Involvement of Protein Kinase C in Pulmonary Surfactant Secretion ...

THEJOURNALOF BIOLOGICAL CHEMISTRY

Vol. 260, No.23, Issue of October 15,pp. 12725-12729 1985in 6 S . A . Printed

Involvement of Protein KinaseC in Pulmonary Surfactant Secretion from Alveolar Type I1 Cells* (Received for publication, March 4,1985)

Kimihiko Sanos, Dennis R. Voelker, and Robert J. Mason From the Pulmonary Division, Department of Medicine, National JewishHospital and Research CenterlNationul Asthma Center, Denver, Co6rado 80206

The purpose of this study is to clarify the involve- see Refs. 1 and 2). Isolation and primary culture of alveolar ment of protein kinase C in pulmonary surfactant se- type I1 cells permit examinationof the effect of various agents cretion from adult rat alveolar type I1 cells in primary on surfactant secretion in vitro (3, 4). At least two different culture. Surfactant secretion in vitro is stimulated by types of secretagogues have been shown to stimulate surfacat least two classes of compounds. One class,(e.g. ter- tant secretion in vitro. One group of agonists consists of cyclic butaline) increases intracellular cyclic AMP, whereas AMP-elevating agents, such as p-adrenergic agonists (5-7), theother class (e.g. 12-0-tetradecanoylphorbol 13- forskolin (8), and cholera toxin (9), and the other is tumor the acetate (TPA)) does not. TPA has been shown to acti- promotor TPA’ (10). The precise mechanism of action of vate protein kinase C in other cell systems. In our (OAG), which is TPA is unknown, but recent evidence suggests that protein studies, 1-oleoyl-2-acetyl-sn-glycerol a direct activator of protein kinase C, stimulated [3H] kinase C is one of the intracellular targets of TPA (for a phosphatidylcholine secretion by alveolar type I1 cells review, see Ref. 11). TPA was shown to directly activate in a dose- and time-dependent manner. Tetracaine, protein kinase C (12), and itsbinding to protein kinase C was which is an inhibitor of protein kinase C, inhibited the shown to be Ca2+- and phosphatidylserine-dependent (13). TPA-induced secretion of [SH]phosphatidylcholine The role of protein kinase C in cellular functions has been from alveolar type I1 cells in a dose-dependent manner. extensively studied with platelets (12,14,15). Tetracaine and However, tetracaine had no effect on terbutaline-in- other amphiphilic cationic drugs inhibitprotein kinase C duced secretion. The effects of terbutaline and OAG activity in vitro by competing with phospholipids that are upon surfactant secretion were significantly more than essential to protein kinase C activity. These drugs have no additive, but those of TPA and OAG were less than effect upon cyclic AMP- or cyclic GMP-dependent protein additive. The specific activity of protein kinase C was kinases (16). Synthetic 1-oleoyl-2-acetyl-sn-glycerol(OAG) 6-fold higher than cyclic AMP-dependent protein kinase found in type I1 cells when both kinases were has been shown to be intercalated into membranes and diassayed using lysine-rich histone as a common phos- rectly activate protein kinase C in intact cells (17). Using phate acceptor. Ninety-four per cent of protein kinase OAG, investigators have studied the role of protein kinase C C activity was recovered in the cytosolic fraction of in thesecretory response of platelets (17,18),leukocytes (19), unstimulated type I1 cells, and 40% of activity in cy- and mast cells (20) and in thecellular proliferation in fibrotosolic fraction wastranslocated to particulate fractionblasts (21-23). In thispaper, we have attempted to clarify the upon treatment with TPA. As observed in other tissues, role of protein kinase C in pulmonary surfactant secretion protein kinase C of alveolar type I1 cells was highly from alveolar type I1 cells using an inhibitor (tetracaine) and activated by 1,2-dioleoyl-sn-glycerolor TPA in the activator (OAG) of protein kinase C. We demonstrate that presence of Ca2+and phosphatidylserine. These results rat alveolar type I1 cells contain a high concentration of suggest that pulmonary surfactant secretion in vitro is protein kinase C and that thisenzyme is activated by unsatstimulated by both protein kinase C and cyclic AMP- urated diacylglycerol or TPA in the presence of Ca2+ and dependent protein kinase. phospholipid. EXPERIMENTALPROCEDURES

