effect of inorganic lead on some functions of the cerebral microvessel endothelium

TOXICOLOGY AND APPLIED PHARMACOLOGY t&$389-399 (1986)

Effect of Inorganic Lead on Some Functions of the Cerebral Microvessel Endothelium

KAREN MAXWELL, HARRY V. VINTERS, JUDITH A. BERLINER, JAMES V. BREADY, AND PASQUALE A. CANCILLA

Department of Pathology, University of Calijornia, Los Angeles, California 90024

Received July 22, 1985; accepted February I 4, 1986

Effect of Inorganic Lead on Some Functions of the Cerebral Microvessel Endothelium. MAX- WELL, K., VINTERS, H. V., BERLINER, J. A., BREADY, J. V., AND CANCILLA, P. A. (1986). Toxicol.

Appl. Pharmacol. 84,389-399. The effect of inorganic lead on two functions of cerebral microvessel endothelium, cell division and glucose analog uptake, was investigated. Lead concentrations considered to be toxic in humans inhibited both functions in cultured endothelial cells. Both effects were dependent on the length of lead exposure and dose over the range of 10e4 to 10e6 M lead acetate. After 4 days of exposure there were 76% fewer cells in 10e4 M lead-exposed cultures relative to control cultures. After 4 days of exposure to 10d5 M lead there were 55% fewer cells, and after 1 Om6 M lead exposure there were 15% fewer cells. Two days after 10m4 M lead exposure [methyl-‘Hlthymidine incorporation into endothelial cells was inhibited by 7 1%. Incorporation was inhibited 47% by IO-’ M lead but 10e6 M lead did not inhibit incorporation after 2 days of exposure. Glucose analog uptake was inhibited in both contact-inhibited and log-phase cells; however, the latter were more sensitive to lead and this increased sensitivity correlated with a higher lead content in this cell population. Both the specific carrier-mediated and the nonspecific components of glucose analog uptake were inhibited by exposure of the endothelial cells to lead. A lead exposure of 40 min produced a significant effect on the uptake mechanism. In order to manifest its effects the lead had to be present in serum-containing medium, suggesting that some serum component was necessary to present the lead to the endothelial cells. These findings imply that the initial target of inorganic lead in the CNS may be the plasma membrane of the capillary endothelial cells, and that lead may act by altering the physiological function of these membranes. 0 1986 Academic Press, Inc.

It has been proposed that the pathological dothelium were then studied (Goldstein et al., changes seen in the brain after acute lead ex- 1977; Kolber et al., 1980; Lefauconnier et al., posure, i.e., edema and hemorrhage, are the 1980). These models, however, present various result of cerebral capillary dysfunction (Gold- problems in assessing endothelial function. stein et al., 1977; Kolber et al., 1980; Lefau- Animal-to-animal variations in lead absorp- cannier et al., 1980; Silbergeld et al., 1980). tion and metabolism exist. In microvessel Neuronal changes are considered to occur preparations, nonviable cells and cell types secondarily to the vascular alterations (Pents- other than endothelium may be present. With chew and Garro, 1966). Previously, to define the availability of an isolated line of mouse the mechanism of lead toxicity at the cerebral cerebral microvessel endothelial cells, we were capillary level, both whole animals and iso- able to study the effects of lead on the cerebral lated brain microvessels have been exposed to endothelium in a controlled and defined en- lead and various functions of the capillary en- vironment, thus avoiding some of the prob-

0041-008X/86 $3.00

389 Copyright 0 1986 by Academic Press.. Inc. All riglm of reproduction in any foonn reserved.

390 MAXWELL ET AL.

lems that have complicated interpretation of data from other models. The effect of lead on cell division and glucose analog uptake in these cultured endothelial cells is the subject of this report.

METHODS

Cell culture. The cells used in these studies were from a line of mouse cerebral microvessel endothelium (ME- ly) that is similar in morphology, function, and derivation to a previously characterized line of cerebral microvessel endothelium, the ME-2 cell line (De Bault, 1982; De Bault and Cancilla, 1980; De Bault et al., 1979, 198 1). The cells were maintained in modified Lewis medium (MLM) sup plemented with 20% heat-inactivated fetal bovine serum in Costar T-75 tissue culture flasks (De Bault et al.. 1979).

