Substrate Transport by the Pulmonary Alveolar Macrophage Effects of Smoke Components 1

2

ROBERT B. LOW and CATHERINE A. BULMAN

SUMMARY An initial analysis was made of the transport of two amino acid analogues (cycloleucine and aaminoisobutyrate) and one glucose analogue *(3-0-methylglucose) by rabbit pulmonary alveolar macrophages. This was followed by an assessment of the effects of two cigarette smoke components, aqueous extracts and acrolein, on transport of these substrate analogues. The cycloleucine, aaminoisobutyrate, and 3-O-methylglucose each demonstrated carrier-mediated transport on the basis of saturation kinetics and competition. The transport of each analogue was competitively inhibited by the appropriate naturally occurring substrate (leucine, alanine, and glucose, respectively). Studies comparing the effects on transport of a number of other naturally occurring substrates indicated that the transport systems for cycloleucine and a-aminoisobutyrate were distinct and were similar to those previously described in the literature for other cell types. The transport system for 3-O-methylglucose appeared to be the same as that for glucose and another analogue, 2deoxyglucose. Finally, the aqueous extracts of cigarette smoke and acrolein had inhibitory effects on cycloleucine and a-aminoisobutyrate transport, and the aqueous extracts also inhibited 3-Omethylglucose transport. These transport effects of smoke components further illustrated their differential effects on pulmonary alveolar macrophage function but were insufficient on a quantitative basis to explain previously described inhibitory effects of the smoke components on amino acid incorporation into protein.

Introduction T h e r e are b u t few reports in the literature concerning substrate t r a n s p o r t by macrophages in general a n d by p u l m o n a r y alveolar macrophages (PAM) in particular, despite the likelihood that these cells d e p e n d on the continued availability of substrate (1-4). T s a n a n d Berlin (5) have defined an a m i n o acid transport system for

(Received in original form January 18, 1977 and in revised form May 9,1977) 1 From the Department of Physiology and Biophysics, the University of Vermont, Burlington, Vt. 05401. 2 Supported by Specialized Center of Research Grant No. HL 14212 from the National Heart, Lung and Blood Institute.

PAM, based mainly o n studies of lysine transport, a n d a transport system for 2-deoxyglucose has been described by Gee a n d associates (6). O t h e r systems, however, r e m a i n to be defined, despite the likelihood that P A M transport systems are different from those defined for other cell types (5). W e report here studies aimed at an initial characterization of two other amino acid transport systems, those for cycloleucine, the nonutilizable analogue of leucine (7) a n d for the nonutilizable a m i n o acid analogue, a-aminoisobutyric acid, which utilizes the same transport system as alanine. T h e s e a m i n o acids were chosen o n the basis of the studies of O x e n d e r a n d Christensen (8), which suggest that these substances use two different transport systems to e n t e r cells. W e also examined the transport of 3-0-

AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME 116, 1977

423

424

LOW AND BULMAN

methyl-D-glucose. T h i s particular glucose analogue was chosen because it is not metabolized by those tissues a n d cells in which its transport has been examined (9-12) a n d because it is believed to use the same transport system as glucose (10,13-16). T w o further reasons for e x a m i n i n g P A M substrate transport stemmed from o u r interest in the effects of aqueous cigarette smoke compon e n t s on macrophage m e m b r a n e activities a n d from o u r desire to examine more closely previously r e p o r t e d effects of such extracts on a m i n o acid incorporation into protein (17-19). Materials and Methods T h e methods for isolation and culture of rabbit PAM have been described previously (19). Briefly, calcium and magnesium-free saline lavage was used to collect PAM from lungs of normal, adult, male, New Zealand rabbits. Calcium- and magnesium-free saline was used to allow recovery of adherent PAM rather than free, perhaps effete cells (20). Our PAM preparations are most likely different from those recovered from lung minces, in which interstitial macrophages are also collected. Cells were collected by centrifugation at 500 g for 5 min at room temperature and were washed twice with the same medium before incubations were begun. Cell viability was determined at this time and at the completion of each experiment by measuring trypan blue dye exclusion and/or lactic dehydrogenase release from the cells. Cells were then placed in phosphate-buffered saline (PBS) containing calcium and magnesium salts and incubated at 37° C in 35-mm tissue culture dishes (Falcon No. 3001; approximately 106 cells per dish) for 30 min to allow them to adhere. Amino acid transport was then measured in Hanks-tris medium (17) and 3-O-methylglucose in PBS by a method similar to that described by Hawkins and Berlin (21). Amino acid transport was measured at 37° C in a warm room (temperature control, 37 ± 1 ° ) ; glucose transport, at room temperature (22 — 15°). Uptake was initiated by the addition of 1 ml of prewarmed incubation medium containing radiolabeled substrates (cycloleucine and a-aminoisobutyrate labeled with carbon-14, and 3-O-methylglucose labeled with hydrogen-3). The exact concentrations of substrate used are given in the text and in legends to the appropriate figures and tables. At varying time intervals, the radioactive medium was aspirated (less than 1 sec) and the cell monolayers were passed rapidly through 5 beakers, each containing 200 ml of ice-cold PBS. This procedure successfully eliminated more than 99.99 per cent of the original isotope in the incubation medium without any evidence that intact cells were released. To disrupt the washed cells, 1 ml of 0.1 N sodium hydroxide (NaOH) was