Alveolar type I1 cells are known to secrete pulmonary surface active material, a complex of phospholipids (especially disaturated phosphatidylcholine) and proteins, that prevents the alveoli from collapsing during expiration (for a review,

* This work was supported by National Heart, Lung, and Blood Institute GrantHL-29891 and was performed in the Lord and Taylor Laboratory of Lung Biochemistry and the AnnaPerahiaAdatto Clinical Research Center of the National Jewish Hospital and Research Center/National Asthma Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ T o whom correspondence should be addressed Department of Medicine, Pulmonary Division, National Jewish Hospital and Research Center/National Asthma Center, 3800 East Colfax Avenue, Denver, CO 80206.

Animals and Materials-Pathogen-free Sprague-Dawley rats (200250 g) were obtained from Bantin-Kingman, Inc. (Fremont, CA). [3H] Methylcholine chloride (80 Ci/mmol) and [y3*P]ATP (30-40Ci/ mmol) werepurchased from New England Nuclear. Elastase (porcine pancreas) was obtained from Cooper Biomedical (Malvern, PA). Phosphatidylserine was purchased from Avanti Biochemicals (Birmingham, AL). Lysine-rich histone (type 111-S), 3-isobutyl-1-methylxanthine (IBMX), lysophosphatidylcholine (1-oleoyl), diolein, phospholipase C (type V), leupeptin, and tetracaine hydrochloride were the products of Sigma. Terbutaline sulfate was kindly donated by Merrell Dow Pharmaceuticals Inc. (Cincinnati, OH). TPA and bovine serum albumin (fraction V) were purchased from Consolidated Midland Corporation (Brewster, NY) and Miles Laboratories, Inc.

* The abbreviations used are: TPA, 12-0-tetradecanoylphorbol13acetate; OAG, 1-oleoyl-2-acetyl-sn-glycerol; EGTA, ethylene glycol bis(8-aminoethyl ether)-N,N,N’,N’-tetraacetic acid; IBMX, 3-isobutyl-1-methylxanthine.

