bluuebunny/crossref_metadata_2025 huggingface dataset:
Endogenous IGF-I protects human intestinal smooth muscle cells from apoptosis by regulation of GSK-3β activity
Authors: Kuemmerle John F.
Published: August 2004

We have previously shown that endogenous IGF-I regulates human intestinal smooth muscle cell proliferation by activation of phosphatidylinositol 3 (PI3)-kinase- and Erk1/2-dependent pathways that jointly regulate cell cycle progression and cell division. Whereas insulin-like growth factor-I (IGF-I) stimulates PI3-kinase-dependent activation of Akt, expression of a kinase-inactive Akt did not alter IGF-I-stimulated proliferation. In other cell types, Akt-dependent phosphorylation of glycogen synthase kinase-3β (GSK-3β) inhibits its activity and its ability to stimulate apoptosis. The aim of the present study was to determine whether endogenous IGF-I regulates Akt-dependent GSK-3β phosphorylation and activity and whether it regulates apoptosis in human intestinal muscle cells. IGF-I elicited time- and concentration-dependent GSK-3β phosphorylation (inactivation) that was measured by Western blot analysis using a phospho-specific GSK-3β antibody. Endogenous IGF-I stimulated GSK-3β phosphorylation and inhibited GSK-3β activity (measured by in vitro kinase assay) in these cells. IGF-I-dependent GSK-3β phosphorylation and the resulting GSK-3β inactivation were mediated by activation of a PI3-kinase-dependent, phosphoinositide-dependent kinase-1 (PDK-1)-dependent, and Akt-dependent mechanism. Deprivation of serum induced β-catenin phosphorylation, increased in caspase 3 activity, and induced apoptosis of muscle cells, which was inhibited by either IGF-I or a GSK-3β inhibitor. Endogenous IGF-I inhibited β-catenin phosphorylation, caspase 3 activation, and apoptosis induced by serum deprivation. IGF-I-dependent inhibition of apoptosis, similar to GSK-3β activity, was mediated by a PI3-kinase-, PDK-1-, and Akt-dependent mechanism. We conclude that endogenous IGF-I exerts two distinct but complementary effects on intestinal smooth muscle cell growth: it stimulates proliferation and inhibits apoptosis. The growth of intestinal smooth muscle cells is regulated jointly by the net effect of these two processes.
Accelerated intestinal transit in inbred mice with an increased number of interstitial cells of Cajal
Authors: Bellier Sylvain , Da Silva Nelly R. , Aubin-Houzelstein Geneviève , Elbaz Colette , Vanderwinden Jean-Marie , Panthier Jean-Jacques
Published: August 2004

The interstitial cells of Cajal (ICC) play an important role in coordinating intestinal motility, and structural alterations in ICC are found in several human digestive diseases. Mouse models with defects in ICC allow a better understanding of their functions. We investigated the pattern of intestinal motility and the distribution of ICC in the PRM/Alf inbred mouse strain, characterized by a selective intestinal lengthening. In PRM/Alf mice, the digestive transit time, evaluated by using thermophilic Bacillus subtilis spores, was normal, indicating accelerated transit. The contractility and slow-wave frequency, recorded on isolated segments from the proximal small intestine, were significantly increased. The number of ICC was also significantly higher along the small intestine and the colon. The concomitant increase of the contractility, the slow-wave frequency, and the number of ICC is consistent with the proposal of a role of ICC number increase in the higher intestinal transit speed. The PRM/Alf model should be useful to further investigate the roles of ICC in the control of digestive motility.
Unequal effects of renin-angiotensin system inhibitors in acute cardiac dysfunction induced by isoproterenol
Authors: Ohta Takafumi , Hasebe Naoyuki , Tsuji Shiro , Izawa Kazuma , Jin Yin-Tie , Kido Shinsuke , Natori Syunsuke , Sato Motohiko , Kikuchi Kenjiro
Published: August 2004

Several clinical trials have demonstrated that angiotensin-converting enzyme inhibitor (ACEI) and angiotensin II type 1 receptor blocker (ARB) are equally effective in the treatment of chronic heart failure. However, this has not been confirmed for acute cardiac dysfunction. We examined whether ACEI or ARB prevents isoproterenol-induced acute left ventricular (LV) dysfunction in dogs. LV dysfunction induced by a large dose of isoproterenol (1 μg·kg−1·min−1, 3-h infusion) was compared in dogs treated with ACEI (temocaprilat) or ARB (olmesartan). Atrial pacing induced a constant heart rate and use of adjustable aortic banding provided a nearly constant afterload. LV systolic function (LV dP/d t, fractional shortening, and ejection fraction) and diastolic function (τ and LV end-diastolic pressure) were significantly deteriorated after isoproterenol infusion. The LV dysfunction was almost totally prevented by ARB but was only partially prevented by ACEI. The partial effect of ACEI was complemented by cotreatment with HOE-140, a bradykinin B2 receptor antagonist. At baseline, the response to low doses of isoproterenol was significantly attenuated by ACEI but not by ARB, and the ACEI-induced attenuation was totally abolished by cotreatment with HOE-140. The response to isoproterenol was significantly attenuated after 3 h of excess isoproterenol loading, and it was almost completely preserved by ARB but not by ACEI. In conclusion, acute LV dysfunction and β-adrenergic desensitization induced by excess isoproterenol administration were almost totally prevented by ARB but only partially prevented by ACEI. These differences were attributable at least in part to bradykinin pathways activated by ACEI administration in acute LV dysfunction.
Inhibition of PLC improves postischemic recovery in isolated rat heart
Authors: Asemu Girma , Dhalla Naranjan S. , Tappia Paramjit S.
Published: August 2004

