bluuebunny/crossref_metadata_2025 huggingface dataset:
MEF2 activation in differentiated primary human skeletal muscle cultures requires coordinated involvement of parallel pathways
Authors: Al-Khalili Lubna , Chibalin Alexander V. , Yu Mei , Sjödin Bertil , Nylén Carolina , Zierath Juleen R , Krook Anna
Published: February 2004

The myocyte enhancer factor (MEF)2 transcription factor is important for development of differentiated skeletal muscle. We investigated the regulation of MEF2 DNA binding in differentiated primary human skeletal muscle cells and isolated rat skeletal muscle after exposure to various stimuli. MEF2 DNA binding activity in nonstimulated (basal) muscle cultures was almost undetectable. Exposure of cells for 20 min to 120 nM insulin, 0.1 and 1.0 mM hydrogen peroxide, osmotic stress (400 mM mannitol), or 1.0 mM 5-aminoimidazole-4-carboxamide-1-β- d-ribofuranoside (AICAR) led to a profound increase in MEF2 DNA binding. To study signaling pathways mediating MEF2 activity, we preincubated human skeletal muscle cell cultures or isolated rat epitrochlearis muscles with inhibitors of p38 mitogen-activated protein kinase (MAPK) (10 μM SB-203580), MEK1 (50 μM PD-98059), PKC (1 and 10 μM GF109203X), phosphatidylinositol (PI) 3-kinase (10 μM LY-294002), or AMP-activated protein kinase (AMPK; 20 μM compound C). All stimuli resulted primarily in activation of MEF2D DNA binding. Exposure of cells to osmotic or oxidative stress increased MEF2 DNA binding via pathways that were completely blocked by MAPK inhibitors and partially blocked by inhibitors of PKC, PI 3-kinase, and AMPK. In epitrochlearis muscle, MAPK inhibitors blocked contraction but not AICAR-mediated MEF2 DNA binding. Thus activation of MEF2 in skeletal muscle is regulated via parallel intracellular signaling pathways in response to insulin, cellular stress, or activation of AMPK.
Osmotic swelling-provoked release of organic osmolytes in human intestinal epithelial cells
Authors: Tomassen Sebastian F. B. , Fekkes Durk , de Jonge Hugo R. , Tilly Ben C.
Published: February 2004

Human Intestine 407 cells respond to osmotic cell swelling by the activation of Cl−- and K+-selective ionic channels, as well as by stimulating an organic osmolyte release pathway readily permeable to taurine and phosphocholine. Unlike the activation of volume-regulated anion channels (VRAC), activation of the organic osmolyte release pathway shows a lag time of ∼30–60 s, and its activity persists for at least 8–12 min. In contrast to VRAC activation, stimulation of organic osmolyte release did not require protein tyrosine phosphorylation, active p21rho, or phosphatidylinositol 3-kinase activity and was insensitive to Cl− channel blockers. Treatment of the cells with putative organic anion transporter inhibitors reduced the release of taurine only partially or was found to be ineffective. The efflux was blocked by a subclass of organic cation transporter (OCT) inhibitors (cyanine-863 and decynium-22) but not by other OCT inhibitors (cimetidine, quinine, and verapamil). Brief treatment of the cells with phorbol esters potentiated the cell swelling-induced taurine efflux, whereas addition of the protein kinase C (PKC) inhibitor GF109203X largely inhibited the response, suggesting that PKC is involved. Increasing the level of intracellular Ca2+ by using A-23187- or Ca2+-mobilizing hormones, however, did not affect the magnitude of the response. Taken together, the results indicate that the hypotonicity-induced efflux of organic osmolytes is independent of VRAC and involves a PKC-dependent step.
Immunocytochemical localization of Na+-HCO3− cotransporters and carbonic anhydrase dependence of fluid transport in corneal endothelial cells
Authors: Diecke Friedrich P. J. , Wen Quan , Sanchez Jose M. , Kuang Kunyan , Fischbarg Jorge
Published: February 2004

In corneal endothelium, there is evidence for basolateral entry of HCO3− into corneal endothelial cells via Na+-HCO3− cotransporter (NBC) proteins and for net HCO3− flux from the basolateral to the apical side. However, how HCO3− exits the cells through the apical membrane is unclear. We determined that cultured corneal endothelial cells transport HCO3− similarly to fresh tissue. In addition, Cl− channel inhibitors decreased fluid transport by at most 16%, and inhibition of membrane-bound carbonic anhydrase IV by benzolamide or dextran-bound sulfonamide decreased fluid transport by at most 29%. Therefore, more than half of the fluid transport cannot be accounted for by anion transport through apical Cl− channels, CO2 diffusion across the apical membrane, or a combination of these two mechanisms. However, immunocytochemistry using optical sectioning by confocal microscopy and cryosections revealed the presence of NBC transporters in both the basolateral and apical cell membranes of cultured bovine corneal endothelial cells and freshly isolated rabbit endothelia. This newly detected presence of an apical NBC transporter is consistent with its being the missing mechanism sought. We discuss discrepancies with other reports and provide a model that accounts for the experimental observations by assuming different stoichiometries of the NBC transport proteins at the basolateral and apical sides of the cells. Such functional differences might arise either from the expression of different isoforms or from regulatory factors affecting the stoichiometry of a single isoform.
Reductions in circulating anabolic hormones induced by sustained sleep deprivation in rats
Authors: Everson Carol A. , Crowley William R.
Published: February 2004

