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School of Pharmacy, The University of Queensland, St Lucia Campus, Brisbane, Australia
Address correspondence and reprint requests to Maree T. Smith, PhD, School of Pharmacy, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia. Address e-mail to m.smith{at}pharmacy.uq.edu.au
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Abstract |
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30 s or (b) a sustained increase in [Ca2+]CYT that slowly recovered to baseline. Naloxone-pretreatment decreased the proportion of M3G-responsive neurones by 10%–25%, implicating a predominantly non-opioidergic mechanism. Although the naloxone-insensitive M3G-induced increases in [Ca2+]CYT were completely blocked by N-methyl-D-aspartic acid (NMDA) antagonists and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate antagonist), CNQX did not block the large increase in [Ca2+]CYT evoked by NMDA (as expected), confirming that M3G indirectly activates the NMDA receptor. Additionally, tetrodotoxin (Na+ channel blocker), baclofen (
-aminobutyric acidB agonist), MVIIC (P/Q-type calcium channel blocker), and nifedipine (L-type calcium channel blocker) all abolished M3G-induced increases in [Ca2+]CYT, suggesting that M3G may produce its neuro-excitatory effects by modulating neurotransmitter release. However, additional characterization is required. IMPLICATIONS: Large systemic doses of morphine administered to some patients for cancer pain management have been reported to produce myoclonus and allodynia. Indirect evidence implicates the major morphine metabolite, morphine-3-glucuronide (M3G), in these neuro-excitatory side effects. Hence, this study was designed to gain insight into the cellular mechanism responsible for M3G’s neuro-excitatory actions.
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Introduction |
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Although the mechanistic basis for the neuro-excitatory consequences (myoclonus, allodynia, and seizures) of large-dose systemic morphine and HMOR is unknown, indirect evidence implicates the 3-glucuronide metabolites (2). After a systemic administration, more than 50% of every dose of morphine is metabolized to morphine-3-glucuronide (M3G), a metabolite that has no intrinsic pain relieving effects (3). Instead, intracerebroventricular (icv) M3G and its structural analog, hydromorphone-3-glucuronide (H3G), the major metabolite of HMOR, evoke a range of dose-dependent neuro-excitatory behaviors (4–6) including myoclonus, allodynia, and seizures in rodents. Although large icv doses of the parent opioid, morphine, produce behavioral excitation in rodents (5,7), morphine is at least 10-fold less potent as a neuro-excitant than M3G/H3G.
Importantly, M3G crosses the blood-brain barrier sufficiently that the mean steady-state cerebrospinal fluid (CSF) M3G concentration exceeds that of morphine by two- to 10-fold after chronic oral and subcutaneous morphine dosing in cancer patients (8–11). In the clinical setting, changing from large-dose systemic morphine to small-dose icv morphine ensures resolution of opioid-related neuro-excitatory behaviors in cancer patients. After initiation of small-dose icv morphine, there is an 
50-fold increase in the trough CSF morphine concentrations relative to the steady-state CSF morphine concentrations achieved during previous oral or subcutaneous morphine dosing (10). The icv-dosing route also facilitates clearance of M3G from patient CSF while maintaining pain relief through delivery of large concentrations of morphine into the lateral ventricle, i.e., adjacent to regions of the brain containing large densities of opioid receptors (10). These findings provide indirect evidence to suggest that the neuro-excitatory behaviors associated with large systemic doses of morphine are produced primarily by M3G and not the parent opioid (morphine) because the icv-dosing route bypasses liver/gut wall metabolism, and the brain has a small propensity for glucuronidating morphine (3).
Another empirically derived strategy for resolution of opioid-related behavioral excitation in cancer patients is opioid rotation, whereby the opioid medication is rotated from morphine/HMOR to a structurally dissimilar opioid, such as methadone or fentanyl (12,13). This facilitates clearance of M3G/H3G from patient CSF with subsequent resolution of the excitatory behaviors and restoration of analgesia with the dissimilar opioid. Additionally, stopping or markedly reducing the dose of systemic morphine/HMOR often results in temporal resolution of behavioral excitation (12) consistent with the clearance of M3G/H3G from patient CSF.
