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1.  Adenosine Kinase Inhibition Protects The Kidney Against Streptozotocin-Induced Diabetes Through Anti-inflammatory and Anti-oxidant Mechanisms 
Adenosine provides anti-inflammatory effects in cardiovascular disease via the activation of adenosine A2A receptors; however, the physiological effect of adenosine could be limited due to its phosphorylation by adenosine kinase. We hypothesized that inhibition of adenosine kinase exacerbates extracellular adenosine levels to reduce renal inflammation and injury in streptozotocin-induced diabetes. Diabetes was induced in male C57BL/6 mice by daily injection of streptozotocin (50 mg/kg/day, i.p. for 5 days). Control and diabetic mice were then treated with the adenosine kinase inhibitor ABT702 (1.5 mg/kg, i.p two times a week for 8 weeks, n = 7–8/group) or the vehicle (5% DMSO). ABT702 treatment reduced blood glucose level in diabetic mice (~ 20%; p<0.05). ABT702 also reduced albuminuria and markers of glomerular injury, nephrinuria and podocalyxin excretion levels, in diabetic mice. Renal NADPH oxidase activity and urinary thiobarbituric acid reactive substances (TBARS) excretion, indices of oxidative stress, were also elevated in diabetic mice and ABT702 significantly reduced these changes. ABT702 increased renal endothelial nitric oxide synthase expression (eNOS) and nitrate/nitrite excretion levels in diabetic mice. In addition, the diabetic mice displayed an increase in renal macrophage infiltration, in association with increased renal NFB activation. Importantly, treatment with ABT702 significantly reduced all these inflammatory parameters (P< 0.05). Furthermore, ABT702 decreased glomerular permeability and inflammation and restored the decrease in glomerular occludin expression in vitro in high glucose treated human glomerular endothelial cells. Collectively, the results suggest that the reno-protective effects of ABT702 could be attributed to the reduction in renal inflammation and oxidative stress in diabetic mice.
doi:10.1016/j.phrs.2014.05.004
PMCID: PMC4302753  PMID: 24841126
Diabetes; adenosine kinase inhibition; albuminuria; renal injury; oxidative stress; nitric oxide; macrophage infiltration
2.  Depressive behavior in the forced swim test can be induced by TRPV1 receptor activity and is dependent on NMDA receptors 
Blocking, desensitizing, or knocking out transient receptor potential vanilloid type 1 (TRPV1) receptors decreases immobility in the forced swim test, a measure of depressive behavior. We questioned whether enhancing TRPV1 activity promotes immobility in a fashion that is prevented by antidepressants. To test this we activated heat-sensitive TRPV1 receptors in mice by water that is warmer than body temperature (41°C) or a low dose of resiniferatoxin (RTX). Water at 41°C elicited less immobility than cooler water (26°C), indicating that thermoregulatory sites do not contribute to immobility. Although a desensitizing regimen of RTX (3–5 injections of 0.1 mg/kg s.c.) decreased immobility during swims at 26°C, it did not during swims at 41°C. In contrast, low dose of RTX (0.02 mg/kg s.c.) enhanced immobility, but only during swims at 41°C. Thus, activation of TRPV1 receptors, endogenously or exogenously, enhances immobility and these sites are activated by cold rather than warmth. Two distinct types of antidepressants, amitriptyline (10 mg/kg i.p.) and ketamine (50 mg/kg i.p.), each inhibited the increase in immobility induced by the low dose of RTX, verifying its mediation by TRPV1 sites. When desensitization was limited to central populations using intrathecal injections of RTX (0.25 µg/kg i.t.), immobility was attenuated at both temperatures and the increase in immobility produced by the low dose of RTX was inhibited. This demonstrates a role for central TRPV1 receptors in depressive behavior, activated by conditions (cold stress) distinct from those that activate TRPV1 receptors along thermosensory afferents (heat).
doi:10.1016/j.phrs.2013.10.006
PMCID: PMC3947229  PMID: 24200896
TRPV1; depression; desensitization; RTX; forced swim test
3.  Erythroid induction of K562 cells treated with mithramycin is associated with inhibition of raptor gene transcription and mammalian target of rapamycin complex 1 (mTORC1) functions 
Pharmacological Research  2015;91:57-68.
Graphical abstract
Rapamycin, an inhibitor of mTOR activity, is a potent inducer of erythroid differentiation and fetal hemoglobin production in β-thalassemic patients. Mithramycin (MTH) was studied to see if this inducer of K562 differentiation also operates through inhibition of mTOR. We can conclude from the study that the mTOR pathway is among the major transcript classes affected by mithramycin-treatment in K562 cells and a sharp decrease of raptor protein production and p70S6 kinase is detectable in mithramycin treated K562 cells. The promoter sequence of the raptor gene contains several Sp1 binding sites which may explain its mechanism of action. We hypothesize that the G + C-selective DNA-binding drug mithramycin is able to interact with these sequences and to inhibit the binding of Sp1 to the raptor promoter due to the following results: (a) MTH strongly inhibits the interactions between Sp1 and Sp1-binding sites of the raptor promoter (studied by electrophoretic mobility shift assays, EMSA); (b) MTH strongly reduces the recruitment of Sp1 transcription factor to the raptor promoter in intact K562 cells (studied by chromatin immunoprecipitation experiments, ChIP); (c) Sp1 decoy oligonucleotides are able to specifically inhibit raptor mRNA accumulation in K562 cells. In conclusion, raptor gene expression is involved in mithramycin-mediated induction of erythroid differentiation of K562 cells and one of its mechanism of action is the inhibition of Sp1 binding to the raptor promoter.
