Serum albumin decreases transendothelial permeability to macromolecules

MICROVASCULAR

Serum

42, 91-102 (1991)

RESEARCH

Albumin

HAZEL The Albany

LUM, Medical

Decreases Transendothelial Macromolecules’ ALMA College

Permeability

SIFLINGER-BIRNBOIM, FRANK BLUMENSTOCK, ASRAR B. MALIK of Union University, Albany, New Received

Department York 12208

October

of Physiology

to

AND

and Cell Biology,

25, 1990

We examined the effects of serum albumin and other serum proteins on the fluxes of tracer rZ51-albumin (MW 69 kDa) and ‘*‘I-haptoglobin (MW 100 kDa) across the pulmonary artery endothelial monolayer in vitro to test the role of serum proteins in modulating the endothelial barrier function. Replacement of control complete culture medium (20% fetal calf serum in DMEM) with DMEM alone increased the transendothelial ‘251-albumin clearance rate (a measure of ‘2SI-albumin permeability) by 83% of the control value. Repletion with 50% calf serum or with 2.0 g% albumin (i.e., the albumin concentration in 50% serum) decreased “‘I-albumin permeability to the control value. This effect of serum or albumin was concentration-dependent since neither 12.5% serum nor 0.5 g% albumin (i.e., albumin concentration in 12.5% serum) altered “‘I-albumin permeability from control values. The ammonium sulfate-precipitated serum protein fraction rich in albumin decreased “SI-albumin permeability from the control DMEM value, whereas serum fractions containing predominantly y-globulin or depleted of protein did not significantly alter “‘I-albumin permeability. Other serum proteins that have been proposed to reduce endothelial permeability, q-acid glycoprotein (0.035-0.14 g/l00 ml) and fibronectin (5 mg/lOO ml), did not decrease “‘Ialbumin permeability from DMEM values. The endothelial permeability of ‘251-haptoglobin of 4.63 t 0.53 x 10mh cm/set in the presence of DMEM was 30% of the ‘ZSI-albumin permeability value. The addition of 2.0 g% albumin or 50% serum decreased “‘1-haptoglobin permeability to 57 and 31%, respectively, of the DMEM value. These results indicate the critical role of serum albumin in regulating the restrictiveness of the endothelial barrier to tKiCrOttIOk!CUkS.

0 1991 Acadrmx

Press. Inc.

INTRODUCTION Albumin comprises 60% of the total plasma proteins and is the primary determinant of colloid osmotic pressure in plasma and interstitial fluids (Peters, 1975). In addition, albumin is an important carrier protein for the transport of fatty acids, ions, drugs, and hormones into endothelial cells, and these molecules being carried by albumin are removed during the transit of albumin across the endothelial cell (Kragh-Hansen, 1981; Peters, 1975). Albumin transcytosis may be an important mechanism for the transport of albumin across the endothelial barrier (Ghitescu et al., 1986; Milici et al., 1987). Recent studies have indicated I Supported by Grants HL 45638 and HL 17016 from the National Institutes of Health. 91 0026.2862/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.

92

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ET

AL.

another potentially important but poorly understood role of serum albumin in increasing the “restrictiveness” of the endothelial barrier to fluid and solutes (McDonagh, 1983; Michel et al., 1985; Myhre and Steen, 1977; Powers et al., 1989; Schneeberger and Hamelin, 1984). The notion that albumin enhances the restrictiveness of the vascular barrier is supported by the finding that “albuminpoor” (co.33 g%) perfusates caused transvascular leakage of fluorescent albumin (McDonagh, 1983), ferritin (Schneeberger and Hamelin, 1984), and radiolabeled dextran (Myhre and Steen, 1977) and the finding that this effect is reversed by repletion of albumin. Albumin’s effect on the barrier function may require albumin binding to the endothelial cell surface and/or to other components of the cells such as its interendothelial clefts (Bignon et al., 1975; Ghitescu et al., 1986; Michel et al., 1985; Schneeberger and Hamelin, 1984). Haraldsson and Rippe (1985, 1987) have shown that albumin regulates transendothelial water flux, supporting the earlier observation of Curry and Michel (1980), whereas another serum protein, orosomucoid (a,-acid glycoprotein), is a critical determinant of transendothelial albumin permeability. In the present study, we examined the effects of different serum fractions and different serum proteins (i.e., albumin, a,-acid glycoprotein, fibronectin) on the transendothelial flux of ‘2”I-albumin (MW 69 kDa) and ‘251-haptoglobin (MW 100 kDa) across confluent monolayers of bovine pulmonary artery endothelial cells.