added to each flask, and the flask was shaken for 5 min at room temperature on a reciprocal shaker. T h e NaOH samples were then quantitatively transferred to scintillation vials; Aquasol® was added, and radioactivity was measured in a Packard Model 3320 Liquid Scintillation Spectrometer. All counts were corrected for counting efficiency using external and internal standardization procedures and were expressed as disintegrations per min. Two corrections that must be made to measure CYCLOLEUCINE TRANSPORT

1

2

3

4

5

TIME (minutes)

Vmax = 3.64 nM/min/lO 6 cells Km» 2 . 5 X K T 3 M

HDLjmM

B Fig. 1. Time course (A) and kinetic analysis (B) of cycloleucine (CL) transport by pulmonary alveolar macrophages. Transport was measured at 37° C as described in Materials and Methods; 0.6 juCi of cycloleucine labeled with carbon-14 (specific activity, 29.97 mCi per mMole) was present in each flask, at a final concentration of 2 mM for the time course study (A), in which each data point represents the mean ± SD of quadruplicate samples. The kinetic analysis (B), based on initial rates of transport, represents data from 3 experiments, each with different rabbit (variation, ± 1 0 per cent). Measurements in each experiment were made in quadruplicate at 30 sec. Also shown is the competitive inhibition of cycloleucine transport by 2 mAf leucine. DPM = disintegrations per min.

SUBSTRATE TRANSPORT BY PULMONARY ALVEOLAR MACROPHAGE

AIB TRANSPORT 800h 700h

y / ^ ^

600h

y

50o|-

/

^ 4ooh

y

Q

X

30o[20r>

/ /

100K/ 0

L

1

1

1

1_

5

10

15

20

TIME (minutes) A

425

carrier-mediated transport accurately involve computation of accumulated counts due to adsorption and to diffusion (5, 6). These can be made by measuring recovered counts per min (cpm) at zero time or on blank flasks (adsorption) or by measuring recovered cpm in monolayers incubated for the duration of the experiment in the presence of supersaturating concentrations of the transported species (diffusion plus adsorption) (5, 6). We routinely used the latter method to make our corrections. T h e method for measuring transport as described allowed reproducible (±10 per cent) measurement of transport after as short a period as 10 sec. We also analyzed the substrate analogues for purity and metabolic conversion before and after transport studies, using conventional chromatographic procedures. The analogues were judged to be 90 per cent pure, and there was no evidence to suggest metabolic conversion. Preparations of aqueous smoke extracts and acrolein were made as previously described, and in each case they were tested to be sure that they gave the expected inhibition of amino acid incorporation into protein (17-19). Rabbits were obtained from Busy Acres Rabbitry, Middlebury, Vt.; media for PAM isolation and culture, from Grand Island Biological Company, Grand Island, N. Y.; radioisotopes, from New England Nuclear Corp., Boston, Mass.; nonradioactive amino acids and analogues, from Sigma Chemical Co., St. Louis, Mo.; and acrolein, from Eastman Organic Chemicals, Rochester, N. Y. All other chemicals were from Mallinckrodt, St. Louis, Mo.

