bluuebunny/crossref_metadata_2025 huggingface dataset:
Contribution of IL-1β and TNF-α to the initiation of the peripheral lung response to atmospheric particulates (PM10)
Authors: Ishii Hiroshi , Fujii Takeshi , Hogg James C. , Hayashi Shizu , Mukae Hiroshi , Vincent Renaud , van Eeden Stephan F.
Published: March 2004

Alveolar macrophages (AM) play a key role in clearing atmospheric particulates from the lung surface and stimulating epithelial cells to produce proinflammatory mediators. The present study examines the role of “acute response” cytokines TNF-α and IL-1β released by AM exposed to ambient particulate matter with a diameter of <10 μm (PM10) in amplifying the proinflammatory mediator expression by A549 cells and human bronchial epithelial cells (HBEC). The results showed that supernatants from human AM incubated 24 h with PM10(100 μg/ml) contained more TNF-α, IL-1β, granulocyte-macrophage colony stimulating factor, IL-6, and IL-8 than nonexposed AM supernatants. The 3-h treatment of A549 cells with PM10-exposed AM supernatants increased TNF-α, IL-1β, IL-8, regulated on activation normal T-cells expressed and secreted (RANTES), and leukemia inhibitory factor mRNA compared with the treatment with nonexposed AM supernatants and, compared with untreated A549 cells, additionally increased ICAM-1 and monocyte chemotactic protein-1 mRNA. Preincubating PM10-exposed AM supernatants with anti-IL-1β antibodies reduced all the above mediators as well as VEGF mRNA expression ( P < 0.05), while anti-TNF-α antibodies were less effective ( P > 0.05), and the combination of the two antibodies most effective. When HBEC were treated similarly, anti-TNF-α antibodies had the greatest effect. In A549 cells PM10-exposed AM supernatants increased NF-κB, activator protein (AP)-1 and specificity protein 1 binding, while anti-TNF-α and anti-IL-1β antibodies reduced NF-κB and AP-1 binding. We conclude that AM-derived TNF-α and IL-1β provide a major stimulus for the production of proinflammatory mediators by lung epithelial cells and that their relative importance may depend on the type of epithelial cell target.
Phorbol ester-mediated pulmonary artery endothelial barrier dysfunction through regulation of actin cytoskeletal mechanics
Authors: Moy Alan B. , Blackwell Ken , Wang Ning , Haxhinasto Kari , Kasiske Mary K. , Bodmer James , Reyes Gina , English Anthony
Published: March 2004

The mechanisms of phorbol ester- and thrombin-mediated pulmonary artery endothelial barrier dysfunction were compared. Phorbol ester dibutyrate (PDBU) mediated slow force velocity and less force than thrombin. Taxol did not attenuate PDBU-mediated tension, while it reversed nocodazole-mediated tension. PDBU-mediated tension was not affected by acrylamide; PDBU increased cell stiffness and produced greater declines in transendothelial resistance (TER) than acrylamide. Thus PDBU caused a net increase in tension and did not unload microtubule or intermediate filaments. Microfilament remodeling, determined on the basis of immunocytochemistry and actin solubility, lacked the sensitivity and specificity to predict actin-dependent mechanical properties. Thrombin increased myosin light chain (MLC) kinase site-specific MLC phosphorylation, according to peptide map analysis, whereas PDBU did not increase PKC-specific MLC phosphorylation. The initial PDBU-mediated tension development temporally correlated with PDBU-mediated decline in TER and increased low-molecular-weight caldesmon ( l-CaD) phosphorylation. PDBU-mediated tension development and decreases in TER were associated with a temporal loss of endothelial cell-matrix adhesion, based on a numerical model of TER. Although, on the basis of immunocytochemistry, thrombin-mediated tension was associated with actin insolubility, actin reorganization, and gap formation, these changes did not predict thrombin-mediated gap formation, based on TER and time-lapse differential interference contrast microscopy. These data suggest that PDBU may disrupt endothelial barrier function through loss of cell-matrix adhesion through l-CaD-dependent actin contraction.
Hyperoxia-induced emphysematous changes in subacute phase of endotoxin-induced lung injury in rats
Authors: Kohno Mitsutomo , Ishizaka Akitoshi , Sawafuji Makoto , Koh Hidefumi , Hirayama Yoshitaka , Ikeda Eiji , Shiomi Takayuki , Ohashi Akira , Okada Yasunori , Kobayashi Koichi
Published: March 2004

We examined the effects of prolonged hyperoxia (75% O2) on lung structure and collagen metabolism in the subacute phase of lung injury induced by continuous infusion of endotoxin (LPS) in a rat model. Experimental groups included control, endotoxin alone, endotoxin plus hyperoxia, and hyperoxia alone. Endotoxin-treated rats received a bolus of LPS (10 mg/kg iv) followed by 500 μg·kg−1·day−1in continuous infusion for 10 days. The bronchoalveolar lavage (BAL) fluid/plasma albumin concentration ratio, an index of capillary permeability, and neutrophil and macrophage counts in BAL fluid were highest in the endotoxin plus hyperoxia group. On pathological examination, prolonged hyperoxia exacerbated destruction of the alveolar wall and caused most prominent emphysematous changes in the endotoxin plus hyperoxia group. Lung tissue hydroxyproline concentration was significantly decreased in the hyperoxia group and increased in the endotoxin group. The latent forms of MMP-2 and MMP-9 increased in BAL fluid of the endotoxin- and/or hyperoxia-treated groups, whereas the activities of collagenase and gelatinase, and the active form of MMP-2 were all increased in the hyperoxia-treated groups. Added to endotoxin, prolonged hyperoxia degraded collagen, the major structural component of basement membranes, and caused emphysematous changes associated with activation of collagenase and MMP-2. Our observations suggest that, in the subacute phase of endotoxin-induced lung injury, prolonged hyperoxia causes pulmonary emphysematous changes with persistent injury to the alveolar capillary barrier. Collagenase and MMP-2 activated by hyperoxia, together with MMP-9, may play prominent roles in disruption of the alveolar basement membranes and degradation of collagen lining the alveolar walls.
Exposure to ambient particles accelerates monocyte release from bone marrow in atherosclerotic rabbits
Authors: Goto Yukinobu , Hogg James C. , Shih Chih-Horng , Ishii Hiroshi , Vincent Renaud , van Eeden Stephan F.
Published: March 2004

