ABSTRACT
AIMS: To evaluate the effect of the combination of carotegrast methyl with rifampicin, a potent inhibitor of organic anion transporter polypeptide, on the pharmacokinetics (PKs), safety and tolerability of carotegrast methyl. METHODS: In this 2 × 2 crossover study in 20 healthy Japanese adults, 10 subjects received carotegrast methyl 960 mg and rifampicin 600 mg on day 1 and received carotegrast methyl 960 mg on day 8. The subjects in the other sequence received the same treatments but in the opposite order. The 90% confidence interval (CI) of the geometric mean ratio of the Cmax and AUC0-t for carotegrast, the main active metabolite of carotegrast methyl, with/without rifampicin was calculated. If the 90% CI fell within the range of 0.80-1.25, this indicated the absence of any drug-drug interaction. Adverse events (AEs) were monitored. RESULTS: The geometric mean ratios (90% CI) of the Cmax and AUC0-t for carotegrast with/without rifampicin were 4.78 (3.64-6.29) and 5.59 (4.60-6.79), respectively, indicating that carotegrast has a PK interaction with rifampicin. The combination with rifampicin increased the exposure of carotegrast and also that of its metabolites. The incidence of any AEs with/without rifampicin was five (25.0%) and one (5.0%), respectively. CONCLUSIONS: Coadministration of carotegrast methyl with rifampicin significantly increased the exposure of carotegrast compared with carotegrast methyl administration alone. In this single dose study, the incidence of AEs of carotegrast methyl with rifampicin increased compared with carotegrast methyl alone, but the incidence of adverse drug reactions did not increase with combination administration.
Subject(s)
Area Under Curve , Cross-Over Studies , Drug Interactions , Healthy Volunteers , Rifampin , Humans , Rifampin/administration & dosage , Rifampin/adverse effects , Rifampin/pharmacokinetics , Male , Adult , Female , Young Adult , Organic Anion Transporters/antagonists & inhibitorsABSTRACT
AIMS: This study evaluated drug-drug interactions between the CYP3A4 inhibitor carotegrast methyl and the other CYP3A4 substrates, midazolam, atorvastatin and prednisolone. METHODS: A total of 88 healthy volunteers orally received carotegrast methyl 960 mg 3 times daily for 14 days. A single oral (5 mg) or intravenous (0.017 mg kg-1 ) midazolam, oral (5 mg) prednisolone or oral (10 mg) atorvastatin was administered before, with and after carotegrast methyl treatment. When the 90% confidence interval (CI) for the geometric mean ratios of the pharmacokinetic (PK) parameters with coadministration with carotegrast methyl (Day 14) to those before carotegrast methyl administration was between 0.80 and 1.25, no PK interaction were deemed. RESULTS: The Cmax and AUC0-t of oral midazolam before administration of carotegrast methyl were 30.9 ± 9.8 ng mL-1 and 74.5 ± 21.9 ng h mL-1 , respectively. The geometric mean ratio of the Cmax and AUC0-t of midazolam on Day 14 to those on Day -1 was 1.86 (90% CI, 1.64-2.11) and 3.07 (90% CI, 2.81-3.35), which did not fall within the range of 0.80-1.25, suggesting that carotegrast methyl had a PK interaction with midazolam. Similar PK interactions were found for intravenous midazolam and atorvastatin, but not for prednisolone. The inhibitory effect of carotegrast methyl on CYP3A4-mediated metabolism of midazolam and atorvastatin had almost disappeared by 14 days after the end of administration. CONCLUSION: Carotegrast methyl was classified as a moderate CYP3A4 inhibitor in humans. Carotegrast methyl might enhance the action of drugs that are metabolized by CYP3A4.