doi:10.1016/j.phrs.2014.11.005
PMCID: PMC4309890  PMID: 25478892
Raptor, regulatory associated protein of mTOR; Rictor, rapamycin-insensitive companion of mTOR; mTOR, mammalian target of rapamycin; mTORC1, mTOR complex 1; m-TORC2, mTOR complex 2; Sp1, specific protein 1; MTH, mithramycin; RAPA, rapamycin; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum; PBS, phosphate-buffered saline; TBS, tris-buffered saline; HbF, fetal hemoglobin; ODN, oligonucleotide; Raptor; mTOR; Sp1; Mithramycin; Erythroid induction; Fetal hemoglobin
4.  From Evolution to Revolution: miRNAs as Pharmacological Targets for Modulating Cholesterol Efflux and Reverse Cholesterol Transport 
There has been strong evolutionary pressure to ensure that an animal cell maintain levels of cholesterol within tight limits for normal function. Imbalances in cellular cholesterol levels are a major player in the development of different pathologies associated to dietary excess. Although epidemiological studies indicate that elevated levels of high-density lipoprotein (HDL)-cholesterol reduce the risk of cardiovascular disease, recent genetic evidence and pharmacological therapies to raise HDL levels do not support their beneficial effects. Cholesterol efflux as the first and probably the most important step in reverse cholesterol transport is an important biological process relevant to HDL function. Small non-coding RNAs (microRNAs), post-transcriptional control different aspects of cellular cholesterol homeostasis including cholesterol efflux. miRNA families miR-33, miR-758, miR-10b, miR-26 and miR-106b directly modulates cholesterol efflux by targeting the ATP-binding cassette transporter A1 (ABCA1). Pre-clinical studies with anti-miR therapies to inhibit some of these miRNAs have increased cellular cholesterol efflux, reverse cholesterol transport and reduce pathologies associated to dyslipidemia. Although miRNAs as therapy have benefits from existing antisense technology, different obstacles need to be solved before we incorporate such research into clinical care. Here we focus on the clinical potential of miRNAs as therapeutic target to increase cholesterol efflux and reverse cholesterol transport as a new alternative to ameliorate cholesterol-related pathologies.
doi:10.1016/j.phrs.2013.02.005
PMCID: PMC3825518  PMID: 23435093
ABCA1; Cholesterol efflux; HDL; miRNAs
5.  microRNAs as Pharmacological Targets in Endothelial Cell Function and Dysfunction 
Endothelial cell dysfunction is a term which implies the dysregulation of normal endothelial cell functions, including impairment of the barrier functions, control of vascular tone, disturbance of proliferative, migratory and morphogenic capacities of endothelial cells, as well as control of leukocyte trafficking. MicroRNAs (miRNAs) are short non-coding RNAs that have emerged as critical regulators of gene expression acting predominantly at the post-transcriptional level. This review summarizes the latest insights in the identification of endothelial-specific miRNAs and their targets, as well as their roles in controlling endothelial cell functions in both autocrine and paracrine manner. In addition, we discuss the therapeutic potential for the treatment of endothelial cell dysfunction and associated vascular pathophysiological conditions.
doi:10.1016/j.phrs.2013.04.002
PMCID: PMC3752325  PMID: 23603154
Endothelial cells; microRNAs; vascular disease; microRNA therapeutics; endothelial cell dysfunction
6.  MicroRNAs as Pharmacological Targets in Diabetes 
Diabetes is characterized by high levels of blood glucose due to either the loss of insulin-producing beta-cells in the pancreas, leading to a deficiency of insulin in type 1 diabetes, or due to increased insulin resistance, leading to reduced insulin sensitivity and productivity in type 2 diabetes. There is an increasing need for new options to treat diabetes, especially type 2 diabetes at its early stages due to the ineffective control of its development in patients. Recently, a novel class of small noncoding RNAs, termed microRNAs (miRNAs), found to play a key role as important transcriptional and posttranscriptional inhibitors of gene expression in fine-tuning the target messenger RNAs (mRNAs). miRNAs are implicated in the pathogenesis of diabetes and have become an intriguing target for therapeutic intervention. This review focuses on the dysregulated miRNAs discovered in various diabetic models and addresses the potential for miRNAs to be therapeutic targets in the treatment of diabetes.
doi:10.1016/j.phrs.2013.06.005
PMCID: PMC3786207  PMID: 23810798
microRNAs; diabetes; insulin resistance; miRNA therapeutics
7.  A Neurochemical Basis for an Epigenetic Vision of Psychiatric Disorders (1996-2009) 
doi:10.1016/j.phrs.2011.05.026
PMCID: PMC4144181  PMID: 21699980
Schizophrenia; Bipolar Disorder; Epigenetics; GABA; Reelin; Antipsychotics
8.  Endothelin and hepatic wound healing 
Liver wound healing is a coordinated response to injury caused by infections (hepatitis) or toxins (alcohol) or other processes where activation of hepatic stellate cells are a central component. During stellate cell activation, a major phenotypic transformation occurs which leads to increased production of increased extracellular matrix proteins and smooth muscle α-actin the results is organ dysfunction due to gross architectural disruption and impaired blood flow.