Previous behavioral studies in our laboratory indicate that M3G may evoke its neuro-excitatory behaviors via indirect activation of N-methyl-D-aspartic acid (NMDA) receptors (4) in a manner similar to that which occurs secondary to nerve or tissue injury (14). To gain insight into the cellular mechanism mediating the neuro-excitatory pharmacological effects of M3G, we have established primary cultures of embryonic rat hippocampal neurones as a model system, consistent with our behavioral studies in rats showing that the rat hippocampus has a range of subregional sensitivities to M3G (15). As the cytosolic calcium concentration ([Ca2+]CYT) plays a crucial role in neuronal processing, drugs that increase [Ca2+]CYT have the potential to profoundly amplify neurotransmission. Hence, we have used fluo-3 fluorescence digital imaging techniques to qualitatively assess the acute effects of M3G on [Ca2+]CYT in cultured hippocampal neurones as a means of gaining insight into the cellular mechanism(s) through which M3G evokes neuro-excitatory behaviors.
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Methods |
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-conotoxin CVID) was a generous gift from AMRAD Corporation Ltd (Melbourne, Australia). Baclofen (Lioresal® intrathecal) vials (2 mg/mL) were purchased from the Royal Brisbane Hospital Pharmacy (Brisbane, Australia). -Conotoxin MVIIC was purchased from Peninsula Laboratories, Inc (San Carlos, CA). Nifedipine was obtained from Calbiochem (La Jolla, CA). Flunarizine was from BIOMOL Research Laboratories, Inc (PA). Midazolam was a generous gift from Roche Products Pty Ltd (Sydney, Australia). Primary cultures of rat embryonic hippocampal neurones, obtained from SD rats at 18–19 days of gestation, were prepared as described by Brewer et al. (16) with modifications. Briefly, hippocampi were dissociated by treatment with 2 mg/mL of papain/Hibernate-E for 30 min at 30°C, followed by trituration. Cells were pelleted and resuspended in neurobasal plating medium containing: L-glutamine (0.5 mM), glutamate (2.5 µM), 2% B-27®, and neurobasal medium. Cells were plated onto 25-mm round glass coverslips coated with PDL (0.05 mg/mL) at 6–7 x 105 cells/mL. Neurones were grown in a humidified atmosphere of 5% CO2 at 37°C. The medium was changed on Day 3 of culture by exchanging half of the medium with neurobasal feeding medium containing 2% B-27® and L-glutamine (0.5 mM). Thereafter, culture media were changed weekly.
Hippocampal neurones aged 14–18 days in vitro were used in these studies. Because experiments were performed over a long period and involved imaging at high rates of acquisition, our studies, similar to others, used the dye fluo-3 to assess Ca2+ levels in a semiquantitative manner. The use of ratio-metric dyes such as fura-2 would have led to significant phototoxicity in our model system and would not have allowed characterization of high frequency Ca2+ oscillations in individual neurones.
Neurones were loaded with 2 µM of fluo-3 for 45 min at 37°C in HEPES HHSS containing 3 mg/mL of BSA and incubated for an additional 15 min to promote hydrolysis of the AM ester. HHSS comprised the following (in mM): HEPES 20, NaCl 137, CaCl2 1.3, MgSO4 0.4, MgCl2 0.5, KCl 5.0, KH2PO4 0.4, Na2HPO4 0.6, NaHCO3 3.0, glucose 5.6, and 10 µM of glycine; the pH value was adjusted to 7.45 with NaOH. Dye-loaded cells were transferred to the recording chamber of an inverted microscope, and cells were superfused with HHSS, containing 0.9 mM of [Mg2+]o, in the presence or absence of naloxone (1 µM) at a rate of 2.5–3 mL/min for 15 min before each experiment. Because our preliminary studies showed that the acute effects of M3G to increase oscillations in [Ca2+]CYT involved both opioid and non-opioid mechanisms, with the latter predominating, the majority of the studies described herein were undertaken in the presence of an opioid receptor-saturating concentration of naloxone (1 µM) to facilitate characterization of the non-opioid mechanism.
