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Ethanol/naltrexone interactions at the mu-opioid receptor. CLSM/FCS study in live cells.

Vukojević V, Ming Y, D'Addario C, Rigler R, Johansson B, Terenius L - PLoS ONE (2008)

Bottom Line: Compared to the action of specific ligands at MOP, ethanol-induced effects show complex kinetics and point to a biphasic underlying mechanism.Pretreatment with naloxone or naltrexone considerably mitigates the effects of ethanol.Naltrexone exerts opposite effects on MOP sorting at the plasma membrane, thereby countering the effects of ethanol.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden. vladana.vukojevic@ki.se

ABSTRACT

Background: Alcoholism is a widespread chronic disorder of complex aetiology with a significant negative impact on the individual and the society. Mechanisms of ethanol action are not sufficiently well understood at the molecular level and the pharmacotherapy of alcoholism is still in its infancy. Our study focuses at the cellular and molecular level on ethanol-induced effects that are mediated through the micro-opioid receptor (MOP) and on the effects of naltrexone, a well-known antagonist at MOP that is used clinically to prevent relapse in alcoholism.

Methodology/principal findings: Advanced fluorescence imaging by Confocal Laser Scanning Microscopy (CLSM) and Fluorescence Correlation Spectroscopy (FCS) are used to study ethanol effects on MOP and plasma membrane lipid dynamics in live PC12 cells. We observed that relevant concentrations of ethanol (10-40 mM) alter MOP mobility and surface density, and affect the dynamics of plasma membrane lipids. Compared to the action of specific ligands at MOP, ethanol-induced effects show complex kinetics and point to a biphasic underlying mechanism. Pretreatment with naloxone or naltrexone considerably mitigates the effects of ethanol.

Conclusions/significance: We suggest that ethanol acts by affecting the sorting of MOP at the plasma membrane of PC12 cells. Naltrexone exerts opposite effects on MOP sorting at the plasma membrane, thereby countering the effects of ethanol. Our experimental findings give new insight on MOP-mediated ethanol action at the cellular and molecular level. We suggest a new hypothesis to explain the well established ethanol-induced increase in the activity of the endogenous opioid system.

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Related in: MedlinePlus

Effects of ethanol and opioid receptor agonists/antagonists on the MOP surface density in live PC12 cells.A. Confocal fluorescence images showing subcellular localization of MOP-EGFP (green) in PC12 cells under control conditions (left), upon 3 h treatment with DAMGO (1.0 µM; middle) or naloxone (100 nM; right). EGFP fluorescence was excited using the 488 nm line of the Ar laser. Fluorescence emitted in the range 505–540 nm was collected. B. Schematic drawing of a PC12 cell, showing the location of the observation volume element during FCS measurements. FCS measurements were always performed on the apical side of the plasma membrane. C. Typical autocorrelation curve for MOP-EGFP in not stimulated PC12 cells. FCS measurements were performed and analyzed as described in the Materials and Methods section. The dots give the experimental autocorelation curve; the smooth curve is a theoretical autocorrelation curve derived using a two-component model for free 2D-diffusion (eq. 3). Two fractions of MOP-EGFP were identified that could be distinguished by differences in lateral mobility, τD1 = (250±150) µs and τD2 = (2.5±1.5) ms. The majority of MOP-EGFP was characterized by fast mobility, f1 = (0.7±0.2). The amplitude of the autocorrelation curve is reciprocally proportional to the average number of MOP-EGFP molecules in the observation volume element (eq. 3). Autocorrelation curves are the basis for the calculation of relative changes in receptor surface densities as in the graph below. D. Relative changes in MOP-EGFP surface density under stimulation with selected drugs: ethanol (stars), naltrexone (diamonds), naloxone (dots), morphine (triangles) and DAMGO (squares). Selective ligands at MOP caused monotonous increase/decrease of MOP-EGFP surface density. Naloxone and naltrexone, acting as antagonists at MOP monotonously increased the MOP-EGFP surface density. The agonists DAMGO and morphine induced rapid internalization of MOP-EGFP, characterized by an internalization half-time t1/2,agonists = 2.5 min. Ethanol induced an abrupt transient increase in MOP-EGFP surface density, followed by partial internalization of MOP-EGFP, with an apparent internalization half-time of t1/2,ethanol = 25 min.
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pone-0004008-g001: Effects of ethanol and opioid receptor agonists/antagonists on the MOP surface density in live PC12 cells.A. Confocal fluorescence images showing subcellular localization of MOP-EGFP (green) in PC12 cells under control conditions (left), upon 3 h treatment with DAMGO (1.0 µM; middle) or naloxone (100 nM; right). EGFP fluorescence was excited using the 488 nm line of the Ar laser. Fluorescence emitted in the range 505–540 nm was collected. B. Schematic drawing of a PC12 cell, showing the location of the observation volume element during FCS measurements. FCS measurements were always performed on the apical side of the plasma membrane. C. Typical autocorrelation curve for MOP-EGFP in not stimulated PC12 cells. FCS measurements were performed and analyzed as described in the Materials and Methods section. The dots give the experimental autocorelation curve; the smooth curve is a theoretical autocorrelation curve derived using a two-component model for free 2D-diffusion (eq. 3). Two fractions of MOP-EGFP were identified that could be distinguished by differences in lateral mobility, τD1 = (250±150) µs and τD2 = (2.5±1.5) ms. The majority of MOP-EGFP was characterized by fast mobility, f1 = (0.7±0.2). The amplitude of the autocorrelation curve is reciprocally proportional to the average number of MOP-EGFP molecules in the observation volume element (eq. 3). Autocorrelation curves are the basis for the calculation of relative changes in receptor surface densities as in the graph below. D. Relative changes in MOP-EGFP surface density under stimulation with selected drugs: ethanol (stars), naltrexone (diamonds), naloxone (dots), morphine (triangles) and DAMGO (squares). Selective ligands at MOP caused monotonous increase/decrease of MOP-EGFP surface density. Naloxone and naltrexone, acting as antagonists at MOP monotonously increased the MOP-EGFP surface density. The agonists DAMGO and morphine induced rapid internalization of MOP-EGFP, characterized by an internalization half-time t1/2,agonists = 2.5 min. Ethanol induced an abrupt transient increase in MOP-EGFP surface density, followed by partial internalization of MOP-EGFP, with an apparent internalization half-time of t1/2,ethanol = 25 min.

