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Keywords:

  • anandamide;
  • arachidonic acid;
  • glycine transporter

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Methods
  6. Electrophysiological recordings
  7. [3H]Glycine uptake assays
  8. Analysis of kinetic data
  9. Results
  10. Arachidonic acid inhibits glycine transport
  11. Anandamide and R1-methanandamide stimulate glycine transport
  12. Modulation of [3H]glycine uptake by C6 glioma cells
  13. Discussion
  14. Acknowledgements
  15. References

The GLYT1 subtypes of glycine transporter are expressed in glia surrounding excitatory synapses in the mammalian CNS and may regulate synaptic glycine concentrations required for activation of the NMDA subtypes of glutamate receptor. In this report we demonstrate that the rate of glycine transport by GLYT1 is inhibited by arachidonic acid. The cyclo-oxygenase and lipoxygenase inhibitors indomethacin and nordihydroguaiaretic acid, and the protein kinase C inhibitor staurosporine, had no effect on the extent of arachidonic acid inhibition of transport, which suggests that the inhibitory effects of arachidonic acid result from a direct interaction with the transporter. In contrast to arachidonic acid, its amide derivative, anandamide, and the more stable analogue R1-methanandamide stimulate glycine transport. This stimulation is unlikely to be a secondary effect of cannabinoid receptor stimulation because the cannabinoid receptor agonist WIN 55 212–2 had no effect on transport. We suggest that the stimulatory effects of anandamide on GLYT1 are due to a direct interaction with the transporter.

Abbreviations used
DMSO

dimethyl sulfoxide

NDGA

nordihydroguaiaretic acid

NFPS

N[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine

GLYT

glycine transporter

PLA2

phospholipase A2

The amino acid glycine is a co-agonist with glutamate at the NMDA subtype of glutamate receptors and the concentrations of glycine within excitatory glutamatergic synapses are thought to be regulated, at least in part, by the GLYT1 subtypes of glycine transporter (Attwell et al. 1993; Berger et al. 1998; Bergeron et al. 1998; Roux and Supplisson 2000). A number of isoforms of the GLYT1 subclass of glycine transporters have been identified, which differ in their amino terminal and carboxy terminal sequences, and arise through use of alternative promoters and alternative splicing (Guastella et al. 1992; Liu et al. 1992, 1993; Smith et al. 1992; Borowsky et al. 1993; Kim et al. 1994; Hanley et al. 2000). The Na+/Cl-dependent GLYT1 subtypes of transporter are located in glial cells surrounding the synapse (Zafra et al. 1995) and, from the stoichiometry of the coupled fluxes of two Na+ ions to one Cl ion for each glycine molecule transported, it has been predicted that resting synaptic glycine concentrations are below 150 nm (Attwell et al. 1993; Roux and Supplisson 2000).

Phospholipase A2 (PLA2)-stimulated liberation of arachidonic acid is an established component of several neurotransmitter systems (glutamatergic, serotonergic, dopaminergic and muscarinic) throughout the brain (Lazarewicz et al. 1988; Dumuis et al. 1990; Kanterman et al. 1990; Attwell et al. 1993). Once released, arachidonic acid modulates the function of a number of ion channels and transporters through two types of mechanism: a direct interaction with the protein; and through second messenger actions of arachidonic acid metabolites produced by cyclo-oxygenases, lipoxygenases and epoxygenases (Attwell et al. 1993; Ordway et al. 1991). Arachidonic acid potentiates the actions of glutamate at NMDA receptors (Miller et al. 1992), and modulates both the transport of glutamate (Volterra et al. 1992, 1994; Trotti et al. 1995; Zerangue et al. 1995) and conductances associated with glutamate transporters (Fairman et al. 1998; Tzingounis et al. 1998; Poulsen and Vandenberg 2001). Glycine uptake into C6 glioma cells, which contain a mixture of high- and low-affinity glycine transporters, is also inhibited by arachidonic acid (Zafra et al. 1990). Arachidonic acid is therefore an important regulator of excitatory neurotransmission.

Anandamide (arachidonylethanolamide), the ethanolamide derivative of arachidonic acid and partial cannabinoid receptor agonist, also appears to modulate the activity of a variety of receptors and ion channels involved in neurotransmission. (Hampson et al. 1998; Vaughan et al. 2000; Zygmunt et al. 2000). Recent evidence to support non-cannabinoid receptor-mediated effects of anandamide has come from cannabinoid receptor knockout mice, in which administration of anandamide still exerts cannabimimetic activity (Di Marzo et al. 2000). These observations suggest that at least some of the behavioural effects of anandamide may be mediated by its interaction with other receptor systems and ion channels. Anandamide has been reported to stimulate NMDA receptors (Hampson et al. 1998), vanilloid receptors (Vaughan et al. 2000; De Petrocellis et al. 2001) and muscarinic receptors (Christopoulos and Wilson 2001).

