276

Riochimica et Biophysics 0 Eisevier/~~rth-Holland

Acta, 573 (1979) Biomedical Press

276-295

BBA 57358

CELL LINE A549 AS A MODEL OF THE TYPE II PNEUMOCYTE PHOSPHOLIPID BIOSYNTHESIS FROM NATIVE AND ORGANOMETALLIC PRECURSORS

LINDA

L. NARDONE

a*b** and S. BRIAN

a Section of Cell Biology and b Department Medicine, New Haven, CT 06510 (U.S.A.) (Received July 31st, 1978) (Revised manuscript received

Key words: Pneumocyte

December

ANDREWS

a

of Anesthesiology,

27th,

Yale University

School

of

1978)

model; P~~~spholi~~d synthesis;

Urg~nomeia~~~c precursor

Summary 1. A549 is a continuous cell line derived from a human pulmonary adenocarcinoma. To evaluate the suitability of this cell line as a model of the type II pneumocyte, the morphology and the composition and biosynthesis of phosphatidylcholine was examined under control culture conditions and during fatty acid supplementation with palmitate. A number of the ultrastructural characteristics of A549 cells were similar to the in situ type II pneumocyte and were unchanged by fatty acid supplemen~tion. The phospholipid composition of the ceil line was similar to that of primary isolates of type II cells in total phosphatidylcholine, disaturated phosphatidylcholine, and palmitate and saturated fatty acid. Phospholipid biosynthetic results were also consistent with those reported for isolated type II cell models. These included: (i) the pattern of incorporation of choline, palmitate and acetate into phosphatidylcholines; {ii) the effect of palmitate supplementation, which resulted in stimulation of the rate of phosphatidylcholine biosynthesis and in increased percentage of labeled precursor in disaturated phosphatidylcholine; and (iii) the preferential synthesis from labeled choline and palmitate of a highly disaturated phosphatidylcholine in short-term incubations. 2. The incorporation of an organomet~lic palmitate analog, lZ,lZ-dimethyl12-stannahexadecanoate, into A549 cell lipids was examined and compared to * Permanent address: Department of Pathology, New York Medical College, Valhalla, NY 10595. U.S.A Abbreviations: Ci 6:~) or 16 : 0, palmitic acid; SnC, 5:” or St115 : 0, 12,12~imethyl-12-stannahexadecanoic acid.

277

that of palmitate. These data demonstrate for the first time the incorporation of an organometallic substrate into the phospholipids of a mammalian cell line. This analog substitutes selectively for the native fatty acid at a rate similar to that of the native fatty acid with no cytotoxic effects. The organotin probe, coupled with spectroscopic detection and electron microscopy, may be useful for examining ultrastructural aspects of phospholipid synthesis, translocation and assembly.

Introduction The cellular heterogeneity of the lung and the difficulties it imposes upon investigation of pulmonary biochemistry and metabolism have prompted the development of in vitro model systems consisting of relatively homogeneous populations of lung cells. The type II pneumocyte is considered to be the cell primarily responsible for the synthesis of surfactant, the major component of which is dipalmitoyl phosphatidylcholine [l-4]. Methods for obtaining preparations of type II cells from normal lung of various species include density gradient centrifugation [k-8], cloning [ 9,101, attachment of dispersed cells to a three-dimensional matrix [ 111, and differential adherence in primary culture [ 12,131. In addition, two tumor models have been reported for the study of surfactant synthesis: a urethane-induced adenoma [14-171 and A549, a continuous cell line derived from a human pulmonary adenocarcinoma [l&19]. Highly purified populations of isolated type II cells from normal lung represent the most physiologically relevant isolated cell system in that these have shortly before been mature, functioning cells in situ. Nevertheless, a continuous cell line that expresses differentiated biochemical characteristics of the type II pneumocyte would be an attractive complementary cell model because of the versatility and convenience that long-term cell culture systems offer. The original report of A549 indicated that this cell line retained in culture the most striking morphological characteristic of the type II pneumocyte, the lamellar body. Like surfactant, lamellar bodies isolated from whole lung have been shown to contain a palmitate-rich phosphatidylcholine [20]. Some aspects of phosphatidylcholine biosynthesis and secretion have also been examined in cell line A549 [13,19,21-231, but these studies have not established a concensus regarding the justified use of A549 as a type II cell model. The presence of phospholipid-rich lamellar inclusions in A549 cells suggested this cell line as a suitable model for further exploring the feasibility of phospholipid labeling with the organotin fatty acid developed in this laboratory [ 241. We had previously demonstrated the incorporation of this formal palmitate analog, 12,12dimethyl-12-stannahexadecanoic acid (A), into the the plasma CH3 CH,(CH2)1gn(cH,)I~COOH