Endothelin-1 (ET-1) is produced in increased amounts and the cellular source of ET-1 shifts from endothelial cells to stellate cells during liver injury thus setting a feedback loop which accentuates further activation, stellate cell proliferation, and production of extracellular matrix proteins. Therapy directed at intervening the ET-1 signaling pathway has significant therapeutic potential in patients with liver disease.
doi:10.1016/j.phrs.2011.03.005
PMCID: PMC4129451  PMID: 21421048
9.  Age and gender dependent bioavailability of R- and R,S-α-lipoic acid: A pilot study 
Lipoic acid (LA) shows promise as a beneficial micronutrient toward improving elder health. Studies using old rats show that (R)-α-LA (R-LA) significantly increases low molecular weight antioxidants that otherwise decline with age. Despite this rationale for benefiting human health, little is known about age-associated alterations in absorption characteristics of LA, or whether the commercially available racemic mixture of LA (R,S-LA) is equally as bioavailable as the naturally occurring R-enantiomer. To address these discrepancies, a pilot study was performed to establish which form of LA is most effectively absorbed in older subjects relative to young volunteers. Young adults (average age = 32 years) and older adults (average age = 79 years) each received 500 mg of either R- or R,S-LA. Blood samples were collected for 3 h after supplementation. After a washout period they were given the other chiral form of LA not originally ingested. Results showed that 2 out of 6 elder males exhibited greater maximal plasma LA and area under the curve for the R-form of LA versus the racemic mixture. The elder subjects also demonstrated a reduced time to reach maximal plasma LA concentration following R-LA supplementation than for the racemic mixture. In contrast, young males had a tendency for increased bioavailability of R,S-LA. Overall, bioavailability for either LA isoform was much more variable between older subjects compared to young adults. Plasma glutathione levels were not altered during the sampling period. Thus subject age, and potential for varied response, should be considered when determining an LA supplementation regimen.
doi:10.1016/j.phrs.2012.05.002
PMCID: PMC4084596  PMID: 22609537
Glutathione; Pharmacokinetics; Lipoic acid
10.  Defining the blanks – Pharmacochaperoning of SLC6 transporters and ABC transporters? 
Pharmacological Research  2014;83(100):63-73.
Graphical abstract
SLC6 family members and ABC transporters represent two extremes: SLC6 transporters are confined to the membrane proper and only expose small segments to the hydrophilic milieu. In ABC transporters the hydrophobic core is connected to a large intracellular (eponymous) ATP binding domain that is comprised of two discontiguous repeats. Accordingly, their folding problem is fundamentally different. This can be gauged from mutations that impair the folding of the encoded protein and give rise to clinically relevant disease phenotypes: in SLC6 transporters, these cluster at the protein–lipid interface on the membrane exposed surface. Mutations in ABC-transporters map to the interface between nucleotide binding domains and the coupling helices, which provide the connection to the hydrophobic core. Folding of these mutated ABC-transporters can be corrected with ligands/substrates that bind to the hydrophobic core. This highlights a pivotal role of the coupling helices in the folding trajectory. In contrast, insights into pharmacochaperoning of SLC6 transporters are limited to monoamine transporters – in particular the serotonin transporter (SERT) – because of their rich pharmacology. Only ligands that stabilize the inward facing conformation act as effective pharmacochaperones. This indicates that the folding trajectory of SERT proceeds via the inward facing conformation. Mutations that impair folding of SLC6 family members can be transmitted as dominant or recessive alleles. The dominant phenotype of the mutation can be rationalized, because SLC6 transporters are exported in oligomeric form from the endoplasmic reticulum (ER). Recessive transmission requires shielding of the unaffected gene product from the mutated transporter in the ER. This can be accounted for by a chaperone-COPII (coatomer protein II) exchange model, where proteinaceous ER-resident chaperones engage various intermediates prior to formation of the oligomeric state and subsequent export from the ER. It is likely that the action of pharmacochaperones is contingent on and modulated by these chaperones.