In this report, we have further investigated the effects of arachidonic acid, and a variety of arachidonic acid derivatives including anandamide, on the glycine transporter GLYT1. The results presented suggest that arachidonic acid is a potent and directly acting inhibitor of glycine transport, whereas anandamide stimulates transporter activity. If these observations are relevant in vivo, then dynamic regulation of glycine transporter activity by these compounds may provide an added degree of complexity to the control of excitatory neurotransmission under physiological and pathological conditions.

Materials

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Methods
  6. Electrophysiological recordings
  7. [3H]Glycine uptake assays
  8. Analysis of kinetic data
  9. Results
  10. Arachidonic acid inhibits glycine transport
  11. Anandamide and R1-methanandamide stimulate glycine transport
  12. Modulation of [3H]glycine uptake by C6 glioma cells
  13. Discussion
  14. Acknowledgements
  15. References

Arachidonic acid, melittin, arachidonic acid ethyl ester, indomethacin, nordihydroguaiaretic acid (NDGA), oleic acid and docosahexaenoic acid were purchased from Sigma (Sydney, NSW, Australia). Anandamide, R1-methanandamide, 2-arachidonyl glycerol, AM404 and WIN 55 212–2 were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Staurosporine was from Alamone labs (Jerusalem, Israel), SR141617A was provided by Dr Ernie Jennings, Department of Pharmacology, University of Sydney, and N[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine (NFPS) was supplied by NPS Allelix Inc. (Mississauga, ON, Canada). All other chemicals were of analytical grade and obtained from Sigma unless otherwise stated. Glycine stock solutions (20 mm) were made in MilliQ water (Millipore, Sydney, Australia). Arachidonic acid (sodium arachidonate) and arachidonic acid ethyl ester stock solutions (200 mm) were supplied in ethanol. Melittin was kept as a stock solution (1 mg/mL) in MilliQ water at − 20°C. Indomethacin (20 mm) and NDGA (20 mm) stock solutions were prepared in ethanol, oleic acid (200 mm) in methanol, docosahexaenoic acid (200 mm) in dimethyl sulfoxide (DMSO) and staurosporine (10 mm) in DMSO. Anandamide (100 mm) and R1- methanandamide (100 mm) were supplied in ethanol and 2-arachidonyl glycerol (26 mm) was supplied in acetonitrile. All lipid stock solutions were stored at − 80°C in 200-µL aliquots. Fresh aliquots of the frozen lipid stock solutions were thawed, placed on ice and then sonicated for 1 min immediately before dilution and application to oocytes. All samples were used within 1 h of removal from the freezer.

Methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Methods
  6. Electrophysiological recordings
  7. [3H]Glycine uptake assays
  8. Analysis of kinetic data
  9. Results
  10. Arachidonic acid inhibits glycine transport
  11. Anandamide and R1-methanandamide stimulate glycine transport
  12. Modulation of [3H]glycine uptake by C6 glioma cells
  13. Discussion
  14. Acknowledgements
  15. References

Female Xenopus laevis frogs were anaesthetized with 0.17% tricaine and ovarian sacs were removed surgically. Ovarian sacs were placed in OR-2 buffer (82.5 mm NaCl, 2 mm KCl, 1 mm MgCl2 and 5 mm HEPES, pH 7.5) and oocytes were liberated from the overlying follicle cells by agitation for 2–3 h in 2 mg/mL collagenase A (Boehringer Mannheim, USA). After rinsing in ND96 (96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2 1 mm MgCl2 and 5 mm HEPES, pH 7.5) stage V–VI oocytes were isolated and stored in tissue culture dishes in ND96 supplemented with 0.1% gentamicin, 2.5 mm pyruvate and 0.5 mm theophylline until injection. All procedures were in accordance with the Australian National Health and Medical Research Council guidelines for the prevention of cruelty to animals. GLYT1a, GLYTb and GLYTc cDNAs were obtained from Dr Marc Caron and subcloned into the plasmid, pOTV (Arriza et al. 1994), and RNA was prepared using the mMessage mMachine In Vitro Transcription Kit (Ambion, Austin, Texas USA) according to the manufacturer's instructions. Oocytes were injected with 50 nL cRNA, and stored at 17°C in ND96.

Electrophysiological recordings

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Methods
  6. Electrophysiological recordings
  7. [3H]Glycine uptake assays
  8. Analysis of kinetic data
  9. Results
  10. Arachidonic acid inhibits glycine transport
  11. Anandamide and R1-methanandamide stimulate glycine transport
  12. Modulation of [3H]glycine uptake by C6 glioma cells
  13. Discussion
  14. Acknowledgements
  15. References

Oocytes were placed in a recording chamber and superfused in ND96 at a rate of 8.5–10 mL/min. Whole-cell currents were measured using standard two-electrode voltage-clamp techniques using a Geneclamp 500 amplifier (Axon instruments, Foster City, CA, USA) interfaced with a MacLab2e chart recorder (ADInstruments, Sydney, NSW, Australia) with electrodes (0.5–1.0 MΩ) filled with 3 m KCl. The effects of ethanol, methanol, acetonitrile and DMSO on the holding current at − 60 mV were negligible at maximal concentrations of drug.