(A)

LH, membrane of the microorganism Acholeplasma laidlawii and had described the detection of this probe by energy-dispersive X-ray microanalysis using a scan-

218

ning electron microscope [24]. Since A. laidlawii employs only limited and simple mechanisms for fatty acid metabolism, experiments with a differentiated mammalian cell, particularly one in which phospholipid metabolism is specialized, would more critically test the ability of this organometallic to substitute for native fatty acids in functional membranes and thus to serve as an ultrastructural probe for phospholipid synthesis and translocation. The present report describes experiments to assess ultrastructural, compositional, and biosynthetic characteristics of A549 under defined cell culture conditions and under conditions of supplementation with palmitate or the organometallic palmitate analog. A portion of this work has appeared previously [25, 261. Methods

and Materials

Cell culture and incubation. The initial culture of cell line A549 was obtained from Dr. A. Gazdar (National Cancer Institute, Bethesda, MD). The cells were received at passage 120 and had been maintained in a Roswell Park Memorial Institute (RPMI) media. Upon receipt, cells were adapted to complete F12K media, which consisted of F12K nutrient media [27] supplemented with 10% fetal calf serum and contained the following final concentrations of antibiotics: penicillin G, 100 units/ml; streptomycin sulfate, 100 pg/ml, and Fungizone, 0.25 pg/ml. All of the above materials were obtained from GIBCo, Grand Island, NY; all fetal calf serum was from the same lot. Cells were cultured in 55-cm2 culture dishes (Falcon, Oxnard, CA) at 37°C in 95% sir/5% CO* in a humidified tissue culture incubator. Mycoplasma-free cultures were plated at a density of approximately lo6 cells/dish and the complete media was changed 24 h later and approximately every other day thereafter. Cultures reached confluence 7-8 days following plating. The cells used in this study were between 20 and 30 passages after adaptation to F12K. At lo-11 days after plating, cultures were incubated for 24 h, under the same conditions as described above for cell culture, in either of three media: (1) Unsupplemented (-IJ) media containing 8.0 ml of complete F12K plus 10% serum, 5 DCi [Me-3H]choline (spec. act. 4.2 Ci/mmol, diluted by media choline (80 PM) to a final activity of 12.5 Ci/mol) and 2.0 ml Krebs-Henseleit bicarbonate [ 28]/2% bovine serum albumin (Pentex Fraction V, fatty acid free, Miles Research Products, Elkhart, IN); (2) Palmitate-supplemented media (- +C,6:o), essentially identical to tl::) unsupplemented media, but containing 3.0 pmol of unlabeled palmitate (Sigma, St. Louis, MO) or ]1-14C]palmitate (spec. act. 10.0 Ci/mol) diluted to a working activity of 1.25 Ci/mol complexed to the albumin according to Rhoades [29] (0.33 mM final concentration) and (3) Organotin-supplemented media (’ +SnCiSZO), similar in composition and concentration (0.30 mM) to the palmitate-supplemented media, but formulated with the organometallic analog as the albumin-bound fatty acid. Throughout the growth and incubation periods for the cultures, the choline concentration was dictated by the culture medium, viz. 80-100 PM, in incorporation experiments the addition of trace amounts of labeled choline did not constitute a supplementation. With regard to fatty acid concentrations, the cell growth media and the incubation media were found to contain 0.07-0.10 mM

