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The following is the abstract of the article discussed in the subsequent letter:
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Lasnier, Joseph M., David H. Ingbar, Ethan P. Carter, Kirk Wilson, Scott McKnite, Keith G. Lurie, and O. Douglas
Wangensteen. Perfusion technique determines alveolar fluid
resorption rate in the isolated perfused rat lung. J. Appl.
Physiol. 84(2): 740-745, 1998.
The isolated perfused lung
(IPL) preparation is a well-established model for the study of alveolar
epithelial sodium transport. We noted that preparations of normal
fluid-filled rat lungs with recirculated perfusate reproducibly lost
weight, whereas preparations in which the perfusate was discarded after
a single pass through the lungs had a variable and lesser weight
change. To confirm this, we performed IPL experiments by using male
Sprague-Dawley specific-pathogen-free rats (175-225 g). In 10 IPLs, perfusate initially was discarded after passing through the lungs
and then was recirculated continuously. During the single-pass period, the rate of weight change was +0.7 ± 2.0 mg/min compared with 
9.0 ± 1.3 mg/min for the recirculating period. Adenosine 3',5'-cyclic monophosphate (cAMP) accumulated during recirculation. The weight loss
induced by recirculation was reproduced by perfusion with 8-bromoadenosine 3',5'-cyclic monophosphate or terbutaline in single-pass fashion and blocked when the kinase inhibitor H-8 or
phosphodiesterase was present in the recirculating perfusate. In
summary, perfusate recirculation in the IPL stimulates fluid resorption
at least partially via cAMP. This should be factored into the design
and interpretation of IPL experiments.
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Perfusion Techniques for Determining Alveolar Fluid Resorption Rate
To the Editor: In an interesting manuscript by Lasnier et al. (6), which appeared recently in this journal, the authors compared the fluid resorption rates of isolated rat lungs perfused in either a recirculating or a single-pass mode. However, I was concerned about several aspects of the data as presented and about the conclusions drawn from the data.First, no experiments were reported in which phosphodiesterase (PDE) or H-8 was used in single-pass perfused lungs. If PDE or H-8 reduces clearance (i.e., weight loss) by an equivalent amount in those lungs, it would be an argument against an effect of the increased cAMP seen in the reperfused lungs.
Second, the effect of 8-bromoadenosine 3',5'-cyclic monophosphate or terbutaline in single-pass lungs reported in the study by Lasnier et al. (6) approximates the effect reported by other investigators using terbutaline or isopreterenol in recirculated lungs (4, 5, 7, 9), suggesting that the effect of accumulating cAMP in the perfusate presents at most a minor level of partial stimulatory activity.
Third, the tracings presented (Figs. 2, 3, and 5 in Ref. 6) do not support the authors' proposed mechanism that the effect is due to accumulating cAMP in recirculating lungs. The rate of weight loss appears to be linear over time, as compared with the continuous accumulation and increasing concentration of cAMP over time. If the cAMP in the perfusate (or an unknown mediator increasing intracellular cAMP) were a major contributor to clearance rates, I would expect an initial rate of weight loss similar to that of single-pass lungs, which then progressively increased as cAMP levels increased.
Although the two events discussed in this manuscript, namely, accumulation of cAMP in the recirculating model and a greater rate of weight loss, occur simultaneously, the data as presented do not convince me that there is a cause-and-effect relationship between these events.
Finally, I am considerably disturbed by a statement in the last
paragraph of the discussion. The authors assert that "investigators using the isolated perfused lung (IPL) model should bear in mind that
use of the recirculating technique does not measure the true baseline rate of alveolar fluid resorption" (emphasis mine). I have used a number of different IPL models (1-3, 8) and have never
assumed that I was measuring a "true baseline" for any parameter, only that the baseline was valid for the model being used. I certainly hope that the authors do not believe they are measuring the
"real" baseline rate for fluid absorption in rat lungs in their
single-pass perfused lung modelit is simply a baseline that is
different from the one measured in the recirculating model. Although
the comparison of the two models poses interesting questions regarding the nature of why they are different, until techniques are developed allowing truly noninvasive measurement of fluid resorption rates, the
"true baseline" will remain unknown.
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1.
Barnard, M. L.,
and
S. Matalon.
Mechanism of extracellular reactive oxygen species injury to the pulmonary microvasculature.
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Barnard, M. L.,
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Activated polymorphonuclear leukocytes increase low-level chemiluminescence of isolated perfused rat lungs.
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Barnard, M. L.,
W. G. Olivera,
D. M. Rutschman,
A. M. Bertorello,
A. I. Katz,
and
J. I. Sznajder.
Dopamine stimulates sodium transport and liquid clearance in rat lung epithelium.
Am. J. Respir. Crit. Care Med.
