Kinetics of pulmonary surfactant phosphatidylcholine metabolism in the lungs of silica-treated rats

TOXlCOLOGYANDAPPLIEDPHARMACOLOGY

Kinetics of Pulmonary

9&l-11(1989)

Surfactant Phosphatidylcholine Lungs of Silica-Treated Rats

Metabolism

B. GILMORE,$ANDGARY

LLOYDA.DETHLOFF,*"BETHC.GLADEN,~LINDA

in the

E.R. HOOKS

*Laboratory ofPulmonary Pathobiology and the tStatistics and Biomathematics Branch, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709, and *The Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 275 14

Received February 8,1988; accepted October 18, I 988 Kinetics of Pulmonary Surfactant Phosphatidylcholine Metabolism in the Lungs of SilicaTreatedRats. DETHLOFF, L. A., GLADEN, B.C., GILMORE, L.B., AND HooK,G.E.R.(I~~~). Toxicol. Appl. Pharmacol. 98, l-l 1. Exposure of rats to silica by intratracheal injection increased the intra- and extracellular compartments of pulmonary surtactant phospholipid. These changes were dose and time dependent, but both pools were not affected equally. Seven days after the instillation of 10 mg of silica, the intracellular pool increased 13.3-fold, from 1.49 +_ 0.30 to 19.86 -+ 0.77 mg of surfactant phospholipid per pair of lungs, and the extracellular pool increased 7.4-fold, from 1.87 f 0.79 to 13.79 _+0.72 mg of surfactant phospholipid per pair of lungs. To investigate the physiologic processes responsible for these massive accumulations of surfactant, [‘%Z]choline was injected into the tail veins of control and silica-treated rats and the specific activity of surfactant phospholipids within the intracellular and extracellular pools was determined at various times between 0 and 26 hr after injection. All of the processes measured were increased in response to silica exposure, but not to the same extent. At 1 hr, incorporation of [%]choline into the intracellular surfactant pool was increased 12.6-fold above controls, from 4.8 + 1.1 X lo3 to 60.6 f 26.6 X lo3 dpm. The flux of [“‘Clcholine-labeled phospholipid from the intracellular to the extracellular pool was increased 7.3-fold, from 102 + 10 to 749 + 39 rgfhr in silica-treated animals, but its disappearance from the extracellular pool was increased only 5.0-fold, from 87 + 8 to 434 +- 21 &hr. The half-life of [‘4C]choline-labeled phospholipids in the intracellular pool of surfactant was increased from 10.1 f 1.Oto 18.3 + 5.3 hr and that in the extracellular surfactant pool from 14.8 + 1.4 to 2 1.9 + 4.9 hr. Expansion of the intra- and extracellular pools of surfactant phospholipids may be explained on the basis of a metabolic imbalance in which the intracellular production of surfactant is increased above its secretion rate into the extracellular compartment, and the secretion rate is elevated above the rate at which surfactant phospholipids are cleared from the alveoli. o 1989 Academic PRSS, IIK.

The surfactant system of the lungs exists within two anatomically distinct pools, or compartments (Pawlowski et al., 1971). The intracellular pool is synthesized and stored within the alveolar type II epithelial cells where it consists primarily of discrete cytoplasmic organelles called lamellar bodies. Upon the appropriate physiologic stimuli,

the contents of these storage structures are secreted onto the alveolar surface. Anatomically, the extracellular compartment lies within the pulmonary extracellular lining that overlies the alveolar and bronchiolar epithelium. Although the regulatory mechanisms governing the metabolism of surfactant are not well understood, it is clear that these two compartments are linked within a strictly controlled precursor-product relationship (Jacobs et al., 1982; 1983) such that

’ Supported in part by a predoctoral training grant, National Research Service Award 5T32 ES-07 126. 1

0041-008X/89

$3.00

Copyright 0 1989 by Academic Press, Inc All rights of reproduction in any Corm reserved

