This study used the D2-preferring dopamine antagonist, haloperidol (3mg) and D1-D2 antagonist, fluphenazine (3mg) to investigate the roles of D1 and D2 receptors in d-amphetamine (20-mg) reinforcement in humans with (9 M; 7 F) and without (12 M; 4 F) an addictive disorder, in a placebo-controlled, between-within counterbalanced design. To preclude neurotoxicity, pathological gamblers served to evaluate effects of addiction status. Incentive motivation (e.g., Desire to Gamble), hedonic impact (e.g., Liking) and risky decision-making were assessed. Haloperidol reduced Desire to Gamble in controls, whereas fluphenazine reduced Desire in gamblers. Both antagonists reduced hedonic impact in both groups, with fluphenazine exhibiting stronger effects in gamblers. Both antagonists decreased risky decisions in controls but increased risky decisions in gamblers. Results suggest that D1 mediates amphetamine-induced motivation to gamble; D2 mediates amphetamine’s hedonic effects; D1 function is deficient in gamblers; and D2 blockade may reverse a restorative effect of amphetamine in addicted individuals.

The endothelium plays an essential role in the regulation of vascular homeostasis and a state of endothelial dysfunction, which develops in the presence of cardiovascular risk factors, may contribute to the development and progression of cardiovascular disease. As such, the measurement of endothelial function, beyond being an experimental tool, may serve as an important tool to complement current risk assessment algorithms in the identification of high-risk patients. Flow-mediated dilation (FMD) is a non-invasive measure of peripheral conduit artery endothelial function that holds great promise. Presently, FMD suffers from methodological heterogeneity and a poor understanding of the various biological components involved in eliciting the dilatory response to a given shear stimulus. We compared both traditional and alternative methods of arterial diameter characterization with regards to their repeatability, nitric oxide-dependency, and their sensitivity in distinguishing between normal and dysfunctional endothelial responses. Our findings emphasize the importance of continuous arterial diameter measurement and suggest that the time to peak FMD is not a useful adjunctive measure of the FMD response. Given that endothelial dysfunction may be of clinical importance, strategies to correct it or prevent it from occurring may be of benefit. The 3-hydroxy-3-methylglutaryl coenzyme A inhibitors are agents that have demonstrated marked cholesterol-independent, endothelial-protective effects. We investigated the ability of rosuvastatin and atorvastatin to protect against endothelial dysfunction associated with ischemia and reperfusion (IR) injury, and chronic nitrate therapy. Using the FMD technique, we demonstrated, for the first time in humans, that acute rosuvastatin administration protects against IR-induced conduit artery endothelial dysfunction. Additionally, we demonstrated that this effect likely occurred by a cyclooxygenase-2-dependent mechanism, which may provide mechanistic insight into the observed cardio-toxicity with cyclooxygenase-2 inhibitors. In contrast, we observed that this endothelial-protective effect was lost upon sustained rosuvastatin administration, which may have important implications regarding the generation of sustained cardioprotective phenotypes. Finally, we demonstrated that atorvastatin co-administration prevented the development of tolerance and endothelial dysfunction associated with continuous transdermal nitroglycerin therapy in humans, likely through an antioxidant mechanism. Future studies are needed in disease patients to determine whether the concept of nitrate tolerance needs reconsideration in the presence of vascular-protective agents.

