Exposure to chlorine (Cl(2)) damages airway and alveolar epithelia, resulting in acute lung injury and reactive airway dysfunction syndrome. We evaluated the efficacy and mechanisms by which arformoterol, a long-term β(2)-agonist, administered after exposure, mitigated the extent of this injury. Exposure of C57BL/6 mice to 400 ppm Cl(2) for 30 minutes increased respiratory system resistance and airway responsiveness to aerosolized methacholine (assessed by FlexiVent) up to 6 days after exposure, and decreased Na(+)-dependent alveolar fluid clearance (AFC). Inducible Nitric Oxide Synthase (iNOS) knockout mice developed similar degrees of airway hyperreactivity as wild-type controls after Cl(2) exposure, indicating that reactive intermediates from iNOS do not contribute to Cl(2)-induced airway dysfunction in our model. Intranasal administration of arformoterol mitigated the Cl(2) effects on airway reactivity and AFC, presumably by increasing lung cyclic AMP level. Arformoterol did not modify the inflammatory responses, as evidenced by the number of inflammatory cells and concentrations of IL-6 and TNF-α in the bronchoalveolar lavage. NF-κB activity (assessed by p65 Western blots and electrophoretic mobility shift assay) remained at control levels up to 24 hours after Cl(2) exposure. Our results provide mechanistic insight into the effectiveness of long-term β(2)-agonists in reversing Cl(2)-induced reactive airway dysfunction syndrome and injury to distal lung epithelial cells.

Recent studies suggest that the activity of epithelial sodium channels (ENaC) is increased by phosphatidylinositides, especially phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)). Stimulation of phospholipase C by either adenosine triphosphate (ATP)-activation of purinergic P2Y receptors or epidermal growth factor (EGF)-activation of EGF receptors reduces membrane PI(4,5)P(2), and consequently decreases ENaC activity. Since ATP and EGF may be trapped in cysts formed by the distal tubule, it is possible that ENaC inhibition induced by ATP and EGF facilitates cyst formation in polycystic kidney diseases (PKD). However, some results suggest that ENaC activity is increased in PKD. In contrast to P2Y and EGF receptors, stimulation of insulin-like growth factor-1 (IGF-1) receptor by aldosterone or insulin produces PI(3,4,5)P(3), and consequently increases ENaC activity. The acute effect of aldosterone on ENaC activity through PI(3,4,5)P(3) possibly accounts for the initial feedback for blood volume recovery after hypovolemic hypotension. PI(4,5)P(2) and PI(3,4,5)P(3), respectively, interacts with the N terminus of beta-ENaC and the C terminus of gamma-ENaC. However, whether ENaC selectively binds to PI(4,5)P(2) and PI(3,4,5)P(3) over other anionic phospholipids remains unclear.

