NAC-1 is a brain POZ/BTB protein that can prevent cocaine-induced sensitization in the rat.

Levels of the mRNA NAC-1 are increased in the rat forebrain weeks after cocaine exposure. This long-term neuroadaptation occurs during the expression of behavioral sensitization, a model of psychostimulant-induced paranoia. NAC-1, the protein encoded by this cocaine-regulated mRNA, contains a Pox virus and zinc finger/bric-a-brac tramtrack broad complex (POZ/BTB) motif, which mediates interactions among several transcriptional regulators. The present studies demonstrate that NAC-1 acts as a transcription factor. NAC-1 was localized to the nucleus of neurons in the brain. Transfection of NAC-1 in cell culture repressed transcription of a reporter gene. NAC-1 was also able to affect the actions of other POZ/BTB proteins in mammalian two-hybrid studies; these interactions required the presence of the POZ/BTB domain. However, NAC-1 appears to be a unique POZ/BTB transcriptional regulator because it does not contain any zinc finger regions found in these other DNA-binding proteins. Adenoviral-mediated overexpression of NAC-1 protein in the rat nucleus accumbens prevented the development but not the expression of behavioral sensitization produced by repeated administration of cocaine. Thus, NAC-1 may modify the long-term behaviors of psychostimulant abuse by regulating gene transcription in the mammalian brain.

Regulation of the Na+/K(+)-ATPase pump in vitro after long-term exposure to cocaine: role of serotonin.

Long-term exposure to cocaine can cause persistent behavioral changes and alterations in neuronal function. One cocaine-regulated mRNA in the rat brain is the beta-1 subunit of the Na+/K(+)-ATPase pump. We examined both Na+/K(+)-ATPase function and expression after cocaine treatment of pheochromocytoma cells. One-hour exposure to cocaine did not alter Na+/K(+)-ATPase activity, as measured by the ouabain-sensitive component of rubidium uptake. Four days of cocaine resulted in an approximately 30% decrease in Na+/K(+)-ATPase activity. Western blot analyses demonstrated an approximately 25% decrease in levels of the beta-1 isoform, without changes in pump total alpha subunit levels. Treatment with dopamine type 1 or type 2 receptor agonists for the same period did not affect Na+/K(+)-ATPase activity. The serotonin-selective reuptake inhibitor paroxetine caused an approximately 45% decrease in rubidium uptake after 4 days, whereas pump function was not altered after treatment with either the dopamine-selective reuptake blocker nomifensine or the norepinephrine-selective reuptake blocker desipramine. Chronic treatment with both cocaine and LY 278,584, a serotonin type 3 receptor antagonist, did not replicate the cocaine-associated decrease in pump function. Long-term cocaine exposure regulates expression and function of the Na+/K(+)-ATPase pump in neuronal-like cells; this regulation is mediated in part via the serotonin type 3 receptor. Similar Na+/K(+)-ATPase pump regulation in vivo may selectively alter neuronal function in the mammalian brain.

NAC-1, a rat brain mRNA, is increased in the nucleus accumbens three weeks after chronic cocaine self-administration.

Chronic cocaine use leads to biochemical and behavioral changes that can persist for weeks to months after drug administration is discontinued. Alterations in gene expression in the mammalian CNS may contribute to these long-term neural consequences of cocaine abuse. A combined in situ transcription-PCR amplification strategy was used to isolate a novel mRNA, NAC-1, from the nucleus accumbens of rats 3 weeks after discontinuing 3 weeks of intravenous cocaine self-administration. In rats that self-administered cocaine, levels of NAC-1 were increased approximately 50% in the nucleus accumbens but not in the dorsal striatum or hippocampus, when compared with levels from yoked-saline controls. In situ hybridization analysis demonstrated increased numbers of NAC-1-expressing cells in the nucleus accumbens of rats who had self-administered cocaine. NAC-1 mRNA exists as one form, approximately 4400 nucleotides (nt) in size, and also is present at much lower amounts in non-neural tissues. A full-length cDNA clone was isolated from a whole brain library. The predicted polypeptide sequence contains a POZ domain in the first 120 amino acids; the same POZ domain sequence mediates protein-protein interactions among some transcriptional regulators. NAC-1 mRNA levels were also increased in the nucleus accumbens 1 week after 6 d of noncontingent cocaine treatments. Regulation of NAC-1 mRNA in the nucleus accumbens demonstrates a long-term effect of cocaine use on cellular function that may be relevant in behavioral sensitization or cocaine self-administration.

Opioid peptide receptor studies. 4. Antisense oligodeoxynucleotide to the delta opioid receptor delineates opioid receptor subtypes.

Prior work in our laboratory has identified putative subtypes of delta (delta cx-1, delta cx-2, delta ncx-1, delta ncx-2) and kappa 2 (kappa 2a and kappa 2b) receptors. Previous studies showed that chronic (three day) i.c.v. administration of antisense oligodeoxynucleotide to the cloned delta opioid receptor selectively decreased [3H][D-Ala2,D-Leu5]enkephalin binding to the delta ncx site, not the delta cx-2 site. The present study extends this work by demonstrating that delta antisense DNA selectively affects the delta ncx-2 site sparing the other putative delta receptor subtypes and kappa 2 receptor subtypes. This selectivity is not due to anatomically specific effects of delta antisense DNA since autoradiograms show that delta binding is reduced in all regions of the brain after chronic i.c.v. administration of delta antisense DNA. These data strongly suggest that the delta cx-1, delta cx-2, delta ncx-1, kappa 2a and kappa 2b binding sites are different proteins than the delta ncx-2 binding site, which, based on its sensitivity to delta antisense DNA, is synonymous to the cloned delta opioid receptor. Viewed collectively, these data suggest that administration of delta antisense DNA, and by extension other receptor-selective antisense DNA, is a powerful approach to distinguishing between postulated receptor subtypes.