Cells were passaged twice weekly at a split ratio of 1:2. All cells used in these studies were between passage 13 and 20.

Lead solutions. Fresh stock solutions (1 O-* M) of lead acetate and sodium acetate were made in boiling double- distilled water and were diluted into complete MLM or serum-free MLM to the desired final lead or sodium concentration ( 1O-4 to IO+’ M).

Measurement of cell number. ME-ly cells were plated at a density of 10,000 cells/cm* in Costar 6-well multiwell culture dishes in complete MLM. After 24 hr the medium was changed to complete MLM containing either lead or sodium acetate ( 10e4 to 1Oa M). The cells were then grown from 1 to 4 days at which time they were trypsinized and counted in a Couher counter. All cell counts were per- formed on triplicate sets of similarly treated cells.

Thymidine incorporation. Cells were plated in 6-well dishes at a density of 10,000 cells/cm* in complete MLM. Twenty-four hours after seeding the cells were washed with sterile phosphate-buffered saline (PBS) and then incubated in serum-free MLM for 48 hr to establish baseline quies- cence with respect to cell division. The medium was then changed to lead- or sodium-containing complete MLM, and the cells were grown from 1 to 4 days, at which time the cells were pulsed with 0.2 &i/well of [methyl-

‘Hlthymidine for 6 hr. The cell layer was then washed twice with 4 ml warm PBS, incubated for 10 min with ice-cold 10% trichloroacetic acid (TCA), and washed again with 4 ml of warm PBS. This treatment determined the amount of [methyl-3H]thymidine incorporated into the TCA precipitable fraction of the cell, which was used as a measure of DNA synthesis. After the washes the cells were solubilized in 0.2 N NaOH overnight at room tem- perature. Aliquots were taken for the determination of radioactivity and protein content. Radioactivity was determined in a Beckman LS7800 scintillation counter with

automatic quench control, and protein concentrations were determined using the method of Lowry et al. (195 1). All determinations of [methyl-3H]thymidine incorporation were done on triplicate sets of cells.

Glucose analog uptake. Cells were plated into Costar multiwell dishes in complete MLM and when they had reached a confluent density the medium was changed to complete MLM containing lead or sodium for 2 days. For studies examining the effects of lead on contact-inhibited cells versus log-phase cells, cells were plated so that they were at various densities when they received the 2- day exposure to lead or sodium. In studies done to determine the length of lead exposure necessary to produce an effect on glucose uptake, confluent cultures of Me-ly cells were exposed to lead or sodium for periods of time varying from 20 min to 48 hr before glucose analog uptake was measured. To measure ghtcose analog uptake, monolayers of cells were washed with PBS and incubated in 1 ml of buffer (0. I % bovine serum albumin (BSA), 1 mM pyruvate, 4 mM KCI. 4.2 mM CaC12, 1.2 mM MgQ, 150 mM NaCl, 15 mM Hepes, pH 7.4) for 20 min at 37°C. The buffer was then changed to buffer containing isotope and the cells were incubated for varying times. The isotope solution consisted of the appropriate ‘H-labeled glucose analog (5 &i/ml) and the corresponding nonradioactive glucose analog (5 IIIM), usually 3-O-methylglucose (3-OMG), in the same buffer as that used in the preincubation step. [‘4C]Sucrose or L-[‘4C]glucose (0.5 &i/ml) was used as an extracellular and nonspecific uptake marker. The uptake was stopped with the addition of ice-cold 0.02 mM cyto- chalasin B in PBS. The monolayer was then washed three times with ice-cold PBS and was solubilized in 0.2 N

NaOH. Aliquots were taken for determination of radioactivity and protein concentration.

Materials. 3-O-[mefhyl-3H]D-Glucose (80 Ci/mmol), [U-“‘C]sucrose (673 mCi/mmol), [methyl-‘Hlthymidine (6.7 Ci/mmol), and ~-[l-~~C]glucose (47 mCi/mmol) were obtained from New England Nuclear. 3-O-[methyl-3H]s

Glucose (50 Ci/mmol) and [U-‘4C]sucrose (590 mCi/ mmol) were purchased from ICN Pharmaceuticals. Lead acetate was from Eastman Kodak Company (Lot CO 15) and sodium acetate was from J. T. Baker Chemical Com- pany (Lot 242 124).