The onset of the effects of some compounds, such as arachidonic acid, was gradual over the course of approximately 30 s to 1 min and in such cases current measurements were taken after the current had stabilized (see Fig. 1).

image

Figure 1. Arachidonic acid inhibits glycine transport. (a) Current trace from a representative oocyte, voltage clamped at − 60 mV. Application of 30 µm glycine (gly) (solid bar) generates an inward current and co-application of 30 µm glycine with 10 µm arachidonic acid (AA) (open bar) reduces the inward current. After 10 min of perfusion in ND96, 30 µm glycine was re-applied demonstrating that the inhibition is fully reversible. The arrow represents the point at which arachidonic acid inhibition of transport current was measured for Fig. 1(b) and (c), and Fig. 2. (b) Dose–response curve of glycine-elicited currents at − 60 mV in the absence (▪) and presence (•) of 30 µm arachidonic acid. Points represent mean ± SEM from 10 cells. Curves represent data fitted to the modified Michaelis–Menten equation. (c) Dose–response curve for arachidonic acid-mediated inhibition of glycine-elicited transport currents at − 60 mV. 100 µm glycine was co-applied with increasing concentrations of arachidonic acid. Data represent mean ± SEM normalized current, with separate cells used for each arachidonic acid dose (n = 4–6 for each dose). (d) Effects of arachidonic acid on uptake of [3H]glycine by oocytes expressing GLYT1a. Uptake of 20 µm[3H]glycine in the absence (gly) and presence of 100 µm arachidonic acid (gly + AA). Non-specific uptake was determined by conducting parallel experiments on H2O-injected control oocytes. Variation in expression levels is observed between batches of oocytes; the data shown represent pooled data from three separate experiments using separate batches of oocytes (10 per batch) and are normalized to the rates of uptake measured in the absence of arachidonic acid. Results presented are mean ± SEM.

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[3H]Glycine uptake assays

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Methods
  6. Electrophysiological recordings
  7. [3H]Glycine uptake assays
  8. Analysis of kinetic data
  9. Results
  10. Arachidonic acid inhibits glycine transport
  11. Anandamide and R1-methanandamide stimulate glycine transport
  12. Modulation of [3H]glycine uptake by C6 glioma cells
  13. Discussion
  14. Acknowledgements
  15. References

[3H]glycine uptake by oocytes was measured at room temperature (22°C) in ND96. Oocytes were incubated in the presence or absence of 100 µm arachidonic acid or 100 µm R1-methanandamide for 1 min followed by the addition of 20 µm[3H]glycine for 5 min. Although the electrophysiological measurements were taken after approximately 1 min exposure of the compounds it was necessary to use 5 min as the time for [3H]glycine uptake to obtain a sufficient signal-to-noise ratio. The oocytes were then transferred through three cold washes in ND96 solution (total transfer time < 20 s) to terminate the reaction. Non-specific uptake was determined by performing parallel experiments with H2O-injected oocytes. [3H]glycine was quantified by scintillation counting after dissolving oocytes in 50 µL 50 mm NaOH and addition of scintillant. Variation in expression levels was observed between batches of oocytes and the data shown represent pooled data from three separate experiments normalized to the rates of uptake measured in the absence of arachidonic acid or R1-methanandamide. In all batches of oocytes the extent of inhibition, or stimulation, was similar.

[3H]glycine uptake by C6 glioma cells was also measured. C6 gliomas were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 10% penicillin/streptomycin. Twenty-four hours before measuring uptake, the cells were transferred to 24-well Cultur plates (Packard Instruments Co. Inc., Meridan, CT, USA) and allowed to grow until confluent. Phosphate-buffered saline supplemented with 1 mm MgCl2 and 1.25 mm CaCl2 was used to wash the cells three times immediately before the start of the assay. The proportion of glycine transport mediated by GLYT1 in C6 gliomas was estimated by measuring the rate of uptake in the presence of 1 µm NFPS and also 300 µm sarcosine. Cells were pretreated with either NFPS or sarcosine for 1 min before addition of 2 µm[3H]glycine and uptake was allowed to proceed for 15 min. [3H]glycine uptake was terminated after 15 min by washing the wells three times in ice-cold phosphate-buffered saline supplemented with 1 mm MgCl2 and 1.25 mm CaCl2. The rate of uptake was compared to control rates measured in the absence of NFPS or sarcosine. For the arachidonic acid assay, cells were pretreated with 10 µm indomethacin and 100 µm NDGA for 10 min, and then with 10 µm arachidonic acid for 1 min before addition of 2 µm[3H]-glycine. [3H]glycine uptake was measured as described above. The rate of uptake was compared with control rates measured in the absence of arachidonic acid, but in the presence of indomethacin and NDGA. For the R1-methanandamide assay, cells were pretreated with the cannabinoid receptor antagonist SR141617A (3 µm) for 10 min followed by 10 µm R1-methanandamide for 1 min before addition of 2 µm[3H]glycine. The rate of uptake was measured as described above and compared with control rates measured in the absence of R1-methanandamide, but in the presence of SR141617A.

