bluuebunny/crossref_metadata_2025 huggingface dataset:
Na+/Ca2+exchanger overexpression impairs frequency- and ouabain-dependent cell shortening in adult rat cardiomyocytes
Authors: Bölck Birgit , Münch Götz , Mackenstein Peter , Hellmich Martin , Hirsch Ingo , Reuter Hannes , Hattebuhr Nadja , Weig Hans-Jörg , Ungerer Martin , Brixius Klara , Schwinger Robert H. G.
Published: June 2004

The Na+/Ca2+exchanger (NCX) may influence cardiac function depending on its predominant mode of action, forward mode or reverse mode, during the contraction-relaxation cycle. The intracellular Na+concentration ([Na+]i) and the duration of the action potential as well as the level of NCX protein expression regulate the mode of action of NCX. [Na+]iand NCX expression have been reported to be increased in human heart failure. Nevertheless, the consequences of altered NCX expression in heart failure are still a matter of discussion. We aimed to characterize the influence of NCX expression on intracellular Ca2+transport in rat cardiomyocytes by adenoviral-mediated gene transfer. A five- to ninefold (dose dependent) overexpression of NCX protein was achieved after 48 h by somatic gene transfer (Ad.NCX.GFP) versus control (Ad.GFP). NCX activity, determined by Na+gradient-dependent45Ca2+-uptake, was significantly increased. The protein expressions of sarco(endo)plasmic reticulum Ca2+-ATPase, phospholamban, and calsequestrin were unaffected by NCX overexpression. Fractional shortening (FS) of isolated cardiomyocytes was significantly increased at low stimulation rates in Ad.NCX.GFP. After a step-wise enhancing frequency of stimulation to 3.0 Hz, FS remained unaffected in Ad.GFP cells but declined in Ad.NCX.GFP cells. The positive inotropic effect of the cardiac glycoside ouabain was less effective in Ad.NCX.GFP cells, whereas the positive inotropic effect of β-adrenergic stimulation remained unchanged. In conclusion, NCX overexpression results in a reduced cell shortening at higher stimulation frequencies as well as after inhibition of sarcolemmal Na+-K+-ATPase, i.e., in conditions with enhanced [Na+]i. At low stimulation rates, increased NCX expression enhances both intracellular systolic Ca2+and contraction amplitude.
Cell transplantation for treatment of acute myocardial infarction: unique capacity for repair by skeletal muscle satellite cells
Authors: Horackova M. , Arora R. , Chen R. , Armour J. A. , Cattini P. A. , Livingston R. , Byczko Z.
Published: June 2004

An adult heart injured by an ischemic episode has a limited capacity to regenerate. We administered three types of adult guinea pig cells [cardiomyocytes (CMs), cardiac fibroblasts (CFs), and skeletal myoblasts (Mbs)] to compare their suitability for repair of acute myocardial infarction. We used confocal fluorescent microscopy and a variety of specific immunomarkers and echocardiography to provide anatomic evidence for the viability of such cells and their possible functional beneficial effects. All cells were transfected with adenovirus-containing β-galactosidase gene so that migration from the injection sites could be traced. Both freshly isolated CMs as well as CFs were found concentrated in the infarcted zone; these cells survived for at least 2 wk posttransplantation. Transplanted CMs were regularly striated and grew long projections that could form gap junctions with native CMs, which was evidenced by connexin43 labeling. In addition, CM transplantation resulted in increased angiogenesis in the infarcted areas. In contrast, transplanted CFs did not appear to make any gap junctional contacts with native CMs nor did they enhance local angiogenesis. Mbs cultured for 7 days and transfected Mbs were identified 7 days posttransplantation in the infarcted area. During that time and thereafter, Mbs proliferated and differentiated into myotubes that formed new, regularly striated myofibers that occupied most (50–70%) of the infarcted area by 2–3 wk. These newly formed myofibers maintained their Mb skeletal muscle origin as evidenced by their capacity to express myogenin and fast skeletal myosin. This skeletal phenotype appeared to downregulate with time, and Mbs partially transdifferentiated into a cardiac phenotype as indicated by labeling for cardiac-specific troponin T and cardiac myosin heavy chain. By the third week posttransplantation, new myofibers formed apparent contacts with the native CMs via putative gap junctions that expressed connexin43. Myocardial performance of animals that were successfully transplanted with Mbs was improved.
5-HT-mediated inhibition of cardiovagal baroreceptor reflex response during defense reaction in the rat
Authors: Comet Marie-Anne , Sévoz-Couche Caroline , Hanoun Naïma , Hamon Michel , Laguzzi Raul
Published: June 2004

Previous studies showed that the cardiac response of the baroreceptor reflex (bradycardia) is inhibited during the defense reaction evoked by direct electrical or chemical stimulation of the periaqueductal gray (dPAG) in the rat. Whether central serotonin and nucleus tractus solitarius (NTS) serotonin3 (5-HT3) receptors might participate in this inhibition was investigated in urethane-anesthetized and atenolol-pretreated rats. Our results showed that both electrical and chemical stimulation of the dPAG produced a drastic reduction of the cardiovagal component of the baroreceptor reflex triggered by either intravenous administration of phenylephrine or aortic nerve stimulation. This inhibitory effect of dPAG stimulation on the baroreflex bradycardia was not observed in rats that had been pretreated with p-chlorophenylalanine (300 mg/kg ip daily for 3 days) to inhibit serotonin synthesis. Subsequent 5-hydroxytryptophan administration (60 mg/kg ip), which was used to restore serotonin synthesis, allowed the inhibitory effect of dPAG stimulation on both aortic and phenylephrine-induced cardiac reflex responses to be recovered in p-chlorophenylalanine-pretreated rats. On the other hand, in nonpretreated rats, the inhibitory effect of dPAG stimulation on the cardiac baroreflex response could be markedly reduced by prior intra-NTS microinjection of granisetron, a 5-HT3 receptor antagonist, or bicuculline, a GABAA receptor antagonist. These results show that serotonin plays a key role in the dPAG stimulation-induced inhibition of the cardiovagal baroreceptor reflex response. Moreover, they support the idea that 5-HT3 and GABAA receptors in the NTS contribute downstream to the inhibition of the baroreflex response caused by dPAG stimulation.
Expression of epithelial sodium channel α-subunit mRNAs with alternative 5′-untranslated regions in the developing human lung
Authors: Banasikowska Katharine , Post Martin , Cutz Ernest , O'Brodovich Hugh , Otulakowski Gail
Published: June 2004