DETHLOFF

2

in normal adult lungs the relationship between the intracellular and the extracellular compartments appears constant (Dethloff et al., 1986a). Therefore, a steady state must exist in which the rate at which surfactant is synthesized equals the rate at which it is secreted into the alveoli and the rate at which it is removed from the alveoli. In the absence of steady-state conditions, surfactant either would be depleted or would accumulate within the lungs. In certain instances of pulmonary disease, this balance between surfactant synthesis, secretion, and clearance may be disrupted. Numerous examples have been reported in which animals exposed to toxicants appeared to have altered levels of pulmonary surfactant. Some toxicants, such as paraquat (Malmquist, 1980) and hyperbaric oxygen (Beckman and Weiss, 1969), appear to deplete the extracellular pool of surfactant, while others, such as nitrogen dioxide (Blank et al., 1978) and bleomycin (Aso et al., 1976), appear to increase it. One of the most effective stimulators of the pulmonary surfactant system is silica (Gabor et al., 1978; Dethloff et al., 1986a,b; Lewis et al., 1986). Exposure of the lungs to silica dust results in the accumulation of surlactant within both intracellular and extracellular compartments to the extent that surfactant levels may be increased as much as lOO-fold (Dethloff et al., 1986b). Expansion of intraand extracellular surfactant phospholipid pools in the lungs of silica-treated rats must arise from severe disturbances in the balance that normally exists between biosynthesis, secretion, and clearance of surfactant. To test this hypothesis, we studied, in intact silicatreated and untreated rats, the incorporation of [14C]choline into intra- and extracellular surfactant phospholipids. METHODS Chemicals used were as follows: silica in the form of Berkeley Min-U-Sil (particle size < 5 pm; Pennsylvania Glass Sand Corp., Pittsburgh, PA); density gradient-

ET AL. grade sucrose (Schwan/Mann, Orangeburg, NY); Aquasol scintillation cocktail, and [methyl-[‘4C]choline chloride, 50.5 mCi/mmol (New England Nuclear, Boston, MA); bovine serum albumin (Miles Laboratories, Inc., Shawnee, KS). All other chemicals were of analytical grade. Dosing of animals. Suspensions of silica were prepared in sterile 0.9% NaCl as described previously (Dethloff et al., 1986b). Adult male rats ofthe Sprague-Dawley strain (225-250 g; Charles River, Wilmington, MA) were anesthetized by injecting, intramuscularly, mixtures of ketamine (8.6 mg, Parke-Davis, Morris Plains, NJ) and xylazine (0.28 mg, Miles Laboratories) in a volume of 100 ~1. The animals were held in a vertical position and intratracheal injections were made by inserting a curved cannula (18-gauge, 3-in., 2.25-mm ball) into the trachea until just above the tracheal bifurcation, and the silica, suspended in 0.5 ml sterile 0.9% NaCl, was injected rapidly into the lungs. The treated rats received various amounts of silica as indicated in the figure legends while control rats received 0.5 ml sterile saline only. All animals were allowed free accessto food and water. Isolation of extracellular pulmonary surfactant. The rats were killed by intraperitoneal injections of 50 mg of sodium pentobarbital. Lungs were excised quickly with their tracheae intact. Extraneous tissue was removed, the trachea was cannulated, and the lungs were filled to capacity with ice-cold 0.9% NaCI. Lavage effluents were then collected, and the procedure was repeated seven times, each with fresh 0.9% NaCI. The lavage effluents from each animal were combined and centrifuged at 580gfor 10 min at 4°C to sediment cells (Sorvall RT 6000 Refrigerated Centrifuge, H 1OOOB rotor). The supematants were aspirated and stored at -2o’C until extracted for phospholipid quantitation and scintillation counting. We have previously demonstrated that our lavage procedure recovers greater than 95% of the total available phospholipid and that these recoveries are not affected by treatment of the lungs with silica (Dethloff et al., 1987). Isolation of intracellular pulmonar surfactant. The intracellular pool of pulmonary surfactant was isolated by sucrose density gradient centrifugation according to the method of Duck-Chong (1978) as modified by Young et al. (198 I). Briefly, lavaged lungs were homogenized in 1.O M sucrose by using a Potter-Elvehjem homogenizer with a Teflon pestle (0.004-0.006 in. clearance). Twenty passes of the pestle were made and the homogenate was diluted to 9.0 ml with 1.O M sucrose. One milliliter each of0.9, 0.8, 0.7, 0.6, 0.5,0.4, 0.3, and 0.2 M sucrose was layered on top of the homogenate and the gradients were centrifuged for I5 min at 2008 (Beckman L3-50 Ultracentrifuge, SW 28.1 rotor). Without interruption, the gradients were then accelerated to 116,OOOgfor 180 min. The lamellar bodies appeared as an opaque band at a density of approximately 1.054. This fraction was aspirated and stored frozen at -20°C until extracted for phospholipid quantitation and scintillation counting. This in-

