American Journal of Physiology最新文献

Heterogeneity of intestinal D-glucose transport is demonstrated using pig jejunal brush-border membrane vesicles in the presence of 100/0 (out/in) mM gradients each of NaCl, NaSCN, and KSCN. Two D-glucose transport systems are kinetically distinguished: high-affinity, low-capacity system 1, which is equivalent to the symporter SGLT1; and low-affinity, high-capacity system 2, which is not a member of the SGLT family but is a D-glucose and D-mannose transporter exhibiting no preference for Na(+) over K(+). A nonsaturable D-glucose uptake component has also been detected; uptake of this component takes place at rates 10 times the rate of components characterizing the classical diffusion marker L-glucose. It is also shown that, in this kinetic work, 1) use of D-glucose-contaminated D-sorbitol as an osmotic replacement cannot cause the spurious appearance of nonexistent transport systems and 2) a large range (>/=50 mM) of substrate concentrations is required to correctly fit substrate saturation curves to distinguish between low-affinity transport systems and physical diffusion.

An understanding of cardiac health and disease requires knowledge of the various factors that control coronary capillary blood flow. An analysis of coronary capillary blood flow based on a complete set of actual data on the capillary anatomy and elasticity does not exist. Previously, a complete set of data on the branching pattern and the vascular geometry of the pig coronary capillary network were obtained in our laboratory. In the present study, we obtained distensibility data on the coronary capillary blood vessels on the epicardial surface in the form of a pressure-diameter relationship using intravital microscopy. A mathematical model of the coronary capillary blood flow was then constructed on the basis of measured anatomic and elasticity data of the coronary capillary network, rheology of blood, physical laws governing blood flow, and appropriate boundary conditions. The constructed model was used to examine the heterogeneity of the spatial distribution of coronary blood flow, which is an important issue in coronary physiology. One interesting result of the model is that the dispersions of pressure and flow are significantly reduced in the presence of capillary cross-connections, and the resistance to flow is reduced as well. Finally, we found that the compliance of the epicardial surface capillary vessels is so small that its effect on the blood pressure drop is negligible in the diastolic state. However, the compliance of the intramyocardial capillaries remains unknown, and the interaction of the muscle contraction and blood vessel elasticity in systole remains to be studied.

Oxidative stress plays a central role in the pathogenesis of acute and chronic pulmonary diseases. Safe sequestration of iron, which participates in the formation of the hydroxyl radical, is crucial in the lung's defense. We used a mouse line defective in the major iron transport protein transferrin to investigate the effect of aberrant iron metabolism on the lung's defense against oxidative injury. The tolerance to hyperoxic lung injury was greater in the hypotransferrinemic than in wild-type mice as documented by histopathology and biochemical indexes for lung damage. There was no increase in the levels of intracellular antioxidants, inflammatory cytokines, and heme oxygenase-1 in the hypotransferrinemic mouse lung compared with those in wild-type mice. However, there were elevated expressions of ferritin and lactoferrin in the lung of hypotransferrinemic mice, especially in the alveolar macrophages. Our results suggest that pulmonary lactoferrin and ferritin protect animals against oxidative stress, most likely via their capacity to sequester iron, and that alveolar macrophages are the key participants in iron detoxification in the lower respiratory tract.

Previously, we isolated a 624-bp cDNA encoding for a 115-amino acid preprosomatostatin containing [Tyr7,Gly10]-somatostatin (SS)-14 (now designated PPSS-II') obtained from the endocrine pancreas (Brockmann bodies) of rainbow trout. In this study we have characterized a second cDNA obtained from trout pancreas that is 600-bp in length and encodes for a 111-amino acid precursor containing [Tyr7,Gly10]-SS-14 (PPSS-II''). The nucleotide and amino acid identity between the two cDNAs is 82.3 and 80.5%, respectively. Both PPSS-II' and PPSS-II'' mRNA were present in esophagus, pyloric ceca, stomach, upper and lower intestine, and pancreas, whereas only SS-II" mRNA was present in brain. PPSS-II'' mRNA was more abundant than PPSS-II' mRNA in pancreas, whereas PPSS-II' mRNA was more abundant than PPSS-II" mRNA in stomach. Fasting increased pancreatic PPSS-II'' mRNA levels but had no effect on the levels of PPSS-II' mRNA. These results indicate the existence of two nonallelic pancreatic SS-II genes that are differentially expressed, both in terms of distribution among tissues and in terms of relative abundance within the tissues.