Results Kinetics of transport. It is essential to measure initial, linear rates of transport if one wishes to analyze the kinetics of any carrier-mediated transport system. T h e time course of u p t a k e a n d the kinetic analysis of transport of cycloleucine, a-aminoisobutyrate, a n d 3-O-methylglucose by P A M , are shown in figures 1 to 3. U p t a k e in [AIB] mM each case was linear only for a short time: approxB imately 1 m i n in the case of cycloleucine, 2 m i n Fig. 2. Time course (A) and kinetic analysis (B) of in the case of a-aminoisobutyrate, a n d 15 sec a-aminoisobutyrate (AIB) transport by pulmonary alin the case of 3-O-methylglucose, even though veolar macrophages. Transport was measured at 37° C measurements in this last case were at 22° C. as described in Materials and Methods. 0.6 JJLCI of aT h i s has been the usual finding for the transaminoisobutyrate labeled with carbon-14 (specific activity, 60 mCi per mMole) was present in each flask, p o r t of these substrates (5, 8,13). at a final concentration of 5 mM for the time course T h e kinetic parameters for the transport of study (A), in which each data point represents the mean these substrate analogues (figures 1-3) were ± SD of quadruplicate samples. The kinetic analysis (B), similar to those r e p o r t e d in the literature (5, 8, based on initial rates of transport, represents data from 13). T h e fact that the transport of each ana3 experiments, each with a different rabbit (variation, logue was inhibited by the corresponding natu± 1 0 per cent). Measurements in each experiment were rally occurring substrate supports the relevance made in quadruplicate at 30 sec. Also shown is the competitive inhibition of a-aminoisobutyrate transport of such analogue studies. by 5 mM alanine. DPM = disintegrations per min. Competition studies. T h e kinetic d a t a offer

426

LOW AND BULMAN

3-0-MG TRANSPORT

CYCLOLEUCINE TRANSPORT COMPETITION

(Inhibitor) X 10"3 M Vmax= 13.2 nM/min/lO6 cells Km= 10.0X10"3M

(3-0-MG] mM

B Fig. 3. Time course (A) and kinetic analysis (B) of 3-0-methylglucose (3-0-MG) transport by pulmonary alveolar macrophages. Transport was measured at 22° C, as described in Materials and Methods; 10 juGi of 3-0-methyl-D-glucose labeled with hydrogen-3 (specific activity, 3.62 Gi per mole) was present in each flask, at a final concentration of 10 mM for the time course study (A), in which each data point represents the mean ± SD of quadruplicate samples. The kinetic analysis (B), based on initial rates of transport, represents the data from 3 experiments, each with a different rabbit (variation, ± 1 0 per cent). Measurements in each experiment were made in quadruplicate at 15 sec. Also shown is the competitive inhibition of 3-0methylglucose transport by 40 mM glucose. DPM = disintegrations per min.

Fig. 4. Competitor effects on cycloleucine transport. Cycloleucine transport was measured as described in the legend to figure 1, in the presence of varying concentrations of the competitors indicated (AIB-aaminoisobutyrate). The variation of triplicate samples was ± 1 2 percent. e n t extents of inhibition caused by the different competitors tested does allow a preliminary assessment of the specificity of the system for each of the analogues tested (5; see Discussion). Effects of aqueous smoke extracts and acrolein on substrate transport. W e next determined the effect of aqueous smoke extracts a n d acrolein on the transport of cycloleucine, a-aminoisobutyrate, a n d 3-O-methylglucose (tables 1 to 3). Cycloleucine transport was diminished to a small extent by aqueous smoke extracts within 15 min, after which inhibition changed b u t little. T h e effect of aqueous smoke extracts also showed litAIB TRANSPORT COMPETITION

Leucine Phenylalanine

evidence that carrier-mediated t r a n s p o r t was b e i n g observed. A n o t h e r a p p r o a c h to demonstrate this is to examine competitive aspects of the t r a n s p o r t p h e n o m e n a using several amino acids or sugars. T h i s also offers the o p p o r t u n i t y to make an initial assessment of the relationship between each of the systems examined. Data obtained from such competitor studies are presented in figures 4 to 6. T h e transport systems defined for cycloleucine, a-aminoisobutyrate, a n d 3-O-methylglucose showed the competition expected of carrier-mediated processes. T h e differ-

[Inhibitor] X 10"3 M

Fig. 5. Competitor effects on a-aminoisobutyrate (AIB) transport, a-aminoisobutyrate transport was measured as described in the legend to figure 2, in the presence of varying concentrations of the competitors indicated. The variation of triplicate samples was ± 1 0 per cent.

