Potentiating Effects Of Ethanol Research Articles

X-Ray Structures of an Interfacial Potentiating Site for Alcohols and Anesthetics in a Pentameric Ligand-Gated Ion Channel

Alcohol consumption produces a variety of undesirable behavioural and physiological effects in animals and humans and can eventually lead to addiction. Converging evidences suggest that its molecular mechanisms of action involve specific protein targets. Among these, pentameric ligand-gated ion channels (pLGICs) and especially GABA-A Receptor have been shown to be one of the main targets of ethanol in the central nervous system. Here we report the first atomic-resolution structure at 2.8 A of ethanol bound to a member of the pLGIC family, the pH-gated prokaryotic homolog GLIC variant F14'A. This GLIC variant is potentiated by concentrations of ethanol similar to the ones effective in the vertebrate Glycine and GABA-A receptors (R. Howard et al., 2012). Comparison the ethanol-bound and apo structures of GLIC F14'A gives a rational and simple explanation to the potentiating effect of ethanol on these receptors by stabilizing the open form. Multiple-sequence alignments and homology structure models suggest that the ethanol binding-pocket identified in GLIC is also present in human Glycine and GABA-A receptors.

The potentiating effects of ethanol on response of aortic strips to stimulant drugs.

The potentiating effects of ethanol on response of aortic strips to stimulant drugs.

The Mechanism for Prejunctional Enhancement of Neuromuscular Transmission by Ethanol in the Mouse

Ethanol has been shown to have both presynaptic and postsynaptic effects on synaptic transmission. However, the mechanisms by which ethanol affects evoked neurotransmitter release have not been studied at the mouse neuromuscular junction, a synapse at which binomial analysis of neurotransmitter release and measurements of prejunctional ionic currents can be made. Ethanol (400 mM) increased neurotransmitter release independently of both the cAMP and phorbol ester/Munc13 signaling pathways. Binomial analysis of neurotransmitter release revealed that ethanol increases the average probability of secretion without an effect on the immediately available store of the neurotransmitter. Application of ethanol also resulted in an inhibition of potassium currents in the motor nerve endings. These results suggest that the potentiating effects of ethanol on neurotransmitter release at the skeletal neuromuscular junction are mediated by an inhibition of the delayed rectifier potassium current, thus increasing both calcium entry into the nerve ending and the probability of neurotransmitter release. Identifying the mechanism through which ethanol enhances neurotransmitter release at the neuromuscular junction may be useful in determining the processes underlying the enhancement of neurotransmitter release at other synapses.

Ethanol potentiates dopamine uptake and increases cell surface distribution of dopamine transporters expressed in SK-N-SH and HEK-293 cells

Ethanol increases dopaminergic release in the reward and reinforcement areas of the brain. The primary protein responsible for terminating dopamine (DA) neurotransmission is the plasma membrane-bound dopamine transporter (DAT). In vitro electrophysiological and biochemical studies in Xenopus laevis oocytes have previously shown ethanol potentiates DAT function and increases transporter-binding sites. The potentiating effect of ethanol on the transporter is eliminated in Xenopus oocytes by the DAT mutation glycine 130 to threonine. However, ethanol's action on DAT functional regulation has yet to be examined in mammalian cell expression systems. To further understand the molecular mechanisms of ethanol's action on DAT, we determined the direct mechanistic action of short-term (≤2 h) ethanol exposure on transporter function and cell surface distribution in non-neuronal human embryonic kidney cells-293 (HEK-293) and neuronal SK-N-SH neuroblastoma cells expressing the transporter. Wild-type or G130T mutant DAT were overexpressed in HEK-293 and SK-N-SH cells. Ethanol potentiated DAT mediated [ 3H]DA uptake in a dose (25, 50, 100 mM), but not time dependent manner in cells expressing wild-type DAT. Ethanol-induced potentiation of uptake was significantly reduced in cells expressing the G130T mutant. Analysis of DA uptake kinetic parameters indicates 100-mM ethanol exposure increased [ 3H]DA uptake velocity ( V max), while affinity for DA ( K m) remained unchanged. The effect of ethanol on wild-type DAT surface expression was measured by biotinylation cell surface labeling. DAT surface expression increased 40%–50% after 1-h, 100-mM ethanol exposure. These studies show ethanol potentiates DAT functional regulation in both neuronal and non-neuronal cells, suggesting a direct mechanistic action of ethanol on transporter trafficking in mammalian systems. Our findings demonstrate ethanol's action on DAT function and regulation is consistent across multiple model systems.

