Reutilization of surfactant phosphatidylcholine in adult rabbits

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Eiochimica et Biophjka Acta 837 (1985) 71-84 Elsevier BBA 52037 R~utili~ati~~ of surfactant ph~sphatidylchoIi~~ in adult rabbits Harris C. Jacobs *, Machiko Ikegami, Alan H. Jabe, David D. Berry and Sally Jones Perinatal Research Laboratories, Harbw UCLA Medical Center, UCLA School of Medicine. 1000 W. Corson Street. Torrance, CA 90509 (U. S. A.) (Received December 13th, 1984) (Revised manuscript received July 4th. 1985) 32P-s41turaed pb~~at~dy~~~iine was added to ~3H~~ho~~I~~~a~~~ natural surfaetmt and the mixture was injected intratracheahy into 87 adult rabbits. The rabbits were also given I %fpalmitate ~o~avenousty at the same time, Rabbits were killed in groups from 10 min to 72 h after injection. Iln each rabbit we measured the total recovered j3H]phosphatidylcholine (PC) in the alveolar wash, the ratio of j3H]PC to j3PjPC in the alveolar wash, and the specific activity of [ C]PC in the alveolar wash and lameltar bodies. Values were averaged for all rabbits killed at the same times and smooth curves were fit to the data by computer, From the intravenous [ C]palmitate data we calculated a turnover time for alveolar PC of 6.0 h. From the intratracheal labeling data, we calculated a turnover time for alveolar PC of 5.7 h and determined that alveolar PC was reutilized at an efficiency of only 23%. We also concluded that this reutilization occurred as intact molecules. fntroduction Reutilization of pulmonary surfactant by the type II cell has been directly or indirectly ad- dressed in several studies using developing and mature animals [l-6]. Studies done by injecting radiolabeled precursors of surfactant in- travenously into developing and mature animals have provided estimates of the rate of secretion of surfactant phosphatidylchol~e into the alveolar space [2,6,7]. From these studies. it was possible to show that reutilization of surfactant phosphati- dylcholine must occur, but no quantitative esti- mates could be made [2]. Other studies have as- sessed the clearance of surfactant from the airways by administering some form of radiolabeled * To whom correspondence should be addressed at: Yale University School of Medicine, Division of Perinataf Medi- cine. Department af Pediatrics, P.O. Box 3333. New Haven, CT 06510. II.S.A. surfactant into the airways of laboratory animals [1,3,5,8-111. Most of these studies only provided qualitative information about the clearance of re- utilization of surfactant. An estimation of the quantitative significance of reutilization is vital to an understanding of normal function of the pulmonary surfactant system. We previously re- ported that developing rabbits reutilized surfactant phosphatidylcholine at greater than 90% efficiency f3f. The study reported here used the same meth- odology to estimate the rate of reutilization of surfactant phosphatidylcholille by mature rabbits, Methods Animals 87 adult female New Zealand White rabbits weighing 1.16 -t_ 0.016 kg (mean + S.E.) were used in the experiment. The rabbits were allowed free access to both food and water for the duration of the study and all were healthy at the time of killing. ~0~~-~?6~/~j/~~3.3~ 8 1985 Elsevier Science Publishers B.V, (Biomedieat Division) 7x Each rabbit was injected intratracheaily with a mixture of [~~z~~~~~-~H]choline-labeled natural surfactant and P-saturated phosphatidylcholine. [ HJCholine-labeled natural surfactant was pre- pared by methods reported previously [I 21. [ H]Methyl iodide (2 Ci/mmol. ICN) was used to synthesize choline by methylating unlabeled ethanolamine (Aldrich) according to the procedure of Smith et al. [13]. By using free ethanolamine rather than phosphatidylethanolamine we pm- duced free choline as the final product. Choline was separated from reactants and by-products by thin-layer chromatography with chloroform/ methanol/acetic acid/water (65 : 25 : 8 : 4. v/v) as the solvent on silica gel H TLC plates using un- labeled compounds as standards (Aldrich and Sigma). The [HIcholine had a final specific activ- ity of 1 Ci/mmol. We injected each of eight 3-day- old rabbits via the trachea with 1 mCi of [ H]choline dissolved in a 1 : 1 mixture of Lactated Ringers Inj. (Travenol) and distilled water [12]. The trachea was exposed through a midline neck incision under local anesthesia and the choline solution was injected into the distal trachea via a 30 gauge needle while occluding the trachea prox- imally with fine forceps. About 40 h later. the rabbits were killed and their surfactant was col- lected by an alveolar wash procedure followed by several centrifugation steps [14]. Over 90qJ of the lipid soluble radioactivity in this surfactant was in phosphatidylcholine. 