RESULTS

Lead Efect on Cell Division

The effect of 10e4 M lead acetate on cerebral endothelial cell number is presented in Fig. 1. Beginning 1 day after lead exposure there was a significant decrease in cell number (p < 0.01). After 3 days of exposure there was a

EFFECT OF LEAD ON CEREBRAL ENDOTHELIUM 391

Time of Lead Exposure (days)

FIG. 1. Effect of 10m4 M lead on cell number. Cells were plated at 10,000 cells/cm* in MLM and changed to 1O-4 M lead- or sodium-containing MLM 24 hr later (Day 0). The cells were grown in 10m4 M lead or sodium for the indicated period of time, then counted with a Coulter counter. The number of cells present in lead-treated and control cultures is given. (All standard deviations were less than 15s.) With 10e4 M sodium acetate (0); with 10e4 M lead acetate (0). Data were analyzed by f test.

highly significant decrease in cell number (p < 0.001). There was a dose-dependent decrease in cell number when the endothelial cells were exposed to 10m4, 10m5, and 10e6 M lead acetate (Fig. 2). A two-way analysis of variance (ANOVA) analyzing the ratio of number of lead-treated cells to control cells by

0’ 1 2 3 4 Time of Lead Exposure (days)

FIG. 2. Lead effect on cell number. Cells were plated at 10,000 cells/cm’ in MLM and changed to lead- or sodium- containing MLM 24 hr later (Day 0). The cells were grown in the lead or sodium medium for the indicated period of time and then counted in a Coulter counter. The ratio of cell number with lead to cell number in sodium acetate- treated control cultures is indicated. 10d6 M lead (0), 10m5 M lead (Cl), 10m4 M lead (A).

length of exposure and concentration of lead indicated that there was a significant effect of both time and concentration of lead on cell number (r = 0.825, p < 0.000) (Table 1). There was also a significant interaction between

TABLE 1

ANALYSIS OF VARIANCE TABLE FOR FIG. 2 BY RATIO, DAY, CONCENTRATION

Source of variation Sum of squares df

Mean square F

Significance ofF

Main effects Day Concentration

Two-way interactions Day Concentration

Explained

Residual

Total

5.266 2.397 2.869

0.535 0.535

5.800

0.580

6.380

5 3 2

6 6

11

96

107

1.053 174.399 0.000 0.799 132.323 0.000 1.434 237.513 0.000

0.089 14.754 0.000 0.089 14.754 0.000

0.527 87.320 0.000

0.006

392 MAXWELL ET AL.

length of exposure and concentration of lead. Further analysis of the data by the least significant difference (LSD) procedure was done to determine which of the individual means differed significantly from each other. This analysis showed that after 1 and 2 days of exposure to lead, cells treated with 10e4 and 10m5 M lead differed from cells treated with 1O-6 M

lead. After 3 and 4 days, cells treated with lop4 M lead differed from cells treated with 10e5 and 10e6 M lead, and cells treated with 10e5 M lead differed from lop6 M lead-treated cells (p < 0.05). The difference in cell number seen in lead-treated cultures cannot be attributed to increased detachment of cells from the culture dish since the number of unattached cells in control and lead cultures did not differ.

In addition to inhibiting cell number, lead acetate also inhibited [methyl-3H]thymidine incorporation into cultured endothelial cells. The effect of lead acetate on [methyl- 3H]thymidine incorp oration was both time and dose dependent, creating the same pat- terns that were seen when cell number was studied. Figure 3 shows the effect of lead on

IO-“M Pb

FIG. 3. Effect of lead on thymidine incorporation. Thy- midine incorporation after 2 days of growth in lead medium was measured in cells as described under Methods. Data are expressed as cpm/mg protein to account for dif- ferences in cell number.