The main systemic disorders resulting from prolonged sleep deprivation in laboratory animals are a negative energy balance, low circulating thyroid hormones, and host defense impairments. Low thyroid hormones previously have been found caused by altered regulation at the level of the hypothalamus with possible pituitary involvement. The present studies investigated the effects of sleep deprivation on other major anabolic hormonal systems. Plasma growth hormone (GH) concentrations and major secretory bursts were characterized. Insulin-like growth factor I (IGF-I) was evaluated as an integrative marker of peripheral GH effector activity. Prolactin (PRL) was assessed by basal concentrations and by stimulating the pituitary with exogenous thyrotropin-releasing hormone. Leptin was studied for its linkage to metabolic signs of sleep loss and its correspondence to altered neuroendocrine regulation in other disease states. Last, plasma corticosterone was measured to investigate the degree of hypothalamic-pituitary-adrenal activation. Sleep deprivation was produced by the disk-over-water method, a well-established means of selective deprivation of sleep and noninterference with normal waking behaviors. Hormone concentrations were determined in sham comparisons and at intervals during baseline and experimental periods lasting at least 15 days in partially and totally sleep-deprived rats. The results indicate that high-amplitude pulses of GH were nearly abolished and that concentrations of GH, IGF-I, PRL, and leptin all were suppressed by sleep deprivation. Corticosterone concentration was relatively unaffected. Features of these results, such as low GH and low IGF-I, indicate failed negative feedback and point to hypothalamic mechanisms as containing the foci responsible for peripheral signs.
The Na+/H+exchanger isoform 2 is the predominant NHE isoform in murine colonic crypts and its lack causes NHE3 upregulation
Authors: Bachmann O. , Riederer B. , Rossmann H. , Groos S. , Schultheis P. J. , Shull G. E. , Gregor M. , Manns M. P. , Seidler U.
Published: February 2004

The Na+/H+exchanger isoform NHE2 is highly expressed in the intestinal tract, but its physiological role has remained obscure. The aim of this study was to define its expression, location, and regulatory properties in murine colon and to look for the compensatory changes in NHE2 (−/−) colon that allow normal histology and absorptive function. To this end, we measured murine proximal colonic surface and crypt cell NHE1, NHE2, and NHE3 expression levels, transport rates in response to acid, hyperosmolarity and cAMP in murine proximal colonic crypts, as well as changes in transcript levels and acid-activated NHE activity in NHE2 (−/−) crypts. We found that NHE2 was expressed most abundantly in crypts, NHE1 equally in crypts and surface cells, and NHE3 much stronger in surface cells. NHE2, like NHE1, was activated by low intracellular pH (pHi), hyperosmolarity, and cAMP, whereas NHE3 was activated only by low pHi. Crypts isolated from NHE2 (−/−) mice displayed increased acid-activated NHE1- and NHE3-attributable Na+/H+exchange activity, no change in NHE1 expression, and NHE3 expression levels twice as high as in normal littermates. No change in cellular ultrastructure was found in NHE2 (−/−) colon. Our results demonstrate high NHE2 expression in the crypts and suggest a role for NHE2 in cryptal pHiand volume homeostasis.
Proapoptotic function of protein kinase CK2α" is mediated by a JNK signaling cascade
Authors: Hilgard Philip , Czaja Mark J. , Gerken Guido , Stockert Richard J.
Published: February 2004

Protein kinase CK2 (formerly casein kinase II) is a tetrameric enzyme constitutively expressed in all eurakyotic tissues that plays a significant role in the regulation of cell proliferation, malignant transformation, and apoptosis. The catalytic α-subunit of the enzyme is known to exist in three isoforms CK2α, CK2α′ and CK2α". CK2α" is highly expressed in liver compared with other tissues and is required for the normal trafficking of several hepatocellular membrane proteins. Initial studies of dengue virus infection indicated that the CK2α"-deficient membrane trafficking mutant cell line (Trf1) was resistant to virus-induced cell death compared with the parental human hepatoma (HuH)-7 hepatoma line. Expression of recombinant CK2α" in Trf1 was capable of reverting this resistant phenotype. This study was extended to TNF-α in addition to other stimuli of cell death in an attempt to uncover common death pathways that might be modulated by CK2α". Evaluation of different pathways involved in death signaling suggest that the regulation of a critical proapoptotic step in HuH-7 cells by CK2α" is mediated by a JNK signaling cascade.
Glucagon-like peptide-2 induces intestinal adaptation in parenterally fed rats with short bowel syndrome
Authors: Martin Gary R. , Wallace Laurie E. , Sigalet David L.
Published: February 2004

Glucagon-like peptide-2 (GLP-2) is an intestinal trophic enteroendocrine peptide that is associated with intestinal adaptation following resection. Herein, we investigate the effects of GLP-2 in a total parenteral nutrition (TPN)-supported model of experimental short bowel syndrome. Juvenile Sprague-Dawley rats underwent a 90% small intestinal resection and jugular catheter insertion. Rats were randomized to three groups: enteral diet and intravenous saline infusion, TPN only, or TPN + 10 μg·kg−1·h−1GLP-2. Nutritional maintenance was isocaloric and isonitrogenous. After 7 days, intestinal permeability was assessed by quantifying the urinary recovery of gavaged carbohydrate probes. The following day, animals were euthanized, and intestinal tissue was processed for morphological and crypt cell proliferation (CCP) analysis, apoptosis (caspase-3), and expression of SGLT-1 and GLUT-5 transport proteins. TPN plus GLP-2 treatment resulted in increased bowel and body weight, villus height, intestinal mucosal surface area, CCP, and reduced intestinal permeability compared with the TPN alone animals ( P < 0.05). GLP-2 treatment induced increases in serum GLP-2 levels and intestinal SGLT-1 expression ( P < 0.01) compared with either TPN or enteral groups. No differences were seen in the villus apoptotic index between resection groups. Enterally fed resected animals had a significant decrease in crypt apoptotic indexes compared with nontreated animals. This study demonstrates that GLP-2 alone, without enteral feeding, stimulates indexes of intestinal adaptation. Secondly, villus hypertrophy associated with adaptation was predominantly due to an increase in CCP and not to changes in apoptotic rates. Further studies are warranted to establish the mechanisms of action and therapeutic potential of GLP-2.
Localized transient increases in endothelial cell Ca2+in arterioles in situ: implications for coordination of vascular function
Authors: Duza Tasmia , Sarelius Ingrid H.
Published: February 2004

