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Currently, there are no FDA approved medications for treatment of cocaine addiction underscoring the dire need to develop such a product. There is an accumulating body of evidence that l-tetrahydropalmatine (l-THP), a non-selective dopamine antagonist, can be used for the treatment of cocaine addiction. Indeed, the FDA recently approved its usage in a Phase I study in cocaine abusers and it was indispensable to develop a simple and sensitive method for the simultaneous determination of l-THP and cocaine in human plasma. We developed a UPLC-FLD method for quantitation of these molecules using an ACQUITY BEH C18 column (2.1 mm × 50mm, 1.7 μm) and a mobile phase that consisted of 10mM ammonium phosphate (pH=4.75), methanol, and acetonitrile (v:v:v, 78:16:6). Venlafaxine was used as the internal standard while hexane was used for the liquid-liquid extraction. The flow rate was 0.4 mL/min with fluorescence detection using an excitation wavelength of 230 nm and emission detection wavelength of 315 nm. This method was selective, linear and sensitive with a lower limit of quantification of 2.5 ng/mL for both cocaine and l-THP. The intra-day precision of cocaine and l-THP was

UNLABELLED Photodynamic therapy (PDT) is an effective local cancer treatment when aphotosensitizer is administered and the tumor is irradiated with light. We examined the effect of PDT using HMME as the photosensitizer, and the 630nm diode laser on human cholangiocarcinoma cell line QBC939. Cell viability was determined by MTT assay. The percentage of apoptotic cell was determined by flow cytometry following annexin V/PI staining. Two methods were used for the determination of apoptosis: terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling assay and laser scanning confocal microscope detection. The procaspase-3 and cytochrome cwere measured by western blot. In vitro PDT showed excellent cytotoxicity that was afunction of laser energy and drug concentration to the QBC939 cell lines. PDT-mediated cell death occurred predominantly by apoptosis in vitro. Furthermore, this treatment initiates early cytochrome crelease, followed by late procaspase-3 activation. Our study demonstrates that PDT using HMME and the diode laser induces apoptosis that is mediated by cytochrome crelease and caspase activation in human cholangiocarcinoma cell lines. It is expected that this therapy would be clinically useful for the treatment of patients with cholangiocarcinoma. KEYWORDS Hematoporphyrin monomethyl ether, photodynamic therapy, apoptosis, cholangiocarcinoma.

A general strategy for the design of dual labeled peptides was developed, and derivatives of the delta opioid receptor (DOR) selective antagonist TIPP (Tyr-Tic-Phe-PheOH) containing both an affinity label and biotin were prepared by solid-phase synthesis. Tyr-Tic-Phe-Phe(p-X)-Asp-NH(CH2CH2O)2-CH2CH2NH-biotin (where X = N=C=S or NHCOCH2Br) exhibit nanomolar DOR affinity. The ability to detect receptors labeled with these peptides following solubilization and SDS-PAGE demonstrate the applicability of this design approach for dual labeled peptide derivatives.

This study was to characterize the effects of a point-mutant at C-terminal of mu opioid receptor (MOR), namely MORT394A, in chronic opioid-induced cellular responses. After 18 h of exposure to [D-Ala 2 , N-Me-Phe 4 , Gly-ol 5 ] enkephalin (DAMGO), adenylyl cyclase (AC) superactivation, a hallmark for the cellular adaptive response after chronic opioid stimulation, was observed in the cells expressing wild-type receptor, but was totally abolished in the cells expressing MOR T394A. Receptor phosphorylation was also attenuated in cells with MOR T394A after prolonged preexposure to agonist. Furthermore, MAP kinase kinase-1 (MKKI) overexpression was able to rescue AC superactivation in cells with MOR T394A, but showed no effect in the wild-type MOR-expressing cells. These results indicated that the amino acid T394 at C-terminus of MOR played a critical role in chronic agonist-induced AC superactivation and receptor phosphorylation.

Protein kinase C interacting protein/histidine triad nucleotide binding protein 1 (PKCI/HINT1) is a member of the histidine triad protein family. Although this protein is widely expressed in the mammalian brain including mesocorticolimbic and mesostriatal regions, its physiological function in CNS remains unknown. Recent microarray studies reported decreased mRNA expression of PKCI/HINT1 in the frontal cortex of individuals with schizophrenia, suggesting the possible involvement of this protein in the pathophysiology of the disease. In view of the documented link between dopamine (DA) transmission and schizophrenia, the present study used behavioral and neurochemical approaches to examine the influence of constitutive PKCI/HINT1 deletion upon: (i) basal and amphetamine (AMPH)-evoked locomotor activity; (ii) DA dynamics in the dorsal striatum, and (iii) postsynaptic DA receptor function. PKCI/HINT1(-/-) (KO) mice displayed lower spontaneous locomotion relative to wild-type (WT) controls. Acute AMPH administration significantly increased locomotor activity in WT mice; nonetheless, the effect was enhanced in KO mice. Quantitative microdialysis studies revealed no alteration in basal DA dynamics in the striatum or nucleus accumbens of KO mice. The ability of acute AMPH to increase DA levels was unaltered indicating that function in presynaptic DA neurotransmission in these regions do not underlie the behavioral phenotype of KO mice. In contrast to WT mice, systemic administration of the direct-acting DA receptor agonist apomorphine (10 mg/kg) significantly increased locomotor activity in KO mice suggesting that postsynaptic DA function is altered in these animals. These results demonstrate an important role of PKCI/HINT1 in modulating the behavioral response to AMPH. Furthermore, they indicate that the absence of this protein may be associated with dysregulation of postsynaptic DA transmission.