The Ca2+-dependent PLC converts phosphatidylinositol 4,5-bisphosphate to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Because these products modulate Ca2+ movements in the myocardium, PLC may also contribute to a self-perpetuating cycle that exacerbates cardiomyocyte Ca2+-overload and subsequent cardiac dysfunction in ischemia-reperfusion (I/R). Although we have reported that I/R-induced changes in PLC isozymes might contribute to cardiac dysfunction, the present study was undertaken to examine the beneficial effects of the PLC inhibitor, U-73122, as well as determining the role of Ca2+ on the I/R-induced changes in PLC isozymes. Isolated rat hearts were subjected to global ischemia 30 min, followed by 5 or 30 min of reperfusion. Pretreatment of hearts with U-73122 (0.5 μM) significantly inhibited DAG and Ins(1,4,5)P3 production in I/R and was associated with enhanced recovery of cardiac function as indicated by measurement of left ventricular (LV) end-diastolic pressure (EDP), LV diastolic pressure (LVDP), maximum rate of pressure development (+dP/d tmax), and maximum rate of LV pressure decay (−dP/d tmax). Verapamil (0.1 μM) partially prevented the increase in sarcolemmal (SL) PLC-β1 activity in ischemia and the decrease in its activity during the reperfusion phase as well as elicited a partial protection of the depression in SL PLC-δ1 and PLC-γ1 activities during the ischemic phase and attenuated the increase during the reperfusion period. Although these changes were associated with an improved myocardial recovery after I/R, verapamil was less effective than U-73122. Perfusion with high Ca2+ resulted in the activation of the PLC isozymes studied and was associated with a markedly increased LVEDP and reduced LVDP, +dP/d tmax, and −dP/d tmax. These results suggest that inhibition of PLC improves myocardial recovery after I/R.
Increased tissue Po2and decreased O2delivery and consumption after 80% exchange transfusion with polymerized hemoglobin
Authors: Cabrales Pedro , Tsai Amy G. , Intaglietta Marcos
Published: August 2004

The O2-carrying blood substitute based on polymerized bovine hemoglobin (PBH) was used to determine efficacy in maintaining tissue Po2after an 80% isovolemic blood exchange leading to a hematocrit of 19% [5.4 g Hb/dl from red blood cells (RBCs) and 6.3 g Hb/dl from PBH]. Effects were studied in terms of O2delivery, O2extraction, and tissue Po2at the microcirculatory level at 1, 12, and 24 h after exchange transfusion in awake hamsters prepared with a window chamber model. At 1 h after exchange, arteriolar and venular diameters were decreased compared with baseline. Arteriolar diameter did not fully recover at 12 h after exchange, but venular diameter returned to normal. At 24 h after exchange, arteriolar and venular diameters were not different from baseline. Combining diameter and flow velocity data allowed us to calculate arteriolar and venular flows. At 1 h after exchange, arteriolar and venular flow was reduced compared with baseline. Arteriolar flow was lower at 12 h after exchange and recovered after 24 h. The number of capillaries with RBC passage [functional capillary density (FCD)] at 1 h after exchange with PBH was significantly lower than baseline. FCD remained decreased at 12 h; at 24 h after exchange transfusion, FCD was fully recovered. Tissue Po2was maximal at 1 h after exchange and decreased progressively at 12 and 24 h after exchange. O2release to the tissue was minimal at 1 h and increased at 12 and 24 h after exchange. These results suggest the impairment of tissue O2metabolism after introduction of PBH into the circulation, which is mitigated as PBH concentration declines.
RhoA and Rac1 are both required for efficient wound closure of airway epithelial cells
Authors: Desai Leena P. , Aryal Ashish M. , Ceacareanu Bogdan , Hassid Aviv , Waters Christopher M.
Published: August 2004

Repair of the airway epithelium after injury is critical for restoring normal lung. The reepithelialization process involves spreading and migration followed later by cell proliferation. Rho-GTPases are key components of the wound healing process in many different types of tissues, but the specific roles for RhoA and Rac1 vary and have not been identified in lung epithelial cells. We investigated whether RhoA and Rac1 regulate wound closure of bronchial epithelial cells. RhoA and Rac1 proteins were efficiently expressed in a cell line of human bronchial epithelial cells (16HBE) by adenovirus-based gene transfer. We found that both constitutively active RhoA and dominant negative RhoA inhibited wound healing, suggesting that both activation and inhibition of RhoA interfere with normal wound healing. Overexpression of wild-type Rac1 induced upregulation of RhoA, disrupted intercellular junctions, and inhibited wound closure. Dominant negative Rac1 also inhibited wound closure. Inhibition of the downstream effector of RhoA, Rho-kinase, with Y-27632 suppressed actin stress fibers and focal adhesion formation, increased Rac1 activity, and stimulated wound closure. The activity of both RhoA and Rac1 are influenced by the polymerization state of microtubules, and cell migration involves coordinated action of actin and microtubules. Microtubule depolymerization upon nocodazole treatment led to an increase in focal adhesions and decreased wound closure. We conclude that coordination of both RhoA and Rac1 activity contributes to bronchial epithelial wound repair mechanisms in vitro, that inhibition of Rho-kinase accelerates wound closure, and that efficient repair involves intact microtubules.
Therapeutic effect of in vivo transfection of transcription factor decoy to NF-κB on septic lung in mice
Authors: Matsuda Naoyuki , Hattori Yuichi , Takahashi Yoshika , Nishihira Jun , Jesmin Subrina , Kobayashi Masanobu , Gando Satoshi
Published: August 2004

