Psychiatric patients frequently exhibit long-chain n-3 (LCn-3) fatty acid deficits and elevated triglyceride (TAG) production following chronic exposure to second generation antipsychotics (SGA). Emerging evidence suggests that SGAs and LCn-3 fatty acids have opposing effects on stearoyl-CoA desaturase-1 (SCD1), which plays a pivotal role in TAG biosynthesis. Here we evaluated whether low LCn-3 fatty acid status would augment elevations in rat liver and plasma TAG concentrations following chronic treatment with the SGA risperidone (RSP), and evaluated relationships with hepatic SCD1 expression and activity indices. In rats maintained on the n-3 fatty acid-fortified (control) diet, chronic RSP treatment significantly increased liver SCD1 mRNA and activity indices (18:1/18:0 and 16:1/16:0 ratios), and significantly increased liver, but not plasma, TAG concentrations. Rats maintained on the n-3 deficient diet exhibited significantly lower liver and erythrocyte LCn-3 fatty acid levels, and associated elevations in LCn-6/LCn-3 ratio. In n-3 deficient rats, RSP-induced elevations in liver SCD1 mRNA and activity indices (18:1/18:0 and 16:1/16:0 ratios) and liver and plasma TAG concentrations were significantly greater than those observed in RSP-treated controls. Plasma glucose levels were not altered by diet or RSP, and body weight was lower in RSP- and VEH-treated n-3 deficient rats. These preclinical data support the hypothesis that low n-3 fatty acid status exacerbates RSP-induced hepatic steatosis by augmenting SCD1 expression and activity.
Omega-3 fatty acids; Risperidone; Atypical antipsychotic; Stearoyl-CoA desaturase-1; Oleic acid; Polyunsaturated fatty acids; Triglycerides; Glucose; Liver; Rat
Tea contains a variety of bioactive chemicals, such as catechins and other polyphenols. These compounds are thought to be responsible for the health benefits of tea consumption by affecting the function of many cellular targets, not all of which have been identified. In a high-throughput screen for small molecule antagonists of the EphA4 receptor tyrosine kinase, we identified five tea polyphenols that substantially inhibit EphA4 binding to a synthetic peptide ligand. Further characterization of theaflavin monogallates from black tea and epigallocatechin-3,5-digallate from green tea revealed that these compounds at low micromolar concentrations also inhibit binding of the natural ephrin ligands to EphA4 and several other Eph receptors in in vitro assays. The compounds behave as competitive EphA4 antagonists, and their inhibitory activity is affected by amino acid mutations within the ephrin binding pocket of EphA4. In contrast, the major green tea catechin, epigallocatechin-3-gallate (EGCG), does not appear to be an effective Eph receptor antagonist. In cell culture assays, theaflavin monogallates and epigallocatechin-3,5-digallate inhibit ephrin-induced tyrosine phosphorylation (activation) of Eph receptors and endothelial capillary-like tube formation. However, the wider spectrum of Eph receptors affected by the tea derivatives in cells suggests additional mechanisms of inhibition besides interfering with ephrin binding. These results show that tea polyphenols derived from both black and green tea can suppress the biological activities of Eph receptors. Thus, the Eph receptor tyrosine kinase family represents an important class of targets for tea-derived phytochemicals.
angiogenesis; small molecule; antagonist; epigallocatechin-3-gallate; epigallocatechin-3; 5-digallate; theaflavin monogallates
Fatty-acid amide hydrolase (FAAH) catalyzes the intracellular hydrolysis of the endocannabinoid anandamide and other bioactive lipid amides. In the present study, we conducted a comparative characterization of the effects of the newly identified brain-impermeant FAAH inhibitor, URB937 ([3-(3-carbamoylphenyl)-4-hydroxy-phenyl] N-cyclohexylcarbamate), in various rodent models of acute and persistent pain. When administered by the oral route in mice, URB937 was highly active (median effective dose, ED50, to inhibit liver FAAH activity: 0.3 mg-kg−1) and had a bioavailability of 5.3%. The antinociceptive effects of oral URB937 were investigated in mouse models of acute inflammation (carrageenan), peripheral nerve injury (chronic sciatic nerve ligation) and arthritis (complete Freund’s adjuvant). In all models, URB937 was as effective or more effective than standard analgesic and anti-inflammatory drugs (indomethacin, gabapentin, dexamethasone) and reversed pain-related responses (mechanical hyperalgesia, thermal hyperalgesia, and mechanical allodynia) in a dose-dependent manner. ED50 values ranged from 0.2 to 10 mg-kg−1, depending on model and readout. Importantly, URB937 was significantly more effective than two global FAAH inhibitors, URB597 and PF-04457845, in the complete Freund’s adjuvant model. The effects of a combination of URB937 with the non-steroidal anti-inflammatory agent, indomethacin, were examined in the carrageenan and chronic sciatic nerve ligation models. Isobolographic analyses showed that the two compounds interacted synergistically to attenuate pain-related behaviors. Furthermore, URB937 reduced the number and severity of gastric lesions produced by indomethacin, while exerting no ulcerogenic effect when administered alone. The results indicate that the peripheral FAAH inhibitor URB937 is more effective than globally active FAAH inhibitors at inhibiting inflammatory pain. Our findings further suggest that FAAH and cyclooxygenase inhibitors interact functionally in peripheral tissues, to either enhance or hinder each other’s actions.
