bluuebunny/crossref_metadata_2025 huggingface dataset:
Impaired expression and insulin-stimulated phosphorylation of Akt-2 in muscle of obese patients with atypical diabetes
Authors: Gosmanov Aidar R. , Umpierrez Guillermo E. , Karabell Ana H. , Cuervo Ruben , Thomason Donald B.
Published: March 2004

Although a pharmacological dose of insulin produces a dramatic increase in phosphorylation and activity of Akt isoforms 1 and 2 in mammalian skeletal muscle, few studies have examined the effect of physiological concentrations of insulin on the phosphorylation of Akt-1 and -2 in normal and diabetic tissue. This study examined the patterns of insulin-stimulated Akt isoform phosphorylation and protein expression in muscle biopsies obtained from obese patients with atypical diabetes immediately after a hyperglycemic crisis and again after near-normoglycemic remission. In obese patients with new-onset diabetes mellitus presenting with hyperglycemic crisis (plasma glucose 30.5 ± 4.8 mM), in vitro stimulation of vastus lateralis muscle biopsies with 100 μU/ml (0.6 nM) insulin increased insulin receptor phosphorylation threefold and Akt-1 phosphorylation on Ser473twofold, whereas Akt-2 phosphorylation was not stimulated. After 10-wk intensive insulin therapy that led to near-normoglycemic remission and discontinuation of insulin therapy, both Akt-2 expression and insulin-stimulated Akt-2 Ser474phosphorylation doubled. Hyperglycemic crisis did not affect insulin-stimulated threonine phosphorylation of either Akt-1 or Akt-2. The decreased Akt-2 expression at presentation was accompanied by reduced GLUT4 protein expression and increased expression of enzymes counterregulatory to insulin action. Thus a physiological concentration of insulin stimulated Akt-1 and Akt-2 phosphorylation in human skeletal muscle in the absence of hyperglycemia, but Akt-2 expression and stimulation appeared to be impaired in muscle of obese patients with atypical diabetes presenting with severe hyperglycemia.
Elevated circulating adiponectin levels in liver cirrhosis are associated with reduced liver function and altered hepatic hemodynamics
Authors: Tietge Uwe J. F. , Böker Klaus H. W. , Manns Michael P. , Bahr Matthias J.
Published: March 2004

Adiponectin is a novel adipocytokine negatively correlated with parameters of the metabolic syndrome, such as body mass index (BMI), body fat mass (BFM), and circulating insulin levels. Furthermore, metabolic actions directly on the liver have been described. The aim of the present study was to characterize circulating adiponectin levels, hepatic turnover, and the association of adiponectin with key parameters of hepatic as well as systemic metabolism in cirrhosis, a catabolic disease. Circulating adiponectin levels and hepatic turnover were investigated in 20 patients with advanced cirrhosis. Hepatic hemodynamics [portal pressure, liver blood flow, hepatic vascular resistance, indocyanine green (ICG) half-life], body composition, resting energy expenditure, hepatic free fatty acids (FFA) and glucose turnover, and circulating levels of hormones (catecholamines, insulin, glucagon) and proinflammatory cytokines (IL-1β, TNF-α, IL-6) were also assessed. Circulating adiponectin increased dependently on the clinical stage in cirrhosis compared with controls (15.2 ± 1.7 vs. 8.2 ± 1.1 μg/ml, respectively, P < 0.01), whereas hepatic extraction decreased. Adiponectin was negatively correlated with parameters of hepatic protein synthesis (prothrombin time: r = −0.62, P = 0.003; albumin: r = −0.72, P < 0.001) but not with transaminases or parameters of lipid metabolism. In addition, circulating adiponectin increased with portal pressure ( r = 0.67, P = 0.003), hepatic vascular resistance ( r = 0.60, P = 0.008), and effective hepatic blood flow (ICG half-life: r = 0.69, P = 0.001). Adiponectin in cirrhosis was not correlated with BMI, BFM, parameters of energy metabolism, insulin levels, hepatic FFA and glucose turnover, and circulating proinflammatory cytokines. These results demonstrate that 1) adiponectin plasma levels in cirrhosis are significantly elevated, 2) the liver is a major source of adiponectin extraction, and 3) adiponectin levels in cirrhosis do not correlate with parameters of body composition or metabolism but exclusively with reduced liver function and altered hepatic hemodynamics.
Angiotensin II type 1 receptor interaction is an important regulator for the development of pancreatic fibrosis in mice
Authors: Nagashio Yoshikuni , Asaumi Hiroshi , Watanabe Shiro , Nomiyama Yoko , Taguchi Masashi , Tashiro Mitsuo , Sugaya Takeshi , Otsuki Makoto
Published: March 2004

The renin-angiotensin system (RAS) plays important roles in various pathophysiological processes. However, the role of the RAS in pancreatic fibrosis has not been established. We investigated the role of angiotensin II (ANG II)-ANG II type 1 (AT1) receptor pathway in the development of pancreatic fibrosis with AT1areceptor-deficient [AT1a(−/−)] mice. To induce pancreatic fibrosis, AT1a(−/−) and wild-type (WT) mice were submitted to three episodes of acute pancreatitis induced by six intraperitoneal injections of 50 μg/kg body wt cerulein at hourly intervals, per week, for four consecutive weeks. Pancreatic fibrosis was assessed by histology and hydroxyproline content. Pancreatic stellate cell (PSC) activation and the localization of AT1receptors were assessed by Western blot analysis for α-smooth muscle actin and immunostaining. Transforming growth factor-β1(TGF-β1) mRNA expression in the pancreas was assessed by RT-PCR. Six intraperitoneal injections of cerulein induced acute pancreatitis in both AT1a(−/−) and WT mice. There were no significant differences between two groups with regard to serum amylase and histological changes. Pancreatic fibrosis induced by repeated episodes of acute pancreatitis was significantly attenuated in AT1a(−/−) mice compared with that in WT mice. This finding was accompanied by a reduction of activated PSCs. Dual-immunofluorescence staining in WT mice revealed that activated PSCs express AT1receptors. The level of TGF-β1mRNA was lower in AT1a(−/−) mice than in WT mice. Our results demonstrate that the ANG II-AT1receptor pathway is not essential for the local pancreatic injury in acute pancreatitis but plays an important role in the development of pancreatic fibrosis through PSC activation and proliferation.
Neutrophils augment recovery of porcine ischemia-injured ileal mucosa by an IL-1β- and COX-2-dependent mechanism
Authors: Shifflett Donnie E. , Bottone Frank G. , Young Karen M. , Moeser Adam J. , Jones Samuel L. , Blikslager Anthony T.
Published: March 2004