In preparation for birth, lung epithelia must switch from net fluid secretion, required for lung development, to net absorption, which prepares the lungs for postnatal gas exchange. The apical membrane amiloride-sensitive epithelial Na channel (ENaC) is the rate-limiting step for Na+ and fluid absorption. Expression of α-ENaC mRNA has been detected in human lung as early as the embryonic stage of development. However, humans express multiple transcripts for α-ENaC, containing differing 5′-untranslated regions (UTR) with unknown effects on protein translation, and different ontogenies for individual transcripts could provide a novel mechanism for developmental regulation of ENaC function. To assess the relative expression of the two most abundant α-ENaC transcripts (α-ENaC1 and α-ENaC2) during lung development, we performed nonradioactive in situ hybridization using probes specific to the alternative 5′-UTRs. Both transcripts were expressed throughout intrauterine lung development (8 to 40 wk gestation), and expression was localized to the surface epithelial cells of the conductive and respiratory airways in both ciliated cells and nonciliated Clara cells. α-ENaC mRNA expression was also identified in the serous cells of the submucosal glands surrounding the proximal airways. In the mature prenatal lung, subsets of alveolar type II (ATII) cells expressed one or both of the α-ENaC transcripts. Our observations demonstrate that a developmentally regulated switch between α-ENaC 5′-UTR variants is not the trigger by which the developing human lung becomes a fluid-absorbing organ at birth, that individual ATII cells express neither, one, or both of the α-ENaC transcripts, and that the overall expression is linked to epithelial cell differentiation and lung maturation.
Modulation of airway inflammation and bacterial clearance by epithelial cell ICAM-1
Authors: Humlicek Alicia L. , Pang Liyi , Look Dwight C.
Published: June 2004

Many cell types in the airway express the adhesive glycoprotein for leukocytes intercellular adhesion molecule-1 (ICAM-1) constitutively and/or in response to inflammatory stimuli. In this study, we identified functions of ICAM-1 on airway epithelial cells in defense against infection with Haemophilus influenzae. Initial experiments using a mouse model of airway infection in which the bacterial inoculum was mixed with agar beads that localize inflammation in airways demonstrated that ICAM-1 expression was required for efficient clearance of H. influenzae. Airway epithelial cell ICAM-1 expression required few or no leukocytes, suggesting that epithelial cells could be activated directly by interaction with bacteria. Specific inhibition of ICAM-1 function on epithelial cells by orotracheal injection of blocking antibodies resulted in decreased leukocyte recruitment and H. influenzae clearance in the airway. Inhibition of endothelial cell ICAM-1 resulted in a similar decrease in leukocyte recruitment but did not affect bacterial clearance, indicating that epithelial cell ICAM-1 had an additional contribution to airway defense independent of effects on leukocyte migration. To assess this possibility, we used an in vitro model of neutrophil phagocytosis of bacteria and observed significantly greater engulfment of bacteria by neutrophils adherent to epithelial cells expressing ICAM-1 compared with nonadherent neutrophils. Furthermore, bacterial phagocytosis and killing by neutrophils after interaction with epithelial cells were decreased when a blocking antibody inhibited ICAM-1 function. The results indicate that epithelial cell ICAM-1 participates in neutrophil recruitment into the airway, but its most important role in clearance of H. influenzae may be assistance with neutrophil-dependent bacterial killing.
Net absorption of IgG via FcRn-mediated transcytosis across rat alveolar epithelial cell monolayers
Authors: Kim Kwang-Jin , Fandy Tamer E. , Lee Vincent H. L. , Ann David K. , Borok Zea , Crandall Edward D.
Published: June 2004

We characterized immunoglobulin G (IgG) transport across rat alveolar epithelial cell monolayers cultured on permeable supports. Unidirectional fluxes of biotin-labeled rat IgG (biot-rIgG) were measured in the apical-to-basolateral ( ab) and opposite ( ba) directions as functions of [rIgG] in upstream fluids at 37 and 4°C. We explored specificity of IgG transport by measuring fluxes in the presence of excess Fc, Fab, F(ab′)2, or chicken Ig (IgY). Expression of the IgG receptor FcRn and the effects of dexamethasone on FcRn expression and biot-rIgG fluxes were determined. Results show that ab flux of biot-rIgG is about fivefold greater than ba flux at an upstream concentration of 25 nM biot-rIgG at 37°C. Both ab and ba fluxes of rIgG saturate, resulting in net absorption with half-maximal concentration and maximal flow of 7.1 nM and 1.3 fmol·cm−2·h−1. At 4°C, both ab and ba fluxes significantly decrease, nearly collapsing net absorption. The presence of excess unlabeled Fc [but not Fab, F(ab′)2, or IgY] significantly reduces biot-rIgG fluxes. RT-PCR demonstrates expression of α- and β-subunits of rat FcRn. Northern analysis further confirms the presence of α-subunit of rat FcRn mRNA of ∼1.6 kb. Dexamethasone exposure for 72 h decreases the steady-state level of mRNA for rat FcRn α-subunit and the ab (but not ba) flux of biot-rIgG. These data indicate that IgG transport across alveolar epithelium takes place via regulable FcRn-mediated transcytosis, which may play an important role in alveolar homeostasis in health and disease.
A “virtual gland” method for quantifying epithelial fluid secretion
Authors: Irokawa Toshiya , Krouse Mauri E. , Joo Nam Soo , Wu Jin V. , Wine Jeffrey J.
Published: June 2004