Nuclear factor-κB (NF-κB) plays a key role in regulating expression of several genes involved in the pathophysiology of endotoxic shock. We investigated whether in vivo introduction of synthetic double-stranded DNA with high affinity for the NF-κB binding site could block expression of genes mediating pulmonary vascular permeation and thereby provide effective therapy for septic lung failure. Endotoxic shock was induced by an intravenous injection of 10 mg/kg Escherichia coli endotoxin in mice. We introduced NF-κB decoy oligodeoxynucleotide (ODN) in vivo 1 h after endotoxic shock by using a gene transfer kit. At 10 h, blood samples were collected for measurement of histamine and for blood-gas analysis. Gene and protein expression levels of target molecules were determined by means of Northern and Western blot analyses, respectively. The transpulmonary flux of125I-labeled albumin was used as an index of lung vascular permeability. Administration of endotoxin caused marked increases in plasma histamine and gene and protein expressions of histidine decarboxylase, histamine H1receptors, and inducible nitric oxide synthase in lung tissues. Elevated lung vascular permeability was also found. Blood-gas analysis showed concurrent decreases in arterial Po2, Pco2, and pH. All of these events induced by endotoxin were significantly inhibited by transfection of NF-κB decoy ODN but not by its mutated (scrambled) form (used as a control). Our results indicate for the first time the potential usefulness of NF-κB decoy ODN for gene therapy of endotoxic shock.
Airway injury in lung disease pathophysiology: selective depletion of airway stem and progenitor cell pools potentiates lung inflammation and alveolar dysfunction
Authors: Reynolds Susan D. , Giangreco Adam , Hong Kyung U. , McGrath Kathleen E. , Ortiz Luis A. , Stripp Barry R.
Published: August 2004

Identification of early events that contribute to the establishment of chronic lung disease has been complicated by the variable involvement of the airway and alveolar compartments in the complex physiology of end-stage disease. In particular, the impact of airway injury on alveolar integrity and function has not been addressed and would be facilitated by development of animal models of lung disease that specifically target a single cell type within the airway epithelium. We have previously demonstrated that ganciclovir treatment of CCtk transgenic mice, which express the herpes simplex thymidine kinase gene under regulation of the mouse Clara cell secretory protein (CCSP) promoter, results in elimination of the airway progenitor and stem cell pools and a consequent failure of airway regeneration that is associated with rapid morbidity and mortality. In this study, we used the CCtk model to test the hypothesis that selective airway injury initiates profound lung dysfunction through mechanisms that compromise alveolar integrity. Results demonstrate that elimination of the CCSP-expressing cell population results in secondary alveolar inflammation, edema, and depletion of the alveolar type II cell population. On the basis of these data we conclude that selective airway injury can serve as the inciting injury in diseases characterized by severely compromised alveolar function.
Estrogen regulates pulmonary alveolar formation, loss, and regeneration in mice
Authors: Massaro Donald , Massaro Gloria DeCarlo
Published: August 2004

Lung tissue elastic recoil and the dimension and number of pulmonary gas-exchange units (alveoli) are major determinants of gas-exchange function. Loss of gas-exchange function accelerates after menopause in the healthy aged and is progressively lost in individuals with chronic obstructive pulmonary disease (COPD). The latter, a disease of midlife and later, though more common in men than in women, is a disease to which women smokers and never smokers may be more susceptible than men; it is characterized by diminished lung tissue elastic recoil and presently irremediable alveolar loss. Ovariectomy in sexually immature rats diminishes the formation of alveoli, and estrogen prevents the diminution. In the present work, we found that estrogen receptor-α and estrogen receptor-β, the only recognized mammalian estrogen receptors, are required for the formation of a full complement of alveoli in female mice. However, only the absence of estrogen receptor-β diminishes lung elastic tissue recoil. Furthermore, ovariectomy in adult mice results, within 3 wk, in loss of alveoli and of alveolar surface area without a change of lung volume. Estrogen replacement, after alveolar loss, induces alveolar regeneration, reversing the architectural effects of ovariectomy. These studies 1) reveal estrogen receptors regulate alveolar size and number in a nonredundant manner, 2) show estrogen is required for maintenance of already formed alveoli and induces alveolar regeneration after their loss in adult ovariectomized mice, and 3) offer the possibility estrogen can slow alveolar loss and induce alveolar regeneration in women with COPD.
Ultrafine carbon black particles stimulate proliferation of human airway epithelium via EGF receptor-mediated signaling pathway
Authors: Tamaoki Jun , Isono Kazuo , Takeyama Kiyoshi , Tagaya Etsuko , Nakata Junko , Nagai Atsushi
Published: August 2004

