Chronic ethanol inhibits the anandamide transport and increases extracellular anandamide levels in cerebellar granule neurons

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Abstract

Ethanol increases extracellular anandamide levels in neuronal cells. However, the molecular mechanisms by which this occurs are unknown. Chronic exposure of cerebellar granule neurons to ethanol increased the levels of anandamide accumulated in the cellular medium. Anandamide uptake was saturable and was inhibited (30% at 3 min) in response to chronic exposure to ethanol. Chronic ethanol treatment did not alter the Km, but significantly decreased Vmax of anandamide transport (33%) (P<0.0001). Fatty acid amide hydrolase activity was not affected by chronic ethanol treatment. Anandamide transport processes are independent of cannabinoid CB1 receptor, as cannabinoid CB1 receptor knockout mice exhibited time-dependent anandamide transport and cannabinoid CB1 receptor antagonists did not alter the effects of chronic ethanol on anandamide transport. Furthermore, anandamide transport was inhibited by acute ethanol in a time- and dose-dependent manner. Interestingly, acute ethanol-induced inhibition of anandamide transport was abolished in neurons exposed to chronic ethanol, suggesting that the anandamide transport processes may play a role in the development of long-term cellular tolerance to ethanol.

Introduction

Chronic exposure to ethanol has been shown to modulate the endogenous cannabinoid (endocannabinoid) system in central nervous system (Basavarajappa and Hungund, 2002). Manipulation of endocannabinoid receptor function has been found to alter drinking behavior in rodents Arnone et al., 1997, Colombo et al., 1998, Colombo et al., 2002, Gallate and McGregor, 1999, Gallate et al., 1999, Rodriguez de Fonseca et al., 1999, Freedland et al., 2001, suggesting that endocannabinoid receptor system may be a potential mechanism for alcoholism. To date, two endogenous cannabinoid substances that mimic the pharmacological actions of Δ9-tetrahydrocannabinol, the active ingredient of marijuana and other synthetic agonists (Mechoulam and Fride, 1995), have been isolated and characterized. These include anandamide and 2-arachidonylglycerol Devane et al., 1992, Mechoulam et al., 1995, Stella et al., 1997. Both anandamide and 2-arachidonylglycerol have been shown to bind specifically to cannabinoid CB1 receptors in the brain Devane et al., 1992, Mechoulam et al., 1995, Sugiura et al., 1995. Recent evidence indicates that the cannabinoid CB1 receptor signal transduction system plays a role in the pharmacological actions of ethanol, including voluntary ethanol consumption Arnone et al., 1997, Basavarajappa et al., 1998, Colombo et al., 1998, Basavarajappa and Hungund, 1999a, Basavarajappa and Hungund, 1999b, Basavarajappa and Hungund, 2002, Gallate and McGregor, 1999, Gallate et al., 1999, Rodriguez de Fonseca et al., 1999. However, the exact molecular mechanism by which cannabinoid CB1 receptors regulate ethanol consumption remains unknown. Recently, we demonstrated that chronic exposure to ethanol enhances the formation of the endogenous cannabinoid CB1 receptor agonists anandamide and 2-arachidonylglycerol in neuronal cells Basavarajappa and Hungund, 1999a, Basavarajappa et al., 2000. In these studies, the majority of the synthesized anandamide and 2-arachidonylglycerol (85–90%) remained in the medium even after 72 h exposure to ethanol Basavarajappa and Hungund, 1999a, Basavarajappa et al., 2000. Anandamide has a variety of regulatory functions, including mediating retrograde signals from depolarized postsynaptic neurons to presynaptic terminals, thereby reducing neurotransmitter release (Ohno-Shosaku et al., 2001).