cerebral endothelial cell [methyl-3H]thymidine incorporation after 2 days of exposure to lead acetate. Lead at lop4 M inhibited [methyl- 3H]thymidine in co rp oration by 7 1% as compared to control values, and 10W5 M lead inhibited incorporation by 47% (Fig. 3). This inhibition corresponds to a decrease in cell number of 22 and 16%, respectively, after 2 days growth in 1O-4 and 10m5 M lead medium (Fig. 2). Thymidine incorporation into cells treated with all three concentrations of sodium acetate was virtually the same so all of the values were combined and used to determine the mean and standard deviation of the control. An ANOVA comparing thymidine incorporation at the different lead concentrations showed that overall the means were significantly different (p < 0.0000) (Table 2). Further analysis of the data by the LSD procedure showed that both 1O-4 and lo-’ M lead-treated cells differed significantly from cells treated with 10m6 M lead and control cells (p < 0.05).

Eflect of Lead on Glucose Analog Uptake

Figure 4 illustrates a typical uptake curve for 3-OMG and ~-glucose in a confluent culture of cells exposed to 1 Om4 M lead for 2 days. ~-Glucose or sucrose was used to correct for extracellular adhesion and nonspecific uptake of glucose analogs since they are not trans- ported by the glucose carrier (Cancilla and De Bault, 1983; Renkawek et al., 1978). Lead at 10e4 M inhibited both the specific carrier-mediated (3-OMG) and the nonspecific (L&U-

case) uptake of glucose analogs into the cells (Fig. 4). An ANOVA comparing glucose analog uptake by treatment (lead vs sodium), incubation time, and substrate (3-OMG vs L- glucose) indicated that the population means were significantly different (r = 0.8 19, p < 0.000). There was also a significant interaction between incubation time and substrate used (a < 0.000) (Table 3). Independent group mean comparisons for each substrate showed that the lead-treated groups differed signifi-


TABLE 2

ANALYSIS OF VARIANCE TABLE FOR FIG. 3 BY VARIABLE UPTAKE AND VARIABLE CONCENTRATION

Source df

Between groups 3 Within groups 20

Total 23

Sum of squares

367.008 1 101.9155

468.9236

Mean squares

122.3360 5.0958

F ratio

24.0073

F probability

o.oooo

cantly from the sodium-treated groups for each incubation time studied (p < 0.0000). Figure 4 shows that the nonspecific uptake of L-glu-

case was inhibited by 10U4 M lead by up to 50% (Fig. 4 and Table 4). We have also shown that the uptake of sucrose is inhibited to the same degree by 1 OP4 M lead (Table 4). Analysis of variance comparing the uptake by treatment (lead vs sodium), incubation time, and substrate (L-glucose vs sucrose) showed that the population means were significantly different (Y = 0.808, p < 0.000) (Table 5). In addition, there was an interaction between time and

Incubation Time (mid

FIG. 4. Effect of lead on glucose analog uptake. Con- fluent cultures of cells were exposed to lo-” M lead for 2 days before glucose analog uptake was measured. Data are expressed as nmoles substrate/mg protein taken up after varying times of incubation with radiolabeled substrate. 3-[3H]OMG uptake with lead (m), and without lead (Ci); L-[‘4C]glucose uptake with lead (0) and without lead (0).

substrate (p -C 0.02). Independent group mean comparisons showed that the lead-treated group mean was significantly different from the sodium-treated group mean for each incubation time studied. This inhibition does not account for all of the inhibition of specific uptake, however, since the nonspecific component was normally 25% of the overall glucose uptake but was only 14% of the overall uptake when the cells were treated with lead.

The inhibition of glucose analog uptake was dose dependent (Fig. 5). In confluent cultures, whereas 1 Om4 M lead inhibited 3-OMG uptake by 39%, 10s5 M lead only inhibited uptake by 19% and there was no significant inhibition of uptake by 10T6 M lead. A two-way ANOVA comparing uptake by treatment (lead vs sodium) showed that there was a significant difference among the population means when the lead was in serum-containing medium (r = 0.538, p < 0.000) (Table 6). When the individual group means were compared by the LSD test, it was shown that the 10m4 M lead- treated cells differed from the 10m5 and 10e6 M lead-treated cells (p < 0.05). In addition the cells treated with 1 OP4 M lead differed from the cells treated with lop4 M sodium (p -C 0.001) and the cells treated with lop5 M lead differed from cells treated with 10m5 M sodium (p < 0.009). The ability of lead to inhibit 3-OMG uptake required the presence of serum in the medium since 1O-4 M lead in serum-free medium had no effect on the glucose uptake mechanism (Fig. 5) (p < 0.696). The effect of lead on the cells was also dependent on time