Subject(s)
Cytochrome P-450 CYP3A , Midazolam , Phenylalanine/analogs & derivatives , Quinazolinones , Adult , Humans , Midazolam/pharmacokinetics , Atorvastatin/pharmacology , Cytochrome P-450 CYP3A/metabolism , Cytochrome P-450 CYP3A Inhibitors/pharmacology , Prednisolone , Drug Interactions , Area Under CurveABSTRACT
We examined the impact of a history of coronary artery disease (CAD) or cerebrovascular disease (CVD) and physical activity habits on functional disability among community-dwelling Japanese adults. This population-based retrospective cohort study included 10,661 people aged 39-98 years in Japan (5054, men). Median follow-up was 3.7 years. During the study period, 209 functional disabilities occurred in the overall study population. In multivariable analysis, a history of CVD (hazard ratio [HR] 1.57 [95% CI: 1.00-2.45]) and no physical activity habit (HR 1.74 [1.27-2.39]) presented increased risks for functional disability. HRs for functional disability among patients with a CVD history with and without a physical activity habit were 1.68 (0.75-3.74) and 2.65 (1.49-4.71), respectively, compared with individuals without a history of CVD with a physical activity habit. Similar results were observed for CAD. We found no significant difference in the incidence of functional disability between the group with a history of CAD or CVD and physical activity habits and the group with no history of CAD or CVD and without physical activity habits. Physical activity habits had a favorable influence on avoiding functional disability regardless of a history of CAD or CVD. Future prospective studies are needed to clarify these associations.
Subject(s)
Cardiovascular Diseases , Cerebrovascular Disorders , Coronary Artery Disease , Adult , Male , Humans , Cardiovascular Diseases/epidemiology , Retrospective Studies , Incidence , Risk Factors , Coronary Artery Disease/epidemiology , HabitsABSTRACT
Carotegrast-methyl (brand name: CAROGRA® Tablets) is a new chemical entity created by Ajinomoto Pharmaceuticals Co., Ltd. (currently EA Pharma Co., Ltd.) as an α4 integrin inhibitor. In vivo, it exerts an anti-inflammatory effect by inhibiting the functions of both α4ß1 integrin and α4ß7 integrin expressed on the surface of inflammatory cells such as lymphocytes. Under the joint development of EA Pharma Co., Ltd. and Kissei Pharmaceutical Co., Ltd., the efficacy and safety of carotegrast methyl were confirmed in patients with moderate active ulcerative colitis. Carotegrast-methyl, the Japan-originated, world-first orally available α4 integrin antagonist, was approved in March and launched in May 2022 in Japan. Patients who had inadequate response or intolerance to the basic treatment with 5-ASA preparations for ulcerative colitis, have widely desired an orally available treatment with the new mechanism of actions. Carotegrast methyl can be a treatment option that meets that unmet medical need and has the potential to greatly contribute to the treatment of ulcerative colitis based on the thorough practice of proper use. This article mainly introduces the pharmacological properties and clinical trial results of carotegrast methyl.
Subject(s)
Colitis, Ulcerative , Humans , Integrin alpha4 , Quinazolinones , IntegrinsABSTRACT
The present study investigated histological subtypes of lymphoma in patients newly diagnosed with malignant lymphoma in the human T-cell leukemia virus type 1 (HTLV-1) endemic area of Japan, and further analyzed the clinicopathological features and clinical outcomes of patients with primary sinonasal lymphoma. We retrospectively examined 151 patients aged 18-90 years in Fukuoka, Japan. Subtypes of lymphoma were determined according to the WHO classification. Among the 151 patients, 104 were diagnosed with malignant lymphoma, including 96 at the time of initial diagnosis. Ninety-two of the 96 lymphomas (96%) were non-Hodgkin lymphoma. Mature B-cell neoplasms comprised 78% (n = 75). Primary lymphoma of the sinonasal cavity was found in six patients (6%). The histological subtype of sinonasal lymphoma was diffuse large B-cell lymphoma (DLBCL) in all six tumors. Furthermore, overall survival was significantly different among three distinct DLBCL patient groups, including primary sinonasal lymphoma patients (p = 0.0016; 3-year overall survival: sinonasal DLBCL group, 53%; DLBCL of the CNS group, 0%; other DLBCL group, 83%). Our study suggests that primary DLBCL of the sinonasal tract is a distinct disease entity of DLBCL.
