INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

THE MORPHOLOGICAL FEATURES of the intercellular junctions of the mesothelium appear to be similar in the pleura (2, 29) and the pericardium (8, 15, 17) of various species (human, ox, sheep, pig, rabbit, and rat). On this basis, the diffusional permeability (P) to small hydrophilic solutes of the mesothelium is likely to be similar in the two tissues. P to small solutes of the pericardium is not known, whereas P to sucrose (P_suc) of stripped specimens of sheep visceral pleura has been found to be 12.8 × 10⁵ cm/s by Kim et al. (16). Values nearly three times greater have been found in stripped specimens of canine visceral and parietal pleura by Payne et al. (21). These values are one order of magnitude greater than P_suc of the endothelium of lung, muscle, and skin capillaries determined with the indicator-diffusion technique, ~10⁵ cm/s (5). On the other hand, mesothelial cells may lose mutual contact when they are irritated (6), are easily detached during handling of the tissue (8), and their intercellular junctions widen on simple exposure to air (23, 27); moreover, shear stress has been found to increase the permeability to solutes of mesothelial cells in culture (30). Pleura stripping involves considerable stretching, manipulation, and exposure to air: it seems, therefore, probable that P found in stripped specimens of pleura is greater than that under physiological conditions. To the end of assessing P of the mesothelium in conditions closer to the physiological ones, we looked for a place where the pleura or the pericardium is loosely connected to the underlying tissues to get specimens without stripping, little handling, and short exposure to air. When the chest wall of rabbits is opened by sternotomy, the sternal part of the parietal pericardium appears free, except for some fat patches. This suggests that specimens of the sternal part of the pericardium may be obtained in conditions closer to the physiological ones than specimens of pleura, provided fat patches may be easily removed.

The first purpose of this research is, therefore, that of determining P_suc and P to Na⁺ (P_Na⁺) of specimens of the sternal part of rabbit parietal pericardium. It is not known whether an active Na⁺ transport occurs in the pericardium. On the other hand, if it does occur, active Na⁺ flux would be very small relative to passive flux; indeed, the active Na⁺ flux that seems to occur in the pleura of rabbits, 0.1 µeq · h¹ · cm² (1), is two orders of magnitude smaller than the passive Na⁺ flux of leaky epithelia of rabbits (9, 18). Hence, active Na⁺ transport should not affect the measurement of P. The second purpose of this research is that of repeating the above experiments after addition of phospholipids to the solution facing the luminal or the interstitial side of the specimen. This was planned because it has been found that 1) in vivo phospholipids are adsorbed on the luminal side of the mesothelium of pleura (11, 13) and pericardium (12); and 2) adsorbed phospholipids decrease the permeability of epithelia (10). The third purpose of this research is that of determining P_suc in specimens in which the mesothelium was scraped away. This was done to measure P of the connective tissue of the pericardium and thus to compute P of the mesothelium alone.

	METHODS

Top Abstract Introduction Methods Results Discussion References

The experiments were performed in 60 giant rabbits, most of which were female (body wt 5-7 kg, age 7-11 mo). The animals were anesthetized with a solution (2 ml/kg iv) containing pentobarbital sodium (10 mg/ml, Sigma Chemical) and urethan (250 mg/ml, Sigma Chemical).