Polymorphonuclear neutrophils (PMNs) play a critical role in intestinal mucosal injury and repair. To study effects of PMNs on acutely injured mucosa, we applied PMNs isolated from circulation or peritoneal fluid from animals with chemically induced peritonitis to ischemia-injured porcine ileal mucosa. In preliminary experiments, PMNs enhanced recovery of transepithelial electrical resistance (TER), and this action was inhibited by pretreatment with the nonselective cyclooxygenase (COX) inhibitor indomethacin. Because COX-2 is upregulated by inflammatory mediators such as IL-1β, which is released by PMNs, we postulated that PMNs enhance recovery of ischemia-injured mucosa by a pathway involving IL-1β and COX-2. Application of 5 × 106PMNs to the serosal surface of ischemia-injured mucosa significantly enhanced recovery of TER ( P < 0.05), an effect that was inhibited by the selective COX-2 inhibitor NS-398 (5 μM) and by an IL-1β receptor antagonist (0.1 mg/ml). Addition of 10 ng/ml IL-1β to the serosal surface of injured tissues caused a significant increase in TER ( P < 0.05) that was inhibited by pretreatment with NS-398. Western blot analysis of mucosal homogenates revealed dramatic upregulation of COX-2 in response to IL-1β or peritoneal PMNs, and the latter was inhibited by an IL-1β receptor antagonist. Real-time PCR revealed that increased mRNA COX-2 expression preceded increased COX-2 protein expression in response to IL-1β. We concluded that PMNs augment recovery of TER in ischemia-injured ileal mucosa via IL-1β-dependent upregulation of COX-2.
In vitro activation of murine DRG neurons by CGRP-mediated mucosal mast cell degranulation
Authors: De Jonge F. , De Laet A. , Van Nassauw L. , Brown J. K. , Miller H. R. P. , van Bogaert P.-P. , Timmermans J.-P. , Kroese A. B. A.
Published: March 2004

Upregulation of CGRP-immunoreactive (IR) primary afferent nerve fibers accompanied by mastocytosis is characteristic for the Schistosoma mansoni-infected murine ileum. These mucosal mast cells (MMC) and CGRP-IR fibers, which originate from dorsal root (DRG) and nodose ganglia, are found in close apposition. We examined interactions between primary cultured MMC and CGRP-IR DRG neurons in vitro by confocal recording of intracellular Ca2+concentration ([Ca2+]i). The degranulatory EC50for the mast cell secretagogue compound 48/80 (C48/80; 10 μg/ml) and the neuropeptides CGRP (2.10−8M) and substance P (SP; 3.10−8M) were determined by measurement of extracellular release of the granule chymase, mouse mast cell protease-1. Application of C48/80 (10 μg/ml) and CGRP and SP (both 10−7M) to Fluo-4-loaded MMC induced a transient rise in [Ca2+]iafter a lag time, indicative of mast cell degranulation and/or secretion. The CGRP response could be completely blocked by pertussis toxin (2 μg/ml), indicating involvement of Giproteins. Application of MMC juice, obtained by C48/80 degranulation of MMC, to Fluo-4-loaded DRG neurons induced in all neurons a rise in [Ca2+]i, indicative of activation. Degranulation of MMC by C48/80 in culture dishes containing Fluo-4-loaded DRG neurons also caused activation of the DRG neurons. In conclusion, these results demonstrate a bidirectional cross-talk between cultured MMC and CGRP-IR DRG neurons in vitro. This indicates that such a communication may be the functional relevance for the close apposition between MMC and CGRP-IR nerve fibers in vivo.
Reversible effects of isoproterenol-induced hypertrophy on in situ left ventricular function in rat hearts
Authors: Kitagawa Yutaka , Yamashita Daisuke , Ito Haruo , Takaki Miyako
Published: March 2004

The aim of the present study was to evaluate specifically left ventricular (LV) function in rat hearts as they transition from the normal to hypertrophic state and back to normal. Either isoproterenol (1.2 and 2.4 mg·kg−1·day−1 for 3 days; Iso group) or vehicle (saline 24 μl·day−1 for 3 days; Sa group) was infused by subcutaneous implantation of an osmotic minipump. After verifying the development of cardiac hypertrophy, we recorded continuous LV pressure-volume (P-V) loops of in situ ejecting hypertrophied rat hearts. The curved LV end-systolic P-V relation (ESPVR) and systolic P-V area (PVA) were obtained from a series of LV P-V loops in the Sa and Iso groups 1 h or 2 days after the removal of the osmotic minipump. PVA at midrange LV volume (PVAmLVV) was taken as a good index for LV work capability ( 13 , 15 , 20 , 21 ). However, in rat hearts during remodeling, whether PVAmLVV is a good index for LV work capability has not been determined yet. In the present study, in contrast to unchanged end-systolic pressure at midrange LV volume, PVAmLVV was significantly decreased by isoproterenol treatment relative to saline; however, these measurements were the same 2 days after pump removal. Simultaneous treatment with a β1-blocker, metoprolol (24 mg·kg−1·day−1), blocked the formation of cardiac hypertrophy and thus PVAmLVV did not decrease. The reversible changes in PVAmLVV reflect precisely the changes in LV work capability in isoproterenol-induced hypertrophied rat hearts mediated by β1-receptors. These results indicate that the present approach may be an appropriate strategy for evaluating the effects of antihypertrophic and antifibrotic modalities.
Leukotriene B4is an indirectly acting vasoconstrictor in guinea pig aorta via an inducible type of BLT receptor
Authors: Bäck Magnus , Qiu Hong , Haeggström Jesper Z. , Sakata Kiyoto
Published: March 2004

