Istituto di Fisiologia Umana I, Università di Milano, 20133 Milano, Italy
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ABSTRACT |
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Apparent permeability to albumin
(Palb) was measured with
125I-albumin in specimens of rabbit parietal pericardium
from lumen to interstitium (L-I) and from interstitium to lumen (I-L).
With albumin concentration (Calb) 0.5%,
Palb (× 105 cm/s) L-I at 37°C
was 0.172 ± 0.019 SE; it decreased to 0.092 ± 0.022 I-L at
37°C, 0.089 ± 0.021 L-I at 12°C, and 0.084 ± 0.018 I-L
at 12°C. These findings provide evidence for an active transport L-I,
likely transcytosis. With Calb 2.5%, 0.05%, and
0.005%, Palb L-I at 37°C was 0.188 ± 0.023, 0.156 ± 0.021, and 0.090 ± 0.021, respectively; at
12°C it was 0.089 ± 0.017, 0.083 ± 0.019, and 0.087 ± 0.026, respectively. Hence, active albumin transport ceases with
Calb 0.005%; Palb values I-L at
12°C and with Calb 0.005% are similar and provide
diffusional permeability. With physiological Calb (~1%),
active albumin flux was ~5 × 10
4
µmol · h
1 · cm
2. Apparent
permeability to FITC-dextran 70 (Pdx) was also
measured. Pdx (× 10
5 cm/s) L-I at
37°C with Calb 0.5% was 0.095 ± 0.018; it
decreased to 0.026 ± 0.004 I-L (37°C, Calb 0.5%),
0.038 ± 0.007 at 12°C (L-I, Calb 0.5%), 0.030 ± 0.009 with Calb 0.005% (L-I, 37°C), and 0.032 ± 0.011 with nocodazole (L-I, 37°C, Calb 0.5%).
These findings provide evidence for transcytosis and confirm
conclusions drawn from Palb. Vesicular liquid
flow, computed from vesicular dextran flux (fluid-phase only), was
~3.5 µl · h
1 · cm
2.
Transcytosis seems a relevant mechanism, removing protein and liquid
from serous cavities.
albumin concentration and transcytosis; dextran; diffusional permeability; vesicular albumin flux; vesicular liquid flow
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INTRODUCTION |
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MORPHOLOGICAL EVIDENCE of free vesicles in the cytoplasm of mesothelial cells of peritoneum, pericardium, and pleura has been provided for a long time (17, 22, 25, 29, 32, 40, 51, 52). Moreover, morphological evidence for vesicular transport of macromolecules from the luminal to the interstitial side of mesothelium has been provided in rat parietal pericardium (28) and mice parietal peritoneum (17, 18, 23). This information, however, is insufficient compared with the wealth of information on vesicular transport available for endothelium (12, 19, 33, 34, 45-47, 50), which, however, has been challenged (42) even recently (43). By analogy to what is known about the endothelium, and on the basis of some hints on the pleural mesothelium, morphologists (23, 51, 52) suggested that vesicles of the pleural mesothelium might provide transcytosis. On the other hand, the prevailing view among physiologists about protein exit from the pleural space is that most of it occurs through the lymphatic stomata of the parietal pleura (35, 37-39, 48). This view is based on the finding that, after ligation of the right lymphatic duct and thoracic duct, only a small fraction of the labeled albumin injected into the pleural space reaches the blood (13, 35). This finding, however, does not prove that most labeled albumin leaves the pleural space through the lymphatic stomata, because albumin leaving the pleural space outside the stomata is eventually drained by the lymphatics of the interstitial space (3). Therefore, following the suggestions of morphologists, it has been proposed that proteins could leave the pleural space also by transcytosis, in addition to lymphatic drainage through the stomata of the parietal mesothelium and solvent drag through the visceral mesothelium (3). Recently, we determined the permeability (P) of the mesothelium to small, medium, and large molecules and determined the equivalent radius of the "small pores" of its intercellular junctions (2, 8, 55). P to albumin was markedly greater than predicted by the relationship between P and Stokes-Einstein radius (a) of the solutes. This suggested that albumin transfer across the mesothelium should mainly occur through "large pores" and/or transcytosis. Indirect evidence for transcytosis was provided by analyzing, in a way similar to that used by Renkin (41) for capillary endothelium, the P-a relationship of macromolecules through the mesothelium (8).
