RNA interference for α-ENaC inhibits rat lung fluid absorption in vivo

2006 ◽  
Vol 290 (4) ◽  
pp. L649-L660 ◽  
Author(s):  
Tianbo Li ◽  
Hans G. Folkesson
Keyword(s):  
Protein Expression ◽  
Conclusive Evidence ◽  
Organ Specificity ◽  
Response Analysis ◽  
Na Channel ◽  
Fluid Absorption ◽  
Alveolar Epithelial ◽  
Lung Fluid ◽  
Stimulation Of

We used siRNA against the α-ENaC (epithelial Na channel) subunit to investigate ENaC involvement in lung fluid absorption in rats by the impermeable tracer technique during baseline and after β-adrenoceptor stimulation by terbutaline. Terbutaline stimulation of lung fluid absorption increased fluid absorption by 165% in pSi-0-pretreated rat lungs (irrelevant siRNA-generating plasmid). Terbutaline failed to increase lung fluid absorption in rats given the specific α-ENaC siRNA-generating plasmid (pSi-4). pSi-4 pretreatment reduced baseline lung fluid absorption by ∼30%. α-ENaC was undetectable in pSi-4-pretreated lungs, regardless of condition but was normal in pSi-0-pretreated lungs. We carried out a dose-response analysis where rats were given 0–200 μg/kg body wt pSi-4, and α-ENaC mRNA and protein expressions were analyzed. To reach IC50for α-ENaC mRNA expression, 32 μg/kg body wt pSi-4 was needed, and to reach IC50for α-ENaC protein expression, 59 μg/kg body wt pSi-4 was needed. We tested for lung tissue specificity and found no changes in β-ENaC expression, at either mRNA or protein level, as well as no changes in α1-Na-K-ATPase protein expression. We isolated alveolar epithelial type II cells 24 h after in vivo pSi-4 pretreatment. In these cells, α-ENaC mRNA was undetectable, demonstrating that alveolar epithelial ENaC expression was attenuated after intratracheal α-ENaC siRNA-generating plasmid DNA instillation. We tested for organ specificity and found no changes in kidney α- and β-ENaC mRNA and protein expression. Thus we provide conclusive evidence that β-adrenoceptor stimulation of lung fluid absorption is critically ENaC dependent, whereas baseline lung fluid absorption seemed less ENaC dependent.

Involvement of αENaC and Nedd4-2 in the conversion from lung fluid secretion to fluid absorption at birth in the rat as assayed by RNA interference analysis

2007 ◽  
Vol 293 (4) ◽  
pp. L1069-L1078 ◽  
Author(s):  
Tianbo Li ◽  
Shyny Koshy ◽  
Hans G. Folkesson
Keyword(s):  
Extravascular Lung Water ◽  
Fluid Secretion ◽  
Alveolar Epithelial Cells ◽  
Na Channel ◽  
Fluid Absorption ◽  
Lung Water ◽  
Fourfold Increase ◽  
Lung Fluid ◽  
Near Term

To explore interactions between the epithelial Na channel (ENaC) and neural precursor expressed, developmentally downregulated protein 4-2 (Nedd4-2) at the conversion of the rat lung from fluid secretion to absorption at birth, we used small-interfering RNA (siRNA) against αENaC and Nedd4-2. siRNA-generating plasmid DNA (pDNA) was administered via trans-thoracic intrapulmonary (ttip) injection 24 h before ENaC and Nedd4-2 expression, extravascular lung water, and mortality were measured. αENaC mRNA and protein were specifically reduced by ∼65% after pSi-4 injection. Nedd4-2 mRNA and protein were reduced by ∼60% after pSi-N1 injection. Interestingly, αENaC and βENaC mRNA and protein expression were increased after Nedd4-2 silencing. Extravascular lung water was significantly increased after αENaC silencing and reduced after Nedd4-2 silencing. αENaC silencing resulted in a fourfold increase in newborn mortality, whereas silencing Nedd4-2 did not affect mortality. We also isolated distal lung epithelial (DLE) cells after in vivo αENaC or Nedd4-2 silencing and measured αENaC or Nedd4-2 expression in freshly isolated DLE cells. In these DLE cells, there were attenuated αENaC or Nedd4-2 mRNA and protein, thus demonstrating that αENaC and Nedd4-2 silencing occurred in alveolar epithelial cells after ttip injection. We also looked for pDNA by PCR to determine pDNA presence in the lungs and found strong evidence for pDNA presence in both lungs. Thus we provide evidence that ENaC and Nedd4-2 are involved in the transition from lung fluid secretion to fluid absorption near term and at birth.