We developed a new apparatus, the virtual gland (VG), for measuring the rate of fluid secretion ( Jv), its composition, and the transepithelial potential (TEP) in cultured epithelial cells under open circuit. The VG creates a 10-μl chamber above the apical surface of epithelial cells on a Costar filter with a small hole leading to an oil-filled reservoir. After the chamber is primed with a fluid of choice, secreted fluid is forced through the hole into the oil, where it forms a bubble that is monitored optically to determine Jv and collected for analysis. Calu-3 cells were mounted in the VG with a basolateral bath consisting of Krebs-Ringer bicarbonate buffer at 37°C. Basal Jv was 2.7 ± 0.1 μl·cm−2·h−1 ( n = 42), and TEP was −9.2 ± 0.6 mV ( n = 33); both measures were reduced to zero by ouabain ( n = 6). Jv and TEP were stimulated 64 and 59%, respectively, by 5 μM forskolin ( n = 10), 173 and 101% by 1 mM 1-ethyl-2-benzimidazolinone ( n = 5), 213 and 122% by 333 nM thapsigargin ( n = 5), and 520 and 240% by forskolin + thapsigargin ( n = 6). Basal Jv and TEP were inhibited to 82 and 63%, respectively, with 10 μM bumetanide ( n = 5), 71 and 82% with 100 μM acetazolamide ( n = 5), and 47 and 56% with 600 μM glibenclamide ( n = 4). Basal Jv and TEP were 52 and 89% of control values, respectively, after HCO3− replacement with HEPES ( n = 16). The net HCO3− concentration of the secreted fluid was close to that of the bath (25 mM), except when stimulated with forskolin or VIP, when it increased (∼80 mM). These results validate the use of the VG apparatus and provide the first direct measures of Jv in Calu-3 cells.
Surfactant lipid synthesis and lamellar body formation in glycogen-laden type II cells
Authors: Ridsdale Ross , Post Martin
Published: June 2004

Pulmonary surfactant is a lipoprotein complex that functions to reduce surface tension at the air liquid interface in the alveolus of the mature lung. In late gestation glycogen-laden type II cells shift their metabolic program toward the synthesis of surfactant, of which phosphatidylcholine (PC) is by far the most abundant lipid. To investigate the cellular site of surfactant PC synthesis in these cells we determined the subcellular localization of two key enzymes for PC biosynthesis, fatty acid synthase (FAS) and CTP:phosphocholine cytidylyltransferase-α (CCT-α), and compared their localization with that of surfactant storage organelles, the lamellar bodies (LBs), and surfactant proteins (SPs) in fetal mouse lung. Ultrastructural analysis showed that immature and mature LBs were present within the glycogen pools of fetal type II cells. Multivesicular bodies were noted only in the cytoplasm. Immunogold electron microscopy (EM) revealed that the glycogen pools were the prominent cellular sites for FAS and CCT-α. Energy-filtering EM demonstrated that CCT-α bound to phosphorus-rich (phospholipid) structures in the glycogen. SP-B and SP-C, but not SP-A, localized predominantly to the glycogen stores. Collectively, these data suggest that the glycogen stores in fetal type II cells are a cellular site for surfactant PC synthesis and LB formation/maturation consistent with the idea that the glycogen is a unique substrate for surfactant lipids.
Nitric oxide increases IL-8 gene transcription and mRNA stability to enhance IL-8 gene expression in lung epithelial cells
Authors: Sparkman Loretta , Boggaram Vijayakumar
Published: June 2004

Interleukin (IL)-8, a C-X-C chemokine, is a potent chemoattractant and an activator for neutrophils, T cells, and other immune cells. The airway and respiratory epithelia play important roles in the initiation and modulation of inflammatory responses via production of cytokines and surfactant. The association between elevated levels of nitric oxide (NO) and IL-8 in acute lung injury associated with sepsis, acute respiratory distress syndrome, respiratory syncytial virus infection in infants, and other inflammatory diseases suggested that NO may play important roles in the control of IL-8 gene expression in the lung. We investigated the role of NO in the control of IL-8 gene expression in H441 lung epithelial cells. We found that a variety of NO donors significantly induced IL-8 mRNA levels, and the increase in IL-8 mRNA was associated with an increase in IL-8 protein. NO induction of IL-8 mRNA was due to increases in IL-8 gene transcription and mRNA stability. NO induction of IL-8 mRNA levels was not inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and KT-5823, inhibitors of soluble guanylate cyclase and protein kinase G, respectively, and 8-bromo-cGMP did not increase IL-8 mRNA levels. This indicated that NO induces IL-8 mRNA levels independently of changes in the intracellular cGMP levels. NO induction of IL-8 mRNA was significantly reduced by inhibitors of extracellular regulated kinase and protein kinase C. IL-8 induction by NO was also reduced by hydroxyl radical scavengers such as dimethyl sulfoxide and dimethylthiourea, indicating the involvement of hydroxyl radicals in the induction process. NO induction of IL-8 gene expression could be a significant contributing factor in the initiation and induction of inflammatory response in the respiratory epithelium.
Effects of intraduodenal fatty acids on appetite, antropyloroduodenal motility, and plasma CCK and GLP-1 in humans vary with their chain length
Authors: Feltrin Kate L. , Little Tanya J. , Meyer James H. , Horowitz Michael , Smout Andre J. P. M. , Wishart Judith , Pilichiewicz Amelia N. , Rades Thomas , Chapman Ian M. , Feinle-Bisset Christine
Published: June 2004