Subject(s)
Human T-lymphotropic virus 1 , Lymphoma, Large B-Cell, Diffuse , Paranasal Sinus Neoplasms , Adolescent , Adult , Aged , Aged, 80 and over , Disease-Free Survival , Female , Humans , Japan/epidemiology , Lymphoma, Large B-Cell, Diffuse/diagnosis , Lymphoma, Large B-Cell, Diffuse/mortality , Lymphoma, Large B-Cell, Diffuse/pathology , Lymphoma, Large B-Cell, Diffuse/therapy , Male , Middle Aged , Paranasal Sinus Neoplasms/diagnosis , Paranasal Sinus Neoplasms/mortality , Paranasal Sinus Neoplasms/pathology , Paranasal Sinus Neoplasms/therapy , Retrospective Studies , Survival RateABSTRACT
Dietary restriction (DR), such as calorie restriction (CR) or methionine (Met) restriction, extends the lifespan of diverse model organisms. Although studies have identified several metabolites that contribute to the beneficial effects of DR, the molecular mechanism underlying the key metabolites responsible for DR regimens is not fully understood. Here we show that stimulating S-adenosyl-l-methionine (AdoMet) synthesis extended the lifespan of the budding yeast Saccharomyces cerevisiae The AdoMet synthesis-mediated beneficial metabolic effects, which resulted from consuming both Met and ATP, mimicked CR. Indeed, stimulating AdoMet synthesis activated the universal energy-sensing regulator Snf1, which is the S. cerevisiae ortholog of AMP-activated protein kinase (AMPK), resulting in lifespan extension. Furthermore, our findings revealed that S-adenosyl-l-homocysteine contributed to longevity with a higher accumulation of AdoMet only under the severe CR (0.05% glucose) conditions. Thus, our data uncovered molecular links between Met metabolites and lifespan, suggesting a unique function of AdoMet as a reservoir of Met and ATP for cell survival.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Longevity , S-Adenosylmethionine/metabolism , Adenosine Triphosphate/metabolism , Caloric Restriction , Epistasis, Genetic , Genes, Dominant , Glucan 1,3-beta-Glucosidase/genetics , Glucan 1,3-beta-Glucosidase/metabolism , Metabolic Networks and Pathways , Methionine/metabolism , Mutation , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolismABSTRACT
A new monoterpenoid indole alkaloid, kopsiyunnanine K, was isolated from Kopsia arborea. Its intriguing rearranged structure and absolute configuration, which were inferred from spectral data and a possible biosynthetic pathway, were determined on the basis of a 13-step asymmetric total synthesis.
ABSTRACT
Two new indole alkaloids, kopsiyunnanines J1 and J2, were isolated from Yunnan Kopsia arborea, and their structures were determined by spectroscopic analyses. Kopsiyunnanines J1 and J2 are unprecedented Strychnos-type indole alkaloids having an additional C1 unit in the secologanin moiety of the molecule.
Subject(s)
Apocynaceae/chemistry , Indole Alkaloids/isolation & purification , Secologanin Tryptamine Alkaloids/isolation & purification , Indole Alkaloids/chemistry , Molecular Structure , Secologanin Tryptamine Alkaloids/chemistryABSTRACT
PURPOSE: Patients in the early stage of retinitis pigmentosa (RP) suffer from night blindness and, therefore, have mobility problems at night. To assist such patients with walking in the dark, we developed a wearable visual aid utilizing a see-through display upon which assistive images from a high-sensitivity video camera are superimposed. We evaluated the efficacy of our new visual aid for RP patients. METHODS: The device is equipped with a camera with a minimum illuminance of 0.08 lux and a view angle of 53° × 40°. The experiment was conducted in a room with dimmed light (illuminance level 0.2-1.2 lux). Eight subjects with RP were instructed to arrive at a goal 16 m away from the starting point, both with and without the device, passing through four 1.5-m-wide gates consisting of pairs of black square carpet pieces, white poles, red and white traffic cones and cardboard boxes with and without the device in a darkened room. Three gates, except for the boxes, which were nearest the goal, were randomly arranged along the x-axis at each trial. The number of trial failures and the time required to walk the course were assessed as outcomes. RESULTS: Seven of the 8 subjects could walk with the aid of the device without any failure. With the device, the number of trial failures significantly decreased in number (p < 0.05) in all subjects. CONCLUSIONS: This device enabled the subjects to see objects that could not be recognized by the unaided eye. Our visual aid effectively assisted RP patients with night blindness.
