Zhan Jiang

Coordinated Movement of RACK1 with Activated βIIPKC

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 βIIPKC, determines the localization and functional activity of βIIPKC. However, the mechanism by which RACK1 localizes activated βIIPKC 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 thein situ association and movement of RACK1 and βIIPKC 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 βIIPKC is required for the movement of both proteins. Our results suggest that RACK1 is a PKC shuttling protein that moves βIIPKC from one intracellular site to another.

βγ Dimers Mediate Synergy of Dopamine D2 and Adenosine A2 Receptor-Stimulated PKA Signaling and Regulate Ethanol Consumption

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.

Adenosine A2a blockade prevents synergy between μ-opiate and cannabinoid CB1 receptors and eliminates heroin-seeking behavior in addicted rats

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 A2a receptors (A2a) in the brain. We have been investigating the postsynaptic signaling mechanisms of μ-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 A2a. Importantly, an A2a 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 A2a antagonists might be effective therapeutic agents in the management of abstinent heroin addicts.

Addicting drugs utilize a synergistic molecular mechanism in common requiring adenosine and Gi-βγ dimers

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 δ-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 Cα translocation and increases cAMP-dependent gene expression. Activation of PKA signaling is mediated by Gi-βγ 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 βγ 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.

Nav1.5-dependent persistent Na+ influx activates CaMKII in rat ventricular myocytes and N1325S mice

Late Na(+) current (I(NaL)) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are both increased in the diseased heart. Recently, CaMKII was found to phosphorylate the Na(+) channel 1.5 (Na(v)1.5), resulting in enhanced I(NaL). Conversely, an increase of I(NaL) would be expected to cause elevation of intracellular Ca(2+) and activation of CaMKII. However, a relationship between enhancement of I(NaL) and activation of CaMKII has yet to be demonstrated. We investigated whether Na(+) influx via Na(v)1.5 leads to CaMKII activation and explored the functional significance of this pathway. In neonatal rat ventricular myocytes (NRVM), treatment with the I(NaL) activators anemone toxin II (ATX-II) or veratridine increased CaMKII autophosphorylation and increased phosphorylation of CaMKII substrates phospholamban and ryanodine receptor 2. Knockdown of Na(v)1.5 (but not Na(v)1.1 or Na(v)1.2) prevented ATX-II-induced CaMKII phosphorylation, providing evidence for a specific role of Na(v)1.5 in CaMKII activation. In support of this view, CaMKII activity was also increased in hearts of transgenic mice overexpressing a gain-of-function Na(v)1.5 mutant (N(1325)S). The effects of both ATX-II and the N(1325)S mutation were reversed by either I(NaL) inhibition (with ranolazine or tetrodotoxin) or CaMKII inhibition (with KN93 or autocamtide 2-related inhibitory peptide). Furthermore, ATX-II treatment also induced CaMKII-Na(v)1.5 coimmunoprecipitation. The same association between CaMKII and Na(v)1.5 was also found in N(1325)S mice, suggesting a direct protein-protein interaction. Pharmacological inhibitions of either CaMKII or I(NaL) also prevented ATX-II-induced cell death in NRVM and reduced the incidence of polymorphic ventricular tachycardia induced by ATX-II in rat perfused hearts. Taken together, these results suggest that a Na(v)1.5-dependent increase in Na(+) influx leads to activation of CaMKII, which in turn phosphorylates Na(v)1.5, further promoting Na(+) influx. Pharmacological inhibition of either CaMKII or Na(v)1.5 can ameliorate cardiac dysfunction caused by excessive Na(+) influx.

Inhibition of aldehyde dehydrogenase-2 suppresses cocaine seeking by generating THP, a cocaine use-dependent inhibitor of dopamine synthesis

There is no effective treatment for cocaine addiction despite extensive knowledge of the neurobiology of drug addiction. Here we show that a selective aldehyde dehydrogenase-2 (ALDH-2) inhibitor, ALDH2i, suppresses cocaine self-administration in rats and prevents cocaine- or cue-induced reinstatement in a rat model of cocaine relapse-like behavior. We also identify a molecular mechanism by which ALDH-2 inhibition reduces cocaine-seeking behavior: increases in tetrahydropapaveroline (THP) formation due to inhibition of ALDH-2 decrease cocaine-stimulated dopamine production and release in vitro and in vivo. Cocaine increases extracellular dopamine concentration, which activates dopamine D2 autoreceptors to stimulate cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC) in primary ventral tegmental area (VTA) neurons. PKA and PKC phosphorylate and activate tyrosine hydroxylase, further increasing dopamine synthesis in a positive-feedback loop. Monoamine oxidase converts dopamine to 3,4-dihydroxyphenylacetaldehyde (DOPAL), a substrate for ALDH-2. Inhibition of ALDH-2 enables DOPAL to condense with dopamine to form THP in VTA neurons. THP selectively inhibits phosphorylated (activated) tyrosine hydroxylase to reduce dopamine production via negative-feedback signaling. Reducing cocaine- and craving-associated increases in dopamine release seems to account for the effectiveness of ALDH2i in suppressing cocaine-seeking behavior. Selective inhibition of ALDH-2 may have therapeutic potential for treating human cocaine addiction and preventing relapse.

