Surfactant treatments alter endogenous metabolism in rabbit lungs SIDARTO Department
OETOMO, GAMI, AND
BAMBANG OETOMO, JIM LEWIS, MACHIKO IKEGAMI, AND ALAN of Pediatrics, Harbor- UCLA Medical Center, Torrance, California 90509
SIDARTO BAMBANG, JIM ALAN H. JOBE. Surfactant
LEWIS,
MACHIKO
IKE-
treatments alter endogenous surfactant metabolism in rabbit lungs. J. Appl. Physiol. 68(4): 1590-1596, 1990.-The effect of exogenous surfactant on endogenous surfactant metabolism was evaluated using a single-lobe treatment strategy to compare effects of treated with untreated lung within the same rabbit. Natural rabbit surfactant, Survanta, or 0.45% NaCl was injected into the left main stem bronchus by use of a Swan-Ganz catheter. Radiolabeled palmitic acid was then given by intravascular injection at two times after surfactant treatment, and the ratios of label incorporation and secretion in the left lower lobe to label incorporation and secretion in the right lung were compared. The treatment procedure resulted in a reasonably uniform surfactant distribution and did not disrupt lobar pulmonary blood flow. Natural rabbit surfactant increased incorporation of palmitate into saturated phosphatidylcholine (Sat PC) -2fold (P < O.Ol), and secretion of labeled Sat PC increased ~2.5 fold in the surfactant-treated left lower lobe relative to the right lung (P < 0.01). Although Survanta did not alter incorporation, it did increase secretion but not to the same extent as rabbit surfactant (P < 0.01). Alteration of endogenous surfactant Sat PC metabolism in vivo by surfactant treatments was different from that which would have been predicted by previous in vitro studies. surfactant distribution; phatidylcholine
surfactant
secretion;
saturated
phos-
SURFACTANT can be successfully used to treat neonatal respiratory distress syndrome (5,8,9,15), and surfactant treatments are being evaluated for the treatment of other pulmonary disorders such as the adult respiratory distress syndrome (3). However, possible effects of surfactant treatments on endogenous surfactant and lung phosphatidylcholine metabolism have not been thoroughly evaluated. Feedback inhibition of endogenous surfactant metabolism could occur, because the most widely used treatment dose of surfactant of 100 mg/kg contains -10 times the amount of phosphatidylcholine present in the alveolar surfactant pool of an adult rabbit (13) and may be an even larger relative dose in humans (22). In vitro studies demonstrate effects of surfactant on the surfactant metabolism in isolated type II cells (6, 23, 27). Thakur et al. (27) found that surfactant and surfactant components inhibited radiolabeled precursor incorporation into phosphatidylcholine in type II cells, and there are several reports of the inhibition of surfactant secretion in the presence of surfactant that contains surfactant protein A (SP-A), the surfactant specific 29- to 36-
1590
surfactant
0161-7567/90 $1.50 Copyright
0
H. JOBE
kDa protein (6, 23). Surfactant phospholipid and SP-A uptake by type II cells also was stimulated by what may be a receptor-specific mechanism (24). Stewart-DeHaan et al. (25) and our laboratory previously reported no changes in endogenous surfactant metabolism after surfactant treatments to either adult or preterm animals (11, 20). However, the multiple effects that have been documented in vitro stimulated us to develop an in vivo model to overcome some of the limitations of previous experiments that could have contributed to the negative results. MATERIALS
AND
METHODS
Surfactants. Natural rabbit surfactant was recovered from alveolar washes of fresh rabbit lungs (20). Surfactant was pelleted at 5,000 g for 30 min. The pellet was resuspended, and surfactant was isolated as the interface over 0.7 M sucrose in 0.9% NaCl after centrifugation at 34,000 g for 30 min. The surfactant was brought to a total lipid concentration of 25 mg/ml in 0.45% NaCl. To study surfactant distribution in the lungs, the surfactant was radiolabeled with [“C]choline-labeled dipalmitoylphosphatidylcholine (DPPC) in the form of liposomes that were associated with the surfactant (10) and/or with 57Co-labeled 15-pm microspheres (New England Nuclear, Boston, MA). To study the uptake and clearance of surfactant phosphatidylcholine, a trace amount of 3zPlabeled natural rabbit surfactant was added to the rabbit surfactant and to the 0.45% NaCl that was given to the group of control animals. Natural rabbit surfactant was radiolabeled by intratracheal injection of 1 mCi of [32P]orthophosphate into each of five 3-day-old rabbits (20), and 24 h later the large-aggregate surfactant was obtained by centrifugation of alveolar washes as above. This radiolabeled surfactant was suspended in 0.45% NaCl and mixed with the natural rabbit surfactant. Approximately 70% of the 32P was found in phosphatidylcholine, and each animal received -1 &i of 32P. Survanta was made by the Abbott Laboratories according to the procedure described by Fujiwara (8) and refrigerated. This surfactant was similar in phospholipid composition to natural surfactant, but the SP-A was removed by processing through organic solvents while the lipophilic surfactant-specific proteins were retained. To radiolabel Survanta, liposomes of organic solvent extracts of 32P-labeled natural rabbit surfactant were mixed with Survanta before the surfactant treatments. These liposomes were prepared by adding 0.45% NaCl to
1990 the American
Physiological
Society
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SURFACTANT
TREATMENT
the dried lipids and stirring into suspension with glass beads for 20 min. Each animal treated with Survanta received -1 &i of ‘*P. Surfactant distribution studies. In the initial study, healthy 2-kg adult rabbits (n = 5) were anesthetized with pentobarbital sodium (30 mg/kg body wt iv) and placed in the supine position. With the aid of a 4-mm flexible laryngoscope, each rabbit was orotracheally intubated and 100% oxygen was administered via the endotracheal tube for 5 min. A 4-Fr Swan-Ganz catheter was directed fluoroscopically into the left main stem bronchus, and the balloon was inflated. Collapse of the left lung after balloon inflation could be clearly visualized on the fluoroscopy monitor. Five minutes after balloon inflation, when complete lung collapse had occurred, 3 ml of surfactant (total lipid cone 25 mg/ml) and 0.5 &i of 57Co and 0.25 &i of [14C]DPPC were slowly injected through the lumen of the Swan-Ganz catheter, and 10 ml of air were injected to reinflate the lung as could be assessed by fluoroscopy. The balloon then was deflated, the rabbits were extubated, and supplemental oxygen was given for 30 min. The animals recovered quickly from the anesthesia without respiratory distress. To assess the pulmonary blood flow, we injected 0.5 &i of 15-pm 46Sclabeled microspheres intravenously 30 min after the intratracheal injection and killed the animals with an overdose of pentobarbital 1.5 h later. The lungs were removed, separated into lobes, and frozen on dry ice. The lobes of the right lung and the left upper lobe were cut into 5 pieces (loo-250 mg), and the lower lobe was cut into 100 pieces (lo-50 mg). The .57Coand ““SC-labeled microsphere radioactivity was measured in each piece. The lung pieces then were disrupted by sonication in water, and an aliquot was taken for protein measurement according to Lowry et al. (17). The rest of the lung suspension was extracted with organic solvent (4)) and the lipid-associated radioactivity was measured by liquid scintillation counting. For the second distribution study in five other rabbits that underwent the oxygen-induced atelectasis procedure, the volume of surfactant was increased by dilution with 0.45% NaCl to 6 ml, resulting in a surfactant suspension containing 12.5 mg/ml. The balloon was deflated 1 min after surfactant injection, and each animal then was given 10 manual lung inflations of 100% oxygen with an anesthesia bag. 57Co-labeled microspheres were mixed with the surfactant. The lungs were cut similarly into 120 pieces, and the radioactivity was measured. Surfactant metabolism studies. Four groups of six -2kg rabbits were studied (Fig. 1). The unmanipulated controls (group 1) did not undergo anesthesia or the lung collapse procedure. The three experimental groups were anesthetized, and left lower lobe instillation procedures were carried out according to the technique described for the second distribution study. Group 2 received 6 ml of 0.45% NaCl, group 3 was given 6 ml of 32P-labeled Survanta, and group 4 received 6 ml of “*P-labeled natural rabbit surfactant. To evaluate lobar pulmonary blood flow distribution, 0.05 &i of 5’Co-labeled microspheres was injected intravenously 30 min after surfactant treatment. Endogenously synthesized phosphatidyl-
1591
EFFECTS Endotracheal !