Subject(s)
Eyeglasses , Night Blindness/rehabilitation , Photography/instrumentation , Retinitis Pigmentosa/rehabilitation , Sensory Aids , Aged , Equipment Design , Female , Humans , Male , Middle Aged , Motor Activity , Night Blindness/physiopathology , Retinitis Pigmentosa/physiopathology , Visual Acuity/physiology , WalkingABSTRACT
Hog1 of Saccharomyces cerevisiae is activated by hyperosmotic stress, and this leads to cell-cycle delay in G1, but the mechanism by which cells restart from G1 delay remains elusive. We found that Whi3, a negative regulator of G1 cyclin, counteracted Hog1 in the restart from G1 delay caused by osmotic stress. We have found that phosphorylation of Ser-568 in Whi3 by RAS/cAMP-dependent protein kinase (PKA) plays an inhibitory role in Whi3 function. In this study we found that the phosphomimetic Whi3 S568D mutant, like the Δwhi3 strain, slightly suppressed G1 delay of Δhog1 cells under osmotic stress conditions, whereas the non-phosphorylatable S568A mutation of Whi3 caused prolonged G1 arrest of Δhog1 cells. These results indicate that Hog1 activity is required for restart from G1 arrest under osmotic stress conditions, whereas Whi3 acts as a negative regulator for this restart mechanism.
Subject(s)
G1 Phase Cell Cycle Checkpoints , Mitogen-Activated Protein Kinases/metabolism , Osmotic Pressure , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Cyclins/metabolism , G1 Phase Cell Cycle Checkpoints/genetics , Mitogen-Activated Protein Kinases/genetics , Mutation , RNA-Binding Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Up-RegulationABSTRACT
BACKGROUND: Effect of bicarbonated Ringer's solution (BRS) on the liver function of rats with metabolic acidosis was compared with that of lactated Ringer's solution (LRS) and Ringer's solutions (RS). Furthermore, the effect of acidosis on the plasma protein binding ratio of propofol was examined. METHODS: Partial hepatectomized rats were divided into three groups, and BRS, LRS or RS was intravenously administered at 20 ml x kg(-1) x hr(-1) with propofol (45 mg x kg(-1) hr(-1)) for 90 min. Immediately after the administration, indocyanine-green (ICG) was infused. Blood sampling was done at 30, 60, 90 and 120 min after the ICG infusion, and the ICG concentration in plasma was measured. Propofol was added to plasma of rats with ketoacidosis or normal rats (final conc. 5 microg x ml(-1)), and incubated for 30 min at 37 degrees C. After incubation, plasma propofol concentrations were measured. RESULTS: The reduction of ICG in plasma of BRS group was the fastest among the three groups. Plasma protein binding ratio of propofol in plasma samples of rats with ketoacidosis tended to be lower than those of normal rats samples. CONCLUSIONS: These results suggest that metabolic acidosis induced the delay of awakening from anesthesia, suggesting that BRS accelerates recovery from anesthesia through amelioration of acidosis.