Exposure to air pollution [particulate matter, particles <10 μm (PM10)] causes a systemic inflammatory response that includes stimulation of the bone marrow (BM) and progression of atherosclerosis. Monocytes are known to play a key role in atherogenesis by migration into subendothelial lesions where they appear as foam cells. The present study was designed to quantify the BM monocyte response in Watanabe heritable hyperlipidemic (WHHL) rabbits after PM10exposure. WHHL rabbits were given twice weekly intrapharyngeal instillations of 5 mg of PM10for 4 wk to a total of 40 mg and compared with control WHHL or New Zealand White (NZW) rabbits. The thymidine analog 5′-bromo-2′-deoxyuridine was used to label dividing cells in the BM and a monoclonal antibody to identify monocytes in peripheral blood. The transit time of monocytes through the BM was faster in WHHL than in NZW rabbits (30.4 ± 1.9 h vs. 35.2 ± 0.9 h, WHHL vs. NZW; P < 0.05). PM10instillation exposure increased circulating band cell counts, caused rapid release of monocytes from the BM, and further shortened their transit time through the BM to 23.2 ± 1.6 h ( P < 0.05). The percentage of alveolar macrophages containing particles in the lung correlated with the BM transit time of monocytes (r2= 0.45, P <0.05). We conclude that atherosclerosis increases the release of monocytes from the BM, and PM10exposure accelerates this process in relation to the amount of particles phagocytosed by alveolar macrophages.
Perfluorocarbon attenuates response of concanavalin A-stimulated mononuclear blood cells without altering ligand-receptor interaction
Authors: Haufe Dirk , Luther Thomas , Kotzsch Matthias , Knels Lilla , Koch Thea
Published: March 2004

Intrapulmonary application of perfluorocarbons (PFC) in acute lung injury is associated with anti-inflammatory effects. A direct impact on leukocytic function may be involved. To further elucidate PFC effects on cellular activation, we compared in an in vitro model the response of concanavalin A (ConA)-stimulated lymphocytes and monocytes exposed to perfluorohexane. We hypothesized that perfluorohexane attenuates the action of the lectin ConA by altering stimulant-receptor interaction on the cell surface. Mononuclear blood cells were stimulated by incubation with ConA in the presence of different amounts of perfluorohexane. The response of lymphocytes and monocytes was determined by means of IL-2 secretion and tissue factor (TF) expression, respectively. The influence of perfluorohexane on cell-surface binding of fluorescence-labeled ConA was studied using flow cytofluorometry and fluorescence microscopy. Perfluorohexane itself did not induce a cellular activation but significantly inhibited both monocytic TF expression and, to a far greater extent, IL-2 secretion of ConA-stimulated mononuclear blood cells. The effect of perfluorohexane was due neither to an alteration of cell viability nor to a binding of the stimulant. The amount of cell surface-bound ConA was not altered by perfluorohexane, and the overall pattern of ConA receptor rearrangement did not differ between controls and treated cells. In the present study, we provide further evidence for an anti-inflammatory effect of PFC that might be beneficial in states of pulmonary hyperinflammation. A PFC-induced alteration of stimulant-receptor interaction on the surface membrane does not seem to be the cause of attenuated cell activation.
Protein kinase A attenuates endothelial cell barrier dysfunction induced by microtubule disassembly
Authors: Birukova Anna A. , Liu Feng , Garcia Joe G. N. , Verin Alexander D.
Published: March 2004

Cross talk between the actin cytoskeleton and the microtubule (MT) network plays a critical role in regulation of endothelial permeability. We have previously demonstrated that MT disruption by nocodazole results in increases in MLC phosphorylation, actomyosin contraction, cell retraction, and paracellular gap formation, cardinal features of endothelial barrier dysfunction (Verin AD, Birukova A, Wang P, Liu F, Becker P, Birukov K, and Garcia JG. Am J Physiol Lung Cell Mol Physiol 281: L565–L574, 2001; Birukova AA, Smurova K, Birukov KG, Usatyuk P, Liu F, Kaibuchi K, Ricks-Cord A, Natarajan V, Alieva A, Garcia JG, and Verin AD. J Cell Physiol. In press.). Although activation of PKA opposes barrier-disrupting effects of edemagenic agents on confluent EC monolayers, information about the molecular mechanisms of PKA-mediated EC barrier protection is limited. Our results suggest that MT disassembly alters neither intracellular cAMP levels nor PKA enzymatic activity; however, elevation of cAMP levels and PKA activation by either cholera toxin or forskolin dramatically attenuates the decline in transendothelial electrical resistance induced by nocodazole in human pulmonary EC. Barrier-protective effects of PKA on EC were associated with PKA-mediated inhibition of nocodazole-induced stress fiber formation, Rho activation, phosphorylation of myosin phosphatase regulatory subunit at Thr696, and decreased MLC phosphorylation. In addition, forskolin pretreatment attenuated MT disassembly induced by nocodazole. These results suggest a critical role for PKA activity in stabilization of MT cytoskeleton and provide a novel mechanism for cAMP-mediated regulation of Rho-induced actin cytoskeletal remodeling, actomyosin contraction, and EC barrier dysfunction induced by MT disassembly.
Effects of obesity on the relationship of leptin mRNA expression and adipocyte size in anatomically distinct fat depots in mice
Authors: Guo Kai-Ying , Halo Patricia , Leibel Rudolph L. , Zhang Yiying
Published: March 2004