394 MAXWELL ET AL.

TABLE 3

ANALYSIS OF VARIANCE TABLE FOR FIG. 4 BY UPTAKE, TREATMENT, TIME, AND SUBSTRATE

Source of variation Sum of squares df

Mean square F

Significance ofF

Main effects 1138.059 Treatment 130.482 Time 186.697 Substrate 820.880

Two-way interactions 201.425 Treatment Time 1.205 Treatment Substrate 0.235 Time Substrate 199.984

Three-way interactions 7.984 Treatment Time Substrate 7.984

Explained 1347.468

Total 1390.063 47 29.576

42.595

5 1 3 1

7 3 1 3

3 3

15

32

221.612 170.997 0.000 130.482 98.027 0.000 62.232 46.753 0.000

820.880 6:6.698 0.000

28.775 21.618 0.000 0.402 0.302 0.824 0.235 0.177 0.677

66.66 1 50.080 0.000

2.66 1 1.999 0.134 2.661 1.999 0.134

89.83 1 67.487 0.000

1.331

of lead exposure. The duration of lead expo- 3-OMG uptake. The degree of inhibition sure necessary to develop a significant inhi- caused by lead increased with exposure time bition of 3-OMG in confluent cultures uptake for 3 hr, after which period the maximum in- was quite short. Forty minutes after exposure hibition of 3-OMG uptake, 3 1 %, was observed to lop4 M lead there was a 12% inhibition of (Fig. 6). An ANOVA comparing the ratio of

TABLE 4

EFFECT OF LEAD ON NONSPECIFIC UPTAKE OF SUBSTRATE

Substrate Presence Incubation time Substrate uptake Ratio (uptake with of lead (min) (nliters/mg protein) lead/control)

L-Glucose L-Glucose ~-Glucose ~-Glucose

Sucrose Sucrose Sucrose Sucrose

+ 10 334 f 139” - 10 835 + 177 0.40 + 20 368 + 102 - 20 1252 f 122 0.29

+ 10 750 + 259 - 10 1554 k 231 0.48 + 20 627 + 133 - 20 1327 + 270 0.47

Note. Confluent cultures of cells were treated with 10e4 M lead for 2 days. ’ The values are X + SD. The number of samples in each group was three.

EFFECT OF LEAD ON CEREBRAL ENDOTHELB-JM 395

TABLE 5

ANALYSIS OF VARIANCE TABLE FOR TABLE 4 BY UPTAKE, TREATMENT, TIME, AND SUBSTRATE

Source of variation

Main effects Treatment Time Substrate

Two-way interactions Treatment Time Treatment Substrate Time Substrate

Three-way interactions Treatment Time Substrate

Explained

Residual

Total

Sum of squares

3942703.500 3129148.167

3952.667 809602.667

274600.333 29260.167

5340.167 240000.000

89060.167 89060.167

4306364.000

5738 13.333

4880177.333

4 Mean square

3 1314234.500 1 3129148.167 1 3952.667 1 809602.667

3 91533.444 1 29260.167 1 5340.167 1 240000.000

1 89060.167 1 89060.167

7 615194.857

16 35863.333

23 212181.623

F

36.646 87.252 0.110

22.575

2.552 0.816 0.149 6.692

2.483 2.483

17.154

Significance

ofF

0.000 0.000 0.744 0.000

0.092 0.380 0.705 0.020

0.135 0.135

0.000

3-OMG uptake, in lead- vs sodium-treated cells, by time of exposure to lead showed that there was a significant effect of time on uptake (r = 0.828, p < 0.000) (Table 7).