279

serum-derived fatty acid (approximately 30% each of palmitate, stearate and oleate) as determined after extraction of free fatty acids by the calorimetric method of Itaya and Ui [30] and by gas-liquid chromatographic analysis of the fatty acid methyl esters as described below. At day 10, the added fatty acid in the palmitate- and organotin fatty acid-supplemented media constituted 7590% of the total exogenous fatty acids. In certain studies 10 &i of [1,2-‘“C,Jacetate (spec. act. 56.4 Ci/mol, final concentration 18 PM) .was added to each culture. All radiochemicals were purchased from New England Nuclear, Boston, MA; the organotin fatty acid was synthesized as previously described [ 241. Incorporation studies of 1 h duration were carried out on cultures grown in 18-cm* dishes as described above. These were of similar cell density to that for 24-h incubations. The cells were rinsed of culture media and preincubated for 30 min at 37°C in 122 mM NaCl, 5 mM KCl, 1.2 mM KH2P04, 1.2 mM MgS04, 0.9 mM CaCl,, and 10 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes), pH 7.4. Subsequently the cultures were rinsed and incubated in a similar salt solution which also contained 5 mM D-glucose, 80 PM [Me-3H]choline (spec. act. 6.2 Ci/mol) and 0.33 mM albumin-bound [l-14C]palmitate (spec. act. 0.6 Ci/mol). Analytical methods. At the termination of incubations, media were decanted and cultures were washed three times with isotonic saline, pH 7.4. Cells were harvested by scraping with a rubber policeman or trypsinization with 0.1% trypsin (Difco 1 : 250, GIBCo) in isotonic salt solution; the method of harvesting did not affect the results. Cell numbers and viability were determined in removed media and trypsinized cell cultures by hemacytometer counting and trypan blue exclusion [31]. Harvested cells were sonicated and aliquots were assayed for protein (bovine serum albumin as standard) by the method of Lowry et al. [32] and DNA content (calf thymus DNA as standard, Sigma) using a fluorescence method based on mithramycin binding [33] as modified by Wagner et al. [34]; separate aliquots were extracted for lipid according to Bligh and Dyer [ 351. Trypsinization harvest allowed three estimates of cell mass: cell number, protein content and DNA content. This provided a correlation of cell number to the other cellular parameters. Thus, data are expressed per 1 * lo6 cells, and have been calculated using an average cell number based on separate determinations of at least two parameters. Lipids were fractionated into lipid classes by silicic acid column chromatography as described by Rooney et al. [36] or into individual phospholipid species by thin-layer chromatography. TLC was carried out on Quantum LQD plates (Quantum Industries, Fairfield, NJ) or on 0.25 mm silica gel 60F-254 (E. Merck, Darmstadt, F.R.G.) using chloroform/methanol/7 M ammonia (60 : 35 : 5, by vol.) as eluent; this solvent system resolved choline-containing phospholipids and phosphatidylethanolamine from other phospholipids. Phospholipids were visualized on chromatograms by iodine vapor or rhodamine G, and were quantitated by inorganic phosphate assay [ 371. Phosphatidylcholine isolated by TLC for subsequent fatty acid analysis or disaturated phosphatidylcholine determination was eluted from the silica gel as described [36]. Phospholipid standards for these procedures were obtained from Serdary, London, Ontario, Canada, or Sigma.

280

Lipid samples for radioactivity determinations were scraped from TLC plates and counted in 1.0 ml water and 10 ml Aquasol (NEN) in a Packard Tri-Carb liquid scintillation spectrometer equipped for double isotope analysis and external standard quench correction. Efficiency and quench corrections were performed on a Hewlett-Packard 9815A programmable calculator interfaced to the scintillation spectrometer. Disaturated phosphatidylcholine was separated by TLC from total phosphatidylcholines following osmium tetroxide treatment as described by Mason et al. [ 381, In each experiment dipalmitoyl phospIlatidylc~loline, dioleoyl phosphatidylcholine, and mixtures of these were used as standards. The fatty acids of isolated phosphatidylcholines were converted to their methyl esters by acid-catalyzed (BF,/methanol, Analabs, North Haven, CT) and base-catalyzed (methanolic base, Supelco, Bellefonte, PA) transesterification as described 139,401. These were analyzed by GLC using a Hewlett-Packard 5830A chromato~aph and a Supelco I/S inch X 6 ft column of 10% DEGS-PS on SO/l00 Supelcoport at 195OC (isothermal) and a flow rate of 25 ml/mm (N2). The esters were identified by comparison of their retention times to authentic standards (Supelco and Analabs) and quantitated as the relative percent corrected for response factors. The organotin methyl ester was quantified by difference analysis as previously described 1241. Proton nuclear magnetic resonance (NMR) spectra (270 MHz, ZO”C, internal *H lock) were obtained on 95%, as determined by trypan blue exclusion. At the point of termination of incubations in unsupplemented media, a small number of cells (2-3 . 104) with decreased viability (50-60%) could be seen freely floating in the media when observed by phase microscopy. In fatty acid-supplemented cultures this number was generally doubled; in all cases, unattached cells represented 70

in amounts

* * Difference ***

by

: 0 **

Percent * * above.

less than

in percent of

total

0.5%.

