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Relapse is the most serious limitation of effective medical treatment of opiate addiction. Opiate-related behaviors appear to be modulated by cannabinoid CB1 receptors (CB1) through poorly understood cross-talk mechanisms. Opiate and CB1 receptors are coexpressed in the nucleus accumbens (NAc) and dorsal striatum. These regions also have the highest density of adenosine [A.sub.2A] receptors ([A.sub.2A]) in the brain. We have been investigating the postsynaptic signaling mechanisms of/[micro]-opiate receptors (MORs) and CB1 receptors in primary NAc/striatal neurons. In this article, we present evidence that MOR and CB1 act synergistically on cAMP/PKA signaling in NAc/striatal neurons. In addition, we find that synergy requires adenosine and [A.sub.2A]. Importantly, an [A.sub.2A] antagonist administered either directly into the NAc or indirectly by i.p. injection eliminates heroin-induced reinstatement in rats trained to self-administer heroin, a model of human craving and relapse. These findings suggest that [A.sub.2A] antagonists might be effective therapeutic agents in the management of abstinent heroin addicts. PKA | addiction | nucleus accumbens | gene activation

The nucleus accumbens (NAc) is central to heroin addiction. Activation of opiate receptors in the NAc dissociates [G.sub.i/o] into [alpha] and [beta][gamma] subunits. G[[alpha].sub.i] inhibits cAMP production, but [beta][gamma] regulates several molecular pathways, including protein kinase A (PKA). We show in NAc/striatal neurons that opiates paradoxically activate PKA signaling by means of [beta][gamma] dimers. Activation requires G[[alpha].sub.i3] and an activator of G protein signaling 3 (AGS3). AGS3 competes with [beta][gamma] for binding to G[[alpha].sub.i3]-GDP and enhances the action of unbound/3% AGS3 and G[[alpha].sub.i3] knockdown prevents opiate activation of PKA signaling. In rats self-administering heroin, AGS3 antisense in the NAc core, but not shell, eliminates reinstatement of heroin-seeking behavior, a model of human relapse. Thus, G[[alpha].sub.i3]/[beta][gamma]/AGS3 appears to mediate [mu] opiate receptor activation of PKA signaling as well as heroin-seeking behavior. [mu] opiate receptor | nucleus accumbens core | addiction | [beta][gamma] subunits | G[[alpha].sub.i3]

The mesolimbic dopamine system and cAMP-dependent/protein kinase A (PKA) pathways are strongly implicated in addictive behaviors. Here we determine the role of dopamine D2 receptors (D2) in PKA signaling responses to [delta]-opioid (DOR) and cannabinoid (CB1) receptors. We find in NG108-15/D2 cells and in cultured primary neurons that a brief exposure to saturating concentrations of DOR and CB1 agonists increases cAMP, promotes PKA C[delta] translocation and increases cAMP-dependent gene expression. Activation of PKA signaling is mediated by Gi-/[beta][gamma] dimers. Importantly, subthreshold concentrations of DOR or CB1 agonists with D2 agonists, which ate without effect when added separately, together activate cAMP/PKA signaling synergistically. There is also synergy between DOR of CB1 with ethanol, another addicting agent. In all instances, synergy requires adenosine activation of adenosine A2 receptors and is mediated by [beta][gamma] dimers. Synergy by this molecular mechanism appears to confer hypersensitivity to opioids and cannabinoids while simultaneously increasing the sensitivity of D2 signaling when receptors are expressed on the same cells. This mechanism may account, in part, for drug-induced activation of medium spiny neurons in the nucleus accumbens.

