bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural characterization, tissue distribution, and functional expression of murine aminoacylase III
Authors: Pushkin Alexander , Carpenito Gerardo , Abuladze Natalia , Newman Debra , Tsuprun Vladimir , Ryazantsev Sergey , Motemoturu Srilakshmi , Sassani Pakan , Solovieva Nadezhda , Dukkipati Ramnath , Kurtz Ira
Published: March 2004

Many xenobiotics are detoxified through the mercapturate metabolic pathway. The final product of the pathway, mercapturic acids ( N-acetylcysteine S-conjugates), are secreted predominantly by renal proximal tubules. Mercapturic acids may undergo a transformation mediated by aminoacylases and cysteine S-conjugate β-lyases that leads to nephrotoxic reactive thiol formation. The deacetylation of cysteine S-conjugates of N-acyl aromatic amino acids is thought to be mediated by an aminoacylase whose molecular identity has not been determined. In the present study, we cloned aminoacylase III, which likely mediates this process in vivo, and characterized its function and structure. The enzyme consists of 318 amino acids and has a molecular mass (determined by SDS-PAGE) of ∼35 kDa. Under nondenaturing conditions, the molecular mass of the enzyme is ∼140 kDa as determined by size-exclusion chromatography, which suggests that it is a tetramer. In agreement with this hypothesis, transmission electron microscopy and image analysis of aminoacylase III showed that the monomers of the enzyme are arranged with a fourfold rotational symmetry. Northern analysis demonstrated an ∼1.4-kb transcript that was expressed predominantly in kidney and showed less expression in liver, heart, small intestine, brain, lung, testis, and stomach. In kidney, aminoacylase III was immunolocalized predominantly to the apical domain of S1 proximal tubules and the cytoplasm of S2 and S3 proximal tubules. The data suggest that in kidney proximal tubules, aminoacylase III plays an important role in deacetylating mercapturic acids. The predominant cytoplasmic localization of aminoacylase III may explain the greater sensitivity of the proximal straight tubule to the nephrotoxicity of mercapturic acids.
Increased vascular endothelial growth factor may account for elevated level of plasminogen activator inhibitor-1 via activating ERK1/2 in keloid fibroblasts
Authors: Wu Yidi , Zhang Qunzhou , Ann David K. , Akhondzadeh Anita , Duong Hai S. , Messadi Diana V. , Le Anh D.
Published: March 2004

Keloids are characterized as an “overexuberant” healing response in which disequilibrium between production and catabolism of extracellular matrix (ECM) occurs. Previous studies from our laboratory and others demonstrate an intrinsically higher level of plasminogen activator inhibitor-1 (PAI-1) expression in keloid tissues and cultured fibroblasts compared with normal bordering skin. These findings support the concept that an altered balance of activator and inhibitor activities in the plasminogen system, in particular, an overexpression of PAI-1, may partly contribute to keloid formation and tissue fibrosis. Vascular endothelial growth factor (VEGF) has been implicated as a critical factor in regulating angiogenesis and inflammation under both physiological and pathological conditions. This study was designed to assess whether VEGF plays a role in keloid fibrosis. We report that VEGF was expressed at higher levels in keloid tissues and their derived fibroblasts compared with their associated normal skin. We have further demonstrated that VEGF stimulated the expression of PAI-1, but not urokinase plasminogen activator (uPA), in keloid fibroblasts at both mRNA and protein levels, in a dose- and time-dependent manner. However, treatment of normal skin fibroblasts with VEGF exerted little effects on PAI-1 gene expression. Additionally, we have characterized for the first time that the extracellular signal-regulated kinase (ERK)1/2 signaling pathway is mainly involved in VEGF-induced PAI-1 expression and have demonstrated its potential as a target molecule for modulation of scar fibrosis. These findings suggest that VEGF may play an important role in keloid formation by altering ECM homeostasis toward a state of impaired degradation and excessive accumulation.
Oxidative stress decreases pHi and Na+/H+ exchange and increases excitability of solitary complex neurons from rat brain slices
Authors: Mulkey Daniel K. , Henderson Richard A. , Ritucci Nick A. , Putnam Robert W. , Dean Jay B.
Published: March 2004

Putative chemoreceptors in the solitary complex (SC) are sensitive to hypercapnia and oxidative stress. We tested the hypothesis that oxidative stress stimulates SC neurons by a mechanism independent of intracellular pH (pHi). pHi was measured by using ratiometric fluorescence imaging microscopy, utilizing either the pH-sensitive fluorescent dye BCECF or, during whole cell recordings, pyranine in SC neurons in brain stem slices from rat pups. Oxidative stress decreased pHi in 270 of 436 (62%) SC neurons tested. Chloramine-T (CT), N-chlorosuccinimide (NCS), dihydroxyfumaric acid, and H2O2 decreased pHi by 0.19 ± 0.007, 0.20 ± 0.015, 0.15 ± 0.013, and 0.08 ± 0.002 pH unit, respectively. Hypercapnia decreased pHi by 0.26 ± 0.006 pH unit ( n = 95). The combination of hypercapnia and CT or NCS had an additive effect on pHi, causing a 0.42 ± 0.03 ( n = 21) pH unit acidification. CT slowed pHi recovery mediated by Na+/H+ exchange (NHE) from NH4Cl-induced acidification by 53% ( n = 20) in [Formula: see text]-buffered medium and by 58% ( n = 10) in HEPES-buffered medium. CT increased firing rate in 14 of 16 SC neurons, and there was no difference in the firing rate response to CT with or without a corresponding change in pHi. These results indicate that oxidative stress 1) decreases pHi in some SC neurons, 2) together with hypercapnia has an additive effect on pHi, 3) partially inhibits NHE, and 4) directly affects excitability of CO2/H+-chemosensitive SC neurons independently of pHi changes. These findings suggest that oxidative stress acidifies SC neurons in part by inhibiting NHE, and this acidification may contribute ultimately to respiratory control dysfunction.
CYP450 dietary inhibitors attenuate TNF-α-stimulated endothelial molecule expression and leukocyte adhesion
Authors: Sasaki Makoto , Elrod John W. , Jordan Paul , Itoh Makoto , Joh Takashi , Minagar Alireza , Alexander J. Steven
Published: March 2004