METHODS Reagents and Solutions

The following solutions were prepared with Dulbecco’s modified Eagle medium (DMEM): 20% fetal calf serum and 50% calf serum (GIBCO, Grand Island, NY); 2.0 and 0.5 g% ml bovine serum albumin (BSA) (Sigma Chemical Co., St. Louis, MO); 0.035, 0.070, and 0.14 g/l00 ml a,-acid glycoprotein (Sigma), and 4.7 mg/lOO ml sheep fibronectin [purified in our laboratory (Daudi et al., 1989)]. All solutions contained 20 mM Hepes (pH 7.4). Calf serum was fractionated using 40, 70, and 100% saturation of (NHJ2S04 to precipitate proteins (Green and Hughes, 1955). The three protein fractions (labeled F40, F70, and FlOO) were dialyzed extensively to remove (NHJ2S04 and passed through cellulose acetate filters (0.5~pm pore diameter). Each fraction was diluted to 50% with DMEM and contained 20 mM Hepes (pH 7.4) before use in the transendothelial permeability assay system described below. The protein concentration of each undiluted fraction was determined with SDS-gel electrophoresis and quantified by densitometry. Permeability

Measurements

The transendothelial tracer fluxes were determined using cultured monolayers of pulmonary artery endothelial cell monolayers as described (Cooper et al., 1987; Siflinger-Birnboim et al., 1987). Briefly, this system measures the diffusive flux of tracers across cell monolayers in the absence of hydrostatic and oncotic pressure gradients. The system consisted of luminal and abluminal compartments between which a polycarbonate micropore filter is “sandwiched” (0.8-pm pore diameter). Confluent monolayers of bovine pulmonary artery endothelial cells (cell line CCL-

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TABLE PHYSICAL

Albumin Haptoglobin

CHARACTERISTICS

OF ALBUMIN

93

MACROMOLECULES

1 AND

HAFTOGLOBIN

TRACERS

D,,

(ix)

(2)

( X lo-’ cm’/sec)

PI

66 100

35.5 46.0

9.3 7.2

4.7 4.1

Note. MW, molecular weight in kilodaltons; r,, Stokes-Einstein diffusion coefficient of solute in water at 37”; ~1, isoelectric point.

radius in Angstroms (A); D,,, free

209 from American Type Culture Collection, Rockville, MD) were grown onto the luminal surface of the filter, previously gelatin- and fibronectin-coated and sterilized under UV light. These cells have been characterized as endothelial cells by the presence of angiotensin-converting enzyme activity, Factor VIII-related antigen, and Weibel-Palade bodies (Del Vecchio et al., 1980). The cells were seeded at 100,000 cells/filter and were used at 4 days postseeding when they had formed a confluent monolayer. Both luminal and abluminal compartments contained the same media (pH 7.4) at volumes of 700 ~1 and 25 ml, respectively. The luminal compartment was fitted with a Styrofoam outer ring and “floated” onto the abluminal media so that fluid levels were equal and remained equal after repeated samplings from the continuously stirred abluminal compartment. The entire system was kept at 37” by a thermostatically regulated water bath. Tracer molecule clearance was determined as the volume of luminal chamber activity “cleared” of the radioactive tracer to the abluminal chamber. The change in volume over time provided clearance rate in pl/min was determined by the weighted least-squares nonlinear regression (BMDP statistical software, Berkeley, CA) (Cooper et al., 1987). Crystallized and lyophilized BSA was further purified by gel chromatography. Lyophilized human haptoglobin was >99% pure as assessed by gel electrophoresis. Both proteins were labeled with 1251using the chloramine-T procedure (Bocci, 1964). Noncovalently bound I251 was removed by extensive dialysis against 0.1 M NaI in pH 7.4 phosphate-buffered saline. The molecular weights, Stokes-Einstein radii, and free diffusion coefficient (&) for solutes in water at 37” of albumin and haptoglobin are given in Table 1 (Renkin and Curry, 1979). Protocols