  • image

using Kaleidagraph 3.05 for Windows (Synergy Software), where I is the current, Imax is the maximal current, [S] is the substrate concentration and K0.5 is the concentration of substrate that generates half-maximal current. The inhibition of glycine transport currents by arachidonic acid was fit to the related equation

  • image

where [AA] is the concentration of arachidonic acid, IC50 is the concentration eliciting half-maximal inhibition, and R is the residual current remaining in the presence of a maximal dose of arachidonic acid. The stimulation of transport currents by anandamide was fit to the equation

  • image

where [AEA] is the concentration of anandamide and K0.5is the concentration eliciting half-maximal stimulation.

All values presented are mean ± SEM and significance tests were performed using the two-tailed Student's t-test. p < 0.05 was taken as statistically significant.

Arachidonic acid inhibits glycine transport

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Methods
  6. Electrophysiological recordings
  7. [3H]Glycine uptake assays
  8. Analysis of kinetic data
  9. Results
  10. Arachidonic acid inhibits glycine transport
  11. Anandamide and R1-methanandamide stimulate glycine transport
  12. Modulation of [3H]glycine uptake by C6 glioma cells
  13. Discussion
  14. Acknowledgements
  15. References

Application of glycine to oocytes expressing GLYT1 generates dose-dependent inward currents due to the co-transport of glycine with two Na+ ions and one Cl ion into the cell (Aubrey et al. 2000; Roux and Supplisson 2000). Co-application of 10 µm arachidonic acid with 30 µm glycine to oocytes expressing GLYT1a, GLYT1b or GLYT1c significantly reduced the current generated by glycine alone (Fig. 1a). The onset of inhibition of glycine transport currents was gradual over the course of about 30 s to 1 min and in further analysis of these effects we have presented results at time points once the degree of inhibition had stabilized. Although similar results were observed for all three GLYT1 subtypes we have only presented results for the GLYT1a isoform. Co-application of low doses of arachidonic acid (< 3 µm) with glycine resulted in a reversible reduction in the transport current, but at higher doses inhibition was only partially reversible and reduced transport currents persisted for at least 10 min after washout of arachidonic acid from the bath solution. Dose-dependent glycine transport currents were measured before and after exposure to 30 µm arachidonic acid. Under these conditions, 30 µm arachidonic acid reduced the maximal glycine transport by 50 ± 2% with a small, but significant, reduction in EC50 for glycine from 15 ± 1 to 7 ± 1 µm (Fig. 1b).

Transport currents were also measured in the presence of increasing concentrations of arachidonic acid (up to 30 µm) and a fixed concentration of glycine (100 µm). As the effects of arachidonic acid were not fully reversible at high doses, separate cells were used for each arachidonic acid concentration tested. The arachidonic acid concentration that generated half-maximal inhibition of glycine-mediated transport currents was 2.1 ± 0.7 µμ (n = 5) with a maximal inhibition of 51 ± 4% (Fig. 1c).

The effects of arachidonic acid on [3H]glycine uptake were also examined to establish whether the inhibition of transport currents by arachidonic acid also reflected a decrease in glycine translocation through GLYT1a. In the presence of arachidonic acid, the rate of [3H]glycine uptake by oocytes expressing GLYT1a was reduced by 47 ± 4% (n = 10) (Fig. 1d), which correlates with the extent of inhibition of glycine transport currents. It should be noted that the [3H]glycine uptake was measured after 5 min, chosen because a better signal-to-noise ratio was obtained with the longer exposure time, although qualitatively similar results were obtained at shorter exposure times. None the less, the result suggests that arachidonic acid inhibits glycine transport currents by reducing the rate of translocation of glycine through GLYT1a.

Activation of PLA2 leads to arachidonic acid release from lipid membranes, and the arachidonic acid may diffuse through the lipid membrane and interact with various receptors, ion channels, transporters and enzymes. Application of the neurotoxin, melittin, directly stimulates PLA2 activity and causes release of arachidonic acid. As such, melittin provides an effective way of causing localized increases in arachidonic acid levels as an alternative to bath application (Volterra et al. 1992), which may represent a more physiological method for arachidonic acid delivery to the cell surface. Application of 0.2 µg/mL melittin for 10 min to oocytes expressing GLYT1a caused a 33 ± 4% (n = 4) reduction in glycine transport currents compared with glycine transport current measurements made before melittin application (Fig. 2a). Thus, arachidonic acid produced by the endogenous PLA2 in oocytes causes a reduction in glycine transport currents.

image

Figure 2. Arachidonic acid interacts directly with the GLYT1a transporter protein. (a) Melittin-mediated inhibition of glycine transport currents. Xenopus oocytes expressing GLYT1a were voltage clamped at − 60 mV, and 100 µm glycine was applied before (gly) and after either melittin treatment (0.2 µg/mL) or mock (buffer) treatment for 10 min. Data represent mean ± SEM (n = 5). *p < 0.05 versus gly (two-tailed Student's t-test). (b) Arachidonic acid ethyl ester (AAEE; 100 µm) had no significant effect on transport currents generated by 100 µm glycine, whereas the inhibition produced by 100 µm arachidonic acid after 5 min of 10 µm indomethacin (I + AA), 100 µm NDGA (NDGA + AA) or 0.5 µm staurosporine (STAU + AA) treatment was comparable to that produced by 100 µm arachidonic acid alone (AA). Data represent mean ± SEM (n = 5–7). (c) In GLYT1a-expressing oocytes voltage clamped at − 60 mV, the extent of inhibition of the current generated by 100 µm glycine observed with oleic acid (OA) 100 µm and docosahexanoic acid (DA) 100 µm, was comparable to that observed upon arachidonic acid (AA) application. Conversely, the amide derivative of arachidonic acid anandamide (AEA) 100 µm stimulated transport currents generated by 100 µm glycine. Data represent mean ± SEM (n = 5).