Enhanced expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and other endothelial cell adhesion molecules (ECAMs) are associated with the onset and progression of inflammatory bowel disease (IBD). We show in this study that two cytochrome P-450 (CYP450) inhibitors from Citrus paradis (grapefruit), bergamottin, and 6′,7′-dihydroxybergamottin (DHB) block tumor necrosis factor (TNF)-α-stimulated expression of MAdCAM-1 in cultured endothelial cells and also reduce α4β7-dependent lymphocyte adhesion. Bergamottin (20–50 μM) or DHB (10–30 μM) pretreatment dose-dependently reduced TNF-α-mediated expression of MAdCAM-1 and lymphocyte adhesion. Bergamottin and DHB also prevented expression of two other ECAMs, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 (but not E-selectin). SKF-525a, a specific CYP450 inhibitor, also blocked the expression of MAdCAM-1 mediated by TNF-α. Similar to SKF-525a (20 μM), bergamottin (20 μM) and DHB (20 μM) directly inhibited the activity of CYP450 3A4. These results suggest that natural CYP450 inhibitors may be effective in reducing ECAM expression and leukocyte adhesion and therefore be useful in the clinical treatment of inflammatory states like IBD.
Description of glucose transport in isolated bovine mammary epithelial cells by a three-compartment model
Authors: Xiao Changting , Quinton V. Margaret , Cant John P.
Published: March 2004

Initial rates of glucose entry into isolated bovine mammary epithelial cells display moderate degrees of asymmetry and cooperative interactions between export and import sites. The present study examined the hypothesis that these kinetic features are due to compartmentalization of intracellular glucose. Net uptake of 3- O-methyl-d-[1-3H]glucose (3-OMG) by isolated bovine mammary epithelial cells was measured at 37°C. The time course of 3-OMG net uptake was better fitted by a double-exponential equation than by a single- or triple-exponential equation. Compartmental analysis of the time course curve suggested that translocated 3-OMG is distributed into two compartments with fractional volumes of 32.6 ± 5.7% and 67.4 ± 5.7%, respectively. The results support the view that glucose transport in bovine mammary epithelial cells is a multistep process consisting of two serial steps: fast, carrier-mediated, symmetric translocation of sugar across the cell plasma membrane into a small compartment and subsequent slow exchange of posttranslocated sugar between two intracellular compartments. A three-compartment model of this system successfully simulated the observed time course of 3-OMG net uptake and the observed dependence of unidirectional entry rates on intra- and extracellular 3-OMG concentrations. Simulations indicated that backflux of radiolabeled sugar from the small compartment to extracellular space during 15 s of incubation gives rise to the apparent asymmetry, trans-stimulation, and cooperativity of mammary glucose transport kinetics. The fixed-site carrier model overestimated the rate of glucose accumulation in cells, and its features can be accounted for by the compartmentalization of intracellular sugar.
Accumulation of βm, a structural member of X,K-ATPase β-subunit family, in nuclear envelopes of perinatal myocytes
Authors: Zhao Hao , Pestov Nikolay B. , Korneenko Tatyana V. , Shakhparonov Mikhail I. , Modyanov Nikolai N.
Published: March 2004

Recently discovered muscle-specific βm protein is structurally closely related to the X,K-ATPase β-subunits. However, it has a number of unique properties such as predominant localization in intracellular stores and lack of association with known X,K-ATPase α-subunits on heterologous coexpression. In this study, the primary structure of mouse βm was determined and developmental regulation of the gene (ATP1B4) was analyzed. The expression is first detected at day 14 of gestation, is sharply increased at day 16, and reaches its maximum at day 18. After birth, the expression quickly decreases and is hardly detectable in adult mice. A more detailed subcellular localization study was undertaken, and its results indicate that βm not only is located in sarcoplasmic reticulum but is concentrated in nuclear envelopes of both prenatal and postnatal skeletal muscles. Immunohistochemical studies show that βm is specific to myocytes and, at the subcellular level, many nuclear envelopes are intensively labeled in both fetal and newborn skeletal muscles. Accordingly, βm is detected by immunoblotting in purified nuclei and nuclear membranes from neonatal skeletal muscles. On transfection of human rhabdomyosarcoma cell line RD, green fluorescent protein-tagged βm resides intracellularly with significant enrichment in nuclear envelopes, whereas βm with transmembrane domain deleted localizes in both cytoplasm and nucleoplasm. Nuclear βm apparently is not in association with Na,K-ATPase because we never detected its α-subunit in myonuclear membranes. These results indicate that βm has a specialized function in mammalian perinatal myocytes, different from functions of other X,K-ATPase β-subunits. The unique temporospatial distribution of βm protein expression suggests its important role in development of growing skeletal muscle.
Adaptation of the nematode Caenorhabditis elegans to extreme osmotic stress
Authors: Lamitina S. Todd , Morrison Rebecca , Moeckel Gilbert W. , Strange Kevin
Published: March 2004