Leukotriene B4(LTB4) is a potent leukocyte chemoattractant recently implicated in the pathogenesis of atherosclerosis. The aim of this study was to assess the effects of LTB4on isolated aortic preparations. Rings of guinea pig aorta were challenged with LTB4for recording mechanical responses and measurements of mediator release, and LTB4receptor (BLT1) expression was assessed by RT-PCR. Single concentrations of LTB4induced concentration-dependent contractions that were inhibited by treatment with antihistamines, indomethacin, or the thromboxane receptor antagonist BAYu3405 as well as by denudation of endothelium. In addition, LTB4increased the release of histamine and thromboxane in the bath. The contractions induced by LTB4were inhibited by either the unselective BLT receptor antagonist ONO-4057 or the selective BLT1receptor antagonist U-75302. Pretreatment with all -trans-retinoic acid enhanced the contractions and the release of histamine induced by LTB4, without affecting either the contractions induced by histamine or the histamine release evoked by calcium ionophore A23187. Analysis by RT-PCR indicated the expression of a BLT1receptor in the guinea pig aorta and that BLT1receptor mRNA was upregulated after treatment with retinoic acid. These results suggest that LTB4contracts the guinea pig aorta via an indirect mechanism involving the release of histamine and thromboxane and that this BLT1receptor-mediated response can be upregulated by all -trans-retinoic acid.
Angiotensin II receptor blockade corrects altered expression of gap junctions in vascular endothelial cells from hypertensive rats
Authors: Kansui Yasuo , Fujii Koji , Nakamura Keiichiro , Goto Kenichi , Oniki Hideyuki , Abe Isao , Shibata Yosaburo , Iida Mitsuo
Published: March 2004

Blockade of the renin-angiotensin system improves the impaired endothelium-dependent relaxations associated with hypertension and aging, partly through amelioration of endothelium-derived hyperpolarizing factor (EDHF)-mediated responses. Although the nature of EDHF is still controversial, recent studies have suggested the involvement of gap junctions in EDHF-mediated responses. Gap junctions consist of connexins (Cx), and we therefore tested whether the expression of Cx in vascular endothelial cells would be altered by hypertension and antihypertensive treatment. Spontaneously hypertensive rats (SHR) were treated with either the angiotensin II type 1 receptor antagonist candesartan or the combination of hydralazine and hydrochlorothiazide for 3 mo from 5 to 8 mo of age. Confocal laser scanning microscopy after immunofluorescent labeling with antibodies against Cx37, Cx40, and Cx43 revealed that the expression of Cx37 and Cx40 in endothelial cells of the mesenteric artery was significantly lower in SHR than in WKY. Treatment with candesartan, but not the combination of hydralazine and hydrochlorothiazide, significantly increased the expression of Cx37 and Cx40, although blood pressure decreased similarly. On the other hand, the expression of Cx43, though scarce and heterogeneous, was increased in SHR compared with WKY, and candesartan treatment lowered the expression of Cx43. These findings suggest that renin-angiotensin system blockade corrects the decreased expression of Cx37 and Cx40 in arterial endothelial cells of hypertensive rats, partly independently of blood pressure, whereas the expression of Cx43 changed in the opposite direction. It remains to be clarified whether these changes in Cx37 and Cx40 are related to endothelial function, particularly that attributable to EDHF.
Quantitative microcomputed tomography analysis of collateral vessel development after ischemic injury
Authors: Duvall Craig L. , Taylor W. Robert , Weiss Daiana , Guldberg Robert E.
Published: March 2004

Transgenic mouse models are increasingly being used to investigate the functions of specific growth factors or matrix proteins to design therapeutic strategies for controlling blood vessel growth. However, the available methodologies for evaluating angiogenesis and arteriogenesis in these models are limited by animal size, user subjectivity, the power to visualize the three-dimensional vessel networks, or the capability to employ a vigorous quantitative analysis. In this study, we employed contrast-enhanced microcomputed tomography imaging to assess collateral development after induction of hindlimb ischemia in the mouse. The morphological parameters vessel volume, connectivity, number, thickness, thickness distribution, separation, and degree of anisotropy were evaluated in control and surgery limbs 0, 3, and 14 days postsurgery. Results indicate that the vascular volume of the surgically manipulated limb was reconstituted as early as 3 days after femoral artery excision through development of a series of highly connected, small caliber, closely spaced, and isotropically oriented collateral vessels. Parametric analyses were completed to assess the sensitivity of the calculated morphological parameters to variations in image binarization threshold and voxel size. Images taken at the 36-μm voxel size were found to be optimal for evaluating collateral vessel formation, whereas 8- to 16-μm voxel sizes were needed to resolve smaller vascular structures. This study demonstrates the utility of microcomputed tomography as a robust method for quantitative, three-dimensional analysis of blood vessel networks. Whereas these initial efforts focused on the mouse hindlimb ischemia model, the developed techniques may be applied to a variety of model systems to investigate mechanisms of angiogenesis and arteriogenesis.
Exercise-induced and nitroglycerin-induced myocardial preconditioning improves hemodynamics in patients with angina
Authors: Crisafulli Antonio , Melis Franco , Tocco Filippo , Santoboni Uberto M. , Lai Carlo , Angioy Giovanni , Lorrai Luigi , Pittau Gianluigi , Concu Alberto , Pagliaro Pasquale
Published: March 2004

