Page 1 of 35 Articles in PresS. Am J Physiol Lung Cell Mol Physiol (May 5, 2006). doi:10.1152/ajplung.00426.2005
Helms, Self, et al.
TITLE:
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Dopamine Activates Amiloride-Sensitive Sodium Channels in Alveolar Type 1 Cells in Lung Slice Preparations.
AUTHORS: My N. Helms1,3, Julie Self1,3, Hui Fang Bao1,3, Lauren C. Job1,3, Lucky Jain,1,2,3, and Douglas C. Eaton1,2,3 Departments of Physiology1 and Pediatrics2 and The Center for Cell and Molecular Signaling3 Emory University School of Medicine Atlanta, GA 30322 RUNNING TITLE: Dopamine regulates ENaC in ATI cells
ADDRESS ALL CORRESPONDENCE TO:
Douglas C. Eaton Emory University School of Medicine Department of Physiology Whitehead Biomedical Research Building 615 Michael Street Atlanta, GA 30322 Phone: (404) 727-4533 Fax: (404) 727-0329 E-mail:
[email protected]
Keywords: lung slice / AT1 cells / ENaC / single channel recording / Erythrina crystagalli lectin / dopamine / D1, D2 receptors
Copyright © 2006 by the American Physiological Society.
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ABSTRACT Active Na reabsorption by alveolar epithelial cells generates the driving force used to clear fluids from the air space.
Using single channel methods, we
examined epithelial sodium channel (ENaC) activity of ATI cells from live 250300µm sections of lung tissue, circumventing concerns of whether protracted cell isolation procedures will compromise the innate transport properties of native lung cells. We used fluorescein-labeled Erythrina crystagalli lectin to positively identify ATI cells for single channel patch clamp analysis. We demonstrate for the first time single channel recordings of highly selective and non-selective, amiloride-sensitive ENaC channels (HSC and NSC) from ATI cells in situ with mean conductances of 8.2+2.5 pS and 22+3.2 pS, respectively. Additionally, 25nM amiloride in the patch electrode blocked Na channel activity in ATI cells. Immunohistochemical studies demonstrated the presence of dopamine D1 and D2 receptors on the surface of ATI cells, and single channel recordings show that 10µM dopamine increased Na channel activity (NPo) from 0.31+0.19 to 0.60+0.21 (p95% of the peripheral lung. The role that ATII cells play in surfactant production and ion transport have been extensively investigated (12; 13; 19; 29).
However, the general functions of ATI cells,
including the role that they play in alveolar ion/fluid transport remains unclear. Evidence for ENaC in AT1 cells.
Epithelial sodium channels (ENaC) are
members of the degenerin family of extracellular-ligand-gated channels. Specifically, ENaC is a heteromultimeric channel composed of homologous α,β, and γ subunits (8). Recently, using enzymatic digestion and mechanical separation of ATI cells from adult rat lungs, Johnson et al. (20) have been able to show that ATI cells take up Na in an amiloride-inhibitable fashion and that these cells express proteins for all three ENaC subunits. In addition, Borok et al. (5) have immunohistochemically identified α-ENaC and amplified α-ENaC message from AT1 cells. Under normal circumstances, ENaC is the rate limiting step for normal Na reabsorption in epithelial cells and ENaC activity
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controls alveolar salt and fluid absorption in the alveoli. This fact is underscored by the observation that transgenic α-ENaC knock-out mice die within 40 hours of birth due to an inability to clear lung fluid (14; 18).
The conventional wisdom has been that only
AT2 cells transport sodium, but, since both ATI and ATII cells express ENaC, both cell types might be responsible for sodium transport and maintenance of airspace fluid homeostasis.
We have shown that there are functional ENaC in ATI cells that were
isolated by enzymatic digestion and immunoaffinity purification, but we worried that the isolation procedure might alter the properties and prevalence of ion channels. It was also sometimes difficult to unequivocally determine the apical surface of the cells. However, in this study we examined the specific channel characteristics of ENaC in a lung slice tissue preparation, circumventing concerns of whether ATI cell membrane are damaged during the isolation procedure and confirming the presence in ATI cells of highly Na selective (HSC) and nonselective (NSC) cation channels, which are blocked by amiloride and activated by dopamine, in an environment close to the native environment of AT1 cells in the lung. Catecholamines can increase lung fluid clearance.
Dopamine (DA) has been
reported to increase Na channel and Na,K-ATPase activity in the alveolar epithelium in order to facilitate lung edema clearance (2-4; 10). The effects of DA appear to be mediated via dopamine D1 receptor (apically located) and basolateral D2 subtypes. These two receptors exert their biological actions by coupling to and activating different G protein complexes. The D1 receptor interacts with the Gs complex to activate adenylyl cyclase, whereas the D2 interacts with either Gi or Gq to inhibit cAMP production or activate phospholipase C (7). We have recently observed in L2 cells (rat ATII cell line)
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that activation of D1 receptors was responsible for increasing ENaC activity in cellattached patches via a cAMP-mediated signaling pathway when DA was applied to the apical surface of cells (16), and we were, therefore, interested in determining whether AT1 cells also responded to DA. Thus, we examined the effect of dopamine treatment on Na channel activity in ATI cells, and whether dopamine-D1 or -D2 receptors might be involved in putative dopamine induced effects. In this report, we show, for the first time, single channel recordings of amiloridesensitive epithelial Na channels from ATI cells in lung slice preparations. We also show a dopamine-induced increase in ENaC channel activity in ATI cells via dopamine-D1 receptor activation. These findings provide evidence of a definitive role for ATI cells in maintaining fluid/ion homeostasis in the alveolar spaces and a responsiveness of AT1 cells to catecholamine treatment.