Schizophrenia may result from altered gene expression leading to abnormal neurodevelopment. In a search for genes with altered expression in schizophrenia, cDNA library subtractive hybridization experiments using post-mortem human frontal cerebral cortices from schizophrenia individuals and neurological controls were performed. I found the mRNA of two neurodevelopmentally important genes, Nogo (RTN4) and calcium/calmodulin-dependent protein kinase II beta (CaMKIIβ), to be overexpressed in post-mortem frontal cortex tissues from patients who suffered with schizophrenia. I used the quantitative real-time polymerase chain reaction method to determined the mRNA levels of these genes in tissues from age- and sex-matched individuals. Nogo is a myelin-associated protein which inhibits the outgrowth of neurites and nerve terminals. The gene produces three splice variants, Nogo-A, B and C. I found Nogo-C mRNA to be overexpressed by 26% in schizophrenia. I also found a 17% reduction of Nogo-B mRNA in samples from individuals who had been diagnosed with severe depression. Furthermore, I showed that there is a direct correlation between the expression of both Nogo-A and -C and the presence of a CAA insert in the 3’UTR of the Nogo gene. CaMKII is a kinase localized at the postsynaptic density. The holoenzyme is primarily composed of the subunits α and β, encoded by two separate genes. It influences the expression of many neuroreceptors, in particular receptors of the glutamatergic pathway. CaMKII also mediates neural maturation during puberty, a time of onset of schizophrenia. The expression of CaMKIIα was elevated 29% in frontal cortex tissues of patients who suffered from depression. The expression of CaMKIIβ was elevated 27% in tissues of schizophrenia patients and 36% in tissues of patients diagnosed with depression. Upregulation of CaMKIIβ was associated with the presence of the CAA insert in at least one copy of the Nogo gene in a group containing both healthy subjects and patients with mental illness, possibly linking the CaMKII and Nogo pathways. The values for the expression of Nogo, CaMKIIα and CaMKIIβ were normalized to β-glucuronidase expression to minimize the effects of mRNA degradation. These results confirm that upregulation of Nogo-C and CaMKIIβ is likely associated with schizophrenia.

ABC drug transporter, MDR1, is a drug flippase that moves a variety of hydrophobic molecules from the inner to the outer leaflet of the plasma membrane. We have previously reported that MDR1 can function as a glycolipid flippase, being one of the mechanisms responsible for the translocation of glucosylceramide into the Golgi for neutral, but not acidic, glycosphingolipids (GSLs) synthesis. The interplay between GSLs and MDR1 could provide a whole new spectrum of innovative therapeutic options. We found that cell surface MDR1 partially co-localized with globotriaosyl ceramide (Gb3) in MDR1 transfected cells. Inhibition of GSL biosynthesis results in the loss of drug resistance and of cell surface MDR1. We speculated that an association of MDR1 and cell surface GSLs, in particular Gb3, may be functional at the cell surface, as MDR1 partitions into plasma membrane lipid rafts regulating MDR1 function. We therefore tested adamantyl Gb3 (adaGb3), a water soluble analog of Gb3, on MDR1 functions. AdaGb3 was able to inhibit MDR1-mediated rhodamine 123 drug efflux from MDR1 expressing cells, like cyclosporin A (CsA), a classical MDR1 inhibitor. AdaGb3 was also able to reverse vinblastine drug resistance in cell culture, whereas adamantyl galactosylceramide had no effect on drug resistance. The strong MDR1 reversal effects of adaGb3, as well as its favourable in vivo features make it a possible choice for inhibition of MDR1 to increase bioavailability of drugs across the intestinal epithelium (De Rosa et al., 2008). Thus, specific GSL analogs provide a new approach to MDR reversal. We have previously shown that MDR1 inhibitor CsA depletes Fabry cell lines of Gb3, the characteristic GSL accumulated in this disease, by preventing its de novo synthesis, and can also deplete Gaucher lymphoid cell lines of accumulated GlcCer (Mattocks et al., 2006). Liver and heart sections of Fabry mice treated with third generation MDR1 inhibitors showed significantly less Gb3 than liver and heart sections of untreated Fabry mice. Thus, MDR1 inhibition offers a potential alternative therapeutic approach not only for Fabry disease given the extraordinary cost of conventional enzyme replacement therapy, but also for other neutral GSL storage diseases, such as Gaucher disease.