The gastrointestinal effects of intraluminal fats may be critically dependent on the chain length of fatty acids released during lipolysis. We postulated that intraduodenal administration of lauric acid (12 carbon atoms; C12) would suppress appetite, modulate antropyloroduodenal pressure waves (PWs), and stimulate the release of cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1) more than an identical dose of decanoic acid (10 carbon atoms; C10). Eight healthy males (19–47 yr old) were studied on three occasions in a double-blind, randomized fashion. Appetite perceptions, antropyloroduodenal PWs, and plasma CCK and GLP-1 concentrations were measured during a 90-min intraduodenal infusion of 1) C12, 2) C10, or 3) control (rate: 2 ml/min, 0.375 kcal/min for C12/C10). Energy intake at a buffet meal, immediately after completion of the infusion, was also quantified. C12, but not C10, suppressed appetite perceptions ( P < 0.001) and energy intake (control: 4,604 ± 464 kJ, C10: 4,109 ± 588 kJ, and C12: 1,747 ± 632 kJ; P < 0.001, C12 vs. control/C10). C12, but not C10, also induced nausea ( P < 0.001). C12 stimulated basal pyloric pressures and isolated pyloric PWs and suppressed antral and duodenal PWs compared with control ( P < 0.05 for all). C10 transiently stimulated isolated pyloric PWs ( P = 0.001) and had no effect on antral PWs but markedly stimulated duodenal PWs ( P = 0.004). C12 and C10 increased plasma CCK ( P < 0.001), but the effect of C12 was substantially greater ( P = 0.001); C12 stimulated GLP-1 ( P < 0.05), whereas C10 did not. In conclusion, there are major differences in the effects of intraduodenal C12 and C10, administered at 0.375 kcal/min, on appetite, energy intake, antropyloroduodenal PWs, and gut hormone release in humans.
Vagal cooling and concomitant portal norepinephrine infusion do not reduce net hepatic glucose uptake in conscious dogs
Authors: Cardin Sylvain , Pagliassotti Michael J. , Moore Mary Courtney , Edgerton Dale S. , Lautz Margaret , Farmer Ben , Neal Doss W. , Cherrington Alan D.
Published: June 2004

We examined the role of efferent neural signaling in regulation of net hepatic glucose uptake (NHGU) in two groups of conscious dogs with hollow perfusable coils around their vagus nerves, using tracer and arteriovenous difference techniques. Somatostatin, intraportal insulin and glucagon at fourfold basal and basal rates, and intraportal glucose at 3.8 mg·kg−1·min−1 were infused continuously. From 0 to 90 min [ period 1 ( P1)], the coils were perfused with a 37°C solution. During period 2 [ P2; 90–150 min in group 1 ( n = 3); 90–180 min in group 2 ( n = 6)], the coils were perfused with −15°C solution to eliminate vagal signaling, and the coils were subsequently perfused with 37°C solution during period 3 ( P3). In addition, group 2 received an intraportal infusion of norepinephrine at 16 ng·kg−1·min−1 during P2. The effectiveness of vagal suppression was demonstrated by the increase in heart rate during P2 (111 ± 17, 167 ± 16, and 105 ± 13 beats/min in group 1 and 71 ± 6, 200 ± 11, and 76 ± 6 beats/min in group 2 during P1–P3, respectively) and by prolapse of the third eyelid during P2. Arterial plasma glucose, insulin, and glucagon concentrations; hepatic blood flow; and hepatic glucose load did not change significantly during P1–P3. NHGU during P1-P3 was 2.7 ± 0.4, 4.1 ± 0.6, and 4.0 ± 1.2 mg·kg−1·min−1 in group 1 and 5.0 ± 0.9, 5.6 ± 0.7, and 6.1 ± 0.9 mg·kg−1·min−1 in group 2 (not significant among periods). Interruption of vagal signaling with or without intraportal infusion of norepinephrine to augment sympathetic tone did not suppress NHGU during portal glucose delivery, suggesting the portal signal stimulates NHGU independently of vagal efferent flow.
Involvement of G protein-coupled receptor kinase 4 and 6 in rapid desensitization of dopamine D1receptor in rat IEC-6 intestinal epithelial cells
Authors: Fraga Sónia , Jose Pedro A. , Soares-da-Silva Patrício
Published: June 2004

Dopamine-induced inhibition of Na+-K+-ATPase has been suggested to play a role in the regulation of Na+absorption at the intestinal level, and these effects were mediated by dopamine D1-like receptors. The aim of this work was to evaluate the effect of the activation of the D1-like receptors on the activity of the Na+/H+exchanger (NHE) in the rat intestinal epithelial cell line IEC-6. The presence of D1receptors was confirmed by immunoblotting. The dopamine D1-like receptor agonist SKF-38393 produced a concentration-dependent inhibition of NHE activity and stimulation of adenylyl cyclase (AC), this being antagonized by the D1selective antagonist SKF-83566. Effects of SKF-38393 on NHE and AC activities were maximal at 5 min of exposure to the agonist and rapidly diminished with no effect at 25 min. Exposure of cells for 25 min to dibutyryl-cAMP (0.5 mM) or to the AC activator forskolin (3 μM) effectively inhibited NHE activity. Pretreatment of cells with heparin (1 μM), a nonselective G protein-coupled receptor kinase (GRK) inhibitor, prevented the loss of effects on NHE activity after 25 min exposure to SKF-38393. The presence of GRK4, GRK6A, and GRK6B was confirmed by immunoblotting. Overnight treatment with the anti-GRK4–6 antibody complexed with Lipofectin was also effective in preventing loss of the effects of SKF-38393 on NHE and AC activities. It is concluded that dopamine D1receptors in IEC-6 rapidly desensitize to D1-like agonist stimulation and GRK4 and 6 appear to be involved in agonist-mediated responsiveness and desensitization.
Dietary phosphorus-responsive genes in the intestine, pyloric ceca, and kidney of rainbow trout
Authors: Sugiura Shozo H. , Ferraris Ronaldo P.
Published: June 2004

