| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Guest Access | Sign In via User Name/Password
|
|
|
|||||||
Guest Access | Sign In via User Name/Password |
|||||||||
* From Pulmonary and Critical Care Medicine, Evanston Hospital, Evanston, IL; Northwestern University Medical School, Chicago, IL; and Technion, Israel Institute of Technology, Haifa, Israel.
Correspondence to: Phillip Factor, DO, FCCP, Pulmonary and Critical Care Medicine, Evanston Northwestern Healthcare, 2650 Ridge Rd, Evanston, IL 60201; e-mail: pfactor{at}northwestern.edu
Pulmonary edema is removed from the airspace as a consequence of active Na+ transport by alveolar epithelial transport proteins, including basolaterally located Na,K-adenosine triphosphatases (ATPases) and apically positioned Na+ entry pathways such as the epithelial Na+ channel (ENaC). These transporters actively extrude Na+ from the airspace to generate a transepithelial osmotic gradient that causes movement of fluid from the airspace via transcellular and paracellular pathways. Accumulating data suggest that in some forms of acute lung injury active, Na+ transport is impaired, possibly due to downregulation of alveolar solute transport pathways.1 2 3 4 5 6 7 Thus, methods that improve alveolar Na+ transport could prove useful for the treatment of pulmonary edema.
We have reported that adenoviral-mediated transfer of Na,K-ATPase subunit genes increases active Na+ transport in human and rat lung epithelial cells.1 8 9 We have also noted that Na,K-ATPase overexpression increases alveolar fluid clearance (AFC) by > 100% in normal rats,8 mitigates oxidant mediated decreases in active Na+ transport in rat fetal distal lung epithelial cells,10 and increases AFC and survival of rats exposed to 100% oxygen.1 These studies demonstrate that upregulation of epithelial active Na+ transport can moderate acute lung injury. They also provide support for the development of therapeutic strategies that improve the ability of the lung to keep itself dry.
ß-Adrenergic agonists increase active Na+ transport in alveolar epithelial cells and normal and injured animal lungs by upregulating both apical Na+ entry pathways (including the epithelial Na+ channel, ENaC) and basally positioned Na,K-ATPases. These effects, which are principally effected by ß2-adrenergic receptors (ß2AR),11 include new message transcription, enhanced trafficking of transport proteins to the cell membrane, and increased activity of solute transporters in the cell membrane.12 13 14 These attributes caused us to hypothesize that overexpression of a ß2AR in the alveolar epithelium of normal rats could improve ß2AR function and enhance catecholamine-responsive active Na+ transport.
To test this hypothesis, we used a surfactant-based delivery system to infect spontaneously breathing adult, male Sprague-Dawley rats with E1a-/E3- adenoviruses that contain expression cassettes with a human immediate-early cytomegalovirus promoter-enhancer element and either a human ß2AR complementary DNA (adß2AR) or no complementary DNA (adNull). Rats were infected with 4 x 109 plaque forming units of adenovirus and then allowed 7 days of recovery to allow vector-induced host responses to subside. We have previously reported that this delivery scheme yields widespread transduction of the alveolar epithelium of normal and injured rat lungs.1 8 To confirm that our vector was capable of gene transfer and transgene activation, total RNA and protein were prepared from whole-lung homogenates for reverse transcriptase-polymerase chain reaction and Western blot analyses using human ß2AR-specific primers and antibodies. These studies demonstrated high-level expression of human ß2AR messenger RNA and protein only in adß2AR-infected lungs. Immunohistochemistry using 5-µm sections localized the transgene to the alveolar epithelium. Confirmation of appropriate trafficking of the transgenic ß2AR to the cell membrane was obtained via Western blot analysis of whole-cell membrane fractions isolated from the peripheral 2 to 3 mm of the lung. Membrane-bound human ß2AR was noted only in adß2AR-infected lungs. Interestingly, expression of a human ß2AR did not appear to affect membrane-bound levels of endogenous (rat) ß2AR. Transgene function in the cell membrane was assayed by measuring cyclic adenosine monophosphate production by cell membranes in the presence and absence of the highly ß2AR-specific agonist procaterol (106mol/L x 30 min). ß2AR function in receptor-overexpressing lungs was increased up to fivefold as compared to membranes from control lungs. These experiments indicate that adenoviral-mediated transfer can produce overexpression of a functional, human ß2AR in the alveolar epithelium of normal rats.
To test if alveolar ß2AR overexpression has a physiologically relevant effect on alveolar solute transport, we infected rats, described above, and measured AFC using a fluid-filled, perfused, isolated lung preparation.1 8 Clearance in these animals was 100% greater than sham and adNull-infected control lungs (n = 5/group) and 40% greater than control lungs treated with high-dose procaterol (106 mol/L via the alveolar instillate and vascular perfusate) during the isolated lung measurements. Treatment with procaterol did not increase clearance further in ß2AR-overexpressing lungs, suggesting that receptor overexpression maximally upregulates ß2AR-sensitive AFC.
To develop an understanding of how ß2-receptor
overexpression increases active solute transport, we assayed apical Na+
channel function by instilling the Na+ channel blocker amiloride
(106 mol/L; n = 4/group) into the alveolar
airspace during the isolated lung studies, and observed that
ß2AR-overexpressing lungs were 100% more
sensitive to amiloride than were sham and adNull-infected control
lungs. We coupled these functional measurements with Western blot
analysis of apical membrane fractions that were isolated from the
peripheral lung and observed a nearly 100% increase in membrane-bound
expression of the 
subunit of the epithelial Na+ channel in
adß2AR-infected lungs. In parallel studies, we
measured ouabain-sensitive adenosine triphosphate hydrolysis by
basolateral cell membranes isolated from the peripheral lung. Doing so,
in the presence of high [Na+], low [K+], produces an index of
functional, membrane-bound Na,K-ATPase number. These experiments
demonstrated that receptor overexpression increases basolateral
membrane Na,K-ATPase abundance by > 300%. These data indicate that
augmentation of alveolar epithelial ß2AR
function upregulates both apical Na+ entry pathways (ie,
ENaC) and basolateral Na,K-ATPases.
Because AFC was increased without catecholamine treatment, we postulated that receptor overexpression may be affecting responsiveness to endogenous catecholamines. To test this hypothesis, we measured AFC in adrenalectomized and propranolol-treated (1 mg/kg tid for 5 days) rats following sham or adß2AR infection. ß2AR overexpression did not increase AFC in these lungs to the same degree as that seen in normal rats, suggesting that receptor overexpression improves responsiveness to endogenous catecholamines.
This study demonstrates that overexpression of ß2AR gene in the alveolar epithelium can augment alveolar ß2AR function and positively affect AFC. These effects are mediated by upregulation of solute transport proteins on both the apical and basolateral cell membrane domains and via enhanced responsiveness to endogenous catecholamines. Taken together, these findings indicate that ß2AR overexpression is a novel application of gene transfer that markedly upregulates active Na+ transport in the alveolus of normal rats. We believe that ß2AR gene transfer may eventually prove useful for the treatment of patients with pulmonary edema.
 |
Footnotes |
|---|
Supported by the American Heart Association, Evanston Northwestern Healthcare Research Institute, HL-48129, and HL-66211.
|
References |
|---|
 TOP References |
|---|
This article has been cited by other articles:
|
|
 |
|
  L. Jain and D. C. Eaton Alveolar fluid transport: a changing paradigm Am J Physiol Lung Cell Mol Physiol, April 1, 2006; 290(4): L646 - L648. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