doi:10.1016/j.phrs.2013.11.009
PMCID: PMC4059943  PMID: 24316454
ABC transporters, ATP-binding cassette containing transporters; DAT, dopamine transporter; ER, endoplasmic reticulum; GAT, GABA transporter with 4 isoforms (GAT-1, GAT-2, GAT-3, GAT-4); NBD, nucleotide binding domains; NET, norepinephrine transporter; SLC6, solute carrier-6; SERT, serotonin transporter; ABC transporters; Chaperones; ER export; Folding-deficient mutants; Pharmacochaperoning; SLC6 transporters
11.  DHA Supplementation: Current Implications in Pregnancy and Childhood 
Dietary supplementation with (ω)-3 long chain fatty acids including docosahexaenoic acid (DHA) has increased in popularity in recent years and adequate DHA supplementation during pregnancy and early childhood is of clinical importance. Some evidence has been built for the neuro-cognitive benefits of supplementation with long chain polyunsaturated fatty acids (LCPUFA) such as DHA during pregnancy; however, recent data indicate that the anti-inflammatory properties may be of at least equal significance. Adequate DHA availability in the fetus/infant optimizes brain and retinal maturation in part by influencing neurotransmitter pathways. The anti-inflammatory properties of LCPUFA are largely mediated through modulation of signaling either directly through binding to receptors or through changes in lipid raft formation and receptor presentation. Our goal is to review the current findings on DHA supplementation, specifically in pregnancy and infant neurodevelopment, as a pharmacologic agent with both preventative and therapeutic value. Given the overall benefits of DHA, maternal and infant supplementation may improve neurological outcomes especially in vulernable populations. However, optimal composition of the supplement and dosing and treatment strategies still need to be determined to lend support for routine supplementation.
doi:10.1016/j.phrs.2012.12.003
PMCID: PMC3602397  PMID: 23266567
DHA; long chain fatty acids; natural products; lipids; omega-3
12.  Ion channel remodeling in vascular smooth muscle during hypertension: Implications for novel therapeutic approaches 
Ion channels are multimeric, transmembrane proteins that selectively mediate ion flux across the plasma membrane in a variety of cells including vascular smooth muscle cells (VSMCs). The dynamic interplay of Ca2+ and K+ channels on the plasma membrane of VSMCs plays a pivotal role in modulating the vascular tone of small arteries and arterioles. The abnormally-elevated arterial tone observed in hypertension thus points to an aberrant expression and function of Ca2+ and K+ channels in the VSMCs. In this short review, we focus on the three well-studied ion channels in VSMCs, namely the L-type Ca2+ (CaV1.2) channels, the voltage-gated K+ (KV) channels, and the large-conductance Ca2+-activated K+ (BK) channels. First, we provide a brief overview on the physiological role of vascular CaV1.2, KV and BK channels in regulating arterial tone. Second, we discuss the current understanding of the expression changes and regulation of CaV1.2, KV and BK channels in the vasculature during hypertension. Third, based on available proof-of-concept studies, we describe the potential therapeutic approaches targeting these vascular ion channels in order to restore blood pressure to normotensive levels.
doi:10.1016/j.phrs.2013.01.008
PMCID: PMC3607210  PMID: 23376354
Hypertension; vascular smooth muscle cell; ion channel; calcium channel; potassium channel; gene therapy
13.  Developing a metagenomic view of xenobiotic metabolism 
The microbes residing in and on the human body influence human physiology in many ways, particularly through their impact on the metabolism of xenobiotic compounds, including therapeutic drugs, antibiotics, and diet-derived bioactive compounds. Despite the importance of these interactions and the many possibilities for intervention, microbial xenobiotic metabolism remains a largely underexplored component of pharmacology. Here, we discuss the emerging evidence for both direct and indirect effects of the human gut microbiota on xenobiotic metabolism, and the initial links that have been made between specific compounds, diverse members of this complex community, and the microbial genes responsible. Furthermore, we highlight the many parallels to the now well-established field of environmental bioremediation, and the vast potential to leverage emerging metagenomic tools to shed new light on these important microbial biotransformations.
doi:10.1016/j.phrs.2012.07.009
PMCID: PMC3526672  PMID: 22902524
Metagenomics; microbiome; microbiota; reduction; hydrolysis; xenobiotics
14.  Cyclooxygenase metabolism mediates vasorelaxation to 2-arachidonoylglycerol (2-AG) in human mesenteric arteries 
Pharmacological Research  2014;81(100):74-82.
Graphical abstract
Objective
The vasorelaxant effect of 2-arachidonoylglycerol (2-AG) has been well characterised in animals. 2-AG is present in human vascular cells and is up-regulated in cardiovascular pathophysiology. However, the acute vascular actions of 2-AG have not been explored in humans.
Approach
Mesenteric arteries were obtained from patients receiving colorectal surgery and mounted on a myograph. Arteries were contracted and 2-AG concentration–response curves were carried out. Mechanisms of action were characterised pharmacologically. Post hoc analysis was carried out to assess the effects of cardiovascular disease/risk factors on 2-AG responses.
Results
2-AG caused vasorelaxation of human mesenteric arteries, independent of cannabinoid receptor or transient receptor potential vanilloid-1 activation, the endothelium, nitric oxide or metabolism via monoacyglycerol lipase or fatty acid amide hydrolase. 2-AG-induced vasorelaxation was reduced in the presence of indomethacin and flurbiprofen, suggesting a role for cyclooxygenase metabolism 2-AG. Responses to 2-AG were also reduced in the presence of Cay10441, L-161982 and potentiated in the presence of AH6809, suggesting that metabolism of 2-AG produces both vasorelaxant and vasoconstrictor prostanoids. Finally, 2-AG-induced vasorelaxation was dependent on potassium efflux and the presence of extracellular calcium.