427

SUBSTRATE TRANSPORT BY PULMONARY ALVEOLAR MACROPHAGE

TABLE 2

3-0-MG TRANSPORT COMPETITION

EFFECT OF AQUEOUS SMOKE EXTRACT AND ACROLEIN ON a-AMINOISOBUTYRATE TRANSPORT % Control Smoke Component

2-DOG

Aqueous smoke extract m l / flask 0.10

99 ± 5 (9)

D-Glucose [inhibitor) X 10" M

TABLE 1 EFFECT OF AQUEOUS SMOKE E X T R A C T A N D A C R O L E I N ON C Y C L O L E U C I N E TRANSPORT % Control Smoke Component Aqueous smoke extract, m l / flask 0.10

15min

30 min

f

85 ± 1 (9)

0.15*

88±2f (6)

83±2t (9)

76±2f (9)

0.30

84 ± 2 * (6)

76 ± 2 + (9)

74±3f (9)

0.40

83±4f (6)

77±2t (9)

74±2t (9)

99 + 1 (12)

92 ± 2 (12)

12.0

99 ± 2 (12)

30.0 60.0

87±5f (9)

94 ± 6 (9)

sots1"

83 15 1 "

8011*

(9)

(9)

(9)

1

78 i S " (9)

75±2 (9)

83±3f (12)

86 ± 3 (12)

f

88 12 1 " (12)

12.0

76±4f (12)

86 ± 2 (12)

f

73±1* (12)

30.0

78 ±3* (12)

79 ± 3 * (12)

73111(12)

60.0

73 ± 2 (12)

69±2f (12)

64±3f (12)

0.40 Acrolein, /xg/flask 6.0*

60 min

88±3f (6)

Acrolein, mg/flask 0.6*

0.30

60 min

96 ± 3 (9)

102 ± 2 (9) 89 ± 4 (9)

86±2f (9)

0.15* 3

Fig. 6. Competitor effects on 3-0-methylglucose (3-0MG) transport. 3-0-methylglucose (3-0-MG) transport was measured as described in the legend to figure 3, in the presence of varying concentrations of the competitors indicated (2-DOG-2-deoxyglucose). The variation of triplicate samples was ± 1 0 per cent.

30 min

15 min

f

t

71 ± 1 * (9)

Cell monolayers were preincubated with aqueous smoke extract or acrolein f o r the times indicated. Transport was measured as described in Materials and Methods and the legend t o figure 2. Results are expressed as the mean of at least 3 separate experiments ± SE. Numbers in parentheses represent the number of measurements made. * A m o u n t of smoke component that produced 50 per cent inhibition of amino acid (leucine) incorporation into protein (17-19). "•> < 0.01 by one-tailed t test.

tie dose dependence. There was a lag before cyf

83 ± 21" (12)

cloleucine transport was depressed by acrolein,

84 ± l t (12)

78±2f (12)

especially at higher concentrations of acrolein,

98 ± 1 (12)

84 1 1 1 " (12)

72±3f (12)

at higher concentrations of aqueous smoke ex-

98 ± 2 (12)

77±2r (12)

69±3f (12)

15 min and then plateauing. The data were sim-

Cell monolayers were preincubated with aqueous smoke extract or acrolein for the times indicated.Transport was then measured as described in Materials and Methods and the legend t o figure 1. Results are expressed as the mean of at least t w o separate experiments ± SE. Numbers in parentheses indicate number of measurements made. * A m o u n t of smoke component that produced 50 per cent inhibition of amino acid (leucine) incorporation into protein (17-19). "•"P < 0.01 by one-tailed t test.