Probes for narcotic receptor-mediated phenomena. 20. Alteration of opioid receptor subtype selectivity of the 5-(3-hydroxyphenyl)morphans by application of the message-address concept: preparation of delta-opioid receptor ligands.

Derivatives of racemic and optically active 5-(3-hydroxyphenyl)-2-methylmorphan (5-(3-hydroxyphenyl)-2-methyl-2-azabicyclo[3.3.1]nonane, 1) were synthesized containing additional aromatic moieties, as an application of the message-address concept targeted at producing delta-opioid receptor selective ligands. In vitro radioreceptor binding studies in rat brain revealed that both of the parent enantiomers, (-)- and (+)-1, had a high affinity for the mu-opioid receptor (21 nM), a slight affinity for kappa 1-opioid receptors (approximately 800-900 nM), and less than 1000 nM affinity for the delta-opioid receptor (mu/delta IC50 ratio of < 0.02 for both). A derivative of (-)-1 containing an indole moiety fused at the C6-C7 position of the phenylmorphan nucleus, (-)-11, displayed a > 180-fold increase in affinity for the delta-opioid receptor with an IC50 value of 6 nM. The parent compound (-)-1 had only 26% agonist activity at 30 microM in the mouse vas deferens (delta) bioassay, whereas compound (-)-11 had an IC50 of 393 nM in this preparation, indicating the importance of the indole moiety in imparting delta-opioid agonist activity to the phenylmorphan (-)-11. A structure-activity relationship (SAR) study of N-alkyl derivatives of the racemic nor 11 indicated similarities between the interaction of various derivatives with the mu- and delta- but not the kappa 1-opioid receptor. As studies on the molecular basis of the interaction of opioid ligands with their respective receptors continue to gain momentum, the SAR data described herein for the synthetic phenylmorphans will prove useful for further studies.

Opioid peptide receptor studies. 1. Identification of a novel delta-opioid receptor binding site in rat brain membranes.

Our laboratory was among the first to propose the existence of delta receptor subtypes: a delta site thought to be associated with a mu-delta-opioid receptor complex termed the delta cx binding site and delta site not associated with the mu-delta-opioid receptor complex, termed the delta ncx site. In previous studies, we assayed the delta cx site with [3H][D-Ala2,D-Leu5]enkephalin using rat brain membranes depleted of delta ncx sites by pretreatment with the site-directed acylating agent, (+)-trans-SUPERFIT. In the present study, we investigated, using (+)-trans-SUPERFIT-pretreated membranes, the possibility of heterogeneity of the delta cx binding site. Two sites were resolved: the delta cx-1 site at which mu ligands are potent noncompetitive inhibitors and delta ligands are weak competitive inhibitors, and the delta cx-2 site where delta ligands are potent and mu ligands are weak, mixed competitive-noncompetitive inhibitors. Although the delta cx-2 site has a delta-like ligand-selectivity profile, several experiments distinguished it from the delta ncx site. Two lines of evidence suggest that the delta ncx site corresponds to the cloned delta receptor. One, the delta receptor was cloned from the NG108-15 cell line, and this receptor, like the delta ncx binding site, irreversibly binds SUPERFIT and (+)-trans-SUPERFIT. Secondly, administration of delta-antisense DNA selectively decreases delta ncx binding. Viewed collectively, the major finding of this study is the discovery of a novel SUPERFIT-insensitive and delta-antisense-insensitive delta cx-2 binding site.

A marked change of receptor affinity of the 2-methyl-5-(3-hydroxyphenyl)morphans upon attachment of an (E)-8-benzylidene moiety: synthesis and evaluation of a new class of sigma receptor ligands.

The (E)-8-benzylidene and (E)-8-(3,4-dichlorobenzylidene), 7-ketone derivatives, 5 and 6, of the synthetic opiate 2-methyl-5-(3-hydroxyphenyl)morphan [5-(3-hydroxyphenyl)-2-methyl-2-azabicyclo[3.3.1]nonane, 1], were synthesized from the 7-ketone derivatives 2 or 4 via the Claisen-Schmidt reaction. The corresponding enantiomers of 5 and 6 were obtained in > 99% optical purity from the optical isomers of 4, resolved with the O,O'-dibenzoyltartaric acids. The absolute configurations of the enantiomers of 4 were determined by conversion, via Clemmensen reduction, to the enantiomers of 1, the configurations of which are known. The determination of the regioisomer and configurational isomer of 5, with respect to the introduced benzylidene group, was determined from a single-crystal X-ray analysis. 1H NMR data was used to confirm that 6 possessed the same configuration as 5. Radioreceptor binding studies in rat and guinea pig brain preparations revealed that (-)-(1S,5S)-5 displayed an 11-fold decrease in affinity for the opioid mu receptor and an increase in affinity for sigma receptors of 81-fold (low nanomolar affinity) relative to the ketone precursor (+)-(1S,5S)-4. An analogous, albeit less dramatic, trend was seen with compound (-)-(1S,5S)-6. Compounds (-)-(1S,5S)-5 and (-)-(1S,5S)-6 are distinct from the typical sigma-opiates in that they have very low affinity for either PCP sites or muscarinic receptors. The high affinity and selectivity of these novel sigma receptor ligands suggests that they will be valuable for the elucidation of the functional roles of sigma receptors.