In humans, regional myocardial dysfunction during ischemia may be improved by ischemic and pharmacological preconditioning. We assessed the possibility that exercise- and nitroglycerin-induced myocardial preconditioning may improve global cardiac performance during subsequent efforts in patients with angina. Ten patients suffering from chronic stable angina and ten healthy volunteers were studied. Through impedance cardiography we assessed hemodynamics during a maximal exercise test, which was used as a baseline (Bas test) and considered as a preconditioning exercise. The Bas test was followed by a sequence of maximal efforts performed during the first (FWOP; 30 min after the Bas test) and second (SWOP; 48 h after the Bas test) windows of protection conferred by ischemic preconditioning. Hemodynamics was further evaluated during maximal exercise performed 48 h later with pharmacologically induced SWOP (PI-SWOP) obtained by transdermal administration of 10 mg of nitroglycerin. In the angina patients, FWOP, SWOP, and PI-SWOP delayed the time to ischemia and allowed them to achieve higher workloads compared with the Bas test. Furthermore, heart rate and cardiac output at peak exercise were enhanced during all the preconditioning phases with respect to the Bas test. However, only SWOP and PI-SWOP increased myocardial contractility and stroke volume. No changes in hemodynamics were detectable in the control subjects. This study demonstrates that in patients with stable angina, although hemodynamics during exercise can be positively improved during both FWOP and SWOP, differences exist between these two phases. Furthermore, the mimicking of exercise-induced SWOP by PI-SWOP with transdermal nitroglycerin may represent an important clinical aspect.
In vivo and in vitro evidence for ACh-stimulated l-arginine uptake
Authors: Parnell Melinda M. , Chin-Dusting Jaye P. F. , Starr Jennifer , Kaye David M.
Published: March 2004

Whereas l-arginine is clearly recognized as the precursor for nitric oxide synthesis, and its entry into endothelial cells via system y+ transport is established, few data exist regarding the acute regulation of this transport process. We specifically investigated the effect of ACh and isoprenaline (Iso) on l-arginine uptake in the human forearm and in cultured bovine aortic endothelial cells (BAEC). Sixteen healthy males were studied. During a steady-state intra-arterial infusion of [3H]l-arginine (100 nCi/min), the effects of ACh (9.25 and 37 μg/min), Iso (25–50 and 200 μg/min), and sodium nitroprusside (SNP) (1–2 and 8 μg/min) on forearm plasma flow (FPF), l-[3H]arginine uptake, and l-[3H]citrulline release were determined. In parallel experiments, the effects of ACh, Iso, and SNP on l-[3H]arginine uptake were studied in BAEC. l-Arginine uptake was inversely related to FPF ( r = −0.50; P < 0.005). At a similar FPF (ACh 56.82 ± 9.25, Iso 58.49 ± 5.56, SNP 57.92 ± 4.96 ml/min; P = ns), intra-arterial ACh significantly increased forearm uptake of l-[3H]arginine (54,655 ± 8,018 dpm/min), compared with that observed with either Iso (40,517.23 ± 6,841 dpm/min; P = 0.01) or SNP (36,816 ± 4,650 dpm/min; P = 0.011). This was associated with increased ACh-induced l-[3H]citrulline release compared with Iso and SNP ( P = 0.046). Similarly, in BAEC, ACh significantly increased l-[3H]arginine uptake compared with control, Iso, or SNP (ACh 12.0 × 107 ± 1.83 × 107 vs. control 6.67 × 107 ± 1.16 × 107 vs. Iso 7.35 × 107 ± 1.63 × 107 vs. SNP 6.01 × 107 ± 1.11 × 107 fmol·min−1·mg−1 at 300 μmol/l l-arginine; P = 0.043). Taken together, these data indicate that ACh stimulates l-arginine uptake in cultured endothelial cells and in human forearm circulation, indicating the potential for acute modulation of endothelial l-arginine uptake.
Total heart volume variation throughout the cardiac cycle in humans
Authors: Carlsson Marcus , Cain Peter , Holmqvist Catarina , Stahlberg Freddy , Lundback Stig , Arheden Hakan
Published: March 2004

Variations in total heart volume (atria plus ventricles) during a cardiac cycle affect efficiency of cardiac pumping. The goals of this study were to confirm the presence, extent, and contributors of total heart volume variation during the cardiac cycle in healthy volunteers with the use of MRI. Eight healthy volunteers were examined by MRI at rest. Changes in total cardiac volume throughout the cardiac cycle were calculated using the following methods: 1) planimetry derived from gradient-echo cine images and 2) flow-sensitive sequences to quantify flow in all vessels leading to and from the heart. The maximum total heart volume diminished during systole by 8.2 ± 0.8% (SEM, range 4.8–10.6%) measured by method 1 and 8.8 ± 1.0% (SEM, range 5.6–11.8%) by method 2 with good agreement between the methods [difference according to Bland-Altman analysis −0.6% ± 1.0% (SD), intraclass correlation coefficient = 0.999]. This decrease in volume is predominantly explained by variation at the midcardiac level at the widest diameter of the heart with a left-sided predominance. In the short axis of the heart, the change of slice volume was proportional to the end-diastolic slice volume. The present study has confirmed the presence of total heart volume variation that predominantly occurs in the region of atrioventricular plane movement and on the left side. The total heart volume variation may relate to the efficiency of energy use by the heart to minimize displacement of surrounding tissue while accounting for the energy required to draw blood into the atria during ventricular systole.
Extracellular superoxide enhances 5-HT-induced murine pulmonary artery vasoconstriction
Authors: Liu John Q. , Folz Rodney J.
Published: March 2004