Mentions: Cells derived from the rat pheochromocytoma (PC12) cell line were stably transformed to express the mu-opioid receptor (MOP) tagged with the Enhanced Green Fluorescent Protein (EGFP). The model is described and characterized in details in reference [22], where it was verified that the cloned construct MOP-EGFP exhibits properties similar to the native MOP–that the construct inserted in the plasma membrane, is sensitive and selective to its specific ligands and coupled to the cellular trafficking machinery (Fig. 1A).


Ethanol/naltrexone interactions at the mu-opioid receptor. CLSM/FCS study in live cells.

Vukojević V, Ming Y, D'Addario C, Rigler R, Johansson B, Terenius L - PLoS ONE (2008)

Effects of ethanol and opioid receptor agonists/antagonists on the MOP surface density in live PC12 cells.A. Confocal fluorescence images showing subcellular localization of MOP-EGFP (green) in PC12 cells under control conditions (left), upon 3 h treatment with DAMGO (1.0 µM; middle) or naloxone (100 nM; right). EGFP fluorescence was excited using the 488 nm line of the Ar laser. Fluorescence emitted in the range 505–540 nm was collected. B. Schematic drawing of a PC12 cell, showing the location of the observation volume element during FCS measurements. FCS measurements were always performed on the apical side of the plasma membrane. C. Typical autocorrelation curve for MOP-EGFP in not stimulated PC12 cells. FCS measurements were performed and analyzed as described in the Materials and Methods section. The dots give the experimental autocorelation curve; the smooth curve is a theoretical autocorrelation curve derived using a two-component model for free 2D-diffusion (eq. 3). Two fractions of MOP-EGFP were identified that could be distinguished by differences in lateral mobility, τD1 = (250±150) µs and τD2 = (2.5±1.5) ms. The majority of MOP-EGFP was characterized by fast mobility, f1 = (0.7±0.2). The amplitude of the autocorrelation curve is reciprocally proportional to the average number of MOP-EGFP molecules in the observation volume element (eq. 3). Autocorrelation curves are the basis for the calculation of relative changes in receptor surface densities as in the graph below. D. Relative changes in MOP-EGFP surface density under stimulation with selected drugs: ethanol (stars), naltrexone (diamonds), naloxone (dots), morphine (triangles) and DAMGO (squares). Selective ligands at MOP caused monotonous increase/decrease of MOP-EGFP surface density. Naloxone and naltrexone, acting as antagonists at MOP monotonously increased the MOP-EGFP surface density. The agonists DAMGO and morphine induced rapid internalization of MOP-EGFP, characterized by an internalization half-time t1/2,agonists = 2.5 min. Ethanol induced an abrupt transient increase in MOP-EGFP surface density, followed by partial internalization of MOP-EGFP, with an apparent internalization half-time of t1/2,ethanol = 25 min.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2602977&req=5

