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1.  Endothelin-1 Regulates Cardiac L-Type Calcium Channels via NAD(P)H Oxidase-Derived Superoxide 
It has been shown that reactive oxygen species (ROS) are involved in the intracellular signaling response to G-protein coupled receptor stimuli in vascular smooth muscle cells and in neurons. In the present study, we tested the hypothesis that NAD(P)H oxidase-derived ROS are involved endothelin-1 (ET-1)-induced L-type calcium channel activation in isolated cardiac myocytes. ET-1 (10 nM) induced a 2-fold increase in L-type calcium channel open-state probability (NPo). This effect of ET-1 was abolished by the ETA receptor antagonist cyclo(D-Trp-D-Asp-Pro-D-Val-Leu) [BQ-123 (1 μM)] but was not altered in the presence of an ETB receptor antagonist N-cis-2,6-dimethylpiperidinocarbonyl-b-tBu-Ala-D-Trp(1-methoxycarbonyl)-D-Nle-OH [BQ-788 (1 μM)]. Pre-treatment of cells with the ROS scavenger tempol (100 μM), polyethylene glycol-superoxide dismutase (SOD, 25 U/ml), or the NAD(P)H-oxidase inhibitor gp91ds-tat ([H]RKKRRQRRR-CSTRIRRQL[NH3]) (5 μM) significantly attenuated ET-1-induced increases in calcium channel NPo. Tempol, SOD, and gp91ds-tat alone had no effect on basal calcium channel activity. In addition, ET-1 significantly increased NAD(P)H oxidase activity and elevated intracellular superoxide levels in cultured cardiac myocytes. The superoxide generator, xanthine-xanthine oxidase (10 mM, 20 mU/ ml), also increased calcium channel NPo in cardiac myocytes, mimicking the effect of ET-1. These observations provide the first evidence that ET-1 induces the activation of L-type Ca2+ channels via stimulation of NAD(P)H-derived superoxide production in cardiac myocytes.
doi:10.1124/jpet.108.140301
PMCID: PMC4323092  PMID: 18539650
2.  Annexin A1 Mediates Hydrogen Sulfide Properties in the Control of InflammationS 
Hydrogen sulfide (H2S) is a gaseous mediator synthesized in mammalian tissues by three main enzymes—cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate-sulfurtransferase—and its levels increase under inflammatory conditions or sepsis. Since H2S and H2S-releasing molecules afford inhibitory properties in leukocyte trafficking, we tested whether endogenous annexin A1 (AnxA1), a glucocorticoid-regulated inhibitor of inflammation acting through formylated-peptide receptor 2 (ALX), could display intermediary functions in the anti-inflammatory profile of H2S. We first investigated whether endogenous AnxA1 could modulate H2S biosynthesis. To this end, a marked increase in CBS and/or CSE gene products was quantified by quantitative real-time polymerase chain reaction in aortas, kidneys, and spleens collected from AnxA1−/− mice, as compared with wild-type animals. When lipopolysaccharide-stimulated bone marrow-derived macrophages were studied, H2S-donor sodium hydrosulfide (NaHS) counteracted the increased expression of inducible nitric oxide synthase and cyclooxygenase 2 mRNA evoked by the endotoxin, yet it was inactive in macrophages harvested from AnxA1−/− mice. Next we studied the effect of in vivo administration of NaHS in a model of interleukin-1β (IL-1β)–induced mesenteric inflammation. AnxA1+/+ mice treated with NaHS (100 μmol/kg) displayed inhibition of IL-1β-induced leukocyte adhesion/emigration in the inflamed microcirculation, not observed in AnxA1−/− animals. These results were translated by testing human neutrophils, where NaHS (10–100 μM) prompted an intense mobilization (>50%) of AnxA1 from cytosol to cell surface, an event associated with inhibition of cell/endothelium interaction under flow. Taken together, these data strongly indicate the existence of a positive interlink between AnxA1 and H2S pathway, with nonredundant functions in the control of experimental inflammation.
doi:10.1124/jpet.114.217034
PMCID: PMC4256431  PMID: 25077524
3.  Tetrathiomolybdate Protects against Bile Duct Ligation-Induced Cholestatic Liver Injury and Fibrosis 
Tetrathiomolybdate (TM), a potent copper-chelating drug, was initially developed for the treatment of Wilson’s disease. Our working hypothesis is that the fibrotic pathway is copper-dependent. Because biliary excretion is the major pathway for copper elimination, a bile duct ligation (BDL) mouse model was used to test the potential protective effects of TM. TM was given in a daily dose of 0.9 mg/mouse by means of intragastric gavage 5 days before BDL. All the animals were killed 5 days after surgery. Plasma liver enzymes and total bilirubin were markedly decreased in TM-treated BDL mice. TM also inhibited the increase in plasma levels of tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β1 seen in BDL mice. Cholestatic liver injury was markedly attenuated by TM treatment as shown by histology. Hepatic collagen deposition was significantly decreased, and it was paralleled by a significant suppression of hepatic smooth muscle α-actin and fibrogenic gene expression in TM-treated BDL mice. Although the endogenous antioxidant ability was enhanced, oxidative stress as shown by malondialdehyde and 4-hydroxyalkenals, hepatic glutathione/oxidized glutathione ratio, was not attenuated by TM treatment, suggesting the protective mechanism of TM may be independent of oxidative stress. In summary, TM attenuated BDL-induced cholestatic liver injury and fibrosis in mice, in part by inhibiting TNF-α and TGF-β1 secretion. The protective mechanism seems to be independent of oxidative stress. Our data provide further evidence that TM might be a potential therapy for hepatic fibrosis.
