase cytoplasmic calcium concentrations in cultured spinal cord neurons, Nature 321 (1986) 519–522. [7] T. Narahash, A. Tsunoo, M. Yoshii, Characterisation of two types of calcium channels in mouse neuroblastoma cells, J. Physiol. 383 (1987) 231–249. [8] A. Neil, Affinities of some opioid analgesics towards four binding sites in mouse brain, Naunyn-schmiedeberg’s, Arch. Pharmacol. 328 (1984) 24–29. [9] R.A. North, Opioid receptors types and membrane ion channels, Trends Neurosci. 11 (1986) 114–117. [10] N. Shimoyama, M. Shimoyama, K.J. Eilloitt, C.E. Inturrissi, dMethadone is antinociceptive in rat Formalin tests, J. Pharmacol. Exp. Ther. 283 (1997) 648–652.