after which inhibition again tended to plateau, a-aminoisobutyrate transport was depressed only tracts, with the effect

again occurring within

ilar for the effect of acrolein on a-aminoisobutyrate transport, except that inhibition was noted even at the lowest dose tested. Aqueous smoke extracts

had

no

effect

on

3-O-methylglucose

transport for at least 15 min, after which transport was inhibited at higher doses. Acrolein, on the other hand, had no effect on 3-O-methylglucose transport

except

at higher doses, in

which case transport was slightly stimulated. We measured the effect of a competing sub-

428

LOW AND BULMAN

TABLE 3 EFFECT OF AQUEOUS SMOKE EXTRACT AND ACROLEIN ON 3-0 METHYL-D-GLUCOSE TRANSPORT % Control Smoke Component

15 min

97 ± 4 (6)

30 min

60 min

94 ± 4 (9)

96 ± 2 (9)

110±9 (6)

94±3f (9)

85±2f (9)

98 ± 8 (6)

82±2t (9)

77±2f (9)

95 ± 3 (6)

77±2f (9)

71 ± 2 f (9)

97 ± 2 (12)

92 ± 3 (12)

98 ± 3 (12)

94 ± 4 (12)

92 ± 4 (12)

99 ± 3 (12)

101 ± 3 (12)

99 ± 4 (12)

104 ± 4 (12)

104 ± 2 (12)

120 ± 8 (12)

114±2 (12)

Acrolein, iig/f\ask

Cell monolayers were preincubated with aqueous smoke extract or acrolein for the times indicated. Transport was measured as described in Materials and Methods and the legend to figure 3. Results are expressed as the mean of at least 2 separate experiments + SE. Numbers in parentheses represent number of measurements made. A m o u n t of smoke component that produces 50 per cent inhibition of amino acid (leucine) incorporation into protein (17-19). "•"P < 0.01 by one-tailed t test.

strate on transport in the presence and absence of smoke components to distinguish between effects of aqueous smoke extracts on noncompetitive (e.g., difFusion) and carrier-mediated transport. The competitor amino acid, methionine, had quantitatively the same inhibitory effect on cycloleucine and a-aminoisobutyrate in the presence or absence of smoke components, as did glucose on the transport of 3-O-methylglucose (table 4). This result was the same in 3 separate experiments and indicates that balanced alterations in carrier-mediated transport and simple diffusion (e.g., transport decrease together with diffusion increase) had not occurred. Discussion

We present evidence for carrier-mediated trans-

port of 3 analogues of naturally occurring substrates, with the competition between naturally occurring substrates and analogues indicating the physiologic import of the systems we have defined. The data for cycloleucine and a-aminoisobutyrate are similar to those of other investigators involving studies of single cells (7, 8, 22). The cycloleucine system does not operate against a gradient and has been characterized as facilitated diffusion (7, 8). The a-aminoisobutyrate studies are not substantially different from those reported for Ehrlich cells (8) and PAM (22). Distribution ratios obtained during longer time periods (unpublished data) are comparable, suggesting uphill and, hence, active transport. Our competition experiments indicate different specificities, in a fashion very similar to that described for Ehrlich ascites cells (8). The competition between cycloleucine and lysine was also expected from the studies of Tsan and Berlin (5). Our data on 3-O-methylglucose transport are in close agreement with those obtained by Crane and associates (13) in studies of Ehrlich ascites cells, and indicate a reversible, first-order process that does not operate against a concentration gradient. Our competition experiments suggest that the 3-O-methylglucose transport system is similar to, if not identical to, that for glucose and 2-deoxyglucose. Gee and associates (6) also reported that PAM do not transport 2-deoxyglucose against a gradient; however, they reported linear uptake for at least 30 min, whereas we found linearity to last less than 1 min (figure 7), as reported for other cells (13). We are not sure of the reason for this difference. One possibility is that Gee and associates (6) measured transport in cell suspensions; we used monolayers. Cells incubated under these two conditions behave differently in a number of ways (19). Pofit and Strauss (23) have shown that rabbit PAM transport lysine and adenosine at slower rates when incubated in suspension than when incubated as adherent monolayers. Clearly, incubation conditions must be carefully defined when reporting such studies, as also must the time during which uptake exhibits maximal initial rates. Further studies are necessary to characterize more fully the transport systems we have described. Measurements of sodium and potassium dependence would be helpful (5). Our preliminary experiments suggest that the cycloleucine and 3-O-methylglucose transport systems do