The ability to control osmotic balance is essential for cellular life. Cellular osmotic homeostasis is maintained by accumulation and loss of inorganic ions and organic osmolytes. Although osmoregulation has been studied extensively in many cell types, major gaps exist in our molecular understanding of this essential process. Because of its numerous experimental advantages, the nematode Caenorhabditis elegans provides a powerful model system to characterize the genetic basis of animal cell osmoregulation. We therefore characterized the ability of worms to adapt to extreme osmotic stress. Exposure of worms to high-salt growth agar causes rapid shrinkage. Survival is normal on agar containing up to 200 mM NaCl. When grown on 200 mM NaCl for 2 wk, worms are able to survive well on agar containing up to 500 mM NaCl. HPLC analysis demonstrated that levels of the organic osmolyte glycerol increase 15- to 20-fold in nematodes grown on 200 mM NaCl agar. Accumulation of glycerol begins 3 h after exposure to hypertonic stress and peaks by 24 h. Glycerol accumulation is mediated primarily by synthesis from metabolic precursors. Consistent with this finding, hypertonicity increases transcriptional expression of glycerol 3-phosphate dehydrogenase, an enzyme that is rate limiting for hypertonicity-induced glycerol synthesis in yeast. Worms adapted to high salt swell and then return to their initial body volume when exposed to low-salt agar. During recovery from hypertonic stress, glycerol levels fall rapidly and glycerol excretion increases approximately fivefold. Our studies provide the first description of osmotic adaptation in C. elegans and provide the foundation for genetic and functional genomic analysis of animal cell osmoregulation.
Na+ pump α2-isoform specifically couples to contractility in vascular smooth muscle: evidence from gene-targeted neonatal mice
Authors: Shelly Daniel A. , He Suiwen , Moseley Amy , Weber Craig , Stegemeyer Michelle , Lynch Ronald M. , Lingrel Jerry , Paul Richard J.
Published: March 2004

The relative expression of α1 - and α2-Na+/K+-ATPase isoforms found in vascular smooth muscle is developmentally regulated and under hormonal and neurogenic control. The physiological roles of these isoforms in vascular function are not known. It has been postulated that the α1-isoform serves a “housekeeping” role, whereas the α2-isoform localizes to a subsarcolemmal compartment and modulates contractility. To test this hypothesis, isoform-specific gene-targeted mice in which the mRNA for either the α1- or the α2-Na+/K+-ATPase isoform was ablated were utilized. Both of these knockouts, [Formula: see text] and [Formula: see text], are lethal; the latter dies at birth, which allows this neonatal aorta to be studied. Isometric force in [Formula: see text]-aorta was more sensitive to contractile agonists and less sensitive to the vasodilators forskolin and sodium nitroprusside (SNP) than wild-type (WT) aorta; [Formula: see text]-aortas had intermediate values. In contrast, neonatal [Formula: see text]-aorta was similar to WT. Western blot analysis indicated a population of 70% α1- and 30% α2-isoforms in the WT. Thus in terms of the total Na+/K+-ATPase protein, the [Formula: see text]-aorta (at 70%) would be similar to the [Formula: see text]-aorta (at 65%) but with a dramatically different phenotype. These data suggest that individual α-isoforms of the Na+/K+-ATPase differ functionally and that the α2-isoform couples more strongly to activation-relaxation pathways. Three-dimensional image-acquisition and deconvolution analyses suggest that the α2-isoform is distributed differently than the α1-isoform. Importantly, these isoforms do not localize to the same regions.
Evidence for nucleotide receptor modulation of cross talk between MAP kinase and NF-κB signaling pathways in murine RAW 264.7 macrophages
Authors: Aga Mini , Watters Jyoti J. , Pfeiffer Zachary A. , Wiepz Gregory J. , Sommer Julie A. , Bertics Paul J.
Published: March 2004

Extracellular nucleotides such as ATP are present in abundance at sites of inflammation and tissue damage, and these agents exert a potent modulatory effect on macrophage/monocyte function via the nucleotide receptor P2X7. In this regard, after exposure to bacterial LPS, P2X7 activation augments expression of the inducible nitric oxide (NO) synthase and production of NO in macrophages. Because P2X7 has been reported to stimulate certain members of the MAP kinase family (ERK1/2) and can enhance the DNA-binding activity of NF-κB, we tested the hypothesis that LPS and nucleotides regulate NF-κB-dependent inflammatory events via cross talk with MAPK-associated pathways. In this regard, the present studies revealed that cotreatment of macrophages with LPS and the P2X7-selective ligand 2′-3′- O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP) results in the cooperative activation of NF-κB DNA-binding activity and a sustained attenuation of levels of the NF-κB inhibitory protein IκBα. Interestingly, a persistent reduction in IκBα levels is also observed when the MEK1/2 inhibitor U0126 is coadministered with LPS, suggesting that components of the MEK/ERK pathway are involved in regulating IκBα protein expression and/or turnover. The observation that U0126 and BzATP exhibit overlapping actions with respect to LPS-induced changes in IκBα levels is supported by the finding that Ras activation, which is upstream of MEK/ERK activation, is reduced upon macrophage cotreatment with BzATP and LPS compared with the effects of BzATP treatment alone. These data are consistent with the concept that the Ras/MEK/ERK pathways are involved in regulating NF-κB/IκB-dependent inflammatory mediator production and suggest a previously unidentified mechanism by which nucleotides can modulate LPS-induced action via cross talk between NF-κB and Ras/MEK/MAPK-associated pathways.
Colistin interactions with the mammalian urothelium
Authors: Lewis Jamie R. , Lewis Simon A.
Published: March 2004