SURFACTANT

KINETICS

tracellular surfactant has been characterized previously (Dethloff et al., 1986a). Injection of radiolabeled phospholipid precursors. Seven days after receiving intratracheal injections of 10 mg ofsilica, the rats were dosed with 3.3 &i [‘4C]choline contained in 0.3 ml of 3.0% bovine serum albumin in 0.9% NaCl. The animals were restrained gently and the isotope solution was injected slowly via the lateral tail vein. At various times from 1 to 26 hr after injections of isotope solution, the animals were killed and the intraand extracellular surfactant phospholipid pools were isolated. Biochemical analyses and scintillation counting. Total lipid was extracted from the intracellular and extracellular surfactant pools with chloroform-methanol (2: 1) according to the method of Folch et al. (1957) and their phospholipid contents were determined by assayof phospholipid phosphorus (Shin, 1962). Phosphorus was assumed to constitute by weight 4% of the phospholipid (Shin, 1962). Chloroform was evaporated from the extracts in 20-ml scintillation vials and the lipid that remained was dissolved in 10 ml of Aquasol. Radioactivity was determined in a Beckman LS 8100 scintillation counter. All data were corrected for counting efficiencies determined by using internal [14C]toluene standards (New England Nuclear). The standard errors of counting were less than 2%. Surfactant lipids consist almost entirely of phospholipids (approximately 90%), of which phosphatidylcholine accounts for 80-90% [see, for example, Harwood et al. ( 1975) and Rooney et al. ( 1974)]. Of these phospholipids only phosphatidylcholine and sphingomyelin incorporate choline, and because sphingomyelin contributes only 3% by weight to surfactant phospholipids (Dethloff el al.. 1986a), incorporation of [‘4C]choline into nonphosphatidylcholine phospholipids was, therefore, considered negligible. In addition, labeling studies by Lewis et al. (1986) have demonstrated that 97% of intravenously administered [“‘Clcholine incorporated into surfactant lipids is recovered in phosphatidylcholine.

RESULTS

3

AND SILICA

the intracellular pool increased 9.8-fold, from 1.7 + 0.4 to 16.4 + 3.5 mg phospholipid per pair of lungs, and the extracellular pool increased 2.1-fold, from 1.4 f 0.6 to 3.0 rt 0.9 mg phospholipid per pair lungs (p < 0.01). The intracellular pool was affected to a larger degree than the extracellular pool at all time points studied. Intracellular surfactant levels increased linearly until 28 days after dosing. At this time intracellular surfactant levels were increased to 145 -t 54 mg phospholipid per pair of lungs, which represented an 80.1fold increase above controls. After 28 days the intracellular pool continued to increase but not at the same rate. After the first 6 days, the increase in the extracellular pool was approximately linear throughout the course of the experiment. Twenty-eight days after dosing, the extracellular pool had increased 2 I .6fold above controls to 25.1 f 7.1 mg phospholipid per pair of lungs. Groups of four control rats were also examined at each time point; their intra- and extracellular pool sizes did not differ significantly from the control values obtained at 3 days (data not shown). The effect of silica on the sizes of the surfactant pools was also dose dependent (Fig. 1B). Both pools showed linear increases with dose of silica and the intracellular pool was affected to a higher degree than the extracellular pool. Six days after dosing with 50 mg of silica, the intracellular pool had expanded 16.7-fold from 2.0 + 0.4 to 33.2 + 4.4 mg phospholipid per pair of lungs. No significant increases were detected in either pool at a dose of 1 mg silica.

Eflect of Silica on Intracellular and ExtracelMar Surfactant Phospholipids

Incorporation of [‘4C]Choline Phospholipids

The effects of intratracheal injections of silica on the sizes of the intra- and extracellular pools of surfactant phospholipids are shown in Fig. 1. The response of the surfactant system to silica was rapid (Fig. 1A); both pools were significantly elevated above controls 3 days after a single intratracheal injection of silica (50 mg). During this first 3&y period