Uncoupling of βIIPKC from its targeting protein RACK1 in response to ethanol in cultured cells and mouse brain

ABSTRACT Protein kinase C (PKC) is involved in many neuroadaptive responses to ethanol in the nervous system. PKC activation results in translocation of the enzyme from one intracellular site to another. Compartmentalization of PKC isozymes is regulated by targeting proteins such as receptors for activated C kinase (RACKs). It is possible, therefore, that ethanol‐induced changes in the function and compartmentalization of PKC isozymes could be due to changes in PKC targeting proteins. Here we study the response of the targeting protein RACK1 and its corresponding kinase βIIPKC to ethanol, and propose a novel mechanism to explain how ethanol modulates signaling cascades. In cultured cells, ethanol induces movement of RACK1 to the nucleus without affecting the compartmentalization of βIIPKC. Ethanol also inhibits βIIPKC translocation in response to activation. These results suggest that ethanol inhibition of βIIPKC translocation is due to miscompartmentalization of the targeting protein RACK1. Similar events occurred in mouse brain. In vivo exposure to ethanol caused RACK1 to localize to nuclei in specific brain regions, but did not affect the compartmentalization of βIIPKC. Thus, some of the cellular and neuroadaptive responses to ethanol may be related to ethanol‐induced movement of RACK1 to the nucleus, thereby preventing the translocation and corresponding function of βIIPKC.— Ron, D., Vagts, A. J., Dohrman, D. P., Yaka, R., Jiang, Z., Yao, L., Crabbe, J., Grisel, J. E., Diamond, I. Uncoupling of βIIPKC from its targeting protein RACK1 in response to ethanol in cultured cells and mouse brain. FASEB J. 14, 2303–2314 (2000)

Dopamine and ethanol cause translocation of epsilonPKC associated with epsilonRACK: cross-talk between cAMP-dependent protein kinase A and protein kinase C signaling pathways.

We found previously that neural responses to ethanol and the dopamine D2 receptor (D2) agonist 2,10,11-trihydroxy-N-propylnorapomorphine hydrobromide (NPA) involve both ϵ protein kinase C (ϵPKC) and cAMP-dependent protein kinase A (PKA). However, little is known about the mechanism underlying ethanol- and D2-mediated activation of ϵPKC and the relationship to PKA activation. In the present study, we used a new ϵPKC antibody, 14E6, that selectively recognized active ϵPKC when not bound to its anchoring protein ϵRACK (receptor for activated C-kinase), and PKC isozyme-selective inhibitors and activators to measure PKC translocation and catalytic activity. We show here that ethanol and NPA activated ϵPKC and induced translocation of both ϵPKC and its anchoring protein, ϵRACK to a new cytosolic site. The selective ϵPKC agonist, pseudo-ϵRACK, activated ϵPKC but did not cause translocation of the ϵPKC/ϵRACK complex to the cytosol. These data suggest a step-wise activation and translocation of ϵPKC after NPA or ethanol treatment, where ϵPKC first translocates and binds to its RACK and subsequently the ϵPKC/ϵRACK complex translocates to a new subcellular site. Direct activation of PKA by adenosine-3′,5′-cyclic monophosphorothioate, Sp-isomer (Sp-cAMPS), prostaglandin E1, or the adenosine A2A receptor is sufficient to cause ϵPKC translocation to the cytosolic compartment in a process that is dependent on PLC activation and requires PKA activity. These data demonstrate a novel cross-talk mechanism between ϵPKC and PKA signaling systems. PKA and PKC signaling have been implicated in alcohol rewarding properties in the mesolimbic dopamine system. Cross-talk between PKA and PKC may underlie some of the behaviors associated with alcoholism.

Up-Regulation of AGS3 during Morphine Withdrawal Promotes cAMP Superactivation via Adenylyl Cyclase 5 and 7 in Rat Nucleus Accumbens/Striatal Neurons

Effective medical treatment of opiate addiction is limited by a high relapse rate in abstinent addicts. Opiate withdrawal causes cAMP superactivation, but the underlying molecular mechanisms are not clear. Recent evidence implicates an activator of G-protein signaling 3 (AGS3) in opiate addiction. We found previously that during a 10-min activation of opioid receptors, AGS3 binds Gαi-GDP to promote free Gβγ stimulation of adenylyl cyclase (AC) 2 and 4, and/or inactivate Gαi inhibitory function, thereby transiently enhancing cAMP-dependent protein kinase A (PKA) activity. In contrast, we report here that in nucleus accumbens/striatal neurons, morphine withdrawal induces cAMP superactivation, which requires up-regulation of AGS3. cAMP increases as a function of withdrawal time, by approximately 20% at 10 min and 75% at 5 h. However, cAMP superactivation does not require Gβγ. Instead, adenosine A2A receptor activation of Gαs/olf seems to initiate cAMP superactivation and promote AGS3 up-regulation. Elevated AGS3 binds to Gαi to prevent its inhibition on AC activation. Moreover, withdrawal-induced increases in cAMP/PKA activate phospholipase C and ϵ protein kinase C to further stimulate AC5 and AC7, causing cAMP superactivation. Our findings identify a critical role for AC 5 and 7 and A2A receptors for up-regulation of AGS3 in morphine withdrawal-induced cAMP superactivation.

Intracellular Na+ overload causes oxidation of CaMKII and leads to Ca2+ mishandling in isolated ventricular myocytes.

An increase of late Na(+) current (INaL) in cardiac myocytes can raise the cytosolic Na(+) concentration and is associated with activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and alterations of mitochondrial metabolism and Ca(2+) handling by sarcoplasmic reticulum (SR). We tested the hypothesis that augmentation of INaL can increase mitochondrial reactive oxygen species (ROS) production and oxidation of CaMKII, resulting in spontaneous SR Ca(2+) release and increased diastolic Ca(2+) in myocytes. Increases of INaL and/or of the cytosolic Na(+) concentration led to mitochondrial ROS production and oxidation of CaMKII to cause dysregulation of Ca(2+) handling in rabbit cardiac myocytes.