Mechanisms and sequelae of increased alveolar fluid clearance in hyperoxic rats

1997 ◽  
Vol 272 (3) ◽  
pp. L407-L412 ◽  
Author(s):  
G. Yue ◽  
S. Matalon
Keyword(s):  
Alveolar Epithelial Cells ◽  
Na Channels ◽  
Alveolar Fluid Clearance ◽  
Na Transport ◽  
Alveolar Epithelial ◽  
Lung Fluid ◽  
Isotonic Fluid ◽  
Epithelial Na Channels ◽  
Alveolar Edema

We instilled 4 ml isotonic fluid containing trace amounts of fluorescently labeled dextran (molecular mass 150 kDa) in the lungs of rats exposed to either 85% O(2) for 7 days or to 85% O(2) for 7 days and 100% O(2) for 3 days. We withdrew the fluid every hour for a 3-h period and calculated alveolar fluid clearance (AFC) from changes in dextran concentration. Postinstillation (3 h), AFC values in the control and the two hyperoxic groups were 51 +/- 1, 63 +/- 2, and 62 +/- 3 (SE), respectively (%instilled volume; n > or = 5; P < 0.05). Addition of either 1 mM amiloride or N-ethyl-N-isopropyl amiloride (EIPA) in the instillate decreased the AFC values in all groups 3 h later to approximately 30% of instilled volume. Instillation of phenamil, an irreversible blocker of epithelial Na+ channels into the lungs of rats exposed to 85% O(2) for 7 days and 100% O(2) for 2 days, resulted in a significant increase of their extravascular lung fluid volumes 24 h later. These results demonstrate the existence of EIPA-inhibitable Na+ channels in alveolar epithelial cells in vivo and indicate that an increase in Na+ transport plays an important role in limiting the amount of alveolar edema in O(2)-damaged lungs.

Increased fluid absorption and cell volume in isolated rabbit proximal straight tubules after in vivo DOCA administration

1981 ◽  
Vol 241 (5) ◽  
pp. F502-F508 ◽  
Author(s):  
M. A. Knepper ◽  
M. B. Burg
Keyword(s):  
Proximal Tubule ◽  
Cell Volume ◽  
Fluid Transport ◽  
Sodium Intake ◽  
Plasma Concentrations ◽  
Dietary Sodium ◽  
Fluid Absorption ◽  
Direct Stimulation ◽  
Stimulation Of

To investigate whether mineralocorticoids affect the intrinsic capacity of the proximal tubule to absorb sodium and fluid, rabbits were chronically treated a number of ways to systematically vary plasma concentrations of mineralocorticoid hormones. The rate of fluid absorption and tubule dimensions were measured in superficial S2 segments from these rabbits. Chronic administration of deoxycorticosterone acetate (DOCA) was associated with a 67% increase in fluid absorption and a 29% increase in cell volume per unit tubule length. However, neither adrenalectomy nor low sodium diet significantly affected either fluid absorption or cell volume. Furthermore, marked dietary sodium restriction prevented the response to DOCA. We conclude that the DOCA-induced increases in fluid absorption and cell volume do not result from a direct stimulation of the proximal tubular cells by the steroid but more likely are responses to systemic effects of DOCA administration that are dependent on the level of sodium intake. Thus, we find no evidence for a direct mineralocorticoid stimulation of sodium and fluid transport by the S2 portion of the proximal tubule.