Subject(s)
Acidosis/metabolism , Anesthesia Recovery Period , Isotonic Solutions/administration & dosage , Propofol/metabolism , Animals , Infusions, Intravenous , Male , Propofol/administration & dosage , Propofol/blood , Protein Binding , Rats , Rats, Sprague-Dawley , Ringer's Lactate , Ringer's Solution , Time FactorsABSTRACT
Mechanical stresses regulate physiological and pathological functions of vascular endothelial cells. We examined, in this study, the effects of hypergravity on endothelial functions. Hypergravity (3 G) applied by low speed centrifuge immediately induced a membrane translocation of small G-protein RhoA and tyrosine phosphorylation of 125 kDa FAK in bovine aortic endothelial cells (BAECs). Hypergravity also induced a transient reorganization of actin fibers in 3 min, which was inhibited by Rho-kinase inhibitor (Y27632) and tyrosine kinase inhibitors (herbimycin A and tyrphostin 46). Furthermore, the extracellular ATP concentration ([ATP]o) was increased by 2 G and 3 G hypergravity in 5 min, and the inhibitors of Rho-kinase, tyrosine kinase, and volume-regulated anion channels (VRAC; verapamil, tamoxifen and fluoxetine) significantly suppressed [ATP]o elevation. Application of 3 G hypergravity for 1 h increased the nuclear uptake of BrdU, which was inhibited by Rho-kinase inhibitor and VARC inhibitors. Furthermore, intermittent application of 3 G hypergravity for 1 or 2 h/day stimulated endothelial migration in 5 days, and this was inhibited by suramin, a P2 antagonist. Collectively, these results indicate that hypergravity induces ATP release and actin reorganization via RhoA activation and FAK phosphorylation, thereby activating cell proliferation and migration in BAECs. These also suggest that gravity can be regarded as an extracorporeal signal that could significantly affect endothelial functions.
Subject(s)
Actins/metabolism , Adenosine Triphosphate/metabolism , Endothelial Cells/metabolism , Hypergravity , Tyrosine/metabolism , rhoA GTP-Binding Protein/metabolism , Animals , Cattle , Cell Movement , Cells, Cultured , Endothelial Cells/cytology , Focal Adhesion Protein-Tyrosine Kinases/metabolism , PhosphorylationABSTRACT
The formation of spore clumps of Bacillus coagulans and Bacillus licheniformis during high-pressure carbon dioxide treatment (HCT) was investigated. As the treatment time increased, the number of spore clumps increased. After 120 min, single spore decreased to 20-35% of the population. Addition of a surfactant decreased the hydrophobicity of spore surface and increased both the number of single spores and the rate of inactivation ratio of B. coagulans and B. licheniformis spores.
Subject(s)
Bacillus/physiology , Carbon Dioxide/pharmacology , Pressure , Spores, Bacterial/drug effects , Spores, Bacterial/physiology , Bacterial Adhesion/drug effects , Bacterial Adhesion/physiology , Food Microbiology , Hydrophobic and Hydrophilic Interactions , Surface-Active Agents/pharmacology , Time FactorsABSTRACT
The pyrrolizidine alkaloid plant toxin monocrotaline pyrrole (MCTP) causes pulmonary hypertension in experimental animals. The present study aimed to examine the effects of MCTP on the endothelium-dependent relaxation. We constructed an in vitro disease model of pulmonary hypertension by overlaying MCTP-treated bovine pulmonary artery endothelial cells (CPAEs) onto pulmonary artery smooth muscle cell-embedded collagen gel lattice. Acetylcholine (Ach) induced a relaxation of the control CPAEs-overlaid gels that were pre-contracted with noradrenaline, and the relaxation was inhibited by L-NAME, an inhibitor of NO synthase (NOS). In contrast, when MCTP-treated CPAEs were overlaid, the pre-contracted gels did not show a relaxation in response to Ach in the presence of 0.5 mM l-arginine. Expression of endothelial NOS protein, Ach-induced Ca2+ transients and cellular uptake of l-[3H]arginine were significantly smaller in MCTP-treated CPAEs than in control cells, indicating that these changes were responsible for the impaired NO production in MCTP-treated CPAEs. Since cellular uptake of l-[3H]arginine linearly increased according to its extracellular concentration, we hypothesized that the excess concentration of extracellular l-arginine might restore NO production in MCTP-treated CPAEs. As expected, in the presence of 10 mM l-arginine, Ach showed a relaxation of the MCTP-treated CPAEs-overlaid gels. These results indicate that the impaired NO production in damaged endothelial cells can be reversed by supplying excess l-arginine.