Specimen collection and preparation. Each rabbit was placed supine on a tilting board, 20° head up; the trachea was cannulated to ensure adequate ventilation during the preliminary surgical procedure; and airflow and tidal volume were recorded on a Hewlett-Packard 7418 thermopaper oscillograph. The sternum, for its whole length, and both parasternal regions were exposed by removing skin and superficial muscles down to the intercostal muscles. The rabbit was then killed by an overdose of anesthetic. The sternum was first sectioned along its transversal axis ~1 cm cephalad of the xyphoid process (in this region the underlying pericardium slopes dorsally, and the risk of damaging it is minimal). The cranial stump of the sternum was then lifted, and the ribs (6th to 2nd) were cut along their sternal ends so that the parietal pleura on both sides remained undamaged. After a second transversal section was made near the manubrium, a segment of sternum 5-6 cm long was removed. Because the pericardial sac is only loosely connected to the sternum by connective and adipose tissue, the removal of the sternum leaves the parietal pericardium undamaged. The parts thus exposed consisted of the interstitial faces of the pleurae on either side and the interstitial face of the sternal pericardium in the middle; the sternal part of the pericardium was on a lower plane because of the downward pull of the weight of the heart. The larger fat patches projecting from the exposed pericardium were removed in situ. Then, the cranial and caudal regions of the exposed pericardium were hooked by a second operator with two devices built for this purpose. Each device consisted of a pair of stainless steel hooks fastened ~1 cm apart by a small handle that was kept between the thumb and index finger. While these devices were gently lifted by the second operator and albumin-Ringer solution (see Solutions) was being poured on the pericardium to prevent air exposure of the mesothelium, the first operator pierced the pericardial sac and cut a roughly rectangular specimen of pericardium (~3 cm long and ~2 cm wide). The specimen was never stretched during removal, and the whole procedure of specimen collection was completed within 4 min of the death of the animal. The specimen, held by the hooks, was immediately placed in a petri dish containing albumin-Ringer solution and rinsed. Thereafter, it was moved to another petri dish, with a layer of Sylgard (Dow Corning) adherent to the bottom, and covered by the albumin-Ringer solution, which was bubbled continuously with a 95% O₂-5% CO₂ gas mixture (21). The specimen was pinned to the layer of Sylgard with its interstitial side facing upward and at its in situ length and width. The specimen (always immersed in the solution) was carefully cleaned by removing small vessels, fat patches, and, when present, blood clots with fine scissors and blunt tweezers. Cleaning was considered satisfactory when a transparent area of ~1 × 1.5 cm was obtained. The mesothelium was never touched during the cleaning procedure, which took 20-25 min. In eight experiments (series 6; see Measurement of P) after this procedure, the specimen was turned and pinned with its luminal side facing upward. The albumin-Ringer solution was removed, and the mesothelium was gently scraped away with the edge of a glass slide (31) or the blade of a scalpel. The tissue removed was weighed to check that it did not markedly exceed the amount computed from the area scraped times the thickness of the mesothelium (2 µm; Refs. 26, 29). Finally, in seven experiments (series 7; see Measurement of P) exposure to air was not prevented during the process of collecting and cleaning the specimen (see above). Moreover, Ringer solution, rather than albumin-Ringer solution, was used in rinsing the specimen and in the Ussing apparatus.

Measurement of P. The specimen was mounted as a planar sheet between the frames of an Ussing apparatus (rectangular window: 0.5 cm²). The equal-volume chambers were immediately and simultaneously filled with 4 ml of albumin-Ringer solution without or with the addition of phospholipids, according to the type of experiment (see below). Unidirectional fluxes of sucrose and Na⁺ through the specimen were determined in the following series of experiments: 1) radioactive markers (³H-labeled sucrose and ²²Na⁺; see below) in the solution facing the luminal side; 2) radioactive markers in the solution facing the interstitial side; 3) phospholipids and radioactive markers in the solution facing the luminal side; 4) phospholipids in the solution facing the luminal side and radioactive markers in the solution facing the interstitial side; 5) phospholipids and radioactive markers in the solution facing the interstitial side; 6) [³H]sucrose in the solution facing the luminal side, in experiments with scraped mesothelium; and 7) [³H]sucrose in the solution facing the luminal side, in experiments in which exposure to air was not prevented and Ringer solution, rather than albumin-Ringer solution, was used. A first incubation period of 30 min was allowed for tissue recovery, temperature equilibration, and initial phospholipid adsorption when scheduled. Solutions were preheated at 37°C, and the apparatus was water jacketed to maintain this temperature in both chambers throughout the experiment. The solution in both chambers was oxygenated and stirred throughout the experiment by bubbling the 95% O₂-5% CO₂ gas mixture (21) through ports opening near the bottom of the frame in each chamber. Bubbling was performed at the highest rate (~500 bubbles/min) that did not result in foam reaching the upper edges of the chambers of the Ussing apparatus. At the end of the first incubation period, both chambers were simultaneously emptied, and the recovered liquid was stored for determination of background radioactivity (see below). Simultaneous refilling of the chambers was immediately made with 4 ml of labeled solution in the donor chamber and 4 ml of unlabeled solution in the recipient chamber. A second 30-min incubation period was allowed to attain equilibrium of tracers between the donor chamber and the specimen and to continue phospholipid adsorption when scheduled. At the end of this period, a 50-µl sample was withdrawn from the donor chamber, whereas all the liquid in the recipient chamber was removed. The recipient chamber was immediately refilled with a volume of fresh unlabeled solution equal to that present in the donor chamber after removal of the 50-µl sample (3.95 ml). The time required for liquid withdrawal and replacement was 3-4 s. After 20 min (end of first experimental period), the above procedure was repeated to have a second experimental period (20 min). The procedure of total liquid withdrawal from the recipient chamber was chosen (rather than that of sampling at various times from this chamber) to ensure that the concentration of isotope in the recipient chamber remained negligible relative to that in the donor chamber. This prevents isotope back-diffusion and thus allows measurement of unidirectional (rather than net) fluxes.