0.45%
0
Surfactant
6
Natural
Group
1 k----+-
Group
2 w-
Injections
Intravenous
NaCl TA Rabbit
: : !
Group
3
q
Group
4 t---&-+-m
Surfactant
-I--+
-----t----t--
I-$
-+---t-+-
: --$--:
57
l
+ -t
.- +
+-
Injections
Co Microspheres
v 14 C-Palmitic v 3 H-Palmitic
acid
.- + -
-$-----I
-m: V
+ --+ V
t--t
t
-t- --t--t
-*--
.--j---
--$--
-+---
acid
-+- --A -+
m"+- -t--t--t6
0
.{ -4 ”
t(h)
Death FIG.
1. Schematic
of treatment
protocol
of surfactant
metabolism
studies.
choline was labeled in the four groups of animals by the intravascular injection of [ l-‘4C]palmitic acid (25 &i/ kg, ICN, Irvine, CA) 30 min after surfactant treatment and [9,10-3H]palmitic acid (200 &i/kg, New England Nuclear) 6 h after surfactant treatment. The rabbits were killed 10 h after surfactant injection. The times for injection of the labeled palmitic acid were selected based on the kinetics of secretion of labeled saturated phosphatidylcholine (Sat PC) in rabbits (13) to allow us to evaluate an integrated effect of the surfactant treatments on secretion over 9.5 h and any effects that persisted over the interval from 6 to 10 h after surfactant treatment. Processing of lungs. The lungs were removed from the rabbits for selective alveolar washes of the right lung and the left lower lobe. Because the balloon catheter obstructed the left upper lobe bronchus, the treatment surfactant was confined to the left lower lobe. Therefore the left upper lobe was tied off, and the left main stem bronchus then was clamped to recover the alveolar wash of the right lung. Thereafter the clamp was released and placed on the right main stem bronchus to recover the alveolar wash of the left lower lobe. Subsequently the right lung and left lower lobe were homogenized separately. Aliquots of alveolar washes and lung homogenates were extracted with chloroform-methanol (2:l). The lipid extracts were treated with osmium tetroxide, and Sat PC was recovered by neutral alumina column chromatography (18). An aliquot of the Sat PC was degraded with sulfuric acid at 18O”C, and inorganic phosphorus was measured by the assay of Bartlett (1). Radioactivity in Sat PC was determined in Complete Counting Cocktail 3a70B scintillation fluid (Research Products) with a LKB multichannel analyzer programmed for the measurement of three radioisotopes. Data analysis. All data are expressed as means t SE. Differences between groups were tested by analysis of variance, and significance was assessedby the StudentNewman-Keuls multiple comparison procedure. RESULTS
Distribution of surfactant. We found in the initial study that surfactant was confined predominantly to the left lower lobe. About 7% of the labeled microspheres mixed
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with the surfactant were recovered from the left upper lobe. We found on average ~0.4% of the “7Co-labeled microspheres in the right lung. The radioactivity of the labeled phospholipid and microspheres that were added to the surfactant was expressed as counts per minute per milligram of protein relative to the ratio of the mean counts per minute for each isotope to the mean protein content of the lung pieces. The large variation of the values indicated an inhomogeneous distribution of surfactant to the left lower lobe (Fig. 2A). The percentage of lung pieces that received ~10% of the calculated mean value of radioactivity per milligram of protein was 27 t 10%. The distribution of the ‘Co-labeled microspheres correlated well with the distribution of [14C]DPPC (r = 0.94, Fig. 3). There was no correlation between the relative pulmonary blood flow to the lung pieces and the distribution of surfactant (data not shown). In the second study after increasing the volume of surfactant and modifying the lung reinflation procedure, we found 19% of the 5’Co-labeled microspheres mixed with the surfactant in the left upper lobe and on average 4.7% in the right lung. In the left lower lobe we found a more homogeneous surfactant distribution (Fig. 2B). The percentage of pieces that received 40% of the calculated mean value of radioactivity per milligram of lung tissue was significantly lower at 8.8 t 7.6% (P < 0.05) than for the first group of animals studied, and far fewer pieces received extremely large doses of surfactant. Pulmonary blood flow distribution. The relative lobar blood flow distributions measured 30 min after surfactant or saline injections into the collapsed left lower lobe were the same as those measured in the unmanipulated
EFFECTS
14 Mean’
C-DPPC
75
TABLE
Right
I. 2. 3. 4.
Unmanipulated controls 0.45% NaCl controls Survanta Natural rabbit surfactant
Lung
57.81kl.l 61.2t2.4 61.1t0.9 58.7k1.6
Values are means t SE expressed microspheres in lungs of each animal.
.4
.6
.8 1.0 1.2 1.4 1.6 1.8 2.Oj2.0
35,B
25
OT
t.05
.l
.2
.4
.6
.8 1.0 1.2 1.4 1.6 1.8 2.012.0
Distribution
interval
2. Normalized distribution of ‘Co-labeled microspheres mixed with natural surfactant. All values were calculated as described in MATERIALS AND METHODS and are presented as mean t SE percent of pieces of left lower lobe in 10 rabbits vs. 20% distribution intervals. A: surfactant at 2 1,2 k SE in pmol/kg
body
wt. L/R,
4 > 3 > 291 left lung lobe-to-
control (Table 1). Therefore the lung collapse, surfactant instillation, and reinflation procedures did not have a lasting effect on pulmonary blood flow distribution. Sat PC pool sizes. Alveolar and lung Sat PC pool sizes were measured 10 h after the lung collapse and surfactant treatment procedure. The alveolar surfactant pools as estimated by the Sat PC content were similar in the right lungs of the four groups of animals (Table 2). The alveolar Sat PC pool sizes in the left lower lobes were significantly higher in the groups that received surfactant (P < 0.01). When the Sat PC pool was expressed as micromoles per milligram of protein, the ratios for the left lower lobe to right lung were 1.2 t 0.2 and 1.1 t 0.1 for the unmanipulated and saline control groups, respectively. This result indicated that the alveolar washes of right and left lung were similarly effective. The Sat PC pool sizes of the lung tissue of the right lungs of the animals were comparable in all groups of
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SURFACTANT
TREATMENT
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EFFECTS
rabbits (Table 3). Significantly higher Sat PC pool sizes were found in the lung tissue of the left lower lobes of the rabbits of the groups that received surfactant (P < 0.01).