We recently showed that adenoviral transfer and expression of the [Lps.sup.d]/Ran gene isolated from endotoxin-resistant C3H/HeJ mice could protect endotoxin-sensitive mice from endotoxic shock. Elevation of proinflammatory cytokines, such as tumor necrosis factor [Alpha] (TNF-[Alpha]), is thought to be essential for the development of septic shock. To investigate the extent to which [Lps.sup.d]/Ran affects TNF-[Alpha] production, we transduced primary macrophages from endotoxin-sensitive and -resistant mice with adenoviral vectors expressing the wild-type and the mutant Lps/Ran cDNAs and other control genes, and compared the amount of TNF-[Alpha] produced by these various transduced macrophages. Successful transfer and expression of [Lps.sup.d]/Ran cDNA in endotoxin-sensitive C3H/HeOuJ macrophages reduced TNF-[Alpha] production upon lipopolysaccharide (LPS) stimulation, as compared with macrophages transduced with vectors expressing the wild-type [Lps.sup.n]/Ran cDNA, the green fluorescent protein gene, or the lacZ gene. On the other hand, successful transfer and expression of the wild-type [Lps.sup.n]/Ran cDNA in primary macrophages from endotoxin-resistant C3H/HeJ mice failed to induce TNF-[Alpha] production to any significant extent unless a very high LPS concentration was used. Given our previous demonstration that [Lps.sup.n]/Ran functions effectively in restoring LPS responsiveness in B cells from C3H/HeJ mice, we conclude that Lps/Ran is involved in a CD14-independent signal transduction pathway. This dominant negative down-regulation by [Lps.sup.d]/Ran on TNF-[Alpha] production by macrophages and probably other innate immune responses may be key to the development of an effective gene therapy for endotoxic or septic shock. gene therapy | septic shock | Ran GTPase | adenovirus

C3H/HeJ inbred mice are defective in that they are highly resistant to endotoxic shock as compared with normal responder mice. Their B cells and macrophages do not respond significantly when exposed to lipopolysaccharide (LPS), whereas cells from the responder mice do. Using a functional assay, we previously isolated a cDNA, which encodes for Ran/TC4 GTPase. We now show that this gene is mutated in C3H/HeJ mice, which accounts for their resistance to endotoxin stimulation. Sequence analysis of independent mutant [Lps.sup.d]/Ran cDNAs isolated from splenic B cells of C3H/HeJ mice reveals a consistent single base substitution at position 870, where a thymidine is replaced with a cytidine. In situ hybridization maps the [Lps.sup.d]/Ran cDNA to mouse chromosome 4. By retroviral gene transfer, the wild-type [Lps.sup.n]/Ran cDNA but not the mutant [Lps.sup.d]/Ran cDNA can restore LPS responsiveness of C3H/HeJ cells. Adenoviral gene transfer in vivo with the mutant [Lps.sup.d]/Ran cDNA but not the wild-type [Lps.sup.n]/Ran cDNA rescues endotoxin-sensitive mice from septic shock. Thus Lps/Ran is an important target for LPS-mediated signal transduction, and the [Lps.sup.d]/Ran gene may be useful as a therapeutic sequence in gene therapy for endotoxemia and septic shock.

The mesolimbic dopamine system and cAMP-dependent/protein kinase A (PKA) pathways are strongly implicated in addictive behaviors. Here we determine the role of dopamine D2 receptors (D2) in PKA signaling responses to delta-opioid (DOR) and cannabinoid (CB1) receptors. We find in NG108-15/D2 cells and in cultured primary neurons that a brief exposure to saturating concentrations of DOR and CB1 agonists increases cAMP, promotes PKA C alpha translocation and increases cAMP-dependent gene expression. Activation of PKA signaling is mediated by Gi-beta gamma dimers. Importantly, subthreshold concentrations of DOR or CB1 agonists with D2 agonists, which are without effect when added separately, together activate cAMP/PKA signaling synergistically. There is also synergy between DOR or CB1 with ethanol, another addicting agent. In all instances, synergy requires adenosine activation of adenosine A2 receptors and is mediated by beta gamma dimers. Synergy by this molecular mechanism appears to confer hypersensitivity to opioids and cannabinoids while simultaneously increasing the sensitivity of D2 signaling when receptors are expressed on the same cells. This mechanism may account, in part, for drug-induced activation of medium spiny neurons in the nucleus accumbens.