bluuebunny/crossref_metadata_2025 huggingface dataset:
Glutamine regulates Caco-2 cell tight junction proteins
Authors: Li Nan , Lewis Patricia , Samuelson Don , Liboni Kellym , Neu Josef
Published: May 2004

Intestinal epithelial tight junction (TJ) barrier dysfunction may lead to inflammation and mucosal injury. Glutamine (GLN) plays a role in maintenance of intestinal barrier function in various animal models and critically ill humans. Recent evidence from intestinal cell monolayers indicates that GLN maintains transepithelial resistance and decreases permeability. The mechanisms of these effects remain undefined. We hypothesized that GLN affects proteins involved in the intercellular junctional complex. GLN availability was controlled in Caco-2 monolayers by addition to the medium and treatment with methionine sulfoximine (MSO) to inhibit glutamine synthetase (GS). Expression of TJ proteins, claudin-1, occludin, and zonula occluden (ZO)-1 was measured by immunoblotting. Localization of TJ proteins was evaluated by immunofluorescence light microscopy. Structure of TJ was determined by transmission electron microscopy (TEM). Deprivation of GLN decreased claudin-1, occludin, and ZO-1 protein expression and caused a disappearance of perijunctional claudin-1 and a reduction of occludin but had no effect on ZO-1. TEM revealed that MSO-treated cells in the absence of GLN formed irregular junctional complexes between the apical lateral margins of adjoining cells. These findings indicate that TJ protein expression and cellular localization in Caco-2 cell monolayers rely on GLN. This mechanism may similarly relate to GLN-mediated modulation of intestinal barrier function in stressed animals and humans.
Intracellular signaling pathways involved in Gas6-Axl-mediated survival of endothelial cells
Authors: Hasanbasic Ines , Cuerquis Jessica , Varnum Brian , Blostein Mark D.
Published: May 2004

Gas6 is a γ-carboxylated ligand for the receptor tyrosine kinase Axl. Gas6-Axl interactions can rescue endothelial cells from apoptosis, and this study examined the intracellular signaling mechanisms responsible for this phenomenon. Using flow cytometry, we first confirmed that Gas6 can abrogate apoptosis induced by serum starvation of primary cultures of human umbilical vein endothelial cells (HUVECs). This effect is mediated through phosphorylation of the serine-threonine kinase Akt, with maximal phosphorylation observed after 4 h of treatment with 100 ng/ml Gas6. Inhibition of Akt phosphorylation and abrogation of gas6-mediated survival of HUVECs by wortmannin implicated phosphatidylinositol 3-kinase as the mediator of Akt phosphorylation. Dominant negative Akt constructs largely abrogated the protective effect of Gas6 on HUVECs, underscoring the importance of Akt activation in Gas6-mediated survival. Several downstream regulators of this survival pathway were identified in HUVECs, namely, NF-κB as well as the antiapoptotic and proapoptotic proteins Bcl-2 and caspase 3, respectively. We showed that NF-κB is phosphorylated early after Gas6 treatment as evidenced by doublet formation on Western blotting. As well, the level of Bcl-2 protein increased, supporting the notion that the Bcl-2 antiapoptotic pathway is stimulated. The levels of expression of the caspase 3 activation products p12 and p20 decreased with Gas6 treatment, consistent with a reduction in proapoptotic caspase 3 activation. Taken together, these experiments provide new information about the mechanism underlying Gas6 protection from apoptosis in primary endothelial cell cultures.
Diabetes-related changes in cAMP-dependent protein kinase activity and decrease in relaxation response in rat mesenteric artery
Authors: Matsumoto Takayuki , Wakabayashi Kentaro , Kobayashi Tsuneo , Kamata Katsuo
Published: May 2004

Using superior mesenteric artery rings isolated from age-matched controls and streptozotocin (STZ)-induced diabetic rats, we recently demonstrated that EDHF-type relaxation is impaired in STZ-induced diabetic rats, possibly due to a reduced action of cAMP via increased phosphodiesterase (PDE) activity (Matsumoto T, Kobayashi T, and Kamata K. Am J Physiol Heart Circ Physiol 285: H283–H291, 2003). Here, we investigated the activity and expression of cAMP-dependent protein kinase (PKA), an enzyme that is produced by a pleiotropic and plays key roles in the transduction of many external signals through the cAMP second messenger pathway and in cAMP-mediated vasorelaxation. The relaxation induced by cilostamide, a selective PDE3 inhibitor, was significantly weaker in superior mesenteric artery rings from STZ-induced diabetic rats than in those from age-matched controls. The relaxation responses to 8-bromo-cAMP (8Br-cAMP) and N6, O2-dibutyryl-adenosine-cAMP (db-cAMP), a cell-permeant cAMP analog, were also impaired in the STZ diabetic group. PKA activity in the db-cAMP-treated mesenteric artery was significantly lower in the STZ diabetic group. The expression levels of the mRNA and protein for PKA catalytic subunit Cat-α were significantly decreased in the STZ diabetic group, but those for PKA regulatory subunit isoform RII-β were increased. We conclude that the abnormal vascular relaxation responsiveness seen in STZ-induced diabetic rats may be attributable not only to increased PDE activity but also to decreased PKA activity. Possibly, the decreased PKA activity may result from an inbalance between PKA catalytic and regulatory subunit expressions.
A beneficial role of cardiac P2X4 receptors in heart failure: rescue of the calsequestrin overexpression model of cardiomyopathy
Authors: Yang Alexander , Sonin Dimitry , Jones Larry , Barry William H. , Liang Bruce T.
Published: May 2004