In support of leptin's physiological role as humoral signal of fat mass, we have shown that adipocyte volume is a predominant determinant of leptin mRNA levels in anatomically distinct fat depots in lean young mice in the postabsorptive state. In this report, we investigated how obesity may affect the relationship between leptin mRNA levels and adipocyte volume in anatomically distinct fat depots in mice with genetic ( Lepob/ Lepoband Ay/+), diet-induced, and aging-related obesity. In all of the obese mice examined, tissue leptin mRNA levels relative to the average adipocyte volume were lower in the perigonadal and/or retroperitoneal than in the inguinal fat depots and were lower than those of the lean young mice in the perigonadal fat depot. A close, positive correlation between leptin mRNA level and adipocyte volume was present from small to hypertrophic adipocytes within each perigonadal and inguinal fat pad in the obese mice, but the slopes of the regression lines relating leptin mRNA level to adipocyte volume were significantly lower in the perigonadal than in the inguinal fat pads of the same mice. These results suggest that obesity per se is associated with a decreased leptin gene expression per unit of fat mass in mice and that the positive correlation between leptin mRNA level and adipocyte volume is an intrinsic property of adipocytes that is not disrupted by adipocyte hypertrophy in obese mice.
Postnatal glucocorticoid exposure alters the adult phenotype
Authors: He Jing , Varma Amit , Weissfeld Lisa A. , Devaskar Sherin U.
Published: March 2004

We examined the effect of six doses of dexamethasone (Dex) administered daily (2–7 days of age) to postnatal rats on body weight gain, food and water intake, peripheral hormonal/metabolic milieu, and hypothalamic neuropeptides that regulate food intake. We observed a Dex-induced acute (3 days of age) suppression of endogenous corticosterone and an increase in circulating leptin concentrations that were associated with a decrease in body weight in males and females. Followup during the suckling, postsuckling, and adult stages (7–120 days of age) revealed hypoleptinemia in males and females, and hypoinsulinemia, a relative increase in the glucose-to-insulin ratio, and a larger increase in skeletal muscle glucose transporter (GLUT 4) concentrations predominantly in the males, reflective of a catabolic state associated with a persistent decrease in body weight gain. The increase in the glucose-to-insulin ratio and hyperglycemia was associated with an increase in water intake. In addition, the changes in the hormonal/metabolic milieu were associated with an increase in hypothalamic neuropeptide Y content in males and females during the suckling phase, which persisted only in the 120-day-old female with a transient postnatal decline in α-melanocyte-stimulating hormone and corticotropin-releasing factor. This increase in neuropeptide Y (NPY) during the suckling phase in males and females was associated with a subsequent increase in adult food intake that outweighed the demands of body weight gain. In contrast to the adult hypothalamic findings, cerebral ventricular dilatation was more prominent in adult males. We conclude that postnatal Dex treatment causes permanent sex-specific changes in the adult phenotype, setting the stage for future development of diabetes (increased glucose:insulin ratio), obesity (increased NPY and food intake), and neurological impairment (loss of cerebral volume).
In vivo regulation of SREBP-1c in skeletal muscle: effects of nutritional status, glucose, insulin, and leptin
Authors: Commerford S. Renee , Peng Li , Dubé John J. , O'Doherty Robert M.
Published: March 2004

Sterol regulatory element binding protein-1c (SREBP-1c), a transcription factor that is important for mediating insulin effects on metabolic gene expression in liver during the fasted-to-fed transition, is also expressed in skeletal muscle. However, the regulation and role of SREBP-1c in skeletal muscle are poorly understood. The present study compared the effects of nutritional status, physiological hyperinsulinemic clamps, and adenovirus-mediated hyperleptinemia (HLEP) in rats on expression of SREBP-1c and other metabolic genes in skeletal muscle. Three- and 6-h refeeding of 18-h-fasted animals increased levels of SREBP-1c mRNA and the SREBP-1 protein (full length and mature) in gastrocnemius muscle ( P < 0.05). Fatty acid synthase (FAS) and hexokinase II (HKII) mRNA levels were also increased by refeeding, and uncoupling protein 3 (UCP3) mRNA level was decreased (all P < 0.05). Surprisingly, 3-h hyperinsulinemic clamps did not increase gastrocnemius muscle SREBP-1c and FAS mRNA levels or SREBP-1 protein levels but did increase HKII mRNA levels and decrease UCP3 mRNA levels ( P < 0.05). HLEP reduced refeeding-induced increases of SREBP-1c and FAS mRNA levels but did not reduce the level of SREBP-1 protein. We conclude that 1) skeletal muscle SREBP-1c gene expression is regulated by nutritional status in a fashion similar to that observed in liver and adipose tissue, 2) physiological hyperinsulinemia is not sufficient to imitate the effects of refeeding on SREBP-1c gene expression, and 3) leptin suppresses refeeding effects on SREBP-1c mRNA levels.
Blunted hypothalamic neuropeptide gene expression in response to fasting, but preservation of feeding responses to AgRP in aging male Brown Norway rats
Authors: Wolden-Hanson Tami , Marck Brett T. , Matsumoto Alvin M.
Published: March 2004