We have identified the Dri 27 cDNA on the basis of its upregulated expression during rat intestinal development. It encodes a hydrophobic protein of 430 amino acids that shares significant homology with members of the mammalian zinc transporter family ZnT. The murine homologue of Dri 27 (named ZnT4) was recently associated with the mouse mutation "lethal milk." The primary sequence of Dri 27/ZnT4 displays features characteristic of polytopic membrane proteins. In this paper, we show that Dri 27/ZnT4 is localized in the membrane of intracellular vesicles, the majority of which concentrate in the basal cytoplasmic region of polarized enterocytes. A Dri 27/ZnT4 myc-tagged construct, transiently transfected in intestinal Caco-2 cells, partially colocalizes with the transferrin receptor and with the beta-subunits of the clathrin adaptor complexes AP-1 and AP-2 in a subpopulation of endosomal vesicles. By subcloning distinct portions of the protein in frame with glutathione-S-transferase, we also provide experimental evidence of their function as zinc-binding and protein-protein-interaction domains.

Glutathione (GSH), a ubiquitous tripeptide thiol, is a vital intra- and extracellular protective antioxidant in the lungs. The rate-limiting enzyme in GSH synthesis is gamma-glutamylcysteine synthetase (gamma-GCS). The promoter (5'-flanking) region of the human gamma-GCS heavy and light subunits are regulated by activator protein-1 and antioxidant response elements. Both GSH and gamma-GCS expression are modulated by oxidants, phenolic antioxidants, and inflammatory and anti-inflammatory agents in lung cells. gamma-GCS is regulated at both the transcriptional and posttranscriptional levels. GSH plays a key role in maintaining oxidant-induced lung epithelial cell function and also in the control of proinflammatory processes. Alterations in alveolar and lung GSH metabolism are widely recognized as a central feature of many inflammatory lung diseases including chronic obstructive pulmonary disease (COPD). Cigarette smoking, the major factor in the pathogenesis of COPD, increases GSH in the lung epithelial lining fluid of chronic smokers, whereas in acute smoking, the levels are depleted. These changes in GSH may result from altered gene expression of gamma-GCS in the lungs. The mechanism of regulation of GSH in the epithelial lining fluid in the lungs of smokers and patients with COPD is not known. Knowledge of the mechanisms of GSH regulation in the lungs could lead to the development of novel therapies based on the pharmacological or genetic manipulation of the production of this important antioxidant in lung inflammation and injury. This review outlines 1) the regulation of cellular GSH levels and gamma-GCS expression under oxidative stress and 2) the evidence for lung oxidant stress and the potential role of GSH in the pathogenesis of COPD.

We examined the effects of superoxide anion (O(-2)) on the intracellular Ca(2+) concentration in cultured human nasal epithelial cells. The cells were exposed to O(-2) by pretreatment with xanthine (X) and xanthine oxidase (XO); control cells were treated with X alone. When Ca(2+)-containing Krebs solution was reperfused in the thapsigargin-treated, store-depleted cells, reapplication-induced intracellular Ca(2+) concentration elevation was significantly smaller in X/XO-treated cells than in the control cells, suggesting that O(-2) impairs Ca(2+) release-activated Ca(2+) entry (CRAC). Bath application of ATP induced a steep Ca(2+) transient in both control and X/XO-treated cells. However, the concentration-response curve of the ATP-induced Ca(2+) transient was shifted to a higher concentration in X/XO-treated cells. The impairments of CRAC and ATP-induced Ca(2+) transient induced by X/XO were reversed by superoxide dismutase. Furthermore, all these X/XO-induced effects were also observed in cells pretreated with pyrogallol, also an O(-2) donor. These results indicate that O(-2) impairs at least two mechanisms involved in Ca(2+) mobilization in human nasal epithelial cells, i.e., CRAC and ATP-induced Ca(2+) release.