The purpose of this research is, therefore, to provide evidence for transcytosis in the mesothelium from lumen to interstitium (L-I). To this end, we used specimens of parietal pericardium because specimens of this serosa may be obtained with less tissue damage than specimens of pleura (55). First, we determined albumin transfer at 37°C and albumin concentration (Calb) 0.5% in the direction interstitium to lumen (I-L; i.e., opposite to that previously measured; Ref. 8) to ascertain the occurrence of a net albumin flux in the direction L-I. Second, we determined albumin transfer in both directions at 12°C and Calb 0.5%, because it has been shown in other tissues that at about this temperature, transcytosis ceases (24). Third, we determined albumin transfer at 37 and 12°C with Calb lower or higher than that which was previously used (0.5%) in an attempt to ascertain whether Calb affects transcytosis and whether the vesicular transport of albumin occurs, besides in fluid phase, also through binding with the vesicular surface as in capillary endothelium (45). Fourth, we determined the transfer of dextran 70 in the direction I-L (i.e., opposite to that which was previously measured; Ref. 8) at 37°C and Calb 0.5%, L-I at 12°C and Calb 0.5%, and at low Calb and 37°C, to extend the study to a different molecule, which should not be catabolized during the transfer and should be transferred only in fluid phase. This allows the determination of its vesicular concentration and, hence, the computation of the vesicular liquid flow. Fifth, we determined the transfer of dextran 70 under the conditions used in the previous measurements (8) after the addition of an inhibitor of transcytosis (nocodazole; Ref. 16).
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METHODS |
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Specimen collection and preparation. Specimens of the retrosternal parietal pericardium were obtained from 117 rabbits (body wt 4-6 kg, age 5-10 mo). Rabbits were purchased from bmg farm (Cividate al Piano, Bergamo). Animal experimentation was authorized by the Ministry of Health by decree N. 36/94A according to decree law 116/92, in compliance with guidelines 86/609/CE. The animals were anesthetized with a solution containing pentobarbital sodium (Sigma, 10 mg/ml) and urethane (Sigma, 250 mg/ml), 2 ml/kg iv, or with a solution containing ketamine hydrochloride (Sigma, 10 mg/ml) and xylazine hydrochloride (Sigma, 3 mg/ml), 2 ml/kg iv. They were placed supine on a tilting board 20° head up. The trachea was cannulated to ensure adequate ventilation during the preliminary surgical procedure, and air flow and tidal volume were recorded on a 7418 Hewlett-Packard thermopaper oscillograph. Collection and preparation of the specimens were performed with the procedure previously described, which minimizes manipulation and air exposure of the mesothelium (55). Briefly, after killing the rabbit by an overdose of anesthetic, we removed a segment of sternum, leaving undamaged the underlying parietal pericardium, which, in this region, should be free of lymphatic stomata (25). While albumin-Ringer solution was being poured on the pericardium to prevent air exposure of the mesothelium, a roughly rectangular specimen of pericardium (~3 × 2 cm) was hooked and excised. The specimen was never stretched during removal, and the whole procedure was completed within 4 min after the death of the animal. The specimen, covered by albumin-Ringer solution, was pinned with its interstitial side facing upward, at its in situ length and width, to a layer of Sylgard (Dow Corning) adhering to the bottom of a petri dish. The solution was bubbled continuously with a 95% O2-5% CO2 gas mixture. Small vessels, fat patches, and, when present, blood clots were removed from the interstitial side of the specimen until a transparent area of ~1 × 1.5 cm was obtained; the mesothelium of the central part of the specimen was never touched. The cleaning procedure took 20-25 min.
Solutions and labeled molecules.
The composition of the Ringer solutions used during specimen collection
and preparation, as well as during the measurements (see
below), was (in mM): 139 Na+, 5 K+,
1.25 Ca2+, 0.75 Mg2+, 119 Cl, 29 HCO
5 to 4.3 × 10
5
µmol). This amount is negligible relative to Calb, except
when the latter is 0.005%, because in this case the ICN albumin added is 2-6% of Calb. Unbound 125I is present
in the labeled albumin solution, and a correction for the inherent
radioactivity was made (see below). To check whether a
relatively high concentration of unbound 125I affected the
results despite the correction, labeled albumin solution was filtered
before use in part of the experiments (see below and
RESULTS). Filtration was made by centrifuging at 5,000 g for 30 min at 4°C through low protein binding
membrane (Millipore) with 30-kDa nominal molecular weight cut off
(8). FITC-dextran was used at a concentration of 0.78 × 10
2 µmol/ml. Unlabeled dextran was added at the same
concentration in the recipient chamber. To minimize the concentration
of free FITC, solutions containing FITC-dextran were dialyzed for ~16 h at ambient temperature before the experiments.