The P2X4 purinergic receptor (P2X4R) is a ligand-gated ion channel. Its activation by extracellular ATP results in Ca2+ influx. Transgenic cardiac overexpression of the human P2X4 receptor showed an in vitro phenotype of enhanced basal contractility. The objective here was to determine the in vivo cardiac physiological role of this receptor. Specifically, we tested the hypothesis that this receptor plays an important role in modulating heart failure progression. Transgenic cardiac overexpression of canine calsequestrin (CSQ) showed hypertrophy, heart failure, and premature death. Crossing the P2X4R mouse with the CSQ mouse more than doubled the lifespan (182 ± 91 days for the binary CSQ/P2X4R mouse, n = 35) of the CSQ mouse (71.3 ± 25.4 days, n = 50, P < 0.0001). The prolonged survival in the binary CSQ/P2X4R mouse was associated with an improved left ventricular weight-to-body weight ratio and a restored β-adrenergic responsiveness. The beneficial phenotype of the binary mouse was not associated with any downregulation of the CSQ level but correlated with improved left ventricular developed pressure and ±dP/d t. The enhanced cardiac performance was manifested in young binary animals and persisted in older animals. The increased contractility likely underlies the survival benefit from P2X4 receptor overexpression. An increased expression or activation of this receptor may represent a new approach in the therapy of heart failure.
A constitutive formulation of arterial mechanics including vascular smooth muscle tone
Authors: Zulliger Martin A. , Rachev Alexander , Stergiopulos Nikos
Published: May 2004

A pseudo-strain energy function (pseudo-SEF) describing the biomechanical properties of large conduit arteries under the influence of vascular smooth muscle (VSM) tone is proposed. In contrast to previous models that include the effects of smooth muscle contraction through generation of an active stress, in this study we consider the vascular muscle as a structural element whose contribution to load bearing is modulated by the contraction. This novel pseudo-SEF models not only arterial mechanics at maximal VSM contraction but also the myogenic contraction of the VSM in response to local increases in stretch. The proposed pseudo-SEF was verified with experimentally obtained pressure-radius curves and zero-stress state configurations from rat carotid arteries displaying distinct differences in VSM tone: arteries from normotensive rats displaying minimal VSM tone and arteries from hypertensive rats exhibiting significant VSM tone. The pressure-radius curves were measured in three different VSM states: fully relaxed, maximally contracted, and normal VSM tone. The model fitted the experimental data very well ( r2> 0.99) in both the normo- and hypertensive groups for all three states of VSM activation. The pseudo-SEF was used to illustrate the localized reduction of circumferential stress in the arterial wall due to normal VSM tone, suggesting that the proposed pseudo-SEF can be of general utility for describing stress distribution not only under passive VSM conditions, as most SEFs proposed so far, but also under physiological and pathological conditions with varying levels of VSM tone.
Increases in CSF [Na+] precede the increases in blood pressure in Dahl S rats and SHR on a high-salt diet
Authors: Huang Bing S. , Van Vliet Bruce N. , Leenen Frans H. H.
Published: May 2004

In Dahl salt-sensitive (S) and salt-resistant (R) rats, and spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats, at 5–6 wk of age, a cannula was placed in the cisterna magna, and cerebrospinal fluid (CSF) was withdrawn continuously at 75 μl/12 h. CSF was collected as day- and nighttime samples from rats on a regular salt intake (0.6% Na+; R-Na) and then on a high salt intake (8% Na+; H-Na). In separate groups of rats, the abdominal aorta was cannulated and blood pressure (BP) and heart rate (HR) measured at 10 AM and 10 PM, with rats first on R-Na and then on H-Na. On H-Na, CSF [Na+] started to increase in the daytime of day 2 in Dahl S rats and of day 3 in SHR. BP and HR did not rise until day 3 in Dahl S rats and day 4 in SHR. In Dahl R and WKY rats, high salt did not change CSF [Na+], BP, or HR. In a third set of Dahl S rats, sampling of both CSF and BP was performed in each individual rat. Again, significant increases in CSF [Na+] were observed 1–2 days earlier than the increases in BP and HR. In a fourth set of Dahl S rats, BP and HR were recorded continuously by means of radiotelemetry for 5 days on R-Na and 8 days on H-Na. On H-Na, BP (but not HR) increased first in the nighttime of day 2. In another set of Dahl S rats, intracerebroventricular infusion of antibody Fab fragments binding ouabain-like compounds (OLC) with high affinity prevented the increase in BP and HR by H-Na but further increased CSF [Na+]. Finally, in Wistar rats on H-Na, intracerebroventricular infusion of ouabain increased BP and HR but decreased CSF [Na+]. Thus, in both Dahl S and SHR on H-Na, increases in CSF [Na+] preceded the increases in BP and HR, consistent with a primary role of increased CSF [Na+] in the salt-induced hypertension. An increase in brain OLC in response to the initial increase in CSF [Na+] appears to attenuate further increases in CSF [Na+] but at the “expense” of sympathoexcitation and hypertension.
Effect of shear stress on microvessel network formation of endothelial cells with in vitro three-dimensional model
Authors: Ueda Akinori , Koga Masaki , Ikeda Mariko , Kudo Susumu , Tanishita Kazuo
Published: May 2004

