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Adenosine is an important mediator of ethanol intoxication. In vitro, ethanol stimulates adenosine signaling by inhibiting the type 1 equilibrative nucleoside transporter (ENT1), whereas chronic ethanol exposure downregulates ENT1. It is not known, however, whether ENT1 is important for ethanol intoxication or consumption in vivo. Here we report that ENT1-null mice show reduced hypnotic and ataxic responses to ethanol and greater consumption of alcohol as compared with their wild-type littermates. These features are associated with a decrease in adenosine tone, as measured indirectly as a reduction in A(1) receptor-mediated inhibition of glutamate excitatory postsynaptic currents (EPSCs) in the nucleus accumbens, leading to increased phosphorylation of CRE-binding protein (CREB) in the striatum. Treatment with an A(1) receptor agonist decreases EPSC amplitude and reduces ethanol consumption in ENT1-null mice. Our results indicate that ENT1 has a physiological role in ethanol-mediated behaviors and suggest that decreased A(1) adenosine receptor function promotes alcohol consumption.

Omegaven is associated with restoration of liver function in patients with SBS and advanced liver disease. Parenteral omega-3 fatty acids, such as Omegaven, have the potential to fundamentally alter the paradigm of neonatal SBS from one of early death or transplantation from liver failure to a more chronic disease. More children with SBS should achieve full enteral tolerance and those who do not have the capacity for intestinal adaptation should be able to survive and receive an intestinal graft when older.

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Recent advances in neuroscience have made it possible to investigate the pathophysiology of alcoholism at a cellular and molecular level. Evidence indicates that ethanol affects hormone- and neurotransmitter-activated signal transduction, leading to short-term changes in regulation of cellular functions and long-term changes in gene expression. Such changes in the brain probably underlie many of the acute and chronic neurological events in alcoholism. In addition, genetic vulnerability also plays a role in alcoholism and, perhaps, in alcoholic medical disorders.

Protein kinase C (PKC) isozymes move upon activation from one intracellular site to another. PKC-binding proteins, such as receptors for activated C kinase (RACKs), play an important role in regulating the localization and diverse functions of PKC isozymes. RACK1, the receptor for activated ␤IIPKC, determines the localization and functional activity of ␤IIPKC. However, the mechanism by which RACK1 localizes activated ␤IIPKC is not known. Here, we provide evidence that the intracellular localization of RACK1 changes in response to PKC activation. In Chinese hamster ovary cells transfected with the dopamine D2L receptor and in NG108-15 cells, PKC activation by either phorbol ester or a dopamine D2 receptor agonist caused the movement of RACK1. Moreover, PKC activation resulted in the in situ association and movement of RACK1 and ␤IIPKC to the same intracellular sites. Time course studies indicate that PKC activation induces the association of the two proteins prior to their co-movement. We further show that association of RACK1 and ␤IIPKC is required for the movement of both proteins. Our results suggest that RACK1 is a PKC shuttling protein that moves ␤IIPKC from one intracellular site to another.Specific intracellular localization of signaling proteins such as PKC 1 is important for the regulation of complex signal transduction cascades (1). PKC is a family of 10 isozymes that are localized to specific intracellular sites in unstimulated cells. Upon activation, each PKC isozyme moves to a different intracellular site (2). Localization of inactive or activated PKC isozymes is mediated, at least in part, by interaction with anchoring proteins (3, 4). For example, inactive PKC isozymes appear to be localized by binding to the scaffolding proteins AKAP-79 and gravin (5, 6). In contrast, activated PKC isozymes are localized by binding to receptors for activated C kinase (RACKs). RACK1 specifically binds the active form of ␤IIPKC (7, 8) thereby regulating PKC function (8 -12). In vitro, RACK1 binds PKC only in the presence of PKC activators and increases PKC kinase activity, presumably by stabilizing its active conformation (13). The RACK1 binding site on PKC is within the C2 region of the regulatory domain providing a direct protein-protein interaction (8). Indeed, RACK1 belongs to the WD40 family of proteins, and the WD40 motif is implicated in mediating protein-protein interactions (14). Furthermore, peptides derived from either PKC and/or RACK1 can alter PKC activity in vitro and in vivo (8,12,15,16).Although RACK1 binds activated PKC and is clearly important for PKC function, the mechanism by which RACK1 localizes ␤IIPKC to its site after activation is not understood. One prediction is that the anchoring protein RACK1 should always be localized to the same site that accepts ␤IIPKC after translocation. We therefore used confocal microscopy to determine whether RACK1 is co-localized with activated ␤IIPKC, whether RACK1 is localized to a specific organelle, and whether the intracellular localization of RACK1 changes...

Dopamine in the nucleus accumbens modulates both motivational and addictive behaviors. Dopamine D1 and D2 receptors are generally considered to exert opposite effects at the cellular level, but many behavioral studies find an apparent cooperative effect of D1 and D2 receptors in the nucleus accumbens. Here, we show that a dopamine-induced enhancement of spike firing in nucleus accumbens neurons in brain slices required both D1 and D2 receptors. One intracellular mechanism that might underlie cooperativity of D1 and D2 receptors is activation of specific subtypes of adenylyl cyclases by G-protein betagamma subunits (Gbetagamma) released from the Gi/o-linked D2 receptor in combination with Galpha(s)-like subunits from the D1 receptor. In this regard, dopaminergic enhancement of spike firing was prevented by inhibitors of protein kinase A or Gbetagamma. Furthermore, intracellular perfusion with Gbetagamma enabled D1 receptor activation but not D2 receptor activation to enhance spike firing. Finally, our data suggest that these pathways may increase spike firing by inhibition of a slow A-type potassium current. These results provide evidence for a novel cellular mechanism through which cooperative action of D1 and D2 receptors in the nucleus accumbens could mediate dopamine-dependent behaviors.

Dopamine release is activated by ethanol and addicting drugs, but molecular mechanisms linking dopaminergic signaling to neuronal responses and drinking behavior are poorly understood. We report that dopamine-D2 receptors induce PKA Calpha translocation and increase CRE-regulated gene expression. Ethanol also activates PKA signaling. Subthreshold concentrations of the D2 agonist NPA and ethanol, without effect alone, together cause synergistic PKA translocation and CRE-mediated gene transcription. D2 or adenosine A2 receptor blockade, pertussis toxin, Rp-cAMPS, or overexpression of dominant-negative peptides that sequester betagamma dimers prevent synergy. Importantly, overexpression of a betagamma inhibitor peptide in the nucleus accumbens strikingly reduces sustained alcohol consumption. We propose that synergy of D2 and A2 confers ethanol hypersensitivity and that betagamma dimers are required for voluntary drinking.