8

Solutions. The composition of the standard solution used during specimen collection and preparation as well as during the experiments was (in mM) 139 Na⁺, 5 K⁺, 1.25 Ca²⁺, 0.75 Mg²⁺, 119 Cl, 29 HCO₃, and 5.6 D-glucose. Rabbit albumin (0.5 g/100 ml, Sigma Chemical) was added to maintain normal permeability (7). Radioactive markers (0.5 µCi/ml) were [³H]sucrose and ²²Na⁺ (Amersham). The phospholipids used were 50% dipalmitoyl phosphatidylcholine (367 µg/ml), 32% dipalmitoyl phosphatidylethanolamine (235 µg/ml), and 18% sphingomyelin (132 µg/ml). That is, they were used in the same proportions as, but at a lower concentration than, that occurring in sheep pleural extracts (13).

Thickness measurements and histological control. The thickness of the specimen of pericardium used in the experiments was determined in 34 of 60 rabbits, and we adapted the method described by Lai-Fook and Kaplowitz (19) to our case. At the end of the measurements of unidirectional fluxes, the specimen in the apparatus was washed repeatedly with unlabeled solution to remove most of the radioactive markers. The specimen within the frames was then removed from the Ussing apparatus, gently blotted to remove excess liquid, and sprinkled on each side with tantalum dust (particle size 5 µm, Sigma Chemical). The specimen within the frames was placed horizontally on the table of a Leitz MPV2 Orthoplan microscope, and a rectangular piece of Plexiglas of the same thickness as one frame was fitted onto the downward-facing part of the window to avoid any sagging of the specimen. The thickness of the specimen was determined by focusing on two tantalum particles, one on each side of the specimen and situated approximately along the same axial line. The magnification used was ×100 because frame thickness prevented use of an objective providing greater magnification. As a consequence, the depth of focus was relatively large (~10 µm); therefore, the positions of the lens were taken when the tantalum particle on each side of the specimen was first focused on during the displacement of the lens in one direction. The thickness of a particle was then subtracted from the distance between the focusing positions of the lens on either side of the specimen. The smallest fine-focusing micrometer scale was 2 µm. The measurement was repeated in 10 different sites of the specimen. The mean of these measurements was taken as the average thickness of the specimen.

Statistics. Data are expressed as means ± SE. Statistical significance of differences among groups was assessed by analysis of variance.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

The mean values of P_suc and P_Na⁺ of the sternal part of the parietal pericardium obtained in the experiments without phospholipids and in those with phospholipids in the solution facing the luminal or the interstitial side of the specimen are reported in Table 1. Because the values for the lumen-to-interstitium direction were similar to those for the opposite direction, the overall means are also reported. When phospholipids were added to the solution facing the luminal side of the specimen, P_suc decreased by 3.4 times (P < 0.01) and P_Na⁺ decreased by 1.9 times (P < 0.01, Table 1). Instead, when phospholipids were added to the solution facing the interstitial side, P_suc and P_Na⁺ did not change (Table 1). In the experiments in which air exposure was not prevented, and Ringer solution rather than albumin-Ringer solution was used (see METHODS), P_suc was 9.28 ± 1.63 × 10⁵ cm/s (n = 7), that is, about four times greater than that in the experiments of series 1 and 2 (see METHODS) (2.39 ± 0.31 × 10⁵ cm/s; Table 1).