Shear stress stimulus is expected to enhance angiogenesis, the formation of microvessels. We determined the effect of shear stress stimulus on three-dimensional microvessel formation in vitro. Bovine pulmonary microvascular endothelial cells were seeded onto collagen gels with basic fibroblast growth factor to make a microvessel formation model. We observed this model in detail using phase-contrast microscopy, confocal laser scanning microscopy, and electron microscopy. The results show that cells invaded the collagen gel and reconstructed the tubular structures, containing a clearly defined lumen consisting of multiple cells. The model was placed in a parallel-plate flow chamber. A laminar shear stress of 0.3 Pa was applied to the surfaces of the cells for 48 h. Promotion of microvessel network formation was detectable after ∼10 h in the flow chamber. After 48 h, the length of networks exposed to shear stress was 6.17 (±0.59) times longer than at the initial state, whereas the length of networks not exposed to shear stress was only 3.30 (±0.41) times longer. The number of bifurcations and endpoints increased for networks exposed to shear stress, whereas the number of bifurcations alone increased for networks not exposed to shear stress. These results demonstrate that shear stress applied to the surfaces of endothelial cells on collagen gel promotes the growth of microvessel network formation in the gel and expands the network because of repeated bifurcation and elongation.
Inotropic effects of ETBreceptor stimulation and their modulation by endocardial endothelium, NO, and prostaglandins
Authors: Leite-Moreira Adelino F. , Brás-Silva Carmen
Published: May 2004

Endothelin (ET)-1 acts on ETAand ETBreceptors. The latter include ETB1(endothelial) and ETB2(muscular) subtypes, which mediate opposite effects on vascular tone. This study investigated, in rabbit papillary muscles ( n = 84), the myocardial effects of ETBstimulation. ET-1 (10−9M) was given in the absence or presence of BQ-123 (ETAantagonist). The effects of IRL-1620 (ETB1agonist, 10−10–10−6M) or sarafotoxin S6c (ETBagonist, 10−10–10−6M) were evaluated in muscles with intact or damaged endocardial endothelium (EE); intact EE, in the presence of NG-nitro-l-arginine (l-NNA); and intact EE, in the presence of indomethacin (Indo). Sarafotoxin S6c effects were also studied in the presence of BQ-788 (ETB2antagonist). ET-1 alone increased 64 ± 18% active tension (AT) but decreased it by 4 ± 2% in the presence of BQ-123. In muscles with intact EE, sarafotoxin S6c alone did not significantly alter myocardial performance. Sarafotoxin S6c (10−6M) increased, however, AT by 120 ± 27% when EE was damaged and by 39 ± 8% or 23 ± 6% in the presence of l-NNA or Indo, respectively. In the presence of BQ-788, sarafotoxin S6c decreased AT (21 ± 3% at 10−6M) in muscles with intact EE, an effect that was abolished when EE was damaged. IRL-1620 also decreased AT (22 ± 3% at 10−6M) in muscles with intact EE, an effect that was abolished when EE was damaged or in the presence of l-NNA or Indo. In conclusion, the ETB-mediated negative inotropic effect is presumably due to ETB1stimulation, requires an intact EE, and is mediated by NO and prostaglandins, whereas the ETB-mediated positive inotropic effect, observed when EE was damaged or NO and prostaglandins synthesis inhibited, is presumably due to ETB2stimulation.
Myocardial insulin resistance associated with chronic hypertriglyceridemia and increased FFA levels in Type 2 diabetic patients
Authors: Monti Lucilla D. , Landoni Claudio , Setola Emanuela , Galluccio Elena , Lucotti Pietro , Sandoli Emilia P. , Origgi Anna , Lucignani Giovanni , Piatti PierMarco , Fazio Ferruccio
Published: May 2004

We evaluated the influence of chronic hypertriglyceridemia and endothelial dysfunction on myocardial glucose uptake (MGU) in Type 2 diabetic patients without coronary heart disease. Patients were divided into two groups according to fasting triglyceride (TG) levels: 5.4 ± 1.1 and 1.5 ± 0.3 mmol/l for high- and normal-TG groups, respectively. Five subjects were assigned to the high-TG group and 11 to the normal-TG group. Age, gender, body mass index, systolic and diastolic blood pressure, glucose, insulin, HbA1c, cholesterol, and HDL cholesterol were similar in the two groups, whereas free fatty acid (FFA) levels were higher in the high-TG group basally and at the end of the clamp. Furthermore, five healthy subjects were subjected to the same protocol and used as the control group. MGU was assessed by using 18F-labeled 2-fluoro-2-deoxy-d-glucose under hyperglycemic-hyperinsulinemic conditions. Basal endothelin-1 and nitric oxide levels were significantly higher in the high-TG group than in the normal-TG and control groups, and cGMP and maximal postischemic vasodilation were significantly decreased in the high-TG group compared with the normal-TG and control groups. However, significant alterations were found in the same parameters in the normal-TG group compared with the control group. By the end of the hyperglycemic clamp, in the high-TG group, MGU was ∼40 and 65% of that in the normal-TG and control groups. MGU negatively correlated with TG, FFA, and endothelin-1, whereas a positive correlation was found with cGMP and maximal postischemic vasodilation. In conclusion, increased TG and FFA levels are risks, in addition to Type 2 diabetes mellitus, for myocardial insulin resistance, endothelial dysfunction, and alteration of nitric oxide/cGMP levels.
17β-Estradiol inhibits cyclic strain-induced endothelin-1 gene expression within vascular endothelial cells
Authors: Juan Shu-Hui , Chen Jin-Jer , Chen Cheng-Hsien , Lin Heng , Cheng Ching-Feng , Liu Ju-Chi , Hsieh Ming-Hsiung , Chen Yen-Ling , Chao Hung-Hsing , Chen Tso-Hsiao , Chan Paul , Cheng Tzu-Hurng
Published: May 2004