Ethanol Potentiation of GABAergic Synaptic Transmission May Be Self-Limiting: Role of Presynaptic GABABReceptors

Ethanol enhances GABAergic synaptic inhibition, and this interaction contributes to many of the behavioral and cognitive effects of this drug. Most studies suggest that ethanol enhances GABAergic neurotransmission via an allosteric potentiation of the postsynaptic GABA(A) receptors that mediate fast synaptic inhibition in the mammalian CNS. Despite widespread acceptance of this hypothesis, direct support for such a mechanism has been difficult to obtain. Ethanol does not enhance GABA(A) receptor function in all brain regions or under all experimental conditions, and factors responsible for this variability remain mostly unknown. Notably, blockade of GABA(B) receptors dramatically enhances ethanol potentiation of hippocampal GABA(A) IPSPs and IPSCs, suggesting that some unknown GABA(B) receptor mechanism limits the overall potentiating effect of ethanol on GABAergic synapses. In this study, we demonstrate that, at perisomatic synapses in the rat hippocampus, ethanol enhances presynaptic GABA(B) autoreceptor function and that this interaction reduces the overall potentiating effect of ethanol at these synapses. We further show that ethanol significantly elevates basal presynaptic GABA(B) receptor tone, possibly via an increase in spontaneous GABA release, and that pretreatment with a subthreshold concentration of the GABA(B) receptor agonist baclofen blocks ethanol but not flunitrazepam or pentobarbital potentiation of GABA(A) IPSCs. These data suggest that an interaction between ethanol and presynaptic GABA(B) autoreceptor activity regulates the ethanol sensitivity of GABAergic synapses. Given that the in vitro ethanol sensitivity of these synapses correlates with in vivo ethanol responsiveness in a number of rodent lines, our data further suggest that presynaptic GABA(B) receptor activity may play a role in regulating behavioral sensitivity to ethanol.

Phosphotidylethanol mimics ethanol modulation of p42/44 mitogen-activated protein kinase signalling in hepatocytes.

Although long-term exposure of hepatocytes to ethanol results in agonist-selective potentiation of p42/44 mitogen-activated protein kinase (MAPK) activation, mediators of this effect of ethanol are not known. We examined the role of phosphatidylethanol (PEth), a novel phospholipid formed exclusively in the presence of ethanol. PEth accumulated in primary cultures of rat hepatocytes treated with ethanol. Exogenously added PEth potentiated angiotensin II-stimulated p42/44 MAPK similarly to that observed with ethanol treatment of cells for 24 h, a condition where PEth accumulates. PEth levels remained elevated 2 h after ethanol removal subsequent to a 24-h exposure, and the potentiating effects of ethanol were also present. PEth did not potentiate p42/44 MAPK activation by either epidermal growth factor or vasopressin, thus further mimicking the known agonist selectivity for this ethanol effect. These results offer a novel role for PEth as a mediator in the ethanol modulation of p42/44 MAPK cascade in hepatocytes.

Low dose ethanol potentiates indomethacin induced inhibition of wound re-epithelialization in duodenal monolayers

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce gastroduodenal mucosal injury and ulceration, and delay ulcer healing. In contrast, the effects of low dose ethanol in induction of gastroduodenal mucosal injury, and the subsequent wound repair remains unclear. The aim of this study was to determine, using an in-vitro duodenal epithelial wound model, whether low clinically relevant doses of ethanol or indomethacin interfere with the wound re-epithelialization of duodenal epithelial monolayers. The possible potentiating effect of ethanol on indomethacin modulation of duodenal re-epithelialization was also examined. In-vitro epithelial wounds were created in confluent IEC-6 duodenal epithelial monolayers by a razor blade scrape. Ethanol at low concentrations (0.25, 0.5, 0.75%) did not have significant effect on duodenal wound re-epithelialization. Similarly, low doses of indomethacin (.01, .05, 0.1 mM) also did not have a significant effect on wound re-epithelialization. However, the combination of ethanol (0.5 or 0.75%) and indomethacin (0.1mM) produced a marked inhibition of IEC-6 re-epithelialization. At the low doses used, ethanol and indomethacin (individually or in combination) did not have direct cytotoxic effect on IEC-6 cells. Ethanol or indomethacin (at the studied concentrations) had only minimal effect on the actin stress fibers in the cells at the migration front. However, in combination, they almost completely abolished the actin stress fibers at the migration front. These findings demonstrate that while low clinically relevant doses of ethanol and indomethacin individually do not affect re-epithelialization of wounded duodenal epithelial monolayers, in combination they produce a significant inhibition.