inflammation; anandamide; neuropathic pain; gastric lesions; cannabinoid receptors
The O-arylcarbamate URB937 is a potent inhibitor of fatty-acid amide hydrolase (FAAH), an intracellular serine hydrolase responsible for the deactivation of the endocannabinoid anandamide. URB937 is unique among FAAH inhibitors in that is actively extruded from the central nervous system (CNS), and therefore increases anandamide levels exclusively in peripheral tissues. Despite its limited distribution, URB937 exhibits marked analgesic properties in rodent models of pain. Pharmacological evidence suggests that the extrusion of URB937 from the CNS may be mediated by the ABC membrane transporter ABCG2 (also called Breast Cancer Resistance Protein, BCRP). In the present study, we show that URB937 is a substrate for both mouse and human orthologues of ABCG2. The relative transport ratios for URB937 in Madin-Darby canine kidney (MDCKII) cells monolayers over-expressing either mouse Abcg2 or human ABCG2 were significantly higher compared to parental monolayers (13.6 and 13.1 vs 1.5, respectively). Accumulation of the compound in the luminal/apical side was prevented by co-administration of the selective ABCG2 inhibitor, Ko-143. In vivo studies in mice showed that URB937 (25 mg-kg−1) readily entered the brain and spinal cord of Abcg2-deficient mice following intraperitoneal administration, whereas the same dose of drug remained restricted to peripheral tissues in wild-type mice. By identifying ABCG2 as a transport mechanism responsible for the extrusion of URB937 from the CNS, the present results should facilitate the rational design of novel peripherally restricted FAAH inhibitors.
Fatty-acid amide hydrolase; URB937; Breast cancer resistance protein; blood-brain barrier; central nervous system; Abcg2-deficient mice
Haptoglobin (Hp) is a hemoglobin (Hb) binding protein whose major function is to prevent heme-iron mediated oxidation. The polymorphic nature of the Hp gene results in varying levels of antioxidant function associated with the protein products. Multiple clinical studies have now determined that the Hp 2-2 genotype is associated with an increased risk of developing vascular complications in patients suffering from diabetes. The mechanism for this phenomenon is a decrease in antioxidant capability associated with the Hp 2-2 protein. Specifically, heme iron associated with the Hp2-2/Hb complex is more redox active than other Hp type complexes and has been shown in a number of systems to lead to increased levels of oxidative stress in the form of oxidized lipids and decreased lipoprotein function. In addition, Hp 2-2/Hb complexes are cleared less efficiently from the circulation, leading to a buildup of iron in the plasma and in tissues. Recent analyses from clinical studies utilizing vitamin E treatment have shown beneficial results specifically in the diabetic Hp 2-2 genotype population. The use of vitamin E in the treatment of Hp 2-2 diabetics has the potential to greatly reduce medical costs and improve quality of life in the ever-growing diabetic population.