20 mCi of carrier free [~~P]orthophosphate (ICN) were diluted into a 1 : 1 mixture of Lactated Ringers Inj. and distilled water and injected into the airways of four 3-day-old rabbits via the trachea as above. The rabbits were killed 40 h later and the lungs were removed and homogenized in 0.15 M saline. The homogenate was extracted once with 3 volumes of 2 : 1 chloroform/methanol and the chloroform phase was collected. P-saturated PC with a specific activity of 11 mCi/mmol saturated PC was isolated from the lipid extract according to Mason et al. [15]. The P-saturated PC was suspended in Lactated Ringers Inj. by sonication for four I-min cycles using the large probe of a Fisher Sonic Dismembrator (Model 300) at 60% maximum out- put. This was mixed with the H-labeled natural surfactant. vortexed and centrifuged for 70 min at 14000 x R. The pellet XX suspended in a 1 : 1 mixture of Lactated Ringers Inj. and distilled water. The final concentration of this solution w;is 0.45 pmol phosphatidylcholine/ml and contained 483 000 counts/min of [ H]phosphatidqloholine and 187000 counts/min of P-saturated PC,imI of solution. [ C]Palmitic acid (52 mCi/mmol) w\as purchased from New England Nuclear as the free acid and complexed to bovine serum albumin as before [16]. This was used to prepare a solution for intravenous injection which contained [l-C.]- palmitic acid at a concentration of 50 ;~C.i~~ml. All rabbits were injected with both solutions. Each rabbit was anesthetized with diethyl ether and held on its back with anesthesia maintained by use of a small beaker containing ether soaked paper towels. A 14 gauge needle was percuta- neously inserted into the traceal lumen pointing distally. A 3.5 Fr. polyeth3flene catheter was passed through &he needle into the distal airways until resistance was met. The catheter was withdrawn about 0.5 cm and 1 ml of the intratracheal injec- tion solution was given through the catheter after which the needle and catheter were withdrawn together. Preliminary injections in three rabbits containing india ink rather than isotope demon- strated a segmental distribution of the injection solution with staining of the airways and parenchyma out to the pleural surface. While re- covering from the anesthesia, each rabbit was in- jected with 0.6 ml of the ~14C]palmitate solution via a marginal ear vein. The entire procedure took about 2--3 mm/rabbit. Frwtion isolution Rabbits were killed in groups of five or six at one of fifteen predetermined times from 10 min to 72 h after injection which were chosen to maxi- mize accuracy in the calculation of percent reutili- zation of surfactant phosphatidylcholitle. Each rabbit was killed by an overdose of pentobarbital followed by exsanguination. The chest was opened, the trachea cannulated and the lungs were filled to total lung capacity with 0.15 M saline at room temperature. This saline was rinsed in and out 19 three times with as much as possible withdrawn the third time. The recovered saline was placed in a graduated cylinder and the process was repeated four more times with fresh saline. The final volume was recorded and an aliquot was stored at - 20C for further analysis. These samples, subsequently referred to as alveolar washes, contain more than 90% of the surfactant recoverable by a wash proce- dure [17]. The washed lungs were weighed and homogenized using the large probe of a Tekmar Ultra Turrex homogenizer at 30% maximum speed in 25 ml of 0.32 M sucrose, 0.01 M Tris-HCl, 0.15 M NaCl, 0.001 M CaCl,, 0.001 M MgSO,, and 0.0001 M EDTA at pH 7.4. 2 ml of the homo- genate were stored at -20C for further analysis and the remainder was used to isolate a lamellar body fraction. This was done by a series of dif- ferential and sucrose density gradient centrifuga- tions [16]. The lamellar body fractions was that fraction which was isolated between 0.45 and 0.55 M sucrose. Phospholipid ana!vsis Lipids in the alveolar wash, lamellar body and lung homogenate samples, as well as those in a sample of the intratracheal injection solution were extracted according to Folch et al. [18], and con- centrated under N, at 50C. Phosphatidylcholine was isolated in duplicate from each sample by one-dimensional thin-layer chromatography [2]. One spot was assayed for phosphate according to Bartlett [19] and the duplicate for radioactivity in Aquasol- scintillation fluid (New England Nuclear). All 3H, 14C and 32P counts/min were corrected for cross-channel contamination using pure standards. The standards were also added to several blanks and samples which were then re- counted to insure that