In mammalian cells, termination of anandamide signaling at the cannabinoid CB1 receptors occurs through an uptake mechanism that transports anandamide into the cell where it subsequently undergoes rapid degradation by fatty acid amidohydrolase Cravatt et al., 1996, Beltramo et al., 1997, Hillard et al., 1997, Piomelli et al., 1999. Current evidence suggests that anandamide uptake is a carrier-mediated process that is time- and temperature-dependent, saturable, and inhibited with unique pharmacologic agents Di Marzo et al., 1994, Beltramo et al., 1997, Hillard et al., 1997, Hillard and Jarrahian, 2000, Rakhshan et al., 2000. Fatty acid amidohydrolase and cannabinoid CB1 receptors that have been shown to be co-localized in brain may indicate fatty acid amidohydrolase's role in anandamide signaling and uptake (Egertova et al., 1998). Thus, chronic ethanol-induced increases in extracellular anandamide could result in a decrease in anandamide influx, an increase in anandamide efflux from the cell, and/or altered intracellular metabolism. In the present study, we have investigated the chronic and acute effects of ethanol on anandamide transport in cerebellar granular neurons. We found that chronic exposure to ethanol leads to increases in extracellular anandamide. After prolonged exposure to ethanol, cells become tolerant to these effects, such that anandamide uptake is no longer inhibited by ethanol. These data suggest that ethanol-induced inhibition of anandamide uptake may in part be responsible for ethanol-induced increase in extracellular anandamide.

Section snippets

Materials

All culture plastic supplies were purchased from Falcon Labware (VWR Scientific, -Piscataway, NJ). Basal Eagle's medium (BEM), heat-inactivated fetal calf serum (FCS), streptomycin, and penicillin solutions were obtained from Sigma (St. Louis, MO). Liquid scintillation cocktail (INSTA-FLUOR) was purchased from Packard (Meriden, CT). N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide) (SR 141716A) was a kind gift from Sanofi Pharmaceuticals, (Montpellier,

Extracellular anandamide

In our previous studies, ethanol-induced increases in the formation of [3H]anandamide in human neuroblastoma cells was shown to be dependent on the dose of ethanol and the duration of exposure (Basavarajappa and Hungund, 1999a). In these studies, most of the synthesized [3H]anandamide (60–70%) was released from the cells and accumulated in the media (Basavarajappa and Hungund, 1999a). In the present study, when cerebellar granular neurons were incubated with various concentration of ethanol for

Discussion

We have recently demonstrated that chronic ethanol exposure increases the extracellular levels of endocannabinoids in human neuroblastoma cells and in primary cultures of cerebellar granular neurons Basavarajappa and Hungund, 1999a, Basavarajappa et al., 2000. In the present study, we investigated the effect of ethanol on anandamide transport processes as a possible mechanism underlying ethanol-induced increases in extracellular anandamide levels.

Anandamide transport was higher in older

Acknowledgments

This study was supported in part by funds from New York State Psychiatric Institute and NIH grant, AA13003 and NARSAD independent investigator award.

References (44)

  • T. Ohno-Shosaku et al.

    Endogenous cannabinoids mediate retrograde signals from depolarized postsynaptic neurons to presynaptic terminal

    Neuron

    (2001)
  • R.L. Omeir et al.

    Arachidonoyl ethanolamide-[1,2-14C] as a substrate for anandamide amidase

    Life Sci.

    (1995)
  • P.K. Smith et al.

    Measurement of protein using bicinchoninic acid

    Anal. Biochem.

    (1985)
  • T. Sugiura et al.

    2-Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain

    Biochem. Biophys. Res. Commun.

    (1995)
  • N. Ueda et al.

    The fatty acid amide hydrolase (FAAH)

    Chem. Phys. Lipids

    (2000)
  • M. Arnone et al.

    Selective inhibition of sucrose and ethanol intake by SR 141716, an antagonist of central cannabinoid (CB1) receptors

    Psychopharmacology

    (1997)
  • B.S. Basavarajappa et al.

    Chronic ethanol increases the cannabinoid receptor agonist, anandamide and its precursor N-arachidonyl phosphatidyl ethanolamine in SK-N-SH cells

    J. Neurochem.

    (1999)
  • M. Beltramo et al.

    Functional role of high-affinity anandamide transport, as revealed by selective inhibition

    Science

    (1997)
  • E. Bligh et al.

    A rapid method of total lipid extraction and purification

    Can. J. Biochem. Physiol.

    (1959)
  • G. Colombo et al.

    Reduction of voluntary ethanol intake in ethanol-preferring sP rats by the cannabinoid antagonist SR-141716

    Alcohol Alcohol.

    (1998)
  • G. Colombo et al.

    Stimulation of voluntary ethanol intake by cannabinoid receptor agonists in ethanol-preferring sP rats

    Psychopharmacology (Berl)

    (2002)
  • B.F. Cravatt et al.

    Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides

    Nature

    (1996)
  • Cited by (0)

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