G protein-coupled receptors (GPCRs) are known to exist as oligomers, but there is much uncertainty over the oligomeric size, the number of interacting G proteins and the stability of that interaction. The present approach to these questions has been threefold. Monomers of the M2 muscarinic receptor were purified from Spodoptera frugiperda (Sf9) cells and reconstituted in phospholipid vesicles, where they spontaneously formed tetramers. The size of the reconstituted complex was determined from its electrophoretic mobility after cross-linking and inferred from a quantitative, model-based assessment of cooperative effects in the binding of two muscarinic antagonists: N-methylscopolamine and quinuclidinylbenzilate. Binding of the agonist oxotremorine-M to receptor reconstituted with purified G proteins revealed at least three classes of sites that interconverted from higher to lower affinity upon the addition of guanylylimidotriphosphate (GMP-PNP). The binding properties resemble those of muscarinic receptors in myocardial preparations, thereby implying the existence of tetramers in native tissues. G proteins that copurify with the M2 receptor from cardiac membranes also were found to exist as oligomers, some of which contain both alpha(o) and alpha(i2), and the purified complexes contained receptor and G protein in near-equal amounts. A tetrameric receptor implies a tetramer of G proteins, a conclusion that is supported by the distribution of sites between different states identified in the binding of [35S]GTPgammaS to the purified complex. Covalent adducts of a GPCR fused to a Galpha-subunit provide a model system in which the relationship between receptor and G protein complex is defined with respect to stability and composition. Such a fusion of the M2 receptor and Galpha(i1) underwent a cleavage near the amino terminus of the alpha-subunit, however, flagging the likelihood of similar effects in other such adducts. Truncation of the amino terminus prior to fusion generated a stable product that revealed GMP-PNP-sensitive, biphasic binding of oxotremorine-M and noncompetitive interactions between N-methylscopolamine and quinuclidinylbenzilate. A covalent RG complex therefore exhibits the functional properties of M2 receptors in native systems. These observations are consistent with the notion that signaling through the M2 receptor occurs via cooperative interactions within a stable complex that comprises four receptors and four G proteins.

Cholinesterase inhibitors (ChEIs) are the first line pharmacotherapy for the symptoms of Alzheimer’s disease (AD). Though ChEIs offer modest cognitive benefits in early AD, literature addressing their continued use in severe AD is scarce. This study assessed the safety of discontinuing ChEIs in institutionalized moderate-severe AD patients. Twenty-six patients were randomized, double-blind to ChEI continuation or placebo for 8-weeks. Vitals, weight (kg) and adverse events (AEs) were monitored biweekly. Chi-square test revealed no significant association between semi-blinded treatment allocation and AE occurrence (χ²=(1,26)=0.99, p=0.32). Groups showed no differences on clinically significant weight loss (χ²=(1,26) =1.9, p=0.17), mean weight loss (F=.531, p= .473), pulse rate (F=.624, p=.437), or side effects (F=.224, p=.640). Preliminary results suggest that either ChEIs are well tolerated or that these drugs are no longer providing therapeutic benefit. Study completion (recruitment of 60 patients and unblinding) will generate more comprehensive data for determination of safe ChEI discontinuation guidelines.

Nicotine, the main addictive compound in tobacco, is metabolically inactivated to cotinine primarily by the hepatic enzyme CYP2A6. Substantial genetic variation in the CYP2A6 gene contributes to large variation in nicotine metabolism which alters numerous smoking behaviours. The goal of this study was to identify and characterize novel CYP2A6 variants. The CYP2A6 gene from African American phenotypically slow nicotine metabolizers was sequenced. Seven novel non-synonymous variants were identified: 468G>A (V68M), 1767C>G (I149M), 3515G>A (R265Q), 3524T>C (I268T), 4406C>T (T303I), 5661G>A (E390K), 6531T>C (L462P). They were introduced into a cDNA expression construct where they displayed lower protein expression, reduced nicotine metabolism to cotinine, and/or reduced stability as evaluated by western blotting and enzymatic activity. Genotyping assays were developed and assessed in 512 African Americans. Allelic frequencies ranged from 0.1-0.6% with a collective genotype frequency of 3.2%. Here we identified novel variants with reduced/loss of CYP2A6 activity, increasing our understanding of CYP2A6 variability.