Exposure to ambient ultrafine particles induces airway inflammatory reactions and tissue remodeling. In this experiment, to determine whether ultrafine carbon black (ufCB) affects proliferation of airway epithelium and, if so, what the mechanism of action is, we studied human primary bronchial epithelial cell cultures. Incubation of cells in the serum-free medium with ufCB increased incorporations of [3H]thymidine and [3H]leucine into cells in a time- and dose-dependent manner. This effect was attenuated by Cu- and Zn-containing superoxide dismutase (Cu/Zn SOD) and apocynin, an inhibitor of NADPH oxidase, and completely inhibited by pretreatment with the epidermal growth factor receptor (EGF-R) tyrosine kinase inhibitors AG-1478 and BIBX-1382, and the mitogen-activated protein kinase kinase inhibitor PD-98059. Transfection of a dominant-negative mutant of H-Ras likewise abolished the effect ufCB. Stimulation with ufCB also induced processing of membrane-anchored proheparin-binding (HB)-EGF, release of soluble HB-EGF into the medium, association of phosphorylated EGF-R and Shc with glutathione- S-transferase-Grb2 fusion protein, and phosphorylation of extracellular signal-regulated kinase (ERK). Pretreatment with AG-1478, [Glu52] Diphtheria toxin, a specific inhibitor of HB-EGF, neutralizing HB-EGF antibody, Cu/Zn SOD, and apocynin each inhibited ufCB-induced ERK activation. These results suggest that ufCB causes oxidative stress-mediated proliferation of airway epithelium, involving processing of HB-EGF and the concomitant activation of EGF-R and ERK cascade.
Regulation of alveolar epithelial cell phenotypes in fetal sheep: roles of cortisol and lung expansion
Authors: Flecknoe Sharon J. , Boland Rochelle E. , Wallace Megan J. , Harding Richard , Hooper Stuart B.
Published: August 2004

Our aim was to determine whether cortisol's effect on alveolar epithelial cell (AEC) phenotypes in the fetus is mediated via a sustained alteration in lung expansion. Chronically catheterized fetal sheep were exposed to 1) saline infusion, 2) cortisol infusion (122–131 days' gestation, 1.5–4.0 mg/day), 3) saline infusion plus reduced lung expansion, or 4) cortisol infusion plus reduced lung expansion. The proportions of type I and II AECs were determined by electron microscopy, and surfactant protein (SP)-A, -B, and -C mRNA levels were determined by Northern blot analysis. Cortisol infusions significantly increased type II AEC proportions (to 38.2 ± 2.2%), compared with saline-infused fetuses (23.8 ± 2.4%), and reduced type I AEC proportions (to 59.0 ± 2.2%), compared with saline-infused fetuses (70.4 ± 2.4%). Reduced lung expansion also increased type II AEC proportions (to 52.9 ± 3.5%) and decreased type I AEC proportions (to 34.2 ± 3.7%), compared with control, saline-infused fetuses. The infusion of cortisol into fetuses exposed to reduced lung expansion tended to further increase type II (to 60.3 ± 2.1%, P = 0.066) and reduce type I AEC (to 26.6 ± 2.3%, P = 0.07) proportions. SP-A, -B, and -C mRNA levels changed in parallel with the changes in type II AEC proportions. These results indicate that cortisol alters the proportion of type I and type II AECs via a mechanism unrelated to the degree of fetal lung expansion. However, reductions in fetal lung expansion appear to have a greater impact on the proportion of AECs than cortisol.
Connective tissue growth factor expression and induction by transforming growth factor-β is abrogated by simvastatin via a Rho signaling mechanism
Authors: Watts Keira L. , Spiteri Monica A.
Published: August 2004

Connective tissue growth factor (CTGF), a potent profibrotic mediator, acts downstream and in concert with transforming growth factor (TGF)-β to drive fibrogenesis. Significant upregulation of CTGF has been reported in fibrogenic diseases, including idiopathic pulmonary fibrosis (IPF), and is partly responsible for associated excessive fibroblast proliferation and extracellular matrix deposition, but no effective therapy exists for averting such fibrogeneic events. Simvastatin has reported putative antifibrotic actions in renal fibroblasts; this study explores such actions on human IPF-derived and normal lung fibroblasts and examines associated driving mechanisms. Simvastatin reduces basal CTGF gene and protein expression in all fibroblast lines, overriding TGF-β induction through inhibition of the cholesterol synthesis pathway. Signaling pathways driving simvastatin's effects on CTGF/TGF-β interaction were evaluated using transient reporter transfection of a CTGF promoter construct. Inhibition of CTGF promoter activity by simvastatin was most marked at 10 μM concentration, reducing activity by 76.2 and 51.8% over TGF-β-stimulated cultures in IPF and normal fibroblasts, respectively. We also show that geranylgeranylpyrophosphate (GGPP), but not farnesylpyrophosphate, induces CTGF promoter activity following simvastatin inhibition by 55.3 and 31.1% over GGPP-negative cultures in IMR90 and IPF-derived fibroblasts, respectively, implicating small GTPase Rho involvement rather than Ras in these effects. Indeed, the specific Rho inhibitor C3 exotoxin significantly ( P < 0.05) suppressed TGF-β-induced CTGF promoter activity in transfected lung fibroblasts, a finding further supported by transfection of dominant-negative and constitutively active RhoA constructs, thus demonstrating that simvastatin through a Rho signaling mechanism in lung fibroblasts can modulate CTGF expression and interaction with TGF-β.
Erectile dysfunction: an early marker for hypertension? A longitudinal study in spontaneously hypertensive rats
Authors: Behr-Roussel Delphine , Gorny Diane , Mevel Katell , Compagnie Sandrine , Kern Patrick , Sivan Virgine , Bernabé Jacques , Bedigian Martin P. , Alexandre Laurent , Giuliano François
Published: August 2004