12725

12726

Protein KinaseC in Alveolar Type 11 Cells

(Elkhart, IN), respectively. 4-Dimethylaminopyridine was the prod- particulate fractions. The cytosolic fraction was directly assayed for uct of Aldrich. protein kinases. Protein kinase C was assayed in the reaction mixture Primary Culture of Alveolar Type ZZ Cells and Secretion of Phos- containing 5 pmol of Tris/HCl at pH7.5, 50 pg of histone (type IIIphatidylcholine-Alveolar type I1 cells were isolated from adult male S), various concentration of CaC12, 10 pg of phosphatidylserine, 0.2 Sprague-Dawley rats by tissue dissociation with elastase and partially pg of diolein, 1.25 pmol of MgCl,, and 2.5 nmol of [y-3ZP]ATP(0.5purified on metrizamide density gradients (5). The cells were sus- 2.0 X lo6 cpm) in a final volume of 0.25 ml for 6 min a t 30 "C. The pended in Dulbecco's modified Eagle's medium supplemented with trichloroacetic acid-precipitable material was collected on a 0.45-pm 10% fetal bovine serum, 2 mM glutamine, 10 pg/ml gentamicin, 100 nitrocellulose membrane filter and then analyzed by liquid scintillaunits/ml penicillin, 50 pg/ml streptomycin, and 1pCi/ml [3H]meth- tion counting (27). CyclicAMP-dependent protein kinase was assayed ylcholine chloride a t a concentration of 1 X IO6 cells/ml. The cells under conditions similar.to those for protein kinase C except for the were routinely plated at 2 X lo6 cells/35-mm dish. After incubation addition of 0.25 nmol of cyclic AMP instead of CaClZ,phosphatidylin an atmosphere of 10% COZ,90% air for 22 h at 37 "C, the dishes serine, and diolein. For the kinetic analysis of calcium and phosphowere washed with 10 ml of Dulbecco's modified Eagle's medium lipid dependence, protein kinase C of alveolar type I1 cells was containing 2.5 mg/ml bovine serum albumin to remove nonadherent partially purified through DEAE-cellulose column chromatography. cells and radioactive materials. Other Procedures-The radioactivity of 3H- and 32P-sampleswas The purity of type I1 cells thus obtained was 93 f 2% (n = 6) as determined using a Beckmann liquid scintillation spectrometer, determined by the modified Papanicolaou stain (10). Model LS 7500. Protein was determined by the method of Lowry et The cells were subsequently incubated with stimuli for the time al. (28) with bovine serum albumin as a standard. Statistical analysis intervals indicated in each experiment. The synthetic compound 1- of the datain Fig. 3 was performed using a one-tailed Wilcoxon signed oleoyl-2-acetyl-sn-glycerol and TPAwere used to examine the protein rank test for a paired sample. kinase C-dependent pathway for surfactant secretion. The CAMPdependent pathway was examined using terbutaline as the agonist in RESULTS conjunction with the phosphodiesterase inhibitor IBMX. Routinely, inclusion of IBMX with terbutaline resulted in greater levels of Stimulatory Effect of OAG on Phosphatidylcholine Secresurfactant secretion than terbutaline alone. The medium was removed tion-Recent studies with platelets have shown that OAG, a and the lipid was extracted by the method of Bligh and Dyer (24). synthetic diacylglycerol, is intercalated into the membrane Routinely, the radioactivity in the totallipid fraction was determined. and directly activates protein kinase C without polyphosUnder these conditions, the phospholipids secreted into themedium are predominantly those in purified surface active material and the phoinositide breakdown or any increase in the intracellular composition of the basal and TPA-stimulated media are the same Ca2+concentration (11, 17, 18). Therefore, OAG is a useful (25). In these current experiments, 98.4% of the radioactivity in the probe to assess the role of protein kinase C in surfactant media from cells stimulated by TPA was phosphatidylcholine, and secretion from alveolar type I1 cells. Fig. 1 shows that OAG 53.1% of the phosphatidylcholine was the disaturated species. The markedly stimulated phosphatidylcholine secretion in a dosecells were harvested from dishes with a rubber policeman and treated and time-dependent manner. This effect of OAG does not in the same manner as the media. A portion of media was subjected to measurement of lactate dehydrogenase (26). Lactate dehydrogen- seem to be a nonspecific action of diacylglycerol because 100 ase released into themedia did not exceed 3% oftotal cellular activity p~ 1,2-diolein did not have any effect on phosphatidylcholine secretion (data not shown). The concentration of OAG rethrough all experimentsin thispaper. Chemical Synthesis of OAG-Preceding synthesis, chloroform, pyr- quired for surfactant secretion is similar to theconcentration idine, and acetic anhydride were redistilled and I-dimethylaminopyr- reported to alter cellular functions in other systems (17-23). idine was recrystallized. Fifty milligrams of l-oleoyllysophosphati- As determined by lactate dehydrogenase release, OAG did not dylcholine was dissolved in a small portion of chloroform and dehy- cause cell damage at the concentrations used in these experdrated with ethyl ether twice under a stream of nitrogen. Then 2 ml of chloroform, 0.5 ml of pyridine, 30 pl of acetic anhydride, and 100 iments. pmol of 4-dimethylaminopyridine were added, and acetylation of the I I sn-2 position of lysophosphatidylcholine was continued overnight. The solvents were dried under vacuum twice, and theresidual material was dissolved in 2 ml of chloroform. The 4-dimethylaminopyridine was removed by washing with 1.8 ml of saline and 2 ml of lot / methanol containing 0.05 N HC1. The sample was subjected to preparative thin layer chromatography using a solvent system of chloroform/methanol/water (65356). Thesilica gel containing l-oleoyl2-acetylphosphatidylcholine was scraped, and thelipid was extracted by the method of Bligh and Dyer (24). A portion of l-oleoyl-2acetylphosphatidylcholine (10 pmol)thus obtained was dried under a stream of nitrogen, and 2 ml of ethyl ether and 0.5 ml of 0.05 M sodium phosphate buffer at pH 7.0 containing 0.1 mM ZnSOc and 9 units of phospholipase C were added. After 2 h of hydrolysis, the ethyl ether was evaporated under nitrogen and then the product "4 4(OAG) was extracted by the method of Bligh and Dyer (24). OAG f +--P-was dissolved in hexane and stored in -70 "C. OAG thus prepared I I I was always pure by thin layer chromatography in a solvent system of 1 2 3 01 40 160 G o 260;) hexane/ethyl ether (9O:lO) and was stable for a t least 3 months under OAG (pM) Hours these conditions. Assay for Protein Kinases and Partial Purification of Protein Kinase FIG. 1. Effect of OAG on phosphatidylcholine secretion.An C from Alveolar Type ZZ Cells-Rat brain or saline-perfused lungs aliquot ofOAG was taken from storage, dried with hexane under nitrogen stream, suspended in media containing 2.5 mg/ml bovine were homogenized in 3 volumes of homogenizing buffer (20 mM Tris/ HCl at pH 7.5 containing 10 mM EGTA, 2 mM EDTA, 50 mM 2- serum albumin, and briefly sonicated just before addition. A, dose mercaptoethanol, and 0.25 M sucrose) with a Teflon-glass homoge- response. Various amounts of OAG, as indicated, were added to the nizer. Eight 100-mm dishes of alveolar type I1 cells in primary culture alveolar type I1 cells, which were prelabeled with [3H]methylcholine chloride. The per cent of total cellular [3H]phosphatidylcholine se(2.8 mgof protein) prepared as described above were scraped in phosphate-buffered saline with a rubber policeman, and thecells were creted into media in 3 h was determined as described under "Experwashed twice with the same buffer and then were sonicated in 2 ml imental Procedures." B, time course. The phosphatidylcholine secreas in A except that 100 p~ OAG of homogenizingbuffer for 1min at theminimum output of a Branson tion was determined in same manner sonicator model W185. The homogenates were centrifuged for 10 min was used and the incubation time was varied. Each point represents at 1,000 X g to pellet cell debris and nuclei, and then the supernatant the mean -C S.E. of triplicate samples from three different experiwas centrifuged for 60 min at 100,000 X g to obtain the cytosolic and ments. 0, control; 0,with OAG.