Accumulating evidence suggests that changes in both 5-hydroxytryptamine (5-HT) receptor activity and in the levels of reactive oxygen species (ROS) play an important role in regulating pulmonary artery (PA) vascular responsiveness, particularly in the setting of pulmonary hypertension. Therefore, we hypothesized that increased levels of superoxide enhance 5-HT-induced PA constriction. With the use of a small-vessel bioassay, 5-HT (0.01–10 μM) induced a concentration-dependent vasoconstriction in isolated wild-type murine intrapulmonary arteries (100–150 μm diameter) that was enhanced by both removal of the endothelium and by treatment with either NG-nitro-l-arginine methyl ester (30 μM) or xanthine (10 μM) + xanthine oxidase (0.005 U/ml). PA isolated from extracellular superoxide dismutase (EC-SOD) knockout mice also showed enhanced constriction. On the other hand, PA constriction to 5-HT was attenuated by either the addition of GR-127935 (0.1 μM, a selective inhibitor of 5-HT1B/1Dreceptor) or copper/zinc-containing superoxide dismutase (Cu/Zn SOD, 150 U/ml) and in PA isolated from transgenic mice overexpressing human EC-SOD. With the use of both oxidative fluorescent confocal microscopy and lucigenin-enhanced chemiluminescence, superoxide levels were increased significantly after 5-HT-induced PA vasoconstriction. This increase in superoxide levels could be blocked by the exogenous addition of Cu/Zn SOD (150 U/ml) or by apocynin (30 μM, an inhibitor of NADPH oxidase) but was not affected by gp91phoxknockout mice. Overall, our results are consistent with 5-HT increasing vascular smooth muscle superoxide production via an NADPH oxidase pathway that is independent of gp91phox, which leads to increases in extracellular superoxide levels, which in turn enhances 5-HT-induced murine pulmonary vasoconstriction.
Myeloperoxidase deficiency enhances inflammation after allogeneic marrow transplantation
Authors: Milla Carlos , Yang Shuxia , Cornfield David N. , Brennan Marie-Luise , Hazen Stanley L. , Panoskaltsis-Mortari Angela , Blazar Bruce R. , Haddad Imad Y.
Published: March 2004

Myeloperoxidase (MPO)-derived oxidants participate in the respiratory antimicrobial defense system but are also implicated in oxidant-mediated acute lung injury. We hypothesized that MPO contributes to lung injury commonly observed after bone marrow transplantation (BMT). MPO-sufficient (MPO+/+) and -deficient (MPO−/−) mice were given cyclophosphamide and lethally irradiated followed by infusion of inflammation-inducing donor spleen T cells at time of BMT. Despite suppressed generation of nitrative stress, MPO−/− recipient mice unexpectedly exhibited accelerated weight loss and increased markers of lung dysfunction compared with MPO+/+ mice. The increased lung injury during MPO deficiency was a result of donor T cell-dependent inflammatory responses because bronchoalveolar lavage fluids (BALF) from MPO−/− mice contained increased numbers of inflammatory cells and higher levels of the proinflammatory cytokine TNF-α and the monocyte chemoattractant protein-1 compared with wild-type mice. Enhanced inflammation in MPO−/− mice was associated with suppressed apoptosis of BALF inflammatory cells. The inflammatory process in MPO−/− recipients was also associated with enhanced necrosis of freshly isolated alveolar type II cells, critical for preventing capillary leak. We conclude that suppressed MPO-derived oxidative/nitrative stress is associated with enhanced lung inflammation and persistent alveolar epithelial injury.
Pseudomonas aeruginosapyocyanin directly oxidizes glutathione and decreases its levels in airway epithelial cells
Authors: O'Malley Yunxia Q. , Reszka Krzysztof J. , Spitz Douglas R. , Denning Gerene M. , Britigan Bradley E.
Published: March 2004

Production of pyocyanin enhances Pseudomonas aeruginosa virulence. Many of pyocyanin's in vitro and in vivo cytotoxic effects on human cells appear to result from its ability to redox cycle. Pyocyanin directly accepts electrons from NADH or NADPH with subsequent electron transfer to oxygen, generating reactive oxygen species. Reduced glutathione (GSH) is an important cellular antioxidant, and it contributes to the regulation of redox-sensitive signaling systems. Using the human bronchial epithelial (HBE) and the A549 human type II alveolar epithelial cell lines, we tested the hypothesis that pyocyanin can deplete airway epithelial cells of GSH. Incubation of both cell types with pyocyanin led to a concentration-dependent loss of cellular GSH (up to 50%) and an increase in oxidized GSH (GSSG) in the HBE, but not A549 cells, at 24 h. An increase in total GSH, mostly as GSSG, was detected in the culture media, suggesting export of GSH or GSSG from the pyocyanin-exposed cells. Loss of GSH could be due to pyocyanin-induced H2O2formation. However, overexpression of catalase only partially prevented the pyocyanin-mediated decline in cellular GSH. Cell-free electron paramagnetic resonance studies revealed that pyocyanin directly oxidizes GSH, forming pyocyanin free radical and O2−·. Pyocyanin oxidized other thiol-containing compounds, cysteine and N-acetyl-cysteine, but not methionine. Thus GSH may enhance pyocyanin-induced cytotoxicity by functioning as an alternative source of reducing equivalents for pyocyanin redox cycling. Pyocyanin-mediated alterations in cellular GSH may alter epithelial cell functions by modulating redox sensitive signaling events.
Effects of matrix metalloproteinase inhibitor on LPS-induced goblet cell metaplasia
Authors: Kim Je Hyeong , Lee Sung Yong , Bak Sang Myeon , Suh In Bum , Lee Sang Yeub , Shin Chol , Shim Jae Jeong , In Kwang Ho , Kang Kyung Ho , Yoo Se Hwa
Published: March 2004