quenching did not occur. From the known dilution at each step, we calcu- lated the total [ 3H]phosphatidylcholine counts/ min in the alveolar wash and lung homogenate for each rabbit. Specific activity of [4C]phosphati- dylcholine was measured in the alveolar wash, lung homogenate and lamellar body fractions for each rabbit and was expressed as counts/min per pmol phosphatidylchoiine. Data analysis The two compartment model shown below was used for all calculations [2,3,6,7]. The flux of surfactant phosphatidylcholine from lamellar bod- ies into the alveolar space was calculated from the intravenous labeling data. Intratracheal labeling data allowed the calculation of all fluxes repre- sented by the arrows in the figure and of the lamellar body pool size [3]. Intravenous label The mean [4C]phosphatidylcholine specific ac- tivity in the lung homogenate versus time after injection was plotted on a semilog axis versus the time after injection. The best fit line to the data was obtained by linear least-squares regression and was used to predict lung homogenate specific activity at each time of death. The alveolar wash and lamellar body specific activity for each rabbit was then corrected by multiplying these values by the ratio of predicted lung homogenate specific activity to measured lung homogenate specific ac- tivity [20]. For the rabbits at each time of killing the mean corrected [I4 Clphosphatidylcholine specific activity in the alveolar wash and the lamel- lar bodies was calculated. Mean values for the alveolar wash and lamellar bodies for the five or six animals killed at each time were then curve-fitted by computer to equations whose forms were sums of exponentials by a process of successive least-squares approximations [3,21]. The area between these two curves from t = 0 to 19 different times after injection was calculated and plotted against the specific activity of the alveolar wash at each time. The slope of the best fit line to these points is a measure of the turnover time of alveolar surfactant [22]. (The turnover time is the time required to fill the alveolar space with surfactant if it were empty.) This number was used along with the measured pool size of alveolar surfactant to calculate the flux of phosphati- dylcholine from lamellar bodies into the alveolar space. Intratracheal label Total [ 3 Hlphosphatidylcholine counts/mm (al- veolar wash + lung homogenate) were obtained for each rabbit killed between 10 min and 24 h after injection. The mean values for rabbits killed at each time were fit to curves as described above, except that each mean was weighted by l/(S.E.M.) [21]. From the describing equation, 80 we calculated expected total [ 3 Hlphosphatidylcho- line counts/min at each time. Total counts/min in the alveolar wash of each of these rabbits was corrected by multiplying the measured value by the ratio of predicted total lung counts/min to measured total lung count/min. The mean cor- rected total alveolar wash counts/min were curve-fitted as described above, again using l/(S.E.M.) as a weighting factor for each mean [21]. The derived equation was used to calculate the fluxes of phosphatidylcholine represented by the arrows in the following two compartment model : Lamellar bodies and the alveolar wash are as- sumed to be two distinct and well-mixed compart- ments. The arrows represent the proposed direc- tions of flux of surfactant phosphatidylcholine. The method was that of Skinner et al. [23] as applied previously to surfactant metabolism [3]. The ratio of [ 3H]phosphatidylcholine counts/ min to [ 32P]phosphatidylcholine counts/min in the alveolar wash of each rabbit killed between 10 min and 24 h was calculated. These ratios were used to determine the metabolic behavior of the added 32 P-disaturated PC relative to the [ 3 H]phos- phatidylcholine of natural surfactant. Results Intravenous labeling Curves of corrected specific activity versus time for [ C]phosphatidylcholine are shown in Fig. 1 A for lamellar bodies and the alveolar wash. As reported previously by ourselves and others, the specific activity rises first in lamellar bodies and subsequently in the alveolar wash [2.6,7.16], and the two curves cross at the peak of the alveolar wash specific activity curve. This is consistent with a precursor-product relationship between lamellar body and alveolar surfactant phosphatidylcholine [22]. The area between the lamellar body and alveolar wash specific activity time curves versus the alveolar wash specific activity was plotted and fit by linear regression (Fig. 1B). The slope of this line gives an alveolar surfactant turnover time of 6 0 15 30 35 60 75 A HOURS 01 0 B I 2 3 PC SP ACT x 10-l Fig. 