Intracellular Ca2+transients were identified in endothelial cells (ECs) in intact blood-perfused arterioles. ECs in cremaster muscle arterioles (diameter ∼45 μm) in anesthetized mice were loaded with the Ca2+indicator fluo 4-AM by intraluminal perfusion, after which blood flow was reestablished. Confocal microscopy was used to visualize Ca2+as a function of fluo-4 intensity in real time. Separate sets of experiments were performed under the following conditions: control, ischemia, during inhibition of P2xor P1purinoreceptors, and with the application of exogenous adenosine. In controls, spontaneous EC Ca2+transients displayed a wide range of activity frequency (1–32 events/min) and about one-third of these transient events were synchronized between adjacent ECs. The increase in Ca2+remained localized and did not spread to encompass the entire cell body. Ca2+transient activity decreased significantly with ischemia (from 9.9 ± 0.6 to 3.1 ± 0.3 events/min, n = 135) but was unaffected by P2xor P1receptor inhibition. Exogenous adenosine significantly increased the frequency of Ca2+transients (to 12.8 ± 0.9 events/min) and increased synchronization so that 50% of all Ca2+events were synchronized between ECs. This response to adenosine was not due to an increase in shear stress. These data indicate that localized Ca2+transients are sensitive to flow conditions and, separately, to metabolically active pathways (exogenous adenosine), although the basal activity occurs independently of P2xor P1receptors. These transients may represent a mechanism by which individual EC responses are integrated to result in coordinated arteriolar responses in situ.
Remodeling of resistance arteries in organoid culture is modulated by pressure and pressure pulsation and depends on vasomotion
Authors: Bakker Erik N. T. P. , Sorop Oana , Spaan Jos A. E. , VanBavel Ed
Published: February 2004

The hypothesis was tested that pressure and pressure pulsation modulate vascular remodeling. Arterioles (∼200 μm lumen diameter) were dissected from rat cremaster muscle and studied in organoid culture. In the first series, arterioles were kept at a stable pressure level of either 50 or 100 mmHg for 3 days. Both groups showed a progressive increase in myogenic tone during the experiment. Arterioles kept at 50 mmHg showed larger endothelium-dependent dilation, compared with vessels kept at 100 mmHg on day 3. Remodeling, as indicated by the reduction in maximally dilated diameter at 100 mmHg, was larger in arterioles kept at 50 mmHg compared with 100 mmHg: 34 ± 4.5 versus 10 ± 4.8 μm ( P < 0.05). In the second series, arterioles were subjected to a stable pressure of 60 mmHg or oscillating pressure of 60 ± 10 mmHg (1.5 Hz) for 4 days. Pressure pulsation induced partial dilation and was associated with less remodeling: 34 ± 4.0 versus 19 ± 4.5 μm ( P < 0.01) for stable pressure versus oscillating pressure. Vasomotion was frequently observed in all groups, and inward remodeling was larger in vessels with vasomotion: 30 ± 2.5 μm compared with vessels that did not exhibit vasomotion: 8.0 ± 5.0 μm ( P < 0.01). In conclusion, these results indicate that remodeling is not enhanced by high pressure. Pressure pulsation causes partial dilation and reduces inward remodeling. The appearance of vasomotion is associated with enhanced inward remodeling.
Endogenous β3-adrenoreceptor activation contributes to left ventricular and cardiomyocyte dysfunction in heart failure
Authors: Morimoto Atsushi , Hasegawa Hiroshi , Cheng Heng-Jie , Little William C. , Cheng Che-Ping
Published: February 2004

The objective of the present study was to test the hypothesis that endogenous β3-adrenoreceptor (AR) activation contributes to left ventricular (LV) and cardiomyocyte dysfunction in heart failure (CHF). Stimulation of the β3-AR inhibits cardiac contraction. In the failing myocardium, β3-ARs are upregulated, suggesting that stimulation of β3-ARs may contribute to depressed cardiac performance in CHF. We assessed the functional significance of endogenous β3-AR activation in 10 conscious dogs before and after pacing-induced CHF. Under normal conditions, L-748,337, a specific β3-AR antagonist, produced a mild increase in LV contractile performance assessed by the slope ( Ees) of the LV pressure-volume relation (18%, 6.2 ± 0.9 vs. 7.3 ± 1.2 mmHg/ml, P < 0.05) and the improved LV relaxation time constant (τ; 28.4 ± 1.9 vs. 26.8 ± 1.0 ms, P < 0.05). After CHF, the plasma norepinephrine concentration increased eightfold, and L-748,337 produced a larger increase in Ees (34%, 3.8 ± 0.7 vs. 5.1 ± 0.8 mmHg/ml, P < 0.05) and a greater decrease in τ (46.4 ± 4.2 vs. 41.0 ± 3.9 ms, P < 0.05). Similar responses were observed in isolated myocytes harvested from LV biopsies before and after CHF. In the normal myocyte, L-748,337 did not cause significant changes in contraction or relengthening. In contrast, in CHF myocytes, L-748,337 produced significant increases in contraction (5.8 ± 0.9 vs. 6.8 ± 0.9%, P < 0.05) and relengthening (33.5 ± 4.2 vs. 39.7 ± 4.0 μm/s, P < 0.05). The L-748,337-induced myocyte response was associated with improved intracellular Ca2+ concentration regulation. In CHF myocytes, nadolol caused a decrease in contraction and relengthening, and adding isoproterenol to nadolol caused a further depression of myocyte function. Stimulation of β3-AR by endogenous catecholamine contributes to the depression of LV contraction and relaxation in CHF.
Protein phosphatase 2A-mediated cross-talk between p38 MAPK and ERK in apoptosis of cardiac myocytes
Authors: Liu Qinghang , Hofmann Polly A.
Published: February 2004