Haptoglobin; Hemoglobin; Oxidative Stress; Vitamin E
β-Adrenoceptor (β-AR)-mediated relaxation plays an important role in the regulation of vascular tone. β-AR-mediated vascular relaxation is reduced in various disease states and aging. We hypothesized that β-AR-mediated vasodilatation is impaired in DOCA-salt hypertension due to alterations in the cAMP pathway. β-AR-mediated relaxation was determined in small mesenteric arteries from DOCA-salt hypertensive and control uninephrectomized (Uni) rats. To exclude nitric oxide (NO) and cyclooxygenase (COX) pathways, relaxation responses were determined in the presence of L-NNA and indomethacin, NO synthase inhibitor and COX inhibitors, respectively. Isoprenaline (ISO)-induced relaxation was reduced in arteries from DOCA-salt compared to Uni rats. Protein kinase A (PKA) inhibitors (H89 or Rp-cAMPS) or adenylyl cyclase inhibitor (SQ22536) did not abolish the difference in ISO-induced relaxation between the groups. Forskolin (adenylyl cyclase activator)-induced relaxation was similar between the groups. The inhibition of IKCa/SKCa channels (TRAM-34 plus UCL1684) or BKCa channels (iberiotoxin) reduced ISO-induced relaxation only in Uni rats and abolished the relaxation differences between the groups. The expression of SKCa channel was decreased in DOCA-salt arteries. The expression of BKCa channel α subunit was increased whereas the expression of BKCa channel β subunit was decreased in DOCA-salt arteries. The expression of receptor for activated C kinase 1 (RACK1), which is a binding protein for BKCa channel and negatively modulates its activity, was increased in DOCA-salt arteries. These results suggest that the impairment of β-AR-mediated relaxation in DOCA-salt mesenteric arteries may be attributable to altered IKCa/SKCa and/or BKCa channels activities rather than cAMP/PKA pathway. Impaired β-AR-stimulated BKCa channel activity may be due to the imbalance between its subunit expressions and RACK1 upregulation.
β-adrenoceptor; calcium-activated potassium channel; DOCA-salt; relaxation; mesenteric artery; RACK1
The biological function of most proteins relies on reversible post-translational modifications, among which phosphorylation is most prominently studied and well recognized. Recently, a growing amount of evidence indicates that acetylation-deacetylation reactions, when applied to crucial mediators, can also robustly affect the function of target proteins and thereby have wide-ranging physiological impacts. Sirtuin 1 (SIRT1), which functions as a nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase, deacetylates a wide variety of metabolic molecules in response to the cellular energy and redox status and as such causes significant changes in metabolic homeostasis. This review surveys the evidence for the emerging role of SIRT1-mediated deacetylation in the control of metabolic homeostasis.
SIRT1; NAD; deacetylation; metabolic homeostasis
Adenosine released during myocardial ischemia mediates cardioprotective preconditioning. Multivalent drugs covalently bound to nanocarriers may differ greatly in chemical and biological properties from the corresponding monomeric agents. Here, we conjugated chemically functionalized nucleosides to poly(amidoamine) (PAMAM) dendrimeric polymers and investigated their effects in rat primary cardiac cell cultures and in the isolated heart. Three conjugates of A3 adenosine receptor (AR) agonists, chain-functionalized at the C2 or N6 position, were cardioprotective, with greater potency than monomeric agonist Cl-IB-MECA. Multivalent amide-linked MRS5216 was selective for A1 and A3ARs, and triazole-linked MRS5246 and MRS5539 (optionally containing fluorescent label) were A3AR-selective. The conjugates protected ischemic rat cardiomyocytes, an effect blocked by an A3AR antagonist MRS1523, and isolated hearts with significantly improved infarct size, rate of pressure product, and rate of contraction and relaxation. Thus, strategically derivatized nucleosides tethered to biocompatible polymeric carriers display enhanced cardioprotective potency via activation of A3AR on the cardiomyocyte surface.
dendrimer; cardiomyocyte; adenosine receptor; ischemia; isolated heart; rat
The ADP-activated P2Y1 receptor is broadly expressed and plays a crucial role in ADP-promoted platelet aggregation. We previously synthesized 2-iodo-N6-methyl–(N)-methanocarba-2′-deoxyadenosine 3′,5′-bisphosphate (MRS2500), as a selective, high affinity, competitive antagonist of this receptor. Here we report utilization of a trimethylstannyl precursor molecule for the multistep radiochemical synthesis of a [125I]-labeled form of MRS2500. [125I]MRS2500 bound selectively to Sf9 insect cell membranes expressing the human P2Y1 receptor but did not specifically bind to membranes isolated from empty vector-infected cells. Binding of [125I]MRS2500 to P2Y1 receptors was saturable with a Kd of 1.2 nM. Known agonists and antagonists of the P2Y1 receptor inhibited [125I]MRS2500 binding to P2Y1 receptor-expressing membranes with potencies in agreement with those previously observed in functional assays of this receptor. A high-affinity binding site for [125I]MRS2500 also was observed on intact human platelets (Kd = 0.61 nM) and mouse platelets (Kd = 1.20 nM) that exhibited the pharmacological selectivity of the P2Y1 receptor. The densities of sites observed were 151 sites/platelet and 229 sites/platelet in human and mouse platelets, respectively. In contrast, specific binding was not observed in platelets isolated from P2Y1 receptor (−/−) mice. Taken together, these data illustrate the synthesis and characterization of a novel P2Y1 receptor radioligand and its utility for examining P2Y1 receptors natively expressed on human and mouse platelets.