The underestimation of P caused by unstirred liquid layers close to the membrane may be computed with the formula of resistances in series (3, 24): 1/P_corr = (1/P_meas)  (d_liq/D), where P_corr is the corrected P, P_meas is the measured P, d_liq is the thickness of the unstirred liquid layers (which may be assumed to be 170 µm: 70 µm on the luminal side and 100 µm on the interstitial side) (3), and D is the diffusion coefficient of the solute in water at 37°C (0.70 × 10⁵ cm²/s for sucrose and 1.80 × 10⁵ cm²/s for Na⁺) (5). According to the above formula, the corrected values of P_suc and P_Na⁺ in the experiments without phospholipids are 2.54 ± 0.33 and 7.75 ± 0.52 × 10⁵ cm/s, respectively. Hence, the underestimation caused by unstirred liquid layers is 6% for P_suc and 7% for P_Na⁺. In the experiments with phospholipids, the corrected values of P_suc and P_Na⁺ are 0.71 ± 0.06 and 3.93 ± 0.18 × 10⁵ cm/s, respectively. Hence, the underestimation caused by unstirred liquid layers is only 1% for P_suc and 4% for P_Na⁺. Finally, in the experiments in which air exposure was not prevented and albumin was not added to Ringer solution, the corrected value of P_suc is 12.0 ± 2.11 × 10⁵ cm/s. Hence, the underestimation caused by unstirred liquid layers is marked (23%): indeed, the effect of unstirred layers increases progressively with the increase in P (3, 24).

The average thickness of the specimens in series 1-5 (see METHODS) was 76.4 ± 3.09 µm (n = 26). There was no relationship between P_suc or P_Na⁺ and the thickness of the specimen (Fig. 1). This finding indicates that the resistance to diffusion is mainly provided by the mesothelium because the variance in the thickness of the specimen depends essentially on the difference in thickness of its connective tissue (see below). The mean value of the P_Na⁺-to-P_suc ratio (P_Na⁺/P_suc) in the experiments without phospholipids, 3.66 ± 0.36 (Table 1), is higher than the corresponding free-diffusion ratio, 2.57 (5). On the other hand, in 9 of 18 specimens the individual value of P_Na⁺/P_suc ranged from 2.20 to 2.96 (Fig. 2, open symbols below dotted line) with a mean of 2.59 ± 0.09, which is similar to the free-diffusion ratio, whereas in the rest of the specimens (Fig. 2, open symbols above dotted line) the value of this ratio is higher (average 4.72 ± 0.50), reaching the lower or middle values obtained in the experiments with phospholipids on the luminal side (Fig. 2, filled symbols). In 9 of the former specimens, P_suc and P_Na⁺ (×10⁵ cm/s) were 3.36 ± 0.34 and 8.52 ± 0.59 × 10⁵ cm/s, respectively; in the other 9 specimens, the values were 1.43 ± 0.23 and 5.92 ± 0.46 × 10⁵ cm/s, respectively. The mean value for P_Na⁺/P_suc in the experiments with phospholipids on the interstitial side (3.66 ± 0.54; Table 1 and Fig. 2, dashed symbols) is lower (P < 0.01) than that in the experiments with phospholipids on the luminal side (5.90 ± 0.42; Table 1 and Fig. 2, filled symbols) and equal to that in the experiments without phospholipids (3.66 ± 0.36; Table 1 and Fig. 2, open symbols).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Diffusional permeability to sucrose (Psuc; top, circles) and to Na+ (PNa+; bottom, diamonds) of parietal pericardium vs. thickness of specimen in Ussing apparatus (n = 26) without (open symbols) and with phospholipids in solution facing luminal (solid symbols) or interstitial (dashed symbols) side of specimen.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   PNa+-to-Psuc ratio (PNa+/Psuc) vs. PNa+ (diamonds, n = 45) or Psuc (circles, n = 45) in experiments without (open symbols) and with phospholipids in solution facing luminal (solid symbols) or interstitial (dashed symbols) side of specimen. Mean value of PNa+/Psuc data below dotted line, 2.49 ± 0.09, is similar to value of free-diffusion ratio, 2.57 (5).