[Ca2+]CYT was measured in the somatic region of neurones. All experiments were performed at room temperature (21°C ± 2°C). Neuronal responses were determined using at least three different coverslips from two different cultures, unless otherwise indicated; n values refer to the number of individual neurones assessed.
The fluorescence excitation wavelength (480 nm) was supplied by a Sutter DG-4 wavelength selector (Sutter Instrument Company, Novato, CA) and emission observed through a 550-nm band-pass filter (Omega Optical, Brattleboro, VT). A 40 x oil immersion objective was used. Data points were collected at 0.6-s intervals during rapid characterization or at 10- to 30-s intervals during prolonged incubation or washout periods to minimize bleaching, depending upon the protocol. Images were acquired using MetafluorTM imaging software (West Chester, PA).
Cells were fixed, and the same areas in which [Ca2+]CYT was determined were examined using fluorescence immunocytochemistry immediately after the measurement of fluo-3 fluorescence. Fixed cells were stained using rabbit polyclonal anti-GFAP and mouse monoclonal anti-synaptophysin. Primary antibodies were then treated with goat anti-mouse IgG (FITC) and goat anti-rabbit IgG (TRITC). Neurones were identified by their positive staining for synaptophysin and by the absence of staining for GFAP. Consistent with previous reports using neurobasal media (16), very few of the cells (<5%) were glia in our culture.
Coverslips containing cultured hippocampal neurones were placed in a microscope chamber for Ca2+ measurements as described above. Test compounds and drugs were added to the chamber either through a gravity-fed perfusion system or via direct addition into the chamber.
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Results |
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76% of cultured neurones perfused with HHSS (0.9 mM of [Mg2+]o) exhibited spontaneous [Ca2+]CYT oscillations, whereas the remaining 24% of cultured neurones were quiescent. The acute (3-min exposure) effects of M3G (5–500 µM) on [Ca2+]CYT in cultured embryonic rat hippocampal neurones were assessed using state of the art high-speed fluo-3 fluorescence digital imaging techniques. Increases in [Ca2+]CYT evident as increases in fluo-3 fluorescence intensity were measured in the soma of neurones. Before the experiments, neurones were treated with a single concentration of M3G (50 µM) to assess their M3G-responsiveness. If the M3G-evoked increase in the amplitude of oscillations (as assessed by fluo-3 intensity) was more than two-fold when compared with pretreatment levels, these neurones were classified as M3G-responsive.
Superfusing M3G (50 µM) for 3 min resulted in a rapid increase in [Ca2+]CYT, which was always reversed upon M3G washout (Fig. 2E). In the experiment shown in Figure 1, three nonspontaneously oscillating neurones were sampled to determine the effects of M3G (50 µM) on [Ca2+]CYT, and the fluo-3 fluorescence intensity from three neuronal regions was plotted against time to show the M3G-evoked increases in [Ca2+]CYT in these neurones (Fig. 1E). Pseudo-color images of these same fluo-3 loaded cells before, during, and after M3G-mediated increases in [Ca2+]CYT are shown in Figure 1A–C, respectively, where hot colors, e.g., yellow-red, indicate a large increase in fluorescence intensity, which in turn reflects a large increase in [Ca2+]CYT. These same cells were then characterized using fluorescence immunocytochemistry, such that neurones were labeled for synaptophysin (green) and glia for GFAP (red) (Fig. 1D). As can be seen, very few (<5%) of the cells in our culture were glia, consistent with previous studies where neurones were cultured using neurobasal media (16). Importantly, any glia present in the culture were clearly stained with GFAP using our staining protocols.