P2Y1 receptor; competitive antagonist; radioligand; MRS2500; platelet
Signaling and internalization of Ste2p, a model G protein-coupled receptor (GPCR) from the yeast Saccharomyces cerevisiae, are reported to be regulated by phosphorylation status of serine (S) and threonine (T) residues located in the cytoplasmic C-terminus. Although the functional roles of S/T residues located in certain C-terminus regions are relatively well characterized, systemic analyses have not been conducted for all the S/T residues that are spread throughout the C-terminus. A point mutation to alanine was introduced into the S/T residues located within three intracellular loops and the C-terminus individually or in combination. A series of functional assays such as internalization, FUS1-lacZ induction, and growth arrest were conducted in comparison between WT- and mutant Ste2p. The Ste2p in which all S/T residues in the C-terminus were mutated to alanine was more sensitive to α-factor, suggesting that phosphorylation in the C-terminus exerts negative regulatory activities on the Ste2p signaling. C-terminal S/T residues proximal to the seventh transmembrane domain were important for ligand-induced G protein coupling but not for receptor internalization. Sites on the central region of the C-terminus regulated both constitutive and ligand-induced internalization. Residues on the distal part were important for constitutive desensitization and modulated the G protein signaling mediated through the proximal part of the C-terminus. This study demonstrated that the C-terminus contains multiple functional domains with differential and interdependent roles in regulating Ste2p function in which the S/T residues located in each domain play critical roles.
Ste2p; C-terminus; Phosphorylation; Internalization; FUS-lacZ; Growth arrest
Tumor necrosis factor alpha (TNF-α) plays a major role in the pathogenesis of many inflammatory diseases. Neutralizing TNF-α by antibodies or antisense oligodeoxynucleotides, alleviate disease symptoms. In this study, we introduce the new generation of gene-silencing molecules, namely the small interfering RNAs (siRNAs) to reduce TNF-α. Although siRNAs of 19–21 base pairs are commonly used, it is reported that longer siRNAs have much higher efficacies. Here, we report the identification of a 27-mer Dicer-substrate siRNA (DsiRNA) against TNF-α mRNA. Primary cells of rat Kupffer cells were transfected with five 27-mer siRNA constructs (si27-1, si27-2 si27-3, si27-4 and si27-5) for 24 h, following which, TNF-α secretion was induced by exposure to LPS (0.1 ug/ml) for 2 h. TNF-α released to the medium was measured by ELISA. Of the five si27 constructs, si27-3 had the highest inhibitory effect on TNF-α secretion. At 10 nM, si27-3 inhibited TNF-α secretion by 80% compared to a 60% inhibition by a 21-mer (SSL3). Following encapsulation in anionic liposomes, si27-3 at 100 µg/kg body weight, on two successive days by intravenous administration, inhibited the secretion of TNF-α by 50%. These data demonstrate the identification of a highly efficacious siRNA formulation, which can be used in the treatment of TNF-α mediated diseases.
TNF alpha; siRNA; Kupffer cells; lipopolysaccharide; liposomes
Sepsis is a disease process that has humbled the medical profession for centuries with its resistance to therapy, relentless mortality, and pathophysiologic complexity. Despite 30 years of aggressive, concerted, well-resourced efforts the biomedical community has been unable to reduce the mortality of sepsis from 30%, nor the mortality of septic shock from greater than 50%. In the last decade only one new drug for sepsis has been brought to the market, drotrecogin alfa-activated (Xigris™), and the success of this drug has been limited by patient safety issues. Clearly a new agent is desperately needed. The advent of recombinant human immune modulators held promise but the outcomes of clinical trials using biologics that target single immune mediators have been disappointing. The complex pathophysiology of the systemic inflammatory response syndrome (SIRS) is self-amplifying and redundant at multiple levels. In this review we argue that perhaps pharmacologic therapy for sepsis will only be successful if it addresses this pathophysiologic complexity; the drug would have to be pleiotropic, working on many components of the inflammatory cascade at once. In this context, therapy that targets any single inflammatory mediator will not adequately address the complexity of SIRS. We propose that Chemically Modified Tetracycline-3, CMT-3 (or COL-3), a non-antimicrobial modified tetracycline with pleiotropic anti-inflammatory properties, is an excellent agent for the management of sepsis and its associated complication of the Acute Respiratory Distress Syndrome (ARDS). The purpose of this review is threefold: 1) to examine the shortcomings of current approaches to treatment of sepsis and ARDS in light of their pathophysiology, 2) to explore the application of COL-3 in ARDS and sepsis, and finally 3) to elucidate the mechanisms of COL-3 that may have potential therapeutic benefit in ARDS and sepsis.