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Arachidonic acid may mediate its effects on GLYT1a through a direct interaction with the transporter; or indirectly via the production of various metabolites of the lipoxygenase and cyclo-oxygenase pathways, by stimulating protein kinase C activity, or by altering the fluidity and organization of the lipid membrane (Asaoka et al. 1992; Attwell et al. 1993). To rule out the effect of an arachidonic acid metabolite, 10 µm indomethacin (a cyclo-oxygenase inhibitor; Laneuville et al. 1994) and 100 µm NDGA (a lipoxygenase inhibitor; Suzuki et al. 1992) were applied to Xenopus oocytes for 5 min, followed immediately by application of 100 µm arachidonic acid together with 100 µm glycine. After treatment with indomethacin for 5 min, arachidonic acid-mediated inhibition of glycine transport currents was 46 ± 4% compared with 49 ± 4% (n = 5) in the absence of indomethacin. Similarly, NDGA did not prevent inhibition of glycine uptake by arachidonic acid: 54 ± 8% (n = 5) inhibition was observed following incubation with NDGA for 5 min and 55 ± 8% inhibition in the absence of NDGA (Fig. 2b). Arachidonic acid may also stimulate protein kinase C (Asaoka et al. 1992), which may subsequently cause a reduction in transporter activity (Sato et al. 1995). To rule out this possibility, oocytes were treated with 0.5 µm staurosporine for 10 min to inhibit protein kinase C activity before addition of arachidonic acid; under these conditions arachidonic acid still caused a 56 ± 2% reduction in transport activity. Arachidonyl ethyl ester is an inactive structural analogue of arachidonic acid, and produces similar effects to arachidonic acid on membrane fluidity (Trotti et al. 1995). Application of 100 µm arachidonyl ethyl ester had no significant effect on glycine transport current inhibition (Fig. 2b). Thus, the inhibitory effects of arachidonic acid on glycine transport are not solely due to alteration of the lipid environment and appear to be mediated by a direct interaction between the fatty acid and the transporter protein.

The inhibition of GLYT1a transport currents caused by 30 µm arachidonic acid was compared with equal concentrations of the unsaturated fatty acids, oleic and docosahexaenoic acids. Oleic acid, which contains one cis double bond in its backbone structure, reduced glycine transport currents by 30 ± 4%, and docosahexaenoic acid, which contains six double bonds in its carbon backbone, inhibited glycine transport currents by 64 ± 6% (n = 5) compared to 55 ± 5% (n = 5) inhibition by arachidonic acid, which contains four cis double bonds (Fig. 2c). These results suggest that the degree of unsaturation, and presumably the flexibility of the fatty acid, is an important determinant of the extent of inhibition of glycine transport currents.

Anandamide and R1-methanandamide stimulate glycine transport

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Methods
  6. Electrophysiological recordings
  7. [3H]Glycine uptake assays
  8. Analysis of kinetic data
  9. Results
  10. Arachidonic acid inhibits glycine transport
  11. Anandamide and R1-methanandamide stimulate glycine transport
  12. Modulation of [3H]glycine uptake by C6 glioma cells
  13. Discussion
  14. Acknowledgements
  15. References

An alternative approach to characterizing the specificity of the interaction between fatty acids and the transporter is to investigate the effects of the related endogenous compounds arachidonyl ethanolamide (also termed anandamide) and 2-arachidonyl glycerol, and also the related synthetic compounds R1-methanandamide and the anandamide transport inhibitor AM404. In contrast to the effects of arachidonic acid and the various other free fatty acids, application of anandamide, R1-methanandamide or 2-arachidonyl glycerol stimulated glycine transport currents. Application of increasing doses of R1-methanandamide or anandamide in the presence of a fixed dose of 100 µm glycine caused dose-dependent increases in transport currents, with EC50 values of 18 ± 6 µm for R1-methanandamide and 13 ± 6 µm for anandamide, and maximal levels of stimulation of 1.67 ± 0.06 fold and 1.56 ± 0.06 fold (Fig. 3b) respectively, over glycine alone. 2-Arachidonyl glycerol also stimulated the current, but to a lesser extent (1.07 ± 0.03 fold stimulation with 100 µm). Dose-dependent glycine transport currents were measured in the presence and absence of 100 µm anandamide and 100 µm R1-methanandamide. Although there was clear stimulation of the maximal current, in both cases there was no significant change in EC50 for glycine (Fig. 3c). In addition, 100 µm R1-methanandamide caused a significant increase in the rate of [3H]glycine uptake by oocytes expressing GLYT1a (Fig. 3d).