Erectile dysfunction (ED) is another manifestation of vascular disease. We evaluated the natural history of ED in the spontaneously hypertensive rat (SHR) and the respective participation of associated pathophysiological modifications, i.e., endothelial dysfunction and tissue remodeling. SHR and their normotensive counterparts [Wistar-Kyoto rats (WKY)] of 6, 12, and 24 wk of age ( n = 12) were used to evaluate erectile function, erectile and aortic tissue reactivity, and remodeling. Erectile responses in SHR are reduced at all ages ( P < 0.001). In both aortic and erectile tissues of SHR and WKY, relaxations to ACh are altered progressively with age, although more markedly in SHR. They are decreased at 12 wk of age in erectile tissue of SHR compared with WKY (maximal relaxation: −19.2 ± 2.8% vs. −28.3 ± 3.9%, P < 0.001) but only at 24 wk of age in aortas (−47.9 ± 6.4% vs. −90.5 ± 2.9%, P < 0.001). Relaxations to sodium nitroprusside are unaltered in aortic rings of both strains but enhanced in erectile tissue of SHR at 12 wk of age. Major modifications in the distribution of collagen I, III, and V in SHR occur in both types of tissue and are detectable sooner in erectile tissue compared with aortic tissue. The onset of ED is detectable before the onset of hypertension in the SHR. Structural and functional alterations, while similar, occur earlier in erectile compared with vascular tissue. If confirmed in humans, ED could be an early warning sign for hypertension, and common therapeutic strategies targeting both ED and hypertension could be investigated.
Left vagal stimulation induces dynorphin release and suppresses substance P release from the rat thoracic spinal cord during cardiac ischemia
Authors: Hua Fang , Ardell Jeffrey L. , Williams Carole A.
Published: August 2004

Electrostimulatory forms of therapy can reduce angina that arises from activation of cardiac nociceptive afferent fibers during transient ischemia. This study sought to determine the effects of electrical stimulation of left thoracic vagal afferents (C8–T1 level) on the release of putative nociceptive [substance P (SP)] and analgesic [dynorphin (Dyn)] peptides in the dorsal horn at the T4 spinal level during coronary artery occlusion in urethane-anesthetized Sprague-Dawley rats. Release of Dyn and SP was measured by using antibody-coated microprobes. While Dyn and SP had a basal release, occlusion of the left anterior descending coronary artery only affected SP release, causing an increase from lamina I-VII. Left vagal stimulation increased Dyn release, inhibited basal SP release, and blunted the coronary artery occlusion-induced release of SP. Dyn release reflected activation of descending pathways in the thoracic spinal cord, because vagal afferent stimulation still increased the release of Dyn after bilateral dorsal rhizotomy of T2–T5. These results indicate that electrostimulatory therapy, using vagal afferent excitation, may induce analgesia, in part, via inhibition of the release of SP in the spinal cord, possibly through a Dyn-mediated neuronal interaction.
Simultaneous imaging of [Ca2+]iand intracellular NO production in freshly isolated uterine artery endothelial cells: effects of ovarian cycle and pregnancy
Authors: Yi Fu-Xian , Magness Ronald R. , Bird Ian M.
Published: August 2004

Pregnancy and the follicular phase of the ovarian cycle show elevation of uterine blood flow and associated increases in uterine artery endothelium (UAE) endothelial nitric oxide (NO) synthase (eNOS) expression. Nonetheless, a role for increased NO production during pregnancy and the follicular phase has only been inferred by indirect measures. The recent development of a uterine artery endothelial cell model further suggests that pregnancy is associated with reprogramming of cell signaling, such that eNOS may become more Ca2+sensitive and be subject to regulation by Ca2+-independent kinases. This study describes for the first time the direct and simultaneous monitoring of NO production and intracellular free Ca2+concentration ([Ca2+]i) in freshly isolated UAE from pregnant, follicular, and luteal sheep. The pharmacological agonists ionomycin (calcium ionophore) and thapsigargin (TG; endoplasmic reticulum Ca2+pump inhibitor) were used to maximally elevate [Ca2+]iand fully activate eNOS as a measure of eNOS expression. NO production stimulated by ionomycin (5 μM) and TG (10 μM) were 1.95- and 2.05-fold, respectively, in pregnant-UAE and 1.34- and 1.37-fold in follicular-UAE compared with luteal-UAE. In contrast, the physiological agonist ATP (100 μM) stimulated a 3.43-fold increase in NO in pregnant-UAE and a 1.90-fold increase in follicular-UAE compared with luteal-UAE, suggesting that pregnancy and follicular phase enhance eNOS activation beyond changes in expression in vivo. 2-aminoethoxydiphenyl borate (APB; an inositol 1,4,5-trisphosphate receptor blocker) totally prevented the ATP-induced [Ca2+]iresponse but only partially inhibited NO production. Thus pregnancy-enhanced eNOS activation in UAE is mediated through [Ca2+]i-insensitive pathways as well as through a greater eNOS sensitivity to [Ca2+]i.
Increased short-term food satiation and sensitivity to cholecystokinin in neurotrophin-4 knock-in mice
Authors: Chi Michael M. , Fan Guoping , Fox Edward A.
Published: August 2004