Mitogen-activated protein kinases (MAPKs) play different regulatory roles in signaling oxidative stress-induced apoptosis in cardiac ventricular myocytes. The regulation and functional role of cross-talk between p38 MAPK and extracellular signal-regulated kinase (ERK) pathways were investigated in cardiac ventricular myocytes in the present study. We demonstrated that inhibition of p38 MAPK with SB-203580 and SB-239063 enhanced H2O2-stimulated ERK phosphorylation, whereas preactivation of p38 MAPK with sodium arsenite reduced H2O2-stimulated ERK phosphorylation. In addition, pretreatment of cells with the protein phosphatase 2A (PP2A) inhibitors okadaic acid and fostriecin increased basal and H2O2-stimulated ERK phosphorylation. We also found that PP2A coimmunoprecipitated with ERK and MAPK/ERK (MEK) in cardiac ventricular myocytes, and H2O2increased the ERK-associated PP2A activity that was blocked by inhibition of p38 MAPK. Finally, H2O2-induced apoptosis was attenuated by p38 MAPK or PP2A inhibition, whereas it was enhanced by MEK inhibition. Thus the present study demonstrated that p38 MAPK activation decreases H2O2-induced ERK activation through a PP2A-dependent mechanism in cardiac ventricular myocytes. This represents a novel cellular mechanism that allows for interaction of two opposing MAPK pathways and fine modulation of apoptosis during oxidative stress.
Ascorbic acid increases cardiovagal baroreflex sensitivity in healthy older men
Authors: Monahan Kevin D. , Eskurza Iratxe , Seals Douglas R.
Published: February 2004

Cardiovagal baroreflex sensitivity (BRS) declines with advancing age in healthy men. We tested the hypothesis that oxidative stress contributes mechanistically to this age-associated reduction. Eight young (23 ± 1 yrs, means ± SE) and seven older (63 ± 3) healthy men were studied. Cardiovagal BRS was assessed using the modified Oxford technique (bolus infusion of 50–100 μg sodium nitroprusside, followed 60 s later by a 100- to 150-μg bolus of phenylephrine hydrochloride) in triplicate at baseline and during acute intravenous ascorbic acid infusion. At baseline, cardiovagal BRS (slope of the linear portion of the R-R interval-systolic blood pressure relation during pharmacological changes in arterial blood pressure) was 56% lower ( P < 0.01) in older (8.3 ± 1.6 ms/mmHg) compared with young (19.0 ± 3.1 ms/mmHg) men. Ascorbic acid infusion increased plasma concentrations similarly in young (62 ± 9 vs. 1,249 ± 72 μmol/l for baseline and during ascorbic acid; P < 0.05) and older men (62 ± 4 vs. 1,022 ± 55 μmol/l; P < 0.05) without affecting baseline blood pressure, heart rate, carotid artery compliance, or the magnitude of change in systolic blood pressure in response to bolus sodium nitroprusside and phenylephrine hydrochloride infusion. Ascorbic acid (vitamin C) infusion increased cardiovagal BRS in older (Δ58 ± 16%; P < 0.01), but not younger (Δ − 4 ± 4%) men. These data provide experimental support for the concept that oxidative stress contributes mechanistically to age-associated reductions in cardiovagal BRS in healthy men.
Comparison of simultaneous measurement of mouse systolic arterial blood pressure by radiotelemetry and tail-cuff methods
Authors: Whitesall Steven E. , Hoff Janet B. , Vollmer Alan P. , D'Alecy Louis G.
Published: February 2004

Radiotelemetry of mouse blood pressure accurately monitors systolic pressure, diastolic pressure, heart rate, and locomotor activity but requires surgical implantation. Noninvasive measurements of indirect systolic blood pressure have long been available for larger rodents and now are being reported more frequently for mice. This study compared mouse systolic arterial blood pressure measurements using implanted radiotelemetry pressure transducer with simultaneous tail-cuff measurements in the same unanesthetized mice. The pressure range for comparison was extended by inducing experimental hypertension or by observations of circadian elevations between 3 AM and 6 AM. Both trained and untrained tail-cuff operators used both instruments. Every effort was made to follow recommended manufacturer's instructions. With the initial flow-based tail-cuff instrument, we made 671 comparisons (89 sessions) and found the slope of the linear regression to be 0.118, suggesting poor agreement. In an independent assessment, 277 comparisons (35 sessions) of radiotelemetry measurements with the pulse based tail-cuff instrument were made. The slope of the linear regression of the simultaneous measurements of systolic pressures was 0.98, suggesting agreement. Bland-Altman analysis also supported our interpretation of the linear regression. Thus although reliable systolic pressure measurements are possible with either tail-cuff or radiotelemetry techniques, in our hands some tail-cuff instruments fail to accurately detect elevated blood pressures. These data, however, do not distinguish whether this instrument-specific tail-cuff failure was due to operator or instrument inadequacies. We strongly advise investigators to obtain an independent and simultaneous validation of tail-cuff determinations of mouse blood pressure before making critical genotyping determinations.
Extracellular norepinephrine reduces neuronal uptake of norepinephrine by oxidative stress in PC12 cells
Authors: Mao Weike , Qin Fuzhong , Iwai Chikao , Vulapalli Raju , Keng Peter C. , Liang Chang-seng
Published: February 2004