We chose to study the effects of IO-mg doses of silica because significant increases in both intra- and extracellular pools were produced within 1 week, but those increases were not so large as to produce excessive dilutions of radiolabel. Seven days after intratracheal injections of 10 mg of silica, rats were administered intra-

into Surftictant

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FIG. 1. Effects of silica on sizes of the intracellular and extracellular pulmonary surfactant phospholipid pools. Rats weighing 225-250 g were injected intratracheally on Day 0 with silica suspended in 0.5 ml 0.9% NaCl. Controls received saline only. (A) Changes in surtactant phospholipid levels with time following a single 50-mg dose of silica. Each point represents the mean f SD of three to five rats. Control values on Day 0 were 1.68 -t 0.35 and 1.42 f 0.55 mg phospholipid per pair of lungs for intracellular and extracellular pools, respectively. Control values did not change significantly during the course of the experiments and are not shown. (B) Changes in surfactant phospholipid levels with dose. Rats were injected intratracheally with doses of silica ranging from 1 to 50 mg on Day 0 and were killed on Day 6.0, Extracellular pool; 0, intracellular pool.

venous injections of 3.3 j&i [ 14C]choline and the appearance and disappearance of intraand extracellular surfactant phospholipid were measured by the changes in radioactivity in the two pools over a 26-hr period. At this time, the intracellular surfactant phospholipid pool in silica-treated animals had increased 13.3-fold above controls, from 1.5 f 0.3 to 19.9 + 0.8 mg per pair of lungs, and the extracellular pool had increased 7.4-fold, from 1.9 f 0.8 to 13.8 f 0.7 mg per pair of lungs. No net increase in pool sizes could be discerned during the 26hr time course and no pool size estimate lay outside one standard deviation of the mean value of all groups combined. Incorporation of [ 14C]choline reached a maximum by 2 hr in the silica-treated rats

and by 4 hr in the control rats (Fig. 2A). At peak labeling, approximately three times more [‘4C]choline was incorporated into the intracellular pool of the silica-treated rats compared to controls. Total incorporation of [ “C]choline into the extracellular pool of surfactant phospholipids reached a maximum at 10 and 12 hr in control and silica-treated rats, respectively (Fig. 2B). Compared to controls, incorporation of [‘4C]choline into the extracellular pool occurred at a higher rate and achieved a higher level (by approximately twofold) in the lungs of silica-treated rats. Changes with time in the specific activity of the intracellular and extracellular radiolabeled surfactant phospholipid are shown in Fig. 3. For both groups, the specific activity of the intracellular pool increased rapidly and

SURFACTANT

67 b 2 Q

KINETICS

80 60 40 20

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pholipid compartments were higher in control rats compared to those in the corresponding pools in silica-treated animals. The areas under the specific activity-versus-time curves were determined by the cutting and weighing method and compared. In the control animals the ratio of these areas (intracellular/extracellular) was 1.O 31 0. I. which indicated that approximately the same amount of radioactivity had passed through each compartment during the 26-hr time course of the experiment. In the silica-treated

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declined well before the peak of labeling was achieved in the extracellular compartment. Maximum specific activities in the intracellular pools were attained by 5 hr after injection of [‘4C]choline, while the peak labeling of the extracellular compartments occurred 5-6 hr later, approximately IO- 12 hr after injection of the radiolabel. The specific activities of both the intracellular and extracellular phos-

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2. Effect of silica on the total incorporation of [“‘Clcholine into the intracellular and extracellular surfactant phospholipid pools of the rat lung. Rats weighing 225-250 g were injected intratracheally on Day 0 with 10 mg silica suspended in 0.5 ml 0.9% NaCl. Controls received saline only. Seven days after being dosed with silica, the animals were injected intravenously with 3.3 PCi [“‘CJcholine. Animals were killed at various times from 0 to 26 hr after injection of radiolabel. Each point represents the mean radioactivity + SE of three or four rats. (A) Total incorporation of [?]choline into the intracellular surfactant pool. (B) Total incorporation of [‘4C]choline into the extracellular surfactant pool. 0. Control rats; 0, silica-treated rats. FIG.

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FIG. 3. Effects of silica on the specific activity of phospholipids in the intracellular and extracellular pools of pulmonary surfactant. Rats were dosed with 10 mg silica and after 7 days with [ Wlcholine as indicated in the legend to Fig. 2. Each point represents the mean specific activity +_ SE of three or four rats. (A) Specific activities of surfactant phospholipid from control rats. (B) Specific activities of surfactant phospholipid from silica-treated rats. l , Intracellular phospholipid; 0, extracellular phospholipid.