Neurotrophin-4 (NT-4) knockout mice exhibited decreased innervation of the small intestine by vagal intraganglionic laminar endings (IGLEs) and reduced food satiation. Recent findings suggested this innervation was increased in NT-4 knock-in (NT-4KI) mice. Therefore, to further investigate the relationship between intestinal IGLEs and satiation, meal patterns were characterized using solid and liquid diets, and cholecystokinin (CCK) effects on 30-min solid diet intake were examined in NT-4KI and wild-type mice. NT-4KI mice consuming the solid diet exhibited reduced meal size, suggesting increased satiation. However, compensation occurred through increased meal frequency, maintaining daily food intake and body weight gain similar to controls. Mutants fed the liquid diet displayed a decrease in intake rate, again implying increased satiation, but meal duration increased, which led to an increase in meal size. This was compensated for by decreased meal frequency, resulting in similar daily food intake and weight gain as controls. Importantly, these alterations in NT-4KI mice were opposite, or different, from those of NT-4 knockout mice, further supporting the hypothesis that they are specific to vagal afferent signaling. CCK suppressed short-term intake in mutants and controls, but the mutants exhibited larger suppressions at lower doses, implying they were more sensitive to CCK. Moreover, devazepide prevented this suppression, indicating this increased sensitivity was mediated by CCK-1 receptors. These results suggest that the NT-4 gene knock-in, probably involving increased intestinal IGLE innervation, altered short-term feeding, in particular by enhancing satiation and sensitivity to CCK, whereas long-term control of daily intake and body weight was unaffected.
Effect of posture on regional gas exchange in pigs
Authors: Altemeier William A. , McKinney Steve , Krueger Melissa , Glenny Robb W.
Published: August 2004

Although recent high-resolution studies demonstrate the importance of nongravitational determinants for both pulmonary blood flow and ventilation distributions, posture has a clear impact on whole lung gas exchange. Deterioration in arterial oxygenation with repositioning from prone to supine posture is caused by increased heterogeneity in the distribution of ventilation-to-perfusion ratios. This can result from increased heterogeneity in regional blood flow distribution, increased heterogeneity in regional ventilation distribution, decreased correlation between regional blood flow and ventilation, or some combination of the above (Wilson TA and Beck KC, J Appl Physiol 72: 2298–2304, 1992). We hypothesize that, although repositioning from prone to supine has relatively small effects on overall blood flow and ventilation distributions, regional changes are poorly correlated, resulting in regional ventilation-perfusion mismatch and reduction in alveolar oxygen tension. We report ventilation and perfusion distributions in seven anesthetized, mechanically ventilated pigs measured with aerosolized and injected microspheres. Total contributions of pulmonary structure and posture on ventilation and perfusion heterogeneities were quantified by using analysis of variance. Regional gradients of posture-mediated change in ventilation, perfusion, and calculated alveolar oxygen tension were examined in the caudocranial and ventrodorsal directions. We found that pulmonary structure was responsible for 74.0 ± 4.7% of total ventilation heterogeneity and 63.3 ± 4.2% of total blood flow heterogeneity. Posture-mediated redistribution was primarily oriented along the caudocranial axis for ventilation and along the ventrodorsal axis for blood flow. These mismatched changes reduced alveolar oxygen tension primarily in the dorsocaudal lung region.
Lower capillary density but no difference in VEGF expression in obese vs. lean young skeletal muscle in humans
Authors: Gavin Timothy P. , Stallings Howard W. , Zwetsloot Kevin A. , Westerkamp Lenna M. , Ryan Nicholas A. , Moore Rebecca A. , Pofahl Walter E. , Hickner Robert C.
Published: August 2004

Obesity is associated with lower skeletal muscle capillarization and lower insulin sensitivity. Vascular endothelial growth factor (VEGF) is important for the maintenance of the skeletal muscle capillaries. To investigate whether VEGF and VEGF receptor [kinase insert domain-containing receptor (KDR) and Flt-1] expression are lower with obesity, vastus lateralis muscle biopsies were obtained from eight obese and eight lean young sedentary men before and 2 h after a 1-h submaximal aerobic exercise bout for the measurement of VEGF, KDR, Flt-1, and skeletal muscle fiber and capillary characteristics. There were no differences in VEGF or VEGF receptor mRNA at rest between lean and obese muscle. Exercise increased VEGF (10-fold), KDR (3-fold), and Flt-1 (5-fold) mRNA independent of group. There were no differences in VEGF, KDR, or Flt-1 protein between groups. Compared with lean skeletal muscle, the number of capillary contacts per fiber was the same, but lower capillary density (CD), greater muscle cross sectional area, and lower capillary-to-fiber area ratio were observed in both type I and II fibers in obese muscle. Multiple linear regression revealed that 49% of the variance in insulin sensitivity (homeostasis model assessment) could be explained by percentage of body fat (35%) and maximal oxygen uptake per kilogram of fat-free mass (14%). Linear regression revealed significant relationships between maximal oxygen uptake and both CD and capillary-to-fiber perimeter exchange. Although differences may exist in CD and capillary-to-fiber area ratio between lean and obese skeletal muscle, the present results provide evidence that VEGF and VEGF receptor expression are not different between lean and obese muscle.
Epithelial cell distribution and abundance in rhesus monkey airways during postnatal lung growth and development
Authors: Winkle Laura S. Van , Fanucchi Michelle V. , Miller Lisa A. , Baker Gregory L. , Gershwin Laurel J. , Schelegle Edward S. , Hyde Dallas M. , Evans Michael J. , Plopper Charles G.
Published: August 2004