The experiments in which the mesothelium was scraped away enabled us to measure P_suc of the connective tissue: it was 13.2 ± 0.76 × 10⁵ cm/s (n = 8). Correcting for the effect of unstirred liquid layers (see above), it became 19.4 ± 1.12 × 10⁵ cm/s (Table 2). Hence, the underestimation is marked (32%). Because the thickness of the unscraped specimens in series 1-5 (see METHODS) was 76.4 µm, and that of the mesothelium is ~2 µm (26, 29), the average thickness of the connective tissue in these specimens is ~74 µm. The average thickness of the scraped specimens was essentially similar (69.9 ± 3.64 µm; n = 8). Because the mesothelium and the connective tissue are placed in series, their resistances to diffusion add up; hence, 1/P_sp = (1/P_mes) + (1/P_con), where sp, mes, and con are unscraped specimen, mesothelium, and connective tissue, respectively. Hence, 1/P_mes = (1/2.54 × 10⁵ cm/s)  (1/19.4 × 10⁵ cm/s); hence, P_mes = 2.92 × 10⁵ cm/s (Table 2). The resistance to diffusion of the mesothelium is, therefore, about seven times greater than that of the connective tissue, despite the fact that the latter is ~35 times thicker than the former. In the experiments with phospholipids, the resistance to diffusion through the connective tissue should be unchanged because P was reduced only when phospholipids were added to the solution facing the mesothelial (not the interstitial) side (Table 1). Therefore, one may compute the resistance to diffusion of sucrose of the mesothelium with phospholipids by subtracting the resistance of the connective tissue from that of the unscraped specimens with phospholipids on the luminal side, that is: 1/P_mes = (1/0.71 × 10⁵ cm/s)  (1/19.4 × 10⁵ cm/s). Hence, with phospholipids, P_mes = 0.74 × 10⁵ cm/s (Table 2), which is essentially similar to P_sp. Indeed, with phospholipids the resistance to diffusion of the mesothelium is so high that the resistance of the connective tissue becomes negligible.

When diffusion through the "pores" is not restricted, P = (D/l) (A_p/A), where l is the membrane thickness and A_p/A is the area of the pores relative to the area of the membrane. Hence, A_p/A = (P · l)/D. The value of P_mes in the nine experiments in which diffusion of sucrose was not restricted (computed from P_sp and P_con, both corrected for the effects of unstirred liquid layers) was 4.56 × 10⁵ cm/s. Hence, A_p/A of the mesothelium is roughly given by (4.56 × 10⁵ cm/s) · (2 × 10⁴ cm)/ (0.7 × 10⁵ cm²/s) = 1.3 × 10³, that is, ~0.1%.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