The human mu-opioid receptor (HmuOR) is a G-protein coupled receptor that mediates analgesia, euphoria and other important central and peripheral neurological functions. In this study, we found in a yeast two-hybrid screen that a protein kinase C-interacting protein (PKCI) specifically interacts with the C terminus of HmuOR. The interaction of PKCI with HmuOR was recapitulated in Chinese hamster ovary cells that express the full-length HmuOR and PKCI proteins. The affinity of HmuOR for an opioid ligand and its ability to mediate the activation of a G-protein were not altered by their interaction. However, the association of PKCI with HmuOR reduced agonist-induced inhibition of adenylyl cyclase and suppressed HmuOR desensitization partially at the G protein level and completely at the adenylyl cyclase level. Furthermore, PMA-induced, but not DAMGO-induced, HmuOR phosphorylation was partially inhibited by the coexpression of PKCI, suggesting that PKCI exerts a selective regulatory effect on HmuOR signaling. This effect was specific to the mu-opioid receptor because delta-opioid receptor desensitization was unaffected by PKCI. In addition, behavioral studies revealed that both basal and morphine-induced analgesia were significantly enhanced in the mutant mice that lacked expression of PKCI gene, and these mice developed a greater extent of tolerance to morphine analgesia. Taken together, these results suggest that PKCI functions as a negative regulator in HmuOR desensitization, phosphorylation, and in mediating morphine analgesia.

Sugar amino acids (SAAs) bearing an amine group on the anomeric position are a challenging class of SAAs to synthesize due to the inherent instability of glycosylamines. We developed a novel synthetic strategy towards both furanoid and pyranoid δ-SAAs of this type, based on a Curtius rearrangement. The latter reaction, which is known to proceed with retention of configuration, was performed on carboxylic acids derived from the oxidation of glycosidic primary hydroxyls. Leu-enkephalin analogs were prepared by replacing the Gly-Gly moiety in the parent Leu-enkephalin pentapeptide with the furanoid and pyranoid δ-SAA. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

By metabolically labeling tissue slices from striatum and thalamus with [32P]orthophosphoric acid and immunoprecipitating the receptor with mu receptor-specific antiserum, we found that the endogenous mu receptor in the brain tissue did undergo phosphorylation. The phosphorylation occurred at basal level (no drug treatment) and was enhanced with DAMGO-treatment. The enhancement of the phosphorylation was blocked by naloxone. Morphine stimulation also increased the phosphorylation, but the amount of enhancement was less than that caused by DAMGO-treatment. Mu receptor phosphorylation in the thalamus was much greater than the striatum, while no phosphorylation of the mu receptor in the cerebellum was detected, even with DAMGO treatment. The extent of mu receptor phosphorylation identified in the thalamus, striatum and cerebellum is consistent with the previous studies of mu receptor distribution. The time course and dose-response studies demonstrated that mu receptor phosphorylation was a rapid event, exhibited a positive dose-dependent response, and was similar to that observed in the cloned mu receptor in CHO cells. Furthermore, we correlated the change of mu receptor phosphorylation with the desensitization of the mu receptor function, specifically, inhibition of adenylyl cyclase activity in the thalamus of morphine-tolerant rats. We found that in the thalamus of rats chronically treated with morphine, the enhancement of mu receptor phosphorylation in basal and DAMGO-treated samples paralleled the desensitization of DAMGO-mediated inhibition of adenylyl cyclase. Our results suggest that mu receptor phosphorylation in vivo may play an important role in the modulation of mu receptor function following both acute exposure to morphine and during the development of morphine tolerance.

Determining which domains and amino acid residues of the mu opioid receptor are phosphorylated is critical for understanding the mechanism of mu opioid receptor phosphorylation. The role of the C-terminus of the receptor was investigated by examining the C-terminally truncated or point-mutated mu opioid receptors in receptor phosphorylation and desensitization. Both wild-type and mutated receptors were stably expressed in Chinese hamster ovary (CHO) cells. The receptor expression was confirmed by receptor radioligand binding and immunoblottting. After exposure to 5 microM of DAMGO, phosphorylation of the C-terminally truncated receptor and the mutant receptor T394A was reduced to 40 and 10% of that of the wild-type receptor, respectively. Mutation effects on agonist-induced desensitization were studied using adenylyl cyclase inhibition assays. The C-terminally truncated receptor and mutant receptor T394A both showed complete loss of DAMGO-induced desensitization, while the mutant T/S-7A receptor only lost part of its ability to desensitize. Taken together, these results suggest that the C-terminus of the mu opioid receptor participates in receptor phosphorylation and desensitization with threonine 394, a crucial residue for both features. DAMGO-induced mu opioid receptor phosphorylation and desensitization are associated and appear to involve both the mu opioid receptor C-terminus and other domains of the receptor.

HINT1 is a mouse histidine triad nucleotide binding protein. Here we report the assignments for the backbone nitrogen, carbon and proton NMR signals.