Identification of phosphorus (P)-responsive genes is important in diagnosing the adequacy of dietary P intake well before clinical symptoms arise. The mRNA abundance of selected genes was determined in the intestine, pyloric ceca, and kidney of rainbow trout fed low-P (LP) or sufficient-P (SP) diet for 2, 5, and 20 days. The LP-to-SP ratio (LP/SP) of mRNA abundance was used to evaluate the difference in gene expression between LP and SP fish, and to compare the response with bone and serum P, which are conventional indicators of P status. The LP/SP of intestinal, cecal, and renal type II sodium-phosphate cotransporter (NaPi-II) mRNA abundance changed from ∼1–2 ( day 2) to ∼1.4–4 ( day 5) and to ∼2–10 ( day 20). The LP/SP of renal NaPi-II, vitamin D 24-hydroxylase, and vitamin D receptor mRNA abundance correlated inversely with serum P on day 5 but not on day 2 and day 20. In another study, differentially expressed genes between LP and SP fish were examined by subtractive hybridization, confirmed by Northern blot, and evaluated by t-test and correlation with serum and bone P concentrations. About 30 genes were identified as dietary P responsive at day 20, including intestinal meprin and cysteinesulfinic acid decarboxylase, renal S100 calcium-binding protein and mitochondrial Pi carrier, and cecal apolipoprotein E, somatomedin B-related protein, and NaPi-II. The LP/SP of mRNA abundance of renal mitochondrial Pi carrier and intestinal cysteinesulfinic acid decarboxylase changed significantly by day 2, and intestinal meprin by day 5. Hence, these genes and NaPi-II are among the earliest steady-response genes capable of predicting P deficiency well before the onset of clinical deficiency.
Human placental taurine transporter in uncomplicated and IUGR pregnancies: cellular localization, protein expression, and regulation
Authors: Roos S. , Powell T. L. , Jansson T.
Published: June 2004

Transplacental transfer is the fetus' primary source of taurine, an essential amino acid during fetal life. In intrauterine growth restriction (IUGR), placental transport capacity of taurine is reduced and fetal taurine levels are decreased. We characterized the protein expression of the taurine transporter (TAUT) in human placenta using immunocytochemistry and Western blotting, tested the hypothesis that placental protein expression of TAUT is reduced in IUGR, and investigated TAUT regulation by measuring the Na+-dependent taurine uptake in primary villous fragments after 1 h of incubation with different effectors. TAUT was primarily localized in the syncytiotrophoblast microvillous plasma membrane (MVM). TAUT was detected as a single 70-kDa band, and MVM TAUT expression was unaltered in IUGR. The PKC activator PMA and the nitric oxide (NO) donor 3-morpholinosydnonimine decreased TAUT activity ( P < 0.05, n = 7–15). However, none of the tested hormones, e.g., leptin and growth hormone, altered TAUT activity significantly. PKC activity measured in MVM from control and IUGR placentas was not different. In conclusion, syncytiotrophoblast TAUT is strongly polarized to the maternal-facing plasma membrane. MVM TAUT expression is unaltered in IUGR, suggesting that the reduced MVM taurine transport in IUGR is due to changes in transporter activity. NO release downregulates placental TAUT activity, and it has previously been shown that IUGR is associated with increased fetoplacental NO levels. NO may therefore play an important role in downregulating MVM TAUT activity in IUGR.
Reduced IP receptors in STZ-induced diabetic rat kidneys and high-glucose-treated mesangial cells
Authors: Nasrallah Rania , Hébert Richard L.
Published: June 2004

Mesangial cells (MG) are an important source of renal PGE2and PGI2. The purpose of this study was to examine the effects of cicaprost (CCP; PGI2analog) on MG function and the expression of IP receptors in streptozotocin (STZ)-diabetic rats and glucose-treated MG cells. CCP increased cellular cAMP in immortalized rat MG cells. Both glucose and anisomycin attenuated CCP-cAMP, but not PMA, angiotensin II, or transforming growth factor-β. Also, IP receptor protein was reduced in response to glucose. While CCP decreased the levels of the cell cycle inhibitor p27, it did not alter thymidine or leucine incorporation. However, CCP reduced fibronectin levels by 40% and increased matrix metalloproteinase-2 levels threefold, a key enzyme in matrix degradation. Finally, IP receptors were significantly reduced in the outer medulla of 4- and 12-wk STZ-diabetic rats and in the cortex, outer, and inner medullary regions in 6-mo uninephrectomized STZ-diabetic rats. The changes in the CCP/IP system observed in this study suggest that IP may serve as an alternate therapeutic target in diabetes.
Angiotensin II directly stimulates activity and alters the phosphorylation of Na-K-ATPase in rat proximal tubule with a rapid time course
Authors: Yingst Douglas R. , Massey Katherine J. , Rossi Noreen F. , Mohanty Madhumita Jena , Mattingly Raymond R.
Published: June 2004