I A

t

li:

-

-

12727

Protein Kinase C in AlveolarType 11 Cells Effect of Tetracaine on Phosphatidylcholine SecretwnBoth terbutaline and TPA have been shown to stimulate phosphatidylcholine secretion from alveolar type I1 cells in vitro (5, 10). The stimulatory effect of terbutaline has been attributed to an increase in cellular cyclic AMP, but the mechanism of action of TPA remained unclear (10). Recently, protein kinase C has been shown to be a cellular target of TPA (11-13). Therefore, we examined if this is the case in alveolar type I1 cells by using tetracaine as an inhibitor of protein kinase C (16). As shown in Fig. 2, preincubation of cells with tetracaine inhibitedTPA-induced phosphatidylcholine secretion in a dose-dependent manner. However, tetracaine had no effect upon the terbutaline plus IBMX-induced response and basal secretion. This shows that the inhibitory effect of tetracaine on TPA-induced secretion is not a nonspecific action of a cationic amphiphilic drug but suggests that this compound directly inhibitsprotein kinase C by competing with membrane phospholipids. Higher concentrations of tetracaine could not be evaluated because of its toxicity, but tetracaine inhibited the cellular response to the same extent as observed previously in platelets (15). Combined Effects of Terbutaline, TPA, and OAG upon Phosphatidylcholine Secretion-Some proteins arephosphorylated by both cyclic AMP-dependent protein kinase and protein kinase C. Therefore, we examined the combined effects of terbutaline, TPA, and OAG on phosphatidylcholine secretion to clarify if both kinasesphosphorylate the same substrate(s) to produce secretion by alveolar type I1 cells. Fig. 3 shows that the effects of terbutaline and TPA were additive and those of terbutaline and OAG were significantly more than additive ( P < 0.05).These results suggest that cyclic AMP-