doi:10.1124/jpet.107.131227
PMCID: PMC4222180  PMID: 18299419
4.  Comparison of Cannabidiol, Antioxidants, and Diuretics in Reversing Binge Ethanol-Induced Neurotoxicity 
Binge alcohol consumption in the rat induces substantial neurodegeneration in the hippocampus and entorhinal cortex. Oxidative stress and cytotoxic edema have both been shown to be involved in such neurotoxicity, whereas N-methyl-D-aspartate (NMDA) receptor activity has been implicated in alcohol withdrawal and excitoxic injury. Because the nonpsychoactive cannabinoid cannabidiol (CBD) was previously shown in vitro to prevent glutamate toxicity through its ability to reduce oxidative stress, we evaluated CBD as a neuroprotectant in a rat binge ethanol model. When administered concurrently with binge ethanol exposure, CBD protected against hippocampal and entorhinal cortical neurodegeneration in a dose-dependent manner. Similarly, the common antioxidants butylated hydroxytoluene and α-tocopherol also afforded significant protection. In contrast, the NMDA receptor antagonists dizocilpine (MK-801) and memantine did not prevent cell death. Of the diuretics tested, furosemide was protective, whereas the other two anion exchanger inhibitors, L-644,711 [(R)-(+)-(5,6-dichloro2,3,9,9a-tetrahydro 3-oxo-9a-propyl-1H-fluoren-7-yl)oxy acetic acid] and bumetanide, were ineffective. In vitro comparison of these diuretics indicated that furosemide is also a potent antioxidant, whereas the nonprotective diuretics are not. The lack of efficacy of L-644,711 and bumetanide suggests that the antioxidant rather than the diuretic properties of furosemide contribute most critically to its efficacy in reversing ethanol-induced neurotoxicity in vitro, in our model. This study provides the first demonstration of CBD as an in vivo neuroprotectant and shows the efficacy of lipophilic antioxidants in preventing binge ethanol-induced brain injury.
doi:10.1124/jpet.105.085779
PMCID: PMC4183207  PMID: 15878999
5.  Oxysterol 22(R)-Hydroxycholesterol Induces the Expression of the Bile Salt Export Pump through Nuclear Receptor Farsenoid X Receptor but Not Liver X Receptor 
Oxysterols are intermediates in the synthesis of bile acids and steroid hormones from cholesterol and function as ligands for liver X receptor (LXR). Bile salt export pump (BSEP) is responsible for canalicular secretion of bile acids and is tightly regulated by its substrates bile acids through nuclear receptor farnesoid X receptor (FXR). In a microarray study using human hepatocytes, BSEP was markedly induced not only by chenodeoxycholic acid (CDCA) but also by oxysterol 22(R)-hydroxycholesterol [22(R)-OHC]. We hypothesized that the expression of BSEP was induced by oxysterols through activation of LXR. To test the hypothesis, human primary hepatocytes or hepatoma cells were treated with 22(R)-OHC, and expression of BSEP was determined. The level of BSEP mRNA was increased as much as 5-fold upon oxysterol induction. In contrast to our hypothesis, the oxysterol-induced up-regulation of BSEP is mediated through FXR but not LXR. BSEP promoter activity was markedly induced by 22(R)-OHC in the presence of FXR but not LXRs. Mutation of the FXR element IR1 in the BSEP promoter significantly reduced its ability to respond to oxysterol induction. To determine whether 22(R)-OHC and CDCA bind to similar structural features of FXR, site-directed mutagenesis was performed in the FXR ligand binding domain. Mutation of residues R331 and I352 abolished activation mediated by CDCA and 22(R)-OHC. In contrast, substitution of residues L340 and R351 differentiated CDCA- and 22(R)-OHC-mediated activation. In conclusion, oxysterol 22(R)-OHC functions as an FXR ligand to induce BSEP expression and differs in the binding with FXR from CDCA.
doi:10.1124/jpet.105.097758
PMCID: PMC4114575  PMID: 16371446
6.  The Hypolipidemic Agent Guggulsterone Regulates the Expression of Human Bile Salt Export Pump: Dominance of Transactivation over Farsenoid X Receptor-Mediated Antagonism 
Conversion of cholesterol to bile acids in the liver is initiated by the rate-limiting enzyme cholesterol 7α-hydroxylase (CYP7A1) and excretion of bile acids from the liver is mediated by the bile salt export pump (BSEP). The expression of CYP7A1 and BSEP is coordinately regulated by a negative feedback and positive feed-forward mechanism, respectively, through bile acid-mediated activation of farsenoid X receptor (FXR). It is well established that hypolipidemic agent guggulsterone is an FXR antagonist and down-regulates FXR target genes. In this study, however, we have demonstrated that guggulsterone synergistically induced the expression of BSEP in cells treated with FXR agonist bile acids. A dissection study located in the BSEP promoter an activating protein (AP)-1 site supporting the action of guggulsterone. Deletion or mutation of the AP-1 element was diminished, whereas insertion of the AP-1 element into a heterologous promoter enhanced activation of the promoter by guggulsterone. Selective c-Jun N-terminal kinase and extracellular signal-regulated kinase inhibitors markedly decreased the transactivation, suggesting an involvement of AP-1 activation pathway in the up-regulation of BSEP by guggulsterone. Consistent with its FXR antagonism, guggulsterone antagonized bile acid-mediated transactivation of BSEP promoter when the AP-1 element was disrupted. In conclusion, guggulsterone regulates BSEP expression through composite mechanisms, and the transactivation through the AP-1 element is dominant over the FXR-mediated antagonism. The up-regulation of BSEP expression by guggulsterone without activating FXR pathway as an FXR agonist to suppress CYP7A1 expression represents a possible mechanism for guggulsterone-mediated hypolipidemic effect.