scholarly journals Nasal potential difference to detect Na+channel dysfunction in acute lung injury

2011 ◽  
Vol 300 (3) ◽  
pp. L305-L318 ◽  
Author(s):  
R. Mac Sweeney ◽  
H. Fischer ◽  
D. F. McAuley
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Ion Channel ◽  
Pulmonary Edema ◽  
Potential Difference ◽  
Na Channel ◽  
Alveolar Epithelial ◽  
Nasal Potential Difference ◽  
Respiratory Epithelial

Pulmonary fluid clearance is regulated by the active transport of Na+and Cl−through respiratory epithelial ion channels. Ion channel dysfunction contributes to the pathogenesis of various pulmonary fluid disorders including high-altitude pulmonary edema (HAPE) and neonatal respiratory distress syndrome (RDS). Nasal potential difference (NPD) measurement allows an in vivo investigation of the functionality of these channels. This technique has been used for the diagnosis of cystic fibrosis, the archetypal respiratory ion channel disorder, for over a quarter of a century. NPD measurements in HAPE and RDS suggest constitutive and acquired dysfunction of respiratory epithelial Na+channels. Acute lung injury (ALI) is characterized by pulmonary edema due to alveolar epithelial-interstitial-endothelial injury. NPD measurement may enable identification of critically ill ALI patients with a susceptible phenotype of dysfunctional respiratory Na+channels and allow targeted therapy toward Na+channel function.

In vivo analysis of fluid transport in cystic fibrosis airway epithelia of bronchial xenografts

1996 ◽  
Vol 270 (5) ◽  
pp. C1326-C1335 ◽  
Author(s):  
Y. Zhang ◽  
J. Yankaskas ◽  
J. Wilson ◽  
J. F. Engelhardt
Keyword(s):  
Cystic Fibrosis ◽  
Fluid Transport ◽  
Xenograft Model ◽  
Na Channel ◽  
Transmembrane Conductance Regulator ◽  
Fluid Absorption ◽  
Cystic Fibrosis Airway ◽  
In Vivo Analysis ◽  
Cf Transmembrane Conductance Regulator

An in vivo human bronchial xenograft model system was used to simultaneously analyze electrolyte and fluid transport defects in fully differentiated human cystic fibrosis (CF) and non-CF proximal airways. CF airways demonstrated three discernible defects when compared with non-CF, including 1) a lack of adenosine 3',5'-cylic monophosphate (cAMP)-inducible Cl- secretion, 2) a fourfold higher basal fluid absorption rate, and 3) an altered regulation of fluid absorption in response to amiloride-stimulated changes in Na+ transport. A unique finding in this study demonstrated that treatment of epithelia with amiloride led to a greater than threefold decrease in the rate of fluid absorption in CF tissues as contrasted to a greater than threefold increase in the rate of fluid absorption in non-CF tissues. The removal of apical Na+ from amiloride-treated non-CF xenografts was capable of ablating this amiloride-induced increase in fluid absorption. In light of the recent interactions demonstrated between CF transmembrane conductance regulator (CFTR) and the rat epithelial, amiloride-sensitive Na+ channel, these findings implicate additional complexities between the Na+ conductance pathways and fluid transport in normal and CF proximal airways. Such findings suggest that CFTR may also regulate amiloride-insensitive Na+ channels.