Measurements of unidirectional flux and permeability. Specimens were mounted as planar sheets between the frames of a Ussing apparatus (rectangular window: 0.5 cm2, chambers vol 4 ml). Both chambers were immediately and simultaneously filled with albumin (0.5%)-Ringer solution. A first incubation period of 30 min was allowed for tissue recovery. Solutions contained in the chambers were oxygenated and stirred throughout the experiment by bubbling 95% O2-5% CO2 through ports opening near the bottom of the frame in each chamber; the apparatus was water-jacketed to maintain the temperature appropriate to the kind of experiment (37 or 12°C, see below), and, accordingly, solutions were heated at 37°C or cooled at 12°C before adding them to the chambers. At the end of the first incubation period, both chambers of the Ussing apparatus were simultaneously emptied and refilled with labeled solution in the donor chamber and unlabeled solution in the recipient chamber. At this stage, the solution with the appropriate Calb for a given experimental series was used in both chambers. A second incubation period of 40 min was allowed to attain steady state of the tracer in the specimen. At the end of this period, a 50-µl sample was withdrawn from the donor chamber while the recipient chamber was emptied and immediately refilled with 3.95 ml of fresh unlabeled solution. At the end of this procedure, which required 3-4 s, the first measurement period started. The duration of the measurement period was 30 min. At the end of this period, a second measurement period of equal duration was performed after the above procedure was repeated. The values of the two experimental periods were averaged except when the second one exceeded the first one by >20%. In these cases (<12%), only the first one was taken. No short-circuit current was applied because no electrical potential difference was found across the in vitro specimens of parietal pericardium (9).
The samples of liquid withdrawn from each chamber at the end of the measurement periods were treated as previously described (55). With both tracers, a correction was made for background radioactivity or fluorescence, measured in samples of liquid before the addition of the labeled molecules. Checks for constant concentration of labeled molecules in the donor chamber throughout the experiment and for their negligible concentration in the recipient relative to the donor chamber (<2%, allowing measurement of unidirectional, rather than net, fluxes) at the end of each measurement period were performed as previously described (55). In the experiments with 125I-albumin,Series of experiments. Labeled albumin flux, Palb, and albumin flux were determined in the following series of experiments. Series 1 was in the direction I-L under the conditions (37°C, Calb 0.5%) previously used for the measurement in the direction L-I (8). This series was performed to ascertain, by comparison with L-I, the occurrence of a net albumin flux. Moreover, four additional experiments L-I, 37°C, Calb 0.5% were done in which labeled albumin solution was filtered before use (see above). Series 2 was in the direction L-I at 12°C with Calb 0.5%. Series 3 was in the direction I-L at 12°C with Calb 0.5%. Series 2 and 3 were performed to ascertain whether the net flux disappears, because it has been shown in other tissues that at ~12°C, transcytosis ceases (24). The values obtained in series 2 and 3 (and in the following series at 12°C, see below) were corrected to bring water viscosity and kinetic energy of solutes to 37°C. Other series were L-I at 37°C with Calb 0.005%, 0.05%, and 2.5% and L-I at 12°C with Calb 0.005%, 0.05%, and 2.5%. These series were performed for the following reasons: 1) to assess whether Calb affects vesicular formation and, hence, vesicular transport and 2) to assess whether the vesicular transport of albumin, besides in fluid phase, also occurs through binding to the vesicular membrane, like in capillary endothelium (45). In this case, a competition for binding should occur between albumin molecules, and, therefore, the vesicular flux of labeled albumin should decrease with increasing Calb. In three experiments of the series I-L, 12°C, Calb 0.5%, and in all of those of the series L-I, 12°C, Calb 0.05%, labeled albumin solution was filtered before use (see above).
Pdx was measured in the following series of experiments: 1) I-L at 37°C with Calb 0.5%; 2) L-I at 12°C with Calb 0.5%; and 3) L-I at 37°C with Calb 0.005%. Moreover, in another series of experiments, L-I, 37°C, and Calb 0.5%, an inhibitor of transcytosis (40 µM nocodazole, Sigma; Refs. 16, 24) was added to the solutions placed in both chambers at the beginning of the second incubation period. Finally, four experiments were added to those previously performed L-I, 37°C, Calb 0.5% (8). Vesicular transport of dextran should be only fluid phase because dextran does not bind to the vesicular surface. Hence, the vesicular concentration of dextran may be determined. This allows the computation of the vesicular liquid flow, which is given by the vesicular flux of dextran divided by its vesicular concentration. The vesicular concentration of dextran is smaller than that in the solution because of steric exclusion, which occurs when the radius of the solute is not negligible relative to that of the vesicle. The effect of the steric exclusion is estimated from the lumen-vesicle partition coefficient (Statistics. Data are expressed as means ± SE. Statistical significance of differences between groups was determined by unpaired t-test.