pone-0004008-g001: Effects of ethanol and opioid receptor agonists/antagonists on the MOP surface density in live PC12 cells.A. Confocal fluorescence images showing subcellular localization of MOP-EGFP (green) in PC12 cells under control conditions (left), upon 3 h treatment with DAMGO (1.0 µM; middle) or naloxone (100 nM; right). EGFP fluorescence was excited using the 488 nm line of the Ar laser. Fluorescence emitted in the range 505–540 nm was collected. B. Schematic drawing of a PC12 cell, showing the location of the observation volume element during FCS measurements. FCS measurements were always performed on the apical side of the plasma membrane. C. Typical autocorrelation curve for MOP-EGFP in not stimulated PC12 cells. FCS measurements were performed and analyzed as described in the Materials and Methods section. The dots give the experimental autocorelation curve; the smooth curve is a theoretical autocorrelation curve derived using a two-component model for free 2D-diffusion (eq. 3). Two fractions of MOP-EGFP were identified that could be distinguished by differences in lateral mobility, τD1 = (250±150) µs and τD2 = (2.5±1.5) ms. The majority of MOP-EGFP was characterized by fast mobility, f1 = (0.7±0.2). The amplitude of the autocorrelation curve is reciprocally proportional to the average number of MOP-EGFP molecules in the observation volume element (eq. 3). Autocorrelation curves are the basis for the calculation of relative changes in receptor surface densities as in the graph below. D. Relative changes in MOP-EGFP surface density under stimulation with selected drugs: ethanol (stars), naltrexone (diamonds), naloxone (dots), morphine (triangles) and DAMGO (squares). Selective ligands at MOP caused monotonous increase/decrease of MOP-EGFP surface density. Naloxone and naltrexone, acting as antagonists at MOP monotonously increased the MOP-EGFP surface density. The agonists DAMGO and morphine induced rapid internalization of MOP-EGFP, characterized by an internalization half-time t1/2,agonists = 2.5 min. Ethanol induced an abrupt transient increase in MOP-EGFP surface density, followed by partial internalization of MOP-EGFP, with an apparent internalization half-time of t1/2,ethanol = 25 min.
Mentions: Cells derived from the rat pheochromocytoma (PC12) cell line were stably transformed to express the mu-opioid receptor (MOP) tagged with the Enhanced Green Fluorescent Protein (EGFP). The model is described and characterized in details in reference [22], where it was verified that the cloned construct MOP-EGFP exhibits properties similar to the native MOP–that the construct inserted in the plasma membrane, is sensitive and selective to its specific ligands and coupled to the cellular trafficking machinery (Fig. 1A).

Bottom Line: Compared to the action of specific ligands at MOP, ethanol-induced effects show complex kinetics and point to a biphasic underlying mechanism.Pretreatment with naloxone or naltrexone considerably mitigates the effects of ethanol.Naltrexone exerts opposite effects on MOP sorting at the plasma membrane, thereby countering the effects of ethanol.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden. vladana.vukojevic@ki.se

ABSTRACT

Background: Alcoholism is a widespread chronic disorder of complex aetiology with a significant negative impact on the individual and the society. Mechanisms of ethanol action are not sufficiently well understood at the molecular level and the pharmacotherapy of alcoholism is still in its infancy. Our study focuses at the cellular and molecular level on ethanol-induced effects that are mediated through the micro-opioid receptor (MOP) and on the effects of naltrexone, a well-known antagonist at MOP that is used clinically to prevent relapse in alcoholism.

Methodology/principal findings: Advanced fluorescence imaging by Confocal Laser Scanning Microscopy (CLSM) and Fluorescence Correlation Spectroscopy (FCS) are used to study ethanol effects on MOP and plasma membrane lipid dynamics in live PC12 cells. We observed that relevant concentrations of ethanol (10-40 mM) alter MOP mobility and surface density, and affect the dynamics of plasma membrane lipids. Compared to the action of specific ligands at MOP, ethanol-induced effects show complex kinetics and point to a biphasic underlying mechanism. Pretreatment with naloxone or naltrexone considerably mitigates the effects of ethanol.

Conclusions/significance: We suggest that ethanol acts by affecting the sorting of MOP at the plasma membrane of PC12 cells. Naltrexone exerts opposite effects on MOP sorting at the plasma membrane, thereby countering the effects of ethanol. Our experimental findings give new insight on MOP-mediated ethanol action at the cellular and molecular level. We suggest a new hypothesis to explain the well established ethanol-induced increase in the activity of the endogenous opioid system.

Show MeSH
Related in: MedlinePlus