Labeled palmitate incorporation. Total incorporation of the intravascularly administered radioisotopes was determined from the radioactivity of recovered isotopes from the right lung and left lower lobe (alveolar wash + lung tissue). There were no differences in total incorporation into the lungs between groups, although there was a large variation of the values, with the standard deviation being 30-40% of the means for the different groups. Therefore we calculated the ratio of incorporation of the isotopes into the left lower lobe to that into the right lung for each animal (Fig. 4). There was a higher ratio for [3H]palmitic acid and [“C]palmitic acid incorporation into Sat PC in the rabbits treated with natural rabbit surfactant than in the groups of rabbits that did not receive surfactant (P < 0.01). Secretion of labeled Sat PC. The percent secretion of the [3H]- and [‘4C]palmitic acid-labeled Sat PC in each lung was calculated as the ratio of the radioactivity in the alveolar wash to the total radioactivity in the lung (alveolar wash + lung tissue) times 100 (Fig. 5). The secretion was similar in the right lungs of the four groups of animals, and the ratio of the secretion of the left lower lobe to that of the right lung did not differ between the control groups. The ratio for the secretion from the left lower lobe to that from the right lung was higher for the 3. Surfactant-saturated phosphatidylcholine in lung tissue 10 h after surfactant treatment TABLE
Right Lung Lobe
I. 2. 3. 4.
Unmanipulated controls 0.45% NaCl controls Survanta Natural rabbit surfactant
Left
11.5k2.2
Lower Lobe
6.3k0.9
16.0t1.2 11.5-eo.3 ll.lkO.8
0 C
1.2
3
& SE in pmol/kg
body
wt. L/R,
0.6kO.l 1.3kO.2 1.5-1-0.1 4 > 3 > 12 left lung lobe-to-
? 2 3
T
4) 1,2,3
0 .-c, dc
FIG.
alveolar Sat PC (A) and in Fig. number
0.9
0.6
0.3
0.0 1 3
2
3
H-Palmitic Sat
1234 Left
1 Lower Lobe
5. Percent secretion expressed as ratio of counts per minute in wash to counts per minute in total lung times 100 of radioactive at 4 and 9.5 h after intravascular injection of [“Hlpalmitic acid [‘4C]palmitic acid (R), respectively. Groups are numbered as 4. Significant differences at P < 0.05 are indicated by group above each column.
4. Recovery of [:‘2PJSat PC from the left lower lobe 10 h after endotracheal injection
1. 0.45%
NaCl
2. Survanta 3. Natural
controls
rabbit
surfactant
P < 0.05 are means
Alveolar Wash
Lung Tissue
13.4t2.7 11.8t2.3 23.8t3.0
20.9t3.2 15.OI1.7 35.3t2.7-
3 > 1,2
2 SE expressed
as percent
3 > 1,2
Total
Lung
34.3t4.7 26.8k6.5 59.1t1.6 3>
of administered
1,2 [“2P]-
4) 1,2,3
b : -I z 4
1234
R ght Lung
Values surfactant.
-I w r
.-m
1 TABLE
L/R
4,3 > 1,2
Values are means right lung lobe ratio.
F_
4)3)1,2 B
0.6kO.l
8.7k1.2 14.321.4 16.9t0.9
P < 0.05
1234
4 acid
PC
1
2
3
’ 4C-Palmitic Sat
4 acid
PC
FIG. 4. Recovery of total lung radioactive Sat PC at 4 h after intravascular injection of [“Hlpalmitic acid 9.5 h after [14C]palmitic acid in unmanipulated control rabbits (group I) or injection into left main stem bronchus of 0.45% NaCl (group 2), Survanta (group 3), or natural rabbit surfactant (group 4). Values are expressed as ratio of counts per minute in left lower lobe to counts per minute in right lung. Significant differences at P < 0.05 are indicated by group number above each column.
Survanta-treated rabbits than for the control groups (P < 0.05). However, the rabbits that were treated with natural rabbit surfactant had higher ratios than was found for any other group (P < 0.01). Recovery of labeled Sat PC. The recovery of [32P]Sat PC in alveolar wash and lung tissue was expressed as the percentage of the administered trace dose of [32P]Sat PC (Table 4). The values for the recovery of [32P]Sat PC from the alveolar washes and lung tissue were higher in the rabbits treated with natural rabbit surfactant than in the other groups (P < 0.01). Although there was more [““PISat PC recovered by alveolar wash in the rabbit surfactant-treated group (Table 4), the net pool size was similar to that measured for the Survanta-treated group (Table 2). This finding probably resulted from the different Sat PC contents of the surfactants (19 pmol/40 mg for rabbit surfactant and 24 pmo1/40 mg for Sur-
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SURFACTANT
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vanta), different effects on secretion of endogenous surfactant by the two treatment surfactants, and inherent variability in Sat PC pool sizes between rabbits. DISCUSSION
The model used in this study was designed specifically to investigate the effects of surfactant treatment on endogenous surfactant metabolism. Direct tracheal injections of small volumes of surfactant have been successfully used to study the fate of the exogenously administered surfactant (12). However, such administration techniques do not result in a uniform distribution of the surfactant at the lobar level, and the distribution is certainly much less uniform at the alveolar level. The lack of effect of treatment doses of exogenous surfactant on endogenous surfactant phosphatidylcholine metabolism in adult rabbits and the subtle changes of the metabolism in 3-day-old rabbits (19, 20) could be explained by the insensitivity of the measurements. These measurements resulted from possible changes in small volumes of lung that received a large amount of surfactant that were lost in the variability of the measurements for the entire lung. Our strategy was to try to reproduce the relative uniform distribution of surfactant that can be accomplished in the fluid-filled lung at birth (14). To that end we initially collapsed the left lower lobe using the technique of oxygen-induced atelectasis and then filled the atelectatic air spaces with 0.45% saline or the surfactant suspensions. Our initial study did not demonstrate good distribution, probably because we rapidly reinflated the lung, forcing air beyond the relatively viscous and small-volume surfactant suspension. In this initial study we did not detect an effect of surfactant on endogenous surfactant metabolism. However, we demonstrated the comparability of 15pm microspheres and radiolabeled DPPC for the measurement of surfactant distribution in small pieces of lung. The distribution of surfactant was significantly improved by use of a larger volume of surfactant suspension and by more gradual reinflation of the lung. Because surfactant distribution was not truly uniform in lo- to 50-mg lung pieces, we must assume a much more nonuniform distribution at the alveolar level. Therefore our conclusions concerning the effects of exogenous surfactant on endogenous surfactant metabolism do not accurately reflect the magnitude of the responsesof individual type II cells. The results do measure the aggregate response of the healthy lung to surfactant and thus form the basis for future studies of the metabolic responses of the injured lung to surfactant treatments. Another source of error in previous experiments was the large variability in radiolabeled precursor incorporation into surfactant phosphatidylcholine (20). To minimize such variability we took advantage of the lobar administration of surfactant by comparing surfactant metabolism in the left and right lung of each animal. However, the collapse, treatment, and reinflation procedure could have altered metabolism in the left lower lobe. We were particularly concerned about pulmonary perfusion because pulmonary blood flow will decrease to atelectatic lung volumes (2, 7). Although transient
EFFECTS