doi:10.1124/jpet.106.113837
PMCID: PMC4114705  PMID: 17135343
7.  Relative Activation of Human Pregnane X Receptor versus Constitutive Androstane Receptor Defines Distinct Classes of CYP2B6 and CYP3A4 Inducers 
Both the human pregnane X receptor (hPXR) and constitutive androstane receptor (hCAR) are capable of regulating CYP3A4 and CYP2B6 gene expression. However, the majority of currently identified CYP3A4 and CYP2B6 inducers are confirmed activators of hPXR but not hCAR. To compare these receptors with respect to their chemical selectivities, 16 drugs known to induce CYP3A4 and/or CYP2B expression were evaluated for relative activation of hPXR versus hCAR. Because of the high basal but low chemical-induced activation of hCAR in immortalized cells, alternative methods were used to evaluate hCAR activation potential. Thirteen of the 16 compounds were classified as moderate to strong hPXR activators. In contrast, carbamazepine (CMZ), efavirenz (EFV), and nevirapine (NVP) were classified as negligible or weak hPXR activators at concentrations associated with efficacious CYP2B6 reporter or endogenous gene induction in primary human hepatocytes, suggesting potential activation of hCAR. Subsequent experiments demonstrated that these three drugs efficiently induced nuclear accumulation of in vivo-transfected enhanced yellow fluorescent protein-hCAR and significantly increased expression of a CYP2B6 reporter gene when hCAR was expressed in CAR−/− mice. In addition, using a recently identified, chemically responsive splice variant of hCAR (hCAR3), the hCAR activation profiles of the 16 compounds were evaluated. By combining results from the hPXR- and hCAR3-based reporter gene assays, these inducers were classified as hPXR, hCAR, or hPXR/hCAR dual activators. Our results demonstrate that CMZ, EFV, and NVP induce CYP2B6 and CYP3A4 preferentially through hCAR and that hCAR3 represents a sensitive tool for in vitro prediction of chemical-mediated human CAR activation.
doi:10.1124/jpet.106.112136
PMCID: PMC4091905  PMID: 17041008
8.  Comparison of Intravenous Buprenorphine and Methadone Self-administration by Recently Detoxified Heroin Abusers 
Although buprenorphine is used worldwide as a safe and effective maintenance medication for opioid dependence, some countries have reported a growing incidence of abuse of this medication. Buprenorphine is considered to have lower abuse potential because of its partial agonist profile, but no studies have directly compared the reinforcing effects of buprenorphine with those of full mu opioid agonists in humans. The present double-blind, placebo-controlled, inpatient study compared the reinforcing and subjective effects of intravenously administered buprenorphine (0.5, 2 and 8 mg) and methadone (5, 10, and 20 mg). Participants (N=6) were detoxified from heroin during the first 1-2 weeks after admission. During subsequent weeks, participants received a sample drug dose and $20 on Monday, and they could self-administer either the sampled dose or $20 during one choice session per day on Thursday and Friday. Participants responded under a modified progressive ratio schedule during each choice session. All active doses maintained higher progressive ratio break points (largest completed ratio) than placebo. There were no significant differences in break point values between buprenorphine and methadone or among the different doses of drug. However, several subjective ratings, including “Good Drug Effect,” “High,” and “Liking” dose-dependently increased after administration of buprenorphine and methadone. The peak ratings for these effects did not significantly differ for the two drugs. These results demonstrate that under these experimental conditions, buprenorphine and methadone were equally effective in producing reinforcing and subjective effects.
doi:10.1124/jpet.105.090423
PMCID: PMC4079467  PMID: 16144974
9.  Self-administration of Intravenous Buprenorphine and the Buprenorphine/Naloxone Combination by Recently Detoxified Heroin Abusers 
Buprenorphine is a partial mu opioid agonist and kappa opioid antagonist currently under development as a maintenance medication for heroin dependence. Because of concerns about illicit diversion of buprenorphine, a combination tablet containing buprenorphine and naloxone has been developed. The present study evaluated the reinforcing effects of intravenously-administered placebo, buprenorphine alone (BUP; 2, 8 mg), and the buprenorphine/naloxone combination (BUP/NX; 2 mg buprenorphine plus 0.5 mg naloxone, 8 mg buprenorphine plus 2 mg naloxone) in recently detoxified heroin abusers during a 6-week inpatient study. Participants (N=6) were detoxified from heroin over approximately 1 week immediately after admission. During the next 5 weeks, the reinforcing effects of placebo, BUP, and BUP/NX were evaluated. Participants first received a dose of drug and $20, and then were given the opportunity to self-administer either the dose or $20 during choice sessions. Progressive ratio break point values were significantly higher after active drug, compared to placebo, but they did not significantly differ as a function of dose or drug. In contrast, positive subjective ratings were higher after administration of BUP compared to BUP/NX, and these ratings increased in a dose-dependent fashion. BUP and the combination had few effects on performance. Relative to placebo, both BUP and BUP/NX decreased pupil diameter, but there were no significant differences in pupil diameter as a function of drug or dose. These results demonstrate that both BUP and BUP/NX served as reinforcers under these conditions, and that they may have similar abuse liability in recently detoxified individuals who abuse heroin.