Sepsis; Shock; Acute Respiratory Distress Syndrome; ARDS; MODS
In vitro and animal studies suggest a possible role for the tetracycline class of drugs in the inhibition of non-enzymatic protein glycation. We conducted a 3-month, randomized placebo-controlled pilot clinical trial of conventional sub-gingival debridement, (periodontal therapy) combined with either a three month regimen of sub-antimicrobial-dose doxycycline (SDD), a two week regimen of antimicrobial-dose doxycycline (ADD), or placebo in 45 patients with long-standing type 2 diabetes (mean duration 9 years) and untreated chronic periodontitis. Subjects were taking stable doses of oral hypoglycemic medications and/or insulin. Treatment response was assessed by measuring hemoglobin A1c (HbA1c),plasma glucose, and clinical periodontal disease measures. At one-month and three-month follow-up, clinical measures of periodontitis were decreased in all groups(data to be presented elsewhere). At three months, mean HbA1c levels in the SDD group were reduced 0.9% unitsfrom 7.2% units ± 2.2(±SD), to 6.3% units ±1.1, which represents a 12.5% improvement. In contrast, there was no significant change in HbA1c in the ADD (7.5%± 2.0 to 7.8%± 2.1) or placebo (8.5%± 2.0 to 8.5%± 2.6) groups. Mean HbA1c change from baseline was significantly greater in the SDD group compared with the ADD group (p=0.04) but not placebo (p=0.22). Moreover, a larger proportion of subjects in the SDD group experienced improvement (p<0.05) compared to the ADD or placebo groups. Mean plasma glucose levels were not significantly different between or within the groups. The results of this pilot study suggest that the treatment of periodontitis with sub-gingival debridement and 3-months of daily sub-antimicrobial-dose doxycycline may decrease HbA1c in patients with type 2 diabetes taking normally prescribed hypoglycemic agents.
sub-antimicrobial-dose doxycycline; antibiotic dose doxycycline; type 2 diabetes; chronic periodontitis; periodontal therapy; HbA1c
Broccoli consumption may reduce the risk of various cancers and many broccoli supplements are now available. The bioavailability and excretion of the mercapturic acid pathway metabolites isothiocyanates after human consumption of broccoli supplements has not been tested. Two important isothiocyanates from broccoli are sulforaphane and erucin. We employed a cross-over study design in which 12 subjects consumed 40 grams of fresh broccoli sprouts followed by a 1 month washout period and then the same 12 subjects consumed 6 pills of a broccoli supplement. As negative controls for isothiocyanate consumption four additional subjects consumed alfalfa sprouts during the first phase and placebo pills during the second. Blood and urine samples were collected for 48 hours during each phase and analyzed for sulforaphane and erucin metabolites using LC-MS/MS. The bioavailability of sulforaphane and erucin is dramatically lower when subjects consume broccoli supplements compared to fresh broccoli sprouts. The peaks in plasma concentrations and urinary excretion were also delayed when subjects consumed the broccoli supplement. GSTP1 polymorphisms did not affect the metabolism or excretion of sulforaphane or erucin. Sulforaphane and erucin are able to interconvert in vivo and this interconversion is consistent within each subject but variable between subjects. This study confirms that consumption of broccoli supplements devoid of myrosinase activity does not produce equivalent plasma concentrations of the bioactive isothiocyanate metabolites compared to broccoli sprouts. This has implications for people who consume the recommended serving size (1 pill) of a broccoli supplement and believe they are getting equivalent doses of isothiocyanates.
glucosinolate; isothiocyanate; sulforaphane; erucin; bioavailability
RhoA protein is involved in the Ca2+ sensitization of bronchial smooth muscle (BSM) contraction, and an upregulation of RhoA in BSMs has been suggested in allergic bronchial asthma. However, the mechanism of upregulation of RhoA remains poorly understood. In the present study, the transcriptional regulation of human RhoA gene was investigated in cultured human BSM cells stimulated with IL-13 and TNF-α, both of which have an ability to upregulate RhoA protein. Luciferase-based assay showed that the RhoA promoter activity was augmented by both IL-13 and TNF-α. The deletion studies revealed a significant level of promoter activity between the 112 bp upstream and the transcription start site, which contains the STAT6 (78–70 bp upstream) and NF-κB (84–74 bp upstream) binding regions. The promoter activity was also decreased significantly by the mutations of these regions. Thus, the current study for the first time characterized the transcriptional regulation of the human RhoA gene. The findings also suggest that STAT6 and NF-κB are important for the upregulation of RhoA in human BSM induced by IL-13 and TNF-α, both of which are major cytokines in the pathogenesis of allergic bronchial asthma.