6

DETHLOFF TABLE 1

EFFECTS OF SILICA ONTHE RATESOF INCORPORATIONOF['~C]CHOLINEINTOPHOSPHOLIPIDSOFTHEINTRACELLULARSURFACTANTPOOLOFRATLUNGS

Control Silica-treated

Appearance of label [dpm/hr (X lo-‘)]

Increase

4.82 1.1” 60.6 + 26.6

12.6-fold

Note. Rats weighing 225-250 g were injected intratracheally on Day 0 with 10 mg silica suspended in 0.5 ml 0.9% NaCl. Controls received saline only. Seven days after being dosed with silica, the animals were injected intravenously with 3.3 PCi [“Cjcholine. Animals were killed 1 hr after injection and the radioactivity incorporated into the intracellular pool of pulmonary surfactant was determined. a Mean + SD (n = 4 individual rats).

animals, however, this ratio was 1.3 -t 0.2 (p < 0.05), which indicated that within the same span of time, the intracellular compartment was not transferring the same relative proportion of phospholipid to the extracellular pool compared to the control rats. Furthermore, the disappearance of radiolabeled phospholipid from the intracellular pool of the silicatreated animals appeared to be slower than that of the control rats.

Surfactant Phospholipid Kinetics The rate of incorporation of [14C]choline into intracellular surfactant phospholipids was estimated from the total amount of radioactivity contained in this pool at 1 hr after intravenous injection of the label. During this initial 1-hr period [ “C]choline incorporation into the intracellular surfactant phospholipids of silica-treated lungs was increased 12.6fold above that of controls (Table 1). This rate is a minimal estimate because we were unable to make reliable estimates at earlier time peIiOdS.

Disappearance of radiolabeled phospholipid from the intracellular and extracellular

ET AL.

surfactant pools occurred logarithmically as shown in Fig. 4. Half-lives of radiolabeled phospholipid in both compartments were derived by linear regression analysis of semilog plots from four individual experiments, and turnover times for each of the pools were calculated by dividing the half-life by 0.69 (Zilversmit et al., 1943). Net fluxes of surfactant phospholipid were then calculated by dividing the intracellular and extracellular pool size estimates by their corresponding turnover times. The half-life of intracellular surfactant phospholipid in the lungs of silicatreated rats was increased 1.8-fold above controls, from 10.1 * 1.0 to 18.3 f 5.3 hr (Table 2). Similarly, the half-life of extracellular surfactant phospholipid was increased 1.5-fold, from 14.8 & 1.4 to 21.9 f 4.9 hr in control and silica-treated lungs, respectively (Table 2). The net fluxes of surfactant phospholipids out of the intracellular and extracellular compartments were calculated from the turnover times of each pool and the pool sizes. The net flux of surfactant phospholipid from the intracellular to the extracellular compartment was increased 7.3-fold, from 102 + 10 pg/hr in control lungs to 749 -t 39 pg/hr in silicatreated lungs (Table 3). However, the flux of surfactant phospholipid out of the extracellular compartment in silica-treated lungs was increased only 5.0-fold above controls, from 87 + 8 to 434 f 2 1 pg/hr. If one assumes that in normal lungs the flux of surfactant phospholipid out of the intracellular compartment equals the rate of its production, then the production rate of surfactant in our control rats was 102 pg/hr. We estimated, based on the total incorporation of [‘4C]choline into the intracellular pool, that in silicatreated lungs production of surfactant was increased 12.6-fold above controls. Thus, the production rate for surfactant phospholipids in the lungs of silica-treated rats would be approximately 1285 pg/hr. DISCUSSION The mechanisms through causes massive accumulations

which silica of surfactant

SURFACTANT

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FIG. 4. Effects of silica on the loss of [‘%Z]choline-labeled phospholipid from the intracellular and extracellular pools of pulmonary surfactant. Rats were dosed as indicated in the legend to Fig. 2. Each point represents the mean ofthree or four rats. (A) Intracellular surfactant pool. (B) Extracellular surfactant pool. 0, Control rats: 0, silica-treated rats.

phospholipids in the lungs (Gabor et al., 1978; Richards and Curtis, 1984; Dethloff et al., 1986) are not known. Our approach to this problem has been to examine the responses of the surfactant system (Dethloff et al., 1986a,b) and to determine the effects of silica on the major rate processes involved in maintaining surfactant levels within the lungs. Two previous studies reported altered phospholipid metabolism in the lungs of silica-treated rats. Heppleston et al. (1974) studied, in whole lungs, incorporation of [3H]palmitate into phospholipids and dipalmitoylphosphatidylcholine of lung tissue but did not examine, specifically, either intra- or extracellular surfactant; however, in their discussion, Heppleston et al. (1974) recognized that dipalmitoylphosphatidylcholine was a major component of surfactant and speculated that the alveolar lipoproteinosis might be due to “an imbalance between formation and removal” of surfactant phospholipids.