changes in pulmonary blood flow probably occurred, there were no differences detected 30 min after the treatment procedure. This result was important because we used intravascularly injected precursors to compare radiolabeled precursor incorporation into Sat PC in the left lower lobes with that in the right lungs. We used two control groups to evaluate our ability to process the lungs separately and to evaluate the influence of the treatment procedure on the endogenous Sat PC metabolism. We found that alveolar washes of the left lower lobes and right lungs could be carried out comparably and consistently, because the alveolar wash phospholipid-to-tissue protein ratios were similar. The alveolar and lung tissue Sat PC pools were not influenced by the lung collapse and vehicle instillation procedure, because the values were similar in the right lungs and in the left lungs and not different from those measured in unmanipulated controls. Furthermore we found that the incorporation of [“Cl - and [“Hlpalmitic acid into Sat PC in the lung tissue and the secretion of the labeled Sat PC from the left lower lobes were not different between the two control groups. Surfactant containing SP-A decreased surfactant secretion by type II cells in vitro in a dose-dependent fashion (16, 23). The most potent inhibitor of the secretory inhibition was SP-A, although surfactant lipids also seemed to decrease secretion (6). Because SP-A also increased uptake of surfactant by type II cells by what appeared to be a receptor-specific mechanism involving endocytosis into multivesicular bodies (24)) the assumption is that these two effects are linked. We anticipated that administration of natural rabbit surfactant at a dose of -40 mg/kg body wt to the left lower lobe, which is equivalent to 100 mg/kg body wt to the total lung, would decrease secretion because it contained SP-A. However, the results clearly demonstrated an almost threefold increase in secretion of surfactant Sat PC that had been synthesized either 9.5 or 4 h before study. The magnitude of the effect was the same for the two different times. The experimental design did not permit us to evaluate the early clearance of the [““PISat PC associated with the rabbit surfactant so that the linkage of reuptake with secretion could not be evaluated by this protocol. However, the overall effect was a striking increase in the accumulation of endogenously labeled Sat PC in the air spaces. A similar but less striking effect on endogenous surfactant secretion resulted from treatments of 3-dayold rabbits with sheep surfactant (11). Survanta was a less potent stimulus to labeled Sat PC accumulation in the air spaces than was rabbit surfactant. A reasonable speculation is that the absence of SPA has decreased the secretory response, although there are other differences in composition and lipoprotein aggregate forms between the two surfactants (26). The striking difference between these in vivo experiments and the results with type II cells could be caused by alterations in the type II cells during isolation or differences in the metabolic state of the cells within the lung vs. that in tissue culture. The apical secretory surface of type II cells probably is bathed with high concentrations of surfactant in vivo, a situation very different from in
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SURFACTANT
TREATMENT
vitro culture conditions. Perhaps the addition of a lofold excess of SP-A and Sat PC stimulated the cells in situ to increase their overall surfactant metabolic activity, resulting in the measured accumulation of de novo synthesized Sat PC in the air spaces.Large and repetitive doses of exogenous surfactant do increase the overall catabolic rate of surfactant phosphatidylcholine in the lung (20, 21). This concept is consistent with the increased incorporation of labeled palmitic acid into Sat PC in the left lower lobes of rabbit surfactant-treated animals. The incorporation of palmitate does not prove increased synthesis because precursor pools could be altered by the surfactant treatments. Our assumption was that the likely result would be increased palmitate pool sizes resulting from catabolism of the surfactant used for treatment (20) and that the surfactant might feedback inhibit Sat PC synthesis, as demonstrated in vitro in type II cells (6). The result of increased incorporation suggestedincreased metabolic activity by the type II cells in the left lower lobe. However, the nature of that effect cannot be determined from these experiments. The surfactant used for treatment or the catabolic products of that surfactant could selectively influence multiple metabolic pathways within the lung. The lack of effect noted with Survanta could indicate that the incorporation response was a SP-A-specific response or that the 7% by weight of free palmitic acid in Survanta interfered with the measurement by altering palmitic acid pool sizes. The clinical implications of these studies are in general positive. The primary goal of surfactant treatment is to deliver biophysically active surfactant to unstable alveoli. A secondary goal should be to preserve endogen.ous surfa.ctant metabolism. These studies indicate that a treatment dose of rabbit surfactant not only did not inhibit endogenous pathways but also stimulated endogenous surfactant Sat PC metabolism. The results were similar but lessstriking with Survanta. These stimulatory effects could be very helpful to the injured lung to help restore normal surfactant metabolism. This research was supported by National Institute of Child Health and Human Development Grant HD-11932; a grant of the Ter Meulen Fund, Royal Netherlands Academy of Arts and Sciences, to S. B. Oetomo; and Canadian Medical Research Council Fellowship 33703 to
Received
1 September
1989; accepted
in final
form
8 December
1989.
503.
9. HORBAR, J. D., R. F. SOLL, J. M. SUTHERLAND, U. KOTAGAL, A. G. S. PHILIP, D. L. KESSLER, G. A. LITTLE, W. H. EDWARDS, D. VIDYASAGAR, T. N. K. RAJU, A. JOBE, M. IKEGAMI, M. MULLET, D. 2. MEYERBERG, T. L. MCAULIFFE, AND J. F. LUCEY. A multicenter randomized, placebo controlled trial of surfactant therapy for respiratory distress syndrome. N. Engl. J. Med. 320: 959-965, 1988. 10.
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G. R. Phosphorus assay in column chromatography. J. Biol. Chem. 234:466-468,1959. 2. BENUMOF, J. L. Mechanism of decreased blood flow to atelectic lung. J. Appl. Physiol. 46: 1047-1048, 1979. 3. BERGGREN, P., B. LACHMANN, T. CURSTEDT, G. GROSSMAN, AND B. ROBERTSON. Gas exchange and lung morphology after surfactant replacement in experimental adult respiratory distress syndrome induced by repeated lung lavage. Acta Anaesthesiol. Stand.
1989.
JACOBS, H. C., M. IKEGAMI, A. JOBE, D. BERRY, AND S. JONES. Reutilization of surfactant phosphatidylcholine in adult rabbits. Biochim. Biophys. Acta 837: 77-84, 1985. 13. JACOBS, H. C., A. JOBE, M. IKEGAMI, AND S. JONES. Surfactant phosphatidylcholine source, fluxes and turnover times in 3-dayold, lo-day-old and adult rabbits. J. Biol. Chem. 257: 1805-1810, 1982. 14.
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JOBE, A., M. IKEGAMI, H. JACOBS, AND S. JONES. Surfactant and pulmonary blood flow distribution following treatment of premature lambs with natural rabbit surfactant. J. Clin. Inuest. 73: 848856,1984.
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KENDIG, J. W., R. H. NOTTER, C. Cox, J. L. ASCHNER, S. BENN, R. M. BERNSTEIN, K. HENDRICKS-MUNOZ, W. M. MANISCALCO, L. A. METLAY, D. L. PHELPS, AND D. L. SHAPIRO. Surfactant replacement therapy at birth: final analysis of a clinical trial and comparisons with similar trials. Pediatrics 82: 756-762, 1988. KUROKI, Y., R. J. MASON, AND D. R. VOELKER. Pulmonary surfactant protein A structure and modulation of surfactant secretion by rat alveolar type II cells. J. Biol. Chem. 263: 3388-3394, 1988. LOWRY, 0. H., N. J. ROSENBROUGH, A. L. FARR, AND R. J. RANDALL. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951. MASON, R. J., J. NELLENBOGEN, AND J. A. CI~EMENTS. Isolation of disaturated phosphatidylcholine using osmium tetroxide. J. Lipid Res. 17: 281-284, 1978. OGUCHI, K., M. IKEGAMI, H. JACOBS, AND A. JOBE. Clearance of large amounts of natural surfactants and liposomes of dipalmitoyl phosphatidylcholine from the lung of rabbits. Exp. Lung Res. 9: 221-235,1985.