METHODS AND MATERALS Lung tissue preparation.
Male Sprague Dawley rats were maintained with
access to standard rat diet and water ad libitum. At 8-12 weeks of age, animals were anesthetized and sacrificed in accordance with Institutional Animal Care/Use Committee guidelines. Following lung perfusion via the pulmonary artery with 75 ml warm (35oC) PBS, warm 2% low melting point agarose in PBS was intratracheally instilled into the lungs to expand the air spaces and provide support for the tissue during the slicing process. Excised lungs were removed en bloc and chilled in cold PBS (4oC) to solidify the agarose, and a small block of tissue was separated from the largest lobe of the lung and mounted using surgical grade cyanoacrylate adhesive onto a VT1000S vibratome
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(Leica Microsystems, IL). The vibratome blade was set to high frequency vibration but was advanced slowly through the continuously chilled tissue to make 250-300µm tissue slices.
Tissue samples were placed in ice cold DMEM/F12 50/50 media (containing
10% FBS, 2mM L-glutamine, 1µM Dex, 84µM gentamycin, and 20 U/ml penicillin and streptomycin) until patch clamp analysis. Lung cells were treated and patched within 4 hours of the initial tissue preparation.
Single channel patch clamp analysis of Na channels in ATI cells. Lung slices were rinsed 3X in recording solution (see below) before patch clamping. Experiments were conducted at 22-23°C and all patch solutions for the cell attached configuration contained (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES at pH 7.4. GigaOhm seals were made between the tip of a fire polished glass microelectrode (back filled with patch solution) and the lung slices were immersed in patch solution. An Axopatch 1-D amplifier (Molecular Devices, CA), interfaced through an analog/digital board to a PC computer, collected single channel data. Channel currents were amplified at 5 kHz and filtered at 1 kHz with a low-pass Bessel filter. For cell-attached patches, voltages are given as the negative of the patch pipette potential (-Vp). This potential is the displacement of the patch potential from the resting potential; positive potentials represent depolarizations, and negative potentials represent hyperpolarizations of the cell membrane away from resting potential. We used the product of the number of channels (N) times the single open probability (Po) as a measure of ENaC activity within a patch, as described in (9).
This product was
calculated using FETCHAN and pCLAMP 6 software (Molecular Devices).
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Chemicals. Dopamine (3-hydroxytyramine, hydrochloride) was purchased from Calbiochem (CA). Amiloride hydrochloride hydrate, sulpiride, and SCH23390 (R-(+)-7chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-H-3-benzazepine hydrochloride) were purchased from Sigma (MO).
Dopamine and amiloride were soluble in patch
clamp solution and prepared immediately before use. Dopamine receptor antagonists (sulpiride and SCH23390) were dissolved in ethanol at .01M stock concentrations. The specified amounts of dopamine and receptor blockers were applied directly to the bath solution, whereas amiloride was back-filled into patch pipettes (described below). Fluorescent microscopy. Fluorescein-labeled lectin from the Cry-Baby Tree, Erythrina crystagalli (ECL), (Vector Labs, CA) could be used as a vital stain for ATI cells in 250-300µm lung slice preparations. When used to label ATI cells for the purpose of recording single channel activity, ECL was diluted 1:1000 directly into the patch solution that the lung tissue was immersed in. For immuno-histochemistry, lung slices were fixed with 4% paraformaldehyde (10 min at room temperature) and then labeled with a 1:1000 dilution (in PBS/BSA with 3% goat serum) of fluorescein-labeled ECL and/or with antibodies. Lung tissue slices were co-labeled with ECL and a final 1:20,000 fold dilution of anti-surfactant protein A antibody (Chemicon, CA) for 1 hour to identify ATI and ATII cells, respectively. Lung slices were then incubated in goat anti-rabbit secondary antibody conjugated to Alexa 568 (1:20,000 dilution; Molecular Probes) for 30 minutes. Dopamine D1 and D2 receptor antibodies, purchased from Chemicon, were used at a 1:1000 fold dilution.
Goat anti-rabbit antibody
conjugated to Alexa 488 (Molecular Probes) was used as the secondary detection antibody for dopamine receptors at a 1:10,000 dilution for 1 hour. All tissue samples
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were mounted onto a glass slide with ProLong antifade reagent (Molecular Probes) and imaged using a Zeiss LSM 510 NLO META laser scanning confocal microscope (Zeiss, NY). Methods for statistical analysis. Statistical significance between two groups was determined by paired or unpaired t-tests, as appropriate. When the comparison between more than one group was required, statistical significance was determined by one-way analysis of variance (ANOVA). P values