156:
709-714,
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Effros, R. M.,
G. R. Mason,
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Fluid resorption and glucose consumption in edematous rat lungs.
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Goodman, B. E.,
K. Kim,
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E. D. Crandall.
Evidence for active sodium transport across alveolar epithelium of isolated rat lung.
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6.
Lasnier, J. M.,
D. H. Ingbar,
E. P. Carter,
K. Wilson,
S. McKnite,
K. G. Lurie,
and
O. D. Wangensteen.
Perfusion technique determines alveolar fluid resorption rate in the isolated perfused rat lung.
J. Appl. Physiol.
84:
740-745,
1998
7.
Lasnier, J. M.,
O. D. Wangensteen,
L. S. Schmitz,
C. R. Gross,
and
D. H. Ingbar.
Terbutaline stimulates alveolar fluid resorption in hyperoxic lung injury.
J. Appl. Physiol.
81:
1723-1729,
1996
8.
Perry, M.,
and
A. E. Taylor.
Phorbol myristate acetate-induced injury of isolated perfused rat lungs: neutrophil dependence.
J. Appl. Physiol.
65:
2164-2169,
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9.
Saldias, F.,
E. Lecuona,
E. Friedman,
M. L. Barnard,
K. M. Ridge,
and
J. I. Sznajder.
Modulation of lung liquid clearance by isoproterenol in rat lungs.
Am. J. Physiol.
274 (Lung Cell. Mol. Physiol. 18):
L694-L701,
1998
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Michele L. Barnard, 21121 Goshen Rd. Gaithersburg, Maryland 20882 |
To the Editor: We reported that IPLs perfused with
recirculation reproducibly resorbed fluid more rapidly than those
perfused in single pass, which had a variable, but slower, rate of
alveolar fluid resorption. In addition, we reported data consistent
with a role for cAMP in this effect.
Our report included experiments in which H-8 was used in single-pass
fashion (see Fig. 7 in Ref. 2). As noted in the discussion, there was
heterogeneity of alveolar fluid resorption rates in IPLs with
single-pass perfusion, and in those IPLs with rapid resorption the
resorption rate could be reduced by the addition of the kinase
inhibitor H-8. This phenomenon of heterogeneity of alveolar fluid
resorption has been documented previously (1, 3). Taken together with
the prevention of the effect of recirculation seen when H-8 or PDE was
present in the recirculating perfusate, we feel that a role for cAMP is
demonstrated. We were careful to qualify our discussion, stating that
the data did not exclude the existence of other mediators.
In addressing Barnard's second and third points, it is true that
terbutaline additionally augmented alveolar fluid resorption in a
recirculating system and that there was an initial increase in alveolar
fluid resorption rate on change to recirculation without subsequent
increase, despite continued accumulation of cAMP. As discussed in our
paper (2), other mediators, in addition to cAMP, are likely present;
extracellular cAMP may rapidly achieve a maximal effect at a low
perfusate concentration, and terbutaline may more efficiently increase
intracellular cAMP in the alveolar epithelium than does accumulation of
extracellular cAMP. These issues notwithstanding, the main point of the
paper remains the demonstration of a reproducible increase in aveolar
fluid resorption on switching to recirculation from single-pass
perfusion; although the mechanism of this effect is of interest, we
consider this a secondary issue.
We agree with Barnard that, since it is not currently possible to
measure alveolar fluid resorption in the unperturbed in vivo setting, a
"true baseline" rate of fluid resorption cannot be defined.
However, it remains possible to compare the effect of one technique
with another. We feel that it is useful to remind investigators of the
effects of choice of technique on their results, as evidenced by the
difference between single-pass and recirculated isolated perfused
lungs, even if the mechanism of the difference is at present only
partially explained.
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1.
Carter, E. P.,
S. Duvick,
C. Wendt,
J. Dunitz,
L. Nici,
O. D. Wangensteen,
and
D. H. Ingbar.
Hyperoxia increases active alveolar Na+ resorption in vivo and type II cell Na, K-ATPase in vitro.
Chest
105:
75S-78S,
1994.
2.
Lasnier, J. M.,
D. H. Ingbar,
E. P. Carter,
K. Wilson,
S. McKnite,
K. G. Lurie,
and
O. D. Wangensteen.
Perfusion technique determines alveolar fluid resorption rate in the isolated perfused lung.
J. Appl. Physiol.
84:
740-745,
1998.
3.
Yue, G.,
and
S. Matalon.
Mechanisms and sequelae of increased alveolar fluid clearance in hyperoxic rats.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L407-412,
1997
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Joseph M. Lasnier, Division of Pulmonary, Allergy and Critical Care Department of Medicine University of Minnesota Minneapolis, Minnesota 55455 |
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