We investigated the mechanisms by which chlorine (Cl 2 ) and its reactive byproducts inhibit Na ؉ -dependent alveolar fluid clearance (AFC) in vivo and the activity of amiloridesensitive epithelial Na ؉ channels (ENaC) by measuring AFC in mice exposed to Cl 2 (0 -500 ppm for 30 min) and Na ؉ and amiloride-sensitive currents (I Na and I amil , respectively) across Xenopus oocytes expressing human ␣-, ␤-, and ␥-ENaC incubated with HOCl (1-2000 M). Both Cl 2 and HOCl-derived products decreased AFC in mice and whole cell and single channel I Na in a dose-dependent manner; these effects were counteracted by serine proteases. Mass spectrometry analysis of the oocyte recording medium identified organic chloramines formed by the interaction of HOCl with HEPES (used as an extracellular buffer). In addition, chloramines formed by the interaction of HOCl with taurine or glycine decreased I Na in a similar fashion. Preincubation of oocytes with serine proteases prevented the decrease of I Na by HOCl, whereas perfusion of oocytes with a synthetic 51-mer peptide corresponding to the putative furin and plasmin cleaving segment in the ␥-ENaC subunit restored the ability of HOCl to inhibit I Na . Finally, I Na of oocytes expressing wild type ␣-and ␥-ENaC and a mutant form of ␤ENaC (S520K), known to result in ENaC channels locked in the open position, were not altered by HOCl. We concluded that HOCl and its reactive intermediates (such as organic chloramines) inhibit ENaC by affecting channel gating, which could be relieved by proteases cleavage.The balance of fluid covering the respiratory and alveolar epithelia is determined in part by the ability of these cells to transport sodium (Na ϩ ) and chloride (Cl Ϫ ) ions in a vectorial fashion. Active Na ϩ reabsorption across lung epithelia requires the coordinated entry of Na ϩ ions through cation-and Na ϩ -selective amiloride-sensitive channels (ENaC) 5 located at the apical membranes, their extrusion across the basolateral membranes by the electrogenic Na ϩ -K ϩ -ATPase, and the passive movement of K ϩ ions through basolateral K ϩ channels. The entry of Na ϩ ions through apical pathways is thought to be the rate-limiting step in this process (1-3). To preserve neutrality, Cl Ϫ ions follow Na ϩ ions both through transcellular and paracellular pathways (4, 5). The coordinated movement of Na ϩ and Cl Ϫ ions creates an oncotic gradient favoring the absorption of alveolar fluid.Injury to either apical or basolateral pathways by partially reduced intermediates may lead to impairment of fluid reabsorption, which in turn may result in pulmonary edema, hypoxemia, and eventually death from respiratory failure (6 -9). One such specie is hypochlorous acid (HOCl) 6 , which may be generated either endogenously or exogenously. Millimolar concentrations of HOCl may be generated by activated neutrophils and eosinophils by the catalytic actions of neutrophil-and eosinophil-derived myeloperoxidases on chloride (Cl Ϫ ) and hydrogen peroxide (H 2 O 2 ) in close proximity of the apical and basolatera...

Background: Multidrug resistance proteins (MRPs) and the cystic fibrosis transmembrane conductance regulator (CFTR) are thermodynamically distinct ATP-binding cassette (ABC) transporters. Results: Structural elements that couple ATP binding to channel opening in the CFTR channel also facilitate MRP drug export. Conclusion: MRPs and CFTR share components of a conserved activation mechanism. Significance: Allosteric hot spots suggest mechanistic similarities between thermodynamically distinct ABC transporters.

Hemorrhagic stroke, including intracerebral hemorrhage (ICH), is a devastating subtype of stroke; yet, effective clinical treatment is very limited. Accumulating evidence has shown that mild to moderate hypothermia is a promising intervention for ischemic stroke and ICH. Current physical cooling methods, however, are less efficient and often impractical for acute ICH patients. The present investigation tested pharmacologically induced hypothermia (PIH) using the second generation neurotensin receptor (NTR) agonist HPI-201 (formerly known as ABS-201) in an adult mouse model with ICH. Acute or delayed administrations of HPI-201 (2 mg/kg bolus injection followed by 2 injections of 1 mg/kg, i.p.) were initiated at 1 or 24 hrs after ICH. HPI-201 induced mild hypothermia within 30 min and maintained body and brain temperatures at 32.7±0.4°C for at least 6 hrs without causing observable shivering. With the 1 hr delayed treatment, HPI-201-induced PIH significantly reduced ICH-induced cell death and brain edema compared to saline-treated ICH animals. When HPI-201-induced hypothermia was initiated 24 hrs after the onset of ICH, it still significantly attenuated brain edema, cell death and blood brain barrier breakdown. HPI-201 significantly decreased the expression of MMP-9, reduced caspase-3 activation, and increased Bcl-2 expression in the ICH brain. Moreover, ICH mice received 1-hr delayed HPI-201 treatment performed significantly better in the neurological behavior test 48 hrs after ICH. All together, these data suggest that systemic injection of HPI-201 is an effective hypothermic strategy that protects the brain from ICH injury with a wide therapeutic window. The protective effect of this PIH therapy is partially mediated through the alleviation of apoptosis and neurovascular damage. We suggest that pharmacological hypothermia using the newly developed neurotensin analogs is a promising therapeutic treatment for ICH.