It has been well documented previously that 17β-estradiol (E2) exerts a protective effect on cardiovascular tissue. The possible role of E2in the regulation of endothelin (ET)-1 production has been previously reported, although the complex mechanisms by which E2inhibits ET-1 expression are not completely understood. The aims of this study were to examine whether E2was able to alter strain-induced ET-1 gene expression and also to identify the putative underlying signaling pathways that exist within endothelial cells. For cultured endothelial cells, E2(1–100 nM), but not 17α-estradiol, inhibited the level of strain-induced ET-1 gene expression and also peptide secretion. This inhibitory effect elicited by E2was able to be prevented by the coincubation of endothelial cells with the estrogen receptor antagonist ICI-182,780 (1 μM). E2also inhibited strain-enhanced NADPH oxidase activity and intracellular reactive oxygen species (ROS) generation as measured by the redox-sensitive fluorescent dye 2′,7′-dichlorofluorescin diacetate and the level of extracellular signal-regulated kinase (ERK) phosphorylation. Furthermore, the presence of E2and antioxidants such as N-acetylcysteine and diphenylene iodonium were able to elicit a decrease in the level of strain-induced ET-1 secretion, ET-1 promoter activity, ET-1 mRNA, ERK phosphorylation, and activator protein-1 binding activity. In summary, we demonstrated, for the first time, that E2inhibits strain-induced ET-1 gene expression, partially by interfering with the ERK pathway via the attenuation of strain-induced ROS generation. Thus this study delivers important new insight regarding the molecular pathways that may contribute to the proposed beneficial effects of estrogen on the cardiovascular system.
Systems analysis of digoxin kinetics and inotropic response in the rat heart: effects of calcium and KB-R7943
Authors: Weiss Michael , Baek Myoungki , Kang Wonku
Published: May 2004

To gain more insight into the mechanistic processes controlling the kinetics of inotropic response of digoxin in the perfused whole heart, an integrated kinetic model was developed incorporating digoxin uptake, receptor binding (Na+-K+-ATPase inhibition), and cellular events linking receptor occupation and response. The model was applied to data obtained in the single-pass Langendorff-perfused rat heart for external [Ca2+] of 0.5 and 1.5 mM under control conditions and in the presence of the reverse-mode Na+/Ca2+ exchange inhibitor KB-R7943 (0.1 μM) in perfusate. Outflow concentration and left ventricular developed pressure data measured for three consecutive doses (15, 30, and 45 μg) in each heart were analyzed simultaneously. While disposition kinetics of digoxin was determined by interaction with a heterogeneous receptor population consisting of a high-affinity/low-capacity and a low-affinity/high- capacity binding site, response generation was >80% mediated by binding to the high-affinity receptor. Digoxin sensitivity increased at lower external [Ca2+] due to higher stimulus amplification. Coadministration of KB-R7943 significantly reduced the positive inotropic effect of digoxin at higher doses (30 and 45 μg) and led to a saturated and delayed receptor occupancy-response relationship in the cellular effectuation model. The results provide further evidence for the functional heterogeneity of the Na+-K+-ATPase and suggest that in the presence of KB-R7943 a reduction of the Ca2+ influx rate via the reverse mode Na+/Ca2+ exchanger might become the limiting factor in digoxin response generation.
Smad and p38-MAPK signaling mediates apoptotic effects of transforming growth factor-β1 in human airway epithelial cells
Authors: Undevia Nidhi S. , Dorscheid Delbert R. , Marroquin Bertha A. , Gugliotta Wendy L. , Tse Roberta , White Steven R.
Published: May 2004

Transforming growth factor-β1 (TGF-β1) belongs to a family of multifunctional cytokines that regulate a variety of biological processes, including cell differentiation, proliferation, and apoptosis. The effects of TGF-β1 are cell context and cell cycle specific and may be signaled through several pathways. We examined the effect of TGF-β1 on apoptosis of primary human central airway epithelial cells and cell lines. TGF-β1 protected human airway epithelial cells from apoptosis induced by either activation of the Fas death receptor (CD95) or by corticosteroids. This protective effect was blocked by inhibition of the Smad pathway via overexpression of inhibitory Smad7. The protective effect is associated with an increase in the cyclin-dependent kinase inhibitor p21 and was blocked by the overexpression of key gatekeeper cyclins for the G1/S interface, cyclins D1 and E. Blockade of the Smad pathway by overexpression of the inhibitory Smad7 permitted demonstration of a TGF-β-mediated proapoptotic pathway. This proapoptotic effect was blocked by inhibition of the p38 MAPK kinase signaling with the inhibitor SB-203580 and was associated with an increase in p38 activity as measured by a kinase assay. Here we demonstrate dual signaling pathways involving TGF-β1, an antiapoptotic pathway mediated by the Smad pathway involving p21, and an apoptosis-permissive pathway mediated in part by p38 MAPK.
Prevention and reversal of pulmonary inflammation and airway hyperresponsiveness by dexamethasone treatment in a murine model of asthma induced by house dust
Authors: Kim Jiyoun , McKinley Laura , Siddiqui Javed , Bolgos Gerry L. , Remick Daniel G.
Published: May 2004