Aging mammals lose the ability to maintain energy balance, exhibiting decreased appetite (anorexia) and impaired ability to maintain body weight. To determine the contribution of hypothalamic neuropeptides, two experiments were performed in male Brown Norway rats. To assess the hypothalamic neuropeptide response to food deprivation, young (Y; 4 mo old), middle-aged (M; 13 mo), and old (O; 25 mo) rats were either ad libitum fed or fasted for 72 h ( n = 10/group) and killed. Hypothalamic levels of agouti-related peptide (AgRP), proopiomelanocortin (POMC), and cocaine-amphetamine-regulated transcript (CART) mRNA were assessed by in situ hybridization. With aging, arcuate AgRP gene expression decreased and CART mRNA increased, but POMC mRNA did not change. Fasting-induced changes in gene expression of all neuropeptides studied were attenuated with aging. To test the food intake response to appetite-stimulating neuropeptides, Y, M, O, and very old (VO; 33 mo) rats ( n = 4–8/group) received one intracerebroventricular injection of each of three treatments: 0.1 nmol AgRP, 2.34 nmol NPY, and saline control. AgRP increased food intake of all groups by 10–20%, compared with saline, and this effect persisted up to 7 days after injection. VO animals were more sensitive to the effects of AgRP than younger animals. In contrast, NPY increased food intake more in Y than in older animals and its effects did not last >24 h. We conclude that the mechanisms by which arcuate nucleus neurons influence appetite are differentially affected by age and speculate that the melanocortin system may be a useful target for treatment of the anorexia of aging.
Entrainment pattern between sympathetic and phrenic nerve activities in the Sprague-Dawley rat: hypoxia-evoked sympathetic activity during expiration
Authors: Dick Thomas E. , Hsieh Y.-H. , Morrison Shaun , Coles Sharon K. , Prabhakar Nanduri
Published: March 2004

Sympathetic and respiratory motor activities are entrained centrally. We hypothesize that this coupling may partially underlie changes in sympathetic activity evoked by hypoxia due to activity-dependent changes in the respiratory pattern. Specifically, we tested the hypothesis that sympathetic nerve activity (SNA) expresses a short-term potentiation in activity after hypoxia similar to that expressed in phrenic nerve activity (PNA). Adult male, Sprague-Dawley (Zivic Miller) rats ( n = 19) were anesthetized (Equithesin), vagotomized, paralyzed, ventilated, and pneumothoracotomized. We recorded PNA and splanchnic SNA (sSNA) and generated cycle-triggered averages (CTAs) of rectified and integrated sSNA before, during, and after exposures to hypoxia (8% O2 and 92% N2 for 45 s). Inspiration (I) and expiration (E) were divided in half, and the average and area of integrated sSNA were calculated and compared at the following time points: before hypoxia, at the peak breathing frequency during hypoxia, immediately before the end of hypoxia, immediately after hypoxia, and 60 s after hypoxia. In our animal model, sSNA bursts consistently followed the I-E phase transition. With hypoxia, sSNA increased in both halves of E, but preferentially in the second rather than the first half of E, and decreased in I. After hypoxia, sSNA decreased abruptly, but the coefficient of variation in respiratory modulation of sSNA was significantly less than that at baseline. The hypoxic-evoked changes in sympathetic activity and respiratory pattern resulted in sSNA in the first half of E being correlated negatively to that in the second half of E ( r = −0.65, P < 0.05) and positively to Te ( r = 0.40, P < 0.05). Short-term potentiation in sSNA appeared not as an increase in the magnitude of activity but as an increased consistency of its respiratory modulation. By 60 s after hypoxia, the variability in the entrainment pattern had returned to baseline. The preferential recruitment of late expiratory sSNA during hypoxia results from either activation by expiratory-modulated neurons or by non-modulated neurons whose excitatory drive is not gated during late E.
Separation of hepatic and gastrointestinal signals from the common “hepatic” branch of the vagus
Authors: Horn Charles C. , Friedman Mark I.
Published: March 2004

Anatomic studies show that the common hepatic branch (CHB) of the vagus contains afferent fibers that innervate sites outside the hepatoportal region, primarily in the gastrointestinal tract. In the current experiments on the anesthetized rat, the source of signals from the CHB was determined by recording CHB neurophysiological responses before and after transection of the gastroduodenal branch (GDB) of the CHB. Serotonin [5-hydroxytryptamine (5-HT)] and CCK-8 were used as probes to stimulate the CHB. Most of the CHB afferent fibers were 5-HT sensitive (56%), and 35% of these were also sensitive to CCK-8. Portal vein vs. jugular vein infusion of 5-HT and CCK-8 and GDB transection showed that 5-HT- and CCK-sensitive fibers innervate the hepatoportal region and areas outside the hepatic hilus (e.g., the gastrointestinal tract). Suppression of basal nerve activity by a 5-HT3 receptor antagonist (Y-25130) suggests that ∼50% of CHB afferent fibers contain 5-HT3 receptors, but none of these fibers appears to be in the hepatoportal region because only in rats with an intact GDB did Y-25130 reduce nerve activity. In summary, these data are in close agreement with anatomic observations on the distribution of the CHB fibers and indicate that neurophysiological studies of the CHB must be carefully evaluated given the prominent role of nonhepatoportal afferent signals recorded from the CHB.
In vitro characterization of aldosterone and cAMP effects in mouse distal convoluted tubule cells
Authors: González-Núñez Daniel , Morales-Ruiz Manuel , Leivas Alberto , Hebert Steven C. , Poch Esteban
Published: March 2004