We present evidence that Na-K-ATPase in the rat proximal tubule is directly activated by ANG II much faster than previously observed. Specifically, we show that a 2-min exposure to 0.1 and 1 nM ANG II slowed the rate of intracellular sodium accumulation in response to an increase in extracellular sodium added in the presence of gramicidin D. From these data, we show that ANG II directly stimulates Na-K-ATPase activity at rate-limiting concentrations of intracellular sodium. Under these same conditions, exposing proximal tubules to ANG II altered the amount of32P incorporated into multiple phosphopeptides generated from a tryptic digest of the α-subunit of Na-K-ATPase. Na-K-ATPase was isolated from whole cell lysates by means of a ouabain-affinity column and then separated into its individual subunits by SDS-PAGE. Na-K-ATPase bound to the column in its E2conformation and was eluted by altering its conformation to E1using Na+ATP. Na-K-ATPase isolated from cells treated with ANG II eluted more easily from the ouabain-affinity column than Na-K-ATPase isolated from control cells, suggesting that ANG II decreased the affinity of Na-K-ATPase for ouabain. Thus ANG II rapidly stimulated the activity of Na-K-ATPase in 2 min or less by a mechanism that could involve changes in phosphorylation and conformation of Na-K-ATPase. We suggest that the physiological role for rapid direct activation of Na-K-ATPase is greater control of intracellular sodium during sodium reabsorption.
Partial bladder outlet obstruction abolishes the receptor- and G protein-dependent increase in calcium sensitivity in rabbit bladder smooth muscle
Authors: Stanton Michaela C. , Delaney Daniel , Zderic Stephen A. , Moreland Robert S.
Published: June 2004

Partial bladder outlet obstruction (PBOO) alters the function of the whole bladder and produces specific alterations in the contractility of the bladder smooth muscle cell. The goal of this study was to test the hypothesis that PBOO affects smooth muscle contraction at the level of the receptor- and G protein-dependent increase in myofilament Ca2+sensitivity. To address this question, we used α-toxin-permeabilized strips of bladder smooth muscle from control animals and animals subjected to 2 wk of PBOO. Increasing free [Ca2+] increased force in permeabilized strips from control animals; the addition of 10 μM carbachol and 10 μM GTP increased both the Ca2+sensitivity of the contractions and the maximal levels of force attained. In contrast, although increases in [Ca2+] increased force in permeabilized strips from PBOO animals, the addition of carbachol and GTP had no additional effects. Myosin light chain phosphorylation levels increased with [Ca2+], and although they tended to be higher in strips from PBOO animals, they did not reach statistical significance. Assessment of G protein activity from both animal models suggests this is not a site responsible for the loss of carbachol and GTP enhancement of myofilament Ca2+sensitivity. The addition of phorbol dibutyrate increased the Ca2+sensitivity of force development in strips from both animal models, suggesting that an alteration in PKC signaling is not involved. Our results are consistent with the hypothesis that PBOO decreases receptor-mediated myofilament calcium sensitization and that the site of action is downstream from either the G proteins or PKC.
Inhibition of ENaC by intracellular Cl−in an MDCK clone with high ENaC expression
Authors: Xie Yi , Schafer James A.
Published: June 2004

We examined the effects of intracellular Cl−concentration ([Cl−]i) on the epithelial Na channel (ENaC) in a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) with a high rate of Na+transport produced by stable retroviral transfection with rENaC subunits (Morris RG and Schafer JA. J Gen Physiol 120: 71–85, 2002). Treatment with cAMP (100 μM 8-cpt-cAMP plus 100 μM IBMX) stimulated ENaC-mediated Na+absorption as well as Cl−secretion via cystic fibrosis transmembrane conductance regulator, which was characterized in α-toxin-permeabilized monolayers to have the anion selectivity sequence NO3−> Br−> Cl−> I−. With the use of FL-MDCK monolayers in which the basolateral membrane was permeabilized by nystatin, the ENaC conductance of the apical membrane [determined from the amiloride-sensitive short-circuit current (AS- Isc) driven by an apical-to-basolateral Na+concentration gradient] was progressively inhibited by increasing the [Cl−] in the basolateral solution (and hence in the cytosol), but it was insensitive to the [Cl−] in the apical solution. This inhibitory effect of [Cl−]ioccurred regardless of the presence or absence of net Cl−transport. However, from fluorometric measurements using the Cl−-sensitive dye 6-methoxy- N-(3-sulfopropyl)-quinolinium in intact FL-MDCK monolayers on permeable supports, cAMP, which activates both Na+absorption and Cl−secretion, produced a decrease of [Cl−]ifrom 76 ± 14 to 36 ± 8 mM ( P = 0.03). Thus it might be expected that activation of Cl−secretion by cAMP would lead to stimulation rather than inhibition of ENaC. In the nystatin-treated monolayers, an increase in [Cl−]ifrom 15 to 145 mM decreased AS- Iscfrom 24.5 ± 1.0 to 10.2 ± 1.6 μA/cm2. This inhibition of ENaC could be attributed to nearly proportional decreases in the density of ENaC in the apical membrane from 1.91 ± 0.16 to 1.32 ± 0.17 fmol/cm2and in the intrinsic channel activity (the average current per ENaC subunit) from 13.3 ± 1.2 to 8.2 ± 1.4 μA/fmol.
Hypertonicity increases CLC-5 expression in mouse medullary thick ascending limb cells
Authors: Pham Phuong-Chi , Devuyst Olivier , Pham Phuong-Thu , Matsumoto Naoko , Shih Remi N. G. , Jo Oak D. , Yanagawa Norimoto , Sun Adam M.
Published: June 2004