Bacterial infections of the lung are known to induce inflammatory responses, which lead to mucus hypersecretion. Moreover, mucin synthesis in the airways has been reported to be regulated by neutrophilic inflammation-induced epidermal growth factor receptor (EGFR) expression and its activation. Furthermore, matrix metalloproteinases (MMPs), especially MMP-9, have been reported to promote the transmigration of activated neutrophils. In this study, we investigated the associations between lipopolysaccharide (LPS)-induced goblet cell (GC) metaplasia and EGFR expression and the effects of MMP inhibitor (MMPI). Various concentrations of LPS were instilled into the tracheas of pathogen-free Sprague-Dawley rats, and airways were examined at different times after LPS instillation. To examine the role of MMP-9, we treated rats 3 days before LPS instillation and daily thereafter with MMPI. Neutrophilic infiltration, Alcian blue/periodic acid-Schiff (AB/PAS) staining, and immunohistochemical staining for MUC5AC, EGFR, and MMP-9 were performed. The instillation of LPS increased AB/PAS and MUC5AC staining in time- and dose-dependent manners, and treatment with MMPI significantly prevented GC metaplasia. The instillation of LPS into the trachea also induced neutrophilic infiltration and EGFR and MMP-9 expression in the airway epithelium, and MMPI was found to significantly prevent neutrophil recruitment, GC metaplasia, and EGFR and MMP-9 expression. This study demonstrates that the MMP-9 and EGFR cascades are associated with LPS-induced mucus hypersecretion.
Contribution of caveolin-1α and Akt to TNF-α-induced cell death
Authors: Ono Koh , Iwanaga Yoshitaka , Hirayama Madoka , Kawamura Teruhisa , Sowa Naoya , Hasegawa Koji
Published: March 2004

We used retrovirus insertion-mediated random mutagenesis to generate tumor necrosis factor-α (TNF-α)-resistant lines from L929 cells. Using this approach, we discovered that caveolin-1α is required for TNF-α-induced cell death in L929 cells. The need for caveolin-1α in TNF-α-induced cell death was confirmed by the restoration of sensitivity to TNF-α after ectopic reconstitution of caveolin-1α/β expression. This caveolin-1α-mutated line was also resistant to H2O2and staurosporine, but not to lonidamine. HepG2 cells are known to lack endogenous caveolins. HepG2 cells stably transfected with caveolin-1α/β were found to be much more sensitive to TNF-α than either parental cells transfected with caveolin-1β or parental cells transfected with an empty vector. In contrast to its extensively documented antiapoptotic effect, the elevated activity of Akt appears to be important in sensitizing caveolin-1-expressing cells to TNF-α, since pretreatment of cells with the phosphatidylinositide 3-kinase (PI3K) inhibitor LY-294002 or wortmannin completely blocked PI3K activation and markedly improved the survival of TNF-α-treated L929 cells. The survival rates of caveolin-1α-normal and caveolin-1α-deficient L929 cells were comparable after treatment with PI3K inhibitor and TNF-α. Similar results were obtained with HepG2 cells that stably expressed caveolin-1α/β or -β and parental cells transfected with an empty vector. In summary, our results indicate that caveolin-1α preferentially sensitizes L929 cells to TNF-α through the activation of a PI3K/Akt signaling pathway.
Differentiation-dependent responsiveness of bronchial epithelial cells to IL-4/13 stimulation
Authors: Kikuchi Tadashi , Shively Jonathan D. , Foley John S. , Drazen Jeffrey M. , Tschumperlin Daniel J.
Published: March 2004

The Th2 cytokines interleukin (IL)-4 and IL-13 are thought to play critical roles in the airway inflammation and hyperresposiveness that characterize asthma. Recent evidence indicates that IL-13 can mediate these effects by acting directly on airway epithelial cells. Here we evaluated early [signal transducer and activator of transcription (STAT)6 phosphorylation] and delayed [granulocyte/macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-β2 (TGF-β2) secretion] responses of airway epithelial cells to IL-4 and IL-13 stimulation and the dependence of these responses on the culture technique employed. As expected, normal human bronchial epithelial cells grown on microporous inserts at an air-liquid interface (ALI) expressed a well-differentiated mucociliary phenotype; in contrast, cells grown on plastic in submerged cultures were poorly differentiated. When stimulated with IL-4 or IL-13, the magnitude and duration of STAT6 phosphorylation under the differing culture conditions were statistically indistinguishable. In contrast, cytokine secretion responses to IL-4 and IL-13 were highly dependent on the culture technique; cells cultured on plastic exhibited significant concentration-dependent increases in GM-CSF and TGF-β2 secretion, whereas cells grown at ALI showed no statistically significant response. These results demonstrate that the coupling between early signal transduction responses to IL-4 and IL-13 and downstream functions such as cytokine secretion may be critically dependent on the cell culture technique employed and the resulting differentiation status of bronchial epithelial cells.
Time course analysis of the mechanism by which silver inhibits active Na+and Cl−uptake in gills of rainbow trout
Authors: Morgan Tammie P. , Grosell Martin , Gilmour Kathleen M. , Playle Richard C. , Wood Chris M.
Published: March 2004