429

SUBSTRATE TRANSPORT BY PULMONARY ALVEOLAR MACROPHAGE

TABLE 4 EFFECT OF COMPETING A M I N O ACIDS ON SUBSTRATE TRANSPORT IN T H E PRESENCE A N D ABSENCE OF SMOKE COMPONENTS % Control Conditions Cycloleucine Control Aqueous smoke extract Acrolein a-Aminoisobutyrate Control Aqueous smoke extract Acrolein 3-O-Methylglucose Control Aqueos smoke extract Acrolein

- Methionine

+ Methionine

- Glucose

+ Glucose

100 82

43 47

-

-

90

47

100 88

37 40

-

-

82

41

-

-

-

-

100 87 87

44 43 44

Transport was measured as described in the legends to tables 1 to 3. Measurements were made after a 60-min preincubation with smoke component. Substrates were present at their Km concentration. The concentrations of methionine, glucose, aqueous smoke extract, and acrolein, when present, were 5 mAf, 20 mM, 0.15 ml, and 6 M9, respectively. Data are expressed as the mean of quadruplicate samples that varied within ± 1 3 per cent.

We found that aqueous smoke extract and acrolein altered transport only to a small degree and only in concentrations larger than those that significantly alter other macrophage activities (17, 28). This indicates that substrate transport functions of the plasma membrane are not a major factor in the interaction of these smoke components with PAM. Lentz and DiLuzio (22) have reported that aqueous smoke extract inhibit a-aminoisobutyrate transport by PAM suspensions. Their transport studies lasted 60 min, during which sub2-D0G TRANSPORT stantial cell:medium ratios of a-aminoisobutyrate were achieved. The concentrations of our aqueous smoke extract preparations were equivalent to doses that they found to be generally inhibitory. Akedo and Christensen (7) pointed out that, at least with rat intestine, one cannot inhibit a-aminoisobutyrate transport with meta• bolic poisons until net uphill accumulation begins to occur. Whether this is also true for aaminoisobutyrate transport in other systems remains to be demonstrated. It indicates, however, that caution must be exercised in attempting to TIME (min) compare our data (no net uphill transport) with Fig. 7. Time course of 2-deoxyglucose (2-DOG) those of Lentz and DiLuzio (22) (measurements transport by pulmonary alveolar macrophages; 10 juCi made during the process of net uphill transof 2-deoxyglucose labeled with hydrogen-3 (specific port). activity, 10 Ci per Mole) was present in each flask at a Our transport studies also permit an initial final concentration of 8.0 mM, and corrections were assessment of whether altered amino acid transmade for phosphorylation of 2-deoxyglucose (6). port could explain previous data indicating that Each point represents the mean ± SD of triplicate smoke components inhibit amino acid incorporsamples; DPM = disintegrations per min.

not require sodium, as expected of facilitated diffusion transport systems, whereas the a-aminoisobutyrate system does have the sodium dependency characteristic of many substrate active transport systems (24-27). There are a number of reasons for believing that aqueous smoke extract and acrolein affect PAM plasma membrane function (see 28). It was, therefore, of some interest to determine whether those agents affect substrate transport.