Here we describe the effect of colistin on the barrier function of the mammalian urinary bladder epithelium. Addition of colistin to the mucosal solution of the rabbit urinary bladder epithelium (urothelium) resulted in an increase in the transepithelial conductance. The magnitude of the increase in transepithelial conductance was dependent on the membrane voltage, concentration of colistin, and presence of divalent cations in the bath solution. The initial site of action of colistin was at the apical membrane. Colistin increased the membrane conductance only when the apical membrane potential was cell interior negative. The more negative the membrane potential, the larger the conductance increase. The concentration dependence of the conductance increase saturated, suggesting a membrane binding site. Divalent cations decreased the magnitude of the conductance increase. This divalent cation action occurred at two sites: one in competition with colistin for a membrane binding site, and the other by rapidly blocking the induced conductance. At short exposure times, the increase in conductance was reversed by either removing colistin from the bath or changing the voltage so that the apical membrane was cell interior positive. At long exposure times, the increase was only partially reversible by voltage or removal from the bath. This finding suggests that at long exposure times, there is a toxic effect of colistin on the urothelium.
A novel mouse-driven ex vivo flow chamber for the study of leukocyte and platelet function
Authors: Hafezi-Moghadam Ali , Thomas Kennard L. , Cornelssen Christian
Published: March 2004

Various in vitro and in vivo techniques exist for study of the microcirculation. Whereas in vivo systems impress with their physiological fidelity, in vitro systems excel in the amount of reduction that can be achieved. Here we introduce the autoperfused ex vivo flow chamber designed to study murine leukocytes and platelets under well-defined hemodynamic conditions. In our model, the murine heart continuously drives the blood flow through the chamber, providing a wide range of physiological shear rates. We used a balance of force approach to quantify the prevailing forces at the chamber walls. Numerical simulations show the flow characteristics in the chamber based on a shear-thinning fluid model. We demonstrate specific rolling of wild-type leukocytes on immobilized P-selectin, abolished by a blocking MAb. When uncoated, the surfaces having a constant shear rate supported individual platelet rolling, whereas on areas showing a rapid drop in shear platelets interacted in previously unreported grapelike conglomerates, suggesting an influence of shear rate on the type of platelet interaction. In summary, the ex vivo chamber amounts to an external vessel connecting the arterial and venous systems of a live mouse. This method combines the strengths of existing in vivo and in vitro systems in the study of leukocyte and platelet function.
Gastric inhibitory polypeptide does not inhibit gastric emptying in humans
Authors: Meier Juris J. , Goetze Oliver , Anstipp Jens , Hagemann Dirk , Holst Jens J. , Schmidt Wolfgang E. , Gallwitz Baptist , Nauck Michael A.
Published: March 2004

The insulinotropic gut hormone gastric inhibitory polypeptide (GIP) has been demonstrated to inhibit gastric acid secretion and was proposed to possess “enterogastrone” activity. GIP effects on gastric emptying have not yet been studied. Fifteen healthy male volunteers (23.9 ± 3.3 yr, body mass index 23.7 ± 2.3 kg/m2) were studied with the intravenous infusion of GIP (2 pmol·kg-1·min-1) or placebo, each administered to the volunteers on separate occasions from -30 to 360 min in the fasting state. At 0 min, a solid test meal (250 kcal containing [13C]sodium octanoate) was served. Gastric emptying was calculated from the 13CO2 exhalation rates in breath samples collected over 360 min. Venous blood was drawn in 30-min intervals for the determination of glucose, insulin, C-peptide, and GIP (total and intact). Statistical calculations were made by use of repeated-measures ANOVA and one-way ANOVA. During the infusion, GIP rose to steady-state concentrations of 159 ± 15 pmol/l for total and 34 ± 4 pmol/l for intact GIP ( P < 0.0001). Meal ingestion further increased GIP concentrations in both groups, reaching peak levels of 265 ± 20 and 82 ± 9 pmol/l for total and 67 ± 7 and 31 ± 9 pmol/l for intact GIP during the administration of GIP and placebo, respectively ( P < 0.0001). There were no differences in glucose, insulin, and C-peptide between the experiments with the infusion of GIP or placebo. Gastric half-emptying times were 120 ± 9 and 120 ± 18 min ( P = 1.0, with GIP and placebo, respectively). The time pattern of gastric emptying was similar in the two groups ( P = 0.98). Endogenous GIP secretion, as derived from the incremental area under the curve of plasma GIP concentrations in the placebo experiments, did not correlate to gastric half-emptying times ( r2 = 0.15, P = 0.15 for intact GIP; r2 = 0.21, P = 0.086 for total GIP). We conclude that gastric emptying does not appear to be influenced by GIP. The secretion of GIP after meal ingestion is not suppressed by its exogenous administration. The lack of effect of GIP on gastric emptying underlines the differences between GIP and the second incretin glucagon-like peptide 1.
Loss of entrainment of high-frequency plasma insulin oscillations in type 2 diabetes is likely a glucose-specific β-cell defect
Authors: Mao Catherine S. , Berman Nancy , Ipp Eli
Published: March 2004