doi:10.1124/jpet.102.038141
PMCID: PMC4079473  PMID: 12388653
10.  Discrete Cell Gene Profiling of Ventral Tegmental Dopamine Neurons after Acute and Chronic Cocaine Self-Administration 
Chronic cocaine administration induces a number of biochemical alterations within the mesolimbic dopamine system that may mediate various aspects of the addictive process such as sensitization, craving, withdrawal, and relapse. In the present study, rats were allowed to self-administer cocaine (0.5 mg/infusion) for 1 or 20 days. Tyrosine hydroxylase immunopositive cells were microdissected from the ventral tegmental area (VTA) using laser capture microdissection, and changes in the abundances of 95 mRNAs were assessed using cDNA macroarrays. Five GABA-A receptor subunit mRNAs (α4, α6, β2, γ2, and δ) were down-regulated at both 1 and 20 days of cocaine self-administration. In contrast, the catalytic subunit of protein phosphatase 2A (PP2α), GABA-A α1, and Gαi2 were significantly increased at both time points. Additionally, calcium/calmodulin-dependent protein kinase IIα mRNA levels were increased initially followed by a slight decrease after 20 days, whereas neuronal nitric-oxide synthase mRNA levels were initially decreased but returned to near control levels by day 20. These results indicate that alterations of specific GABA-A receptor subtypes and other signal transduction transcripts seem to be specific neuroadaptations associated with cocaine self-administration. Moreover, as subunit composition determines the functional properties of GABA-A receptors, the observed changes may indicate alterations in the excitability of dopamine transmission underlying long-term biochemical and behavioral effects of cocaine.
doi:10.1124/jpet.103.054965
PMCID: PMC4048547  PMID: 12966149
11.  Methylnaltrexone Antagonizes Opioid-Mediated Enhancement of HIV Infection of Human Blood Mononuclear Phagocytes 
Opioid abuse has been postulated as a cofactor in the immunopathogenesis of human immunodeficiency virus (HIV) infection and AIDS. We and others have recently demonstrated that opioid enhances HIV infection of human macrophages through modulation of β-chemokines and the CCR5 receptor and that this effect is reversed by naltrexone, a tertiary opioid antagonist. Tertiary opioid antagonists cannot be used in opioid-dependent patients because they precipitate withdrawal or reversal of analgesia. We determined whether the quaternary opioid antagonist methylnaltrexone (MNTX), now in phase III clinical trials for opioid-induced constipation, reverses the opioid-mediated enhancement of HIV infection of macrophages at clinically relevant doses. MNTX completely abrogated morphine-induced HIV Bal strain infection of macrophages. MNTX also inhibited the R5 strain (ADA) envelope-pseudotyped HIV replication induced by morphine. Furthermore, MNTX abolished morphine-mediated up-regulation of CCR5 receptor expression. The ability of MNTX to block opioid-induced CCR5 expression and HIV replication at clinically relevant doses may have additional benefit for opioid abusers with HIV infection, or patients with AIDS pain receiving opioids.
doi:10.1124/jpet.103.056697
PMCID: PMC4016816  PMID: 14560041
12.  Inhibition of Mitochondrial Hydrogen Peroxide Production by Lipophilic Metalloporphyrins 
Many studies have established a role for oxidative stress and mitochondrial dysfunction as an important mechanism in the pathogenesis of neuronal disorders. Metalloporphyrins are a class of catalytic antioxidants that are capable of detoxifying a wide range of reactive oxygen species. The AEOL112 series of glyoxylate metalloporphyrins were designed with increased lipid solubility for better oral bioavailability and penetration of the blood-brain barrier. The goal of this study was to develop an in vitro assay using rat brain mitochondria to reliably detect endogenously released hydrogen peroxide (H2O2) and identify glyoxylate metalloporphyrins based on rank order of potency for removal of physiologically relevant H2O2. A polarographic method was established for the sensitive, accurate, and reproducible detection of low levels of H2O2. The assay identified several potent glyoxylate metalloporphyrins with H2O2 scavenging potencies (IC50) in the nanomolar range. These results provide a simplified in vitro model system to detect physiologically generated mitochondrial H2O2 as a screening tool to predict the biological efficacy of potential therapeutic entities.
doi:10.1124/jpet.107.132134
PMCID: PMC3983961  PMID: 18063723
13.  Food restriction alters pramipexole-induced yawning, hypothermia, and locomotor activity in rats: Evidence for sensitization of dopamine D2 receptor-mediated effects 
Food restriction enhances sensitivity to the reinforcing effects of a variety of drugs of abuse including opiates, nicotine, and psychostimulants. Food restriction has also been shown to alter a variety of behavioral and pharmacological responses to dopaminergic agonists including an increased sensitivity to the locomotor stimulatory effects of direct- and indirect-dopamine agonists, elevated extracellular dopamine levels in responses to psychostimulants, as well as suppression of agonist-induced yawning. Behavioral and molecular studies suggests that augmented dopaminergic responses observed in food-restricted animals result from a sensitization of the dopamine D2 receptor, however, little is known about how food restriction affects dopamine D3 receptor function. The current studies were aimed at better defining the effects of food restriction on D2 and D3 receptor function by assessing the capacity of pramipexole to induce yawning, penile erection (PE), hypothermia, and locomotor activity in free-fed and food-restricted rats. Food restriction resulted in a suppression of pramipexole-induced yawning, a sensitized hypothermic response, and an enhanced locomotor response to pramipexole, effects that are suggestive of an enhanced D2 receptor activity; no effect on pramipexole-induced PE was observed. Antagonist studies further supported a food restriction-induced enhancement of D2 receptor activity as the D2 antagonist, L-741,626, recovered pramipexole-induced yawning to free-fed levels, while yawning and PE were suppressed following pretreatment with the D3 antagonist, PG01037. The results of the current studies suggest that food restriction sensitized rats to the D2-mediated effects of pramipexole while having no effect on the D3-mediated effects of pramipexole.