Airway epithelium explants from cystic fibrosis (CF) patients and non-CF subjects formed monolayered spheres, with the apical ciliated cell membrane facing the bath and the basolateral cell membrane pointing toward a fluid-filled lumen. With the use of two microelectrodes, transepithelial potential difference and changes in potential difference in response to passage of current pulses were recorded, and epithelial resistance and the equivalent short-circuit current were calculated. Non-CF control potential difference and short-circuit current values were significantly lower than the CF values, and amiloride inhibited both values. Fluid transport rates were calculated from repeated measurements of spheroid diameters. The results showed that 1) non-CF and CF spheroids absorbed fluid at identical rates (4.4 microl x cm(-2) x h(-1)), 2) amiloride inhibited fluid absorption to a lower residual level in non-CF than in CF spheroids, 3) Cl(-)-channel inhibitors increased fluid absorption in amiloride-treated non-CF spheroids to a level equal to that of amiloride-treated CF spheroids, 4) hydrochlorothiazide reduced the amiloride-insensitive fluid absorption in both non-CF and CF spheroids, and 5) osmotic water permeabilities were equal in non-CF and CF spheroids ( approximately 27 x 10(-7) cm x s(-1) x atm(-1)).

The vascular effects of carbon monoxide (CO) resemble those of nitric oxide (NO), but it is unknown whether the two messengers converge or exhibit reciprocal feedback regulation. These questions were examined in microdissected perfused renal resistance arteries (RRA) studied using NO-sensitive microelectrodes. Perfusion of RRA with buffers containing increasing concentrations of CO resulted in a biphasic release of NO. The NO response peaked at 100 nM CO and then declined to virtually zero at 10 microM. When a series of 50-s pulses of 100 nM CO were applied repeatedly (150-s interval), the amplitude of consecutive NO responses was diminished. NO release from RRA showed dependence on L-arginine but not D-arginine, and the responses to CO were inhibited by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthases (NOS). CO (100 nM) also suppressed NO release induced by 100 microM carbachol, a potent agonist for endothelial NOS (eNOS). RRA from rats in which endogenous CO production from inducible HO was elevated (cobalt chloride 12 h prior to study) also showed suppressed responses to carbachol. Furthermore, responses consistent with these findings were obtained in juxtamedullary afferent arterioles perfused in vitro, where the vasodilatory response to CO was biphasic and the response to acetylcholine was blunted. Collectively, these data suggest that the CO-induced NO release could be attributed to either stimulation of eNOS or to NO displacement from a cellular storage pool. To address this, direct in vitro measurements with an NO-selective electrode of NO production by recombinant eNOS revealed that CO dose-dependently inhibits NO synthesis. Together, the above data demonstrate that, whereas high levels of CO inhibit NOS activity and NO generation, lower concentrations of CO induce release of NO from a large intracellular pool and, therefore, may mimic the vascular effects of NO.

The effects of copper on tight-junction permeability were investigated in human intestinal Caco-2 cells, monitoring transepithelial electrical resistance and transepithelial passage of mannitol. Apical treatment of Caco-2 cells with 10-100 microM CuCl(2) (up to 3 h) produced a time- and concentration-dependent increase in tight-junction permeability, reversible after 24 h in complete medium in the absence of added copper. These effects were not observed in cells treated with copper complexed to L-histidine [Cu(His)(2)]. The copper-induced increase in tight-junction permeability was affected by the pH of the apical medium, as was the apical uptake of (64)CuCl(2), both exhibiting a maximum at pH 6.0. Treatment with CuCl(2) produced a concentration-dependent reduction in the staining of F actin but not of the junctional proteins zonula occludens-1, occludin, and E-cadherin and produced ultrastructural alterations to microvilli and tight junctions that were not observed after treatment with up to 200 microM Cu(His)(2) for 3 h. Overall, these data point to an intracellular effect of copper on tight junctions, mediated by perturbations of the F actin cytoskeleton.