Spontaneous high-frequency insulin oscillations are easily entrainable to exogenous glucose in vitro and in vivo, but this property is lost in type 2 diabetes (2-DM). We hypothesized that this lack of entrainment in 2-DM would be specific to glucose. This was tested in nine control and ten 2-DM subjects. Serial blood sampling at 1-min intervals was carried out for 60 min in the basal state and for 120 min while small (1–60 mg/kg) boluses of arginine were injected intravenously at exactly 29-min intervals. Samples were analyzed for insulin concentrations, and time series analysis was carried out using spectral analysis. In control subjects, the mean period of basal plasma insulin oscillations was 10.3 ± 1.3 min and was entrained by arginine to a mean period of 14.9 ± 0.6 min ( P < 0.00001 vs. basal). Similarly, in 2-DM subjects, spontaneous insulin oscillations were entrained by arginine; mean basal insulin period was 10.0 ± 1.0 min and 14.5 ± 1.8 min with arginine boluses ( P < 0.00001). All of the primary peaks observed in spectral analysis were statistically significant ( P < 0.05). Percent total power of primary peaks ranged from 17 to 68%. Thus arginine boluses entrain spontaneous high-frequency insulin oscillations in 2-DM subjects. This represents a distinct and striking difference from the resistance of the β-cell to glucose entrainment in 2-DM. We conclude that loss of entrainment of spontaneous high-frequency insulin oscillations in 2-DM is likely a glucose-specific manifestation of β-cell secretory dysfunction.
Gastric ulcers reduce A-type potassium currents in rat gastric sensory ganglion neurons
Authors: Dang K. , Bielefeldt K. , Gebhart G. F.
Published: March 2004

Voltage-dependent potassium currents are important contributors to neuron excitability and thus also to hypersensitivity after tissue insult. We hypothesized that gastric ulcers would alter K+ current properties in primary sensory neurons. The rat stomach was surgically exposed, and a retrograde tracer (1,1′-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanine methanesulfonate) was injected into multiple sites in the stomach wall. Inflammation and ulcers were produced by 10 injections of 20% acetic acid (HAc) in the gastric wall. Saline (Sal) injections served as control. Nodose or T9–10 dorsal root ganglia (DRG) cells were harvested and cultured 7 days later to record whole cell K+ currents. Gastric sensory neurons expressed transient and sustained outward currents. Gastric inflammation significantly decreased the A-type K+ current density in DRG and nodose neurons (Sal vs. HAc-DRG: 82.9 ± 7.9 vs. 46.5 ± 6.1 pA/pF; nodose: 149.2 ± 10.9 vs. 71.4 ± 11.8 pA/pF), whereas the sustained current was not altered. In addition, there was a significant shift in the steady-state inactivation to more hyperpolarized potentials in nodose neurons (Sal vs. HAc: -76.3 ± 1.0 vs. -83.6 ± 2.2 mV) associated with an acceleration of inactivation kinetics. These data suggest that a reduction in K+ currents contributes, in part, to increased neuron excitability that may lead to development of dyspeptic symptoms.
Ischemic preconditioning attenuates ischemia-reperfusion-induced mucosal apoptosis by inhibiting the mitochondria-dependent pathway in rat small intestine
Authors: Wu Bin , Ootani Akifumi , Iwakiri Ryuichi , Fujise Takehiro , Tsunada Seiji , Toda Shuji , Fujimoto Kazuma
Published: March 2004

Ischemic preconditioning provides a way of protecting organs from damage inflicted with prolonged ischemia-reperfusion. In this study, we investigated the mechanism of ischemic preconditioning involved in inhibition of prolonged ischemia-reperfusion-induced mucosal apoptosis in rat small intestine. Ischemic preconditioning was triggered by a transient occlusion of the superior mesenteric artery followed by reperfusion. Ischemia-reperfusion was induced by 60-min occlusion of the superior mesenteric artery followed by 60-min reperfusion in the small intestine. Ischemia-reperfusion alone induced mucosal apoptosis and mitochondrial respiratory dysfunction via promoted reactive oxygen species generation, reduced mitochondrial glutathione oxidation, increased mitochondrial lipid peroxidation, reduced mitochondrial membrane potential, and enhanced release of cytochrome c from mitochondria to activate caspase-9 and caspase-6 in the small intestine. Pretreatment with 20-min ischemia followed by 5-min reperfusion significantly inhibited the prolonged ischemia-reperfusion-induced mucosal apoptosis by 30%. Ischemic preconditioning ameliorated mitochondrial respiratory dysfunction by 50%, reduced reactive oxygen species generation by 38%, and suppressed mitochondrial lipid peroxidation by 36%, resulting in improvement of the mitochondrial membrane potential and prevention of cytochrome c release as well as caspase-6 activation. Results suggest that ischemic preconditioning attenuated ischemia-reperfusion-induced mucosal apoptosis partly by inhibiting the reactive oxygen species-mediated mitochondria-dependent pathway in the rat small intestine.
Restitution of the bullfrog gastric mucosa is dependent on a DIDS-inhibitable pathway not related to \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} ion transport
Authors: Hagen Susan J. , Morrison Sarah W. , Law Christina S. , Yang David X.
Published: March 2004

This study was conducted to determine the contribution of ion transport to restitution after injury in the gastric mucosa. For this, intact sheets of stomach from the bullfrog, Rana catesbeiana, were mounted in Ussing chambers. Restitution was evaluated in the presence or absence of ion transport inhibitors amiloride, DIDS, and bumetanide to block Na+/H+ exchange, [Formula: see text]/[Formula: see text] exchange and [Formula: see text]/[Formula: see text] co-transport, and Na+-K+-2Cl- cotransport, respectively. Ion substitution experiments with Na+-free, Cl--free, and [Formula: see text]-free solutions were also performed. Injury to the mucosa was produced with 1 M NaCl, and restitution was evaluated by recovery of transepithelial resistance (TER), mannitol flux, and morphology. Amiloride, bumetanide, Cl--free, or [Formula: see text]-free solutions did not affect restitution. In Na+-free solutions, recovery of TER and mannitol flux did not occur because surface cells did not attach to the underlying basement membrane. In contrast, all aspects of restitution were inhibited by DIDS, a compound that inhibits Na+-dependent [Formula: see text] transport. Because [Formula: see text]-free solutions did not inhibit restitution, it was concluded that DIDS must block a yet undefined pathway not involved in [Formula: see text] ion transport but essential for cell migration after injury and restitution in the gastric mucosa.
Electrical current-induced atrial and pulmonary vein action potential duration shortening and repetitive activity
Authors: Miyauchi Yasushi , Fishbein Michael C. , Karagueuzian Hrayr S.
Published: March 2004