Richards and Lewis ( 1985) studied the incorporation of [‘4C]choline into phosphatidylcholine and dipalmitoylphosphatidylcholine by tissue slices from the lungs of silica-treated rats and found significant increases with the former but not the latter phospholipid; these investigators did not directly measure the kinetics of phospholipid metabolism in either the intra- or extracellular pools of surfactant. To our knowledge, this study is the first to examine specifically the kinetics of surfactant phospholipid metabolism in the lungs of silica-treated rats. We measured the incorporation of labeled choline into the intra- and extracellular pools of surfactant phospholipids and determined that the rates at which phosphatidylcholine is synthesized, secreted, and cleared from the lungs are all severely disturbed by silica. Our data indicate that each of the measured metabolic rates was increased. However, imbalances between the rates at which

8

DETHLOFF TABLE 2

ET AL.

sons for these apparent differences, the data of Lewis et al. (1987) indirectly support our EFFECTSOF SILICA ON HALF-LIVES OF [ “‘C]CHOLINELABELED PHOSPHOLIPIDSIN THE INTRACELLULAR AND contention that in silica-treated rats, secreEXTRACELLULAR SURFACTANT POOLS OF RAT LUNGS tion of surfactant exceeds its clearance from the alveoli and thus accounts for the increase Half-life (hr) in the extracellular pool. Our data indicate Control Silica-treated Increase that the normal lung’s capacity for clearance of surfactant may be well in excess of that reIntracellular pool 10.1Yk1.0n 18.3 * 5.36 1.8-fold normal levels of surfacExtracellular pool 14.8 f 1.4 21.9 k4.9’ 1.5-fold quired to maintain tant. Pettenazzo et al. (1988) have recently Note. Rats weighing 225-250 g were injected intratramade similar observations in normal rabbits cheally on Day 0 with 10 mg silica suspended in 0.5 ml administered large amounts of phosphatidyl0.9% NaCl. Controls received saline only. Seven days afcholine by intratracheal injection. ter being dosed with silica the animals were injected inThe method of estimating fluxes from travenously with 3.3 &i [‘4C]choline. Animals were semilogarithmic plots is based upon the askilled at various time intervals after injection and the radiolaheled phospholipids were determined. Half-lives sumption that the components studied were were derived from linear regression analysis of semilogapulse labeled, which is only an approximarithmic plots from four experiments. tion as indicated by the gradual accumulaa Mean k SD (n = 4). tion of radioactivity in the intracellular pools b p i 0.05, Student’s t test. of both control and silica-treated animals. This assumption is inherent and considered reasonable in all attempts reported for the surfactant phospholipids were synthesized, secreted, and cleared resulted in different ex- measurement of surfactant phospholipids using radiolabeled precursors in viva (Jobe, pansion rates for each pool. The apparent synthesis of surfactant was increased to a 1979; Coultas et al., 1987; Heppleston et al., 1974; Young et al., 198 1). Deviation from greater extent than was its secretion which, in turn, was increased more than the rate of its the pulse labeling ideal would tend toward clearance from the alveoli. Thus, the rate of expansion of the intracellular pool exceeded TABLE 3 that of the extracellular pool. These data are consistent with our previous finding that the EFFECTSOF SILICA ON FLUXES OF [“‘CICHOLINE-LAresponse of the surfactant system to silica is BELED PHOSPHOLIPIDSFROM THE PULMONARY INTRACELLULAR AND EXTRACELLULAR SURFACTANT POOLS first manifested within the intracellular pool (Dethloff et al., 1986a) and may indicate that Rate of phospholipid the increase in the extracellular pool is largely flux WW a consequence of the expanding intracellular pool. Our findings differ from those of Lewis Control Silica Increase et al. (1987) who were unable to detect any Intracellular pool 102 + 10” 749 + 39’ 7.3-fold effect of silica on the clearance of surfactant Extracellular pool 87+ 8 434k21b 5.0-fold phospholipids. We attribute this discrepancy to the different times following silica treatNote. Male rats weighing 225-250 mg were injected ment; Lewis et al. (1987) made their mea- with 10 mg silica suspended in 0.5 ml 0.9% NaCl on Day surements 5 and 15 weeks after silica whereas 0. Controls received 0.5 ml 0.9% NaCl only. On Day 7 we made ours after only 1 week. Quite possi- rats were injected intravenously with 3.3 &i [‘4C]choand the presence of radiolaheled phospholipid in the bly the effect of silica on clearance may have line pulmonary intra- and extracellular pools of surfactant subsided by 5 weeks. In addition, the dose of was determined. silica that Lewis et al. (1987) used was only n Mean + SD (n = 4). b p =z0.0 1, Student’s t test. half of what we used. Regardless of the rea-

