EDITORIAL FOCUS
Albumin transcytosis in mesothelium

Francesca Bodega, Luciano Zocchi, and Emilio Agostoni

Istituto di Fisiologia Umana I, Università di Milano, 20133 Milano, Italy


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (× 10-5 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP>, and 5.6 D-glucose. Bovine serum albumin (Sigma) was added to the solution to have a 0.1% concentration in the preparation periods, a 0.5% concentration in the first incubation period (see below), and the following concentrations (Calb) in the various series of experiments: 0.005% (a value a little above the minimum required to keep normal permeability; Ref. 15), 0.05%, 0.5% (that which was previously used; Ref. 8), and 2.5%. The labeled molecules used were bovine 125I-albumin (specific activity ~1 mCi/mg, ICN Biomedicals) and 70 kDa FITC-dextran (0.013 mol FITC/mol glucose, Sigma). The mean activity of 125I-albumin in the solution placed in the donor chamber of the Ussing apparatus was ~1 µCi/ml (ranging from 0.7 to 1.8 µCi/ml). Because of the fast decay of 125I-albumin (half-life 59.7 days), increasing amounts of the ICN albumin (which is only partly labeled) had to be added as time elapsed: the amount added to 1 ml of solution ranged from 1 to 3 µg (i.e., 1.4 × 10-5 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, beta -activity of 100- to 400-µl samples was determined as counts per minute (cpm) in a liquid scintillation spectrometer (Minaxi beta  Tri-Carb 4000, Packard Instruments) and expressed as cpm per milliliter to provide values proportional to isotope concentration in a given chamber. A correction for unbound 125I present in the samples was performed by subtracting from the cpm value measured in each sample the value due to unbound 125I; this was obtained by measuring in corresponding samples the radioactivity remaining in the supernatant after protein precipitation with trichloroacetic acid (TCA, 12%) and centrifugation. In the experiments in which the solution with labeled albumin was filtered before use (see below and RESULTS), the percentage of "TCA-soluble radioactivity" relative to total radioactivity before the experiment was 5.0 ± 0.3 before filtration and 1.9 ± 0.1 after filtration. At the end of the experiment, this percentage in the donor chamber was 1.9 ± 0.1, while in the recipient chamber it was 27 ± 3. In the experiments in which filtration before use was not performed, this percentage at the end of the experiment was 8.3 ± 0.4 in the donor chamber and 63 ± 2 in the recipient chamber (see RESULTS). Because small peptides are TCA soluble, the occurrence of albumin catabolism during transcytosis may introduce an error in the correction for unbound 125I owing to the subtraction of a value that also includes small labeled peptides. This leads to an underestimation of albumin transfer in the experiments with transcytosis if this involves albumin catabolism.

The unidirectional flux of labeled albumin through the specimen was measured as cpmR/At, where cpmR is cpm in the recipient chamber (corrected as mentioned above), A is the surface area of the window, and t the duration of the measurement period. Because cpm in the donor chamber changed a little among experiments, the flux of labeled albumin was normalized: cpmR of each experiment was multiplied by the ratio between cpm in the donor chamber (corrected as mentioned above, cpmD) of that experiment and the average cpmD of all experiments. The apparent permeability to albumin (Palb) was computed as Palb = cpmR/[(cpmD/ml)At]. The values of Palb were corrected for the effect of liquid unstirred layers (USL) close to the membrane by the equation of Barry and Diamond (7), as previously done (55). The unidirectional flux of albumin was computed as Palb · Calb, except for the series with nominal Calb 0.005% in which actual Calb was computed, taking into account the amount of ICN albumin added that is, in this case, not negligible (see above).

Proteases released from damaged mesothelial cells could catabolize albumin in the luminal chamber, and the diffusion of peptides could lead to an overestimation of albumin transfer. The following tests were, therefore, performed to rule out the occurrence of an appreciable amount of peptides in the luminal chamber (8). In three experiments without labeled albumin, with Calb 0.5%, samples from the luminal chamber were taken after a time corresponding to the end of the experimental period and were filtered through a 30-kDa membrane. Albumin concentration before filtration and peptide concentration in the filtrate were measured with the Lowry micromethod. Peptide concentration in the filtrate was ~0.4% of albumin concentration before filtration; this percentage was similar to that found in the solution before any contact with the specimen. This indicates that albumin catabolism in the luminal chamber was essentially nil.

In the experiments with FITC-dextran, fluorescence intensity (proportional to FITC concentration) of the samples was measured in a fluorescence spectrometer (LS50, Perkin-Elmer; excitation 494 nm and emission 525 nm). The relationship between the concentration of labeled dextran 70 and its fluorescence in arbitrary units was linear within the range used. The unidirectional flux of dextran 70 was computed from the amount of dextran 70 entered in the receiving chamber during the experimental period divided by At. The apparent permeability to dextran (Pdx) was computed from the unidirectional flux of dextran 70 divided by its concentration in the donor chamber. The values of Pdx were corrected for the effect of USL, as described above.