Conclusions
We have shown for the first time that 2-AG causes vasorelaxation of human mesenteric arteries. Vasorelaxation is dependent on COX metabolism, activation of prostanoid receptors (EP4 & IP) and ion channel modulation. 2-AG responses are blunted in patients with cardiovascular risk factors.
doi:10.1016/j.phrs.2014.02.001
PMCID: PMC3992009  PMID: 24548820
2-AG, 2-arachidonoylglcerol; AEA, anandamide; ANOVA, analysis of variance; CB1, cannabinoid receptor one; CB2, cannabinoid receptor two; COX-1, cyclooxygenase one; COX-2, cyclooxygenase two; ECS, endocannabinoid system; FAAH, fatty acid amide hydrolase; KPSS, high potassium physiological saline solution; l-NAME, NG-nitro-l-arginine methyl ester; MAGL, monoacylglycerol lipase; PPAR, peroxisome proliferator activated receptor; PSS, physiological saline solution; SEM, standard error of mean; TRPV1, transient receptor potential vanilloid-1; Endocannabinoid; 2-Arachidonoylglycerol; Vasorelaxation; Cyclooxygenase; Prostanoid; Human
15.  Cannabinoid Agonists Increase the Interaction between β-Arrestin 2 and ERK1/2 and Upregulate β-Arrestin 2 and 5-HT2A Receptors 
We have recently reported that selective cannabinoid 2 (CB2) receptor agonists upregulate 5-HT2A receptors by enhancing ERK1/2 signaling in prefrontal cortex (PFCx). Increased activity of cortical 5-HT2A receptors has been associated with several neuropsychiatric disorders such as anxiety and schizophrenia. Here we examine the mechanisms involved in this enhanced ERK1/2 activation in rat PFCx and in a neuronal cell model. Sprague-Dawley rats treated with a non-selective cannabinoid agonist (CP55940, 50 μg/kg, 7 days, i.p.) showed enhanced co-immunoprecipitation of β-Arrestin 2 and ERK1/2, enhanced pERK protein levels, and enhanced expression of β-Arrestin 2 mRNA and protein levels in PFCx. In a neuronal cell line, we found that selective CB2 receptor agonists upregulate β-Arrestin 2, an effect that was prevented by selective CB2 receptor antagonist JTE-907 and CB2 shRNA lentiviral particles. Additionally, inhibition of clathrin-mediated endocytosis, ERK1/2, and the AP-1 transcription factor also prevented the cannabinoid receptor-induced upregulation of β-Arrestin 2. Our results suggest that sustained activation of CB2 receptors would enhance β-Arrestin 2 expression possibly contributing to its increased interaction with ERK1/2 thereby driving the upregulation of 5-HT2A receptors. The CB2 receptor-mediated upregulation of β-Arrestin 2 would be mediated, at least in part, by an ERK1/2-dependent activation of AP-1. These data could provide the rationale for some of the adverse effects associated with repeated cannabinoid exposure and shed light on some CB2 receptor agonists that could represent an alternative therapeutic because of their minimal effect on serotonergic neurotransmission.
doi:10.1016/j.phrs.2012.11.002
PMCID: PMC3562593  PMID: 23174265
Cannabinoid receptors; 5-HT2A receptor; ERK1/2; β-Arrestin 2; prefrontal cortex
16.  Blueberry polyphenol-enriched soybean flour reduces hyperglycemia, body weight gain and serum cholesterol in mice 
Defatted soybean flour (DSF) can sorb and concentrate blueberry anthocyanins and other polyphenols, but not sugars. In this study blueberry polyphenol-enriched DSF (BB-DSF) or DSF were incorporated into very high fat diet (VHFD) formulations and provided ad libitum to obese and hyperglycemic C57BL/6 mice for 13 weeks to investigate anti-diabetic effects. Compared to the VHFD containing DSF, the diet supplemented with BB-DSF reduced weight gain by 5.6%, improved glucose tolerance, and lowered fasting blood glucose levels in mice within 7 weeks of intervention. Serum cholesterol of mice consuming the BB-DSF-supplemented diet was 13.2% lower than mice on the diet containing DSF. Compounds were eluted from DSF and BB-DSF for in vitro assays of glucose production and uptake. Compared to untreated control, doses of BB-DSF eluate containing 0.05 – 10 μg/μL of blueberry anthocyanins significantly reduced glucose production by 24% - 74% in H4IIE rat hepatocytes, but did not increase glucose uptake in L6 myotubes. The results indicate that delivery of blueberry polyphenols stabilized in a high-protein food matrix may be useful for the dietary management of pre-diabetes and/or diabetes.