doi:10.1124/jpet.107.133181
PMCID: PMC3893827  PMID: 18305018
14.  Differential Role of Nicotinic Acetylcholine Receptor Subunits in Physical and Affective Nicotine Withdrawal Signs 
The Journal of pharmacology and experimental therapeutics  2008;325(1):10.1124/jpet.107.132977.
It has been suggested that the negative effects associated with nicotine withdrawal promote continued tobacco use and contribute to the high relapse rate of smoking behaviors. Thus, it is important to understand the receptor-mediated mechanisms underlying nicotine withdrawal to aid in the development of more successful smoking cessation therapies. The effects of nicotine withdrawal are mediated through nicotinic acetylcholine receptors (nAChRs); however, the role of nAChRs in nicotine withdrawal remains unclear. Therefore, we used mecamylamine-precipitated, spontaneous, and conditioned place aversion (CPA) withdrawal models to measure physical and affective signs of nicotine withdrawal in various nAChR knockout (KO) mice. β2, α7, and α5 nAChR KO mice were chronically exposed to nicotine through surgically implanted osmotic minipumps. Our results show a loss of anxiety-related behavior and a loss of aversion in the CPA model in β2 KO mice, whereas α7 and α5 KO mice displayed a loss of nicotine withdrawal-induced hyperalgesia and a reduction in somatic signs, respectively. These results suggest that β2-containing nAChRs are involved in the affective signs of nicotine withdrawal, whereas non-β2-containing nAChRs are more closely associated with physical signs of nicotine withdrawal; thus, the nAChR subtype composition may play an important role in the involvement of specific subtypes in nicotine withdrawal.
doi:10.1124/jpet.107.132977
PMCID: PMC3821841  PMID: 18184829
15.  Enhancement of Cisplatin [cis-Diammine Dichloroplatinum (II)] Cytotoxicity by O6-Benzylguanine Involves Endoplasmic Reticulum StressS 
O6-Benzylguanine (BG) enhances cisplatin [cis-diammine dichloroplatinum (II)]-induced cytotoxicity and apoptosis in head and neck cancer cell lines by an unknown mechanism. We investigated the effect of cisplatin with and without BG on two targets of damage: DNA and the endoplasmic reticulum (ER). We chose three cancer cell lines to ascertain the mechanism of BG-enhanced cytotoxicity: SQ20b head and neck and SKOV-3x ovarian cancer cell lines, where BG enhanced cisplatin cytotoxicity, and A549 nonsmall cell lung cancer line, where BG did not enhance cisplatin cytotoxicity. All three lines had an increase in DNA damage when BG was added to cisplatin treatment, as evidenced by increased platination and phosphorylated histone H2AX formation. The increase in cisplatin-induced DNA damage after treatment with BG plus cisplatin is not sufficient to increase cytotoxicity or apoptosis in A549 cells. We evaluated the effect of cisplatin on the ER and observed increased caspase 12 cleavage in SQ20b and SKOV-3x cells, but not in A549 cells, after treatment with BG plus cisplatin versus cisplatin alone. Growth arrest and DNA damage inducible (GADD) 153, an ER stress-response gene, is up-regulated after treatment with BG plus cisplatin compared with cisplatin alone in SQ20b and SKOV-3x cells, but not in A549 cells. ER stress-induced apoptosis is an integral part of the mechanism by which BG enhances cisplatin. Inhibition of ER stress in the SQ20b cell line by salubrinal, an inhibitor of eIF2α dephosphorylation, or GADD153 small interfering RNA, abrogated BG-enhancement of cisplatin cytotoxicity and apoptosis through caspase 3 and 12 cleavage. These data indicate GADD153 up-regulation plays an important role in BG-enhanced cisplatin cytotoxicity and apoptosis.
doi:10.1124/jpet.108.141291
PMCID: PMC3785988  PMID: 18664592
16.  Induction of apoptosis in Neurofibromatosis Type 1 malignant peripheral nerve sheath tumor cell lines by a combination of novel farnesyl transferase inhibitors and lovastatin 
Neurofibromatosis Type 1 (NF1) is a genetic disorder that is driven by the loss of neurofibromin (Nf) protein function. Nf contains a Ras GTPase activating domain (Ras-GAP), which directly regulates Ras signaling. Numerous clinical manifestations are associated with the loss of Nf and increased Ras activity. Ras proteins must be prenylated in order to traffic and functionally localize with target membranes. Hence, Ras is a potential therapeutic target for treating NF1. We have tested the efficacy of two novel farnesyl transferase inhibitors (FTI), 1 and 2, alone or in combination with lovastatin, on two NF1 malignant peripheral nerve sheath tumor (MPSNT) cell lines, NF90-8 and ST88-14. Single treatments of 1, 2, or lovastatin had no effect on MPNST cell proliferation. However, low micromolar combinations of 1 or 2 with lovastatin (FTI/lovastatin) reduced Ras prenylation in both MPNST cell lines. Further, this FTI/lovastatin combination treatment reduced cell proliferation and induced an apoptotic response as shown by morphological analysis, pro-caspase-3/-7 activation, loss of mitochondrial membrane potential, and accumulation of cells with sub G1 DNA content. Little to no detectable toxicity was observed in normal rat Schwann cells following FTI/lovastatin combination treatment. These data support the hypothesis that combination FTI plus lovastatin therapy may be a potential treatment for NF1 MPNSTs.