P_suc of the sternal part of the parietal pericardium in rabbits in the experiments with albumin-Ringer solution, 2.39 ± 0.31 × 10⁵ cm/s, is about one order of magnitude smaller than that found in stripped specimens of sheep visceral pleura, 12.8 ± 1.4 × 10⁵ cm/s (17), and of dog visceral, 33.1 ± 3.1 × 10⁵ cm/s, and parietal pleura, 37.1 ± 3.5 × 10⁵ cm/s (22). Because the morphological features of the intercellular junctions of the mesothelium appear to be similar in the pleura (2, 29) and the pericardium (8, 15, 17) of various species, these results support the hypothesis, based on morphological findings (see the introduction), that the stretching, handling, and air exposure involved in stripping the specimen of pleura widen the tight junctions between mesothelial cells and may even detach some of the cells. Furthermore, the solution used in the Ussing chambers in the studies on stripped specimens of pleura (16, 21) was protein free, and it has been shown that a protein-free solution increases the permeability of the capillary endothelium (7). In the experiments in which air exposure was not prevented and albumin was not added to the Ringer solution, P_suc of the pericardium, corrected for the effects of unstirred liquid layers (12.0 × 10⁵ cm/s), is about five times greater than the corrected value (2.54 × 10⁵ cm/s) of series 1 and 2 (the uncorrected value is 2.39 × 10⁵ cm/s, Table 1). Hence, part of the difference in P_suc between stripped specimens of pleura and unstripped specimens of pericardium may be explained by air exposure and lack of albumin. The rest is likely due to the stretching involved in stripping. This factor, however, cannot be tested because one does not know quantitatively the tension that has to be applied to strip the pleural specimen.

One could argue that the difference in P_suc between stripped specimens of pleura and unstripped specimens of pericardium is due to the smaller thickness of pleural specimens (16, 21). This, however, is not the case because there is no relationship between P and the thickness of the specimens (Fig. 1). Moreover, the findings obtained in the experiments in which the mesothelium was scraped away from the specimen show that most of the resistance to diffusion in the pericardium is provided by the mesothelium (86% in the experiments without phospholipids and 96% in the experiments with phospholipids on the luminal side). For this reason a twofold change in specimen thickness does not produce an appreciable change in P (Fig. 1). The finding that P_suc of scraped specimens is similar to that found in stripped specimens of sheep visceral pleura (16), and smaller than that found in stripped specimens of dog visceral or parietal pleura (21), indicates that 1) the mesothelium of stripped specimens of pleura was damaged so much that its resistance to diffusion was nearly nil; and 2) P of the connective tissue of the pleura is smaller in sheep than in dogs, and/or in the latter species or experiments P not only of the mesothelium but also of the connective tissue was increased by stripping. In conclusion, although we cannot rule out that P of the pleura to small solutes is a little higher than that of the pericardium or that there are small species differences, the above findings indicate that P of the pleura should be on the same order of magnitude as that presently found in the pericardium. In the sternal part of rabbit pericardium, cribriform zones and milky spots (Kampmeier foci) are fewer and smaller than in the parietal pleura (26). Because these areas are likely more permeable than is the rest of the mesothelium, the permeability of the sternal part of the pericardium could be smaller than that of the parietal pleura but greater than that of the visceral pleura (which lacks cribriform zones and milky spots). Considering also that our specimens of pericardium may have been damaged, although markedly less so than were the stripped specimens of pleura (16, 21), the value of P obtained in the experiments with phospholipids on the luminal side (0.70 × 10⁵ cm/s; Table 1) is likely still higher than that occurring under physiological conditions. If this is the case, our finding for P_suc of the pericardium supports the value of P to mannitol of the pleura, indirectly assessed in anesthetized rabbits (0.5 × 10⁵ cm/s) (35). The latter value was obtained from the time course of the concentration of labeled mannitol in a hydrothorax, the diffusional outflux of mannitol from the hydrothorax, an estimate of mannitol concentration in the interstitium of the area of apposition between right and left pleural space, and the area of pleural surface (35). Finally, P_suc of the pericardium in the experiments with phospholipids (0.70 ± 0.06 × 10⁵ cm/s), is somewhat larger than or similar to P_suc of the gallbladder, 0.38 ± 0.14 (25) and 0.76 ± 0.14 × 10⁵ cm/s (28), the wall of which consisted of a leaky epithelium, connective tissue, and smooth muscles (the mesothelium was likely destroyed by the manipulations required by the experiment).