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RESULTS |
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Albumin.
The value of Palb of the parietal pericardium in
the direction I-L at 37°C was 0.092 × 105 cm/s,
i.e., 53% (P < 0.02) of that in the direction L-I,
0.172 × 10
5 cm/s (Table
1). Because the catabolism of albumin in
the luminal chamber was essentially nil (see METHODS), the
greater albumin flux in the direction L-I than in I-L cannot be due to
a diffusion of peptides in the direction L-I. Therefore, most of the
net albumin flux should represent an active transport in the L-I
direction. The value of Palb L-I at 12°C
(corrected to bring water viscosity and kinetic energy of solutes to
37°C) was 0.089 × 10
5 cm/s, i.e., 52%
(P < 0.02) of that L-I at 37°C (see Table 1). The
value of Palb I-L at 12°C (corrected as
before) was 0.084 × 10
5 cm/s, i.e., similar to that
L-I at the same temperature (Table 1). The finding that at 12°C the
net albumin flux disappears shows that it is due to an active
transport, likely transcytosis, because at this temperature the
vesicles do not occur (24) and because of the results
obtained with dextran 70 (see Dextran).
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Dextran.
The value of Pdx of parietal pericardium I-L at
37°C and Calb 0.5% was 0.026 × 105
cm/s, i.e., lower (P < 0.01) than that L-I at the same
temperature and Calb, 0.095 × 10
5 cm/s
(Table 4). The value of
Pdx L-I at 12°C and Calb 0.5%
(corrected to bring water viscosity and kinetic energy of solutes to
37°C) was 0.038 × 10
5 cm/s, i.e., lower
(P < 0.05) than that at 37°C (Table 4). Moreover, the value of Pdx with 40 µM nocodazole L-I,
37°C, Calb 0.5% (n = 10) was 0.032 (± 0.012) × 10
5 cm/s, i.e., lower (P < 0.02) than that without nocodazole L-I, 37°C, Calb
0.5% and similar to those I-L, 37°C, Calb 0.5% and L-I,
12°C, Calb 0.5%. These findings show the occurrence of
transcytosis in the mesothelium from L to I. The value of
Pdx L-I at 37°C and Calb 0.005%
was 0.030 × 10
5 cm/s, i.e., lower
(P < 0.02) than that with Calb 0.5%
(Table 4). This finding shows that transcytosis in mesothelium ceases when Calb is 0.005%.
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DISCUSSION |
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The results show the occurrence of an active transport of albumin
and dextran from the luminal to the interstitial side of the
mesothelium of the parietal pericardium. The findings that this active
transport involves dextran and ceases at 12°C or with nocodazole show
that this transport is due to transcytosis, in line with our previous
indirect evidence (8). Moreover, the results show that
transcytosis decreases progressively at low Calb and
eventually vanishes when Calb 0.005%. This suggests that
this vesicular transport is not constitutive, but appears to be
activated by albumin. With Calb similar to that which
occurs under physiological conditions in the pleural or pericardial
liquid, ~1% or 0.14 µmol/ml (20, 36, 44),
Jalb,ves through the mesothelium should be
~5 × 10
4
µmol · h
1 · cm
2. This is
likely an underestimation because no correction was made for
TCA-soluble peptides formed during transcytosis (see METHODS and RESULTS). Vesicular liquid flow
(Jliq,ves), computed from the vesicular flux of
dextran 70 (which is only fluid phase) with Calb 0.5%,
should be ~3.5
µl · h
1 · cm
2.
In a study on the absorption of colloidal thorium dioxide from the pericardial cavity of rats, morphological evidence suggested that it is carried by vesicles through the mesothelium of the parietal pericardium and, to a much smaller extent, of the visceral pericardium (28). Thorotrast was then shown to be drained by lymphatics (30). Moreover, radioiodinated serum albumin placed in the pericardial cavity of rabbits was found to leave through the parietal pericardium and to be drained by lymphatics (21). Finally, the lymphatic drainage from the pericardial cavity of conscious sheep, estimated from the egress of labeled serum albumin, was found to be 0.04 ml/(h kg) (10).