doi:10.1016/j.phrs.2012.11.008
PMCID: PMC3833590  PMID: 23220243
metabolic syndrome; diabetes; blueberry; soybean; anthocyanins; polyphenols
17.  Aorta from angiotensin II hypertensive mice exhibit preserved nitroxyl anion mediated relaxation responses 
Hypertension is a disorder affecting millions worldwide, and is a leading cause of death and debilitation in the United States. It is widely accepted that during hypertension and other cardiovascular diseases the vasculature exhibits endothelial dysfunction; a deficit in the relaxatory ability of the vessel, attributed to a lack of nitric oxide (NO) bioavailability. Recently, the one electron redox variant of NO, nitroxyl anion (NO−) has emerged as an endothelium-derived relaxing factor (EDRF) and a candidate for endothelium-derived hyperpolarizing factor (EDRF). NO− is thought to exist protonated (HNO) in vivo, which would make this species more resistant to scavenging. However, no studies have investigated the role of this redox species during hypertension, and whether the vasculature loses the ability to relax to HNO. Thus, we hypothesize that aorta from angiotensin II (AngII)-hypertensive mice will exhibit a preserved relaxation response to Angeli’s Salt, an HNO donor. Male C57Bl6 mice, aged 12–14 weeks were implanted with mini-osmotic pumps containing AngII (90ng/min, 14 days plus high salt chow) or sham surgery. Aorta were excised, cleaned and used to perform functional studies in a myograph. We found that aorta from AngII-hypertensive mice exhibited a significant endothelial dysfunction as demonstrated by a decrease in acetylcholine (ACh)-mediated relaxation. However, vessels from hypertensive mice exhibited a preserved response to Angeli’s Salt (AS), the HNO donor. To confirm that relaxation responses to HNO were maintained, concentration response curves (CRCs) to ACh were performed in the presence of scavengers to both NO and HNO (carboxy-PTIO and L-cys, resp.). We found that ACh-mediated relaxation responses were significantly decreased in aorta from sham and almost completely abolished in aorta from AngII-treated mice. Vessels incubated with L-cys exhibited a modest decrease in ACh-mediated relaxations responses. These data demonstrate that aorta from AngII-treated hypertensive mice exhibit a preserved relaxation response to AS, an HNO donor, regardless of a significant endothelial dysfunction.
doi:10.1016/j.phrs.2011.07.002
PMCID: PMC3908541  PMID: 21767645
nitroxyl anion; HNO; vascular; aorta; angiotensin II hypertension
18.  Alterations in endocannabinoid tone following chemotherapy-induced peripheral neuropathy: Effects of endocannabinoid deactivation inhibitors targeting fatty-acid amide hydrolase and monoacylglycerol lipase in comparison to reference analgesics following cisplatin treatment 
SUMMARY
Cisplatin, a platinum-derived chemotherapeutic agent, produces mechanical and cold allodynia reminiscent of chemotherapy-induced neuropathy in humans. The endocannabinoid system represents a novel target for analgesic drug development. The endocannabinoid consists of endocannabinoids (e.g. anandamide (AEA) and 2-arachidonoylglycerol (2-AG)), cannabinoid receptors (e.g. CB1 and CB2) and the enzymes controlling endocannabinoid synthesis and degradation. AEA is hydrolyzed by fatty-acid amide hydrolase (FAAH) whereas 2-AG is hydrolyzed primarily by monoacylglycerol lipase (MGL). We compared effects of brain permeant (URB597) and impermeant (URB937) inhibitors of FAAH with an irreversible inhibitor of MGL (JZL184) on cisplatin-evoked behavioral hypersensitivities. Endocannabinoid modulators were compared with agents used clinically to treat neuropathy (i.e. the opioid analgesic morphine, the anticonvulsant gabapentin and the tricyclic antidepressant amitriptyline). Cisplatin produced robust mechanical and cold allodynia but did not alter responsiveness to heat. After neuropathy was fully established, groups received acute intraperitoneal (i.p.) injections of vehicle, amitriptyline (30 mg/kg), gabapentin (100 mg/kg), morphine (6 mg/kg), URB597 (0.1 or 1 mg/kg), URB937 (0.1 or 1 mg/kg) or JZL184 (1, 3 or 8 mg/kg). Pharmacological specificity was assessed by coadministering each endocannabinoid modulator with either a CB1 (AM251 3 mg/kg), CB2 (AM630 3 mg/kg), TRPV1 (AMG9810 3 mg/kg) or TRPA1 (HC030031 8 mg/kg) antagonist. Effects of cisplatin on endocannabinoid levels and transcription of receptors (CB1, CB2, TRPV1, TRPA1) and enzymes (FAAH, MGL) linked to the endocannabinoid system were also assessed. URB597, URB937, JZL184 and morphine reversed cisplatin-evoked mechanical and cold allodynia to pre-cisplatin levels. By contrast, gabapentin only partially reversed the neuropathy while amitriptyline, administered acutely, was ineffective. CB1 or CB2 antagonist completely blocked the anti-allodynic effects of both FAAH (URB597, URB937) and MGL (JZL184) inhibitors to mechanical and cold stimulation, while TRPV1 antagonist AMG9810 blocked only the anti-allodynic efficacy of both FAAH inhibitors, but not the MGL inhibitor,. By contrast, the TRPA1 antagonist HC30031 did not attenuate anti-allodynic efficacy of any endocannabinoid modulator. When the levels of endocannabinoids were examined, cisplatin increased both anandamide (AEA) and 2-arachidonoylglycerol (2-AG) levels in the lumbar spinal cord and decreased 2-AG levels (but not AEA) in dorsal hind paw skin. RT-PCR showed that mRNA for FAAH, but not other markers, was upregulated by cisplatin treatment in dorsal root ganglia. The present studies demonstrate that cisplatin alters endocannabinoid tone and that inhibition of endocannabinoid hydrolysis alleviates chemotherapy-induced mechanical and cold allodynia. The anti-allodynic effects of FAAH and MGL inhibitors are mediated by CB1 and CB2 cannabinoid receptors, whereas TRPV1, but not TRPA1, -dependent mechanisms contribute to the anti-allodynic efficacy of FAAH (but not MGL) inhibitors. Strikingly, endocannabinoid modulators potently suppressed cisplatin-evoked allodynia with a rapid onset and showed efficacy that equaled or exceeded that of major classes of anti-neuropathic pain medications used clinically. Thus, inhibition of endocannabinoid hydrolysis, via FAAH or MGL inhibitors, represents an efficacious pharmacological approach for suppressing chemotherapy-induced neuropathic pain.