Ethanol preexposure attenuates the interaction of ethanol and cocaine in taste aversion learning

Although the potentiating effects of ethanol and cocaine have been well documented, little has been reported regarding the effects of ethanol or cocaine history on this interaction. In the present study, female Long–Evans rats received five exposures to ethanol (3.5 g/kg ip) or vehicle prior to taste aversion conditioning in which a novel saccharin solution was paired with either ethanol (0.56 g/kg ip), cocaine (25 mg/kg sc) or the combination (or the drugs' vehicle) for a total of five conditioning trials. Nonpreexposed subjects conditioned with the ethanol/cocaine combination displayed aversions, drinking levels significantly less than nonpreexposed subjects conditioned with either drug alone. Further, the aversions produced by the combination were greater than the sum of the aversions produced by ethanol and cocaine, alone. Ethanol-preexposed animals conditioned with the combination displayed an attenuated aversion, drinking significantly greater amounts of saccharin than nonpreexposed conditioned subjects and not differing from controls. Although the basis for the attenuation by ethanol of the aversions induced by the drug combination is not known, the present findings may have implications for the use and abuse of the combination in that alcohol history may reduce the subsequent toxicity of the combination that in turn may affect its acceptability.

Bombesin Promotes Synergistic Stimulation of DNA Synthesis by Ethanol and Insulin in Fibroblasts

In NIH 3T3 fibroblasts and several other cellular systems, ethanol (50–80 mM) was previously shown to greatly enhance the mitogenic effects of insulin particularly in the presence of zinc. Here we report that in NIH 3T3 fibroblasts the combined stimulatory effects of ethanol and insulin on DNA synthesis can be further increased by bombesin both in the absence and presence of zinc. Bombesin also enhanced insulin-plus-ethanol-induced DNA synthesis in mouse Swiss 3T3 and Balb/c 3T3 fibroblasts, but in these cells bombesin was effective only in the presence of zinc. In NIH 3T3 fibroblasts, the potentiating effects of ethanol on insulin-induced DNA synthesis by the zinc-dependent and bombesin-dependent mechanisms were additive. Wortmannin, an inhibitor of phosphatidylinositol 3′-kinase (PI3K), prevented the comitogenic effect of ethanol in the presence of bombesin but not in the presence of zinc. Furthermore, bombesin, but not ethanol, was found to enhance the stimulatory effect of insulin on PI3K activity. Rapamycin, an indirect inhibitor of p70 S6 kinase actions, inhibited the comitogenic effects of ethanol in the presence of both zinc and bombesin. However, only ethanol, but not bombesin, enhanced the stimulatory effect of insulin on p70 S6 kinase activity; this effect of ethanol was zinc-dependent. Neither ethanol nor bombesin enhanced the stimulatory effects of insulin on the phosphorylation (activation) of p38/p42/p44 mitogen-activated protein kinases. The results suggest that in mouse fibroblasts maximal stimulation of DNA synthesis by physiologically relevant concentrations of ethanol occurs if both PI3K and p70 S6 kinase are activated. These data suggest a mechanism by which ethanol may affect growth in affected human tissues during its tumor promoting actions.

The role of Src kinase in the potentiation by ethanol of cytokine- and endotoxin-mediated nitric oxide synthase expression in rat hepatocytes.

This study demonstrates that exposure of primary rat hepatocytes or mouse BNL Cl.2 liver cell line to ethanol causes potentiation of tumor necrosis factor-alpha (TNF-alpha)- and lipopolysaccharide (LPS)-stimulated nitrite accumulation. The potentiating effect of ethanol (0.02-2 mM) appears to be time and concentration dependent. Consistent with nitrite production, the amount of inducible nitric oxide synthase (iNOS) mRNA and protein is initially detected at 4 hr after treatment with TNF-alpha/LPS/ethanol. Furthermore, the capability of these agents to induce iNOS expression is primarily determined by the age of the animals. Interestingly, antioxidants such as N-acetylcysteine (NAC), ascorbic acid, or alpha-tocopherol fail to inhibit TNF-alpha/LPS/ethanol-induced increase in iNOS protein. In addition, several kinase inhibitors, including staurosporine, genistein, curcumin, and herbimycin A, were used to examine their effects on this induction. Among them, only herbimycin A potently inhibits the accumulation of nitrite and iNOS expression. In vitro kinase assay verifies that Src tyrosine kinase is rapidly activated with a peak at 1 hr after treatment with TNF-alpha/LPS/ethanol but is not activated by these agents singly or doubly. As expected, herbimycin A can block Src kinase activity under circumstances in which iNOS expression is also inhibited. However, our results do not indicate that the mitogen-activated protein kinase is activated after treatment with these agents. The study results suggest that Src tyrosine kinase plays a prominent role in transducing the signal to induce iNOS expression in hepatocytes treated with TNF-alpha/LPS/ethanol.