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IKEGAMI, M., A. JOBE, AND G. DUANE. Liposomes of dipalmitoylphosphatidylcholine associated with natural surfactant. Biochim. Biophys. Acta 835: 352-359, 1985. IKEGAMI, M., A. JOBE, T. YAMADA, A. PRIESTLY, L. RUFFINI, E. RIDER, AND S. SEIDNER. Surfactant metabolism in surfactanttreated preterm ventilated lambs. J. Appl. Physiol. 67: 429-437,
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REFERENCES 1. BARTLETT,
1595
5. COLLABORATIVE EUROPEAN MULTICENTER STUDY GROUP. Surfactant replacement therapy for severe neonatal respiratory distress syndrome: an international randomized clinical trial. Pediatrics 82: 683-691, 1988. 6. DOBBS, L. G., J. R. WRIGHT, S. HAWGOOD, R. GONZALEZ, K. VENSTROM, AND J. NELLENBOGEN. Pulmonary surfactant and its components inhibit secretion of phosphatidylcholine from cultured rat alveolar type II cells. Proc. Natl. Acad. Sci. USA 48: 1010-1014, 1987. 7. ENJETI, S., J. T. O’NEILL, P. B. TERRY, H. A. MENKES, AND R. J. TRAYSTMAN. Sublobar atelectasis and regional pulmonary blood flow. J. Appl. Physiol. 47: 1245-1250, 1979. 8. FUJIWARA, T. Surfactant replacement therapy in neonatal RDS. In: Pulmonary Surfactant, edited by B. Robertson, L. M. G. Van Golde, and J. J. Batenburg. Amsterdam: Elsevier, 1984, p. 479-
J. Lewis.
Address for reprint requests: A. H. Jobe, Dept. of Pediatrics, HarborUCLA Medical Center, A-17 Annex, 1000 W. Carson St., Torrance, CA 90509.
EFFECTS
24.
PETTENAZZO, A., M. IKEGAMI, S. SEIDNER, AND A. JOBE. Clearance of surfactant phosphatidylcholine from adult rabbit lungs. J. Appl. Physiol. 64: 120-127, 1988. PETTENAZZO, A., A. JOBE, M. IKEGAMI, E. RIDER, S. SEIDNER, AND T. YAMADA. Cumulative effects of repeated surfactant treatments in the rabbit. Exp. Lung Res. In press. PRE, J., G. PERRET, AND D. BLADIER. Lecithin content eximate of human alveolar lining larger: comparison with mouse, rat and rabbit. Comp. Biochem. Physiol. A Comp. Physiol. 76: 393-395, 1983. RICE, W. R., G. F. Ross, F. M. SINGLETON, S. DINGLE, AND J. A. WHITSETT. Surfactant-associated protein inhibits phospholipid secretion from type II cells. J. Appl. Physiol. 63: 692-698, 1987. RYAN, R. M., R. E. MORRIS, W. R. RICE, G. CIRAOLO, AND J. E. WHITSETT. Binding and uptake of pulmonary surfactant protein
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 17, 2019.
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SURFACTANT
(SP-A) by pulmonary 25.
26.
type II epithelial cells. J. Histochem.
TREATMENT Cyto-
them. 37: 429-440,1989. STEWART-DEHAAN, P. J., I. L. METCALF, ENHORNING, AND F. POSSMAYER. Effect
P. G. R. HARDING, G. of birth and surfactant treatment on x>hospholit$d svnthesis in the Dremature rabbit. BioL. Neonate TAUSCH,
38: 2i8-2i7,
1480.
u
H. W., K. M. W. KEOUGH,
M. WILLIAMS,
R. SLAVIN,
E.
EFFECTS
A. S. LEE. D. PHELPS. N. KARIEL. J. FLORES. AND M. E. ’ Characterizat#ion of bovine surfakant for infants with respiratory distress syndrome. Pediatrics 77: 572481, 1986. 27. THAKUR, N. R., M. TESAN, N. E. TYLER, AND J. E. BLEASDALE. Altered lipid synthesis in type II pneumocytes exposed to lung surfactant. Biochem. J. 240: 679-690,1986. STEELE. AVERY.
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OETOMO, GAMI, AND
BAMBANG OETOMO, JIM LEWIS, MACHIKO IKEGAMI, AND ALAN of Pediatrics, Harbor- UCLA Medical Center, Torrance, California 90509
SIDARTO BAMBANG, JIM ALAN H. JOBE. Surfactant
LEWIS,
MACHIKO
IKE-
treatments alter endogenous surfactant metabolism in rabbit lungs. J. Appl. Physiol. 68(4): 1590-1596, 1990.-The effect of exogenous surfactant on endogenous surfactant metabolism was evaluated using a single-lobe treatment strategy to compare effects of treated with untreated lung within the same rabbit. Natural rabbit surfactant, Survanta, or 0.45% NaCl was injected into the left main stem bronchus by use of a Swan-Ganz catheter. Radiolabeled palmitic acid was then given by intravascular injection at two times after surfactant treatment, and the ratios of label incorporation and secretion in the left lower lobe to label incorporation and secretion in the right lung were compared. The treatment procedure resulted in a reasonably uniform surfactant distribution and did not disrupt lobar pulmonary blood flow. Natural rabbit surfactant increased incorporation of palmitate into saturated phosphatidylcholine (Sat PC) -2fold (P < O.Ol), and secretion of labeled Sat PC increased ~2.5 fold in the surfactant-treated left lower lobe relative to the right lung (P < 0.01). Although Survanta did not alter incorporation, it did increase secretion but not to the same extent as rabbit surfactant (P < 0.01). Alteration of endogenous surfactant Sat PC metabolism in vivo by surfactant treatments was different from that which would have been predicted by previous in vitro studies. surfactant distribution; phatidylcholine
surfactant
secretion;
saturated
phos-
SURFACTANT can be successfully used to treat neonatal respiratory distress syndrome (5,8,9,15), and surfactant treatments are being evaluated for the treatment of other pulmonary disorders such as the adult respiratory distress syndrome (3). However, possible effects of surfactant treatments on endogenous surfactant and lung phosphatidylcholine metabolism have not been thoroughly evaluated. Feedback inhibition of endogenous surfactant metabolism could occur, because the most widely used treatment dose of surfactant of 100 mg/kg contains -10 times the amount of phosphatidylcholine present in the alveolar surfactant pool of an adult rabbit (13) and may be an even larger relative dose in humans (22). In vitro studies demonstrate effects of surfactant on the surfactant metabolism in isolated type II cells (6, 23, 27). Thakur et al. (27) found that surfactant and surfactant components inhibited radiolabeled precursor incorporation into phosphatidylcholine in type II cells, and there are several reports of the inhibition of surfactant secretion in the presence of surfactant that contains surfactant protein A (SP-A), the surfactant specific 29- to 36-
1590
surfactant
0161-7567/90 $1.50 Copyright
0
H. JOBE
kDa protein (6, 23). Surfactant phospholipid and SP-A uptake by type II cells also was stimulated by what may be a receptor-specific mechanism (24). Stewart-DeHaan et al. (25) and our laboratory previously reported no changes in endogenous surfactant metabolism after surfactant treatments to either adult or preterm animals (11, 20). However, the multiple effects that have been documented in vitro stimulated us to develop an in vivo model to overcome some of the limitations of previous experiments that could have contributed to the negative results. MATERIALS
AND
METHODS
Surfactants. Natural rabbit surfactant was recovered from alveolar washes of fresh rabbit lungs (20). Surfactant was pelleted at 5,000 g for 30 min. The pellet was resuspended, and surfactant was isolated as the interface over 0.7 M sucrose in 0.9% NaCl after centrifugation at 34,000 g for 30 min. The surfactant was brought to a total lipid concentration of 25 mg/ml in 0.45% NaCl. To study surfactant distribution in the lungs, the surfactant was radiolabeled with [“C]choline-labeled dipalmitoylphosphatidylcholine (DPPC) in the form of liposomes that were associated with the surfactant (10) and/or with 57Co-labeled 15-pm microspheres (New England Nuclear, Boston, MA). To study the uptake and clearance of surfactant phosphatidylcholine, a trace amount of 3zPlabeled natural rabbit surfactant was added to the rabbit surfactant and to the 0.45% NaCl that was given to the group of control animals. Natural rabbit surfactant was radiolabeled by intratracheal injection of 1 mCi of [32P]orthophosphate into each of five 3-day-old rabbits (20), and 24 h later the large-aggregate surfactant was obtained by centrifugation of alveolar washes as above. This radiolabeled surfactant was suspended in 0.45% NaCl and mixed with the natural rabbit surfactant. Approximately 70% of the 32P was found in phosphatidylcholine, and each animal received -1 &i of 32P. Survanta was made by the Abbott Laboratories according to the procedure described by Fujiwara (8) and refrigerated. This surfactant was similar in phospholipid composition to natural surfactant, but the SP-A was removed by processing through organic solvents while the lipophilic surfactant-specific proteins were retained. To radiolabel Survanta, liposomes of organic solvent extracts of 32P-labeled natural rabbit surfactant were mixed with Survanta before the surfactant treatments. These liposomes were prepared by adding 0.45% NaCl to
1990 the American
Physiological
Society
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 17, 2019.