1. Specific activity-time curves and area plot from in- travenous [t4C]palmitate. (A) Mean + S.E. specific activity in the alveolar wash (0) and in lamellar bodies (0) for rabbits killed at the indicated times. Error bars not shown fall withtn the data point in this and subsequent figures. Included curves are computer generated smooth approximations to the means. (B) Each point represents the area between the curves for lamellar body specific activity and alveolar wash specific activ- ity shown in part A from f = 0 to 19-times after injection plotted against the alveolar wash specific activity at that time. The included tine was determined by linear least-squares regression on the point and has a slope of 6.0 h which is the turnover time for alveolar surfactant. h. The points in this figure do not fall on a single line which would be expected for a precursor- product relationship. This was due to the times of killing (see Discussion). Intratracheal labeling The mean of the corrected total [Hlphos- phatidylcholine counts/min in the alveolar wash for each group was fitted by computer to an equation whose form was a linear sum of exponen- tials (Fig. 2). For this equation and the measured alveolar wash pool size of 11.25 + 0.64 pmol of phosphatidylcholine, we calculated the values given 81 9 0 ; 9 b Y ,r 0 0 5 IO I5 20 25 HOURS Fig. 2. Total [ 3 Hlphosphatidylcholine counts/min recovered in the alveolar wash. Each point represents the mean f SE. of the total recovered [ Hlphosphatidylchdine counts/min in the al- veolar wash for rabbits killed at the indicated times. The included curve was determined by computer and is described by the equation y = 807716 e-19 + 98642 e-0.24 + 35 258 e.- 0 067, in the table and the pool size of lamellar body phosphatidylcholine [3]. The calculated turnover time of alveolar phosphatidylcholine and the calculation of the flux of surfactant phosphati- dylcholine from lamellar bodies into the alveolar space were similar for the intratracheal and in- travenous labeling experiments. This suggests that all the values are reasonable estimates. The lamel- lar body phosphatidylcholine pool size in these rabbits was 60% larger than the measured alveolar wash phosphatidylcholine pool size. The saturated phosphatidylcholine which was 5 n * 1 0. 2 \ ,I 3. P - ?i 0 2 c Q CL 1 ! 0 5 10 15 20 25 HOURS Fig. 3. Ratio of [HI- to [ P]phosphatidylchohne counts/min in the alveolar wash. Each point represents the mean f SE. of the rate of [ 3 HI- to [ 32 PJphosphatidylcholine counts/min in the alveolar wash for all rabbits killed at the indicated times. The line was determined by linear least-squares regression on the means and has a slope which does not differ from 0 (P > 0.05). mixed with the natural surfactant prior to injec- tion was labeled with 32P. We assessed the ratio of [ 3 Hlphosphatidylcholine to [ 32 Plphosphatidylcho- line two ways. A linear regression analysis on the mean of this ratio for all rabbits killed at each time versus the time after injection produced a line with a slope which was not different from 0 (P > 0.05) (Fig. 3). We also compared this ratio for rabbits killed at each time to the same ratio mea- sured in the injection solution in triplicate by an unpaired Students t-test; no differences were found. Thus, the saturated phosphatidylcholine mixed with the natural surfactant behaved in a fashion which was indistinguishable from the phosphatidylcholine of the natural surfactant and indicated that reutilization of surfactant phos- phatidylcholine in adult rabbits occurs as intact molecules. Discussion The calculations reported here are based on the model shown above. This model assumes that lamellar body phos- phatidylcholine is the precursor for alveolar TABLE I CALCULATED VALUES FROM KINETIC DATA BASED ON THE TWO-COMPARTMENT MODEL These were values calculated from the curves of specific activity versus time (Fig 1A) or the curve of total recovered counts/mm versus time (Fig. 2). The percent reutilization is the flux from alveolar space into lamellar bodies divided by the flux from lamellar bodies into the alveolar space. Results are *SE. Intratracheal Intravenous 3H natural [4C]palmitic surfactant acid PC flux-lamellar bodies into alveolar space (pmol/h) 1.97 f 1.85 1.88 PC flux-alveolar space into lamellar bodies ( c mol/h) 0.45 f 0.94 - Rate of new synthesis ( P mol/h) 1.42+0.80 - Alveolar wash PC l turnover time (h) 5.7 +5.1 6.0 PC reutilization (%) 23.0 f3.2 _ 82 surfactant phosphatidylcholine and that the in- tratracheally injected label functions as a true tracer. That is, once injected, labeled surfactant molecules are indistinguishable from endogenously produced surfactant. It