At the beginning of the experiment, the luminal compartment with the attached monolayer was “floated” onto the abluminal media and filled with media containing -6 pCi/ml of the tracer. Abluminal samples of 400 ~1 were obtained at 5-min intervals for 30 min, and the radioactivity was determined using a gamma counter (Packard Instrument Co., Dowers Grove, IL). At the termination of experiments, the percentage of free “‘1 in both luminal and abluminal compartments was determined by 12% trichloroacetic acid precipitation. These values were used to correct the clearance rates for the amount of free “‘1 “cleared” into the abluminal compartment. The tracer clearance rates were used to calculate the endothelial permeability values, which are reported as values normalized to the free diffusion coefficient for solutes in water at 37” (Table l)(PJ&).

94

LUM

F E > 2

0.40

‘I

0.30

-

0.20

-

ET AL.

2 z 8 g ii ,Y v .r

g P a L z

O.lO-

OComplete Culture Medium

DMEM

0.5g% Albumin

2.Og% Albumin

12.5% Serum

50% Serum

FIG. 1. Effects of serum and albumin on the transendothelial ‘251-aIbumin clearance rates. The 0.5 g% albumin is the approximate albumin concentration present in 12.5% serum; the 2.0 g% albumin is the approximate albumin concentration present in 50% serum. Complete culture medium contained 20% fetal calf serum. DMEM, Dulbecco’s modified Eagle medium. Values are mean f SEM; asterisks indicate a difference when compared to the complete culture medium group, P < 0.01. Number of endothelial monolayers for each group ranged from 16 to 30.

Statistics Analysis of variance (BMDP Statistical Software) was used to determine the significance of the data (Snedecor and Cochran, 1967) with significance set at P < 0.05. The data are presented as mean k SEM.

RESULTS The ‘251-albumin transendothelial clearance rate of cultured monolayers of bovine pulmonary artery endothelial cells in control complete culture media containing 20% fetal calf serum (FCS) in DMEM was 0.16 k 0.02 pl/min (Fig. 1). Replacement of the control medium with DMEM alone increased the ‘251-albumin clearance rate by 83% of the control value (Fig. 1). Addition of 2.0 g% albumin in DMEM lowered the ‘251-albumin clearance rate to near the control value of the complete culture media (0.18 + 0.01 pl/min), whereas 0.5 g% albumin was ineffective in lowering the clearance rate (Fig. 1). Addition of 50% serum in DMEM (the approximate serum concentration containing 2.0 g% albumin) also decreased the 1251-albumin clearance rate to control values (0.14 k 0.021 &min), whereas 12.5% serum (the serum concentration containing 0.5 g% albumin) did not decrease the clearance rate (Fig. 1). Higher concentrations of albumin of up to 4.0 g% added to the medium resulted in 1251-albumin clearance rates (0.17 + 0.01 pl/min at 4.0 g% albumin) similar to those observed with endothelial cells exposed to 2.0 g% albumin. The effects of different albumin and serum concentrations on the i2?-albumin transport of filters alone are shown in Table 2. The transfilter 1251-albumin clearance rates determined in the presence of DMEM containing either albumin or

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TABLE l*‘I-A~~~~~~

CLEARANCE

95

MACROMOLECULES

2 RATES ACROSS FILTERS

Medium condition

“‘I-albumin clearance rate (Nmin)

Complete culture media (20% FCS in DMEM) DMEM 0.5 g% albumin 2.0 g% albumin 12.5% serum 50% serum

1.02 k 0.09* 1.34 1.07 1.29 1.41 1.22

+ ? 2 IT +

0.09 0.11 0.08 0.09 0.02

FCS, fetal calf serum. * Values are mean k SEM.