Subject(s)
Arginine/pharmacology , Endothelium, Vascular/physiology , Monocrotaline/analogs & derivatives , Muscle, Smooth, Vascular/physiology , Animals , Blotting, Western , Calcium/metabolism , Cattle , Cell Count , Cell Size , Cells, Cultured , Endothelial Cells/drug effects , Endothelial Cells/ultrastructure , Endothelium, Vascular/drug effects , Enzyme Inhibitors/pharmacology , Monocrotaline/toxicity , Muscle Relaxation/drug effects , Muscle, Smooth, Vascular/drug effects , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/biosynthesis , Nitric Oxide Synthase Type IIIABSTRACT
High-pressure CO2 treatment has been studied as a promising method for inactivating bacterial spores. In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment. Spores of Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, and Geobacillus stearothermophilus were subjected to CO2 treatment at 30 MPa and 35 degrees C, to high-hydrostatic-pressure treatment at 200 MPa and 65 degrees C, or to heat treatment at 0.1 MPa and 85 degrees C. All of the bacterial spores except the G. stearothermophilus spores were easily inactivated by the heat treatment. The highly heat- and pressure-resistant spores of G. stearothermophilus were not the most resistant to CO2 treatment. We also investigated the influence of temperature on CO2 inactivation of G. stearothermophilus. Treatment with CO2 and 30 MPa of pressure at 95 degrees C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95 degrees C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95 degrees C for 120 min had little effect. The activation energy required for CO2 treatment of G. stearothermophilus spores was lower than the activation energy for heat or pressure treatment. Although heat was not necessary for inactivationby CO2 treatment of G. stearothermophilus spores, CO2 treatment at 95 degrees C was more effective than treatment at 95 degrees C alone.
Subject(s)
Bacillaceae/drug effects , Bacillaceae/physiology , Carbon Dioxide/pharmacology , Spores, Bacterial/drug effects , Bacillaceae/growth & development , Culture Media , Hot Temperature , Hydrostatic Pressure , Spores, Bacterial/growth & development , Spores, Bacterial/physiology , Sterilization/methodsABSTRACT
We have investigated the relationship between Ca2+ mobilization and the cellular production of nitric oxide (NO) by using fura-2 and diaminofluorescein-2 (DAF-2), an NO-sensitive dye, in bovine aortic endothelial cells (BAEC). High concentrations of ATP (100 microM) or thapsigargin (1 micro M) depleted intracellular Ca2+ store sites with a single Ca2+ transient, and induced an increase in DAF-2 fluorescence even in Ca2+-free solution, thereby indicating that store depletion leads to NO production. The same level of increase in DAF-2 fluorescence was elicited by low concentrations of ATP (1 micro M), which induced Ca2+ oscillations but did not deplete store sites, only in the presence of extracellular Ca2+. Furthermore, inhibition of ATP (1 micro M)-induced Ca2+ entry with La3+ suppressed DAF-2 fluorescence. ATP (0.3 micro M), applied in Ca2+-free, Mn2+-containing solution induced Mn2+ entry-coupled fura-2 quenching, repeating shortly after each oscillation peak. These results indicate that NO is produced preferentially by entered Ca2+, and that Ca2+ oscillations, which are induced by low levels of stimulation, play a significant role in NO production by strongly modulating Ca2+ entry.