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overestimation of half-lives but this is unavoidable in view of our lack of knowledge concerning the distribution of label into the intracellular pool. Changing pool sizes may also affect half-life estimates; however, during the limited time course (26 hr) of our labeling studies, changes in pool sizes were below the limits of detectability and any such influence on half-life estimates should have been mitigated. We based our analysis on a simple twocompartment model in which surfactant phospholipids are synthesized, enter the intracellular pool, and are secreted into and then cleared from the extracellular pool. We assume that this standard model remains appropriate when applied to a situation involving pulmonary damage or disease. Evidence that the lamellar bodies of the alveolar type II epithelial cell are the sole and immediate precursors of the extracellular surfactant appears certain (Jacobs, 1983). In the present study, several criteria, important in establishing a precursor-product relationship as described by Zilversmit et al. ( 1943), were satisfied in both silica-treated and control animals. The specific activity-time curve of the first pool peaked and declined before the peak of the second pool. The specific activity-time curve of the first pool approximately intersected the peak of the specific activity-time curve of the second pool and the areas under the specific activity-time curves of both pools were nearly equal. The latter criterion was true for the control animals as would be expected, but in the silica-treated group the area under the intracellular compartment curve was 20-30% greater than that of the extracellular compartment. We interpret this to indicate that phospholipid was being synthesized and stored intracellularly and was not being transported to the extracellular compartment at an equivalent rate. The half-lives for phosphatidylcholine (PC) in the intra- and extracellular pools of control rats were similar to those reported by Toshima et al. (1972 [extracellular PC halflife. 10.3 hr]) and Tierney et al. (1967 [extra-

AND SILICA

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cellular PC half-life, 14 hr]), although longer than those measured by Young et al. (198 1) (half-lives for intracellular and extracellular disaturated phosphatidylcholine were 6.3 and 7.5 hr, respectively). In the lungs of silicatreated rats, the half-lives of PC in both intracellular and extracellular pools increased by approximately 50%. Increases in the half-life of intracellular PC cannot be accounted for by simply more type II cells synthesizing and secreting more surfactant at normal cellular rates. For the half-life to change, metabolic rates must be altered within type II cells or some other source of phosphatidylcholine must be invoked such as the presence of substantial amounts of cell debris. Although cell debris is to be expected in the alveoli because of the presence of damaged cells (Dethloff et a/., 1986c), this material appears to make a minor contribution because compositional changes in the phospholipid composition of surfactant were undetected in both intra- and extracellular pools (Dethloff et al., 1986a). Recently, we have shown that the number of type II cells in the lungs increases in response to silica (Miller et al., 1987a). However, increased numbers of normal type II cells cannot account for the increased levels of surfactant phospholipids in the lungs of silica-treated rats because normal type II cells do not contain sufficient surfactant (Dethloff et ul., 1986b) and increased numbers of normal type II cells could not account for changes in the half-life of intracellular surfactant phosphatidylcholine. It seems likely that surfactant metabolism must be changed within the type II cells themselves. In the lungs of silica-treated rats some, but not all, type II cells increase in size and contain more surfactant than normal (Miller et al., 1986, 1987a.b). Recently we have shown that those hypertrophic type II cells incorporate phospholipid precursors at a significantly faster rate than normal type II cells. It seems highly likely that the kinetic alterations in the metabolism of surfactant phospholipids in the lungs of silica-treated rats is due to the presence of this hypertrophic type II cell.

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ACKNOWLEDGMENT We acknowledge the skilled technical assistance of Sara Short.

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