Cardiac norepinephrine (NE) uptake activity is reduced in congestive heart failure. Our studies in intact animals suggest that this effect on the cardiac sympathetic nerve endings is caused by oxidative stress and/or NE toxic metabolites derived from NE. In this study, we investigated the direct effects of NE on neuronal NE uptake activity and NE transporter (NET), using undifferentiated PC12 cells. Cells were incubated with NE (1–500 μM) either alone or in combination of Cu2+sulfate (1 μM), which promotes free radical formation by Fenton reaction for 24 h. NE uptake activity was measured using [3H]NE. Cell viability was determined with the use of Trypan blue exclusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay, and cellular oxidative stress by dichlorodihydrofluorescein fluorescence and the GSH/GSSG ratio. Cell viability was reduced by NE >100 μM. At lower doses, NE produced oxidative stress and a dose-dependent reduction of NE uptake activity without affecting cell viability significantly. Cu2+, which has no direct effect on NE uptake activity, potentiated oxidative stress and reduction of NE uptake activity produced by NE. This decrease of NE uptake activity was associated with reductions of NE uptake binding sites and NET protein expression by using the radioligand assay and Western blot analysis, but no changes in NET gene expression. In addition, the free-radical scavenger mannitol, and antioxidant enzymes superoxide dismutase and catalase, reduced oxidative stress and attenuated the reductions of NE uptake activity and NET protein produced by NE/Cu. Thus our results support a functional role of oxidative stress in mediating the neuronal NE uptake reducing effect of NE and that this effect of NE on NET is a posttranscriptional event.
PKC activates BKCachannels in rat pulmonary arterial smooth muscle via cGMP-dependent protein kinase
Authors: Barman Scott A. , Zhu Shu , White Richard E.
Published: February 2004

Normally, signaling mechanisms that activate large-conductance, calcium- and voltage-activated potassium (BKCa) channels in pulmonary vascular smooth muscle cause pulmonary vasodilatation. BKCa-channel modulation is important in the regulation of pulmonary arterial pressure, and inhibition (decrease in the opening probability) of the BKCachannel has been implicated in the development of pulmonary vasoconstriction. Protein kinase C (PKC) causes pulmonary vasoconstriction, but little is known about the effect of PKC on BKCa-channel activity in pulmonary vascular smooth muscle. Accordingly, studies were done to determine the effect of PKC on BKCa-channel activity using patch-clamp studies in pulmonary arterial smooth muscle cells (PASMCs) of the Sprague-Dawley rat. The PKC activators phorbol myristate acetate (PMA) and thymeleatoxin opened BKCachannels in single Sprague-Dawley rat PASMC. The activator response to both PMA and thymeleatoxin on BKCa-channel activity was blocked by Gö-6983, which selectively blocks PKC-α, -δ, -γ, and -ζ, and by rottlerin, which selectively inhibits PKC-δ. In addition, the specific cyclic GMP-dependent protein kinase antagonist KT-5823 blocked the responses to PMA and thymelatoxin, whereas the specific cyclic AMP-dependent protein kinase blocker KT-5720 had no effect. In isolated pulmonary arterial vessels, both PMA and forskolin caused vasodilatation, which was inhibited by KT-5823, Gö-6983, or the BKCa-channel blocker tetraethylammonium. The results of this study indicate that activation of specific PKC isozymes increases BKCa-channel activity in Sprague-Dawley rat PASMC via cyclic GMP-dependent protein kinase, which suggests a unique signaling mechanism for vasodilatation.
Heparin affects signaling pathways stimulated by fibroblast growth factor-1 and -2 in type II cells
Authors: Newman Donna R. , Li Cheng-Ming , Simmons Rebecca , Khosla Jody , Sannes Philip L.
Published: February 2004

Undersulfation of the basement membrane matrix of alveolar type II (AT2) cells compared with that of neighboring type I cells is believed to account for some of the known morphological and functional differences between these pneumocytes. Heparin, a model for sulfated components of basement membrane matrices, is known to inhibit fibroblast growth factor (FGF)-2-stimulated DNA synthesis as well as gene expression of FGF-2 and its receptor in AT2 cells. To determine whether these end points result from specific effects of heparin on FGF-related signaling pathways, isolated rat AT2 cells were treated with 100 ng/ml FGF-1 or FGF-2 in the presence of up to 500 μg/ml heparin. In addition, experiments were done on cells grown in the presence of 20 mM sodium chlorate (sulfation inhibitor). High-dose heparin reduced FGF-1- or FGF-2-stimulated phosphorylation of mitogen-activated protein kinase kinases (MEK1/2), p44/42 mitogen-activated protein kinases (MAPK/ERK1/2), stress-activated protein kinase/c-Jun NH2-terminal kinase, Akt/protein kinase B, and p90RSK. FGF-2-stimulated signaling was more sensitive to heparin's effects than was signaling stimulated by FGF-1. Heparin had an additive effect on the reduced [3H]thymidine incorporation in FGF-2-treated AT2 cells caused by inhibition of the MEK/ERK pathway by the MEK inhibitor PD-98059. The data suggest that heparin's known capacity to alter DNA synthesis and, possibly, other biological end points is realized via cross talk between multiple signaling pathways.
Roles for early response cytokines duringEscherichia colipneumonia revealed by mice with combined deficiencies of all signaling receptors for TNF and IL-1
Authors: Mizgerd Joseph P. , Lupa Michal M. , Hjoberg Josephine , Vallone Joseph C. , Warren Henry B. , Butler James P. , Silverman Eric S.
Published: February 2004