RhoA; STAT6; NF-κB; Airway hyperresponsiveness; IL-13; TNF-α
Diazepam Binding Inhibitor; Dopamine; Opioid peptides; NGF; GM1; Trk
Ranolazine is a clinically approved drug for treating cardiac ventricular dysrhythmias and angina. Its mechanism(s) of protection is not clearly understood but evidence points to blocking the late Na+ current that arises during ischemia, blocking mitochondrial complex I activity, or modulating mitochondrial metabolism. Here we tested the effect of ranolazine treatment before ischemia at the mitochondrial level in intact isolated hearts and in mitochondria isolated from hearts at different times of reperfusion. Left ventricular (LV) pressure (LVP), coronary flow (CF), and O2 metabolism were measured in guinea pig isolated hearts perfused with Krebs-Ringer’s solution; mitochondrial (m) O2•−, Ca2+, NADH/FAD (redox state), and cytosolic (c) Ca2+ were assessed on-line in the LV free wall by fluorescence spectrophotometry. Ranolazine (5 µM), infused for 1 min just before 30 min of global ischemia, itself did not change O2•−, cCa2+, mCa2+ or redox state. During late ischemia and reperfusion (IR) O2•− emission and m[Ca2+] increased less in the ranolazine group vs. the control group. Ranolazine decreased c[Ca2+] only during ischemia while NADH and FAD were not different during IR in the ranolazine vs. control groups. Throughout reperfusion LVP and CF were higher, and ventricular fibrillation was less frequent. Infarct size was smaller in the ranolazine group than the control group. Mitochondria isolated from ranolazine-treated hearts had mild resistance to permeability transition pore (mPTP) opening and less cytochrome c release than control hearts. Ranolazine may provide functional protection of the heart during IR injury by reducing cCa2+ and mCa2+ loading secondary to its effect to block the late Na+ current. Subsequently it indirectly reduces O2•− emission, preserves bioenergetics, delays mPTP opening, and restricts loss of cytochrome c, thereby reducing necrosis and apoptosis.
mitochondrial Ca2+; oxidative stress; permeability transition pore; ranolazine
Endothelin-1 (ET-1) is a pain mediator, elevated in skin after injury, which potentiates noxious thermal and mechanical stimuli (hyperalgesia) through the activation of ETA (and, perhaps, ETB) receptors on pain fibers. Part of the mechanism underlying this effect has recently been shown to involve potentiation of neuronal TRPV1 by PKCε. However, the early steps of this pathway, which is recapitulated in HEK 293 cells co-expressing TRPV1 and ETA receptors, remain unexplored. To clarify these steps we investigated the pharmacological profile and signaling properties of native endothelin receptors in immortalized cell lines including HEK 293 and ND-7 model sensory neurons. Previously we showed that in ND7/104, a dorsal root ganglia-derived cell line, ET-1 elicits a rise in intracellular calcium ([Ca2+]in) which is blocked by BQ-123, an ETA receptor antagonist, but not by BQ-788, an ETB receptor antagonist, suggesting that ETA receptors mediate this effect. Here we extend these findings to HEK 293T cells. Examination of the expression of ETA and ETB receptors by RT-PCR and [125I]-ET-1 binding experiments confirms the slight predominance of ETA receptor binding sites and messenger RNA in both ND7/104 and HEK 293T cells. In addition, selective agonists of the ETB receptor (sarafotoxin 6c, BQ-3020 or IRL-1620) do not induce a transient increase in [Ca2+]in. Furthermore, reduction of ETB mRNA levels by siRNA do not abrogate calcium mobilization by ET-1 in HEK 293T cells, corroborating the lack of an ETB receptor role in this response. However, in cells with low endogenous ETA mRNA levels, ET-1 does not induce a transient increase in [Ca2+]in. Observation of the [Ca2+]in elevation in ND7/104 and HEK 293T cells in the absence of extracellular calcium suggests that ET-1 elicits a release of calcium from intracellular stores, and pretreatment of the cells with pertussis toxin or a selective inhibitor of phospholipase C (PLC) point to a mechanism involving Gαq/11 coupling.