image

Figure 3. Anandamide stimulates glycine transport currents. (a) Current trace from a representative oocyte, voltage clamped at − 60 mV, in response to application of 100 µm glycine (gly) (solid bar) followed by co-application of 100 µm glycine and 30 µm R1-methanandamide (R1-AEA) (open bar). (b) Dose–response curves for anandamide (AEA; •) and R1-methanandamide (R1-AEA; ▪) stimulation of transport currents elicited by 100 µm glycine and measured at − 60 mV. Points represent mean ± SEM (n = 6 cells). Curves represent data fitted to the modified Michaelis–Menten equation. (c) Dose–response curve of glycine-elicited currents at − 60 mV (▪) and in the presence of 100 µm R1-methanandamide (◆). Points represent mean ± SEM from six cells. Curves represent data fitted to the modified Michaelis–Menten equation. (d) Effects of R1-methanandamide on the uptake of [3H]glycine by oocytes expressing GLYT1a. Mean uptake of 20 µm[3H]glycine in the absence (Gly) and presence of 100 µm R1-methanandamide (Gly + R1-AEA). Non-specific uptake was determined by conducting parallel experiments on H2O-injected control oocytes. Variation in expression levels is observed between batches of oocytes; the data represent pooled data from three separate batches (10 oocytes in each condition for each batch) and are normalized to the rates of uptake measured in the absence of R1-methanandamide. Results are mean ± SEM. *p < 0.05 versus gly (two-tailed Student's t-test). (e) Application of 50 µm WIN 55 212–2 with 30 µm glycine (Gly + WIN) had no effect on the glycine transport current compared with 30 µm glycine alone (Gly). Data are mean ± SEM (n = 3).

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Anandamide is a potent agonist of the cannabinoid receptor CB1 and it is possible that the stimulation of GLYT1a transport currents is due to a second messenger-mediated effect following CB1 receptor stimulation. We ruled out this possibility by applying the CB1 receptor agonist WIN 55 212–2 to oocytes expressing GLYT1a. WIN 55 212–2 had no effect on oocytes when applied alone and also had no effect on glycine transport currents when co-applied with glycine (Fig. 3e). Although there have been no reports of endogenous CB1 receptors in X. laevis oocytes, application of WIN 55 212–2, under similar conditions to those used here, to oocytes injected with CB1 receptors does elicit biological responses (McAllister et al. 1999). Therefore we can be confident that the stimulation of glycine transport by anandamide is most likely to be mediated by a direct effect on the transporter and not indirectly by stimulation of CB1 receptors.

Modulation of [3H]glycine uptake by C6 glioma cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Methods
  6. Electrophysiological recordings
  7. [3H]Glycine uptake assays
  8. Analysis of kinetic data
  9. Results
  10. Arachidonic acid inhibits glycine transport
  11. Anandamide and R1-methanandamide stimulate glycine transport
  12. Modulation of [3H]glycine uptake by C6 glioma cells
  13. Discussion
  14. Acknowledgements
  15. References

C6 glioma cells express high-affinity glycine transporters (Zafra et al. 1990) and provide an alternative model system for investigating the properties of these transporters. Glycine may be transported by a number of high-affinity transporters including GLYT1, GLYT2, system ASC and system N. The proportion of glycine uptake by the GLYT1 subtypes in C6 glioma cells was first established by pharmacological methods. [3H]glycine uptake by C6 glioma cells was measured in the presence and absence of saturating doses of the GLYT1-selective transport inhibitor NFPS (Aubrey and Vandenberg 2001; Atkinson et al. 2001) and the GLYT1-selective substrate inhibitor sarcosine (Kim et al. 1994; Lopez-Corcuera et al. 1998). NFPS reduced [3H]glycine uptake by C6 gliomas to 35 ± 8% and sarcosine reduced uptake to 39 ± 5% compared with the uptake measured in the absence of these compounds (Fig. 4), demonstrating that approximately 65% of glycine transport under these conditions is due to GLYT1 transporters.

image

Figure 4. Arachidonic acid inhibits, and R1-methanandamide stimulates, glycine transport in C6 glioma cells. C6 glioma cells were treated with either 1 µm NFPS, 300 µm sarcosine (sarc), 10 µm arachidonic acid (AA) or 10 µm R1-methanandamide (R1-AEA) and [3H]glycine (gly) uptake measured. Data presented are relative to control values and are mean ± SEM values from five separate wells of confluent cells for each treatment. The data presented are from a representative experiment. The experiment was repeated three times and similar results were obtained in each case.