A variety of studies have shown that Na 1 reabsorption across epithelial cells depends on the protease-antiprotease balance. Herein, we investigate the mechanisms by which a 1 -antitrypsin (A1AT), a major anti-serine protease in human plasma and lung epithelial fluid and lacking a Kunitz domain, regulates amiloridesensitive epithelial Na 1 channel (ENaC) function in vitro and in vivo. A1AT (0.05 mg/ml 5 1 mM) decreased ENaC currents across Xenopus laevis oocytes injected with human a,b,g-ENaC (hENaC) cRNAs, and human lung Clara-like (H441) cells expressing native ENaC, in a partially irreversible fashion. A1AT also decreased ENaC singlechannel activity when added in the pipette but not in the bath solutions of ENaC-expressing oocytes patched in the cell-attached mode. Incubation of A1AT with peroxynitrite (ONOO 2 ), an oxidizing and nitrating agent, abolished its antiprotease activity and significantly decreased its ability to inhibit ENaC. Intratracheal instillation of normal but not ONOO 2 -treated A1AT (1 mM) in C57BL/6 mice also decreased Na 1 -dependent alveolar fluid clearance to the same level as amiloride. Incubation of either H441 cells or ENaC-expressing oocytes with normal but not ONOO 2 -treated A1AT decreased their ability to cleave a substrate of serine proteases. A1AT had no effect on amiloride-sensitive currents of oocytes injected with hENaC bearing Liddle mutations, presumably because these channels remain at the surface longer than the wild-type channels. These data indicate that A1AT may be an important modulator of ENaC activity and of Na 1 -dependent fluid clearance across the distal lung epithelium in vivo by decreasing endogenous protease activity needed to activate silent ENaC.Keywords: alveolar fluid clearance; serine proteases; H441 cells; Xenopus oocytes; ENaCThe amiloride-sensitive epithelial Na 1 channels (ENaCs) play an important role in Na 1 and fluid homeostasis across a number of epithelial cells, including those in airway and alveolar spaces (1, 2). ENaC mRNA, protein levels, and ENaC activity are regulated by a variety of hormones (such as aldosterone and dexamethasone), second messengers cAMP/protein kinase A, and reactive intermediates (2-4). In addition, endogenous channelactivating proteases (CAPs), as well as proteases released by inflammatory cells (trypsin, elastase), activate ENaC either by cleaving critical amino acids in a-and g-ENaC subunits, or by activating signaling pathways (1, 2, 5-7).There has been considerable interest in identifying the mechanisms by which endogenous and exogenous proteases activate ENaC. Aprotinin, a potent and reversible Kunitz-type inhibitor of several serine proteases, including trypsin, plasmin, and kallikreins (8), has been reported to inhibit sodium transport among a variety of epithelial cells, including A6 (9), human bronchial epithelial (HBE) cells (10, 11), and rat and mouse lung alveolar epithelial cells (1). Other Kunitz-type serine protease inhibitors, such as hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2 (plac...

Cystatin C, together with hs-CRP and homocysteine, is an independent risk factor that is important in the prognosis of patients with systolic heart failure.

We used mouse cortical collecting duct principal cells (mpkCCDc14 cell line) as a model to determine whether statins reduce the harmful effects of cyclosporine A (CsA) on the distal nephron. The data showed that treatment of cells with CsA increased transepithelial resistance and that the effect of CsA was abolished by lovastatin. Scanning ion conductance microscopy showed that CsA significantly increased the height of cellular protrusions near tight junctions. In contrast, lovastatin eliminated the protrusions and even caused a modest depression between cells. Western blot analysis and confocal microscopy showed that lovastatin also abolished CsA-induced elevation of both zonula occludens-1 and cholesterol in tight junctions. In contrast, a high concentration of CsA induced apoptosis, which was also attenuated by lovastatin, elevated intracellular ROS via activation of NADPH oxidase, and increased the expression of p47phox. Sustained treatment of cells with lovastatin also induced significant apoptosis, which was attenuated by CsA, but did not elevate intracellular ROS. These results indicate that both CsA and lovastatin are harmful to principal cells of the distal tubule, but via ROS-dependent and ROS-independent apoptotic pathways, respectively, and that they counteract probably via mobilization of cellular cholesterol levels.