of palmitate

incorporated

content

SnC15

between

: 0 which

can

+SnCl5

: 0 and

be ascribed

+C16

to palmitate

: 0 conditions. substitution

as defined

in

290

lent to the increase predicted by choline incorporation, namely, 16 nmol/lO’ cells. However, this is less than the incorporation of palmitate determined by isotope techniques, and implies that some proportion of the palmitate must have been introduced by redundant palmitate-palmitate exchange processes. Incubation in organotin-supplemented medium produced analogous changes in the fatty acid composition of cellular phosphatidylcholine. The relative percentage of C,, fatty acids showed a dramatic decline, while all others, including oleate, were unaffected or decreased slightly. Thus, at least 70%, and usually much more than that, of the organotin analog incorporated (18% of total fatty acids) could be ascribed to palmitate and palmitoleate substitution. Alternatively, it can be calculated that 27-30% of the Cl6 fatty acids were replaced by the analog. These experiments described in Table IV were apparently plagued by a variable desaturase activity, the effect of which can be best appreciated by focusing on the C,, fatty acid composition. Although the relative proportion of palmitate varied from one experiment to the next, a compensatory change in palmitoleate content consistently occurred as well. Thus, the composition of the sum of palmitate and palmitoleate could be determined with much improved accuracy relative to the composition of either of the individual fatty acids; the improvement in standard error when the results were calculated as the sum can be seen in Table IV. The same effects appeared to operate to a lesser extent with stearate and oleate. Disaturatecl pllos~hatidylcholirle. The results of analyses for disaturdted phosphatidylcholine are shown in Table V. The disaturated fraction of the cellular phosphatidylcholine of A549 was assayed by radioisotope techniques after growing the cultures to confluence in the continual presence of a trace amount of [Me-“HIcholine. In this situtation, the specific activity of the choline moiety of various molecular species of phosphatidylcholine will be in equilibrium. The cellular content of disaturated phosphatidylcholine was 38%. The percentage of total cellular phosphatidylcholine of A549 which is disaturated has been variously reported as 47% [19], 32-48X [21] and approximately 16% [ 13,231. The present result is generally consistent with the higher literature values and only slightly less than those reported for isolated rabbit type II cells, namely, 49% [6] and 50% [23]. Bascad on [Me-“HIcholine incorporation into the disaturated fraction during the standard 24 h incubation period (Table V), unsupplemented cultures of A549 synthesized a phosphatidylcholinr that was 28% disaturated, and this value was elevated to 37% and 57% upon supplementation with palmitate and the organotin fatty acid, respectively *. The enhancement of disaturated phosphatidylcholine synthesis by addition of saturated fatty acid to the incubation has also been observed for the rabbit type II cell [51]. Based on [l-‘4C]palmitate incorporation, the percent of newly synthesized phosphatidylcholine that was disaturated was 35%. Although certain of these data appear reasonable for a valid type I1 cell model, comparable data for an authenticated type II cell system have not been reported. * The

organometallic

unreactive

towards

analog osmium

is opwationally tetroxide.

defined

in

this

assay

as a saturated

fatty

acid.

since

it is

291 TABLE

V

DISATURATED TYPE II CELLS

PHOSPHATIDYI.&HOLINE

SYNTHESIS

BY

AND

COMPOSITION

OF

A549

AND

Data are percent of total phosphatidylcholine. For the 24 h incubation cells were incubated in either of the three culture media described in Methods and Materials. For the short-term incubation the incubation times were as follows: present study, 75 min; Ref. 51, 1 h: Ref. 52, 0.5-3 h. ___Disaturated phosphatidylcholine

A549 U

Total cellular content Synthesis from radiolabeled lrrecursors: 24 h incubation: Choline Palmitate Short-term incubation: Choline Palmitate

cells ‘Cl6

Type II cells -_

:0

+S*C15

38 *

23

37 35 104 ** 72 *+

:0

Ref. 23

Ref. 51

50

49

Ref. 52

57

52 *** 77

74 *** 75 --__

* A549 was grown to confluence (approx. 4.4 * lo6 cells) in an 18 cm* culture dish over a four day period. The culture medium was the unsupplemented, complete F12K described in Methods and Materials which contained 80 nM choline labeled with a trace amount of [Me3H1choline. ** Confluent cultures of approx. 4.4 . lo6 A549 cells. grown as described, were rinsed of cell culture media and incubated for 1 h in a balanced salt solution described in Methods and Materials. The total time of incorporation was 75 min, since an additional 15 min was required to harvest and sonicate the cells prior to lipid extraction. *** This value should be compared to the data for the CC19 : 0 condition of the present study.