The influence of threshold electrical currents (EC) during regular drive on pulmonary vein (PV) and atrial myocardial cell action potential (AP) duration (APD) is unknown. We determined the effects of EC on cellular APD of PV, atria, and ventricles in isolated perfused and superfused male rat hearts (Fisher-344 strain, 3–4 mo old) at 37°C ( n = 14). We determined APD changes caused by subthreshold and threshold EC synchronized with a distant pacing electrode and delivered nearby cells from which transmembrane APs were recorded with a glass microelectrode. Progressive APD shortening ( P < 0.001) and membrane hyperpolarization ( P < 0.05) developed over a 20-s interval in the PV and atrial cells when the EC was delivered at <2 mm but not at >4 mm from the microelectrode. No such effects were seen in ventricular muscle cells. APD fully recovered 25 s after the cessation of EC application. Premature stimuli applied during EC-induced shortening of the APD caused rapid repetitive PV and atrial activity lasting two to five beats. Atropine (2 μM, n = 10) prevented, whereas propranolol (2 μM, n = 5) had no effect, on EC-induced APD shortening or repetitive activity. We conclude that EC shortens the APD and hyperpolarizes the membrane by local release of acetylcholine and causes the steep repolarization gradient in the vicinity of the current source leading to repetitive activity in atrial and PV cells during premature stimulation.
Differential cross-bridge kinetics of FHC myosin mutations R403Q and R453C in heterozygous mouse myocardium
Authors: Palmer Bradley M. , Fishbaugher David E. , Schmitt Joachim P. , Wang Yuan , Alpert Norman R. , Seidman Christine E. , Seidman J. G. , VanBuren Peter , Maughan David W.
Published: March 2004

The kinetic effects of the cardiac myosin point mutations R403Q and R453C, which underlie lethal forms of familial hypertrophic cardiomyopathy (FHC), were assessed using isolated myosin and skinned strips taken from heterozygous (R403Q/+ and R453C/+) male mouse hearts. Compared with wild-type (WT) mice, actin-activated ATPase was increased by 38% in R403Q/+ and reduced by 45% in R453C/+, maximal velocity of regulated thin filament ( VRTF) in the in vitro motility assay was increased by 8% in R403Q/+ and was not different in R453C/+, myosin concentration at half-maximal VRTF was reduced by 30% in R403Q/+ and not different in R453C/+, and the characteristic frequency for oscillatory work production ( b frequency), determined by sinusoidal analysis in the skinned strip at maximal calcium activation, was 27% lower in R403Q/+ and 18% higher in R453C/+. The calcium sensitivity for isometric tension in the skinned strip was not different in R403Q/+ (pCa50 5.64 ± 0.02) and significantly enhanced in R453C/+ (5.82 ± 0.03) compared with WT (5.58 ± 0.02). We conclude that isolated myosin and skinned strips of R403Q/+ and R453C/+ myocardium show marked differences in cross-bridge kinetic parameters and in calcium sensitivity of force production that indicate different functional roles associated with the location of each point mutation at the molecular level.
Heme oxygenase attenuates allergen-induced airway inflammation and hyperreactivity in guinea pigs
Authors: Almolki Abdelhamid , Taillé Camille , Martin Gillian F. , Jose Peter J. , Zedda Christine , Conti Marc , Megret Jerome , Henin Dominique , Aubier Michel , Boczkowski Jorge
Published: March 2004

Heme oxygenase (HO), the heme-degrading enzyme, has shown anti-inflammatory effects in several models of pulmonary diseases. HO is induced in airways during asthma; however, its functional role is unclear. Therefore, we evaluated the role of HO on airway inflammation [evaluated by bronchoalveolar lavage (BAL) cellularity and BAL levels of eotaxin, PGE2, and proteins], mucus secretion (evaluated by analysis of MUC5AC gene expression and periodic acid-Schiff staining), oxidative stress (evaluated by quantification of 4-hydroxynonenal adducts and carbonylated protein levels in lung homogenates), and airway responsiveness to histamine in ovalbumin (OVA)-sensitized and multiple aerosol OVA or saline-challenged guinea pigs (6 challenges, once daily, OVA group and control group, respectively). Airway inflammation, mucus secretion, oxidative stress, and responsiveness were significantly increased in the OVA group compared with the control group. HO upregulation by repeated administrations of hemin (50 mg/kg ip) significantly decreased airway responsiveness in control animals and airway inflammation, mucus secretion, oxidative stress, and responsiveness in OVA animals. These effects were reversed by the concomitant administration of the HO inhibitor tin protoporphyrin-IX (50 μmol/kg ip). Repeated administrations of tin protoporphyrin-IX alone significantly increased airway responsiveness in control animals but did not modify airway inflammation, mucus secretion, oxidative stress, and responsiveness in OVA animals. These results suggest that upregulation of the HO pathway has a significant protective effect against airway inflammation, mucus hypersecretion, oxidative stress, and hyperresponsiveness in a model of allergic asthma in guinea pigs.
In vitro and in vivo effects of ghrelin on luteinizing hormone and growth hormone release in goldfish
Authors: Unniappan Suraj , Peter Richard E.
Published: March 2004