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30 s in the absence (Fig. 2A) and presence (Fig. 2B) of naloxone (1 µM). By contrast, in nonspontaneously oscillating neurones, M3G often increased [Ca2+]CYT, reaching a plateau concentration that slowly recovered back to baseline in the absence (Fig. 2C) and presence (Fig. 2D) of naloxone (1 µM). The amplitude of the M3G-evoked calcium responses in quiescent neurones (Figs. 2, C and D) seemed to exceed that produced in oscillating neurones (Figs. 2, A and B); however, quantitative comparison of these relative responses using fura-2 remains for future studies. The relative response of the same neurone to each of M3G (50 µM) and glutamate (10 µM) is shown in Figure 4C. The effect of M3G on [Ca2+]CYT responses in this neurone is clearly significant because glutamate is well documented to produce increases in [Ca2+]CYT in the micromolar range in hippocampal neurones (17,18).
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In separate experiments, neurones were assessed for their responsiveness to 5, 50, and 500 µM concentrations of M3G in both the absence and presence of an opioid receptor saturating concentration of naloxone (1 µM). For these studies, all neurones tested in the experiments were included. There was a concentration-dependent increase in the percentage of neurones that responded to M3G (Fig. 2F), which was reduced by 10%–25% in the presence of naloxone (1 µM) (Fig. 2F). However, this decrease only reached statistical significance (P < 0.01; 
2 test) for neurones exposed to M3G (50 µM) (Fig. 2F).
Spontaneous [Ca2+]CYT oscillations were completely abolished by 15 min of pretreatment with LY274614 (50 µM), a competitive NMDA receptor antagonist, MK-801 (30 µM), a noncompetitive NMDA antagonist, and CNQX (10 µM), an AMPA/kainate receptor antagonist. Additionally, the ability of M3G (50 µM) to increase [Ca2+]CYT was blocked by pretreatment with these same concentrations of LY274614 (n = 58; Figs. 3,A and B), MK-801 (n = 17; Fig. 3C), and CNQX (n = 34; Fig. 3D). By contrast, CNQX did not prevent NMDA (125 µM)-evoked increases in [Ca2+]CYT, as expected (Fig. 3E; n = 22 neurones from two coverslips in the same culture). Importantly, subsequent removal of the glutamate receptor antagonists from the perfusion medium resulted in neurones again becoming responsive to M3G (50 µM), confirming that the effects of these antagonists were reversible and that viable neurones were studied throughout the experiments (Fig. 3A–E). As expected, the increase in [Ca2+]CYT in our cultured neurones induced by NMDA (125 µM) was blocked by pretreatment with MK-801 (30 µM; data not shown).
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The Na+ channel blocker, TTX (1 µM), abolished spontaneous oscillations in calcium (data not shown), consistent with previous studies showing that nerve terminal depolarization is critical to calcium entry to facilitate presynaptic vesicular release of glutamate (19–21). Additionally, TTX (1 µM) completely abolished M3G (50 µM)-evoked increases in [Ca2+]CYT, an effect that was completely reversible upon TTX washout (Fig. 4B; n = 41 neurones from four coverslips in two cultures).
Exposure of cultured hippocampal neurones to the P/Q-type calcium channel blocker, MVIIC (1 µM), for 15 min abolished spontaneous calcium oscillations (Fig. 4C) as well as completely blocking M3G-evoked increases in [Ca2+]CYT (n = 46; Fig. 4C). Consistent with previous observations in the literature (22), the effects of MVIIC were long lasting.
Preincubation with the N-type calcium channel blocker, AM336 (also known as omega-conotoxin CVID) (1 µM), for 2 min completely abolished spontaneous calcium oscillations (Fig. 4D). AM336 did not prevent but possibly attenuated M3G-evoked increases in [Ca2+]CYT (n = 41; Fig. 4D). Clearly, further quantitative investigation using fura-2 is required to determine the extent to which AM336 attenuates M3G’s actions.