Another factor influencing the effect of lead

! Serum-free

I 1 Media

Pb No Pb No XT=f4 KPM

Pb No lo-’ M

FIG. 5. Dosedependent lead inhibition of &cose analog uptake. Confluent cultures of cells were exposed to the indicated concentrations of lead for 2 days before 3- [‘H]OMG uptake was measured. Data are from a 2-min

incubation with 3-[‘H]OMG and are expressed in nmoles/ mg protein.

on 3-OMG uptake was cell density at the time of lead exposure. Cells exposed to lead when they were at a low density showed a greater inhibition of 3-OMG uptake than cells that were exposed to lead when they were at a higher density (Fig. 7). A two-way ANOVA comparing uptake by cell density and treatment (lead vs sodium) showed that there was a significant difference among population means (r = 0.691, p < 0.00) (Table 8). There was also an interaction between density and treatment. Individual group means which differed significantly were (1) 50,000 cells/cm2 treated with lead differed from those treated with sodium (p < 0.0008); (2) 100,000 cells/ cm2 treated with lead differed from those treated with sodium (p < 0.02); (3) 50,000 cells/cm2 treated with lead differed from 100,000 cells/cm2 treated with lead (p < 0.05); and (4) 50,000 cells/cm2 treated with lead differed from 200,000 cells/cm2 treated with lead (p < 0.05). Glucose uptake itself was not a density-dependent process since control values at various densities showed no significant difference in 3-OMG uptake. Interestingly,

396 MAXWELL ET AL.

TABLE 6

ANALYSIS OF VARIANCE TABLE FOR FIG. 5 BY UPTAKE, CONCENTRATION, AND TREATMENT

Source of variation Sum of squares 4

Mean square F

Significance ofF

Main effects Concentration Treatment

Two-way interactions Concentration Treatment

Explained

Residual

Total

1.388 3 0.463 11.792 0.000 0.374 2 0.187 4.76 1 0.019 1.015 1 1.015 25.854 0.000

0.330 2 0.165 4.207 0.028 0.330 2 0.165 4.207 0.028

1.718 5 0.344 8.758 0.000

0.863 22 0.039

2.582 27 0.096

higher lead levels were found in low-density DISCUSSION cultures than in high-density ones. Lead concentrations measured by inductive coupled

We have shown that inorganic lead pro-

argon plasma were 0.326 mg lead/mg protein foundly inhibits division of cerebral endothe-

in cells exposed to lead when they were at lial cells. In addition, lead significantly inhibits

50,000 cells/cm2; 0.19 1 mg lead/mg protein both the specific and nonspecific mechanisms

in cultures with 100,000 cells/cm2; and 0.057 of glucose analog uptake in rapidly dividing,

mg lead/mg protein in cultures with 200,000 low-density cells. The effect that lead has on the glucose uptake system of these cells is re-

cells/cm2. duced when the cells have reached a high density and are contact inhibited. The greater sensitivity of low-density cells to the presence of lead in the medium may be the result of the higher lead content found in these cells. The mechanism by which lead enters the endothelial cells is unknown, but it has been proposed that lead uses the same carrier as Ca2’ to enter other cell types (Goldstein et al., 1977). The higher lead content in low-density cells suggests to us that the functioning of the carrier utilized by lead for entry into the cell is enhanced in log-phase cells. This type of phenomenon has been observed for amino acid transport in our endothelial cells (Cancilla and De Bault, 1983). These findings provide a basis for previous observations that lead en-

3%

30.

5-

0-I 0.5

T im 3 24

Lead Exposum (hrl

FIG. 6. Exposure-time-dependent inhibition of glucose cephalopathy results from damage to the de- analog uptake. Confluent cultures of cells were exposed to 10e4 M lead for the indicated lengths of time. Uptake

veloping cerebral vascular system of young

was measured after a 5-min incubation with 3-[‘HIOMG. children and neonatal animals (Hertz et al., Data are exnressed as the % inhibition of alucose analog 198 1; Holtzman et al., 1980; Krigman et al., uptake after varying periods of lead exposure. 1977; Lefauconnier et al., 1980; Press, 1977;