Lung development is both a pre- and postnatal process. Although many lung diseases have their origins in early childhood, few quantitative data are available on the normal growth and differentiation of both the conducting airways and the airway epithelium during the postnatal period. We examined rhesus monkey lungs from five postnatal ages: 4–6 days and 1, 2, 3, and 6 mo. Airways increase significantly in both length and circumference as monkeys increase significantly in body weight from 5 days to 6 mo. In this study we asked: as basement membrane surface area increases, does the epithelial cell organization change? To answer this question, we quantified total epithelial cell mass using high-resolution light micrographs and morphometric techniques on sections from defined airway regions: trachea, proximal intrapulmonary bronchus (generations 1 or 2), and distal intrapulmonary bronchus (generations 6–8). Epithelial thickness decreased in the smaller, more distal, airways compared with trachea but did not change with age in the trachea and proximal bronchus. The volume fraction of all cell types measured did not change significantly. Ciliated cells in the distal bronchus and goblet cells in the trachea both decreased in abundance with increasing age. Overall, the epithelial cell populations changed little in terms of mass or relative abundance to each other during this period of active postnatal lung growth. Regarding the proximal conducting airway epithelium, we conclude that 1) the steady-state abundance is tightly regulated to keep the proportion of cell types constant, and 2) establishment of these cell types occurs before 4–6 days postnatal age. We conclude that growth of the proximal airways occurs primarily in length and lags behind that of the lung parenchyma.
Redistribution of pulmonary EC-SOD after exposure to asbestos
Authors: Tan Roderick J. , Fattman Cheryl L. , Watkins Simon C. , Oury Tim D.
Published: August 2004

Inhalation of asbestos fibers leads to interstitial lung disease (asbestosis) characterized by inflammation and fibrosis. The pathogenesis of asbestosis is not fully understood, but reactive oxygen species are thought to play a central role. Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that protects the lung in a bleomycin-induced pulmonary fibrosis model, but its role has not been studied in asbestos-mediated disease. EC-SOD is found in high levels in the extracellular matrix of lung alveoli because of its positively charged heparin-binding domain. Proteolytic removal of this domain results in clearance of EC-SOD from the matrix of tissues. We treated wild-type C57BL/6 mice with 0.1 mg of crocidolite asbestos by intratracheal instillation and euthanized them 24 h later. Compared with saline- or titanium dioxide-treated control mice, bronchoalveolar lavage fluid (BALF) from asbestos-treated mice contained significantly higher total protein levels and increased numbers of inflammatory cells, predominantly neutrophils, indicating acute lung injury in response to asbestos. Decreased EC-SOD protein and activity were found in the lungs of asbestos-treated mice, whereas more EC-SOD was found in the BALF of these mice. The EC-SOD in the BALF was predominantly in the proteolyzed form, which lacks the heparin-binding domain. This redistribution of EC-SOD correlated with development of fibrosis 14 days after asbestos exposure. These data suggest that asbestos injury leads to enhanced proteolysis and clearance of EC-SOD from lung parenchyma into the air spaces. The depletion of EC-SOD from the extracellular matrix may increase susceptibility of the lung to oxidative stress during asbestos-mediated lung injury.
Insights into the role of interleukin-6 in the induction of hepatic injury after trauma-hemorrhagic shock
Authors: Toth Balazs , Yokoyama Yukihiro , Schwacha Martin G. , George Richard L. , Rue Loring W. , Bland Kirby I. , Chaudry Irshad H.
Published: August 2004

Although systemic interleukin-6 (IL-6) level is elevated, hepatocellular function is impaired and liver injury occurs after trauma-hemorrhage (T-H), it remains unknown whether a causal relationship exists between elevated IL-6 levels and liver injury after T-H. We hypothesized that IL-6 is causative in the development of hepatic dysfunction and injury after T-H. To examine this, adult male Sprague-Dawley rats underwent a 5-cm midline laparotomy and were subjected to hemorrhagic shock (blood pressure = 35 mmHg for ∼90 min), followed by resuscitation (Ringer lactate, 4 times the shed blood volume). At 2, 5, and 24 h thereafter, blood samples were collected and the liver isolated and perfused for 60 min. Portal inflow pressure was measured, and perfusate samples were collected to measure IL-6, alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) levels. A significant positive correlation between plasma levels of IL-6 and ALT and perfusate levels of IL-6 and LDH levels was observed. In a second series of experiments, rats were treated with immunoglobulin G (IgG) or antibodies against rat IL-6 (anti-IL-6) at the onset of resuscitation. At 5 h after resuscitation, anti-IL-6 treatment attenuated the T-H induced increases in plasma ALT and thromboxane B2 (a thromboxane A2 metabolite) levels, and bile flow was normalized to sham levels. Perfusion of livers from normal rats with IL-6 did not alter portal pressure; however, perfusion of a stable thromboxane A2 analog dose dependently increased portal pressure. Thus IL-6 plays a significant role in the induction of hepatic dysfunction and liver injury after T-H that appears to be in part mediated by increased thromboxane A2 levels.
Pretranslational markers of contractile protein expression in human skeletal muscle: effect of limb unloading plus resistance exercise
Authors: Haddad F. , Baldwin K. M. , Tesch P. A.
Published: August 2004