These results are consistent with the hypothesis that a certain threshold of ETA receptor expression is necessary to drive a transient [Ca2+]in increase in these cells and that this process involves release of calcium from intracellular stores following Gαq/11 activation.
Endothelin-1; ETA receptor; ETB receptor; HEK 293T; ND7/104; calcium; Gαq/11
Statins, long known to be beneficial in conditions where dyslipidemia occurs by lowering serum cholesterol levels, also have been proposed for use in neurodegenerative conditions, including Alzheimer disease. However, it is not clear that the purported effectiveness of statins in neurodegenerative disorders is directly related to cholesterol-lowering effects of these agents; rather, the pleiotropic functions of statins likely play critical roles.
Moreover, it is becoming more apparent with additional studies that statins can have deleterious effects in preclinical studies and lack effectiveness in various recent clinical trials. This perspective paper outlines pros and cons of the use of statins in neurodegenerative disorders, with particular emphasis on Alzheimer disease.
Dysregulation in signaling of the endocannabinoid 2-arachidonoylglycerol (2-AG) is implicated in hyperresponsiveness to stress. We hypothesized that blockade of monoacylglycerol lipase (MGL), the primary enzyme responsible for 2-AG deactivation in vivo, would produce context-dependent anxiolytic effects in rats. Environmental aversiveness was manipulated by varying illumination of an elevated plus maze. Percentage open arm time and numbers of open and closed arm entries were measured in rats receiving a single intraperitoneal (i.p.) injection of either vehicle, the MGL inhibitor JZL184 (1–8 mg/kg), the benzodiazepine diazepam (1 mg/kg), the cannabinoid CB1 receptor antagonist rimonabant (1 mg/kg), or JZL184 (8 mg/kg) coadministered with rimonabant (1 mg/kg). JZL184 (8 mg/kg) produced anxiolytic-like effects (i.e. increased percentage open arm time and number of open arm entries) under high, but not low, levels of environmental aversiveness. Diazepam produced anxiolytic effects in either context. Rimonabant blocked the anxiolytic-like effects of JZL184, consistent with mediation by CB1. Anxiolytic effects of JZL184 were preserved following chronic (8 mg/kg per day × 6 days) administration. Chronic and acute JZL184 treatment similarly enhanced behavioral sensitivity to an exogenous cannabinoid (WIN55,212-2; 2.5 mg/kg i.p.) 24 or 72 h following the terminal injection, suggesting a pervasive effect of MGL inhibition on the endocannabinoid system. We attribute our results to alterations in emotion rather than locomotor activity as JZL184 did not alter the number of closed arm entries in the plus maze or produce motor ataxia in the bar test. Our results demonstrate that JZL184 has beneficial, context-dependent effects on anxiety in rats, presumably via inhibition of MGL-mediated hydrolysis of 2-AG. These data warrant further testing of MGL inhibitors to elucidate the functional role of 2-AG in controlling anxiety and stress responsiveness. Our data further implicate a role for 2-AG in the regulation of emotion and validate MGL as a therapeutic target.
anxiety; 2-arachidonoylglycerol (2-AG); endocannabinoid; environmental aversiveness; JZL184; monoacylglycerol lipase (MGL)
Experimental studies have consistently shown the inhibitory activities of tea extracts on tumorigenesis in multiple model systems. Epidemiologic studies, however, have produced inconclusive results in humans. A comprehensive review was conducted to assess the current knowledge on tea consumption and risk of cancers in humans. In general, consumption of black tea was not associated with lower risk of cancer. High intake of green tea was consistently associated with reduced risk of upper gastrointestinal tract cancers after sufficient control for confounders. Limited data support a protective effect of green tea on lung and hepatocellular carcinogenesis. Although observational studies do not support a beneficial role of tea intake on prostate cancer risk, phase II clinical trials have demonstrated an inhibitory effect of green tea extract against the progression of prostate pre-malignant lesions. Green tea may exert beneficial effects against mammary carcinogenesis in premenopausal women and recurrence of breast cancer. There is no sufficient evidence that supports a protective role of tea intake on the development of cancers of the colorectum, pancreas, urinary tract, glioma, lymphoma, and leukemia. Future prospective observational studies with biomarkers of exposure and phase III clinical trials are required to provide definitive evidence for the hypothesized beneficial effect of tea consumption on cancer formation in humans.