The μ opioid receptor (MOR) has been shown to desensitize after 1 h of exposure to the opioid peptide, [d-Ala2, N-MePhe4, Gly-ol5]enkephalin (DAMGO), largely by the loss of receptors from the cell surface and receptor down-regulation. We have previously shown that the Thr394 in the carboxyl tail is essential for agonist-induced early desensitization, presumably by serving as a primary phosphorylation site for G protein-coupled receptor kinase. Using a T394A mutant receptor, we determined that Thr394 was also responsible for μ opioid receptor down-regulation. The T394A mutant receptor displayed 50% reduction of receptor down-regulation (14.8%) compared with wild type receptor (34%) upon 1 h of exposure to DAMGO. Agonist-induced T394A receptor down-regulation was unaffected by pertussis toxin treatment, indicating involvement of a mechanism independent of G protein function. Interestingly, pertussis toxin-insensitive T394A receptor down-regulation was completely inhibited by a tyrosine kinase inhibitor, genistein. Tyrosine kinase inhibition blocked wild type MOR down-regulation by 50%, and the genistein-resistant wild type MOR down-regulation was completely pertussis toxin-sensitive. Following DAMGO stimulation, MOR was shown to be phosphorylated at tyrosine residue(s), indicating that the receptor was a direct substrate for tyrosine kinase action. Mutagenesis of the four intracellular tyrosine residues resulted in complete inhibition of the G protein-insensitive MOR internalization. Therefore, agonist-induced MOR down-regulation appears to be mediated by two distinct cellular signal transduction pathways. One is G protein-dependent and GRK-dependent, which can be abolished by pertussis toxin treatment of wild type MOR or by mutagenesis of Thr394. The other novel pathway is G protein-independent but tyrosine kinase-dependent, blocked by genistein treatment, and one in which Thr394 has no regulatory role but phosphorylation of tyrosine residues appears essential.

Previous data obtained with the cloned rat mu opioid receptor demonstrated that the "super-potent" opiates, ohmefentanyl (RTI-4614-4) and its four enantiomers, differ in binding affinity, potency, efficacy, and intrinsic efficacy. Molecular modeling (Tang et al., 1996) of fentanyl derivatives binding to the mu receptor suggests that Asp147, Tyr148, Trp318, and His319 are important residues for binding. According to this model, Asp147 interacts with the positively charged opiate agonist to form potent electrostatic and hydrogen-bonding interactions. In this study, the role of weak electrostatic and hydrogen-bonding "pi-pi" interactions of the O atom of the carbonyl group and the phenyl ring structures of RTI-4614-4 and its four enantiomers with residues Tyr148, Trp318, and His319 were explored via site-directed mutagenesis. Tyr148 (in transmembrane helix 3 {TMH3}), Trp318 (TMH7), and His319 (TMH7) were individually replaced with phenylalanine or alanine. Receptors transiently expressed in COS-7 cells were labeled with [125I]IOXY according to published procedures. Mutation of Tyr148 to phenylalanine reduced the binding affinities of some mu-selective agonists (2-7 fold) but did not alter the affinities of DAMGO, naloxone, and the non-selective opiates etorphine and buprenorphine. In contrast, this mutation significantly increased the binding affinities (decreased the Kd values) of [D-Ala2,D-Leu5]enkephalin, IOXY, and dermorphin. Mutation of Trp318 decreased opioid receptor binding to almost undetectable levels. Substitution of alanine for His319 significantly reduced binding affinities for the opioid ligands tested (1.3- to 48-fold), but did not alter the affinities of naloxone and bremazocine. These results indicate the importance of Tyrl48 and His319 for the binding of fentanyl derivatives to the mu receptor. Functional studies using the mutant receptors will provide additional insight into the mechanism of action of RTI-4614-4 and its four enantiomers.

The orvinols are a class of potent opioids which have been extensively studied, yet little is known about the effects of introducing substituents into the 18- and 19-positions. The etheno bridge of thevinone was hydroxylated to give both the 18- and 19-hydroxyl substituted thevinols. After 3-O-demethylation to the corresponding orvinols, binding and GTPgammaS functional assays indicated that hydroxyl substitution at the 18- and 19-positions differentially affects the mu opioid efficacy of orvinols.

To delineate the cellular sites for the motor effects of opiates acting at the mu opioid receptor (MOR) in the rat caudate-putamen nucleus, we examined the ultrastructural immunogold and immunoperoxidase labeling of an antipeptide antiserum specific for the MOR. We also combined these labeling methods to examine the subcellular relationship between the MOR and the endogenous opioid peptide, Leu5-enkephalin (LE). By light microscopy, MOR-labeling was seen in a heterogeneous patchy distribution. Electron microscopic analysis of these patches showed that more than 80% of the total neuronal profiles (n = 1,586) containing MOR-like immunoreactivity (MOR-IR) were dendrites and dendritic spines. The remaining labeled profiles included a few perikarya and many axon terminals. MOR-IR was predominantly localized to extrasynaptic plasma membranes of dendrites, and to both synaptic vesicles and plasma membranes in terminals. Ten percent of the total MOR-labeled terminals (n = 272) formed asymmetric synapses with unlabeled or MOR-labeled dendritic spines. Terminals containing LE-IR formed synapses, in almost equal proportions, on MOR-labeled dendrites and dendritic spines, while over 80% of the unlabeled terminals formed synapses on MOR-labeled dendritic spines. Moreover, colocalization of MOR- and LE-IR was often seen in both dendrites and terminals. These results indicate that in patch compartments of the caudate-putamen nucleus, the MOR is mainly involved in extrasynaptic modulation of spiny neurons, including those that contain LE. In addition, the findings provide a cellular basis for presynaptic opioid modulation of neurotransmitter release through MOR located on axon terminals.