We studied the in vitro and in vivo effects of octanoylated goldfish ghrelin peptides (gGRL-19 and gGRL-12) on luteinizing hormone (LH) and growth hormone (GH) release in goldfish. gGRL-19 and gGRL-12 at picomolar doses stimulated LH and GH release from dispersed goldfish pituitary cells in perifusion and static incubation. Incubation of pituitary cells for 2 h with 10 nM gGRL-12 and 1 or 10 nM gGRL-19 increased LH-β mRNA expression, whereas only 10 nM gGRL-19 increased GH mRNA expression. Somatostatin-14 abolished the stimulatory effects of ghrelin on GH release from dispersed pituitary cells in perifusion and static culture. The GH secretagogue receptor antagonist d-Lys3-GHRP-6 inhibited the ghrelin-induced LH release, whereas no effects were found on stimulation of GH release by ghrelin. Intracerebroventricular injection of 1 ng/g body wt of gGRL-19 or intraperitoneal injection of 100 ng/g body wt of gGRL-19 increased serum LH levels at 60 min after injection, whereas significant increases in GH levels were found at 15 and 30 min after these treatments. Our results indicate that, in addition to its potent stimulatory actions on GH release, goldfish ghrelin peptides have the novel function of stimulating LH release in goldfish.
Bid activation in kidney cells following ATP depletion in vitro and ischemia in vivo
Authors: Wei Qingqing , Alam Mohammad M. , Wang Mong-Heng , Yu Fushin , Dong Zheng
Published: March 2004

Bid is a proapoptotic Bcl-2 family protein, which on activation translocates to mitochondria and induces damage to the organelles. Activation of Bid depends on its proteolytic processing into truncated forms of tBid. Bid is highly expressed in the kidneys; however, little is known about its role in renal pathophysiology. In this study, we initially examined Bid activation in cultured rat kidney proximal tubular cells following ATP depletion. The cells were depleted of ATP by azide incubation in the absence of metabolic substrates and then returned to normal culture medium for recovery. Typical apoptosis developed during recovery of ATP-depleted cells. This was accompanied by Bid cleavage, releasing tBid of 15 and 13 kDa. Bid cleavage was abolished in cells overexpressing Bcl-2, an antiapoptotic gene. It was also suppressed by caspase inhibitors. Peptide inhibitors of caspase-9 were more effective in blocking Bid cleavage compared with inhibitors of caspase-8 and caspase-3. Provision of glucose, a glycolytic substrate, during azide incubation inhibited Bid cleavage as well, indicating that Bid cleavage was initiated by ATP depletion. Consistently, Bid cleavage was also induced following ATP depletion by hypoxia or mitochondrial uncoupling. Of significance, cleaved Bid translocated to mitochondria, suggesting a role for Bid in the development of mitochondrial defects in ATP-depleted cells. Finally, Bid cleavage was induced during renal ischemia-reperfusion in the rat. Together, these results provide the first evidence for Bid activation in kidney cells following ATP depletion in vitro and renal ischemia in vivo.
Gentamicin traffics retrograde through the secretory pathway and is released in the cytosol via the endoplasmic reticulum
Authors: Sandoval Ruben M. , Molitoris Bruce A.
Published: March 2004

Previous mechanisms describing how aminoglycosides exert their cellular toxicity, including lysosomal accumulation, rupture, and release, cannot account for the rapidity and extent of the observed subcellular and organ effects. Using immunoamplification techniques and colocalization with epitopes of the endoplasmic reticulum (ER), we report rapid retrograde transport of gentamicin to the ER. Additionally, exposure times of 2 and 4 h in LLC-PK1cells produced cytosolic release and nuclear association. Cellular internalization and trafficking of aminoglycoside structural analogs, amine-containing cationic fluorescent dextrans of 3,000 molecular weight, corroborated these findings. However, identical anionic fluorescent dextrans, or larger cationic dextrans, of 10,000 molecular weight, failed to traverse from the ER into the cytosol or localize within the nucleus. These studies suggest that a pathway exists that transports internalized aminoglycosides, and other small-molecular-weight cationic compounds, in a retrograde manner through the Golgi complex and to the ER. From there, these compounds move into the cytosol for delivery throughout the cell. To quantify the potential toxic effects of cytosolic aminoglycoside release, experiments examining mitochondrial membrane potential in the continued presence of extracellular gentamicin were undertaken and demonstrated a significant reduction after 4 and 8 h. These observations provide a mechanism for the rapidly induced known cellular alterations, including aberrant vesicle fusion, mitochondrial toxicity/free radical generation, and decreased protein synthesis either by reduced transcription or translation after aminoglycoside exposure.
Vascular localization of soluble epoxide hydrolase in the human kidney
Authors: Yu Zhigang , Davis Benjamin B. , Morisseau Christophe , Hammock Bruce D. , Olson Jean L. , Kroetz Deanna L. , Weiss Robert H.
Published: March 2004