During infection, inflammation is essential for host defense, but it can injure tissues and compromise organ function. TNF-α and IL-1 (α and β) are early response cytokines that facilitate inflammation. To determine the roles of these cytokines with overlapping functions, we generated mice deficient in all of the three receptors mediating their effects (TNFR1, TNFR2, and IL-1RI). During Escherichia coli pneumonia, receptor deficiency decreased neutrophil recruitment and edema accumulation to half of the levels observed in wild-type mice. Thus these receptors contributed to maximal responses, but substantial inflammation progressed independently of them. Receptor deficiency compromised antibacterial efficacy for some infectious doses. Decreased ventilation during E. coli pneumonia was not affected by receptor deficiency. However, the loss of lung compliance during pneumonia was substantially attenuated by receptor deficiency. Thus during E. coli pneumonia in mice, the lack of signaling from TNF-α and IL-1 decreases inflammation and preserves lung compliance.
Preoptic α1- and α2-noradrenergic agonists induce, respectively, PGE2-independent and PGE2-dependent hyperthermic responses in guinea pigs
Authors: Feleder Carlos , Perlik Vit , Blatteis Clark M.
Published: February 2004

We have shown previously that norepinephrine (NE) microdialyzed into the preoptic area (POA) of conscious guinea pigs stimulates local PGE2release. To identify the cyclooxygenase (COX) isozyme that catalyzes the production of this PGE2and the adrenoceptor (AR) subtype that mediates this effect, we microdialyzed for 6 h NE, cirazoline (α1-AR agonist), and clonidine (α2-AR agonist) into the POA of conscious guinea pigs pretreated intrapreoptically (intra-POA) with SC-560 (COX-1 inhibitor) or nimesulide or MK-0663 (COX-2 inhibitors) and measured the animals' core temperature (Tc) and intra-POA PGE2responses. Cirazoline induced Tcrises promptly after the onset of its dialysis without altering PGE2levels. NE and clonidine caused early falls followed by late rises of Tc; intra-POA PGE2levels were closely correlated with this thermal course. COX-1 inhibition attenuated the clonidine-induced Tcand PGE2falls but not the NE-elicited hyperthermia, but COX-2 inhibition suppressed both the clonidine- and NE-induced Tcand PGE2rises. Coinfused cirazoline and clonidine reproduced the late Tcrise of clonidine but not its early fall and also not the early rise produced by cirazoline; on the other hand, the PGE2responses were similar to those to NE. Prazosin (α1-AR antagonist) and yohimbine (α2-AR antagonist) blocked the effects of their respective agonists. These results indicate that α1- and α2-AR agonists microdialyzed into the POA of conscious guinea pigs evoke distinct Tcresponses: α1-AR activation produces quick, PGE2-independent Tcrises, and α2-AR stimulation causes an early Tcfall and a late, COX-2/PGE2-dependent Tcrise.
Glucocorticoids influence brain glycogen levels during sleep deprivation
Authors: Gip Phung , Hagiwara Grace , Sapolsky Robert M. , Cao Vinh H. , Heller H. Craig , Ruby Norman F.
Published: February 2004

We investigated whether glucocorticoids [i.e., corticosterone (Cort) in rats] released during sleep deprivation (SD) affect regional brain glycogen stores in 34-day-old Long-Evans rats. Adrenalectomized (with Cort replacement; Adx+) and intact animals were sleep deprived for 6 h beginning at lights on and then immediately killed by microwave irradiation. Brain and liver glycogen and glucose and plasma glucose levels were measured. After SD in intact animals, glycogen levels decreased in the cerebellum and hippocampus but not in the cortex or brain stem. By contrast, glycogen levels in the cortex of Adx+ rats increased by 43% ( P < 0.001) after SD, while other regions were unaffected. Also in Adx+ animals, glucose levels were decreased by an average of 28% throughout the brain after SD. Intact sleep-deprived rats had elevations of circulating Cort, blood, and liver glucose that were absent in intact control and Adx+ animals. Different responses between brain structures after SD may be due to regional variability in metabolic rate or glycogen metabolism. Our findings suggest that the elevated glucocorticoid secretion during SD causes brain glycogenolysis in response to energy demands.
In vivo expression profile of a H+-K+-ATPase α2-subunit promoter-reporter transgene
Authors: Zhang Wenzheng , Xia Xuefeng , Zou Lei , Xu Xiangyang , LeSage Gene D. , Kone Bruce C.
Published: February 2004

Because little is known about the molecular basis of transcriptional regulation of the murine H+-K+-ATPase α2(HKα2) gene or other genes whose expression is restricted in part to the collecting duct, especially in vivo, we developed transgenic mice carrying an insertional HKα2promoter-reporter gene construct. In these mice, the region −7,264/+253 of the HKα25′-flanking region controls expression of the reporter gene enhanced green fluorescent protein (EGFP). Patterns of HKα2/EGFP transgene expression were examined by fluorescence microscopy and immunoblotting. Of 10 major organs examined, EGFP immunoreactivity was detected abundantly in the kidney, and to a far lesser extent, in the brain and lung. Within the kidney, EGFP fluorescence was detected exclusively in the collecting ducts of transgenic mice and colocalized with the cellular distribution of both endogenous HKα2and aquaporin-2, consistent with the known expression pattern of endogenous HKα2in principal cells. Surprisingly, no transgene expression was evident by immunoblotting or fluorescence microscopy in the distal colon, the site of the highest endogenous HKα2expression. Although previous studies of steady-state mRNA levels suggested differences in HKα2gene regulation in the kidney and colon, our results provide the first direct evidence of differential transcriptional control of the HKα2gene in these organs and suggest that regions outside the 5′-flanking region or other regulatory factors play a role in HKα2expression in the distal colon.
Neonatal RAS inhibition changes the phenotype of the developing thick ascending limb of Henle
Authors: Lasaitiene Daina , Friberg Peter , Sundelin Birgitta , Chen Yun
Published: February 2004