doi:10.1124/jpet.107.135830
PMCID: PMC3768167  PMID: 18367665
17.  Mapping the Structural Requirements in the CB1 Cannabinoid Receptor Transmembrane Helix II for Signal Transduction 
Amino acid residues in the transmembrane domains of the CB1 receptor are important for ligand recognition and signal transduction. We used site-directed mutagenesis to identify the role of two novel and adjacent residues in the transmembrane helix II domain, Ile2.62 and Asp2.63. We investigated the role of the conserved, negatively charged aspartate at position 2.63 in cannabinoid receptor (CB1) function by substituting it with asparagine (D2.63N) and glutamate (D2.63E). In addition, the effect of the mutant I2.62T alone and in combination with D2.63N (double mutant) on the affinity and potency of structurally diverse ligands was investigated. Recombinant human CB1 receptors, stably expressed in human embryonic kidney 293 cells, were assayed for ligand affinity and agonist-stimulated guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding. The charge-conserved mutant D2.63E behaved similar to wild type. The charge-neutralization mutation D2.63N attenuated the potency of (−)-3-[2-hydroxyl-4-(1,1-dimethylheptyl) phenyl]-4-[3-hydroxylpropyl] cyclohexan-1-ol (CP,55940), (R)-(−)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN55212-2), (−)-11β-hydroxy-3-(1′,1′-dimethylheptyl) hexahydrocannabinol (AM4056), and (−)-11-hydroxyldimethylheptyl-Δ8-tetrahydrocannabinol (HU210) for the stimulation of GTPγS binding, without affecting their binding affinities. Likewise, the I2.62T mutant selectively altered agonist potency without altering agonist affinity. It was surprising to note that the double mutant (I2.62T-D2.63N) displayed a drastic and synergistic increase (by ~50-fold) in the EC50 for agonist-mediated activation. The profound loss of function in the I2.62T-D2.63N double mutant suggests that, although these residues are not obligatory for agonist recognition, they play a synergistic and crucial role in modulating signal transduction.
doi:10.1124/jpet.107.133256
PMCID: PMC3767288  PMID: 18174385
18.  Transglutaminase-catalyzed Transamidation: A Novel Mechanism for Rac1 Activation by 5-HT2A Receptor Stimulation 
Transglutaminase (TGase)-induced activation of small G proteins via 5-HT2A receptor signaling leads to platelet aggregation (Walther et al., 2003). We hypothesize that stimulation of 5-HT2A receptors in neurons activates TGase, resulting in transamidation of serotonin to a small G protein, Rac1, thereby constitutively activating Rac1. Using immunoprecipitation and immunoblotting, we show that in a rat cortical cell line, A1A1v cells, serotonin increases TGase-catalyzed transamidation of Rac1. This transamidation occurs in both undifferentiated and differentiated cells. Treatment with a 5-HT2A/2C receptor agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI), but not the 5-HT1A receptor agonist, 5-hydroxy-2-dipropylamino tetralin (DPAT), increases transamidation of Rac1 by TGase. In A1A1v cells, 5-HT2A receptors mediate the transamidation reaction since expression of 5-HT2C receptors was not detectable and the selective 5-HT2A receptor antagonist blocked transamidation. Time course studies demonstrate that transamidation of Rac1 is significantly elevated after 5 and 15 minutes of serotonin treatment, but returns to control levels after 30 minutes. The activity of Rac1 is also transiently increased following serotonin stimulation. Inhibition of TGase by cystamine or siRNA reduces TGase-modification of Rac1 and cystamine also prevents Rac1 activation. Serotonin itself is bound to Rac1 by TGase following 5-HT2A receptor stimulation as demonstrated by co-immunoprecipitation experiments and a dose-dependent decrease of serotonin-associated Rac1 by cystamine. These data support the hypothesis that Rac1 activity is transiently increased due to TGase-catalyzed transamidation of serotonin to Rac1 via stimulation of 5-HT2A receptors. Activation of Rac1 via TGase is a novel effector and second messenger of the 5-HT2A receptor signaling cascade in neurons.
doi:10.1124/jpet.107.135046
PMCID: PMC3733250  PMID: 18400843
19.  Extracting Global System Dynamics of Corticosteroid Genomic Effects in Rat Liver 
One of the challenges in constructing biological models involves resolving meaningful data patterns from which the mathematical models will be generated. For models that describe the change of mRNA in response to drug administration, questions exist whether the correct genes have been selected given the myriad transcriptional effects that may occur. Oftentimes, different algorithms will select or cluster different groups of genes from the same data set. A new approach was developed that focuses on identifying the underlying global dynamics of the system instead of selecting individual genes. The procedure was applied to microarray genomic data obtained from rat liver after a large single dose of methylprednisolone in 52 adrenalectomized rats. Twelve clusters of at least 30 genes each were selected, reflecting the major changes over time. This method along with isolating the underlying dynamics of the system also extracts and clusters the genes that make up this global dynamic for further analysis as to the contributions of specific mechanisms affected by the drug.