+\

I

A \

t

2o 15

A

B

C

D

E

F

G

FIG. 3. Combined effects of terbutaline, TPA, and OAG on phosphatidylcholine secretion. The alveolar type I1 cells, which were prelabeled with [3H]methylcholinechloride for 22 h, were stimulated for 3 h with various stimulants as indicated below. Phosphatidylcholine secretion was determined as described under “Experimental Procedures.” Each bar represents the mean f S.E. of triplicate samples from five different experiments. A , control; B, with 100 p~ terbutaline and 50 p~ IBMX C, with 50 ng/ml TPA; D, with 200 p~ O A G E, with 100 p~ terbutaline, 50 p~ IBMX, and 50 ng/ml TPA; F, with 100 p~ terbutaline, 50 PM IBMX, and 200 pM O A G G, with 50 ng/ml TPA and200 FM OAG.

TABLEI Specifi activities of cyclic AMP-dependent proteinkinase and protein kinase C in rat brain, lung, and alveolar type II cells Each tissue was homogenized and centrifuged as described under “Experimental Procedures.” The supernatant was assayed for both protein kinases. The data represent the mean of the number of experiments indicated in parentheses. CAMP-dependent orotein kinase nmol Pjminf mg protein 1.05 0.11 ( n = 4) 0.22 0.10 ( n = 2 ) 0.12 (n = 3) 0.75

Protein kinase

Brain Whole lung Type I1 cells

~

‘0

0.5

1

1.5

2

Tetracaine (mM)

FIG. 2. Effect of tetracaine on phosphatidylcholine secretion. The alveolar type I1 cells, which were labeled with [3H]methylcholine chloride for 22 h, were preincubated with various concentrations of tetracaine as indicated for 5 min. After preincubation, the media was aspirated and fresh media containingeither TPA or terbutaline were added. Incubation was continued for 3 h, and the radioactivity of the lipid fraction of both media and the cells was counted as described under “Experimental Procedures.” Each point represents the mean f S.E. of triplicate samples from the number of experiments indicated. 0, control; a, with 50 ng/ml TPA; A, with 100 p~ terbutaline and 50 PM IBMX.

dependent protein kinase and proteinkinase C phosphorylate different protein(s) or different site(s) of the same protein(s) to cause secretory response. The dataalso suggest that cyclic AMP can modulate the action of protein kinase C, and vice versa. The effects of TPA and OAG were less than additive ( P < 0.05),which suggests that both agents act on the same cellular target, protein kinase C. Specific Activities of Cyclic AMP-dependent Protein Kinase and Protein Kinase C-In the next set of experiments, we measured and compared the specific activities of cyclic AMPdependent proteinkinase and protein kinase C of brain, whole lung, and alveolar type I1 cells.Table I shows that thespecific activity of cyclic AMP-dependent protein kinase was not very different among these tissues. Alveolar type I1 cells have a higher specific activity of protein kinase C than whole lung and slightly less than brain. The specific activity of protein kinase C of alveolar type I1 cells was about 6-fold higher than that of cyclic AMP-dependent protein kinase when lysinerich histone was used as a common phosphate acceptor. Translocation of Protein Kinase C from Cytosolic Fraction