The morbidity and mortality from asthma in the Western world have increased 75% in the past 20 years. Recent studies have demonstrated that sensitization to cockroach allergens correlates strongly with the increased asthma morbidity for adults and children. We investigated whether dexamethasone administered before or after allergen challenge would inhibit the pulmonary inflammation and airway hyperresponsiveness in a mouse model of asthma induced by a house dust extract with high levels of cockroach allergens. For the prevention experiment, mice were treated with an intraperitoneal injection of dexamethasone 1 h before each pulmonary challenge, and airway hyperresponsiveness was measured 24 h after the last challenge. Mice were killed 48 h after the last challenge. For the reversal study, airway hyperresponsiveness was measured 24 h after the last challenge, and the mice were treated with dexamethasone. Dexamethasone treatment before allergen challenge significantly reduced the pulmonary recruitment of inflammatory cells, myeloperoxidase activity in the lung, airway hyperreactivity, and total serum IgE levels compared with PBS-treated mice. Additionally, dexamethasone treatment could significantly reduce the airway hyperreactivity of an established asthmatic response. These results demonstrate that dexamethasone not only prevents but also halts the asthmatic response induced by house dust containing cockroach allergens. This model exhibits several features of human asthma that may be exploited in the study of pathophysiological mechanisms and potential therapeutic interventions.
Amylin receptor blockade stimulates food intake in rats
Authors: Reidelberger Roger D. , Haver Alvin C. , Arnelo Urban , Smith D. David , Schaffert Courtney S. , Permert Johan
Published: May 2004

Amylin is postulated to act as a hormonal signal from the pancreas to the brain to inhibit food intake and regulate energy reserves. Amylin potently reduces food intake, body weight, and adiposity when administered systemically or into the brain. Whether selective blockade of endogenous amylin action increases food intake and adiposity remains to be clearly established. In the present study, the amylin receptor antagonist acetyl-[Asn30, Tyr32] sCT-( 8 – 32 ) (AC187) was used to assess whether action of endogenous amylin is essential for normal satiation to occur. Non-food-deprived rats received a 3- to 4-h intravenous infusion of AC187 (60–2,000 pmol·kg−1·min−1), either alone or coadministered with a 3-h intravenous infusion of amylin (2.5 or 5 pmol·kg−1·min−1) or a 2-h intragastric infusion of an elemental liquid diet (4 kcal/h). Infusions began just before dark onset. Food intake and meal patterns during the first 4 h of the dark period were determined from continuous computer recordings of changes in food bowl weight. Amylin inhibited food intake by ∼50%, and AC187 attenuated this response by ∼50%. AC187 dose-dependently stimulated food intake (maximal increases from 76 to 171%), whether administered alone or with an intragastric infusion of liquid diet. Amylin reduced mean meal size and meal frequency, AC187 attenuated these responses, and AC187 administration alone increased mean meal size and meal frequency. These results support the hypothesis that endogenous amylin plays an essential role in reducing meal size and increasing the postmeal interval of satiety.
Fluorescein-methotrexate transport in rat choroid plexus analyzed using confocal microscopy
Authors: Breen Christopher M. , Sykes Destiny B. , Baehr Carsten , Fricker Gert , Miller David S.
Published: May 2004

One function of the vertebrate choroid plexus (CP) is removal of potentially toxic metabolites and xenobiotics from cerebrospinal fluid (CSF) to blood for subsequent excretion in urine and bile. We have used confocal microscopy and quantitative image analysis to follow transport of the large organic anion fluorescein-methotrexate (FL-MTX) from bath (CSF side) to blood vessels in intact rat CP and found concentrative transport from CSF to blood. With 2 μM FL-MTX in the bath, steady-state fluorescence in the subepithelium and vascular spaces exceeded bath levels by 5- to 10-fold, but fluorescence in epithelial cells was below bath levels. FL-MTX accumulation in subepithelium and vascular spaces was reduced by NaCN, Na removal, and by other organic anions, e.g., MTX, probenecid, and estrone sulfate. Increasing medium K 10-fold had no effect. None of these treatments affected cellular accumulation. However, two observations indicated that apical FL-MTX uptake was indeed mediated: first, cellular accumulation was a saturable function of medium substrate concentration; and second, digoxin and MK-571 reduced FL-MTX accumulation in the subepithelial/vascular spaces but also increased cellular accumulation severalfold. In the presence of digoxin and MK-571, cellular accumulation was concentrative, specific, and Na dependent. Thus transepithelial FL-MTX transport involved the following two mediated steps: Na-dependent uptake at the apical membrane and electroneutral efflux at the basolateral membrane, possibly on Oatp2 and Mrp1.
Response of descending vasa recta to luminal pressure
Authors: Zhang Zhong , Pallone Thomas L.
Published: May 2004

We tested whether luminal perfusion and pressurization induce an endothelial cytoplasmic Ca2+([Ca2+]CYT) response in descending vasa recta (DVR). DVR isolated from the rat outer medulla were cannulated and subjected to free-flow microperfusion (5 nl/min); the onset of which increased [Ca2+]CYTfrom a baseline of 76 ± 13 to 221 ± 65 nM. A graded increase in luminal pressure from 0 to 45 mmHg in stopped-flow experiments induced a parallel increase in [Ca2+]CYTfrom a baseline of 74 ± 24 to 194 ± 33 nM at 45 mmHg, with a tendency for [Ca2+]CYTto plateau at pressures >25 mmHg. The removal of extracellular Ca2+and blockade by either La3+(10 μM) or SKF-96365 (100 μM) eliminated the response. Luminal pressurization to 25 mmHg increased nitric oxide (NO) generation, a response blocked by NO synthase inhibition or removal of extracellular Ca2+. The NO generation was not affected by the superoxide dismutase mimetic tempol. We conclude that DVR endothelia are mechanosensitive and respond to luminal pressure by elevating [Ca2+]CYTand generating NO. That response might augment medullary perfusion and saliuresis.
Passive stretch inhibits central corelike lesion formation in the soleus muscles of hindlimb-suspended unloaded rats
Authors: Baewer D. V. , Hoffman M. , Romatowski J. G. , Bain J. L. W. , Fitts R. H. , Riley D. A.
Published: May 2004