cancer prevention; catechins; black tea; epidemiology; green tea; polypehnols
Osteoporosis is a major health problem in the elderly. Epidemiological evidence has shown an association between tea consumption and the prevention of bone loss in the elderly population. Ingestion of green tea and green tea bioactive compounds may be beneficial in mitigating bone loss of this population and decreasing their risk of osteoporotic fractures. This review describes the effect of green tea with its bioactive components on bone health with an emphasis on the following: (i) the etiology of osteoporosis, (ii) evidence of osteo-protective impacts of green tea on bone mass and microarchitecture in various bone loss models in which induced by aging, sex hormone deficiency, and chronic inflammation, (iii) discussion of impacts of green tea on bone mass in two obesity models, (iv) observation of short-term green tea supplementation given to postmenopausal women with low bone mass, (v) possible mechanisms for the osteo-protective effects of green tea bioactive compounds, and (vi) a summary and future research direction of green tea and bone health.
tea polyphenols; antioxidant; bone mineral density; osteoporosis; human; rat; cell
Green tea (Camellia sinensis, Theaceace) is the second most popular beverage in the world and has been extensively studied for its putative disease preventive effects. Green tea is characterized by the presence of a high concentrations of polyphenolic compounds known as catechins, with (−)-epigallocatechin-3-gallate (EGCG) being the most abundant and most well-studied. Metabolic syndrome (MetS) is a complex condition that is defined by the presence of elevated waist circumference, dysglycemia, elevated blood pressure, decrease serum high density lipoprotein-associated cholesterol, and increased serum triglycerides. Studies in both in vitro and laboratory animal models have examined the preventive effects of green tea and EGCG against the symptoms of MetS. Overall, the results of these studies have been promising and demonstrate that green tea and EGCG have preventive effects in both genetic and dietary models of obesity, insulin resistance, hypertension, and hypercholesterolemia. Various mechanisms have been proposed based on these studies and include: modulation of dietary fat absorption and metabolism, increased glucose utilization, decreased de novo lipogenesis, enhanced vascular responsiveness, and antioxidative effects. In the present review, we discuss the current state of the science with regard to laboratory studies on green tea and MetS. We attempt to critically evaluate the available data and point out areas for future research. Although there is a considerable amount of data available, questions remain in terms of the primary mechanism(s) of action, the dose-response relationships involved, and the best way to translate the results to human intervention studies.
catechins; (−)-epigallocatechin-3-gallate; green tea; metabolic syndrome; obesity
There is increasing evidence for a protective effect of tea consumption against cardiovascular disease. This review summarizes the available epidemiological data providing evidence for and against such an effect. We also review observational and intervention studies that investigated an effect of tea and tea extracts on cardiovascular risk factors, including blood pressure, serum lipids, diabetes mellitus, and obesity. Finally, we review potential mechanisms of benefit, including anti-inflammatory, anti-oxidant, and anti-proliferative effects, as well as favorable effects on endothelial function. Overall, the observational data suggest a benefit, but results are mixed and likely confounded by lifestyle and background dietary factors. The weight of evidence indicates favorable effects on risk factors and a number of plausible mechanisms have been elucidated in experimental and translational human studies. Despite the growing body evidence, it remains uncertain whether tea consumption should be recommended to the general population or to patients as a strategy to reduce cardiovascular risk.
Tea; polyphenols; catechins; flavonoids; cardiovascular disease; endothelium
Green tea and its major polyphenols constituents, tea catechins, have been shown to have many health benefits including cancer prevention. Tea catechins and tea catechin metabolites/catabolites are bioavailable in the systemic circulation after oral intake of green tea or green tea catechins. The metabolites/catabolites identified in humans include glucuronide/sulfate conjugates, methylated tea catechin conjugates, and microflora-mediated ring fission products and phenolic acid catabolites. Plasma levels of unchanged tea catechins in humans are mostly in the sub-μM or nM concentration range, which is much lower than the effective concentrations determined in most in vitro studies. However, some of the catechin metabolites/catabolites are present in the systemic circulation at levels much higher than those of the parent catechins. The contribution of catechin derived metabolites/catabolites to the biological effects associated with green tea is yet to be defined. A limited number of chemoprevention trials of green tea or green tea catechins have been conducted to date and have observed potential preventive activity for oral, prostate, and colorectal cancer. Emerging data from multiple ongoing intervention trials will further contribute to defining the cancer preventive activity of green tea or green tea catechins.