Pharmacological interruption of angiotensin II type 1 (AT1) receptor signaling during nephrogenesis in rats perturbs renal tubular development. Perturbed tubulogenesis may contribute to long-term impairment of urinary concentrating ability, which is the main functional irreversible defect. The aim of this study was to further characterize tubular developmental deficits in neonatal rats, focusing on the thick ascending limb of Henle (TALH), known to undergo profound developmental changes and to be involved in urine-concentrating mechanisms. We have carried out immunohistochemistry and Western immunoblotting using antibodies directed against the major histocompatibility complex class II (MHC II) molecule and different TALH-specific markers, namely, cyclooxygenase-2 (COX-2), Tamm-Horsfall glycoprotein (THP), and the bumetanide-sensitive Na+-K+-2Cl−cotransporter (BSC-1/NKCC2). Immunohistochemistry demonstrated expression of MHC II, COX-2, THP, and BSC-1/NKCC2 proteins in normally developing TALH cells. The AT1-receptor antagonist losartan abolished MHC II expression exclusively in the developing TALH cells. Increased expression of COX-2 and THP was observed in the TALH cells of losartan-treated rats. Western immunoblotting confirmed increases in cortical and medullary COX-2 and THP abundance and revealed a decrease in cortical BSC-1/NKCC2 abundance in response to losartan treatment. We conclude that neonatal losartan treatment causes significant changes in the phenotype of the developing TALH in the rat.
H2O2-induced transactivation of EGF receptor requires Src and mediates ERK1/2, but not Akt, activation in renal cells
Authors: Zhougang Shougang , Schnellmann Rick G.
Published: February 2004

Although oxidative stress activates epidermal growth factor receptor (EGFR), ERK1/2, and Akt in a number of cell types, the mechanisms by which oxidative stress activates these kinases are not well defined in renal epithelial cells. Exposure of primary cultures of rabbit renal proximal tubular cells to hydrogen peroxide (H2O2) stimulated Src, EGFR, ERK1/2, and Akt activation in a time-dependent manner as determined by the phosphorylation of each protein. The Src inhibitor PP1 completely blocked EGFR, ERK1/2, and Akt phosphorylation following H2O2exposure. In contrast, blockade of the EGFR by AG1478 inhibited phosphorylation of ERK1/2 but not Src or Akt phosphorylation following H2O2exposure. Exogenous EGF stimulated EGFR, ERK1/2, and Akt activation and the EGFR inhibitor blocked phorphorylation of ERK1/2 and Akt. The presence of PP1, but not AG1478, significantly accelerated H2O2-induced cell death. These results suggest that Src mediates H2O2-induced EGFR transactivation. H2O2- and EGF-induced ERK1/2 activation is mediated by EGFR, whereas Akt is activated by Src independent of EGFR following H2O2exposure. Src-mediated EGFR transactivation contributes to a survival response following oxidative injury.
Ornithine metabolism in male and female rat kidney: mitochondrial expression of ornithine aminotransferase and arginase II
Authors: Levillain Olivier , Hus-Citharel Annette , Garvi Sandra , Peyrol Simone , Reymond Isabelle , Mutin Mireille , Morel François
Published: February 2004

In the kidney, l-ornithine is reabsorbed along the proximal convoluted tubule (PCT), transported by basolateral carriers, and produced by arginase II (AII). Here, the renal metabolic fate of l-ornithine was analyzed in male and female rats. Kidneys and renal zones were dissected and used for Western blot analysis, immunofluorescence, and electron microscopic studies. Ornithine aminotransferase (OAT) and AII were localized using specific antibodies. Ornithine oxidation was determined by incubating microdissected tubules with l-[1-14C] or l-[U-14C]ornithine in the presence or absence of energy-providing substrates. Ornithine decarboxylase (ODC) mRNAs were localized by in situ hybridization. The 48-kDa OAT protein was detected in male and female kidneys, but its level was fourfold higher in the latter. OAT relative distribution increased from the superficial cortex toward the outer medulla to reach its highest level. Almost all OAT protein was localized in cortical and medullary proximal straight tubules (CPST and OSPST, respectively). In proximal straight tubule (PST), AII protein distribution overlapped that of OAT. No gender difference in AII protein level was found. OAT and AII were colocalized within PST mitochondria. l-[1-14C]ornithine decarboxylation occurred in all tubules, but predominantly in proximal tubules. l-[1-14C]ornithine decarboxylation was enhanced when l-[1-14C]ornithine was given to tubules as the sole substrate. The use of l-[U-14C]ornithine demonstrated the complete oxidation of ornithine. In conclusion, the OAT gene was expressed more in female rat proximal tubules than in male. Because OAT and AII proteins overlapped in PST mitochondria, l-arginine-derived ornithine may be preferentially converted to l-glutamate, as proven by ornithine oxidation. However, the coexpression of ODC, glutamate decarboxylase, and glutamine synthetase in PST suggests that l-ornithine can also be metabolized to putrescine, GABA, and l-glutamine. The fate of l-ornithine may depend on the cellular context.
Injections of recombinant human erythropoietin increases lactate influx into erythrocytes
Authors: Connes Philippe , Caillaud Corinne , Mercier Jacques , Bouix Didier , Casties Jean François
Published: February 2004