Hindlimb suspension unloading (HSU) is a ground-based model simulating the effects of microgravity unloading on the musculoskeletal system. In this model, gravity causes the hind foot of the rat to drop, opening the front of the ankle to 90–105° plantar flexion at rest. As HSU proceeds, the normal weight-bearing angle of 30° dorsiflexion is achieved progressively less, and the contraction range of soleus is abbreviated. Our laboratory reported that 12 days of HSU caused central corelike lesions (CCLs) of myofibril breakdown (Riley DA, Slocum GR, Bain JL, Sedlak FR, Sowa TE, and Mellender JW. J Appl Physiol. 69: 58–66, 1990). The present study investigated whether daily stretch of the calf muscles prevents CCL formation. The soleus muscles of HSU Sprague-Dawley male rats (∼287 g) were lengthened by unilateral ankle splinting at 30°. Compared with the nonsplinted side, splinting for 10 or 20 min per day in awake rats significantly decreased CCLs in soleus by 88 and 91%, respectively ( P < 0.01). Compared with control muscle wet weight, 20-min splinting reduced atrophy by 33%, whereas 10-min splinting ameliorated atrophy by 17% ( P < 0.01). Bilateral soleus electromyograph recording revealed higher levels of contractile activity on the splinted side during splinting. To isolate the effects of stretch from isometric contractile activity, contractions were eliminated by whole animal anesthesia with isoflurane during 10-min daily splinting. The percentage of fibers with CCLs was reduced by 57%, and the average lesion size was 29% smaller in the stretched muscle ( P < 0.05). Soleus muscle wet weight and fiber area were unaltered by stretch alone. Loaded contractions during splinting are necessary to prevent muscle fiber atrophy. Passive muscle stretch acts to maintain myofibril structural integrity.
Effects of insulin resistance on substrate utilization during exercise in overweight women
Authors: Braun Barry , Sharoff Carrie , Chipkin Stuart R. , Beaudoin Francesca
Published: May 2004

During exercise, obese individuals oxidize less glycogen and more fat than their lean counterparts, but the shift in substrate use may be mediated by insulin resistance rather than body fat per se. In addition, individuals with Type 2 diabetes are not resistant to contraction-mediated glucose uptake during exercise, but in vivo studies uncomplicated by hyperglycemia are lacking. The purpose of this study was to compare blood glucose uptake and the balance between carbohydrate and fat utilization during exercise in insulin-resistant (IR) and insulin-sensitive (IS) women of equivalent body fatness and maximal oxygen consumption (V̇o2 max). Twelve overweight sedentary women were divided into two groups with similar body mass index (IR = 28.5 ± 1.6, IS = 27.5 ± 1.9), lean mass (IR = 42.4 ± 1.8 kg, IS = 41.5 ± 1.9 kg), and V̇o2 max (IR = 29.7 ± 3.5 ml·kg−1·min−1, IS = 30.7 ± 3.9 ml·kg−1·min−1) but a markedly different composite insulin sensitivity index (IR = 3.0 ± 0.7, IS = 7.7 ± 0.9). Blood glucose kinetics and substrate oxidation were assessed by stable isotope dilution and indirect calorimetry during 50 min of treadmill walking at 45% V̇o2 max. Total carbohydrate oxidation and estimated muscle glycogen use were significantly lower in the IR group. Blood glucose uptake was the same in the IR and IS groups. These data suggest that insulin resistance, independent of body fat, spares muscle glycogen and shifts substrate oxidation toward less carbohydrate use during exercise. Insulin-resistant individuals with normoglycemia appear to have no defect in blood glucose uptake during exercise.
Low-frequency depression of tension in the cat gastrocnemius muscle after eccentric exercise
Authors: Parikh S. , Morgan D. L. , Gregory J. E. , Proske U.
Published: May 2004

Subjecting a muscle to a series of eccentric contractions in which the contracting muscle is lengthened results in a number of changes in its mechanical properties. These include a fall in isometric tension that is particularly pronounced during low-frequency stimulation, a phenomenon known as low-frequency depression (LFD). Reports of LFD have not taken into account the shift in optimum length for active tension generation to longer muscle lengths that takes place after eccentric contractions. Given the length dependence of the stimulation frequency-tension curve, we tested the hypothesis that the change in this relationship after eccentric exercise is due to the shift in optimum length. We measured LFD by recording tension in response to a linearly increasing rate of stimulation of the nerve to medial gastrocnemius of anesthetized cats, over the range 0–100 pulses per second. Tension responses were measured before and after 50 eccentric contractions consisting of 6-mm stretches starting at 3 mm below optimum length and finishing at 3 mm above it. An index of LFD was derived from the tension responses to ramp stimulation. It was found that LFD after the eccentric contractions was partly, but not entirely, due to changes in the muscle's optimum length. An additional factor was the effect of fatigue. These observations led to the conclusion that the muscle length dependence of LFD was reduced by eccentric contractions. All of this means that after eccentric exercise the tension deficit at low rates of muscle activation is likely to be less severe than first thought.
Decreased exhaled nitric oxide as a marker of postinsult immune paralysis
Authors: Attalah Habiba L. , Honoré Stéphanie , Eddahibi Saadia , Marcos Elisabeth , Soussy Claude-James , Adnot Serge , Delclaux Christophe
Published: May 2004