Genetic studies indicated that mutations of the chloride channel CLC-5 in the kidney are responsible for a group of clinical disorders, collectively called Dent's disease. In the kidney, CLC-5 was found to be expressed in the proximal tubule, medullary thick ascending limb (mTAL) of loop of Henle, and intercalated cells of the collecting tubule. In proximal tubular cells, CLC-5 was found to play an important role in receptor-mediated endocytosis. However, the functional roles of CLC-5 in mTAL and collecting tubules remain unclear. Because mTAL is normally exposed to a hypertonic environment, we aimed to examine the effect of hypertonicity on CLC-5 expression in this nephron segment. Our studies revealed that exposure to hypertonicity (up to 550 mosM) increased CLC-5 mRNA and protein levels in a murine mTAL cell line (MTAL) but not in an opossum kidney (OK) proximal tubular cell line. A similar effect was also found in mouse kidneys, where CLC-5 expression was enhanced in renal medulla, but not cortex, after 48 h of water deprivation. We also tested the effect of hypertonicity on endocytotic activity and found that exposure to hypertonicity caused a significant decrease in cellular uptake of FITC-labeled albumin in OK but not in MTAL cells. Our results suggest that CLC-5 expression is upregulated by hypertonicity in mTAL cells but not in proximal tubular cells. We speculate that the increased CLC-5 levels in mTAL may serve to maintain the endocytotic activity in a hypertonic environment.
Acidosis impairs insulin receptor substrate-1-associated phosphoinositide 3-kinase signaling in muscle cells: consequences on proteolysis
Authors: Franch Harold A. , Raissi Sina , Wang Xiaonan , Zheng Bin , Bailey James L. , Price S. Russ
Published: June 2004

Chronic acidosis is a stimulus for proteolysis in muscle in vivo, but the mechanism of this response is unknown. We tested the hypothesis that acidosis or TNF-α, a cytokine whose production increases in acidosis, regulates proteolysis by inhibiting insulin signaling through phosphoinositide 3-kinase (PI3K). In cultured L6 myotubes, acidified (pH 7.1) media did not accelerate the basal protein degradation rate, but it inhibited insulin’s ability to suppress proteolysis. Insulin receptor substrate-1 (IRS-1)-associated PI3K activity was not altered in cells acidified for 10 min but was strongly inhibited in cells incubated at pH 7.1 for 24 h. Phosphorylation of Akt was also suppressed by acidification for 24 h. Acidification did not induce changes in IRS-1 abundance, insulin-stimulated IRS-1 tyrosine phosphorylation, or the amount of PI3K p85 regulatory subunit. In contrast to acidification, TNF-α suppressed proteolysis in the presence or absence of insulin but had no effect on IRS-1-associated PI3K activity. To establish that the PI3K pathway can regulate protein degradation in muscle, we measured proteolysis in cells after inhibition of PI3K activity with LY-294002 or infection with an adenovirus encoding a dominant negative PI3K p85α-subunit. Both approaches inhibited insulin-induced suppression of proteolysis to a degree similar to that seen with acidification. We conclude that acidosis accelerates protein degradation by impairing insulin signaling through PI3K in muscle cells.
Rapid upregulation of pyruvate dehydrogenase kinase activity in human skeletal muscle during prolonged exercise
Authors: Watt Matthew J. , Heigenhauser George J. F. , LeBlanc Paul J. , Inglis J. Greig , Spriet Lawrence L. , Peters Sandra J.
Published: May 2004

Prolonged moderate-intensity exercise is characterized by a progressive reduction in carbohydrate oxidation and concomitant increase in fat oxidation. Pyruvate dehydrogenase (PDH) controls the entry of pyruvate into oxidative pathways and is a rate-limiting enzyme for carbohydrate metabolism. PDH is controlled by the activities of a kinase (PDK, inhibitory) and phosphatase (stimulatory). To test the hypothesis that increased PDK activity was associated with decreased PDH activity and carbohydrate oxidation during an acute exercise bout, seven recreationally active men completed 4 h of cycle exercise at 55% peak oxygen consumption. Muscle samples were obtained before and at 10 min and 4 h of exercise for the measurement of PDH activity and the extraction of intact mitochondria for the measurements of PDK activity and PDK-2 and PDK-4 protein expression. Carbohydrate oxidation was reduced ( P < 0.05) with exercise duration. Muscle glycogen content was lower ( P ≤ 0.05) at 4 h compared with rest and there was no change in muscle pyruvate content from 10 to 240 min during exercise (10 min: 0.28 ± 0.05; 240 min: 0.35 ± 0.09 mmol/kg dry muscle). PDH activity increased ( P < 0.05) above resting values at 10 min (2.86 ± 0.26 mmol·min−1·kg wet muscle−1), but was lower than 10 min after 4 h (2.23 ± 0.24 mmol·min−1·kg wet muscle−1) of exercise. PDK-2 and PDK-4 protein expression was not different from rest at 10 min and 4 h of exercise. PDK activity at rest averaged 0.081 ± 0.016 min−1, was similar at 10 min, and increased ( P < 0.05) to 0.189 ± 0.013 min−1 at 4 h. Although reduced glycolytic flux may have played a role in decreasing carbohydrate oxidation, the results suggest that increased PDK activity contributed to the reduction in PDH activity and carbohydrate oxidation late in prolonged exercise. The increased PDK activity was independent of changes in intra-mitochondrial effectors, and PDK-2 and PDK-4 protein content, suggesting that it was caused by a change in the specific activity of the existing kinases.
Connexin expression and conducted vasodilation along arteriolar endothelium in mouse skeletal muscle
Authors: Looft-Wilson Robin C. , Payne Geoffrey W. , Segal Steven S.
Published: June 2004