Note.

serum were not significantly different from values determined in the presence of DMEM alone. Therefore, increased filter restrictiveness is probably not responsible for the reduction in ‘251-albumin permeability across the endothelial monolayer observed in the presence of 2.0 g% albumin or 50% calf serum (Fig. 1). We examined whether the transendothelial clearance rate of another tracer, ‘*?-haptoglobin (MW 100 kDa), was similarly reduced with the addition of 2.0 g% albumin or 50% serum. To compare the two tracers, endothelial permeability (P,,) values were calculated (Cooper et al., 1987) and normalized to the respective diffusion coefficient of the tracers in water (&)(Table 1). The computed Pec/D37 values for 1251-albumin and 1251-haptoglobin are presented in Table 3. The Pec/Dj7 values for ‘251-haptoglobin in all conditions (DMEM, 2.0 g% albumin, or 50% serum) were lower than the ‘251-albumin Pec/D3, values (P < 0.05). The addition of 2.0 g% albumin or 50% serum lowered the ‘251-haptoglobin permeability to 57 and 31%, respectively, of the DMEM value; whereas, the 1251-albumin permeability was lowered to 37% of the DMEM value in the presence of either 2.0 g% albumin or 50% serum (Table 3). TABLE COMPARISON

BETWEEN “‘I-ALBUMIN

“‘I-albumin Groups DMEM 2.0 g% albumin 50.0% serum

(X

PC, lo-” cm/set)

15.21 ? 4.09 5.62 k 0.46* 5.70 k 0.69*

3

AND ‘251-H~pr~~~~~~~ ENDOTHELIAL MONOLAYERS

permeability

PERMEABILITY

VALUES

“‘1-haptoglobin

ACROSS

permeability

PeclD,, (cm-‘)

PC< (X 10mh cm/set)

PeJDx (cm-‘)

16.37 r 4.39 6.04 k 0.49* 6.13 * 0.75*

4.63 ? 0.53 2.66 + 0.54* 1.42 k O.lS*

6.43 f 0.74 3.69 + 0.75* 1.98 f 0.25*

Note. P, is endothelial permeability (the value has been subtracted for the filter’s contribution to permeability as in Cooper et al. (1987); PJD,, is endothelial permeability normalized to D3, (see Table 1). Values are epxressed as the mean f SEM. * P < 0.05 compared to DMEM.

96

LUM

PROTEIN CONCENTRATIONS

Protein fraction*

Albumin

F40 F70

ET

AL.

TABLE 4 (g/100 ml) OF SERUM FRACTIONS

a-Globulin

P-Globulin

y-Globulin

Total protein

0.4 2.5

0.5 0.3

0.4 0.4

1.6 0

2.8 3.2

0

0

0

0

0

FlOO

Note. *Protein fractions were obtained with 40% (F40), 70% (F70), 100% (FlOO)/(NH,),SO, cipitation of serum (See Methods for details).

pre-

The possibility that the albumin component of serum was responsible for the reduction in 1251-albumin permeability was examined by testing the effects of serum fractions either rich or poor in albumin as obtained by (NHJ2S04 precipitation. Fraction F40 [obtained using 40% (NH&SO41 contained predominantly y-globulin (57% of total protein), F70 [obtained using 70% (NH4)2S04] had predominantly albumin (78% of total protein), and FlOO [using 100% (NH4)2S04] had no detectable protein (Table 4). Only fraction F70 decreased the transendothelial 1251albumin clearance rate across the endothelial monolayer (P < 0.01); the other fractions had no significant effects (Fig. 2). 1251-albumin clearance rate in the presence of We measured transendothelial cY,-acid glycoprotein (orosomucoid). As with serum and albumin, q-acid glycoprotein was added to both luminal and abluminal compartments at 0.035, 0.07, [which is the reported basal human circulating concentration (Putnam, 1975)], and 0.14 g/100 ml. However, none of these concentrations significantly altered the ‘251-albumin clearance rate from the control DMEM value (Fig. 3a). Medium containing q-acid glycoprotein plus albumin (4.0 g% albumin) also did not further lower the ‘251-albumin clearance rate from the value observed with 4.0 g% albumin

0.4 ‘I z E :

: 5 u E E $2

0.31

0.2-

U

.c E 1

O.l-

5 5 oDMEM

F40

F70

FlOO

2. Effects of different protein fractions obtained with (NH&SO, precipitation on transendothelial “51-albumin clearance rate (see Methods). F40 contained predominantly y-globulin, F70 contained predominantly albumin, and FlOO contained no detectable protein. Values are mean ? SEM; the asterisk indicates a difference when compared to the DMEM group, P < 0.01. Numbers of endothehal monolayers are 20, 30, 29, and 29 for DMEM, F40, F70, and FlOO groups, respectively. FIG.