Many of the analgesic effects of opiate drugs and of endogenous opioid ligands, such as Leu5-enkephalin (LE) are thought to be mediated in part by mu-opioid receptors (MOR) in the dorsal horn of the spinal cord. To establish the cellular sites for the spinally mediated analgesic effects of MOR activation and the potential anatomical substrates for interactions with LE, we examined the ultrastructural localization of MOR and LE immunoreactivities in the adult rat cervical spinal cord (C3-C5). Anti-MOR sera recognizing the carboxyl terminal domain of MOR was localized using immunoperoxidase and immunogold-silver methods. mu-opioid receptor-like immunoreactivity (MOR-LI) was observed mainly in the superficial layers of the dorsal horn. Electron microscopy of this region revealed that small unmyelinated axons and axon terminals constituted 48% (91/189) and 15% (28/189), respectively, while dendrites comprised 36% (68/189) of the total population of neuronal profiles containing the MOR. MOR-LI was localized mainly along extrasynaptic portions of the plasma membrane in both axons and dendrites. In sections dually labeled for MOR and LE, 21% (14/68) of the dendrites containing MOR-LI closely apposed or received synaptic contact from axon terminals exhibiting LE reaction product. The results provide the first ultrastructural evidence that within the dorsal horn of the spinal cord, LE, as well as exogenous opiates may alter both axonal release of neurotransmitters and postsynaptic responsiveness of target neurons to afferent input through activation of extrasynaptic MOR.

An herb derived compound, levo-tetrahydropalmatine (L-THP), attenuates self-administration of cocaine and opiates in rodents. Since L-THP mainly antagonizes dopamine D2 receptors (D2R) in the brain, it is likely to regulate other addictive behaviors as well. Here, we examined whether L-THP regulates ethanol drinking in C57BL/6J mice using a two-bottle choice drinking experiment. L-THP treated mice consumed less ethanol compared to vehicle-treated mice during the 15% ethanol drinking session while water consumption remained similar between each group. We then examined the molecular basis underlying the pharmacological effect of L-THP in mice. Our results indicated that a single injection of L-THP increased active phosphorylated forms of PKA, AKT and ERK in the caudate-putamen (CPu), but not in the nucleus accumbens (NAc), of alcohol naive mice. Interestingly, we found that systematic treatment with L-THP for 4 consecutive days while mice were drinking 15% ethanol increased pPKA levels in the CPu, but not in the NAc. In contrast to the effect of acute L-THP treatment, no differences were detected for pAKT or pERK in either striatal regions. Together, our findings suggest that reduction of ethanol drinking by L-THP treatment is possibly correlated with D2R-mediated PKA signaling in the CPu.

To investigate roles of second extracellular loop sequences in peptide and nonpeptide ligand recognition by human opiate receptors, we have constructed a chimeric receptor in which this domain of the human mu opiate receptor has been replaced with that of the human kappa opiate receptor. The chimeric opiate receptor displays dramatically increased affinity for dynorphin peptides. Affinities for dynorphin A-(1-17), dynorphin A-(1-13), and alpha-neoendorphin increase by up to 250-fold when compared with the wild-type human mu opiate receptor. The chimera maintains recognition of the mu-selective ligands morphine and [D-Ala2,MePhe4,Gly-ol5]enkephalin and displays no significant changes in affinity for the kappa-selective small molecule ligand U50,488. The chimeric opiate receptor displays evidence for effective G-protein coupling; 100 nM dynorphin A-(1-17) is as effective as 100 nM morphine at inhibiting forskolin-stimulated adenyl cyclase activity through actions at the chimeric receptor. These data suggest that the putative second extracellular loop contributes substantially to the kappa receptor's selectivity in dynorphin ligand recognition.

The mu opiate receptor is a principal brain site for activities of morphine, other opiate drugs, and opioid peptides in modulating pain and altering mood. Recent cloning of cDNAs encoding rat and human mu receptors reveals charged amino acid residues within putative transmembrane domains (TMs) II, III, and VI, a substantial N-terminal extracellular domain, and a C-terminal intracellular domain. Deletion of 64 N-terminal amino acids produced little effect on receptor function (Wang, J.B., Imai, Y., Eppler, C.M., Gregor, P., Spivak, C.E., and Uhl, G.R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10230-10234). Further deletion of 33 C-terminal amino acids yielded a receptor at which morphine, but not the substituted enkephalin DAMGO ([D-Ala2,MePhe4,Glyol5]enkephalin), inhibited adenylate cyclase. Alanine substitution for each charged TM residue in the N-terminally deleted receptor reduced affinities for morphine, DAMGO, and the opiate antagonist naloxone. Replacement of TM II Asp114 with asparagine or glutamic acid increased mu receptor affinity for naloxone. TM II and TM III glutamic acid substitutions for Asp114 and Asp147 reduced agonist binding affinities but allowed full inhibition of adenylate cyclase at high agonist concentrations. TM VI histidine substitution with alanine yielded a receptor that produced almost twice the cyclase inhibition displayed by the wild type receptor in parallel transient expression assays. These findings underscore the importance of charged residues in TM II, III, and VI for different receptor functions and the modest involvement of extensive portions of N- and C-terminal receptor domains in these processes.

Histidine triad nucleotide binding protein (HINT1) is an intracellular protein that binds purine mononucleotides. Strong sequence conservation suggests that these proteins play a fundamental role in cell biology, however its exact cellular function continues to remain elusive. nuclear magnetic resonance (NMR) studies using STD and HSQC were conducted to observe ligand binding to HINT1. These studies were confirmed using fluorescence spectroscopy titrations. We found that AICAR, the first non-phosphate containing ligand, binds to mouse histidine triad nucleotide binding protein 1 (HINT1). Chemical shift perturbations are mapped onto the X-ray structure showing AICAR binds at the same site as GMP. The NMR results demonstrated that this method will be valuable for the future screening of small molecules that can be used to modulate the function of HINT1.