doi:10.1124/jpet.107.133074
PMCID: PMC3725546  PMID: 18086872
20.  Non-competitive antagonism and inverse agonism as mechanism of action of non-peptidergic antagonists at primate and rodent CXCR3 chemokine receptors 
The chemokine receptor CXCR3 is involved in various inflammatory diseases, such as rheumatoid arthritis, multiple sclerosis, psoriasis and allograft rejection in transplantation patients. The CXCR3 ligands CXCL9, CXCL10 and CXCL11 are expressed at sites of inflammation and attract CXCR3-expressing lymphocytes, thus contributing to the inflammatory process. Here, we characterize 5 non-peptidergic compounds of different chemical classes that block the action of CXCL10 and CXCL11 at the human CXCR3, i.e. VUF10472/NBI-74330, VUF10085/AMG-487, VUF5834, VUF10132 and TAK-779. In order to understand the action of these CXCR3 antagonists in various animal models of disease, the compounds were also tested at rat and mouse CXCR3, as well as at CXCR3 from rhesus macaque, cloned and characterized for the first time in this study. Except for TAK-779, all compounds show slightly lower affinity for rodent CXCR3 than for primate CXCR3. Additionally, we have characterized the molecular mechanism of action of the various antagonists at the human CXCR3 receptor. All tested compounds act as noncompetitive antagonists at CXCR3. Moreover, this non-competitive behavior is accompanied by inverse agonistic properties of all 5 compounds as determined on an identified constitutively active mutant of CXCR3, CXCR3 N3.35A. Interestingly, all compounds except TAK-779 act as full inverse agonists at CXCR3 N3.35A. TAK-779 shows weak partial inverse agonism at CXCR3 N3.35A, and likely has a different mode of interaction with CXCR3 than the other three classes of small molecule inverse agonists.
doi:10.1124/jpet.107.134783
PMCID: PMC3659174  PMID: 18270317
21.  Carnosol and Carnosic Acids from Salvia officinalis Inhibit Microsomal Prostaglandin E2 Synthase-1 
Prostaglandin E2 (PGE2), the most relevant eicosanoid promoting inflammation and tumorigenesis, is formed by cyclooxygenases (COXs) and PGE2 synthases from free arachidonic acid. Preparations of the leaves of Salvia officinalis are commonly used in folk medicine as an effective antiseptic and anti-inflammatory remedy and possess anticancer activity. Here, we demonstrate that a standard ethyl acetate extract of S. officinalis efficiently suppresses the formation of PGE2 in a cell-free assay by direct interference with microsomal PGE2 synthase (mPGES)-1. Bioactivity-guided fractionation of the extract yielded closely related fractions that potently suppressed mPGES-1 with IC50 values between 1.9 and 3.5 μg/ml. Component analysis of these fractions revealed the diterpenes carnosol and carnosic acid as potential bioactive principles inhibiting mPGES-1 activity with IC50 values of 5.0 μM. Using a human whole-blood assay as a robust cell-based model, carnosic acid, but not carnosol, blocked PGE2 generation upon stimulation with lipopolysaccharide (IC50 = 9.3 μM). Carnosic acid neither inhibited the concomitant biosynthesis of other prostanoids [6-keto PGF1α, 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid, and thromboxane B2] in human whole blood nor affected the activities of COX-1/2 in a cell-free assay. Together, S. officinalis extracts and its ingredients carnosol and carnosic acid inhibit PGE2 formation by selectively targeting mPGES-1. We conclude that the inhibitory effect of carnosic acid on PGE2 formation, observed in the physiologically relevant whole-blood model, may critically contribute to the anti-inflammatory and anticarcinogenic properties of S. officinalis.
doi:10.1124/jpet.112.193847
PMCID: PMC3523389  PMID: 22511203
22.  A Novel Human Multidrug Resistance Gene MDR1 Variant G571A (G191R) Modulates Cancer Drug Resistance and Efflux Transport 
The human multidrug resistance gene MDR1 encodes a membrane-bound transporter P-glycoprotein (Pgp) that confers the drug resistance of cancer cells by mediating an ATP-dependent drug efflux transport. We and others have reported a number of functionally significant MDR1 variants, including G1199A and G1199T, that modulate cancer drug resistance and intracellular levels of antivirals. In this report, we describe a novel G571A variant of MDR1 detected in 6.4% of leukemia patients. Because this nucleotide modification gives rise to an amino acid change from Gly to Arg at the 191 amino acid position of Pgp, we have developed and characterized the functional affect of the G571A variant in stable, recombinant cells. Using six chemotherapeutic drugs, doxorubicin HCl, daunorubicin HCl, vinblastine sulfate, vincristine sulfate, taxanes (paclitaxel), and epipodophyllotoxin (etoposide, VP-16), we found that the MDR1571A variant selectively reduced the degree of Pgp-mediated resistance in drug-dependent manner. Although there was a minimal effect on doxorubicin and daunorubicin, the MDR1-dependent resistance on vinblastine, vincristine, paclitaxel, and etoposide was reduced by approximately 5-fold. The increased drug sensitivity in MDR1571A, compared with MDR1wt, paralleled the intracellular drug levels. These data suggest that individuals with this novel MDR1 variant, the 571A genotype, may be more sensitive to the specific anticancer drugs that are Pgp substrates.