A Novel Role for Thrombin in Hematopoietic Stem/Progenitor Cell Homing.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3374-3374
Author(s):  
Neeta Shirvaikar ◽  
Ali Jalili ◽  
Mariusz Z. Ratajczak ◽  
Anna Janowska-Wieczorek
Keyword(s):  
Protein Expression ◽  
Lipid Rafts ◽  
Leading Edge ◽  
Membrane Lipid ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
And Migration ◽  
Stimulation Of

Abstract Thrombin, an important serine protease, not only plays a pivotal role in platelet aggregation and coagulation, but also through activation of its receptor, seven transmembrane, G-protein-coupled receptor PAR-1, elicits numerous cellular responses in platelets and endothelial cells such as induction of adhesion molecules, production of chemokines, activation of matrix metalloproteinase (MMP)-2, cytoskeletal reorganization and migration. Thrombin is also one of the inflammatory molecules elevated during G-CSF mobilization of hematopoietic stem/progenitor cells (HSPC) and their collection by leukapheresis. We recently reported that components of leukapheresis products including thrombin enhance in vitro chemotaxis of CD34+ cells towards an SDF-1 gradient and in vivo homing to bone marrow (BM) niches in a murine model (Blood2005; 105:40). In this study we investigated whether thrombin enhances the homing-related responses of human HSPC (CD34+ cells) through MMPs, especially membrane-type (MT)1-MMP which is known to be localized on the leading edge of migrating cells and both activates latent proMMPs (MMP-2, -9) and itself has strong pericellular proteolytic activity. We found that stimulation of CD34+ cells with thrombin upregulates mRNA for MT1-MMP and MMP-9 as well as MT1-MMP protein expression (Western blot, flow cytometry) and proMMP-2 and proMMP-9 secretion (zymography). Thrombin was also found to (i) prime trans-Matrigel chemoinvasion of CD34+ cells towards a low SDF-1 gradient (20 ng/mL), which was inhibited by epigallocatechin-3-gallate, a potent inhibitor of MT1-MMP, and (ii) activate MMP-2 in of co-cultures of CD34+ cells with stromal cells (BM fibroblasts and HUVEC) which secrete proMMP-2. We also found that SDF-1 upregulates mRNA and protein expression of MT1-MMP. Moreover, using confocal microscopy we demonstrate for the first time that in CD34+ cells, PAR-1, like CXCR4, is localized in the GM1 fraction of lipid rafts and stimulation of these cells with thrombin as well as SDF-1 increases incorporation of MT1-MMP into membrane lipid rafts. Furthermore, disruption of lipid raft formation by the cholesterol-depleting agent methyl-b-cyclodextrin inhibits MT1-MMP incorporation into membrane lipid rafts and also trans-Matrigel chemoinvasion of CD34+ cells towards SDF-1. Thus we conclude that thrombin, through PAR-1 signalling and the SDF-1-CXCR4 axis, upregulates the incorporation of MT1-MMP into membrane lipid rafts and the interaction of these axes enhances the homing-related responses of HSPC towards SDF-1.

Phosphatidylinositol 4,5-bisphosphate stimulates alveolar epithelial fluid clearance in male and female adult rats

2011 ◽  
Vol 301 (5) ◽  
pp. L804-L811 ◽  
Author(s):  
Edgar E. Kooijman ◽  
Stephanie R. Kuzenko ◽  
Denghuang Gong ◽  
Michael D. Best ◽  
Hans G. Folkesson
Keyword(s):  
Female Rats ◽  
Male Rats ◽  
Na Channel ◽  
Alveolar Fluid Clearance ◽  
Membrane Phospholipids ◽  
Adult Rats ◽  
Male And Female ◽  
Alveolar Epithelial ◽  
Male And Female Rats