Subject(s)
Calcium/metabolism , Endothelium, Vascular/metabolism , Nitric Oxide/biosynthesis , Adenosine Triphosphate/pharmacology , Animals , Aorta, Thoracic/metabolism , Calcimycin/pharmacology , Cattle , Cells, Cultured , Enzyme Inhibitors/pharmacology , Fluorescein , Fluorescent Dyes , Thapsigargin/pharmacologyABSTRACT
We examined the effects of superoxide anion (O) generated by xanthine plus xanthine oxidase (X/XO) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) and muscle contractility in cultured bovine aortic smooth muscle cells (BASMC). Cells were grown on collagen-coated dish for the measurement of [Ca(2+)](i). Pretreatment with X/XO inhibited ATP-induced Ca(2+) transient and Ca(2+) release-activated Ca(2+) entry (CRAC) after thapsigargin-induced store depletion, both of which were reversed by superoxide dismutase (SOD). In contrast, Ca(2+) transients induced by high-K(+) solution and Ca(2+) ionophore A-23187 were not affected by X/XO. BASMC-embedded collagen gel lattice, which was pretreated with xanthine alone, showed contraction in response to ATP, thapsigargin, high-K(+) solution, and A-23187. Pretreatment of the gel with X/XO impaired gel contraction not only by ATP and thapsigargin, but also by high-K(+) solution and A-23187. The X/XO-treated gel showed normal contraction; however, when SOD was present during the pretreatment period. These results indicate that O(2)(-) attenuates smooth muscle contraction by impairing CRAC, ATP-induced Ca(2+) transient, and Ca(2+) sensitivity in BASMC.
Subject(s)
Aorta, Thoracic/drug effects , Muscle, Smooth, Vascular/drug effects , Superoxides/pharmacology , Vasoconstriction/drug effects , Adenosine Triphosphate/pharmacology , Animals , Aorta, Thoracic/cytology , Aorta, Thoracic/physiology , Calcium/metabolism , Cattle , Cells, Cultured , Collagen/physiology , Enzyme Inhibitors/pharmacology , Gels , Imidazoles , Intracellular Fluid/metabolism , Ionophores/pharmacology , Luminescent Measurements , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/physiology , Potassium/pharmacology , Pyrazines , Superoxide Dismutase/metabolism , Superoxides/analysis , Superoxides/metabolism , Vasoconstriction/physiology , Xanthine/metabolism , Xanthine Oxidase/metabolismABSTRACT
Mechanical stress induces auto/paracrine ATP release from various cell types, but the mechanisms underlying this release are not well understood. Here we show that the release of ATP induced by hypotonic stress (HTS) in bovine aortic endothelial cells (BAECs) occurs through volume-regulated anion channels (VRAC). Various VRAC inhibitors, such as glibenclamide, verapamil, tamoxifen, and fluoxetine, suppressed the HTS-induced release of ATP, as well as the concomitant Ca(2+) oscillations and NO production. They did not, however, affect Ca(2+) oscillations and NO production induced by exogenously applied ATP. Extracellular ATP inhibited VRAC currents in a voltage-dependent manner: block was absent at negative potentials and was manifest at positive potentials, but decreased at highly depolarized potentials. This phenomenon could be described with a "permeating blocker model," in which ATP binds with an affinity of 1.0 +/- 0.5 mM at 0 mV to a site at an electrical distance of 0.41 inside the channel. Bound ATP occludes the channel at moderate positive potentials, but permeates into the cytosol at more depolarized potentials. The triphosphate nucleotides UTP, GTP, and CTP, and the adenine nucleotide ADP, exerted a similar voltage-dependent inhibition of VRAC currents at submillimolar concentrations, which could also be described with this model. However, inhibition by ADP was less voltage sensitive, whereas adenosine did not affect VRAC currents, suggesting that the negative charges of the nucleotides are essential for their inhibitory action. The observation that high concentrations of extracellular ADP enhanced the outward component of the VRAC current in low Cl(-) hypotonic solution and shifted its reversal potential to negative potentials provides more direct evidence for the nucleotide permeability of VRAC. We conclude from these observations that VRAC is a nucleotide-permeable channel, which may serve as a pathway for HTS-induced ATP release in BAEC.