The distal nephron plays a capital role in the fine regulation of sodium reabsorption. Compared with the cortical collecting duct, much less information is available on the hormonal regulation of sodium transporter genes in the distal convoluted tubule (DCT), where the thiazide-sensitive Na+-Cl-cotransporter (NCC) is the major entry pathway for Na+. The purpose of this study was to characterize the in vitro effects of aldosterone (Aldo; 1 μM) and cAMP (8-BrcAMP; 0.5 mM) on mouse DCT (mDCT) by using an immortalized mDCT cell line. Western blot analysis and semiquantitative RT-PCR were performed to analyze the expression of genes involved in sodium transport. The mDCTcell line expressed the 11β-hydroxysteroid dehydrogenase type 2 gene and both the mineralocorticoid and glucocorticoid receptor genes, suggesting Aldo responsiveness. In this sense, we found that mDCT cells expressed the amiloride-sensitive Na+channel (ENaC) and responded to Aldo by upregulating the α-subunit protein. Similarly, α1Na+-K+-ATPase protein was upregulated by Aldo and 8-BrcAMP. In addition, the Aldo intermediate gene sgk1 mRNA was increased in response to both Aldo and 8-BrcAMP, and the transcription factor HNF–3α mRNA was induced by 8-BrcAMP. With respect to NCC regulation, although Aldo induced NCC protein levels in mice in vivo, neither Aldo nor 8-BrcAMP significantly induced the NCC mRNA or protein levels in mDCT cells. These results suggest that in mDCT, Aldo and cAMP modulate some downstream mediators and effectors in vitro but do not influence the expression of NCC in this cell model.
Protein kinase C-α inhibits the repair of oxidative phosphorylation afterS-(1,2-dichlorovinyl)-l-cysteine injury in renal cells
Authors: Liu Xiuli , Godwin Malinda L. , Nowak Grażyna
Published: March 2004

Previously, we showed that physiological functions of renal proximal tubular cells (RPTC) do not recover following S-(1,2-dichlorovinyl)-l-cysteine (DCVC)-induced injury. This study investigated the role of protein kinase C-α (PKC-α) in the lack of repair of mitochondrial function in DCVC-injured RPTC. After DCVC exposure, basal oxygen consumption (Qo2), uncoupled Qo2, oligomycin-sensitive Qo2, F1F0-ATPase activity, and ATP production decreased, respectively, to 59, 27, 27, 57, and 68% of controls. None of these functions recovered. Mitochondrial transmembrane potential decreased 53% after DCVC injury but recovered on day 4. PKC-α was activated 4.3- and 2.5-fold on days 2 and 4, respectively, of the recovery period. Inhibition of PKC-α activation (10 nM Go6976) did not block DCVC-induced decreases in mitochondrial functions but promoted the recovery of uncoupled Qo2, oligomycin-sensitive Qo2, F1F0-ATPase activity, and ATP production. Protein levels of the catalytic β-subunit of F1F0-ATPase were not changed by DCVC or during the recovery period. Amino acid sequence analysis revealed that α-, β-, and ε-subunits of F1F0-ATPase have PKC consensus motifs. Recombinant PKC-α phosphorylated the β-subunit and decreased F1F0-ATPase activity in vitro. Serine but not threonine phosphorylation of the β-subunit was increased during late recovery following DCVC injury, and inhibition of PKC-α activation decreased this phosphorylation. We conclude that during RPTC recovery following DCVC injury, 1) PKC-α activation decreases F0F1-ATPase activity, oxidative phosphorylation, and ATP production; 2) PKC-α phosphorylates the β-subunit of F1F0-ATPase on serine residue; and 3) PKC-α does not mediate depolarization of RPTC mitochondria. This is the first report showing that PKC-α phosphorylates the catalytic subunit of F1F0-ATPase and that PKC-α plays an important role in regulating repair of mitochondrial function.
ENaC subunit-subunit interactions and inhibition by syntaxin 1A
Authors: Berdiev Bakhrom K. , Jovov Biljana , Tucker Ward C. , Naren Anjaparavanda P. , Fuller Catherine M. , Chapman Edwin R. , Benos Dale J.
Published: March 2004

Amiloride-sensitive epithelial Na+ channels (ENaCs) are subject to modulation by many factors. Recent data have also linked the N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery to this regulation of ENaC, but the molecular mechanisms that underlie this modulation are poorly understood. In this study, we demonstrate that syntaxin 1A physically interacts with ENaC and functionally regulates ENaC activity. Syntaxin 1A was able to coimmunoprecipitate in vitro-translated γ-ENaC, but not α- or β-ENaC. Also, using antibodies raised against α-, β-, or γ-ENaC, we detected syntaxin 1A in immunoprecipitates from Madin-Darby canine kidney cells stably transfected with αβγ-ENaC. In bilayers, syntaxin 1A inhibited ENaC, and this syntaxin 1A modulation of ENaC activity was eliminated by truncations of cytoplasmic domains of the ENaC subunits. Our findings provide evidence for a direct physical interaction between ENaC and syntaxin 1A and suggest involvement of ENaC's cytoplasmic domains in functional modulation of ENaC activity by syntaxin 1A.
Intestinal Na-Picotransporter adaptation to dietary Picontent in vitamin D receptor null mice
Authors: Segawa Hiroko , Kaneko Ichiro , Yamanaka Setsuko , Ito Mikiko , Kuwahata Masahi , Inoue Yoshio , Kato Shigeaki , Miyamoto Ken-ichi
Published: March 2004