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in brain, opens chloride channels through actions on GABAA receptors. We now report base and amino acid sequences of the α1, α2, and α3 subunits from GABAA receptors of audiogenic seizure-prone (DBA/2J) and -resistant (C57BL/6J) inbred strains of mice. Inbreeding had fixed different alleles of the α1 subunit in the two strains, giving five base differences in the cDNAs. None of these affected amino acid sequence, but one did create a NsiI restriction site potentially useful in mapping genomic DNA. No base or amino acid sequence differences between the strains were detected for the other two subunits. Northern blots revealed no apparent strain differences in message levels for these three subunits in whole brains of the mice at 3 weeks of age, the peak of seizure susceptibility in DBA/2J, but did reveal distinct regional and developmental patterns of expression among the subunits in mouse brain.

Expression of brain mRNA or cRNAs in Xenopus oocytes was used to determine what subunits of the GABAA receptor are required for modulation by barbiturates, benzodiazepines, and ethanol. Mouse brain mRNA was hybridized with antisense oligonucleotides complementary to sequences unique to specific subunits and injected into oocytes. Antisense oligonucleotides to the alpha 1, beta 1, gamma 1, gamma 2S + 2L, gamma 2L, or gamma 3 subunits did not alter GABA action or enhancement by pentobarbital. Action of diazepam was prevented by antisense oligonucleotides to gamma 2S + 2L and reduced by antisense sequences to gamma 2L, but was not affected by the other oligonucleotides. Ethanol enhancement of GABA action was prevented only by antisense oligonucleotides to gamma 2L (which differs from gamma 2S by the addition of 8 amino acids). Expression of either the alpha 1 beta 1 gamma 2S or the alpha 1 beta 1 gamma 2L subunit cRNA combination in oocytes resulted in GABA responses that were enhanced by diazepam or pentobarbital, but only the combination containing the gamma 2L subunit was affected by ethanol.

A series of cyclic analogues with a lactam linkage were prepared by solid phase peptide synthesis to explore possible biologically active conformation(s) of nociceptin/orphanin FQ (N/OFQ). cyclo[D-Asp(7),Lys(10)]- and cyclo[Asp (6),Lys(10)]N/OFQ(1-13)NH2 exhibit high affinity (Ki = 0.27 and 0.34 nM, respectively) and high potency in the GTPgammaS assay (EC 50 = 1.6 and 4.1 nM, respectively) at human nociceptin/orphanin FQ peptide (NOP) receptors. These analogues exhibit 2- to 3-fold higher affinity and 2- to 5-fold higher potency than the parent peptide.

Intracerebral hemorrhage (ICH) remains a major medical problem and currently has no effective treatment. Hemorrhaged blood is highly toxic to the brain, and catabolism of the pro-oxidant heme, mainly released from hemoglobin, is critical for the resolution of hematoma after ICH. The degradation of the pro-oxidant heme is controlled by heme oxygenase (HO). We have previously reported a neuroprotective role for HO2 in early brain injury after ICH; however, in vivo data that specifically address the role of HO2 in brain edema and neuroinflammation after ICH are absent. Here, we tested the hypothesis that HO2 deletion would exacerbate ICH-induced brain edema, neuroinflammation, and oxidative damage. We subjected wild-type (WT) and HO2 knockout ((-/-)) mice to the collagenase-induced ICH model. Interestingly, HO2(-/-) mice had enhanced brain swelling and neuronal death, although HO2 deletion did not increase collagenase-induced bleeding; the exacerbation of brain injury in HO2(-/-) mice was also associated with increases in neutrophil infiltration, microglial/macrophage and astrocyte activation, DNA damage, peroxynitrite production, and cytochrome c immunoreactivity. In addition, we found that hemispheric enlargement was more sensitive than brain water content in the detection of subtle changes in brain edema formation in this model. Combined, these novel findings extend our previous observations and demonstrate that HO2 deficiency increases brain swelling, neuroinflammation, and oxidative damage. The results provide additional evidence that HO2 plays a critical protective role against ICH-induced early brain injury.

In neurons, the C terminus of the Mu-opioid receptor (MOR) binds to the protein kinase C-interacting protein/histidine triad nucleotide binding protein 1 (PKCI/HINT1) which in turn binds the regulator of G-protein signalling RGSZ1/Z2 (RGSZ) protein. In this study, we found that intracerebroventricular (icv) administration of morphine recruits PKC isoforms, mostly PKCγ, to the MOR via the HINT1/RGSZ complex. There, diacylglycerol (DAG) activates this PKCγ to phosphorylate the MOR and thus, its signal strength was reduced. When PKCI/HINT1 expression is depressed, morphine produces stronger analgesic effects and neither the PKCγ-MOR complex nor serine phosphorylation of this receptor is detected. This MOR-PKC association involves the cysteine rich domains (CRDs) in the regulatory C1 region of PKC, as well as requiring free zinc ions, HINT1 and RGSZ proteins. Increasing the availability of this metal ion recruits inactive PKCγ to the MOR, while phorbol esters prevent this binding and even disrupt it. The nitric oxide donor (S)-Nitroso-N-acetylpenicillamine (SNAP) foments the association of PKCγ with the MORs, effect that was prevented by the heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), suggesting a role for endogenous zinc and neural nitric oxide synthase. The N-methyl-D-aspartate receptor (NMDAR) antagonist, MK801, also prevented PKCγ recruitment to MORs and serine phosphorylation of the receptors following icv morphine. These results indicate that the NMDAR/nNOS cascade, activated via MORs, provide the free zinc ions required for inactive PKCγ to bind to HINT1/RGSZ complex at the C terminus of the receptor. © 2008 Elsevier Inc. All rights reserved.