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We also used C6 glioma cells to investigate whether glycine transport by endogenous transporters is also modulated by arachidonic acid and anandamide, as observed for Xenopus oocytes over-expressing recombinant GLYT1 transporters. Arachidonic acid (10 µm), with indomethacin and NDGA to inhibit metabolism of arachidonic acid, reduced the rate of [3H]glycine uptake to 40 ± 6% of control values (Fig. 4). R1-methanandamide (10 µm), in the presence of the cannabinoid receptor antagonist SR141617A (3 µm), which has been reported to completely block endogenous cannabinoid receptor stimulation in C6 cells (Sanchez et al. 1998; Jacobsson et al. 2001), caused a 28 ± 2% stimulation of the rate of [3H]glycine uptake (Fig. 4). Although arachidonic acid inhibits, and anandamide stimulates, glycine transport by C6 cells, the extent of inhibition, and stimulation, does not precisely correspond to that observed in X. laevis oocytes.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Methods
  6. Electrophysiological recordings
  7. [3H]Glycine uptake assays
  8. Analysis of kinetic data
  9. Results
  10. Arachidonic acid inhibits glycine transport
  11. Anandamide and R1-methanandamide stimulate glycine transport
  12. Modulation of [3H]glycine uptake by C6 glioma cells
  13. Discussion
  14. Acknowledgements
  15. References

The GLYT1 subtypes of transporter are abundantly expressed in glial cells surrounding excitatory synapses and it has been postulated that these transporters regulate glycine concentrations within the synapse (Berger et al. 1998; Bergeron et al. 1998; Roux and Supplisson 2000). Therefore, mechanisms that modulate GLYT1 activity would be expected to modulate synaptic glycine concentrations, glycine site occupancy of NMDA receptors and excitatory neurotransmission. In this study we have demonstrated that arachidonic acid acutely inhibits GLYT1 activity through a direct interaction with the transporter, whereas the related compound, anandamide, directly and rapidly stimulates GLYT1 activity. These conclusions are based on a number of observations. Inhibition of transport by arachidonic acid is likely to be due to a direct interaction with the transporter because (i) either bath application of arachidonic acid or activation of PLA2 to produce arachidonic acid produced similar levels of inhibition of transport; (ii) various inhibitors of arachidonic acid metabolism did not influence the extent of inhibition; and (iii) non-metabolized derivatives of arachidonic acid, such as docosahexaenoic acid, generated similar levels of inhibition of transport as arachidonic acid. In the case of anandamide stimulation of the transporter, a direct effect is also the most likely explanation of its effect on transport. The structurally related, but more stable, compound R1-methanandamide had similar effects to anandamide on glycine transport in oocytes expressing GLYT1a, but the structurally unrelated CB1 receptor agonist WIN 55 212–2 had no effect, which suggests that indirect CB1 receptor-mediated effects can be ruled out. Furthermore, R1-methanandamide also stimulated [3H]glycine transport in C6 glioma cells in the presence of the CB1 receptor antagonist, SR141617A. Finally, the time course for inhibition by arachidonic acid, or stimulation by anandamide, was rapid, with the full effects apparent after 30 s to 1 min, which is most easily explained by a direct interaction with the transporter and unlikely to be a result of changes in second messenger systems that may be modulated by arachidonic acid or anandamide.

The inhibitory effects of arachidonic acid and stimulatory effects of anandamide are likely to be at least partially mediated by alterations in the transporter–lipid interface. These compounds are lipid soluble and the inhibition of transport by arachidonic acid is only partially reversible, which could be explained by only partial removal of arachidonic acid from a binding site on the transporter that is also exposed to lipids in the cell membrane. A related observation is that the degree of saturation of other lipid molecules appeared to influence the degree of inhibition observed (Fig. 2), which indicates that the degree of flexibility of the hydrophobic tail group of the lipid may be important in determining accessibility to the arachidonic acid binding site at the transporter–lipid interface. Finally, the effects of arachidonic acid are likely to be a result of a specific interaction between the transporter and the lipid, and not due to an artefact of the Xenopus laevis oocyte expression system because inhibition of transport was also observed for GLTY1 expressed in C6 glioma cells (Fig. 4 and also see Zafra et al. 1990). However, it should be noted that differences in the extent of inhibition of transport by arachidonic acid in the two cell systems are observed. Possible explanations for these differences are that differences in lipid composition of C6 cells compared with oocytes may influence accessibility of arachidonic acid for a lipid transporter site and that other glycine transport systems present in C6 cells may also be modulated by arachidonic acid to cause a different level of inhibition of total glycine transport into these cells.

The opposing actions of arachidonic acid and anandamide raise the question of the mechanism of action of these compounds. The lipid compounds tested in this study may be broadly described as having a hydrophobic tail group of varying degrees of saturation and a small hydrophilic head group with different chemical groups. As such, all of the compounds have a propensity to equilibrate rapidly within the cell membrane. A number of explanations are possible from these considerations. The hydrophobic tail portion of the lipid may serve to anchor the compound in the membrane, and the degree of saturation of the lipid may be an important factor in regulating the rate of diffusion within the membrane or the range of conformations that can associate effectively with the transporter. Once the lipid is equilibrated in the membrane it may diffuse through the membrane until the head group comes into contact with a specific site on the transporter and influence the rate of conformational change required for the translocation of glycine through the transporter protein. The different chemical head groups may interact in different ways with the transporter; the carboxylic acid group of arachidonic acid might restrict the conformational changes required for transport, whereas the amide moiety of anandamide might facilitate the conformational changes and increase the rate of glycine translocation.