Recent studies suggest that vitamin D may play a role in intestinal Na+-dependent phosphate transport adaptation to variable levels of dietary Pi. Therefore, the goal of the current study was to assess Na+-dependent Picotransport activity in transgenic mice to determine whether vitamin D is an essential mediator of this process. Intestinal brush-border membrane (BBM), Na+-dependent Picotransport activity was significantly decreased in vitamin D receptor (VDR) null [VDR (−/−)] mice compared with wild-type (VDR+/+) mice. While intestinal Na-Picotransporter (type IIb) mRNA levels were similar in VDR (−/−) and VDR (+/+) mice, type IIb Na-Picotransporter protein expression was markedly suppressed in VDR (−/−) mice compared with VDR (+/+) mice. Furthermore, Na-Picotransport activity in renal BBM was similar in VDR (−/−) and VDR (+/+) mice, but type IIa Na-Picotransporter protein expression was decreased in VDR (−/−) mice. After administration of a low-Pidiet, type IIb protein expression was significantly increased in VDR (+/+) and VDR (−/−) mice, and type IIb protein expression was present in the intestinal BBM of VDR (−/−) mice. These data demonstrate that intestinal Na-Picotransport adaptation to a low-Pidiet occurs independently of vitamin D.
Effects of calcium channel blockers on “dynamic” and “steady-state step” renal autoregulation
Authors: Griffin Karen A. , Hacioglu Rifat , Abu-Amarah Isam , Loutzenhiser Rodger , Williamson Geoffrey A. , Bidani Anil K.
Published: March 2004

Renal autoregulation (AR) mechanisms provide the primary protection against transmission of systemic pressures and hypertensive renal damage. However, the relative merits of the “step” change vs. “dynamic” methods for the assessment of AR capacity remain controversial. The effects of 48–72 h of orally administered amlodipine (L-type) and mibefradil (T-type) calcium channel blockers (CCBs) on step and dynamic AR in Sprague-Dawley rats were compared. Both CCBs significantly impaired “steady-state step” AR (autoregulatory indexes = ∼0.5 vs. ∼0.1 in controls, P < 0.05; n = 9–10/group). By contrast, dynamic AR compensation in separate conscious rats ( n = 12) was not significantly altered by either amlodipine ( n = 10) or mibefradil ( n = 6; fractional gain in admittance ∼0.4–0.5 in all groups at frequencies in the range of 0.0025–0.025 Hz). However, both CCBs tended to attenuate the myogenic resonance peak along with shifting it to a significantly slower frequency ( P < 0.001) during dynamic AR, but no consistent effects were observed on the tubuloglomerular feedback resonance peak. While the reasons for the insensitivity of dynamic vs. steady-state step AR capacity estimates to CCBs remain to be established, the present data indicate that dynamic AR methods may have a limited utility for assessing AR capacity but may provide potentially important insights into the operational characteristics of AR control mechanisms. A strong correlation was also observed between the average conductance and the admittance gain at the heart beat frequency ( r = 0.77, P < 0.001), suggesting that such parameters may provide additional and possibly more meaningful indexes of BP transmission in conscious animals during dynamic AR.
Effect of lactate infusion on M-wave characteristics and force in the rat plantaris muscle during repeated stimulation in situ
Authors: Karelis Antony D. , Marcil Mariannick , Péronnet François , Gardiner Phillip F.
Published: March 2004

It is unclear whether accumulation of lactate in skeletal muscle during exercise contributes to muscle fatigue. The purpose of the present study was to examine the effect of lactate infusion on muscle fatigue during prolonged indirect stimulation in situ. For this purpose, the plantaris muscle was electrically stimulated (50 Hz, for 200 ms, every 2.7 s, 5 V) in situ through the sciatic nerve to perform concentric contractions for 60 min while either saline or lactate was infused intravenously (8 rats/group). Lactate infusion (lactate concentration ∼12 mM) attenuated the reduction in submaximal dynamic force (-49 vs. -68% in rats infused with saline; P < 0.05). Maximum dynamic and isometric forces at the end of the period of stimulation were also higher ( P < 0.05) in rats infused with lactate (3.8 ± 0.3 and 4.4 ± 0.3 N) compared with saline (3.1 ± 0.2 and 3.6 ± 0.2 N). The beneficial effect of lactate infusion on muscle force during prolonged stimulation was associated with a better maintenance of M-wave characteristics compared with control. In contrast, lactate infusion was not associated with any reduction in muscle glycogen utilization or with any reduction of fatigue at the neuromuscular junction (as assessed through maximal direct muscle stimulation: 200 Hz, 200 ms, 150 V).
Age-related changes in finger coordination in static prehension tasks
Authors: Shim Jae Kun , Lay Brendan S. , Zatsiorsky Vladimir M. , Latash Mark L.
Published: March 2004

We studied age-related changes in the performance of maximal and accurate submaximal force and moment production tasks. Elderly and young subjects pressed on six dimensional force sensors affixed to a handle with a T-shaped attachment. The weight of the whole system was counterbalanced with another load. During tasks that required the production of maximal force or maximal moment by all of the digits, young subjects were stronger than elderly. A greater age-related deficit was seen in the maximal moment production tests. During maximal force production tests, elderly subjects showed larger relative involvement of the index and middle fingers; they moved the point of thumb force application upward (toward the index and middle fingers), whereas the young subjects rolled the thumb downward. During accurate force/moment production trials, elderly persons were less accurate in the production of both total moment and total force. They produced higher antagonistic moments, i.e., moment by fingers that acted against the required direction of the total moment. Both young and elderly subjects showed negative covariation of finger forces across repetitions of a ramp force production task. In accurate moment production tasks, both groups showed negative covariation of two components of the total moment: those produced by the normal forces and those produced by the tangential forces. However, elderly persons showed lower values of the indexes of both finger force covariation and moment covariation. We conclude that age is associated with an impaired ability to produce both high moments and accurate time profiles of moments. This impairment goes beyond the well-documented deficits in finger and hand force production by elderly persons. It involves worse coordination of individual digit forces and of components of the total moment. Some atypical characteristics of finger forces may be viewed as adaptive to the increased variability in the force production with age.
Formation of focal adhesions on fibronectin promotes fluid shear stress induction of COX-2 and PGE2release in MC3T3-E1 osteoblasts
Authors: Ponik Suzanne M. , Pavalko Fredrick M.
Published: March 2004