A time course analysis using110mAg,24Na+, and36Cl−examined gill silver accumulation and the mechanism by which waterborne silver (4.0 × 10−8M; 4.3 μg/l) inhibits Na+and Cl−uptake in gills of freshwater rainbow trout. Analyses of gill and body fluxes allowed calculation of apical uptake and basolateral export rates for silver, Na+, and Cl−. To avoid changes in silver bioavailability, flow-through conditions were used to limit the buildup of organic matter in the exposure water. For both Na+and Cl−uptake, apical entry, rather than basolateral export, was the rate-limiting step; Na+and Cl−uptake declined simultaneously and equally initially, with both uptakes reduced by ∼500 nmol·g−1·h−1over the 1st h of silver exposure. There was a further progressive decline in Na+uptake until 24 h. Carbonic anhydrase activity was inhibited by 1 h, whereas Na+-K+-ATPase activity was not significantly inhibited until 24 h of exposure. These results indicate that carbonic anhydrase inhibition can explain the early decline in Na+and Cl−uptake, whereas the later decline is probably related to Na+-K+-ATPase blockade. Contrary to previous reports, gill silver accumulation increased steadily to a plateau. Despite the rapid inhibition of apical Na+and Cl−uptake, apical silver uptake (and basolateral export) increased until 10 h, before decreasing thereafter. Thus silver did not inhibit its own apical uptake in the short term. These results suggest that reduced silver bioavailability is the mechanism behind the pattern of peak and decline in gill silver accumulation previously reported for static exposures to silver.
Increased heat loss affects hibernation in golden-mantled ground squirrels
Authors: Kauffman Alexander S. , Paul Matthew J. , Zucker Irving
Published: March 2004

During hibernation at ambient temperatures (Ta) above 0°C, rodents typically maintain body temperature (Tb) ∼1°C above Ta, reduce metabolic rate, and suspend or substantially reduce many physiological functions. We tested the extent to which the presence of an insulative pelage affects hibernation. Tb was recorded telemetrically in golden-mantled ground squirrels ( Spermophilus lateralis) housed at a Ta of 5°C; food intake and body mass were measured at regular intervals throughout the hibernation season and after the terminal arousal. Animals were subjected to complete removal of the dorsal fur or a control procedure after they had been in hibernation for 3–4 wk. Shaved squirrels continued to hibernate with little or no change in minimum Tb, bout duration, duration of periodic normothermic bouts, and food intake during normothermia. Rates of rewarming from torpor were, however, significantly slower in shaved squirrels, and rates of body mass loss were significantly higher, indicating increased depletion of white adipose energy stores. An insulative pelage evidently conserves energy over the course of the hibernation season by decreasing body heat loss and reducing energy expenditure during periodic arousals from torpor and subsequent intervals of normothermia. This prolongs the hibernation season by several weeks, thereby eliminating the debilitating consequences associated with premature emergence from hibernation.
Rat renal cortical OAT1 and OAT3 exhibit gender differences determined by both androgen stimulation and estrogen inhibition
Authors: Ljubojević Marija , Herak-Kramberger Carol M. , Hagos Yohannes , Bahn Andrew , Endou Hitoshi , Burckhardt Gerhard , Sabolić Ivan
Published: March 2004

In rats, the secretion of p-aminohippurate (PAH) by the kidney is higher in males (M) than in females (F). The role of the major renal PAH transporters, OAT1 and OAT3, in the generation of these gender differences, as well as the responsible hormones and mechanisms, has not been clarified. Here we used various immunocytochemical methods to study effects of gender, gonadectomy, and treatment with sex hormones on localization and abundance of OAT1 and OAT3 along the rat nephron. Both transporters were localized to the basolateral membrane: OAT1 was strong in proximal tubule S2 and weak in the S3 segments, whereas OAT3 was stained in proximal tubule S1 and S2 segments, thick ascending limb, distal tubule, and in principal cells along the collecting duct. Gender differences in the expression of both transporters in adult rats (M > F) were observed only in the cortical tubules. OAT1 in the cortex was strongly reduced by castration in adult M, whereas the treatment of castrated M with testosterone, estradiol, or progesterone resulted in its complete restitution, further depression, or partial restitution, respectively. In adult F, ovariectomy weakly increased, whereas estradiol treatment of ovariectomized F strongly decreased, the expression of OAT1. The expression of OAT3 in the M and F cortex largely followed a similar pattern, except that ovariectomy and progesterone treatment showed no effect, whereas in other tissue zones gender differences were not observed. In prepubertal rats, the expression of OAT1 and OAT3 in the kidney cortex was low and showed no gender differences. Our data indicate that gender differences in the rat renal cortical OAT1 and OAT3 (M > F) appear after puberty and are determined by both a stimulatory effect of androgens (and progesterone in the case of OAT1) and an inhibitory effect of estrogens.
Inhibition of nNOS expression in the macula densa by COX-2-derived prostaglandin E2
Authors: Paliege Alex , Mizel Diane , Medina Carmen , Pasumarthy Anita , Huang Yuning G. , Bachmann Sebastian , Briggs Josephine P. , Schnermann Jurgen B. , Yang Tianxin
Published: March 2004

It is well established that cyclooxygenase-2 (COX-2) and the neuronal form of nitric oxide synthase (nNOS) are coexpressed in macula densa cells and that the expression of both enzymes is stimulated in a number of high-renin states. To further explore the role of nNOS and COX-2 in renin secretion, we determined plasma renin activity in mice deficient in nNOS or COX-2. Plasma renin activity was significantly reduced in nNOS −/− mice on a mixed genetic background and in COX-2 −/− mice on either BALB/c or C57/BL6 congenic backgrounds. In additional studies, we accumulated evidence to show an inhibitory influence of PGE2on nNOS expression. In a cultured macula densa cell line, PGE2significantly reduced nNOS mRNA expression, as quantified by real-time RT-PCR. In COX-2 −/− mice, nNOS mRNA expression in the kidney, determined by real-time RT-PCR, was upregulated throughout the postnatal periods, ranging from postnatal day ( PND) 3 to PND 60. The induction of nNOS protein expression and NOS activity in COX-2 −/− mice was localized to macula densa cells using immunohistochemistry and NADPH-diaphorase staining methods, respectively. Therefore, these findings reveal that the absence of either COX-2 or nNOS is associated with suppressed renin secretion. Furthermore, the inhibitory effect of PGE2on nNOS mRNA expression indicates a novel interaction between NO and prostaglandin-mediated pathways of renin regulation.
Opioid receptor-like 1 stimulation in the collecting duct induces aquaresis through vasopressin-independent aquaporin-2 downregulation
Authors: Hadrup Niels , Petersen Jørgen S. , Praetorius Jeppe , Meier Eddi , Græbe Martin , Brønd Lone , Staahltoft Dennis , Nielsen Søren , Christensen Sten , Kapusta Daniel R. , Jonassen Thomas E. N.
Published: March 2004