Differential effects of ethanol on feline rage and predatory attack behavior: an underlying neural mechanism.

Previous studies have shown that, at certain dose levels, ethanol can exert a powerful, facilitatory effect on aggressive behavior in both animals and humans. In the cat, however, it was discovered that ethanol differentially alters two forms of aggression that are common to this species. Defensive rage behavior is significantly enhanced, whereas predatory attack behavior is suppressed by ethanol administration. One possible mechanism governing alcohol's potentiation of defensive rage behavior is that it acts on the descending pathway from the medial hypothalamus to the midbrain periaqueductal gray (PAG)-an essential pathway for the expression of defensive rage behavior that uses excitatory amino acids as a neurotransmitter. This hypothesis is supported by the finding that the excitatory effects of alcohol on defensive rage behavior are blocked by administration of the N-methyl-D-aspartate antagonist alpha-2-amino-7-phosphoheptanoic acid (AP-7) when microinjected into the periaqueductal gray, a primary neuronal target of descending fibers from the medial hypothalamus that mediate the expression of defensive rage behavior. Thus, the present study establishes for the first time a specific component of the neural circuit for defensive rage behavior over which the potentiating effects of ethanol are mediated.

Ethanol enhances GABA A receptor-activated chloride currents in chick cerebral cortical neurons

Primary cultures of cerebral cortical neurons were prepared from 7- to 8-day-old chick embryos. The effect of ethanol on GABA-activated membrane current was examined using whole-cell voltage-clamp recording in cells maintained for 3–25 days in vitro. In approximately 60% of neurons examined ethanol caused a potentiation of the membrane current elicited by GABA. The threshold concentration of ethanol was 1 mM, and the potentiating effect of ethanol on GABA-activated currents was maximal at 10 mM. In many cells higher concentrations (40–50 mM) of ethanol inhibited GABA-activated currents. These effects of ethanol were all reversible.

The Potentiating Effects of Ethanol on the Blue Light Depolarization of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is the site of two major effects of ethanol. In humans, ethanol produces a slow damped oscillation in the steady electrical potential of the eye, which is generated primarily by the RPE. It has also been shown that ethanol potentiates the reversible, depolarizing effect of blue light on the transepithelial potential (TEP) of the isolated RPE. The present study demonstrates that in addition to the depolarizing effect of blue light on the TEP, a secondary, compensatory potential arises, which functions to maintain the TEP. The magnitude of the secondary response varies somewhat among preparations. It appears that ethanol eliminates or reduces the secondary, compensatory potential change which results in a large depolarization of the TEP when the RPE is irradiated with blue light. Microelectrode studies reveal that ethanol hyperpolarizes both the apical and basal membranes of the RPE with a greater effect noted in the apical membrane. This would account for the corneal positive potential elicited by ethanol in the human eye. Brief exposures (2-3 min) of blue light, after administration of 0.5% ethanol, results in a large (25-30 mV) depolarization of both membrane potentials as compared with 3 to 5 mV in untreated tissue. On the basis of our observations, it is hypothesized that some of the potentiating effects of ethanol in combination with other agents may result from an interference with a cellular adaptive response to impaired respiration rather than an additive effect on a common mechanism.

Ethanol potentiates serotonin stimulated inositol lipid metabolism in primary astroglial cell cultures

Serotonin-stimulated activation of phospholipase C in primary astroglial cell cultures was studied as a mean of evaluating the effect of acute ethanol exposition on this signal transduction system. The addition of 50–150 mM ethanol prior to stimulation with 10 −5M serotonin led to a potentiation of the serotonin-induced [ 3H]-inositol phosphate formation and an increased incorporation of [ 3H]-inositol into the three phosphoinositides studied. This potentiating effect of ethanol was observed only when ethanol was added together with serotonin. No stimulatory effect ofethanol perse was found. Furthermore, ethanol had no effect on arginin-vasopressin, bradykinin or phenylephrine stimulated inositol lipid metabolism.