Mechanical loading of bone is important for the structural integrity of the skeleton and the maintenance of bone mass. Mechanically loading bone generates fluid shear stress (FSS) across the surface of bone cells resulting in the induction of cyclooxygenase-2 (COX-2) and release of prostaglandins, both of which are necessary for mechanically induced bone formation. However, the mechanisms by which cells transduce FSS-induced signals across the membrane and into the cell remain poorly understood. Focal adhesions, which are specialized sites of attachment between cells and the extracellular matrix, play a role in signal transduction and have been proposed to function as mechanosensors. To directly test whether focal adhesions mediate mechanotransduction in bone cells, we inhibited the formation of focal adhesions by 1) culturing MC3T3-E1 osteoblasts on bovine serum albumin (BSA), which does not contain integrin binding sites or by 2) treating cells cultured on fibronectin with soluble Arg-Gly-Asp-Ser (RGDS) peptide to specifically block integrin-fibronectin interactions. We then subjected the cells to FSS and measured COX-2 induction and PGE2release. Both COX-2 induction and PGE2release in response to FSS were significantly decreased when osteoblasts were treated with soluble RGDS peptide compared with controls. However, RGDS peptide treatment did not affect FSS-induced ERK phosphorylation. Interestingly, osteoblasts cultured on BSA to suppress focal adhesion formation secreted fibronectin and increased focal adhesion formation over time, which correlated with the induction of COX-2 in response to FSS. Together, these results suggest that fibronectin-induced formation of focal adhesions promotes FSS-induced PGE2release and upregulation of COX-2 protein.
Pressure gradient, not exposure duration, determines the extent of epithelial cell damage in a model of pulmonary airway reopening
Authors: Kay Sarina S. , Bilek Anastacia M. , Dee Kay C. , Gaver Donald P.
Published: March 2004

The reduction of tidal volume during mechanical ventilation has been shown to reduce mortality of patients with acute respiratory distress syndrome, but epithelial cell injury can still result from mechanical stresses imposed by the opening of occluded airways. To study these stresses, a fluid-filled parallel-plate flow chamber lined with epithelial cells was used as an idealized model of an occluded airway. Airway reopening was modeled by the progression of a semi-infinite bubble of air through the length of the channel, which cleared the fluid. In our laboratory’s prior study, the magnitude of the pressure gradient near the bubble tip was directly correlated to the epithelial cell layer damage (Bilek AM, Dee KC, and Gaver DP III. J Appl Physiol 94: 770–783, 2003). However, in that study, it was not possible to discriminate the stress magnitude from the stimulus duration because the bubble propagation velocity varied between experiments. In the present study, the stress magnitude is modified by varying the viscosity of the occlusion fluid while fixing the reopening velocity across experiments. This approach causes the stimulus duration to be inversely related to the magnitude of the pressure gradient. Nevertheless, cell damage remains directly correlated with the pressure gradient, not the duration of stress exposure. The present study thus provides additional evidence that the magnitude of the pressure gradient induces cellular damage in this model of airway reopening. We explore the mechanism for acute damage and also demonstrate that repeated reopening and closure is shown to damage the epithelial cell layer, even under conditions that would not lead to extensive damage from a single reopening event.
Enhanced insulin action on glucose transport and insulin signaling in 7-day unweighted rat soleus muscle
Authors: O'Keefe Matthew P. , Perez Felipe R. , Sloniger Julie A. , Tischler Marc E. , Henriksen Erik J.
Published: March 2004

Hindlimb suspension (HS), a model of simulated weightlessness, enhances insulin action on glucose transport in unweighted rat soleus muscle. In the present study, we tested the hypothesis that these changes in glucose transport in 3- and 7-day HS soleus of juvenile, female Sprague-Dawley rats were due to increased functionality of insulin signaling factors, including insulin receptor (IR), IR substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI3-kinase), and Akt. Insulin-stimulated (2 mU/ml) glucose transport was significantly ( P < 0.05) enhanced in 3- and 7-day HS soleus by 59 and 113%, respectively, compared with weight-bearing controls. Insulin-stimulated tyrosine phosphorylation of IR and Ser473phosphorylation of Akt was not altered by unweighting. Despite decreased (34 and 64%) IRS-1 protein in 3- and 7-day HS soleus, absolute insulin-stimulated tyrosine phosphorylation of IRS-1 was not diminished, indicating relative increases in IRS-1 phosphorylation of 62 and 184%, respectively. In the 7-day HS soleus, this was accompanied by increased (47%) insulin-stimulated IRS-1 associated with the p85 subunit of PI3-kinase. Interestingly, the enhanced insulin-stimulated glucose transport in the unweighted soleus was not completely inhibited (89–92%) by wortmannin, a PI3-kinase inhibitor. Finally, protein expression and activation of p38 MAPK, a stress-activated serine/threonine kinase associated with insulin resistance, was decreased by 32 and 18% in 7-day HS soleus. These results indicate that the increased insulin action on glucose transport in the 7-day unweighted soleus is associated with increased insulin signaling through IRS-1 and PI3-kinase and decreased p38 MAPK protein expression. However, PI3-kinase-independent mechanisms must also play a small role in this adaptive response to HS.
Ventilatory, cerebrovascular, and cardiovascular interactions in acute hypoxia: regulation by carbon dioxide
Authors: Ainslie Philip N. , Poulin Marc J.
Published: March 2004