Subject(s)
Adenosine Triphosphate/metabolism , Autocrine Communication/physiology , Endothelium, Vascular/metabolism , Ion Channels/metabolism , Paracrine Communication/physiology , Adenosine Diphosphate/metabolism , Animals , Anions/metabolism , Aorta, Thoracic/cytology , Brefeldin A/pharmacology , Cattle , Chlorides/metabolism , Endothelium, Vascular/cytology , Hypotonic Solutions/pharmacology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Models, Biological , Nitric Oxide/metabolism , Osmotic Pressure , Protein Synthesis Inhibitors/pharmacologyABSTRACT
AIM To study the effects of superoxide anion (O.2) on Ca2+ homeostasi and contractility in cultured bovine aortic smooth muscle cell. METHODS Using Fura-2 fluorescence technique to determine Ca2+ level and collagen gel contraction system to analyze muscle contractility. RESULTS ATP (10 μmol*L-1 )-induced Ca2+ transient was smaller in xanthine oxidase treated cells(X/XO) than control. The mean peak increment of [Ca2+]i(△[Ca2+]i peak) and the time integral of the elevated [Ca2+]i(∫△[Ca2+]i dt) for 5 min were decreased from (206.1±10.2) to (147.4±14.7) nmol·L-1,and from (12.2±0.5) to (9.8±0.8) μmol·L-1·s-1. Δ[Ca2+]i peak induced by thapsigargin(1 μmol·L-1 )in Ca2+-free solution was not affected by X/XO, but was decreased from (27.3±1.0) nmol·L-1 to (13.5±1.0) nmol·L-1 in Ca2+-containing solution because of the activation of CRAC(△[Ca2+]i CRAC). X/XO accelerated the velocity of thapsigargin-induced Ca2+ leak from (78.7±3.4) s to (64.8±4.40) s. Gel contraction area in X/XO-treated cells induced by ATP or thapsigargin (in Ca2+ free solution and in Krebs solution)was decreased from 23.6%±4.6% to 7.4%±0.2%, from 3.5%±0.6% to -1.0%±0.5%, and from 7.9%±1.4% to -0.5%±0.7%, respectively. CONCLUTION O.2 attenuats smooth muscle contraction by impairing some of Ca2+ mobilization pathways.
ABSTRACT
AIM To study the effects of superox- ide anion (O ) on Ca2+ homeostasi and contractility in cultured bovine aortic smooth muscle cell. METHODS Using Fura-2 fluorescence technique to determine Ca2+ level and collagen gel contraction system to analyze muscle contractility. RESULTS ATP (10 ?mol? L-1 )-induced Ca2+ transient was smaller in xanthine oxidase treated cells(X/XO) than control. The mean peak increment of [Ca2+ ]i (△[Ca2+ ], peak) and the time integral of the elevated [Ca2+ ], (∫ △[Ca2+ ]i dt) for 5 min were decreased from (206. 1 ? 10.2) to (147.4 ? 14.7) nmol? L-1, and from (12.2 ?0.5) to (9.8 ? 0.8) ?mol?L-1 .s-1. △ [Ca2+ ], peak induced by thapsigargin(1 ?mol. L- 1 ) in Ca2+ -free solution was not affected by X/XO, but was decreased from (27. 3 ? 1 .0) nmol? L-l to (13 .5 ? 1 .0 ) nmol? L- 1 in Ca2+ -containing solution be cause of the activation of CRAC(△[Ca2+ ], CRAC). X/XO accelerated the velocity of thapsigargin-induced Ca2+ leak from (78.7 ? 3.4) s to (64.8 ? 4.40) s. Gel contraction area in X/XO-treated cells induced by ATP or thapsigargin (in Ca2+ free solution and in Krebs solution) was decreased from 23.6% ? 4.6% to 7.4% ?0.2%, from 3.5% ?0.6% to - l.0% ? 0.5%, and from 7.9% ? l.4% to - 0.5% ? 0.7%, respectively. CONCLUTION O2- attenuats smooth muscle contraction by impairing some of Ca2+ mobilization pathways.