also assumes that instanta- neous mixing occurs locally within both compart- ments (see below). Studies using a variety of tech- niques have been published which support the assumption of a precursor-product relationship [2,6,24-291. While the assumption of instanta- neous mixing is not strictly accurate, the slight differences found between the specific activities in various fractions of lamellar bodies and alveolar wash are not very large (4,301. Hence, the results obtained under this assumption are reasonable approximations. The accuracy of our calculations also depended on our ability to separate the lamellar body and alveolar compartments by the isolation techniques used and on uniformity in surfactant kinetics throughout the lung. The isolation techniques used in this study were identical to those used in a previous study specifically designed to test the precursor-product relationship between lamellar body and alveolar surfactant phosphatidylcholine [Z]. That study, which confirmed this relationship, detected no significant confounding of pools dur- ing isolation. Thus, the assumption that these two pools were separable in the present study is rea- sonable. Similarly, the study referred to above would not have been consistent with a precursor- product relationship had surfactant phosphati- dylcholine turnover time varied over different parts of the lung. This supports our assumption of uni- formity of surfactant kinetics throughout the lung. The intratracheal injection solution was not uniformly distributed in these animals. However, if our assumption about uniformity in surfactant kinetics in different part of the lung is valid, then it can be argued that the segmental distribution of the injection solution would not effect our results. The endogenous pool of alveolar surfactant is ob- viously distributed throughout the lung. If one considers progressively larger segments of the total lung, the pool size of alveolar surfactant contained within those segments also increases. Uniforin surfactant kinetics throughout the lung means that, independent of the amount of lung considered, the alveolar surfactant contained within that volume of lung will have the same fractional clearance and reutilization as the alveolar surfactant in any other segment and the same as for the lung taken as a whole. Thus, at a given time after injection of radiolabeled surfactant. the fraction cleared from the lung and the fraction contained within various compartments related to surfactant metabolism (alveolar space and lamellar bodies) would be in- dependent of the distribution of the injection. As mentioned, the proposed model had been confirmed in a previous study [ZJ. We elected to kiil rabbits at times which would maximize accu- racy in the calculation of percent reutilization of surfactant phosphatidylcholine rather than at times which would reconfirm the proposed model. This necessitated the choice of many early times of killing. In fact, half the rabbits were killed within 2 h of injection, a time when alveolar wash specific activity from intravenously injected [C]palmitate was still very low. Furthermore. the peak specific activity in the alveolar wash was poorly defined because of the wide spacing of points beyond 2 h: the peak of the alveolar wash specific activity can be localized only to the time interval between 6 and 24 h after injection. Thus, the accuracy of these data for the calculation of a turnover time, which depends on the area under this curve. is limited. It should also be noted that the points in Fig. 1 B appear to define smooth lines because they were derived from the smooth curves fit to the alveolar wash and lamellar body specific activities (Fig. 1A). The estimated turnover time is the same if the actual mean values are used rather than the defined curves, but the random error would not have been eliminated. When mean values are used. there is still a tendency toward two separate lines as shown in Fig. 1B rather than all the points falling on one line. This is due to the reasons given above. These data are included along with the estimated turnover time. since they provide a rough internal standard to compare with the results from the intratracheal labeling. They were not intended to support or refute the proposed model nor are they adequate for this purpose. This study confirms the results of a previous study which indirectly indicated that surfactant phosphatidylcholine was reutilized by adult rab- bits 121. However, this is the first study designed to quantitate that reutilization. The reutilization rate 83 of 23 ~fr 3% is much less than that measured in 3-day-old rabbits where the value was about 95% 131. We