Previously, it has been shown that the human ground-based model consisting of unilateral limb suspension (ULLS) induces atrophy and reduced strength of the affected quadriceps muscle group. Resistance exercise (RE) involving concentric-eccentric actions, in the face of ULLS, is effective in ameliorating these deficits. The goal of the present study was to determine whether alterations in contractile protein gene expression, e.g., myosin heavy chain and actin, as studied at the pretranslational level, provide molecular markers concerning the deficits that occur in muscle mass/volume during ULLS, as well as its maintenance in response to ULLS plus RE. Muscle biopsies were obtained from the vastus lateralis muscle of 31 middle-aged men and women before and after 5 wk of ULLS, ULLS plus RE, or RE only. The RE paradigm consisted of 12 sessions of 4 sets of 7 concentric-eccentric knee extensions. Our findings show that there were net deficits in total RNA, total mRNA, and actin and myosin heavy chain mRNA levels of expression after ULLS ( P < 0.05), whereas these alterations were blunted in the two groups receiving RE. Additional observations involving IGF-I and its associated receptor and binding proteins suggest that RE postures the skeletal muscle for signaling processes that favor a greater anabolic state relative to that observed in the ULLS group. Collectively, these findings suggest that molecular markers of contractile protein gene expression serve as useful subcellular indicators for ascertaining the underlying mechanisms regulating alterations in muscle mass in human subjects in response to altered loading states.
Effect of pancreatic and leukocyte elastase on hydraulic conductivity in lung interstitial segments
Authors: Houtz P. K. , Jones P. D. , Aronson N. E. , Richardson L. M. , Lai-Fook S. J.
Published: August 2004

Elastase-induced changes in flow were used to quantify the degradation of lung interstitial elastin. Degassed rabbit lungs were inflated with silicon rubber via airways and vessels. The lungs were cut into 1-cm-thick sections. Two chambers were bonded to each section to enclose the interstitium surrounding an arterial segment. Flow of albumin solution (0–5 g/dl) between the chambers was followed by that of the albumin solution with 0.25 g/dl pancreatic elastase solution. Driving pressure was 5 cmH20, and mean interstitial pressure was either 0 or 10 cmH2O. Elastase caused an increase in flow in ∼70% of the interstitial segments and a reduction in flow in the remaining segments. The elastase-induced response in flow was independent of both albumin concentration and mean interstitial pressure. Leukocyte elastase (5 units/dl) produced flow responses similar to those of 0.25 g/dl pancreatic elastase. The increased flow of leukocyte elastase was reduced by a subsequent flow with 0.25 g/dl pancreatic elastase but enhanced by a subsequent flow with a 10-fold lower concentration. A change in the order of the elastase flows reversed the concentration-dependent responses. This behavior suggests a complex interaction among the interstitial fibers after degradation by pancreatic and leukocyte elastase. Endogenous elastase-induced increases in interstitial permeability might affect blood-lymph barrier permeability, whereas elastase-induced cessation of flow might be related to the alveolar septal wall destruction observed in emphysema.
Effect of small flow reversals on aerosol mixing in the alveolar region of the human lung
Authors: Darquenne Chantal , Prisk G. Kim
Published: August 2004

It has been suggested that irreversibility of alveolar flow combined with a stretched and folded pattern of streamlines can lead to a sudden increase in mixing in the lung. To determine whether this phenomenon is operative in the human lung in vivo, we performed a series of bolus studies with a protocol designed to induce complex folding patterns. Boli of 0.5- and 1-μm-diameter particles were inhaled at penetration volumes (Vp) of 300 and 1,200 ml in eight subjects during short periods of microgravity aboard the National Aeronautics and Space Administration Microgravity Research Aircraft. Inspiration was from residual volume to 1 liter above 1 G functional residual capacity. This was followed by a 10-s breathhold, during which up to seven 100-ml flow reversals (FR) were imposed at Vp = 300 ml and up to four 500-ml FR at Vp = 1,200 ml, and by an expiration to residual volume. Bolus dispersion and deposition were calculated from aerosol concentration and flow rate continuously monitored at the mouth. There was no significant increase in dispersion and deposition with increasing FR except for dispersion between 0 and 7 FR at Vp = 300 ml with 0.5-μm-diameter particles, and this increase was small. This suggested that either the phenomenon of stretch and fold did not occur within the number of FR we performed or that it had already occurred during the one breathing cycle included in the basic maneuver. We speculate that the phenomenon occurred during the basic maneuver, which is consistent with the high degree of dispersion and deposition observed previously in microgravity.
Insulin-like growth factor-2 genotype, fat-free mass, and muscle performance across the adult life span
Authors: Schrager Matthew A. , Roth Stephen M. , Ferrell Robert E. , Metter E. Jeffrey , Russek-Cohen Estelle , Lynch Nicole A. , Lindle Rosemary S. , Hurley Ben F.
Published: August 2004

The influence of insulin-like growth factor-2 ( IGF2) genotype on total body fat-free mass (FFM), muscle strength, and sustained power (SP) was evaluated repeatedly at ∼2-yr intervals in two cohorts from the Baltimore Longitudinal Study of Aging. Cohort 1 was comprised of 94 men tested for isometric grip strength and SP. Cohort 2 was comprised of 246 men and 239 women tested for total body FFM and isokinetic peak torque. Subjects were retrospectively genotyped for the IGF2 gene's ApaI polymorphism. Differences between genotype groups for total FFM, strength, and SP at first visit, at peak age (35 yr), at age 65, and across the adult age span were analyzed using either two-sample t-tests or mixed-effects models, depending on the specific comparisons made. Isokinetic arm strength at the time of first visit was lower in A/A men than in G/G men ( P < 0.05). Compared with G/G women, A/A women had lower total body FFM, lower isokinetic arm and leg strength at the time of first visit, and lower values at age 35 (all P < 0.05) for these muscle phenotypes. Furthermore, this difference between the genotype groups was maintained at age 65 and across the adult age span ( P < 0.05). No genotype-associated differences in rates of loss of grip strength or SP were found in cohort 1. These results from cohort 2 support the hypothesis that variation within a gene known to influence developing muscle affects muscle mass and muscle function in later life.