Based on non-competitive binding interactions we suggested that mu and delta receptors associate as a mu/delta receptor complex in rat brain. We hypothesized that the same non-competitive binding interactions observed in rat brain will be seen in CHO cells that co-express mu and delta receptors, but not in cells that express just mu or delta receptors. We used CHO cells expressing the cloned human mu receptor, cloned human delta receptor, or cloned mouse delta/human mu ("dimer cell"). Cell membranes were prepared from intact cells pretreated with 100nM SUPERFIT. [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding assays followed published procedures. SUPERFIT, a delta-selective irreversible ligand, decreased [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to delta receptors by approximately 75% and to mu receptors by approximately 50% in dimer cells. SUPERFIT treatment did not decrease [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to mu cells. The IC(50) values observed in SUPERFIT-treated dimer cells were: [d-Pen(2),d-Pen(5)]enkephalin (1820nM) and morphine (171nM). Saturation binding experiments with SUPERFIT-treated dimer cells showed that [d-Pen(2),d-Pen(5)]enkephalin (5000nM) was a competitive inhibitor. In contrast, morphine (1000nM) lowered the B(max) from 1944fmol/mg to 1276fmol/mg protein (35% decrease). Both [d-Pen(2),d-Pen(5)]enkephalin and morphine competitively inhibited [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to SUPERFIT-treated mu cells. The results indicate that the mu-delta opioid receptor complex defined on the basis of non-competitive binding interactions in rat brain over 20 years ago likely occurs as a consequence of the formation of mu-delta heterodimers. SUPERFIT-treated dimer cells may provide a useful model to study the properties of mu-delta heterodimers.

The recently cloned rat mu opiate receptor cDNA has been expressed in COS and Chinese hamster ovary (CHO) cells to examine the coupling of this receptor to G-protein linked second messenger systems and examine possible coupling to multiple second messenger systems. Morphine (1 microM) reduced both forskolin-stimulated cAMP levels and IP3 levels by 20 +/- 5 and 34 +/- 8% respectively in COS and CHO cell cultures expressing the cloned rat mu receptor cDNA. Both effects could be blocked by naloxone and Gpp(NH)p. These results represent the first clear representation of the second messenger system promiscuity possible with a single cloned opiate receptor.

Striatal extracellular fluid concentrations of dopamine and metabolites in response to direct striatal administration of two L-DOPA boluses administered sequentially were determined in three rhesus monkeys during halothane anesthesia. Whereas in an initial microdialysis run, generation of dopamine was less following the second L-DOPA bolus than the first, in a subsequent run, in which the selective MAO-B inhibitor R(+)-Npropargyl-1-aminoindan (rasagiline) was administered systemically (0.2 mg/ kg s.c.) between the two L-DOPA boluses, generation of dopamine was greater following the second bolus.

Mu opioid receptors are subject to phosphorylation and desensitization through actions of at least two distinct biochemical pathways: agonist-dependent mu receptor phosphorylation and desensitization induced by a biochemically distinct second pathway dependent on protein kinase C activation (1). To better understand the nature of the agonist-induced mu receptor phosphorylation events, we have investigated the effects of a variety of opiate ligands of varying potencies and intrinsic activities on mu receptor phosphorylation and desensitization. Exposure to the potent full agonists sufentanil, dihydroetorphine, etorphine, etonitazine, and [D-Ala2, MePhe4, Glyol5]enkephalin (DAMGO) led to strong receptor phosphorylation, while methadone, l-alpha-acetylmethadone (LAAM), morphine, meperidine, DADL, beta-endorphin(1-31), enkephalins, and dynorphin A(1-17) produced intermediate effects. The partial agonist buprenorphine minimally enhanced receptor phosphorylation while antagonists failed to alter phosphorylation. Buprenorphine and full antagonists each antagonized the enhanced mu receptor phosphorylation induced by morphine or DAMGO. The rank order of opiate ligand efficacies in producing mu receptor-mediated functional desensitization generally paralleled their rank order of efficacies in producing receptor phosphorylation. Interestingly, the desensitization and phosphorylation mediated by methadone and LAAM were disproportionate to their efficacies in two distinct test systems. This generally good fit between the efficacies of opiates in mu receptor activation, phosphorylation, and desensitization supports the idea that activated receptor/agonist/G-protein complexes and/or receptor conformational changes induced by agonists are required for agonist-induced mu receptor phosphorylation. Data for methadone and LAAM suggest possible contribution from their enhanced desensitizing abilities to their therapeutic efficacies.

The activation of μ-opioid receptors in the nucleus accumbens (Acb) produces changes in locomotor and rewarding responses that are believed to involve neurons, including local γ-aminobutyric acid (GABA)ergic neurons. We combined immunogold–silver detection of an antipeptide antiserum against the cloned μ-opioid receptor (MOR) and immunoperoxidase labeling of an antibody against GABA to determine the cellular basis for the proposed opioid modulation of GABAergic neurons in the rat Acb. MOR-like immunoreactivity (MOR-LI) was localized prominently to plasma membranes of neurons having morphological features of both spiny and aspiny cells, many of which contained GABA. Of 351 examples of profiles that contained MOR-LI and GABA labeling, 65% were dendrites. In these dendrites, MOR-LI was seen mainly along extrasynaptic portions of the plasma membrane apposed to unlabeled terminals and/or glial processes. Dually labeled dendrites often received convergent input from GABAergic terminals and/or from unlabeled terminals forming asymmetric excitatory-type synapses. Of all profiles that contained both MOR and GABA immunoreactivity, 28% were axon terminals. MOR-containing GABAergic terminals and terminals separately labeled for MOR or GABA formed synapses with unlabeled dendrites and also with dendrites containing MOR or GABA. Our results indicate that MOR agonists could modulate the activity of GABA neurons in the Acb via receptors located mainly at extrasynaptic sites on dendritic plasma membranes. MOR ligands also could alter the release of GABA onto target dendrites that contain GABA and/or respond to opiate stimulation.