After exposure of neurones to nifedipine, a dihydropyridine L-type calcium channel blocker, (10 µM) for 15 min, spontaneous calcium oscillations in cultured hippocampal neurones were completely abolished, as were M3G-evoked increases in [Ca2+]CYT (n = 28; Fig. 4E).
Pretreatment (15 min) of cultured hippocampal neurones with flunarizine (1 µM) had little or no effect on either spontaneous calcium oscillations or on M3G-evoked increases in [Ca2+]CYT (n = 35; Fig. 4F).
Preincubation of neurones with a -aminobutyric acid (GABA)B agonist, baclofen (10 µM), for 15 min before acute M3G (50 µM) exposure completely inhibited spontaneous calcium oscillations as well as M3G-mediated increases in [Ca2+]CYT (n = 29; Fig. 5A). By contrast, the increases in [Ca2+]CYT induced by exposure of neurones to AMPA (10 µM) were not prevented by pretreatment with baclofen (10 µM) (n = 35; Fig. 5B), as expected.
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Discussion |
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76% of neurones tested exhibiting spontaneous oscillations in [Ca2+]CYT (19,21,23) that were abolished when depolarization of the nerve terminal was prevented by TTX, in accordance with previous reports (19–21). Additionally, synaptic transmission was blocked by removal of extracellular calcium or by increasing extracellular magnesium, consistent with the documented role of NMDA receptors in mediating spontaneous glutamatergic synaptic transmission (24).
After acute (three minutes) exposure to M3G (5–500 µM), the proportion of M3G-responsive neurones increased in a concentration-dependent manner from 30% at 5 µM to 95% at 50 µM (Fig. 2F). Thus, an M3G concentration of 50 µM was chosen for the remainder of our investigations to ensure M3G-responsiveness of the majority of neurones tested. Although this concentration of M3G is 50-fold larger than the mean CSF M3G concentration (1 µM) found in cancer patients dosed chronically with large-dose systemic morphine at the time of ventriculostomy (10), these CSF M3G concentrations would have been significantly underestimated. This is because in many patients, the morphine dose was reduced substantially before ventriculostomy to resolve opioid-related confusion so patients could give informed consent (10). It should also be noted that the typical concentrations of morphine and other opioids (1–30 µM) used to characterize the inhibitory effects of opioids in electrophysiological studies are 25–800 times larger than the mean steady-state CSF morphine concentration (35 nM) determined in patients receiving oral or s.c. morphine for chronic pain (8,9). Almost certainly, the necessity to use larger concentrations of each of M3G and morphine in in vitro studies of the mechanisms underpinning their respective pharmacological effects reflects the fact that integration of simultaneous neuronal responses at multiple levels of the central nervous system occurs in vivo to produce a behavioral response.
The percentage of neurones that responded to M3G in the presence of an opioid receptor saturating concentration of naloxone (1 µM) was reduced by 10%–25% (Fig. 2F), indicating the predominantly non-opioidergic mechanism mediating M3G’s neuro-excitatory actions. Consistent with the low binding affinity of M3G for µ, 
, and 
-opioid receptors (half-maximal inhibitory concentration [IC50] >1 µM) in rat brain homogenate (25,26), a 50-fold increase in the naloxone concentration (from 1 µM to 50 µM), failed to block or attenuate M3G-evoked calcium responses in imaged neuronal cell bodies. These observations confirm our previous in vivo findings in rats that M3G’s neuro-excitatory behavioral effects are mediated by a predominantly non-opioid mechanism (15). Moreover, the naloxone-insensitivity of M3G’s actions has been confirmed in a very recently published electrophysiological study undertaken in rat spinal cord neurones (27). To characterize M3G’s non-opioid mechanism, all of our subsequent experiments were undertaken in the presence of an opioid receptor-saturating concentration of naloxone (1 µM).