Cholinesterase inhibitors (ChEIs) are the first line pharmacotherapy for the symptoms of Alzheimer’s disease (AD). Though ChEIs offer modest cognitive benefits in early AD, literature addressing their continued use in severe AD is scarce. This study assessed the safety of discontinuing ChEIs in institutionalized moderate-severe AD patients. Twenty-six patients were randomized, double-blind to ChEI continuation or placebo for 8-weeks. Vitals, weight (kg) and adverse events (AEs) were monitored biweekly. Chi-square test revealed no significant association between semi-blinded treatment allocation and AE occurrence (χ²=(1,26)=0.99, p=0.32). Groups showed no differences on clinically significant weight loss (χ²=(1,26) =1.9, p=0.17), mean weight loss (F=.531, p= .473), pulse rate (F=.624, p=.437), or side effects (F=.224, p=.640). Preliminary results suggest that either ChEIs are well tolerated or that these drugs are no longer providing therapeutic benefit. Study completion (recruitment of 60 patients and unblinding) will generate more comprehensive data for determination of safe ChEI discontinuation guidelines.

Nicotine, the main addictive compound in tobacco, is metabolically inactivated to cotinine primarily by the hepatic enzyme CYP2A6. Substantial genetic variation in the CYP2A6 gene contributes to large variation in nicotine metabolism which alters numerous smoking behaviours. The goal of this study was to identify and characterize novel CYP2A6 variants. The CYP2A6 gene from African American phenotypically slow nicotine metabolizers was sequenced. Seven novel non-synonymous variants were identified: 468G>A (V68M), 1767C>G (I149M), 3515G>A (R265Q), 3524T>C (I268T), 4406C>T (T303I), 5661G>A (E390K), 6531T>C (L462P). They were introduced into a cDNA expression construct where they displayed lower protein expression, reduced nicotine metabolism to cotinine, and/or reduced stability as evaluated by western blotting and enzymatic activity. Genotyping assays were developed and assessed in 512 African Americans. Allelic frequencies ranged from 0.1-0.6% with a collective genotype frequency of 3.2%. Here we identified novel variants with reduced/loss of CYP2A6 activity, increasing our understanding of CYP2A6 variability.

Adenylyl cyclase (Adcy) is an important early effector of adrenocorticotrophin (ACTH) on the adrenal cortex; however, this enzyme consists of ten isozymes in mammalian cells and the factors governing the expression of different Adcy isozymes have not been well defined. The aim of this study is to investigate the regulation of mouse Adcy4, one of ten isozymes, in Y1 adrenocortical tumor cells and in mutant subclones derived from the Y1 cells. Adcy4 is expressed at a high level in brain but at lower levels in many other tissues including the Y1 cells. Moreover, this isozyme is specifically deficient in Y1 mutants with impaired steroidogenic factor 1 (SF1) activity. These observations support a hypothesis that Adcy4 expression is influenced by both ubiquitously expressed and tissue-specific transcription factors. My sequencing results indicate that mouse Adcy4 is highly homologous to the human and rat counterparts; its gene is located less than 1 kb downstream of Ripk3 and contains 26 exons. Primer extension and in silico analyses suggest that Adcy4 contains a TATA-less promoter and initiates transcription from multiple sites. Luciferase reporter gene assays indicate that Adcy4 promoter activity is mainly stimulated by the proximal GC-rich region but is inhibited by the first intron. This 124 bp GC-rich region is well conserved among several mammalian species and exhibits strong promoter activity in Y1 cells, which is functionally compromised in the Adcy4-deficient mutant. Within this region, three Sp1/Sp3- and one SF1-binding sites have been identified which bind the corresponding proteins Sp1 and Sp3 or SF1 in electrophoretic mobility shift assays (EMSAs). Site-directed mutagenesis reveals that the 5’-most Sp1/Sp3 site enhances Adcy4 promoter activity, whereas the middle Sp1/Sp3 and SF1 sites each repress this activity. In Y1 mutant cells, mutating the SF1 site restores Adcy4 promoter activity and knocking down SF1 with shRNA increases Adcy4 expression. All these data demonstrate that Adcy4 expression is under the control of the ubiquitous factors Sp1 and Sp3 and the tissue-specific factor SF1 and establish that SF1 is a repressor for Adcy4 promoter activity. This study is the first to demonstrate a repressor function for SF1 in certain promoter contexts.