Interleukin-2 (IL-2) was found to have an analgesic effect in the peripheral nervous system. Both electrophysiological and behavioural experiments suggested an anti-noceiceptive effect of IL-2 in animals. Biochemical experiments revealed that IL-2 could displace diprenorphine binding in both NG 108-15 cells and HEK 293 cells transfected with pCDNA3-DOR (delta opioid receptor). Furthermore, IL-2 significantly inhibited cAMP production stimulated by forskolin in both NG 108-15 and transfected HEK 293 cells, indicating that the binding of IL-2 to the delta opioid receptor is functional.

mu opiate receptors, the principal sites for opiate analgesia and reward, can display compensatory responses to opiate agonist drug administration. Agonist-induced K+ channel responses mediated by these receptors desensitize when examined in Xenopus oocyte expression systems. Mechanisms underlying such processes could include phosphorylation events similar to those reported to desensitize other G-protein-linked receptors. We used C-terminally directed anti-mu receptor antibodies to immunoprecipitate a phosphoprotein with size appropriate for the mu receptor from stably expressing Chinese hamster ovary cells. Phosphorylation of this mu opiate receptor protein was enhanced approximately 5-fold by treatment with the mu agonist morphine. The time course and dose-response relationships between mu receptor phosphorylation and agonist-induced desensitization display interesting parallels. Phosphorylation of mu opiate receptor protein is also enhanced approximately 5-fold by treatment with the protein kinase C activator phorbol 12-myristate 13-acetate. The protein kinase inhibitor staurosporine blocked the effect of phorbol 12-myristate 13-acetate on mu receptor phosphorylation. However, staurosporine failed to block morphine-induced phosphorylation. These observations suggest that several biochemical pathways can lead to mu receptor phosphorylation events that may include mechanisms involved in mu receptor desensitization.

The effect of selective inhibition of monoamine oxidase (MAO) subtypes A and B on striatal metabolism of DOPA to dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid; HVA) was studied in halothane-anesthetized rats 3 weeks after unilateral 6-hydroxydopamine lesion of the substantia nigra. Implantation of bilateral microdialysis probes allowed simultaneous quantitation of metabolite production on lesioned and control sides. The DOPA was administered as a 15-min bolus of 1 mM solution in the striatal microdialysate. Rats were pretreated with the selective MAO-A inhibitor clorgyline, or the selective MAO-B inhibitors deprenyl or TVP-101 [2,3-dihydro-N-2-propynyl-1H-inden-1-amine-(1R)-hydrochloride]. Intrastriatal infusion of DOPA caused an increased efflux of DA, DOPAC, and HVA, which was greater on the intact side. Clorgyline, but not deprenyl or TVP-101, increased post-DOPA DA efflux on both intact and lesioned sides. Clorgyline also caused a marked suppression of post-DOPA DOPAC and HVA effluxes, whereas only mild effects were produced by the MAO-B inhibitors. There was no evidence for a differential effect of MAO-B inhibition on efflux of DA or metabolites in the lesioned as compared with the control striatum. The results indicate a major role for MAO-A in DA metabolism both intra- and extraneuronally in the rat striatum.

Opiate receptors play major roles in analgesic and euphoric effects of opiate drugs. Recent cloning of cDNAs encoding the rodent and human mu receptor revealed high homology between the predicted receptors but also some sequence differences. To determine if these sequence differences produced significant changes in ligand-selectivity profiles, we assessed these profiles in expressing COS and CHO cell lines using the agonist ligand [125I]IOXY-AGO (6 beta-[125Iodo]-3,14-dihydroxy-17-methyl-4,5 alpha- epoxymorphinan). This ligand's high specific activity (2,200 Ci/mmol) and high affinity for mu opioid receptors generated high signal-to-noise ratio binding. The resulting ligand-selectivity profiles of the human and rat mu receptors reveal modest differences in affinities for morphine and naloxone in COS cells but not CHO cells. Ligand-selectivity profiles of the rat and human mu receptors were otherwise similar. Interesting differences between these data and data previously obtained with the peptide agonist [3H]DAMGO suggest that the peptide and alkaloid agonists may label different domains of the mu receptor.

Studies with thiol-modifying reagents have suggested that cysteines might play important roles in the function of the dopamine transporter (DAT). To identify DAT cysteines with important thiol groups, we have studied six mutant dopamine transporters in which cysteines were replaced by alanines. Substitutions of cysteines assigned to the DAT's second putative extracellular loop--positions 180 and 189--dramatically decreased the expression of the mutant transporters. Substitutions at positions 90, 242, 305, and 345 had no significant effect in decreasing dopamine uptake, MPP+ uptake, or cocaine analogue binding. Immunostaining COS cells transfected with Cys180 and Cys189 to Ala mutants revealed reduced membrane staining and prominent staining in perinuclear regions consistent with Golgi apparatus. These results suggest that cysteines i the DAT second extracellular loop may provide sulfide residues crucial to full transporter expression, at least in part, through interference with membrane insertion. Conceivably, they might also provide the targets for the influences of thiol-modifying reagents in modifying the function of the wild-type DAT expressed in striatal membranes.

mu opiate receptors recognize morphine with high affinity. A 2.1-kb rat brain cDNA whose predicted translation product displays 63% identity with recently described delta and kappa opiate receptor sequences was identified through polymerase chain reaction and cDNA homology approaches. This cDNA recognizes a 10.5-kb mRNA that is expressed in thalamic neurons. COS-cell expression confers naloxonazine-, Na(+)-, and GTP-sensitive binding of mu but not delta or kappa opioid ligands. Expressing cells bind morphine, [D-Ala2,N-methyl-Phe4,glyol5]enkephalin (DAMGO), and [D-Ala2,D-Leu5]enkephalin (DADLE) with nanomolar or subnanomolar affinities, defining a mu opiate receptor that avidly recognizes analgesic and euphoric opiate drugs and opioid peptides.