SURFACTANT
TREATMENT
the dried lipids and stirring into suspension with glass beads for 20 min. Each animal treated with Survanta received -1 &i of ‘*P. Surfactant distribution studies. In the initial study, healthy 2-kg adult rabbits (n = 5) were anesthetized with pentobarbital sodium (30 mg/kg body wt iv) and placed in the supine position. With the aid of a 4-mm flexible laryngoscope, each rabbit was orotracheally intubated and 100% oxygen was administered via the endotracheal tube for 5 min. A 4-Fr Swan-Ganz catheter was directed fluoroscopically into the left main stem bronchus, and the balloon was inflated. Collapse of the left lung after balloon inflation could be clearly visualized on the fluoroscopy monitor. Five minutes after balloon inflation, when complete lung collapse had occurred, 3 ml of surfactant (total lipid cone 25 mg/ml) and 0.5 &i of 57Co and 0.25 &i of [14C]DPPC were slowly injected through the lumen of the Swan-Ganz catheter, and 10 ml of air were injected to reinflate the lung as could be assessed by fluoroscopy. The balloon then was deflated, the rabbits were extubated, and supplemental oxygen was given for 30 min. The animals recovered quickly from the anesthesia without respiratory distress. To assess the pulmonary blood flow, we injected 0.5 &i of 15-pm 46Sclabeled microspheres intravenously 30 min after the intratracheal injection and killed the animals with an overdose of pentobarbital 1.5 h later. The lungs were removed, separated into lobes, and frozen on dry ice. The lobes of the right lung and the left upper lobe were cut into 5 pieces (loo-250 mg), and the lower lobe was cut into 100 pieces (lo-50 mg). The .57Coand ““SC-labeled microsphere radioactivity was measured in each piece. The lung pieces then were disrupted by sonication in water, and an aliquot was taken for protein measurement according to Lowry et al. (17). The rest of the lung suspension was extracted with organic solvent (4)) and the lipid-associated radioactivity was measured by liquid scintillation counting. For the second distribution study in five other rabbits that underwent the oxygen-induced atelectasis procedure, the volume of surfactant was increased by dilution with 0.45% NaCl to 6 ml, resulting in a surfactant suspension containing 12.5 mg/ml. The balloon was deflated 1 min after surfactant injection, and each animal then was given 10 manual lung inflations of 100% oxygen with an anesthesia bag. 57Co-labeled microspheres were mixed with the surfactant. The lungs were cut similarly into 120 pieces, and the radioactivity was measured. Surfactant metabolism studies. Four groups of six -2kg rabbits were studied (Fig. 1). The unmanipulated controls (group 1) did not undergo anesthesia or the lung collapse procedure. The three experimental groups were anesthetized, and left lower lobe instillation procedures were carried out according to the technique described for the second distribution study. Group 2 received 6 ml of 0.45% NaCl, group 3 was given 6 ml of 32P-labeled Survanta, and group 4 received 6 ml of “*P-labeled natural rabbit surfactant. To evaluate lobar pulmonary blood flow distribution, 0.05 &i of 5’Co-labeled microspheres was injected intravenously 30 min after surfactant treatment. Endogenously synthesized phosphatidyl-
1591
EFFECTS Endotracheal !
0.45%
0
Surfactant
6
Natural
Group
1 k----+-
Group
2 w-
Injections
Intravenous
NaCl TA Rabbit
: : !
Group
3
q
Group
4 t---&-+-m
Surfactant
-I--+
-----t----t--
I-$
-+---t-+-
: --$--:
57
l
+ -t
.- +
+-
Injections
Co Microspheres
v 14 C-Palmitic v 3 H-Palmitic
acid
.- + -
-$-----I
-m: V
+ --+ V
t--t
t
-t- --t--t
-*--
.--j---
--$--
-+---
acid
-+- --A -+
m"+- -t--t--t6
0
.{ -4 ”
t(h)
Death FIG.
1. Schematic
of treatment
protocol
of surfactant
metabolism
studies.
choline was labeled in the four groups of animals by the intravascular injection of [ l-‘4C]palmitic acid (25 &i/ kg, ICN, Irvine, CA) 30 min after surfactant treatment and [9,10-3H]palmitic acid (200 &i/kg, New England Nuclear) 6 h after surfactant treatment. The rabbits were killed 10 h after surfactant injection. The times for injection of the labeled palmitic acid were selected based on the kinetics of secretion of labeled saturated phosphatidylcholine (Sat PC) in rabbits (13) to allow us to evaluate an integrated effect of the surfactant treatments on secretion over 9.5 h and any effects that persisted over the interval from 6 to 10 h after surfactant treatment. Processing of lungs. The lungs were removed from the rabbits for selective alveolar washes of the right lung and the left lower lobe. Because the balloon catheter obstructed the left upper lobe bronchus, the treatment surfactant was confined to the left lower lobe. Therefore the left upper lobe was tied off, and the left main stem bronchus then was clamped to recover the alveolar wash of the right lung. Thereafter the clamp was released and placed on the right main stem bronchus to recover the alveolar wash of the left lower lobe. Subsequently the right lung and left lower lobe were homogenized separately. Aliquots of alveolar washes and lung homogenates were extracted with chloroform-methanol (2:l). The lipid extracts were treated with osmium tetroxide, and Sat PC was recovered by neutral alumina column chromatography (18). An aliquot of the Sat PC was degraded with sulfuric acid at 18O”C, and inorganic phosphorus was measured by the assay of Bartlett (1). Radioactivity in Sat PC was determined in Complete Counting Cocktail 3a70B scintillation fluid (Research Products) with a LKB multichannel analyzer programmed for the measurement of three radioisotopes. Data analysis. All data are expressed as means t SE. Differences between groups were tested by analysis of variance, and significance was assessedby the StudentNewman-Keuls multiple comparison procedure. RESULTS
Distribution of surfactant. We found in the initial study that surfactant was confined predominantly to the left lower lobe. About 7% of the labeled microspheres mixed
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1592
SURFACTANT
TREATMENT
with the surfactant were recovered from the left upper lobe. We found on average ~0.4% of the “7Co-labeled microspheres in the right lung. The radioactivity of the labeled phospholipid and microspheres that were added to the surfactant was expressed as counts per minute per milligram of protein relative to the ratio of the mean counts per minute for each isotope to the mean protein content of the lung pieces. The large variation of the values indicated an inhomogeneous distribution of surfactant to the left lower lobe (Fig. 2A). The percentage of lung pieces that received ~10% of the calculated mean value of radioactivity per milligram of protein was 27 t 10%. The distribution of the ‘Co-labeled microspheres correlated well with the distribution of [14C]DPPC (r = 0.94, Fig. 3). There was no correlation between the relative pulmonary blood flow to the lung pieces and the distribution of surfactant (data not shown). In the second study after increasing the volume of surfactant and modifying the lung reinflation procedure, we found 19% of the 5’Co-labeled microspheres mixed with the surfactant in the left upper lobe and on average 4.7% in the right lung. In the left lower lobe we found a more homogeneous surfactant distribution (Fig. 2B). The percentage of pieces that received 40% of the calculated mean value of radioactivity per milligram of lung tissue was significantly lower at 8.8 t 7.6% (P < 0.05) than for the first group of animals studied, and far fewer pieces received extremely large doses of surfactant. Pulmonary blood flow distribution. The relative lobar blood flow distributions measured 30 min after surfactant or saline injections into the collapsed left lower lobe were the same as those measured in the unmanipulated