doi:10.1016/j.phrs.2012.10.013
PMCID: PMC3525790  PMID: 23127915
anandamide; 2-arachidonoyl glycerol; endocannabinoid; neuropathic pain; cold allodynia and hyperalgesia
19.  Reverse-mode Na+/Ca2+ exchange is an important mediator of venous contraction 
The Na+/Ca2+ exchanger (NCX) is a bi-directional regulator of cytosolic Ca2+, causing Ca2+ efflux in forward-mode and Ca2+ influx in reverse-mode. We hypothesized that reverse-mode NCX is a means of Ca2+ entry in rat aorta (RA) and vena cava (RVC). NCX protein in RA and RVC was confirmed by immunoprecipitation. To assess NCX function, isometric contraction and intracellular Ca2+ was measured in RA and RVC rings in response to low extracellular Na+, endothelin-1 (ET-1), and KCl, in the presence or absence of the NCX antagonist KB-R7943. In RVC, low extracellular Na+ caused vasoconstriction and an increase in intracellular Ca2+ that was attenuated by 10 μM KB-R7943. KB-R7943 (10 μM) attenuated maximal contraction to ET-1 in RVC (53±9% of control), but not RA (91±1% of control). KB-R7943 (10 μM) reduced the maximal contraction to KCl in RA (48±5%) and nearly abolished it in RVC (9±2%), suggesting that voltage-dependent Ca2+ influx may be inhibited by KB-R7943 as well. However, the L-type Ca2+ channel inhibitor nifedipine (1 μM) did not alter ET-1-induced contraction. Our findings suggest that reverse-mode NCX is an important mechanism of Ca2+ influx in RVC but not RA, especially during ET-1-induced contraction. Also, the effects of KB-R7943 on ET-1-induced contraction of RA and RVC are predominantly mediated by reverse-mode NCX inhibition and not due to off-target inhibition of Ca2+ channels.
doi:10.1016/j.phrs.2012.08.004
PMCID: PMC3502721  PMID: 22974823
Na+/Ca2+ Exchanger; vasoconstriction; veins; calcium; endothelin-1
20.  Targeting Autophagy for the Treatment of Liver Diseases 
Autophagy is a lysosomal degradation pathway that can degrade bulk cytoplasm and superfluous or damaged organelles, such as mitochondria, to maintain cellular homeostasis. It is now known that dysregulation of autophagy can cause pathogenesis of numerous human diseases. Here, we discuss the critical roles that autophagy plays in the pathogenesis of liver diseases such as nonalcoholic and alcoholic fatty liver, drug-induced liver injury, protein aggregate-related liver diseases, viral hepatitis, fibrosis, aging and liver cancer. In particular, we discuss the emerging therapeutic potential by pharmacological modulation of autophagy for these liver diseases.
doi:10.1016/j.phrs.2012.07.003
PMCID: PMC3502728  PMID: 22871337
autophagy; mitophagy; liver diseases
21.  Chaperones in autophagy 
Cells continuously turn over proteins through cycles of synthesis and degradation in order to maintain a functional proteome and to exert a tight control in the levels of regulatory proteins. Selective degradation of proteins was initially thought to be an exclusive function of the ubiquitin-proteasome system however, over the years, the contribution of lysosomes to this selective degradation, through the process of autophagy, has become consolidated. In this context, molecular chaperones, classically associated with protein folding, unfolding and assembling, have been revealed as important modulators of selectivity during the autophagic process. Here, we review this relatively new role of chaperones in mediating selective autophagy and comment on how alterations of this function can lead to human pathologies associated to proteotoxicity.
doi:10.1016/j.phrs.2012.10.002
PMCID: PMC3502706  PMID: 23059540
Aging; chaperones; lysosomes; membrane proteins; protein degradation
22.  Targets, trafficking, and timing of cardiac autophagy 
Heart failure is the major case of death in developed countries, and its prevalence is growing worldwide. Autophagy is a fundamental cellular mechanism through which intracellular components can be removed, recycled and repaired. Studies in humans and animal models demonstrate a marked increase in cardiac autophagic activity under a wide range of disease states and in response to diverse stimuli. Recently, autophagy has been widely promoted as a potential therapeutic target for the treatment of cardiovascular disease and heart failure. An important challenge to achieving this goal is the dual nature of cardiac autophagy, sometimes acting to help preserve cardiac function, other times appearing to promote cardiac decline. Numerous control points regulating autophagic activity and cargo selection provide a diversity of opportunities for drug targeting. In addition there is an innate circadian rhythm to the systemic regulation of autophagy that is often overlooked but provides potential opportunities to target and optimize pharmacological intervention.