ENDOTHELIAL

0.5~

PERMEABILITY

TO

97

MACROMOLECULES

a

T

E 9 0.4 Y Kz u 2 e a

0.3

B .E

0.2

5 P 1

0.1

3 0 DMEM

0.035

0.070+

al-Acid

0.140

Glycoprotein (g/lOOml)

3a. Effect of a,-acid glycoprotein (orosomucoid) alone on transendothelial “‘l-albumin clearance rate. The dagger indicates the normal human circulating concentration of 0.070 g/100 ml. None of the groups was significantly different from the DMEM control group. Values are mean 2 SEM. Numbers of cell monolayers are 16, 20,24, and 23 for DMEM. 0.035, 0.07, and 0.14 g/100 ml groups. respectively. FIG.

alone (Fig. 3b). Addition of fibronectin alone (5 mg/lOO ml) or in combination with albumin (2.0 8%) to both luminal and abluminal compartments had no effect in altering the transendothelial ‘251-albumin clearance rate (Fig. 4).

Medium

b

0

Medium+

0.5g%

q-Acid

Glycoprotein

T

T

DMEM

Alone

Albumin

4.Og%

Albumin

3b. Effect of q-acid glycoprotein plus albumin on transendothelial ‘ZSI-albumin clearance rate. cY,-acidglycoprotein concentration in DMEM alone or DMEM with 0.5 g% albumin was O.O7g/lOO ml; whereas the 4.0 g% albumin group contained 0.14 g/100 ml ol,-acid glycoprotein. Values are mean 2 SEM. Asterisks indicate a difference when compared to the DMEM group (P < 0.01). Number of cell monolayers is 20 for each of the three groups. FIG.

LUM

ET

AL.

0.6 2

E ‘3 t

j

0.48 5 b 8 -; 0.21 2 9 a 1 ! ODMEM

5mg/lOOml Fibronectin

29%

Albumin

2g%

Albumin

+

5mg/lOOml Fibronectin

FIG. 4. Effect of fibronectin alone (5 mg/lOO ml) or fibronectin added to 2 g% albumin on transendothelial ‘ZSI-albumin clearance rates. Values are mean ? SEM; asterisks indicate a difference when compared to the DMEM group, P < 0.01. The numbers of cell monolayers are 17 each for DMEM, fibronectin, and fibronectin plus albumin group and 12 for the albumin group.

DISCUSSION Previous studies in intact microvessels and whole organ preparations indicate that circulating proteins in some unknown manner modulate the capillary permeability of water (Mason et al., 1977; Turner et al., 1983) and solutes (McDonagh, 1983; Myhre and Steen, 1977; Schneeberger and Hamelin, 1984). The role of albumin’s interaction with the endothelial cell in reducing water flux is becoming clear (Michel et al., 1985; Powers et al., 1989), but the effects of albumin and other serum proteins in regulating macromolecular flux across the endothelial barrier are poorly understood. In various studies, specific serum proteins (i.e., albumin, a,-acid glycoprotein, and fibronectin) have been reported to increase the “restrictiveness” of the capillary barrier (Haraldsson and Rippe, 1985, 1987; McDonagh, 1983; Myhre and Steen, 1977; Kowalczyk et al., 1990). In light of these data, we examined whether serum contained proteins that are capable of modulating the endothelial barrier function and evaluated the relative contributions of albumin, cr,-acid glycoprotein, and fibronectin in this process. We studied the effects of various concentrations of serum on transendothelial ‘251-albumin permeability using the cultured pulmonary artery endothelial monolayer grown on a microporous filter. The control ‘251-albumin clearance rate of 0.16 ? 0.02 pl/min measured in the presence of complete culture medium (i.e., 20% FCS in DMEM) was increased by 83% with the replacement of the complete culture medium with DMEM. Interestingly, the addition of 50% serum or 2.0 g% albumin (which is the approximate albumin concentration contained in 50% serum) reduced the ‘251-albumin clearance rates from values observed in the presence of DMEM alone (see Fig. 1). These reductions were comparable to those observed in the presence of complete culture medium. The permeability reducing effect of serum or albumin required an interaction of endothelial cells since 50% serum or 2.0