Protein Kinase C in Alveolar Type 11 Cells

12728

TABLE I1 Distribution of protein kinase C in cytosolic ana‘particulate fractions 1c e k following various stimulants of alveolar type 1 Alveolar type I1 cells were plated in 100-mm dishes (20 X lo6 cells/ dish) and incubated for 22 h in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. After washing the plates, cells were further incubated with 50 ng/ml TPA or 200 p M OAG for 30 min a t 37 “C. Soluble and particulate fractions were obtained as described under “Experimental Procedures” except that cells were sonicated in 20 mM Tris/HCl at pH7.5 containing 100 pg/ml leupeptin and 2 mM EDTA. The cytosolic fraction was directly assayed for protein kinase C under the standard condition. The particulate fraction was resuspended in an original volume of the same buffer containing 0.1% Triton X-100, sonicated, and incubated for 30 min a t 4 ”C following centrifugation for 60 min at 100,000 X g (26). The supernatant thus obtained was subjected to protein kinase C assay. The amount of enzyme employed for the assay was 5-10 pg ofprotein for cytosolic fraction and 20-30 pg of protein for particulate fraction. The datarepresent the mean of duplicate samples from two different exoeriments. Protein kinase C activity Treatment cytosolic Particulate fraction

fraction

nmol Pf minf mg protein

46

None TPA 0.28 OAG

0.71 0.25 0.78

I

I

Ca2+ Phosphatidylserine Diofein

3.4

0.3

i?