doi:10.1016/j.phrs.2012.10.001
PMCID: PMC3502698  PMID: 23059539
23.  Autophagy and the Regulation of the Immune Response 
Summary
Autophagy is a highly conserved mechanism of lysosomal-mediated protein degradation that plays a crucial role in maintaining cellular homeostasis by recycling amino acids, reducing the amount of damaged proteins and regulating protein levels in response to extracellular signals. In the last few years specific functions for different forms of autophagy have been identified in many tissues and organs. In the Immune System, autophagy functions range from the elimination infectious agents and the modulation of the inflammatory response, to the selection of antigens for presentation and the regulation of T cell homeostasis and activation. Here, we review the recent advances that have allowed us to better understand why autophagy is a crucial process in the regulation of the innate and adaptive immune responses.
doi:10.1016/j.phrs.2012.10.003
PMCID: PMC3508673  PMID: 23063674
Autophagy; T cell; Dendritic cell; Antigen presentation; Macrophage; Pathogen
24.  Bortezomib enhances antigen-specific cytotoxic T cell responses against immune-resistant cancer cells generated by STAT3-ablated dendritic cells 
Dendritic cell (DC)-based vaccines have received attention as a new therapeutic modality against cancer. However, increased STAT3 activity in the tumor microenvironment makes DCs tolerogenic and suppresses their antitumor activity. In this study, we explored the effects of a combination treatment consisting of a proteasome inhibitor, bortezomib, and an antigen specific STAT3-ablated (STAT3−/−) DC-based vaccine on the control of TC-1(P3) tumors, a p53-degraded immune resistant cancer cells. We found that E7-antigen expressing STAT3−/− DC (E7-DC-1STAT3−/−) vaccination enhanced generation of E7-specific CD8+ T cells, but was not enough to control TC-1(P3) cancer cells. Therefore, we investigated whether bortezomib could create a synergistic effect with E7-DC-1STAT3−/− vaccination. We found that apoptosis via down-regulation of STAT3 and NF-κB and up-regulation of Fas and death receptor 5 (DR5) expression in TC-1(P3) induced by bortezomib was independent of p53 status. We also observed that TC-1(P3) cells pretreated with bortezomib had markedly enhanced anti-tumor effects on E7-specific CD8+ T cells through a Fas/DR5-mediated mechanism. In addition, TC-1(P3) tumor-bearing mice treated with bortezomib prior to vaccination with E7-DC-1STAT3−/− demonstrated enhanced generation of E7-specific CD8+ T cells and prolonged survival compared to those treated with monotherapy. These results suggest that the anti-tumor effects against a p53-degraded immune resistant variant generated by antigen-expressing STAT3-ablated mature DCs may be enhanced by bortezomib via death receptor-mediated apoptosis.
doi:10.1016/j.phrs.2013.02.001
PMCID: PMC3817614  PMID: 23428347
Bortezomib; STAT3; Immune resistance; Cytotoxic T cells; Dendritic cells
25.  The CB2-preferring agonist JWH015 also potently and efficaciously activates CB1 in autaptic hippocampal neurons 
The G protein coupled receptors CB1 and CB2 are targets for the psychoactive constituents of cannabis, chief among them Δ9-THC. They are also key components of the multifunctional endogenous cannabinoid signaling system. CB1 and CB2 receptors modulate a wide variety of physiological systems including analgesia, memory, mood, reward, appetite and immunity. Identification and characterization of selective CB1 and CB2 receptor agonists and antagonists will facilitate understanding the precise physiological and pathophysiological roles of cannabinoid receptors in these systems. This is particularly necessary in the case of CB2 because these receptors are sparsely expressed and problematic to detect using traditional immunocytochemical approaches.
1-Propyl-2-methyl-3-(1-naphthoyl)indole (JWH015) is an aminoalkylindole that has been employed as a “CB2-selective” agonist in more than 40 published papers. However, we have found that JWH015 potently and efficaciously activates CB1 receptors in neurons. Using murine autaptic hippocampal neurons, which express CB1, but not CB2 receptors, we find that JWH015 inhibits excitatory postsynaptic currents with an EC50 of 216 nM. JWH015 inhibition is absent in neurons from CB1−/− cultures and is reversed by the CB1 antagonist, SR141716 [200 nM]. Furthermore, JWH015 partially occludes CB1-mediated DSE (~35% remaining), an action reversed by the CB2 antagonist, AM630 [1 and 3 μM], suggesting that high concentrations of AM630 also antagonize CB1 receptors.
We conclude that while JWH015 is a CB2-preferring agonist, it also activates CB1 receptors at experimentally encountered concentrations. Thus, CB1 agonism of JWH015 needs to be considered in the design and interpretation of experiments that use JWH015 to probe CB2-signaling.
doi:10.1016/j.phrs.2012.08.002
PMCID: PMC3601544  PMID: 22921769
JWH015; Cannabinoid; CB1; CB2; Marijuana; AM630

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