TABLE I

ANALYSIS OF VARIANCE TABLE FOR FIG. 6 BY RATIO AND TIME

Source of variation Sum of squares 4

Mean square F

Significance ofF

Main effects 0.211 I 0.030 11.017 0.000 Time 0.211 7 0.030 11.017 0.000

Explained 0.211 I 0.030 11.017 0.000

Residual 0.044 16 0.003

Total 0.255 23 0.011

Zook et al., 1980). The observation that lead lead (Holtzman et al., 1980; Pentschew and must be in serum-containing medium to exert Garro, 1966). A second mechanism which was its toxic effect is similar to one made by Fil- proposed by Goldstein et al. (1977) involves erman and Berliner (1980) on RLC-GA1 ep- the disruption of calcium homeostasis in the ithelioid cells. In that study it was shown that cell by lead, thus leading to increased cellular lead was soluble in serum, even in the presence calcium. An additional mechanism proposed of excess phosphate which might have caused for lead encephalopathy is the elevation of tis- the lead to precipitate. Therefore it seems pos- sue Cu by lead, leading to the inhibition of sible that there is some factor in serum that cell membrane adenosine triphosphatase with acts to effectively deliver lead to its carrier on the eventual breakdown of cell membrane the cell membrane. properties (Niklowitz, 1977).

Several mechanisms for lead toxicity have been proposed previously. One involves the alteration of cellular energy metabolism by

The findings that lead can inhibit the non- energy requiring process of glucose uptake in our endothelial cells and in isolated microvessels seems to discount altered cellular energy metabolism as the sole mechanism of lead toxicity (Kolber et al., 1980). In fact, it appears as if the mitochondrial changes brought about by lead intoxication may occur only after a prolonged exposure to lead and that other cellular changes precede the mitochondrial ones (Filer-man and Berliner, 1980). The other proposed mechanisms imply that lead leads to the breakdown of membrane functions that serve to maintain the ionic composition of the cytosol. In fact, lead seems to lead to several types of membrane damage. The leakiness of lead-treated red blood cells to potassium ions suggests that there has been some damage to the membrane. Additionally, Filerman and Berliner (1980) have shown that lead causes cell rounding in epithelioid cells and makes these cells more susceptible to osmotic shock. Lead leads to the altered function of three

‘51 2 2 (A) (8) (C)

FIG. 7. Effect of cell density on lead inhibition of glucose analog uptake. Cells were exposed to 10e4 M lead when they were at the following densities: (A) 200,000 cells/ cm*, (B) 100,000 cells/cm2, (C) 50,000 cells/cm2. Afier 2 days of exposure to the lead medium, uptake of 3- [‘H]OMG was measured. Data are from a 2-min incubation with 3-[3H]OMG.

398 MAXWELL ET AL.

TABLE 8

ANALYSIS OF VARIANCE TABLE FOR FIG. 7 BY UPTAKE. DENSITY. AND TREATMENT

Source of variation

Main effects Density Treatment

Two-way Interactions Density Treatment

Explained

Residual

Total

Sum of squares df

Mean square F

Significance ofF

35.972 3 11.991 6.276 2 3.138

29.696 1 29.696

9.268 2 4.634 9.268 2 4.634

45.241 5 9.048

6.81 I 12 0.568

52.052 17 3.062

21.126 0.00 5.528 0.02

52.320 0.00

8.165 0.00 8.165 0.00

15.941 0.00

brush border enzymes in the kidney without any evident cytosolic changes (Nicholls et al., 1983). Finally, the rapidity with which lead affects glucose analog uptake in endothelial cells suggests that the membrane may be lead’s initial target. All of these observations, along with our findings that lead inhibits both the specific and nonspecific uptake of glucose analogs in our cells, suggest that lead affects the plasma membrane, perhaps by altering the fluidity of the membrane bilayer, thus leading to breakdown of the plasma membrane integ- rity. Since the cerebral capillary endothelial cells play such an important barrier function in the brain as the site of the blood-brain barrier, the loss of specific barrier functions in these cell membranes could easily account for the edema and hemorrhage seen in acute lead encephalopathy.

ACKNOWLEDGMENTS

This study was supported by NINCDS Grant NS19279 and the Ontario Heart Foundation. We gratefully thank George V. Alexander for measurement of cellular lead content.

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