The NMDA receptor is a ligand- and voltage-gated channel, which not only requires the binding of glutamate for activation, but also an initial AMPA-receptor mediated depolarization of the cell membrane. Depolarization of the neuronal membrane removes the Mg2+ block from the NMDA receptor ion channel, thereby permitting the influx of Ca2+ and Na+ ions into the cell (28). MK-801 (noncompetitive NMDA antagonist) and LY274614 (competitive NMDA antagonist) completely blocked spontaneous calcium oscillations, consistent with previous reports that these arise from glutamatergic synaptic transmission (19,21,23,24). Furthermore, LY274614 and MK-801 completely blocked the M3G-induced increases in [Ca2+]CYT in both quiescent and spontaneously oscillating cultured hippocampal neurones in accordance with our previous in vivo studies in rats, which showed that LY274614 dose-dependently attenuated the behavioral excitation evoked by icv M3G (4). However, as M3G does not bind to any of the known binding sites on the NMDA receptor complex (25), our current data suggest that M3G increases [Ca2+]CYT by a mechanism that involves indirect rather than direct activation of NMDA receptors. The failure of M3G to increase [Ca2+]CYT in the presence of NMDA antagonists also indicates that M3G does not promote calcium influx by directly opening postsynaptic voltage-gated calcium channels nor does it mobilize calcium from intracellular stores.
As CNQX (AMPA/kainate receptor antagonist) completely blocked M3G-evoked increases in [Ca2+]CYT, but had no effect (as expected) on the increase in [Ca2+]CYT produced by direct exposure of cultured neurones to NMDA (125 µM), this confirms that M3G indirectly activates the NMDA receptor. Additionally, the fact that CNQX blocked M3G-evoked increases in [Ca2+]CYT indicates that M3G does not directly depolarize postsynaptic cells because such depolarization of the postsynaptic cell membrane would have removed the Mg2+ block from the NMDA receptor, thereby allowing Ca2+ influx, which we would have detected as an increase in [Ca2+]CYT. Our findings are consistent with the results of a recent electrophysiological study that showed that M3G did not affect postsynaptic membrane conductance or excitability in slices of rat spinal cord (27).
As the GABAB agonist and P-type calcium channel blocker, baclofen, completely inhibited M3G-evoked increases in [Ca2+]CYT but had no effect on AMPA-induced increases in [Ca2+]CYT, it is clear that M3G does not directly interact with AMPA/kainate receptors, which is consistent with our previous radioligand binding work that showed M3G does not bind to the AMPA/kainate receptor (25). Thus, M3G’s site of action is upstream of the AMPA receptor in the signaling cascade that ultimately leads to indirect activation of AMPA and NMDA receptors. Given that micromolar concentrations of baclofen have been shown to inhibit calcium currents and synaptic transmission in cultured rat hippocampal neurones (29), it is possible that M3G increases [Ca2+]CYT by an augmentation of glutamate release. This proposal is further supported by a previous report showing that activation of presynaptic GABAB receptors reduces neurotransmitter release by inhibition of calcium channels in nerve terminals (30).
Exposure of neurones to TTX, as well as removal of extracellular calcium or increasing the extracellular magnesium (3 mM) concentration (data not shown) abolished M3G-responsiveness, indicating that influx of calcium from the extracellular environment rather than mobilization from intracellular stores is critical for M3G’s pharmacological action. These findings are consistent with the results of a recent study that used whole patch-clamp recordings in substantia gelatinosa neurones in transverse slices of rat spinal cord to show that in the presence of TTX, M3G reduced the miniature inhibitory postsynaptic current (mIPSC) frequency without affecting the amplitude in all cells tested, suggestive of a presynaptic mechanism (27).