The 3.4-fold decrease in P_suc and the 1.9-fold decrease in P_Na⁺ occurring when phospholipids are added to the solution facing the luminal side of the specimen of pericardium (Table 1) are likely due to the adsorption of phospholipids on the luminal side of the mesothelial cells, according to the findings by Hills (11) and Hills et al. (13) in the pleura and Hills and Butler (12) in the pericardium. Indeed, this marked decrease in P does not occur when phospholipids are added to the solution facing the interstitial side of the specimen, where they are not adsorbed (10). As a matter of fact, the layer (or layers) of phospholipids adsorbed on the luminal side of the mesothelial cells provides a further resistance to diffusion. Moreover, the greater decrease in P_suc than in P_Na⁺ (and hence the increase in P_Na⁺/P_suc; see RESULTS) occurring in the experiments with phospholipids in the solution facing the luminal side of the specimen suggests that the layer of phospholipids adsorbed on the mesothelial cells restricts the diffusion of sucrose. Finally, the finding that P_Na⁺/P_suc in the experiments without phospholipids is similar to that occurring in free diffusion in only one-half of the specimens, whereas in the rest it is greater (see RESULTS), suggests that the layer of phospholipids normally adsorbed on the luminal side of the mesothelium is altered to a varied extent in the process of collecting the specimens. Therefore, one should consider only the nine specimens with P_Na⁺/P_suc similar to that in free diffusion to have a more precise indication of the effect of the phospholipids on P. If this is done, it turns out that phospholipids decrease P_suc by 4.8-fold and P_Na⁺ by 2.3-fold.

The finding that P_Na⁺ is similar in both directions suggests that active Na⁺ transport in the pericardium is nil or very small relative to passive Na⁺ flux. Passive Na⁺ flux in the experiments with phospholipids on the luminal side, 18.9 ± 0.9 µmol · h¹ · cm² (Table 1), is smaller than that in renal proximal tubule of rabbits, 55.8 ± 9.6 µmol · h¹ · cm² (18), and is roughly similar to that in rabbit gallbladder, 22.1 ± 0.6 µmol · h¹ · cm² (9). On the other hand, even in the experiments with phospholipids, passive Na⁺ flux through pericardial specimens is ~200 times greater than active Na⁺ flux that seems to occur in the pleura, according to indirect evidence in anesthetized rabbits, 0.1 µmol · h¹ · cm² (1). This situation makes it extremely difficult at present to detect an active Na⁺ transport from the differences between unidirectional Na⁺ fluxes across a specimen of pleura. Nevertheless, as already pointed out by Kim et al. (16), it is possible that active transport occurs across the pleura. The present finding that passive Na⁺ flux through the pericardium is similar to or smaller than that through leaky epithelia displaying a marked active transport suggests that pleural permeability is compatible with a small active transport, which is in line with the various pieces of evidence indirectly obtained in vivo (1, 32-35).

In the experiments with phospholipids on the luminal side of the specimen, P_suc of the mesothelium, 0.74 × 10⁵ cm/s (Table 2), is similar to P_suc of the leaky epithelium of renal proximal tubule, 0.69 × 10⁵ cm/s (14), and roughly similar to (perhaps smaller than) P_suc of the endothelium of lung, muscle, and skin capillaries, ~1 × 10⁵ cm/s (5). The relative pore area of the mesothelium (~0.1%) appears also similar to that of capillary endothelium (~0.1%) (20).

The experiments in which the mesothelium was scraped away from the specimen provided the opportunity to measure the apparent diffusion coefficient (D') of sucrose through the connective tissue of the pericardium. D' is lower than D because 1) only part of the area of the membrane is available for diffusion; 2) the diffusion path is tortuous; and 3) the diffusion may be restricted if the ratio between solute radius and pore radius is >0.01 (22). All three factors affect P; hence, D' can be obtained from P · l. From the above data for P_con (corrected for the effect of unstirred liquid layers) and the corresponding values of l (see RESULTS), one gets D'_con = (19.4 × 10⁵ cm/s) · (74 × 10⁴ cm) = 0.14 × 10⁵ cm²/s. This value is 20% of that in water (0.7 × 10⁵ cm²/s). This difference should mainly depend on the reduction in diffusion area brought forth by the presence of many fibers in the connective tissue of the pericardium (particularly collagen) (26). D' of K⁺ through the connective tissue of frog mesentery has been found to be 37% of that in water (4). D' of sucrose through the relaxed myocardium has been found to be 23% of that in water (25).