An active transport of bovine serum albumin through bullfrog alveolar
epithelium was found by Kim et al. (27). Part of the albumin transferred was intact. Net P in the alveolar to
pleural direction was 0.018 × 105 cm/s, i.e.,
one-fifth of that presently found in rabbit mesothelium (0.084 × 10
5 cm/s; Table 1, from the value of the first line minus
the average value from the second, third, and fourth lines). In this
connection, one has to consider that their measurements were performed
at room temperature. Moreover, an active transport of dog albumin through canine bronchial epithelium was shown by Johnson et al. (26). Most of the albumin transferred was catabolized. Net
P in the L-I direction was 0.049 × 10
5
cm/s, i.e., approximately one-half that presently found in rabbit mesothelium. No marked fall in albumin transport appeared at low Calb.
The time taken by vesicles to form and move to the other side of the
cell (turnover time) has been determined or indirectly computed in
different preparations of capillary endothelium and of pericardial
mesothelium. Renkin (41), after having indirectly determined Jliq,ves through the capillary
endothelium of dog leg, computed the volume of vesicles from estimates
of the volume of the capillary endothelium in his preparation and of
the percentage of this volume occupied by the vesicles. Dividing
vesicle volume by Jliq,ves, he got a turnover
time of ~5 min. This time is similar to that of the vesicular
transport of thorium dioxide through the mesothelium of parietal
pericardium of rats studied by electron microscopy (28).
On the other hand, Milici et al. (34), with immunocytochemical procedures, showed that albumin carried by plasmalemmal vesicles through the capillary endothelium of mouse myocardium appears in the pericapillary space <15 s after the beginning of perfusion, with no gradient from the intercellular junctions. From our data, the turnover time can be obtained following Renkin's approach with only one estimate, because the surface area of
the membrane is known in our experiments. The average thickness of the
mesothelium in rabbit is ~2 µm (51). Hence, the volume
of 1 cm2 of mesothelium is 2 × 104 ml.
From electron micrographs (28, 29), one can estimate that
the vesicles occupy ~15% of this volume. Hence, vesicle volume in 1 cm2 of mesothelium is 3 × 10
5 ml.
Because Jliq,ves through 1 cm2 of
mesothelium is 3.5 µl/h or 9.7 × 10
4 µl/s, the
turnover time should be ~30 s.
To the extent that the data obtained on the specimens of parietal
pericardium can be applied to the pleura under physiological conditions
(for morphological and cytochemical similarity of the mesothelium of
these serosas, see Ref. 49), transcytosis appears to be a
relevant mechanism that removes liquid and protein from the pleural
space. Therefore, we now consider the other mechanisms that are known
to remove liquid and protein from the pleural space to attempt a rough
comparison. 1) The volume of liquid absorbed by the Starling
forces of the capillaries of the visceral pleura was found in dogs to
be 0.5 µl · h1 · cm
2 per
mmHg of driving pressure (4), but under those experimental conditions the mesothelium was damaged because of air exposure and
manipulation (6, 55). This was not a problem for the aim
of that research, but it is if one wants to know the liquid flow
through the visceral mesothelium. Moreover, the driving pressure across
this mesothelium is uncertain and controversial (6, 37).
As a rough estimate, the volume of liquid absorbed by the Starling
forces through the visceral mesothelium in rabbit could be ~1.5
µl · h
1 · cm
2. The amount
of albumin following this flow by solvent drag should be small, but
probably not negligible (3). 2) The lymphatic drainage from the pleural space under physiological conditions has been
estimated in dogs from egress kinetics of labeled albumin injected into
the space and has been found to be 0.02 ml/(h kg) (35).
Taking into account that the surface area of the parietal pleura in
dogs is 110 cm2 · kg
2/3
(Ref. 36, plus an estimate of the surface
area of the costo-phrenic sinus) and that dog weight in the above
research was 17.5 kg, the lymphatic drainage from the pleural space
should be 0.5 µl · h
1 · cm
2.
Considering that the above measurements should be a little
underestimated, as pointed out by the authors (35) and
that the turnover of liquid per unit surface area in rabbits appears to
be greater than in dogs (37), the lymphatic drainage from
the pleural space in rabbits could be ~2
µl · h
1 · cm
2. Because
albumin concentration in the pleural liquid is ~1% or 0.14 µmol/ml
(36, 44), this lymphatic drainage should imply an albumin
removal from the pleural space of 2.8 × 10
4
µmol · h
1 · cm
2. This
value, however, should be greater than that corresponding to the
lymphatic drainage through the stomata of the parietal pleura because
albumin egress from the pleural space (as it has been measured; Ref.