430

LOW AND BULMAN

ation into protein (17-19). This was deemed especially relevant because there is evidence for direct coupling of transport events to amino acid precursor pools for protein synthesis in several cells (29, 30). Our results indicate that altered transport cannot explain altered amino acid incorporation into protein on a quantitative basis. This is because doses of aqueous smoke extract and acrolein that inhibit amino acid (leucine) incorporation into protein by 50 per cent, without any detectable effect on cell viability, have much smaller inhibitory effects on transport, specifically in this case of cycloleucine. It is difficult to exclude the possibility that the smaller alteration in transport is amplified in some fashion so as to cause a sufficient effective decrease in precursor pool specific activity to explain the incorporation data. The possibility certainly exists that the smoke components alter the size of the precursor pool and/or the contribution of amino acid from protein degradation to it in a fashion that would explain altered labeled amino acid incorporation in the absence of a change in the true rate of protein synthesis. Final experimental resolution of this question is complicated by the possibility, for which there is some evidence, that neither the total cellular amino acid pool nor the total cellular amino acyl-transfer ribonucleic acid pool is the immediate precursor pool for the synthesis of all proteins in a given cell (31). We currently are examining this problem directly using methods that have successfully been applied by others (29,30). The transport data also point to some differences between the effects of aqueous smoke extract and acrolein on PAM function, based on the following observations: (1) aqueous smoke extract begins to inhibit cycloleucine transport within 15 min, whereas acrolein does not produce a measurable effect until after that time. (2) High doses of aqueous smoke extract inhibit 3-O-methylglucose transport after 60 min of exposure, whereas, if anything, acrolein at high doses stimulates transport of the analogue. These results are in general agreement with those of Low and associates (28) and Green and associates (32) in pointing to acrolein as one of the active agents in smoke extracts, the effects of which can explain some, though not all, of the effects of those extracts. Previous appreciation of acrolein as a respiratory irritant (35) and as a toxic agent in humans (36) has resulted in the establishment of 0.1 ppm as a threshold limit value (TLV) for

industrial exposure (37). Cigarette smoke contains acrolein at concentrations approximately 1,700 times higher (32, 34) than this TLV, although such a comparison is between a dose received intermittently (smoking) and one received constantly (environmental exposure). An important question is whether acrolein and other water-soluble smoke components reach the alveolus in concentrations that might alter macrophage function, because such components are believed largely to be removed in the upper airways (38). In partial answer to this question, Jakab (39) has found that 24-hour continuous in vivo exposure of mice to acrolein causes significant impairment of pulmonary antibacterial defenses. This is in keeping with other in vivo studies indicating impaired macrophage function after smoke exposure (40-42). A number of smoke components are almost certainly involved and doubtless act synergistically (38). Further studies will be necessary to delineate more carefully the particular nature of these effects and, as well, the general relationship between the effects of in vivo and in vitro cigarette smoke exposure.

References 1. Gee, J. B. L., and Khandwala, A. S.: Oxygen metabolism in the alveolar macrophage: friend and foe, J Reticuloendothel Soc, 1976,19, 229. 2. Karnovsky, M. L.: Metabolic basis of phagocytic activity, Physiol Rev, 1962, 42,143. 3. Oren, R., Farnham, A. E., Saito, K., Milofsky, E., and Karnovsky, M. L.: Metabolic patterns in three types* of phagocytic cells, J Cell Biol, 1963, 17, 487. 4. Ouchi, E., Selvaraj, R. J., and Sbarra, A. J.: The biochemical activities of rabbit alveolar macrophage during phagocytosis, Exp Cell Res, 1965,40,456. 5. Tsan, M. F., and Berlin, R. D.: Membrane transport in the rabbit alveolar macrophage. The specificity and characteristics of amino acid transport systems, Biochem Biophys Acta, 1971, 241,155. 6. Gee, J. B. L., Khandwala, A. S., and Bell, R. W: Hexose transport in alveolar macrophage: kinetics and pharmacologic features, J Reticuloendothel Soc, 1974,15, 394. 7. Akedo, H., and Christensen, H. N.: Transfer of amino acids across the intestine: a new model amino acid, J Biol Chem, 1962, 237,113. 8. Oxender, D. L., and Christensen, H. N.: Distinct mediating systems for the transport of neutral amino acids by Ehrlich cells, J Biol Chem, 1963, 238, 3686. 9. Campbell, P. N., and Davson, H.: Absorption

SUBSTRATE TRANSPORT BY PULMONARY ALVEOLAR MACROPHAGE

10. 11.

12. 13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

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Substrate transport by the pulmonary alveolar macrophage. Effects of smoke components.

Substrate Transport by the Pulmonary Alveolar Macrophage Effects of Smoke Components 1 2 ROBERT B. LOW and CATHERINE A. BULMAN SUMMARY An initial a...
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