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 (phi ), which is given by (1 - a/r)3, where a is the hydrodynamic radius of the solute and r the radius of the vesicle (14). Because a of dextran 70 is 5.77 nm (31) and r of mesothelium vesicles can be estimated to be ~45 nm (18, 51), phi  should be 0.66. Therefore, the concentration of dextran in the vesicles should be 0.78 × 10-2 µmol/ml × 0.66 = 0.51 × 10-2 µmol/ml.

Statistics. Data are expressed as means ± SE. Statistical significance of differences between groups was determined by unpaired t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Albumin. The value of Palb of the parietal pericardium in the direction I-L at 37°C was 0.092 × 10-5 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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Table 1.   Apparent permeability of specimens of parietal pericardium to albumin in direction lumen to interstitium or vice versa, at 37 and 12°C

The values of Palb reported above, like those of the previous research (8), were obtained with a Calb of 0.5% (7.2 × 10-2 µmol/ml). The values of Palb L-I with Calb 0.005%, 0.05%, and 2.5% at 37°C and at 12°C are reported in Table 2. All the values of Palb at 12°C (corrected as before) are similar under the various conditions and similar to that I-L at 37°C (Tables 1 and 2), i.e., they provide the diffusional permeability. The value of Palb L-I at 37°C with Calb 0.005% is lower than those L-I at 37°C with Calb 0.05%, 0.5%, and 2.5% (P < 0.05, 0.02, and 0.01, respectively) and is similar to those providing the diffusional permeability (see above). The above findings suggest that the active transport of albumin through the mesothelium decreases at low Calb and eventually vanishes at Calb ~0.005%.

                              
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Table 2.   Apparent permeability of specimens of parietal pericardium to albumin with various concentrations of albumin at 37 and 12°C in direction lumen to interstitium

In the experiments of the series L-I, 12°C, Calb 0.05%, in all of which labeled albumin solution was filtered before use (see METHODS), the value of Palb, 0.083 × 10-5 cm/s, was similar to that of the other series at 12°C or I-L or Calb 0.005% (Tables 1 and 2), in which labeled albumin solution was not filtered. Moreover, in the three experiments of the series I-L, 12°C, Calb 0.5%, in which labeled albumin solution was filtered before use, Palb, 0.062 (± 0.031) × 10-5 cm/s, was not significantly different from that of the other series at 12°C or I-L or Calb 0.005%. (Tables 1 and 2) in which labeled albumin solution was not filtered. Finally, in the four experiments of the series L-I, 37°C, Calb 0.5% in which labeled albumin solution was filtered before use (see METHODS), Palb, 0.148 (± 0.019) × 10-5 cm/s, was not significantly different from that of the eight experiments of the same series previously done (0.184 ± 0.027 × 10-5 cm/s; Ref. 8) in which labeled albumin solution was not filtered. Therefore, the lower Palb occurring in the experiments of the series at 12°C or I-L or Calb 0.005% cannot be ascribed to an artifact depending on the relatively large value of radioactivity, due to unbound 125I, occurring when labeled albumin solution is not filtered before use (this value has to be subtracted from the total radioactivity to correct for unbound 125I, see METHODS).

In the experiments with previous filtration of labeled albumin solution, the percentage of radioactivity left in the solution after protein precipitation with TCA, relative to total radioactivity in the recipient chamber at the end of the experiment, was 34 ± 4 (n = 4) in those with transcytosis and 24 ± 4 (n = 11) in those without transcytosis. In the experiments without previous filtration, this percentage was 64 ± 3 (n = 26) in those with transcytosis and 62 ± 3 (n = 46) in those without transcytosis. Therefore, the percentage of TCA-soluble radioactivity, relative to total radioactivity in the recipient chamber at the end of the experiment, was not greater without transcytosis than with transcytosis. This finding indicates that albumin catabolism occurs during transcytosis for the following reasons. In the experiments without transcytosis, albumin transfer is smaller, and, therefore, the percentage of TCA-soluble radioactivity (essentially due to unbound 125I), relative to total radioactivity in the recipient chamber at the end of experiment, should be larger than in those with transcytosis if this does not involve albumin catabolism. Conversely, in the experiments with transcytosis involving albumin catabolism, the percentage of TCA-soluble radioactivity, relative to total radioactivity in the recipient chamber at the end of the experiment, may be similar to, or even greater than, that in experiments without transcytosis because TCA-soluble peptides (produced by albumin catabolism during transcellular transfer) may compensate for or overwhelm the greater transfer of albumin. Therefore, the present data of albumin transcytosis are underestimated by an amount corresponding to TCA-soluble peptides formed during transcytosis (see METHODS).