35) includes most of the albumin that has left the pleural
space outside the stomata and is then removed from the interstitium by
the inherent lymphatics (see Introduction). Therefore, if transcytosis
occurs in the mesothelium of the parietal pleura, to get the albumin
flux occurring through the lymphatic stomata of the parietal pleura,
most of the vesicular albumin flux should be subtracted from albumin
egress from the pleural space as it has been measured. Hence, the
lymphatic drainage of albumin through the stomata of the parietal
pleura (no stomata occur in the visceral pleura; Ref. 51)
should be only a fraction of albumin egress from the pleural space, as
it has been measured (35). The same consideration applies
to liquid flow. Finally, the finding that vesicular albumin flux in the
specimens of parietal pericardium, ~5 × 10
4
µmol · h
1 · cm
2, is
greater than albumin egress from the pleural space, ~2.8 × 10
4
µmol · h
1 · cm
2,
indicates that the vesicular albumin flux in the parietal pleura should
be smaller than that found in our specimens of parietal pericardium.
This fits with the morphological evidence of a smaller concentration of
vesicles in the pleural (51, 52) than in the pericardial
mesothelium (28, 29).
Even if vesicular liquid flow in the pleura under physiological
conditions were one-half that found in our specimens of parietal pericardium, i.e., ~1.7 instead ~3.5
µl · h1 · cm
2 (see
above), the contribution of vesicular transport to the removal of
liquid and protein from the pleural space would be substantial, appearing to be greater than that of the other known mechanisms. In
particular, the present findings provide experimental support to one
argument raised (3, 6) against the view that under physiological conditions, the lymphatic drainage through the stomata of
the parietal pleura accounts for 75-80% of liquid removal from the pleural space and that, therefore, it is the main mechanism setting
pleural liquid pressure under physiological conditions (35,
37-39). Indeed, if transcytosis occurred in the pleural mesothelium, the lymphatic drainage through the stomata should not be
considered the main mechanism setting pleural liquid pressure under
physiological conditions. Moreover, even if the vesicular albumin flux
through the mesothelium were one-half that found in our specimens of
parietal pericardium, i.e., ~2.5 instead of ~5 × 10
4
µmol · h
1 · cm
2 (see
above), the role of lymphatic drainage through the stomata in
preventing protein accumulation into the pleural space (1, 37,
48) should be reevaluated. It might be that under physiological conditions, the peculiar function of the lymphatic stomata of the
parietal pleura is that of allowing removal of cells, debris of cells,
and bacteria from the pleural space. This function, which implies a
simultaneous removal of liquid and protein, appears more consistent
with the size of the stomata, which is 2-6 µm or more in
diameter (51). The situation changes when the volume and
the pressure of the pleural liquid increase, because then the lymphatic
drainage from the pleural space may increase >10 times
(11).
We have previously provided indirect evidence of a
Na+-coupled liquid absorption from the pleural space, which
appears to be ~0.7
µl · h1 · cm
2 (5,
6, 53, 54). This absorption of essentially protein-free liquid
increases when Calb in the pleural liquid decreases below its normal value (5, 6). Conversely, the present findings show that transcytosis decreases when Calb is relatively
low (Figs. 1, 3). Because the former mechanism increases
Calb by removing protein-free liquid from the pleural
space, whereas the latter mechanism would decrease Calb if
albumin binding occurred in the vesicles, these two mechanisms could
contribute automatically to the constancy of Calb in the
pleural liquid. Indeed, a decrease in Calb stimulates the
former and inhibits the latter and vice versa.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. D. Cremaschi and K. J. Kim for helpful suggestions and stimulating discussions, Dr. D. Cremaschi for critically reading the paper and for permission to use the fluorescence spectrometer (Dipartimento di Fisiologia e Biochimica generali), and Drs. N. Cascinelli and E. Bombardieri (Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano) for permission to use the facilities of the Divisione di Medicina Nucleare. Finally, we thank R. Galli for skillful technical assistance.
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FOOTNOTES |
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This research was supported by Ministero dell'Università e della Ricerca Scientifica e Tecnologica of Italy, Rome.
Address for reprint requests and other correspondence: E. Agostoni, Istituto di Fisiologia Umana I, Università di Milano, Via Mangiagalli 32, 20133 Milano, Italy (E-mail: emilio.agostoni{at}unimi.it).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 3 May 2001; accepted in final form 2 August 2001.
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