Nitric oxide (NO) regulates neutrophil migration and alveolar macrophage functions such as cytokine synthesis and bacterial killing, both of which are impaired in immune paralysis associated with critical illness. The aim of this study was to determine whether NO is involved in immune paralysis and whether exhaled NO measurement could help to monitor pulmonary defenses. NO production (protein expression, enzyme activity, end products, and exhaled NO measurements) was assessed in rats after cecal ligation and puncture to induce a mild peritonitis (leading to ∼20% mortality rate). An early and sustained decrease in exhaled NO was found after peritonitis (from 1 to 72 h) compared with healthy rats [median (25th–75th percentile), 1.5 parts per billion (ppb) (1.2–1.7) vs. 4.0 ppb (3.6–4.3), P < 0.05], despite increased NO synthase-2 and unchanged NO synthase-3 protein expression in lung tissue. NO synthase-2 activity was decreased in lung tissue. Nitrites and nitrates in supernatants of isolated alveolar macrophages decreased after peritonitis compared with healthy rats, and an inhibitory experiment suggested arginase overactivity in alveolar macrophages bypassing the NO substrate. Administration of the NO synthase-2 inhibitor aminoguanidine to healthy animals reproduced the decreased neutrophil migration toward alveolar spaces that was observed after peritonitis, but l-arginine administration after peritonitis failed to correct the defect of neutrophil emigration despite increasing exhaled NO compared with d-arginine administration [4.8 (3.9–5.7) vs. 1.6 (1.3–1.7) ppb, respectively, P < 0.05]. In conclusion, the decrease in exhaled NO observed after mild peritonitis could serve as a marker for lung immunodepression.
Facilitation of the diaphragm response to transcranial magnetic stimulation by increases in human respiratory drive
Authors: Straus C. , Locher C. , Zelter M. , Derenne J.-P. , Similowski T.
Published: May 2004

The human respiratory neural drive has an automatic component (bulbospinal pathway) and a volitional component (corticospinal pathway). The aim of this study was to assess the effects of a hypercapnia-induced increase in the automatic respiratory drive on the function of the diaphragmatic corticospinal pathway as independently as possible of any other influence. Thirteen healthy volunteers breathed room air and then 5 and 7% hyperoxic CO2. Cervical (cms) and transcranial (tms) magnetic stimulations were performed during early inspiration and expiration. Transdiaphragmatic pressure (Pdi) and surface electromyogram of the diaphragm (DiEMG) and of the abductor pollicis brevis (apbEMG) were recorded in response to cms and tms. During inspiration, Pdi,cms was unaffected by CO2, but Pdi,tms increased significantly with 7% CO2. During expiration, Pdi,cms was significantly reduced by CO2, whereas Pdi,tms was preserved. DiEMG,tms latencies decreased significantly during early inspiration and expiration (air vs. 5% CO2and air vs. 7% CO2). DiEMG,tms amplitude increased significantly in response to early expiration-tms (air vs. 5% CO2and air vs. 7% CO2) but not in response to early inspiration-tms. DiEMG,cms latencies and amplitudes were not affected by CO2whereas 7% CO2significantly increased the apbEMG,cms latency. The apbEMG,tms vs. apbEMG,cms latency difference was unaffected by CO2. In conclusion, increasing the automatic drive to breathe facilitates the response of the diaphragm to tms, during both inspiration and expiration. This could allow the corticospinal drive to breathe to keep the capacity to modulate respiration in conditions under which the automatic respiratory control is stimulated.
Expression of MHC-β and MCT1 in cardiac muscle after exercise training in myocardial-infarcted rats
Authors: Hashimoto Takeshi , Kambara Naoshige , Nohara Ryuji , Yazawa Masayuki , Taguchi Sadayoshi
Published: May 2004

To evaluate the hypothesis that increasing the potential for glycolytic metabolism would benefit the functioning of infarcted myocardium, we investigated whether mild exercise training would increase the activities of oxidative enzymes, expression of carbohydrate-related transport proteins (monocarboxylate transporter MCT1 and glucose transporter GLUT4), and myosin heavy chain (MHC) isoforms. Myocardial infarction (MI) was produced by occluding the proximal left coronary artery in rat hearts for 30 min. After the rats performed 6 wk of run training on a treadmill, the wall of the left ventricle was dissected and divided into the anterior wall (AW; infarcted region) and posterior wall (PW; noninfarcted region). MI impaired citrate synthase and 3-hydroxyacyl-CoA dehydrogenase activities in the AW ( P < 0.01) but not in the noninfarcted PW. No differences in the expression of MCT1 were found in either tissues of AW and PW after MI, whereas exercise training significantly increased the MCT1 expression in all conditions, except AW in the MI rats. Exercise training resulted in an increased expression of GLUT4 protein in the AW in the sham rats and in the PW in the MI rats. The relative amount of MHC-β was significantly increased in the AW and PW in MI rats compared with sham rats. However, exercise training resulted in a significant increase of MHC-α expression in both AW and PW in both sham and MI rats ( P < 0.01). These findings suggest that mild exercise training enhanced the potential for glycolytic metabolism and ATPase activity of the myocardium, even in the MI rats, ensuring a beneficial role in the remodeling of the heart.
Skeletal muscle oxidative metabolism in sedentary humans: 31P-MRS assessment of O2 supply and demand limitations
Authors: Haseler Luke J. , Lin Alexander P. , Richardson Russell S.
Published: May 2004