This study examined the effect of high, normal, and uncontrolled end-tidal Pco2 (PetCO2) on the ventilatory, peak cerebral blood flow velocity ( V̄p), and mean arterial blood pressure (MAP) responses to acute hypoxia. Nine healthy subjects undertook, in random order, three hypoxic protocols (end-tidal Po2 was held at eight steps between 300 and 45 Torr) in conditions of hypercapnia, isocapnia, or poikilocapnia (PetCO2 +7.5 Torr, +1.0 Torr, or uncontrolled, respectively). Transcranial Doppler ultrasound was used to measure V̄p in the middle cerebral artery. The slopes of the linear regressions of ventilation, V̄p, and MAP with arterial O2 saturation were significantly greater in hypercapnia than in both isocapnia and poikilocapnia ( P < 0.05). Strong, significant correlations were observed between ventilation, V̄p, and MAP with each PetCO2 condition. These data suggest that 1) a high acute hypoxic ventilatory response (AHVR) decreases the acute hypoxic cerebral blood flow responses during poikilocapnia hypoxia, due to hypocapnic-induced cerebral vasoconstriction; and 2) in hypercapnic hypoxia, a high AHVR is associated with a high acute hypoxic cerebral blood flow response, demonstrating a linkage of individual sensitivities of ventilation and cerebral blood flow to the interaction of PetCO2 and hypoxia. In summary, the between-individual variability in AHVR is shown to be firmly linked to the variability in V̄p and MAP responses to hypoxia. Individuals with a high AHVR are found also to have high V̄p and MAP responses to hypoxia.
Functional Nicotinic Acetylcholine Receptors on Subplate Neurons in Neonatal Rat Somatosensory Cortex
Authors: Hanganu Ileana L. , Luhmann Heiko J.
Published: March 2004

The establishment of cortical synaptic circuits during early development requires the presence of subplate neurons (SPn's), a heterogeneous population of neurons capable to integrate and process synaptic information from the thalamus, cortical plate, and neighboring SPn's. An accumulation of cholinergic afferents and nicotinic acetylcholine receptors (nAChRs) has been documentated in the subplate around birth. To assess the developmental role of the cholinergic innervation onto SPn's, we used whole cell patch-clamp recordings of visually identified and biocytin-labeled SPn's in neonatal rat somatosensory cortex. Functional nAChRs were present in 92% of the investigated SPn's. Activation of postsynaptic nAChRs by local application of agonists elicited a brief membrane depolarization associated with a barrage of action potentials and large inward currents reversing around 0 mV. According to our pharmacological data, excitation of SPn's is mediated by α4β2 receptors. In contrast, functional α7 nAChRs could not be identified on SPn's. Activation of nAChRs affected neither the spontaneous synaptic activity of SPn's nor the synaptic connections between thalamus and SPn's and within subplate. Nicotine, at concentrations reaching the developing brain by maternal smoking, induced a severe desensitization of nAChRs and an increase in the baseline noise. These results indicate that nAChR-mediated excitation of SPn's may stabilize the developing synaptic circuits and suggest the involvement of nAChRs located on SPn's in the fetal tobacco syndrome.
Dorsal Neck Muscle Vibration Induces Upward Shifts in the Endpoints of Memory-Guided Saccades in Monkeys
Authors: Corneil Brian D. , Andersen Richard A.
Published: March 2004

Producing a movement in response to a sensory stimulus requires knowledge of the body's current configuration, and spindle organs embedded within muscles are a primary source of such kinesthetic information. Here, we sought to develop an animal model of kinesthetic illusions induced by mechanically vibrating muscles as a first step toward a mechanistic understanding of how kinesthesia is integrated into neural plans for action. We elected to examine the effects of mechanical vibration of dorsal neck muscles in head-restrained monkeys performing memory-guided saccades requiring them to look to the remembered location of a flashed target only after an imposed delay. During the delay on one-half of all trials, mechanical vibration (usually 1,500 ms in duration, 200 μm in amplitude, 100 Hz in frequency) was applied to the dorsal aspect on one side of the monkey's neck. We compared the metrics of such vibration saccades to control saccades without vibration during the delay interval. Relative to control saccades, the endpoints of vibration saccades were shifted consistently upward, even though the variability in saccadic endpoints was unaltered. Although the stability of the eye was compromised during the delay interval of vibration trials, as evidenced by an increased incidence of upward drifts and downward microsaccades, vibration saccades displayed different metrics than control saccades, including an upwardly deviated radial direction and increased vertical amplitude. The influence of variations in the duration (500–2,500 ms), amplitude (100–300 μm), or frequency (75–125 Hz) of vibration scaled well with the presumed change in spindle activity entrained by vibration. Comparisons of the profile of these results are made to the human literature. We conclude that neck muscle vibration induces alterations in oculomotor performance in monkeys consistent with a central interpretation of illusory neck flexion and downward gaze deviation due to increased activation in the spindles of neck extensor muscles.
Precise Temporal Responses in Whisker Trigeminal Neurons
Authors: Jones Lauren M. , Lee SooHyun , Trageser Jason C. , Simons Daniel J. , Keller Asaf
Published: March 2004

The ability of rats using their whiskers to perform fine tactile discrimination rivals that of humans using their fingertips. Rats must perform these discriminations rapidly and accurately while palpating the environment with their whiskers. This suggests that whisker-derived inputs produce a robust and reliable code, capable of capturing complex, high-frequency information. The first neural representation of whisker-derived stimulus information is in primary afferent neurons of the trigeminal ganglion. Here we demonstrate that there is a continuum of direction-dependent response profiles in trigeminal neurons and provide the first quantitative analysis of the encoding of complex stimuli by these neurons. We show that all classes of trigeminal ganglion neurons respond with highly reproducible temporal spike patterns to transient stimuli. Such a robust coding mechanism may allow rapid perception of complex tactile features.