EFFECTS
14 Mean’
C-DPPC
75
TABLE
Right
I. 2. 3. 4.
Unmanipulated controls 0.45% NaCl controls Survanta Natural rabbit surfactant
Lung
57.81kl.l 61.2t2.4 61.1t0.9 58.7k1.6
Values are means t SE expressed microspheres in lungs of each animal.
.4
.6
.8 1.0 1.2 1.4 1.6 1.8 2.Oj2.0
35,B
25
OT
t.05
.l
.2
.4
.6
.8 1.0 1.2 1.4 1.6 1.8 2.012.0
Distribution
interval
2. Normalized distribution of ‘Co-labeled microspheres mixed with natural surfactant. All values were calculated as described in MATERIALS AND METHODS and are presented as mean t SE percent of pieces of left lower lobe in 10 rabbits vs. 20% distribution intervals. A: surfactant at 2 1,2 k SE in pmol/kg
body
wt. L/R,
4 > 3 > 291 left lung lobe-to-
control (Table 1). Therefore the lung collapse, surfactant instillation, and reinflation procedures did not have a lasting effect on pulmonary blood flow distribution. Sat PC pool sizes. Alveolar and lung Sat PC pool sizes were measured 10 h after the lung collapse and surfactant treatment procedure. The alveolar surfactant pools as estimated by the Sat PC content were similar in the right lungs of the four groups of animals (Table 2). The alveolar Sat PC pool sizes in the left lower lobes were significantly higher in the groups that received surfactant (P < 0.01). When the Sat PC pool was expressed as micromoles per milligram of protein, the ratios for the left lower lobe to right lung were 1.2 t 0.2 and 1.1 t 0.1 for the unmanipulated and saline control groups, respectively. This result indicated that the alveolar washes of right and left lung were similarly effective. The Sat PC pool sizes of the lung tissue of the right lungs of the animals were comparable in all groups of
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SURFACTANT
TREATMENT
1593
EFFECTS
rabbits (Table 3). Significantly higher Sat PC pool sizes were found in the lung tissue of the left lower lobes of the rabbits of the groups that received surfactant (P < 0.01).
Labeled palmitate incorporation. Total incorporation of the intravascularly administered radioisotopes was determined from the radioactivity of recovered isotopes from the right lung and left lower lobe (alveolar wash + lung tissue). There were no differences in total incorporation into the lungs between groups, although there was a large variation of the values, with the standard deviation being 30-40% of the means for the different groups. Therefore we calculated the ratio of incorporation of the isotopes into the left lower lobe to that into the right lung for each animal (Fig. 4). There was a higher ratio for [3H]palmitic acid and [“C]palmitic acid incorporation into Sat PC in the rabbits treated with natural rabbit surfactant than in the groups of rabbits that did not receive surfactant (P < 0.01). Secretion of labeled Sat PC. The percent secretion of the [3H]- and [‘4C]palmitic acid-labeled Sat PC in each lung was calculated as the ratio of the radioactivity in the alveolar wash to the total radioactivity in the lung (alveolar wash + lung tissue) times 100 (Fig. 5). The secretion was similar in the right lungs of the four groups of animals, and the ratio of the secretion of the left lower lobe to that of the right lung did not differ between the control groups. The ratio for the secretion from the left lower lobe to that from the right lung was higher for the 3. Surfactant-saturated phosphatidylcholine in lung tissue 10 h after surfactant treatment TABLE
Right Lung Lobe
I. 2. 3. 4.
Unmanipulated controls 0.45% NaCl controls Survanta Natural rabbit surfactant
Left
11.5k2.2
Lower Lobe
6.3k0.9
16.0t1.2 11.5-eo.3 ll.lkO.8
0 C
1.2
3
& SE in pmol/kg
body
wt. L/R,
0.6kO.l 1.3kO.2 1.5-1-0.1 4 > 3 > 12 left lung lobe-to-
? 2 3
T
4) 1,2,3
0 .-c, dc
FIG.
alveolar Sat PC (A) and in Fig. number
0.9
0.6
0.3
0.0 1 3
2
3
H-Palmitic Sat
1234 Left
1 Lower Lobe
5. Percent secretion expressed as ratio of counts per minute in wash to counts per minute in total lung times 100 of radioactive at 4 and 9.5 h after intravascular injection of [“Hlpalmitic acid [‘4C]palmitic acid (R), respectively. Groups are numbered as 4. Significant differences at P < 0.05 are indicated by group above each column.
4. Recovery of [:‘2PJSat PC from the left lower lobe 10 h after endotracheal injection
1. 0.45%
NaCl
2. Survanta 3. Natural
controls
rabbit
surfactant
P < 0.05 are means
Alveolar Wash
Lung Tissue
13.4t2.7 11.8t2.3 23.8t3.0
20.9t3.2 15.OI1.7 35.3t2.7-
3 > 1,2
2 SE expressed
as percent
3 > 1,2
Total
Lung
34.3t4.7 26.8k6.5 59.1t1.6 3>
of administered
1,2 [“2P]-
4) 1,2,3
b : -I z 4
1234
R ght Lung
Values surfactant.
-I w r
.-m
1 TABLE
L/R
4,3 > 1,2
Values are means right lung lobe ratio.
F_
4)3)1,2 B
0.6kO.l
8.7k1.2 14.321.4 16.9t0.9
P < 0.05
1234
4 acid
PC
1
2
3
’ 4C-Palmitic Sat
4 acid
PC
FIG. 4. Recovery of total lung radioactive Sat PC at 4 h after intravascular injection of [“Hlpalmitic acid 9.5 h after [14C]palmitic acid in unmanipulated control rabbits (group I) or injection into left main stem bronchus of 0.45% NaCl (group 2), Survanta (group 3), or natural rabbit surfactant (group 4). Values are expressed as ratio of counts per minute in left lower lobe to counts per minute in right lung. Significant differences at P < 0.05 are indicated by group number above each column.