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99

g% albumin did not alter 1251-albumin permeability across the microporous filter alone. Moreover, this effect of serum and albumin was concentration-dependent in that 0.5 g% albumin or 12.5% serum did not reduce ‘25J-albumin clearance rates from the values observed with DMEM alone. To evaluate the potential role of albumin in mediating the serum-induced reduction in transendothelial ‘251-albumin permeability, we determined the effects of the serum protein fractions obtained with ammonium sulphate precipitation. The serum fraction (F70) containing mostly albumin (78%) was the most effective in decreasing ‘251-albumin permeability from the DMEM value. Since the F70 fraction also contained 9.4% a-globulin and 12.5% P-globulin, it is possible that these serum proteins may contribute to the decreased ‘251-albumin permeability; however, fraction F40 containing similar amounts of (Y- and P-globulins but did not have an effect on ‘251-albumin permeability, suggesting that serum albumin was responsible for the observed reduction in endothelial ‘251-albumin permeability. The finding that albumin is capable of decreasing transcapillary ‘251-albumin transport is consistent with observations in the intact vascular preparations (McDonagh, 1983; Schneeberger and Hamelin, 1984). In a rat heart, addition of 2 g% of albumin decreased the fluorescein-labeled albumin “leakage” from the coronary circulation (McDonagh, 1983). Using a fluorocarbon exchange-transfused rat lung model in which plasma proteins were removed, Schneeberger and Hamelin (1984) observed that the loss of circulating albumin was associated with increased permeability to ferritin, suggesting that circulating albumin normally serves to restrict endothelial permeability of macromolecules. Haraldsson and Rippe (1987) proposed a critical role of serum protein, cy,-acid glycoprotein (effective concentrations 0.015 to 0.1 g/100 ml), in modulating transcapillary albumin flux in hindlimb vessels. Our results could be explained on this basis since the commercially available albumin may be contaminated with c-u,-acid glycoprotein (Lima and Salzer, 1981). However, two observations do not support this possible role of &,-acid glycoprotein. First, addition of q-acid glycoprotein to the medium at normal (0.07 g/100 ml), half-normal, or twice the normal plasma concentrations had no effect on endothelial r2’I-albumin permeability. Second, addition of the normal concentration of cy,-acid glycoprotein (0.07 g/100 ml) plus albumin (4.0 g%) did not further decrease “51-albumin permeability relative to the decrease observed with albumin alone (4.0 g%). The different findings of Haraldsson and Rippe (1987) may be attributed to the complexity of the intact hindlimb vessel preparation with includes not only the endothelial cell monolayer lining the vessel, but also the complex interstitial matrix components with which a,-acid glycoprotein can bind (Putnam, 1975), and thereby influence the transcapillary flux of albumin. From the present data, we can make the conclusion that cy,-acid glycoprotein does not influence endothelial ‘251-albumin permeability; however, we cannot rule out the possibility that interaction of this serum protein with extracellular matrix proteins may modulate capillary permeability. We also examined the effects of fibronectin (MW 440 kDa) on transendothelial 12”1-albumin clearance rate because of the suggestion that fibronectin binds to endothelial junctional and surface components (Kowalczyk et al., 1990), thereby reducing endothelial permeability, particularly in conditions where the permeability is elevated (Saba et al., 1983). In the present study, fibronectin alone or in

100

LUM ET AL.