Cell membrane phospholipids, like phosphatidylinositol 4,5-bisphosphate [PI( 4 , 5 )P2], can regulate epithelial Na channel (ENaC) activity. Gender differences in lung ENaC expression have also been demonstrated. However, the effects in vivo on alveolar fluid clearance are uncertain. Thus PI( 4 , 5 )P2 effects on alveolar fluid clearance were studied in male and female rats. An isosmolar 5% albumin solution was intrapulmonary instilled; alveolar fluid clearance was studied for 1 h. Female rats had a 37 ± 19% higher baseline alveolar fluid clearance than male rats. Bilateral ovariectomy attenuated this gender difference. Compared with controls, PI( 4 , 5 )P2 instillation (300 μM) increased alveolar fluid clearance by ∼93% in both genders. Amiloride or the specific αENaC small-interfering RNA inhibited baseline and PI( 4 , 5 )P2-stimulated alveolar fluid clearance in both genders, indicating a dependence on amiloride-sensitive pathways. The fraction of amiloride inhibition was greater in PI( 4 , 5 )P2-instilled rats (male: 64 ± 10%; female: 70 ± 11%) than in controls (male: 30 ± 6%; female: 44 ± 8%). PI( 4 , 5 )P2 instillation lacked additional alveolar fluid clearance stimulation above that of terbutaline, nor did propranolol inhibit alveolar fluid clearance after PI( 4 , 5 )P2 instillation, indicating that PI( 4 , 5 )P2 stimulation was not secondary to endogenous β-adrenoceptor activation. PI( 4 , 5 )P2 amine instillation resulted in an intermediate alveolar fluid clearance stimulation, suggesting that, to reach maximal alveolar fluid clearance stimulation, PI( 4 , 5 )P2 must reside in cell membranes. In summary, PI( 4 , 5 )P2 instillation upregulated in vivo alveolar fluid clearance similar to short-term β-adrenoceptor upregulation of alveolar fluid clearance. PI( 4 , 5 )P2 stimulation was mediated partly by increased amiloride-sensitive Na transport. There exist important gender-related effects suggesting a female advantage that may have clinical implications for resolution of acute lung injury.

scholarly journals β-Adrenergic agonists differentially regulate highly selective and nonselective epithelial sodium channels to promote alveolar fluid clearance in vivo

2012 ◽  
Vol 302 (11) ◽  
pp. L1167-L1178 ◽  
Author(s):  
Charles A. Downs ◽  
Lisa H. Kriener ◽  
Ling Yu ◽  
Douglas C. Eaton ◽  
Lucky Jain ◽  
...  
Keyword(s):  
Single Channel ◽  
Selective Inhibition ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Channel Measurements ◽  
Open Probability ◽  
Alveolar Epithelial ◽  
Lung Fluid ◽  
T1 And T2

β-Adrenergic receptors (β-AR) increase epithelial sodium channel (ENaC) activity to promote lung fluid clearance. However, the effect of selective β-AR agonist on highly selective cation (HSC) channels or nonselective cation (NSC) channels in alveolar type 1 (T1) and type 2 (T2) cells is unknown. We hypothesized that stimulation with β1-AR agonist (denopamine) or β2-AR agonist (terbutaline) would increase HSC and/or NSC channel activity in alveolar epithelial cells. We performed single-channel measurements from T1 and T2 cells accessed from rat lung slices. Terbutaline (20 μM) increased HSC ENaC activity (open probability, NPo) in T1 (from 0.96 ± 0.61 to 1.25 ± 0.71, n = 5, P <0.05) and T2 cells (from 0.28 ± 0.14 to 1.0 ± 0.30, n = 8, P = 0.02). Denopamine (20 μM) increased NSC NPo in T1 cells (from 0.34 ± 0.09 to 0.63 ± 0.14, n = 7, P = 0.02) and in T2 cells (from 0.47 ± 0.09 to 0.68 ± 0.10, P = 0.004). In vivo X-ray imaging of lung fluid clearance and ICI 118,551 selective inhibition of β2-ARs confirmed patch-clamp findings. cAMP concentrations increased following treatment with denopamine or terbutaline ( n = 3, P < 0.002). The effects of systemic (intraperitoneal, IP) and local (intratracheal, IT) modes of delivery on lung fluid clearance were assessed. IT delivery of denopamine promoted alveolar flooding, whereas IP delivery promoted delayed fluid clearance. In summary, β-AR agonists differentially regulate HSC and NSC in T1 and T2 cells to promote lung fluid clearance in vivo, and the mode of drug delivery is critical for maximizing β-AR agonist efficacy.

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