doi:10.1124/jpet.108.138313
PMCID: PMC3477805  PMID: 18723777
23.  Adenosine Receptor Prodrugs: Towards Kidney-Selective Dialkylxanthines 
XAC (xanthine amine congener, 8-{4-[(2-aminoethyl)-aminocarbonylmethyloxy]phenyl}-1,3-dipropylxanthine is a potent adenosine antagonist that reverses the reduction in urine flow, sodium excretion and heart rate produced by the adenosine agonist, N6-cyclohexyladenosine. New derivatives of XAC in which the primary amino group has been condensed to the γ-carboxyl group of glutamic acid have been synthesized as prodrugs. These amino acid-XAC conjugates, which are considerably less potent than XAC in competitive binding assays at A1-adenosine receptors, are designed for selective enzymatic activation in the kidneys. The γ-glutamyl xanthine derivatives are substrates for γ-glutamyl transferase (EC 2.3.2.2) to generate an amine-functionalized xanthine. N-acetyl-γ-L-glutamyl-XAC is not active in vivo, consistent with inability of renal acylase (EC 3.5.1.14) to hydrolyze the acetyl group, a prerequisite step for the production of XAC from this molecule. The xanthine derivatives, γ-L-glutamyl-XAC and γ-L-glutamyl-γ-L-glutamyl-XAC are metabolized to XAC and produce a diuresis in vivo.
PMCID: PMC3476460  PMID: 2746513
24.  MRS2500 [2-Iodo-N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate], a Potent, Selective, and Stable Antagonist of the Platelet P2Y1 Receptor with Strong Antithrombotic Activity in Mice 
The platelet P2Y1 ADP receptor is an attractive target for new antiplatelet drugs. However, because of the lack of strong and stable antagonists, only a few studies have suggested that pharmacological inhibition of the P2Y1 receptor could efficiently inhibit experimental thrombosis in vivo. Our aim was to determine whether the newly described potent and selective P2Y1 receptor antagonist MRS2500 [2-iodo-N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate] could inhibit platelet function ex vivo and experimental thrombosis in mice in vivo. MRS2500 was injected intravenously into mice, and its effect on ex vivo platelet aggregation and in several models of thrombosis in vivo was determined. MRS2500 displayed high potency and stable and selective P2Y1 receptor inhibition ex vivo. Although MRS2500 injection resulted in only moderate prolongation of the bleeding time, it provided strong protection in systemic thromboembolism induced by infusion of a mixture of collagen and adrenaline. MRS2500 also potently inhibited localized arterial thrombosis in a model of laser-induced vessel wall injury with two degrees of severity. Moreover, combination of MRS2500 with clopidogrel, the irreversible inhibitor of the platelet P2Y12 receptor for ADP, led to increased antithrombotic efficacy compared with each alone. These results add further evidence for a role of the P2Y1 receptor in thrombosis and validate the concept that targeting the P2Y1 receptor could be a relevant alternative or complement to current antiplatelet strategies.
doi:10.1124/jpet.105.094037
PMCID: PMC3471153  PMID: 16236815
25.  Induction of Novel Agonist Selectivity for the ADP-Activated P2Y1 Receptor Versus the ADP-Activated P2Y12 and P2Y13 Receptors by Conformational Constraint of an ADP Analog 
ADP is the cognate agonist of the P2Y1, P2Y12, and P2Y13 receptors. With the goal of identifying a high potency agonist that selectively activates the P2Y1 receptor, we examined the pharmacological selectivity of the conformationally constrained non-nucleotide analog (N)-methanocarba-2MeSADP [(1′S,2′R, 3′S,4′R,5′S)-4-[(6-amino-2-methylthio-9H-purin-9-yl)-1-diphosphoryloxymethyl]bicyclo[3.1.0]hexane-2,3-diol] among the three ADP-activated receptors. Each P2Y receptor was expressed transiently in COS-7 cells, and inositol lipid hydrolysis was quantified as a measure of receptor activity. In the case of the Gi-linked P2Y12 and P2Y13 receptors, a chimeric G protein, Gαq/i, was coexpressed to confer a capacity of these Gi-linked receptors to activate phospholipase C. 2MeSADP (2-methylthio-ADP) was a potent agonist at all three receptors exhibiting EC50 values in the sub to low nanomolar range. In contrast, whereas (N)-methanocarba-2MeSADP was an extremely potent (EC50 = 1.2 ± 0.2 nM) agonist at the P2Y1 receptor, this non-nucleotide analog exhibited no agonist activity at the P2Y12 receptor and very low activity at the P2Y13 receptor. (N)-Methanocarba-2MeSADP also failed to block the action of 2MeSADP at the P2Y12 and P2Y13 receptors, indicating that the (N)-methanocarba analog is not an antagonist at these receptors. The P2Y1 receptor selectivity of (N)-methanocarba-2MeSADP was confirmed in human platelets where it induced the shape change promoted by P2Y1 receptor activation without inducing the sustained platelet aggregation that requires simultaneous activation of the P2Y12 receptor. These results provide the first demonstration of a high-affinity agonist that discriminates among the three ADP-activated P2Y receptors, and therefore, introduce a potentially important new pharmacological tool for delineation of the relative biological action of these three signaling proteins.
doi:10.1124/jpet.104.068650
PMCID: PMC3459333  PMID: 15345752

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