combination with albumin had no effect on ‘251-albumin permeability, indicating that fibronectin in the endothelial cell culture medium did not enhance the restrictiveness of the endothelial barrier even though the barrier in vitro is more permeable than the in situ capillary barrier (Cooper et al., 1987). However, it is possible that fibronectin incorporation into basolateral matrix following a several day period of incubation is needed to enhance the restrictiveness of the endothelial monolayer to albumin (Kowalczyk et al., 1990). The permeability decreasing effect of albumin was not limited to 1251-albumin transport. Addition of 2.0 g% albumin also lowered the transendothelial 12’1haptoglobin permeability to 57% of the DMEM value, whereas serum lowered it to 31% of the DMEM value. In addition, Pec/D3, values for ‘251-haptoglobin for all groups (DMEM, 2.0 g% albumin, and 50% serum) were lower than those for 1251-albumin, which is likely due to the fact that haptoglobin is a larger molecule (r, = 46 8, for haptoglobin vs r, = 35.5 A for albumin). The mechanism of albumin-induced restriction of tracer albumin and haptoglobin flux is unclear since the precise nature of the interaction(s) between albumin and the endothelial glycolayx is unknown. Albumin may be adsorbed to the glycocalyx structure at the level of the junctions, and may thereby restrict the transport of macromolecules filtered through junctions. Studies using cultured endothelial cells (Schnitzer et al., 1988; Sage et al., 1984) and intact vessels (Ghitescu et al., 1986; Milici et al., 1987; Schneeberger and Hamelin, 1984) have also shown that albumin binds specifically to plasmalemmal membrane and plasmalemmal vesicles; therefore, “specific” binding of albumin may increase restrictiveness of the barrier if binding occurs at the sites of macromolecular transport. Nonspecific binding, involving electrostatic interactions between positively charged residues on albumin with negative charge sites on the endothelial glycocalyx (Michel et al., 1985; Powers et al., 1989), may be another factor in increasing the restrictiveness of the endothelial monolayer. In summary, these results indicate that a serum component(s), chiefly albumin, decreases tracer ‘2sI-albumin permeability in a concentration-dependent manner. In contrast to albumin, a,-acid glycoprotein and fibronectin had no such effect. Albumin also decreased ‘251-haptoglobin permeability, indicating that albumin also regulates the flux of other macromolecules across the endothelial barrier. This regulatory effect of albumin in reducing ‘2”I-albumin permeability may involve the interaction of albumin with sites on the endothelial cell (e.g., interendothelial junctions) which are responsible for endothelial macromolecule transport. ACKNOWLEDGMENTS The authors thank Ms. Lynn McCarthy for typing the manuscript and Nancy Gertzberg for preparing the illustrations.

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Radiat.Isot. 15, 449-456.

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COOPER,J. A., DEL VECCHIO, P. J., MINNEAR, F. L., BURHOP, K. E., GARCIA, J. G. N., AND MALIK, A. B. (1987). Measurement of albumin permeability across endothelial monolayers in vitro. J. Appl. Physiol. 62, 1076-1083. CURRY, F. E., AND MICHEL, C. C. (1980). A fiber matrix model of capillary permeability. Microvasc. Res. 20, 96-99. DAUDI, I., SABA, T. M., LEWIS, M., LEWIS, E., BLUMENSTOCK, F. A., GUDEWICZ, P., MALIK, A. B., AND FENTON, J. W. (1989). Fibronectin fragments in lung lymph after thrombin-induced lung vascular injury. Lab. Invest. 61, 539-547. DEL VECCHIO, P. J., RYAN, J. W., CHANG, A., AND RYAN, U. S. (1980). Capillaries of the adrenal cortex possess aminopeptidase A and angiotensin-converting enzyme activities. Biochem. J. 186,

605-608. L., FIXMAN, A., SIMIONESCU, M., AND SIMIONESCU,N. (1986). Specific binding sites for albumin restricted to plasmalemmal vesicles of continuous capillary endothelium: Receptor-mediated transcytosis. J. Cell Biol. 102, 1304-1311. GREEN, A. A., AND HUGHES, W. L. (1955). Protein fractionization on the basis of solubility in aqueous solutions of salts and organic solvents. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, Eds.), Vol. 1, pp. 67-82, Academic Press, New York. HARALDSSON,B., AND RIPPE, B. (1987). Orosomucoid as one of the serum components contributing to normal capillary permselectivity in rat skeletal muscle. Acta Physiol. Stand. 129, 127-135. HARALDSSON,B., AND RIPPE, B. (1985). Serum factors other than albumin are needed for the maintenance of normal capillary permselectivity in rat hindlimb muscle. Acta. Physiol. &and. 123, 427436. KOWALCZYK, A. P., TULLOH, R. H., AND MCKEOWN-LONGO, P. J. (1990). Polarized fibronectin secreting and localized matrix assembly sites correlated with subendothelial matrix formation. Blood 75, 2335-2342. KRAGH-HANSEN, U. (1981). Molecular aspects of ligand binding to serum albumin. Pharmacol. Rev. 33, 17-53. LIMA, J. J., AND SALZER, L. B. (1981). Contamination of albumin by cy,-acid glycoprotein. Biochem. GHITESCU,

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