Previously, it was demonstrated in exercise-trained humans that phosphocreatine (PCr) recovery is significantly altered by fraction of inspired O2 (FiO2), suggesting that in this population under normoxic conditions, O2 availability limits maximal oxidative rate. Haseler LJ, Hogan ML, and Richardson RS. J Appl Physiol 86: 2013–2018, 1999. To further elucidate these population-specific limitations to metabolic rate, we used 31P-magnetic resonance spectroscopy to study the exercising human gastrocnemius muscle under conditions of varied FiO2 in sedentary subjects. To test the hypothesis that PCr recovery from submaximal exercise in sedentary subjects is not limited by O2 availability, but rather by their mitochondrial capacity, six sedentary subjects performed three bouts of 6-min steady-state submaximal plantar flexion exercise followed by 5 min of recovery while breathing three different FiO2 (0.10, 0.21, and 1.00). PCr recovery time constants were significantly longer in hypoxia (47.0 ± 3.2 s), but there was no difference between hyperoxia (31.8 ± 1.9 s) and normoxia (30.0 ± 2.1 s) (mean ± SE). End-exercise pH was not significantly different across treatments. These results suggest that the maximal muscle oxidative rate of these sedentary subjects, unlike their exercise-trained counterparts, is limited by mitochondrial capacity and not O2 availability in normoxia. Additionally, the significant elongation of PCr recovery in these subjects in hypoxia illustrates the reliance on O2 supply at the other end of the O2 availability spectrum in both sedentary and active populations.
In Vivo Mammalian Brain Imaging Using One- and Two-Photon Fluorescence Microendoscopy
Authors: Jung Juergen C. , Mehta Amit D. , Aksay Emre , Stepnoski Raymond , Schnitzer Mark J.
Published: May 2004

One of the major limitations in the current set of techniques available to neuroscientists is a dearth of methods for imaging individual cells deep within the brains of live animals. To overcome this limitation, we developed two forms of minimally invasive fluorescence microendoscopy and tested their abilities to image cells in vivo. Both one- and two-photon fluorescence microendoscopy are based on compound gradient refractive index (GRIN) lenses that are 350–1,000 μm in diameter and provide micron-scale resolution. One-photon microendoscopy allows full-frame images to be viewed by eye or with a camera, and is well suited to fast frame-rate imaging. Two-photon microendoscopy is a laser-scanning modality that provides optical sectioning deep within tissue. Using in vivo microendoscopy we acquired video-rate movies of thalamic and CA1 hippocampal red blood cell dynamics and still-frame images of CA1 neurons and dendrites in anesthetized rats and mice. Microendoscopy will help meet the growing demand for in vivo cellular imaging created by the rapid emergence of new synthetic and genetically encoded fluorophores that can be used to label specific brain areas or cell classes.
Purkinje Cell Spike Firing in the Posterolateral Cerebellum: Correlation With Visual Stimulus, Oculomotor Response, and Error Feedback
Authors: Norris Scott A. , Greger Bradley , Hathaway Emily N. , Thach W. Thomas
Published: May 2004

Complex (CS)- and simple-spike (SS) discharge from single Purkinje cells (Pc) in the posterolateral cerebellum of two monkeys was recorded during a visually guided reach-touch task. A visual target appeared (TA) off-gaze at a random location on a screen. On initiation of arm reach, the target disappeared, then reappeared (TR) after a fixed delay. TR was either at the same location (baseline condition) or a shifted location at a fixed distance and direction from TA location (shift condition). Across trials, we observed one or two peaks of CS activity, depending on the reach condition. The first CS (T1 CS) peak was tuned to the location of TA on the screen, following TA by ∼150 ms. The second CS (T2 CS) peak occurred only in the shift condition, was tuned to the shift location of TR, and followed TR by ∼150 ms. The locational preferences of T1 and T2 CS peaks were the same. T1 and T2 CSs preceded saccades to TA and TR at the preferred location and occurred during reaches with either arm. T1 CSs occurred during trials in which the target appeared, and there was a saccade to target, but no subsequent arm reach followed. SS firing varied with TA/TR in the same preferred location as for the accompanying CS. We conclude that posterolateral Pc CS and SS firing changes following an off-gaze visual target appearance in a preferred location when there is a subsequent saccade to that location.
Cardiac transcriptional response to acute and chronic angiotensin II treatments
Authors: Larkin Jennie E. , Frank Bryan C. , Gaspard Renee M. , Duka Irena , Gavras Haralambos , Quackenbush John
Published: May 2004

Exposure of experimental animals to increased angiotensin II (ANG II) induces hypertension associated with cardiac hypertrophy, inflammation, and myocardial necrosis and fibrosis. Some of the most effective antihypertensive treatments are those that antagonize ANG II. We investigated cardiac gene expression in response to acute (24 h) and chronic (14 day) infusion of ANG II in mice; 24-h treatment induces hypertension, and 14-day treatment induces hypertension and extensive cardiac hypertrophy and necrosis. For genes differentially expressed in response to ANG II treatment, we tested for significant regulation of pathways, based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Microarray Pathway Profiler (GenMAPP) databases, as well as functional classes based on Gene Ontology (GO) terms. Both acute and chronic ANG II treatments resulted in decreased expression of mitochondrial metabolic genes, notably those for the electron transport chain and Krebs-TCA cycle; chronic ANG II treatment also resulted in decreased expression of genes involved in fatty acid metabolism. In contrast, genes involved in protein translation and ribosomal activity increased expression following both acute and chronic ANG II treatments. Some classes of genes showed differential response between acute and chronic ANG II treatments. Acute treatment increased expression of genes involved in oxidative stress and amino acid metabolism, whereas chronic treatments increased cytoskeletal and extracellular matrix genes, second messenger cascades responsive to ANG II, and amyloidosis genes. Although a functional linkage between Alzheimer disease, hypertension, and high cholesterol has been previously documented in studies of brain tissue, this is the first demonstration of induction of Alzheimer disease pathways by hypertension in heart tissue. This study provides the most comprehensive available survey of gene expression changes in response to acute and chronic ANG II treatment, verifying results from disparate studies, and suggests mechanisms that provide novel insight into the etiology of hypertensive heart disease and possible therapeutic interventions that may help to mitigate its effects.