It is now convenient to express the results in terms of albumin flux (Jalb) and to quantitate its diffusive and vesicular component (Jalb,dif and Jalb,ves, respectively), because the latter is of particular interest from the physiological point of view (see DISCUSSION). These fluxes at the various values of Calb are reported in Table 3. With Calb 0.5%, the values of flux I-L at 37°C and L-I or I-L at 12°C were similar: 2.39 (± 0.56) × 10-4, 2.31 (± 0.55) × 10-4, and 2.19 (± 0.48) × 10-4 µmol · h-1 · cm-2. Therefore, they were pooled together to provide the diffusive component. Jalb and Jalb,dif are plotted vs. Calb in the log-log diagram of Fig. 1. Vesicular albumin flux is given by the vertical distance between the circles and the dotted line: with Calb 0.005%, vesicular albumin flux is nil. Jalb,ves is plotted vs. Calb in the diagram of Fig. 2. From the data of Jalb,ves with Calb 0.5% and 2.5%, one obtains by interpolation the value of Jalb,ves with Calb 1% (or 0.14 µmol/ml), which is that occurring in the pleural or pericardial liquid under physiological conditions (21, 36, 44). This value is ~5 × 10-4 µmol · h-1 · cm-2.

                              
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Table 3.   Total, diffusive, and vesicular albumin flux lumen to interstitium through specimens of parietal pericardium with various concentrations of unlabeled albumin



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Fig. 1.   Total (circles) and diffusive (broken line) albumin flux through specimens of parietal pericardium vs. albumin concentration in a log-log plot. open circle , lumen to interstitium at 37°C. +, lumen to interstitium at 12°C (corrected to bring water viscosity and kinetic energy of solutes at 37°C). , interstitium to lumen at 37°C. down-triangle, interstitium to lumen at 12°C (corrected as before). Vesicular albumin flux is given by vertical distance between circles and dotted line.



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Fig. 2.   Vesicular albumin flux lumen to interstitium through specimens of parietal pericardium vs. albumin concentration.

If the vesicular transport of albumin, besides in fluid phase, also occurred through binding with a plasmalemmal receptor (as in other tissues; Refs. 24, 45), a competition for binding should occur among albumin molecules, and it should increase with increasing Calb. As a consequence, the vesicular flux of labeled albumin should decrease with the increase in Calb (as in endothelium; Ref. 45). On the contrary, as shown by Fig. 3, the vesicular flux of labeled albumin (which is given by the vertical distance between the continuous line and the broken line) decreases at low Calb and becomes nil at Calb 0.005%, while the diffusive flux of labeled albumin is constant at various Calb, in line with an essentially free diffusion of albumin through large pores (8). Because labeled albumin flux decreases and eventually ceases at low Calb, one cannot detect with the present data whether albumin binding occurs in this vesicular transport.


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Fig. 3.   Total (continuous line) and diffusive (broken line) flux of labeled albumin through specimens of parietal pericardium vs. albumin concentration. Vesicular flux of labeled albumin is given by the vertical distance between continuous line and broken line. Symbols same as in Fig. 1. cpm, Counts per minute.

Dextran. The value of Pdx of parietal pericardium I-L at 37°C and Calb 0.5% was 0.026 × 10-5 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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Table 4.   Apparent permeability and unidirectional flux of dextran 70 through specimens of parietal pericardium

The flux of dextran (Jdx) L-I at 37°C and Calb 0.5% was 2.66 × 10-5 µmol · h-1 · cm-2 (Table 4). Jdx I-L at 37°C and Calb 0.5%, L-I at 12°C and Calb 0.5%, and L-I at 37°C and Calb 0.005% are reported in Table 4. They are not significantly different from each other, and, therefore, they were pooled together to provide the diffusive flux of dextran, ~0.88 (± 0.12) × 10-5 µmol · h-1 · cm-2. This is lower (P < 0.01) than Jdx L-I at 37°C and Calb 0.5% and was subtracted from the latter to obtain the vesicular flux of dextran: ~1.78 × 10-5 µmol · h-1 · cm-2. Jdx with 40 µM nocodazole, L-I, 37°C, Calb 0.5% was 0.91 (± 0.32) × 10-5 µmol · h-1 · cm-2, i.e., lower (P < 0.02) than that without nocodazole, L-I, 37°C, Calb 0.5% and similar to those I-L or at 12°C or with Calb 0.005%. The vesicular transport of dextran is only fluid phase because no dextran binding occurs to the surface of the vesicles. Therefore, at variance with albumin, one can know the concentration of dextran in the vesicles (0.51 × 10-2 µmol/ml, see METHODS). The vesicular flux of dextran divided by its concentration in the vesicles provides the vesicular liquid flow: 3.5 µl · h-1 · cm-2.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 × 10-5 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 × 10-4 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 · h-1 · 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 · h-1 · 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 · h-1 · 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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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DISCUSSION
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