Previous studies showed that erythropoietin not only increases erythrocyte production but is also essential in both the synthesis and the good functioning of several erythrocyte membrane proteins, including band 3. It is still unknown whether anion and/or H+fluxes are modified by erythropoietin. This study aimed to evaluate the effect of recombinant human erythropoietin (rHuEPO) injections on lactate transport into erythrocytes via band 3 and H+-monocarboxylate transporter MCT-1, two proteins involved in lactate exchange. Nine athletes received subcutaneous rHuEPO (50 U/kg body mass 3 times a week for 4 wk), and seven athletes received a saline solution (placebo group). All subjects were also supplemented with oral iron and vitamins B9and B12. Lactate transport into erythrocytes was studied before and after the rHuEPO treatment at different lactate concentrations (1.6, 8.1, 41, and 81.1 mM). After treatment, MCT-1 lactate uptake was increased at 1.6, 41 ( P < 0.01), and 81.1 mM lactate concentration ( P < 0.001) although lactate uptake via band 3 and nonionic diffusion were unchanged. MCT-1 maximal velocity increased in the rHuEPO group ( P < 0.05), reaching higher values than in the placebo group ( P < 0.05) after treatment. Our results show that rHuEPO injections increased MCT-1 lactate influx at low and high lactate concentrations. The increase in MCT-1 maximal velocity suggests that rHuEPO may stimulate MCT-1 synthesis during erythrocyte formation in bone marrow.
Effects of posture and bronchoconstriction on low-frequency input and transfer impedances in humans
Authors: Dellacà Raffaele L. , Black Lauren D. , Atileh Haytham , Pedotti Antonio , Lutchen Kenneth R.
Published: February 2004

We simultaneously evaluated the mechanical response of the total respiratory system, lung, and chest wall to changes in posture and to bronchoconstriction. We synthesized the optimal ventilation waveform (OVW) approach, which simultaneously provides ventilation and multifrequency forcing, with optoelectronic plethysmography (OEP) to measure chest wall flow globally and locally. We applied an OVW containing six frequencies from 0.156 to 4.6 Hz to the mouth of six healthy men in the seated and supine positions, before and after methacholine challenge. We measured mouth, esophageal, and transpulmonary pressures, airway flow by pneumotachometry, and total chest wall, pulmonary rib cage, and abdominal volumes by OEP. We computed total respiratory, lung, and chest wall input impedances and the total and regional transfer impedances (Ztr). These data were appropriately sensitive to changes in posture, showing added resistance in supine vs. seated position. The Ztr were also highly sensitive to lung constriction, more so than input impedance, as the former is minimally distorted by shunting of flow into alveolar gas compression and airway walls. Local impedances show that, during bronchoconstriction and at typical breathing frequencies, the contribution of the abdomen becomes amplified relative to the rib cage. A similar redistribution occurs when passing from seated to supine. These data suggest that the OEP-OVW approach for measuring Ztr could noninvasively track important lung and respiratory conditions, even in subjects who cannot cooperate. Applications might range from routine evaluation of airway hyperreactivity in asthmatic subjects to critical conditions in the supine position during mechanical ventilation.
Effect of tidal volume on distribution of ventilation assessed by synchrotron radiation CT in rabbit
Authors: Porra L. , Monfraix S. , Berruyer G. , Le Duc G. , Nemoz C. , Thomlinson W. , Suortti P. , Sovijärvi A. R. A. , Bayat S.
Published: February 2004

A respiration-gated synchrotron radiation computed tomography (SRCT) technique, which allows visualization and direct quantification of inhaled stable xenon gas, was used to study the effect of tidal volume (Vt) on regional lung ventilation. High-resolution maps (pixel size 0.35 × 0.35 mm) of local washin time constants (τ) and regional specific ventilation were obtained in five anesthetized, paralyzed, and mechanically ventilated rabbits in upright body position at the fourth, sixth, and eighth dorsal vertebral levels with a Vt from 4.9 ± 0.3 to 7.9 ± 0.4 ml/kg (means ± SE). Increasing Vt without an increase in minute ventilation resulted in a proportional increase of mean specific ventilation up to 65% in all studied lung levels and reduced the scattering of washin τ values. The τ values had log-normal distributions. The results indicate that an increase in Vt decreases nonuniformity of intraregional ventilatory gas exchange. The findings suggest that (SRCT) provides a new quantitative tool with high spatial discrimination ability for assessment of changes in peripheral pulmonary gas distribution during mechanical ventilation.
Neural and mechanical responses of the triceps surae muscle group after 1 h of repeated fast passive stretches
Authors: Avela Janne , Finni Taija , Liikavainio Tuomas , Niemelä Elina , Komi Paavo V.
Published: February 2004

Experiments were carried out to examine interaction between mechanical changes of the muscletendon unit and reduced reflex sensitivity after repeated and prolonged passive muscle stretching (RPS). There is some evidence that this interaction might be relevant also during active stretch-shortening cycle type of fatigue tasks. The results demonstrated a clear deterioration of voluntarily and electrically induced muscle contractions after RPS. Maximal voluntary contraction (MVC), average electromyographic activities of the gastrocnemius and soleus muscles, and maximal twitch contraction decreased on average by 13.8, 10.4, 7.6, and 16.8%, respectively. In addition, there was a 14% lengthening in the total duration of the twitch. MVCs measured at different ankle joint angles revealed a downward and rightward shift in the torquefascicle length curve after RPS. Interestingly, there was a crossing in the torque-fascicle length curves while measured at different activation levels but at the same joint angle before and after RPS. Even though no changes were observed in the activation level during MVCs, all the reflex parameters showed a clear reduction after RPS. This study presents evidence that repeated and prolonged passive muscle stretching can lead to some modification of material behavior of the aponeurosis-tendon system, such as stress relaxation and/or plastic deformation. In addition, altered material properties seem to affect proprioceptive feedback and, therefore, the motor unit activation in proportion to the contractile failure.