We have discounted the possibility that M3G may evoke its neuro-excitatory effects by blocking postsynaptic GABAA or glycine receptors for the following reasons. First, we have previously reported that M3G has insignificant binding affinity for either the benzodiazepine or the GABA binding site on the GABAA-benzodiazepine receptor complex or for inhibitory glycine receptors in neuronal tissue (25). Second, the recently published electrophysiological study mentioned above (27) showed that M3G produced a concentration-dependent decrease in the amplitude of the evoked IPSC (27), such that M3G reduced the frequency but not the amplitude of TTX-insensitive mIPSCs, thereby implicating a presynaptic mechanism of action. Third, because midazolam (10 µM) seemed to attenuate, but did not block, M3G-induced increases in [Ca2+]CYT, it is possible that M3G interacts with one or more subtypes of metabotropic glutamate receptors to modulate GABA or glutamate release from nerve terminals (31); this remains for future examination.
Taken together, the previously mentioned findings collectively suggest that M3G does not have direct effects on the postsynaptic cell; rather, the evidence points to an upstream site of action such as the promotion of neurotransmitter release. This proposal is further supported by our findings that M3G-responsiveness of neurones was abolished when nerve terminal depolarization was blocked by TTX, thereby preventing Ca2+ influx into the nerve terminal, a step critical to neurotransmitter release. We further investigated this issue using selective blockers of each of N-, P/Q-, L-, and T-type calcium channels.
Consistent with previous reports (22), MVIIC (1 µM), the selective P/Q-type calcium channel blocker, produced long-lasting inhibitory effects. Specifically, MVIIC blocked spontaneous oscillations in calcium in accordance with reports that P-type calcium channels have an important role in the modulation of glutamatergic synaptic transmission in hippocampal neurones (32) at all stages of neuronal development. Furthermore, our observation that MVIIC also abolished M3G-evoked increases in [Ca2+]CYT, supports the possibility that M3G elicits its neuro-excitatory effects via indirect augmentation of glutamatergic neurotransmission.
AM336, the selective N-type calcium channel blocker (33), abolished spontaneous [Ca2+]CYT oscillations in a reversible manner, in accordance with the report by Verderio et al. (34) that N-type Ca2+ channels have a pivotal role in modulating presynaptic glutamate release in developing rat hippocampal neurones. However, although AM336 (1 µM) seemed to attenuate M3G’s effects, it did not completely block M3G-evoked increases in [Ca2+]CYT. Clearly, additional research is required to quantify AM336’s inhibitory effects.
Although the release of neurotransmitters is generally reported to be under the influence of both N and P/Q-type calcium channels, there are reports that also implicate L-type calcium channels in this process (35,36). In support of this, blockade of L-type calcium channels with nifedipine (10 µM) in the present study, abolished spontaneous oscillations in calcium as well as preventing M3G-evoked increases in [Ca2+]CYT. Furthermore, pretreatment of cultured hippocampal neurones with the T-type calcium channel blocker, flunarizine (1 µM), seemed to have little or no effect on either spontaneous oscillations in calcium or on M3G-induced increases in [Ca2+]CYT.
Thus, collectively, our findings show that selective blockade of N-, P/Q-, and L-type calcium channels abolishes spontaneous calcium oscillations, consistent with the well-accepted view that calcium influx into the nerve terminal via N-, P/Q-, and L-type calcium channels is critical to facilitate presynaptic neurotransmitter release. By contrast, only P/Q- and L-type calcium channel blockers completely prevented M3G-evoked increases in [Ca2+]CYT, suggesting that M3G may act via pathways that primarily rely on P/Q- and L-type calcium channels to indirectly modulate neurotransmitter release. Our findings described herein are consistent with a recent electrophysiological study undertaken in rat spinal cord slices (published while our manuscript was under review) showing that M3G produced a naloxone-insensitive suppression of inhibitory synaptic transmission via a presynaptic mechanism (27).
In summary, our studies in cultured hippocampal neurones show that M3G indirectly activates the NMDA receptor resulting in increased cytosolic calcium concentrations via a predominantly non-opioidergic mechanism. Our findings add further evidence to that published by Moran and Smith (27), who suggest that M3G produces its neuro-excitatory effects via indirect modulation of neurotransmitter release.
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Acknowledgments |
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