0.2 0

n

s

~~~~lactivity in particulate fraction

W

3.1

%

0.06

6

0.08

6

to Particulate Fraction-Recently, the translocation of protein kinase C from the cytosolic fraction to theparticulate fraction following treatment with TPA has been reported in various cell types (29, 30). This phenomenon is assumed to be an initial event in the action of TPA (29). Table I1 shows that normally 94% of protein kinase C activity was recovered in the cytosolic fraction in alveolar type I1 cells and that40% of activity was translocated to the particulate fraction upon treatment with TPA. The precise mechanism and significance of translocation of this enzyme are obscure, but these data provide evidence that TPA does act on protein kinase C in alveolar type I1 cells. OAG did not promote translocation of protein kinase C. The reason is not clear at present, but this might be dueto rapid conversion of OAG to phosphatidic acid (17), whereas TPA is hardly metabolized (31), as discussed below. Partial Purification and Characterization of Protein Kinase C of Alueolar Type II Cells-When the cytosolic fraction of alveolar type I1 cells in primary culture was subjected to DEAE-cellulose column chromatography, protein kinase C activity appeared at anNaCl concentration of approximately 75 mM, as shown in Fig. 4. This elution profile is similar to that of protein kinase C from rat brain prepared in our laboratory (data not shown). Next, we characterized protein kinase C of alveolar type I1 cells in the peak fraction from DEAE-cellulosecolumn chromatography. Table I11 shows that phosphatidylserine stimulated phosphorylation of lysinerich histone by this enzyme to a slight extent. Addition of diolein or TPA toa reaction mixture containing calcium and phosphatidylserine greatly stimulated the phosphorylation of histone by the enzyme isolated from alveolar type I1 cells as reported in other tissues (12, 27).

5

10

15

0

20

Fraction Number FIG. 4. DEAE-cellulose column chromatography of protein kinases in alveolar typeI1 cells in primary culture. The soluble fraction of type I1 cells (2 ml, 2.8 mg of protein) was applied to a DEAE-cellulose column (1 ml) equilibrated with Buffer A (20 mM Tris/HCl at pH 7.5 containing 50 mM 2-mercaptoethanol, 2 mM EDTA, and 5 mM EGTA). After the column was washed with 10 ml of Buffer A, protein kinases were eluted by a 20-ml linear concentration gradient of NaCl (0-0.4 M) in Buffer A. Eachfraction was assayed for protein kinases as described under “Experimental Procedures.” A 50-p1 aliquot of each fraction was employed, and incubation was performed for 20 min. Two different experiments showed same results as shown. 0,with 1.1mM CaC12,10 pg of phosphatidylserine, and 0.2 pg of diolein; 0,with 1p M cyclic AMP;A,with 1mM EGTA; - - -, NaCl concentration. TABLE I11 Activation of protein kinase C by diolein and TPA A 40-pl aliquot of the peak fraction of protein kinase C from DEAE-cellulose column chromatography (fraction 6 in Fig. 4; 1.5 pg of protein) was assayed in thepresence or absence of 0.85 mM CaC12, 2 pg of phosphatidylserine, 0.2 pg of diolein, and 50 ng/ml TPA as described under “Experimental Procedures.” The background count without enzvme is subtracted from each value. Protein kinase activity

Additions

cPm

None Ca2+ Ca2+ diolein Ca2+ TPA Ca2++ phosphatidylserine Ca2++ phosphatidylserine Ca2++ phosphatidylserine

+ +

+ TPA + diolein

150 60 210 280 1030 5030 7260

stimulator of surfactant secretion (lo),the precise mechanism of its action has remained unclear. Four lines of evidence Alveolar type I1 cells store surface active material in discrete suggest that TPA stimulates surfactant secretion through organelles (lamellar bodies) and secrete it by an exocytotic activation of protein kinase C in alveolar type I1 cells. 1) process similar to that of other secretory cells (1,2). In vitro TPA-induced phosphatidylcholine secretion was inhibited by studies revealed that alveolar type I1 cells possess @-adrenergic a specific inhibitor of protein kinase C, tetracaine, as shown receptors and secrete pulmonary surfactant upon stimulation in Fig. 2; 2) TPA caused translocation of protein kinase C by @-adrenergicstimulants (5-7). Although TPA is a potent from the cytosolic fraction to the particulate fraction as is DISCUSSION

Protein KinaseC in Alveolar Type 11 Cells shown in Table11; 3) alveolar type I1 cells contain a significant amount of protein kinase C, and thisenzyme is activated by TPA aswell as diacylglycerol (Tables I and 111);and 4) OAG stimulates phosphatidylcholine secretion from alveolar type I1 cells (Fig. 1). If both TPA and OAG stimulate only protein kinase C, then the effect of both agents added at saturating concentrations should not be additive. Our result shows that the addition of both agents causes a greater response than theaddition of each agent alone (Fig. 3). The exact reason for this result is not clear at present, but it may be assumed that neither TPA nor OAG reaches a concentration in thealveolar type I1 cell membrane for maximal protein kinase C activation, or TPA may have additional effects, e.g. on protein alkylation (32), phospholipase A2 (33), or the relationship between surface receptors and adenylate cyclase (34)as reported in other tissues. However, OAG is not reported to have other actions. As shown in Table 11, protein kinase C was translocated from the cytosolic fraction to the particulate fraction upon stimulation by TPA as reported in other cell types (29, 30). Kraft andAnderson (29) proposed the possibility that protein kinase C loosely binds to plasma membrane in the resting state andthat TPA strengthens the interaction between Ca2+, protein kinase C, and TPAperse. In this case, it is conceivable that diacylglycerolalso promotes association of protein kinase C with the plasma membrane because TPA anddiacylglycerol have the same action on protein kinase C, that is loweringK, for Ca2+and phospholipid (12,27); but OAG did not translocate protein kinase C in alveolar type I1 cells, as shown in Table 11. This isprobably because OAG is rapidly metabolized to phosphatidic acid (17). I n platelets, thrombin does not cause translocation of protein kinase C (30) even though thrombin fully activates protein kinase C (15). Therefore, diacylglycerol might promote association of protein kinase C with plasma membrane transiently during activation of this enzyme. However, the precise mechanism and significance of translocation of this enzyme remain to be explored. In human platelets, the activation process of protein kinase C is coupled to a phosphatidylinositol response induced by various physiological stimuli (14, 15). Thus far, phosphatidylinositol turnover has not been evaluated in pulmonary surfactant secretion. In uiuo agents that increase intracellular levels of cyclic AMP stimulate secretion (2). However, the mechanisms for basal secretion and for secretion during hyperventilation are not known (2, 35). Protein kinase C may be involved in these two processes, but the precise role of protein kinase C in pulmonary surfactant secretion in vivo still remains to be explored. In summary, we reported in this paper that alveolar type I1 cells contain relatively high amounts of protein kinase C and that thisenzyme is involved in surfactantsecretion in addition to cyclic AMP-dependent protein kinase. Acknowledgment-We thank Sandy Subotnick for providing excellent secretarial assistance.

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