Nociceptin, the endogenous ligand of the inhibitory G protein-coupled opioid receptor-like 1 receptor, produces aquaresis (i.e., increases the excretion of solute-free urine) in rats. However, the mechanism underlying this effect has not yet been explained. Using immunohistochemistry, we found the opioid receptor-like 1 receptor in the rat kidney colocalized with the vasopressin-regulated water channel aquaporin-2 in inner medullary collecting ducts. We investigated the aquaretic effect of opioid receptor-like 1 receptor stimulation by infusing the selective nociceptin analog ZP120C; volume depletion was prevented by computer-driven, servo-controlled intravenous volume replacement with 50 mM glucose. ZP120C induced a marked and sustained aquaresis in normal and congestive heart failure rats in the absence of changes in vasopressin plasma concentrations. The ZP120C-induced aquaresis was associated with downregulation of the aquaporin-2 protein level in both rat groups, suggesting that opioid receptor-like 1 receptor stimulation produces aquaresis by inhibiting the vasopressin type-2 receptor-mediated stimulation on collecting duct water reabsorption. However, substantial amounts of PKA-mediated serine 256 phosphorylated aquaporin-2 were still present after 4 h of ZP120C treatment. Furthermore, neither preincubation with nociceptin nor ZP120C inhibited vasopressin-mediated cAMP accumulation in isolated collecting ducts. We conclude that renal opioid receptor-like 1 receptor stimulation in normal and congestive heart failure rats produces aquaresis by a direct renal effect, via aquaporin-2 downregulation, through a mechanism not involving inhibition of vasopressin type-2 receptor-mediated cAMP production.
Downregulation of hepatic acyl-CoA:diglycerol acyltransferase in chronic renal failure
Authors: Vaziri N. D. , Kim C. H. , Dang B. , Zhan Chang-De , Liang K.
Published: March 2004

Chronic renal failure (CRF) is associated with hypertriglyceridemia and elevated plasma VLDL and IDL concentrations. These events can be due to either increased production or depressed catabolism of triglyceride-rich lipoproteins. Several studies have documented downregulation of lipoprotein lipase, hepatic triglyceride lipase, and the VLDL receptor, leading to depressed clearance and elevated plasma concentration of triglyceride-rich lipoproteins and their remnants in CRF. However, the effect of CRF on the triglyceride biosynthetic pathway has not been explored. Diglycerol acyltransferase (DGAT) is a microsomal enzyme that joins acyl-CoA to 1,2 diacylglycerol and, as such, constitutes the final step in triglyceride biosynthesis. Two distinct forms of DGAT (DGAT-1 and -2) have thus far been identified. The present study was undertaken to examine the effect of CRF on DGAT gene expression and activity in the liver, which is the source of endogenous triglycerides in the circulation. Male Sprague-Dawley rats were studied 8 wk after [Formula: see text]nephrectomy (CRF) or sham operation. DGAT-1 and DGAT-2 mRNA abundance and DGAT activity were quantified. The CRF group showed reduced creatinine clearance, elevated plasma triglycerides, and VLDL concentrations. This was accompanied by significant reductions in hepatic DGAT-2 mRNA abundance ( P < 0.01) and total DGAT activity ( P < 0.1), pointing to diminished hepatic triglyceride production capacity in CRF animals. In conclusion, CRF results in significant downregulation of hepatic DGAT gene expression and activity. Given the critical role of DGAT in triglyceride biosynthesis, the present study points to diminished, not increased, hepatic triglyceride synthetic capacity in CRF rats.
Upregulation of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in endothelial cells by nitric oxide deficiency
Authors: Smirnova I. V. , Sawamura T. , Goligorsky M. S.
Published: March 2004

Endothelial cell dysfunction (ECD) is emerging as a common denominator for diverse cardiovascular abnormalities associated with inhibition of endothelial nitric oxide (NO) synthase (eNOS). Elevated levels of asymmetric dimethylarginine (ADMA), a potent eNOS inhibitor, are common in renal failure and may contribute to ECD. Through DNA microarray screening of genes modulated in human umbilical vein endothelial cells (HUVEC) by NG-nitro-l-arginine methyl ester (l-NAME), we found a 1.8-fold increase in low-density lipoprotein receptor-1 (LOX-1) expression. LOX-1 is a major endothelial receptor for oxidized low-density lipoproteins (OxLDL) and is assumed to play a role in the initiation and progression of atherosclerosis. Here, we confirmed the upregulation of LOX-1 mRNA and protein level by quantitative RT-PCR and Western blot analysis. Increased expression of LOX-1 was associated with the accumulation of DiI-labeled OxLDL (DiI-OxLDL) in ADMA- and l-NAME-pretreated HUVEC. To evaluate the contribution of LOX-1 in ADMA-induced accumulation of OxLDL by HUVEC, we used the competitive receptor inhibitor, soluble LOX-1. Treatment of HUVEC with soluble LOX-1 was associated with an approximately two- to threefold inhibition of DiI-OxLDL uptake in l-NAME- or ADMA-treated HUVEC. In conclusion, ADMA- or l-NAME-induced NO deficiency leads to the increased expression of LOX-1 mRNA and protein in HUVEC, which in turn results in the accumulation of OxLDL. Competition with LOX-1-soluble extracellular domain reduces OxLDL accumulation. In summary, elevated ADMA levels, i.e., in patients with renal failure, may be responsible for endothelial accumulation of OxLDL via upregulated LOX-1 receptor, thus contributing to endothelial lipidosis and dysfunction.