feel that the value obtained in the present study for percent reutilization is a reasonable estimate, since the turnover time of alveolar phos- phatidylcholine calculated from data obtained from the intratracheal injections is close to the value calculated from data obtained from the in- travenous injection (5.7 h vs. 6 h). The turnover time of alveolar phosphatidylcholine in adult rab- bits is in the range measured by us previously [2] and in the range of values reported by Baritussio et al. [6]. The pool size of lamellar body phosphati- dyicholine calculated in this study contrasts with previous results obtained in 3-day-old rabbits, in adult rats and in adult rabbits where the lamellar body phosphatidylcholine pool size was de- termined to be 20-80% less than the measured alveolar wash phosphatidylcholine pool size [1,3,7]. The techniques used in the studies on adult animals (including this one) were all different, making further comparison between the results difficult. In calculating the magnitude of the fluxes in- dicated by the arrows in the two-compartment model, we only used data from the intratracheal labeling for rabbits killed between 10 min and 24 h. We expected to find a reutilization rate of about 80-90% [3,4]. The lower reutilization rate found combined with the percentage of the injections solution which reached the alveoli precluded accu- rate measurements of total [ 3H]phosphatidylcho- line in the alveolar wash by 36 h after injection (about 6 turnover time). This study demonstrates a distinct deveiopmen- tal change in the kinetics of surfactant secretion and reutilization with increasing age. Rabbits in- crease their pool of alveolar surfactant from about 1 ymoi at the time of birth (311 to about 6 ymol by 3 days of age [2,3]. This 6-fold increase occurs while the animal only increases in body weight and lung weight less than 2-fold [32]. Since new- born rabbits must be able to ventilate adequately to survive, it is not clear why such a large change in the surfactant pool size relative to growth oc- curs. This is even more confusing in the light of the changes which occur from 3 days of age to maturity when body weight and lung weight in- crease over IO-fold, while the alveolar surfactant pool size only doubles. Furthermore, the 3-day-old rabbit appears to maintain its alveolar pool of phosphatidylcholine by a very efficient reutihza- tion process [3]. The ability of the rabbit to sustain this reutilization diminishes somewhere between a few days of age and maturity about 3-fold. All of this is occurring while the rate at which alveolar surfactant is turned over is increasing (shorter turnover time). These developmental changes seem paradoxical and may reflect as yet unidentified properties of surfactant. Awareness of their ex- istence is important, since the different ways in which developing and adult rabbits maintain a functional pool of surfactant have implications when one tests various inte~entions for their ef- fect on the surfactant pool. For example, let us assume that the mechanism of reutilization is the same for developing and adult rabbits. Then a drug which effectively blocks reutilization may have little effect on the adult rabbit. The develop- ing rabbit, however, would potentially be required to dramatically increase new synthesis to maintain an adequate pool of surfactant. Newborn lambs at a few days of age also have a large pool size of surfactant/kg body weight com- pared to adult sheep 111,141. Glatz et al. [ll], after giving newborn lambs a radiolabeled surfactant intratracheally, found that the radiolabeled surfactant left the alveoli very slowly. While they did not quantitate the degree of reutilization, their data were consistent with a reutilization process that was very efficient. If this is in fact the case, then the developmental changes we have demon- strated in rabbits may well extend to other species, including humans. This work was supported by NIH Grant HD- 11932 from Child Health and Development, De- partment of Health and Human Services, and NIH Research Service Award HL-06544 to H.J. We would also like to thank Dr. Elliot Landaw, with whom we had extensive discussions about the method of analysis. References 1 Hallman, M., Epstein. B.L. and Gluck, L. (1981) J. Chin. Invest. 68, 742-751 84 2 Jacobs, H.C., Jobe, A.H.. Ikegami, M. and Jones, S. (1982) J. Biol. Chem. 257, 180551810 3 Jacobs, H.C.. Jobe. A.H., Ikegami, M. and Conaway. D. (1983) J. Biol. Chem. 258, 4159-4165 4 Magoon, M.W., Wright, J.R., Baritussio, A.. Williams. M.C.. Goerke. J.. Benson, B.J., Hamilton, R.L. and Clements. J.A. (1983) Biochim. Biophys. 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