The difference in the time course of choline and p~mitate incorporation (Figs. 4 and 5) suggested the advisabihty of examining disaturated phosphatidylcholine synthesis during short-term culture. Thus, preliminary, duplicate experiments were carried out by incubation of A549 cultures in a simple and defined medium for 75 min as indicated in Table V. These conditions were as close as possible to those employed by others for studying phosphatidyl~lloline synthesis in primary cultures of type II cells [ 51,523. As assayed by [l-14C]palmitate incorporation, 72% of the radioactivity was found in disaturated phosphatidylcholine of A549 and this is in excellent agreement with the corresponding results for type II cells. The incorporation of choline-derived radioactivity into the disaturated species of phosphatidylcholine was essentially quantitative under these conditions. Although the three experimental models of the type II cell differ somewhat in this last regard, all synthesize a notably high proportion of disaturated phosphatidylcholine. No significance can be attached to these differences in choline incorporation in the absence of information on the relative condition of intracellular choline pools immediately prior to incubation. Discussion The present experiments indicate that the A549 tumor cell line used in this study is faithful model of the pulmonary type II cell in at least five respects, (1) The general ultrastructure of cultured A549 is very similar to that of the

in situ type II 1)ncumocyte, and this comparison is particularly striking with respect to the lamellar inclusions. (2) The total and disaturated phosphatidylcholine coI~~I~(~sitio~lof the two cell types is much the same. (3) The 1)1l~)s~~hatidyl(~~~oli~le of 12539 contains at least as much palmitate and total sat.urated fatty acids as the phosphatidylcholine of the isolated type II cell. (4) The ljattern of incorporation of 1)hos1,hatidylcholille precursors was similar for both (~11 types. as c*onsideretl in more detail in the following paragraph. (5) The proportion of tlisaturated phosphaticiyl(.hoIine synthesized from [ .Sle-31-f] choline and [l-‘4f: Jpal~~~itate is consistent with values reported for type II cells, particularly in short-term culture 151,523. Additionally, two other pieces of supllorting evidence are available. (6) Preliminary experiments have shown that the phospholipids of the isolated and purified lamellar inclusions of A549 contain 56% 1)hos1~hatidylcl~oli~~e~ of which 39% is disaturated (Nardonc, L.L., unpublisheti dal,u). (7) :\549 rclt>ases a ~lisat~~ra~e~1l,h”sliftatidylcl_1oline in response t.o the calcbium ionophore ‘223187 1221; isolated type II ~11s do likewise [ 531. :Zs indicated in Ijoint (4 j above, the results of the present experiments cornl)arc? reasonably well with the recent biochemical data obtained using primary cultures of isolated type II cells from rabbit [ 511 and rat [ 521. The three aforementioned invest.igations employed cells from different species and the cells were incubated under differeIlt conditions; however, the relative a~lo~~nts of choline, l)alnlitat~~ and acetate it~(~~)r1~oratedby these cell types is very similar, and thcl absolute incorporation rates for 125-19 fall squarely between the values palmitate incureported for the two isolated type II (bell systems. Additionally, bation reproduces in A549 the st,imulating effect on choline incorporation found for both tyI)c 1I c*ells and 011 disaturdted 1>hosphatidylchuline synthesis found for t,he rabbit mode1 [51]. Two features of the A549 celi line examined in these studies cannot presently be (*orrelated with known characteristics of type II cells. (1) As originally reported I,y Lieher et, al. 1191, the population of lamellar inclusions in A549 is hett>rogeneous. The reason for this heterogeneity is not known, although the possibility that these ar-e merely different maturational states has not been excfuded. (2) While the coi~~nlitl~~e~t of ,I549 to disat‘urated r~liosl~~lati~1ylcholine synthesis during short-term incubations correlates well with the behavior of type 11 cell systems, the proportion of labeled disaturated phosphatidylcholinc at 24 h was much 1owerJhan the 1 h values. It is possible that this reflects an ccIuilibrium condition for phospholipid components of type II 1)~~~~~l~~locyt~s maintained in culture; ~,~)I~l~arable data from long-term cultures of isolated 1~~leuI~l~~cyteswill be necessary to corroborate this point. Additionally, the apparent proclivity of A519 to convert a substantial proportion of its palmitate to palmitoleate may partially account for this observation. Regarding the biochemical pathways of phosphatidylcholine synthesis in 11549 cells, these studies indicate that c’lloiine and palmitate are incorporated by different, l~~ec1ia~lisrns sintc3 the overall amounts (Tables I and II) and the time dependen~~e of incorporation of the two precursors (Figs. 4 and 5) differ subst.antially. The most attracative c>splanation would invoke one of the documcntrd transesterification mec*hanisms (54,551 to account for at least 50% of the pahnitate incorporation. Other workers have considered this possibility repeatedly (for a review, SW Ref. 3) but only recently has evidence for this