The family of Na(+)- and Cl(-)-dependent, 12 transmembrane domain transporter proteins now includes transporters for neurotransmitter molecules in the brain and for substances important in extraneuronal tissues, including adrenal, kidney, and gut. Transported substrates include monoamine and amino acid neurotransmitters and nonperturbing osmolytes. A common protein topology is predicted and features intracellular N- and C-termini possessing phosphorylation sites and at least one large extramembranous loop with N-linked glycosylation. Using the rat dopamine transporter as a template, molecular modeling of putative transmembrane domains coupled with amino acid sequence conservation analysis indicates amino acid residues potentially involved in substrate and/or ion recognition. Targeting such residues with site-directed mutagenesis will help clarify substrate and ion binding sites and should facilitate rational design of therapeutics to combat depression, locomotor disorders, and substance abuse.

Background Protein kinase C interacting protein (PKCI/HINT1) is a small protein belonging to the histidine triad (HIT) family proteins. Its brain immunoreactivity is located in neurons and neuronal processes. PKCI/HINT1 gene knockout (KO) mice display hyper-locomotion in response to D-amphetamine which is considered a positive symptom of schizophrenia in animal models. Postmortem studies identified PKCI/HINT1 as a candidate molecule for schizophrenia and bipolar disorder. We investigated the hypothesis that the PKCI/HINT1 gene may play an important role in regulating mood function in the CNS. We submitted PKCI/HINT1 KO mice and their wild type (WT) littermates to behavioral tests used to study anti-depressant, anxiety like behaviors, and goal-oriented behavior. Additionally, as many mood disorders coincide with modifications of hypothalamic-pituitary-adrenal (HPA) axis function, we assessed the HPA activity through measurement of plasma corticosterone levels. Results Compared to the WT controls, KO mice exhibited less immobility in the forced swim (FST) and the tail suspension (TST) tests. Activity in the TST tended to be attenuated by acute treatment with valproate at 300 mg/kg in KO mice. The PKCI/HINT1 KO mice presented less thigmotaxis in the Morris water maze and spent progressively more time in the lit compartment in the light/dark test. In a place navigation task, KO mice exhibited enhanced acquisition and retention. Furthermore, the afternoon basal plasma corticosterone level in PKCI/HINT1 KO mice was significantly higher than in the WT. Conclusion PKCI/HINT1 KO mice displayed a phenotype of behavioral and endocrine features which indicate changes of mood function, including anxiolytic-like and anti-depressant like behaviors, in conjunction with an elevated corticosterone level in plasma. These results suggest that the PKCI/HINT 1 gene could be important for the mood regulation function in the CNS.

In order to explore the structural basis of GABAA receptor function, we have expressed murine alpha 1, beta 1, and gamma 2 subunit cDNAs by transient transfection of human 293 cells. Expression of GABAA receptors was measured by ligand binding assay and by electrophysiological analysis. As in other species, expression of the alpha 1 and beta 1 subunits produced a receptor that was insensitive to modulation by benzodiazepines as measured by electrophysiological analysis; however, a small number of flunitrazepam binding sites were detectable. The coexpression of the gamma 2 subunit was found to be essential for this modulation, and also resulted in a dramatic (14-fold) increase in the number of binding sites for flunitrazepam. On the coexpression of all 3 subunit cDNAs, a receptor was produced that exhibited a similar number of binding sites for flunitrazepam and muscimol.

Background The negative consequences of nicotine use are well known and documented, however, abstaining from nicotine use and achieving abstinence poses a major challenge for the majority of nicotine users trying to quit. l-Tetrahydropalmatine (l-THP), a compound extracted from the Chinese herb Corydalis, displayed utility in the treatment of cocaine and heroin addiction via reduction of drug-intake and relapse. The present study examined the effects of l-THP on abuse-related effects of nicotine. Methods Self-administration and reinstatement testing was conducted. Rats trained to self-administer nicotine (0.03 mg/kg/injection) under a fixed-ratio 5 schedule (FR5) of reinforcement were pretreated with l-THP (3 or 5 mg/kg), varenicline (1 mg/kg), bupropion (40 mg/kg), or saline before daily 2-h sessions. Locomotor, food, and microdialysis assays were also conducted in separate rats. Results l-THP significantly reduced nicotine self-administration (SA). l-THP’s effect was more pronounced than the effect of varenicline and similar to the effect of bupropion. In reinstatement testing, animals were pretreated with the same compounds, challenged with nicotine (0.3 mg/kg, s.c.), and reintroduced to pre-extinction conditions. l-THP blocked reinstatement of nicotine seeking more effectively than either varenicline or bupropion. Locomotor data revealed that therapeutic doses of l-THP had no inhibitory effects on ambulatory ability and that l-THP (3 and 5 mg/kg) significantly blocked nicotine induced hyperactivity when administered before nicotine. In in-vivo microdialysis experiments, l-THP, varenicline, and bupropion alone elevated extracellular dopamine (DA) levels in the nucleus accumbens shell (nAcb). Conclusions Since l-THP reduces nicotine taking and blocks relapse it could be a useful alternative to varenicline and bupropion as a treatment for nicotine addiction. Electronic supplementary material The online version of this article (doi:10.1186/s40360-016-0093-6) contains supplementary material, which is available to authorized users.