Epoxyeicosatrienoic acids are cytochrome P-450 metabolites of arachidonic acid with multiple biological functions, including the regulation of vascular tone, renal tubular transport, cellular proliferation, and inflammation. Epoxyeicosatrienoic acids are converted by soluble epoxide hydrolase into the corresponding dihydroxyeicosatrienoic acids, and epoxyeicosatrienoic acid hydration is regarded as one mechanism whereby their biological effects are eliminated. Previous animal studies indicate that soluble epoxide hydrolase plays an important role in the regulation of renal eicosanoid levels and systemic blood pressure. To begin to elucidate the mechanism of these effects, we determined the cellular localization of soluble epoxide hydrolase in human kidney by examining biopsies taken from patients with a variety of non-end-stage renal diseases, as well as those without known renal disease. Immunohistochemical staining of acetone-fixed kidney biopsy samples revealed that soluble epoxide hydrolase was preferentially expressed in the renal vasculature with relatively low levels in the surrounding tubules. Expression of soluble epoxide hydrolase was evident in renal arteries of varying diameter and was localized mostly in the smooth muscle layers of the arterial wall. Western blot analysis and functional assays confirmed the expression of soluble epoxide hydrolase in the human kidney. There were no obvious differences in soluble epoxide hydrolase expression between normal and diseased human kidney tissue in the samples examined. Our results indicate that soluble epoxide hydrolase is present in the human kidney, being preferentially expressed in the renal vasculature, and support an essential role for this enzyme in renal hemodynamic regulation and its potential utility as a target for therapeutic intervention.
Signaling cascade of ANG II-induced inhibition of α-MG uptake in renal proximal tubule cells
Authors: Han Ho Jae , Park Soo Hyun , Lee Yun Jung
Published: March 2004

ANG II and Na+-glucose cotransporter have been reported to be associated with the onset of diverse renal diseases. However, the effect of ANG II on Na+-glucose cotransporter activity was not elucidated. The effects of ANG II on α-methyl-d-[14C]glucopyranoside (α-MG) uptake and its related signal pathways were examined in the primary cultured rabbit renal proximal tubule cells (PTCs). ANG II (>2 h; >10-9M) inhibited α-MG uptake in a time- and concentration-dependent manner and decreased the protein level of Na+-glucose cotransporters, the expression of which was abrogated by both actinomycin D and cycloheximide exposure. ANG II-induced inhibition of α-MG uptake was blocked by losartan, an ANG II type 1 (AT1) receptor blocker, but not by PD-123319, an ANG II type 2 receptor blocker. ANG II-induced inhibition of α-MG uptake was blocked by genistein, herbimycin A [tyrosine kinase (TK) inhibitors], mepacrine, and AACOCF3(phospholipase A2inhibitors), suggesting the role of TK phosphorylation and arachidonic acid (AA). Indeed, ANG II increased AA release, which was blocked by losartan or TK inhibitors. The effects of ANG II on AA release and α-MG uptake also were abolished by staurosporine and bisindolylmaleimide I (protein kinase C inhibitors) or PD-98059 (p44/42 MAPK inhibitor), but not SB-203580 (p38 MAPK inhibitor), respectively. Indeed, ANG II increased p44/42 MAPK activity. ANG II-induced activation of p44/42 MAPK was blocked by staurosporine. In conclusion, ANG II inhibited α-MG uptake via PKC-MAPK-cPLA2signal cascade through the AT1receptor in the PTCs.
Oxidative stress, renal infiltration of immune cells, and salt-sensitive hypertension: all for one and one for all
Authors: Rodríguez-Iturbe Bernardo , Vaziri Nosratola D. , Herrera-Acosta Jaime , Johnson Richard J.
Published: March 2004

Recent evidence indicates that interstitial infiltration of T cells and macrophages plays a role in the pathogenesis of salt-sensitive hypertension. The present review examines this evidence and summarizes the investigations linking the renal accumulation of immune cells and oxidative stress in the development of hypertension. The mechanisms involved in the hypertensive effects of oxidant stress and tubulointerstitial inflammation, in particular intrarenal ANG II activity, are discussed, focusing on their potential for sodium retention. The possibility of autoimmune reactivity in hypertension is raised in the light of the proinflammatory and immunogenic pathways stimulated by the interrelationship between oxidant stress and inflammatory response. Finally, we present some clinical considerations derived from the recognition of this interrelationship.
Ca2+uptake in mitochondria occurs via the reverse action of the Na+/Ca2+exchanger in metabolically inhibited MDCK cells
Authors: Smets Ilse , Caplanusi Adrian , Despa Sanda , Molnar Zsolt , Radu Mihai , vandeVen Martin , Ameloot Marcel , Steels Paul
Published: March 2004

In ischemic or hypoxic tissues, elevated Ca2+levels have emerged as one of the main damaging agents among other Ca2+-independent mechanisms of cellular injury. Because mitochondria, besides the endoplasmic reticulum, play a key role in the maintainance of cellular Ca2+homeostasis, alterations in the mitochondrial Ca2+content ([Ca2+]m) were monitored in addition to changes in cytosolic Ca2+concentration ([Ca2+]i) during metabolic inhibition (MI) in renal epithelial Madin-Darby canine kidney (MDCK) cells. [Ca2+]iand [Ca2+]mwere monitored via, respectively, fura 2 and rhod 2 measurements. MI induced an increase in [Ca2+]ireaching 631 ± 78 nM in ∼20 min, followed by a decrease to 118 ± 9 nM in the next ∼25 min. A pronounced drop in cellular ATP levels and a rapid increase in intracellular Na+concentrations in the first 20 min of MI excluded Ca2+efflux in the second phase via plasma membrane ATPases or Na+/Ca2+exchangers (NCE). Mitochondrial rhod 2 intensities increased to 434 ± 46% of the control value during MI, indicating that mitochondria sequester Ca2+during MI. The mitochondrial potential (ΔΨm) was lost in 20 min of MI, excluding mitochondrial Ca2+uptake via the ΔΨm-dependent mitochondrial Ca2+uniporter after 20 min of MI. Under Na+-free conditions, or when CGP-37157, a specific inhibitor of the mitochondrial NCE, was used, no drop in [Ca2+]iwas seen during MI, whereas the MI-induced increase in mitochondrial rhod 2 fluorescence was strongly reduced. To our knowledge, this study is the first to report that in metabolically inhibited renal epithelial cells mitochondria take up Ca2+via the NCE acting in the reverse mode.