293

been presented for a homogeneous, whole-cell lung system [ 511. Although A549 appears to be capable of de novo fatty acid biosynthesis, as indicated by the incorporation of acetate into phospholipids, exogenous palmitate seems to be a preferred substrate. Furthermore, the high metabolic activity of palmitate is distinctly localized in the phosphatidylcholine pool, and this result cannot be accounted for by heterogeneous metabolic rates for different lipid classes since the preference of palmitate for phosphatidylcholine persists throughout the 24 h incubation. Lastly, the time course of choline incorporation is most straightforwardly interpreted to suggest that this cell type contains a kinetically significant intracellular choline pool. Verification of this point would be of assistance in designing and interpreting biosynthetic studies on type II cells employing choline as a tracer. The experiments in which cultures of A549 were supplemented with the organotin fatty acid demonstrate that this probe is metabolized in a fashion quite analogous to palmitate. The organometallic analog, when incorporated into cellular phosphatidylcholine at levels approaching 20%, exhibited insignificant cytotoxicity and did not alter the ultrastructure of the cells, particularly the cell membranes. Biochemically, the organotin substrate closely paralleled the behavior of palmitate; the absolute rate of utilization of this analog was similar to palmitate, and the organometallic also stimulated the incorporation of choline into total and disaturated phosphatidylcholine. It is interesting that the organotin fatty acid was in fact more active than palmitate in these respects. The organometallic analog showed a high selectivity for palmitate substitution in these experiments, although the A549 cell system may well be grossly biased toward this result. Nevertheless, at least one of two conclusions must be correct: either the organometallic fatty acid prefers to substitute for palmitate or A549 cells selectively metabolize palmitate. In the latter case, rapid turnover of palmitate could result incidentally in significant organotin labeling. In further analogy to palmitate, the organotin fatty acid appeared to substitute for palmitoleate with about the same efficiency that palmitate displayed as a precursor for this fatty acid. The evident difference is that the analog was not a substrate for desaturation. The ability of this compound to serve as a substrate for some enzymes but not others could possibly be exploited in experiments designed to specifically modify the fatty acid composition of cellular lipids. This study offers two general conclusions. The results suggest that A549 deserves serious consideration as a useful model for surfactant biosynthesis and, more specifically, as a model for the isolated type II pneumocyte. Due to the easy manipulation and reproducibility of A549, this cell line might be particularly useful for ‘screening’ experimental prctocols and ideas for preliminary, suggestive results before investing the substantially greater time and effort necessary to carry out similar studies on isolated type II cells. The second conclusion concerns the biochemical compatibility of the organometallic fatty acid. The A549 system is an informative cell model in which to test a fatty acid analog since fatty acid metabolism is so distinctive in these cells. In this context, the data presented here demonstrate that the organotin analog is a biochemically acceptable substrate and provide the foundation for testing this fatty acid as an ultrastructural probe. Electron micro-

294

scopy of tissues labeled with this probe, coupled with analytical methods such as X-ray microanalysis or scattering analysis of high-resolution images potentially provides a unique qualitative and quantitative strategy for approaching the problems of phospholipid and membrane synthesis, modification and assembly. Acknowledgements The authors are grateful to N. Zaehringer for expert technical assistance, to Dr. R.J. Bamnett for his substantial financial and moral support and to Dr. I.M. Armitage for assistance in obtaining 270 MHz NMR spectra. This work was supported by USPHS Grants HL-19591 and AM-03688 and by American Cancer Society Grant ACS-IN-31-Q-11. The authors also acknowledge the American Lung Association, The Charles Hood Foundation and the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research. NMR spectra were obtained on an instrument of the Southern New England High Field NMR Facility, which is supported by NIH Grant l-P07-PRO0798 from the Division of Research Resources. References King.

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