Functional hyperemia requires the coordination of smooth muscle cell relaxation along and between branches of the arteriolar network. Vasodilation is conducted from cell to cell along the arteriolar wall through gap junction channels composed of connexin protein subunits. Within skeletal muscle, it is unclear whether arteriolar endothelium, smooth muscle, or both cell layers provide the cellular pathway for conduction. Furthermore, the constitutive profile of connexin expression within the microcirculation is unknown. We tested the hypothesis that conducted vasodilation and connexin expression are intrinsic to the endothelium of arterioles (17 ± 1 μm diameter) that supply the skeletal muscle fibers in the cremaster of anesthetized C57BL/6 mice. ACh delivered to an arteriole (500 ms, 1-μA pulse; 1-μm micropipette) produced local dilation of 17 ± 1 μm; conducted vasodilation observed 1 mm upstream was 9 ± 1 μm ( n = 5). After light-dye treatment to selectively disrupt endothelium (250-μm segment centered 500 μm upstream, confirmed by loss of local response to ACh while constriction to phenylephrine and dilation to sodium nitroprusside remained intact), we found that conducted vasodilation was nearly abolished (2 ± 1 μm; P < 0.05). Whole-mount immunohistochemistry for connexins revealed punctate labeling at borders of arteriolar endothelial cells, with connexin40 and connexin37 in all branches and connexin43 only in the largest branches. Immunoreactivity for connexins was not apparent in smooth muscle or in capillary or venular endothelium, despite robust immunolabeling for α-actin and platelet endothelial cell adhesion molecule-1, respectively. We conclude that vasodilation is conducted along the endothelium of mouse skeletal muscle arterioles and that connexin40 and connexin37 are the primary connexins forming gap junction channels between arteriolar endothelial cells.
Age-related alterations in NOS and oxidative stress in mesenteric arteries from male and female rats
Authors: Sullivan Jennifer C. , Loomis Elizabeth Dabbs , Collins Matthew , Imig John D. , Inscho Edward W. , Pollock Jennifer S.
Published: June 2004

Epidemiological evidence suggests that advancing age affects the cardiovascular system of men and women differently. The purpose of this study was to determine whether the effects of aging on nitric oxide synthase (NOS), oxidative stress, and vascular function are different in males and females. Mesenteric arteries from young (3 mo) and old (24 mo) male and female Fischer 344/Brown Norway rats were studied. Western blot analysis and NOS activity were performed on the homogenized mesenteric arterial bed separated into cytosolic and membrane-associated fractions. Plasma 8-isoprostane measurements assessed oxidative stress. Vascular reactivity was determined by using a wire myograph in the absence and presence of a NOS inhibitor, Nω-nitro-l-arginine, to examine endothelial function and basal and stimulated nitric oxide release. In additional arteries, reactivity was performed in the presence of polyethylene glycol-SOD to assess the impact of superoxide on vascular function. Among females, aging was associated with a decline in membrane-associated NOS activity and membrane-associated NOS III protein expression. Advancing age in males was associated with increased cytosolic NOS III protein expression. Among both males and females, advancing age resulted in increased oxidative stress. Vascular function was maintained with age in arteries from both males and females, and there was no difference in either basal or stimulated nitric oxide release with age. Despite sex-specific effects of advancing age on the NOS system and increases in markers of oxidative stress, vascular function is maintained in mesenteric arteries from aged Fischer 344/Brown Norway rats. These data suggest that age-related alterations in the resistance vasculature are complex and likely involve multiple compensating vasoactive pathways.
Passive dynamics change leg mechanics for an unexpected surface during human hopping
Authors: Moritz Chet T. , Farley Claire T.
Published: June 2004

Humans running and hopping maintain similar center-of-mass motions, despite large changes in surface stiffness and damping. The goal of this study was to determine the contributions of anticipation and reaction when human hoppers encounter surprise, expected, and random changes from a soft elastic surface (27 kN/m) to a hard surface (411 kN/m). Subjects encountered the expected hard surface on every fourth hop and the random hard surface on an average of 25% of the hops in a trial. When hoppers on a soft surface were surprised by a hard surface, the ankle and knee joints were forced into greater flexion by passive interaction with the hard surface. Within 52 ms after subjects landed on the surprise hard surface, joint flexion increased, and the legs became less stiff than on the soft surface. These mechanical changes occurred before electromyography (EMG) first changed 68–188 ms after landing. Due to the fast mechanical reaction to the surprise hard surface, center-of-mass displacement and average leg stiffness were the same as on expected and random hard surfaces. This similarity is striking because subjects anticipated the expected and random hard surfaces by landing with their knees more flexed. Subjects also anticipated the expected hard surface by increasing the level of EMG by 24–76% during the 50 ms before landing. These results show that passive mechanisms alter leg stiffness for unexpected surface changes before muscle EMG changes and may be critical for adjustments to variable terrain encountered during locomotion in the natural world.
Effect of thermal stress on the vestibulosympathetic reflexes in humans
Authors: Wilson Thad E. , Ray Chester A.
Published: May 2004

Both heat stress and vestibular activation alter autonomic responses; however, the interaction of these two sympathetic activators is unknown. To determine the effect of heat stress on the vestibulosympathetic reflex, eight subjects performed static head-down rotation (HDR) during normothermia and whole body heating. Muscle sympathetic nerve activity (MSNA; peroneal microneurography), mean arterial blood pressure (MAP), heart rate (HR), and internal temperature were measured during the experimental trials. HDR during normothermia caused a significant increase in MSNA (Δ5 ± 1 bursts/min; Δ53 ± 14 arbitrary units/min), whereas no change was observed in MAP, HR, or internal temperature. Whole body heating significantly increased internal temperature (Δ0.9 ± 0.1°C), MSNA (Δ10 ± 3 bursts/min; Δ152 ± 44 arbitrary units/min), and HR (Δ25 ± 6 beats/min), but it did not alter MAP. HDR during whole body heating increased MSNA (Δ16 ± 4 bursts/min; Δ233 ± 90 arbitrary units/min from normothermic baseline), which was not significantly different from the algebraic sum of HDR during normothermia and whole body heating (Δ15 ± 4 bursts/min; Δ205 ± 55 arbitrary units/min). These data suggest that heat stress does not modify the vestibulosympathetic reflex and that both the vestibulosympathetic and thermal reflexes are robust, independent sympathetic nervous system activators.