An important consideration when predicting the physiological or pathological implications of these results is the concentrations of arachidonic acid and anandamide likely to be present in vivo. Although it is difficult precisely to measure concentrations of these compounds, some clues as to the concentrations reached may be obtained from estimates of the affinities of these compounds for other receptors and transporters directly involved in arachidonic acid and anandamide biology. Arachidonic acid has been reported to modulate the activity of a wide range of membrane proteins at concentrations up to 1 mm. However, it is highly unlikely that such high free acid concentrations will be reached under physiological or even pathological conditions because the critical micelle concentration for arachidonic acid is approximately 30 µm, and concentrations above this value are likely to cause non-specific membrane disruption. However, concentrations up to 30 µm are likely based on the affinity estimates of arachidonic acid binding to cyclo-oxygenases and lipoxygenases (reviewed by Attwell et al. 1993). The concentrations of arachidonic acid required to inhibit glycine transport (IC50 = 2.1 µm) are therefore well within the expected physiological concentration range. However, it should be noted that at the highest concentrations used in this study, the effects of arachidonic acid were only partially reversible. Although partial irreversible inhibition of transport is unlikely to be physiological, this process may take place under pathological conditions, when higher concentrations of arachidonic acid are likely to be produced and cause prolonged increases in synaptic glycine concentrations. The EC50 for anandamide stimulation of cannabinoid receptors has been estimated to be within the range of 100 nm to 1 µm (Zygmunt et al. 2000) and the EC50 for anandamide transport by various cell types is within the range of 300 nm to 40 µm (Hillard 2000). The concentration of anandamide required to stimulate GLYT1 activity (EC50 = 13–18 µm) is at the higher end of the possible physiological concentration range and as such the endogenous effects of anandamide on GLYT1 may only be apparent under conditions of increased anandamide release. It should be noted that concentrations of anandamide required to modulate other receptors and ion channels (Vaughan et al. 2000; Christopoulos and Wilson 2001) are comparable to or even higher than those required to stimulate GLYT1.

Anandamide modulation of neurotransmitter transporter activity has not been reported previously, but arachidonic acid does modulate the activity of other Na+/Cl-dependent neurotransmitter transporters, in particular the dopamine transporter (reviewed by Reith et al. 1997). Arachidonic acid causes both short- and long-term changes in dopamine transporter function. Long-term effects of arachidonic acid (45–60 min exposure) are likely to result from changes in surface expression levels (Zhang and Reith 1996) and as such are likely to be mediated by very different processes to those observed in this study, in which 1–5-min exposures to arachidonic acid were measured. However, short-term exposure to arachidonic acid causes an increase in dopamine transport activity (Zhang and Reith 1996) and also causes acute changes in the electrophysiological properties of dopamine transporters (Ingram and Amara 2000). Although the modulatory effects of arachidonic acid on dopamine transporters are different to those observed in this study, these two studies indicate that both classes of neurotransmitter transporters interact directly with arachidonic acid and it is an intriguing possibility that similar binding sites for arachidonic acid may exist.

Arachidonic acid has been suggested to play an important physiological role in the induction of long-term potentiation (Williams et al. 1989) and also in the pathogenesis of excitotoxicity (Kolko et al. 1996). Arachidonic acid modulates the properties of a variety of membrane proteins within, or in close proximity to, excitatory synapses. Arachidonic acid potentiates the action of glutamate at NMDA receptors (Miller et al. 1992; Casado and Ascher 1998), inhibits AMPA receptor activity (Chabot et al. 1998; Kovalchuk et al. 1994), and both inhibitory and stimulatory effects on glutamate transport have been reported (Barbour et al. 1989; Volterra et al. 1992, 1994; Zerangue et al. 1995; Fairman et al. 1998; Tzingounis et al. 1998). The inhibitory actions of arachidonic acid on GLYT1 are also consistent with a stimulatory effect on excitatory synaptic transmission. Arachidonic acid may increase synaptic glycine concentrations and increase glycine site occupancy of NMDA receptors, and thereby provide an additional positive feedback mechanism. Anandamide also modulates the activity of various proteins in the glutamatergic synapse. Anandamide inhibits voltage-dependent Ca2+ channels (Mackie et al. 1993), stimulates K+ channels (Maingret et al. 2001), and stimulation of cannabinoid receptors leads to a reduction in NMDA receptor activity (Hampson et al. 1998). As such, if anandamide is released into the synaptic cleft in sufficient concentrations its effects are likely to suppress excitatory neurotransmission. The actions of arachidonic acid and anandamide on GLYT1 are therefore likely to have important roles in modulating the plasticity of glutamatergic neurotransmission.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Methods
  6. Electrophysiological recordings
  7. [3H]Glycine uptake assays
  8. Analysis of kinetic data
  9. Results
  10. Arachidonic acid inhibits glycine transport
  11. Anandamide and R1-methanandamide stimulate glycine transport
  12. Modulation of [3H]glycine uptake by C6 glioma cells
  13. Discussion
  14. Acknowledgements
  15. References
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