Survanta-treated rabbits than for the control groups (P < 0.05). However, the rabbits that were treated with natural rabbit surfactant had higher ratios than was found for any other group (P < 0.01). Recovery of labeled Sat PC. The recovery of [32P]Sat PC in alveolar wash and lung tissue was expressed as the percentage of the administered trace dose of [32P]Sat PC (Table 4). The values for the recovery of [32P]Sat PC from the alveolar washes and lung tissue were higher in the rabbits treated with natural rabbit surfactant than in the other groups (P < 0.01). Although there was more [““PISat PC recovered by alveolar wash in the rabbit surfactant-treated group (Table 4), the net pool size was similar to that measured for the Survanta-treated group (Table 2). This finding probably resulted from the different Sat PC contents of the surfactants (19 pmol/40 mg for rabbit surfactant and 24 pmo1/40 mg for Sur-
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1594
SURFACTANT
TREATMENT
vanta), different effects on secretion of endogenous surfactant by the two treatment surfactants, and inherent variability in Sat PC pool sizes between rabbits. DISCUSSION
The model used in this study was designed specifically to investigate the effects of surfactant treatment on endogenous surfactant metabolism. Direct tracheal injections of small volumes of surfactant have been successfully used to study the fate of the exogenously administered surfactant (12). However, such administration techniques do not result in a uniform distribution of the surfactant at the lobar level, and the distribution is certainly much less uniform at the alveolar level. The lack of effect of treatment doses of exogenous surfactant on endogenous surfactant phosphatidylcholine metabolism in adult rabbits and the subtle changes of the metabolism in 3-day-old rabbits (19, 20) could be explained by the insensitivity of the measurements. These measurements resulted from possible changes in small volumes of lung that received a large amount of surfactant that were lost in the variability of the measurements for the entire lung. Our strategy was to try to reproduce the relative uniform distribution of surfactant that can be accomplished in the fluid-filled lung at birth (14). To that end we initially collapsed the left lower lobe using the technique of oxygen-induced atelectasis and then filled the atelectatic air spaces with 0.45% saline or the surfactant suspensions. Our initial study did not demonstrate good distribution, probably because we rapidly reinflated the lung, forcing air beyond the relatively viscous and small-volume surfactant suspension. In this initial study we did not detect an effect of surfactant on endogenous surfactant metabolism. However, we demonstrated the comparability of 15pm microspheres and radiolabeled DPPC for the measurement of surfactant distribution in small pieces of lung. The distribution of surfactant was significantly improved by use of a larger volume of surfactant suspension and by more gradual reinflation of the lung. Because surfactant distribution was not truly uniform in lo- to 50-mg lung pieces, we must assume a much more nonuniform distribution at the alveolar level. Therefore our conclusions concerning the effects of exogenous surfactant on endogenous surfactant metabolism do not accurately reflect the magnitude of the responsesof individual type II cells. The results do measure the aggregate response of the healthy lung to surfactant and thus form the basis for future studies of the metabolic responses of the injured lung to surfactant treatments. Another source of error in previous experiments was the large variability in radiolabeled precursor incorporation into surfactant phosphatidylcholine (20). To minimize such variability we took advantage of the lobar administration of surfactant by comparing surfactant metabolism in the left and right lung of each animal. However, the collapse, treatment, and reinflation procedure could have altered metabolism in the left lower lobe. We were particularly concerned about pulmonary perfusion because pulmonary blood flow will decrease to atelectatic lung volumes (2, 7). Although transient
EFFECTS
changes in pulmonary blood flow probably occurred, there were no differences detected 30 min after the treatment procedure. This result was important because we used intravascularly injected precursors to compare radiolabeled precursor incorporation into Sat PC in the left lower lobes with that in the right lungs. We used two control groups to evaluate our ability to process the lungs separately and to evaluate the influence of the treatment procedure on the endogenous Sat PC metabolism. We found that alveolar washes of the left lower lobes and right lungs could be carried out comparably and consistently, because the alveolar wash phospholipid-to-tissue protein ratios were similar. The alveolar and lung tissue Sat PC pools were not influenced by the lung collapse and vehicle instillation procedure, because the values were similar in the right lungs and in the left lungs and not different from those measured in unmanipulated controls. Furthermore we found that the incorporation of [“Cl - and [“Hlpalmitic acid into Sat PC in the lung tissue and the secretion of the labeled Sat PC from the left lower lobes were not different between the two control groups. Surfactant containing SP-A decreased surfactant secretion by type II cells in vitro in a dose-dependent fashion (16, 23). The most potent inhibitor of the secretory inhibition was SP-A, although surfactant lipids also seemed to decrease secretion (6). Because SP-A also increased uptake of surfactant by type II cells by what appeared to be a receptor-specific mechanism involving endocytosis into multivesicular bodies (24)) the assumption is that these two effects are linked. We anticipated that administration of natural rabbit surfactant at a dose of -40 mg/kg body wt to the left lower lobe, which is equivalent to 100 mg/kg body wt to the total lung, would decrease secretion because it contained SP-A. However, the results clearly demonstrated an almost threefold increase in secretion of surfactant Sat PC that had been synthesized either 9.5 or 4 h before study. The magnitude of the effect was the same for the two different times. The experimental design did not permit us to evaluate the early clearance of the [““PISat PC associated with the rabbit surfactant so that the linkage of reuptake with secretion could not be evaluated by this protocol. However, the overall effect was a striking increase in the accumulation of endogenously labeled Sat PC in the air spaces. A similar but less striking effect on endogenous surfactant secretion resulted from treatments of 3-dayold rabbits with sheep surfactant (11). Survanta was a less potent stimulus to labeled Sat PC accumulation in the air spaces than was rabbit surfactant. A reasonable speculation is that the absence of SPA has decreased the secretory response, although there are other differences in composition and lipoprotein aggregate forms between the two surfactants (26). The striking difference between these in vivo experiments and the results with type II cells could be caused by alterations in the type II cells during isolation or differences in the metabolic state of the cells within the lung vs. that in tissue culture. The apical secretory surface of type II cells probably is bathed with high concentrations of surfactant in vivo, a situation very different from in
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SURFACTANT
TREATMENT
vitro culture conditions. Perhaps the addition of a lofold excess of SP-A and Sat PC stimulated the cells in situ to increase their overall surfactant metabolic activity, resulting in the measured accumulation of de novo synthesized Sat PC in the air spaces.Large and repetitive doses of exogenous surfactant do increase the overall catabolic rate of surfactant phosphatidylcholine in the lung (20, 21). This concept is consistent with the increased incorporation of labeled palmitic acid into Sat PC in the left lower lobes of rabbit surfactant-treated animals. The incorporation of palmitate does not prove increased synthesis because precursor pools could be altered by the surfactant treatments. Our assumption was that the likely result would be increased palmitate pool sizes resulting from catabolism of the surfactant used for treatment (20) and that the surfactant might feedback inhibit Sat PC synthesis, as demonstrated in vitro in type II cells (6). The result of increased incorporation suggestedincreased metabolic activity by the type II cells in the left lower lobe. However, the nature of that effect cannot be determined from these experiments. The surfactant used for treatment or the catabolic products of that surfactant could selectively influence multiple metabolic pathways within the lung. The lack of effect noted with Survanta could indicate that the incorporation response was a SP-A-specific response or that the 7% by weight of free palmitic acid in Survanta interfered with the measurement by altering palmitic acid pool sizes. The clinical implications of these studies are in general positive. The primary goal of surfactant treatment is to deliver biophysically active surfactant to unstable alveoli. A secondary goal should be to preserve endogen.ous surfa.ctant metabolism. These studies indicate that a treatment dose of rabbit surfactant not only did not inhibit endogenous pathways but also stimulated endogenous surfactant Sat PC metabolism. The results were similar but lessstriking with Survanta. These stimulatory effects could be very helpful to the injured lung to help restore normal surfactant metabolism. This research was supported by National Institute of Child Health and Human Development Grant HD-11932; a grant of the Ter Meulen Fund, Royal Netherlands Academy of Arts and Sciences, to S. B. Oetomo; and Canadian Medical Research Council Fellowship 33703 to
Received
1 September
1989; accepted
in final
form
8 December
1989.
503.
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