Experimental Lung Research
ISSN: 0190-2148 (Print) 1521-0499 (Online) Journal homepage: http://www.tandfonline.com/loi/ielu20
Protein Kinase C Activation Modulates Arachidonic Acid Metabolism in Cultured Alveolar Epithelial Cells Marc Peters-Golden, Kathie Coburn & James B. Chauncey To cite this article: Marc Peters-Golden, Kathie Coburn & James B. Chauncey (1992) Protein Kinase C Activation Modulates Arachidonic Acid Metabolism in Cultured Alveolar Epithelial Cells, Experimental Lung Research, 18:4, 535-551, DOI: 10.3109/01902149209064344 To link to this article: http://dx.doi.org/10.3109/01902149209064344
Published online: 02 Jul 2009.
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Date: 21 May 2016, At: 22:50
Protein Kinase C Activation Modulates Arachidonic Acid Metabolism in Cultured Alveolar Epithelial Cells
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Marc Peters-Golden, Kathie Coburn, and James B. Chauncey
ABSTRACT: Cultured alveolar type 11 cells can liberate esterified arachidonic acid (AA)and metabolize it predominantly via the cyclooxygenasepathway, and their capacity to do so increases as they alter their phenotpe over time in culture. Little is known, however, about the regulation of AA metabolism in alveolar pneumocytes. We have examined the effects of protein kinase C (PKC) activation on arachidonate metabolism in primary cultures of rat alveolar epithelial cells studied at 2 and 7 days following isolation. B e potent PKC activator phorbol myristate acetate (PMA) stimulated dose-dependent increases in free A A levels in both day 2 and day 7 cultures, with optimal stimulation at 50 nM. Greater stimulation was demonstrated for day 7 cells, and this was associated with greater prostanoid synthesis in response to PMA by day 7 than by day 2 cells. The capacity of PMA to “prime”epithelial cells for augmented AA liberation and metabolism in response to calcium ionophore A23187 (5 fl)was examined also. Significant priming by PMA was observed i n both day 2 and day 7 cells; once again, augmentation of both free A A levels as well as prostaglandin E, levels was greater for day 7 cells than for day 2 cells. That the capacity of PAL4 to modulate AA metabolism was mediated by activation of PKC was confirmed by demonstrating that (I) phorbol didecanoate, which lacks the ability to activate PKC, failed to activate AA metabolism; (2) pretreatment for 18 h with 1 f l PMA,which depletes cellular PKC, abolished subsequent AA metabolism activated by 50 nM PMA; and (3) the PKC inhibitor staurosporine abrogated increases in the quantities of both fyee AA and prostaglandin E2 in response to PMA. We conclude that activation of PKC increases the availability of AA for prostanoid synthesis in alveolar pneumocytes, and that this ejfict is more evident as t p e 11 cell differentiation is modeled during prolonged cultivation.
From the Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan and Kterans Affairs Medical Centers, Ann Arbor, Michigan. Address all correspondence to Marc Peters-Golden, MD, Division of Pulmonary and Critical Care Medicine, 3916 Taubman Center, University of Michigan Medical Center, Ann Arbor, MI 48109-0360. Received 17 July 1991; accepted 12 November 1991.
Experimental Lung Research 18:535-551 (1992) Copyright 0 1992 by Hemisphere Publishing Corporation
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INTRODUCTION Immune and inflammatory processes occurring within the pulmonary alveolar space are thought to be of primary importance in the initiation and eventual outcome of acute lung injury, as well as of chronic lung disorders such as pulmonary fibrosis. Traditional “effector” cells, including macrophages, lymphocytes, and neutrophils, have been studied extensively in an effort to delineate the cellular mechanisms of alveolar inflammation. However, the participation of the alveolar epithelium itself in such events is poorly understood. This is true despite the fact that alveolar epithelial cells, or pneumocytes, are situated in a position where they have the potential to respond to inhaled agents, as well as to interact with traditional effector cells via the elaboration of mediators. In this regard, previous studies from our laboratory have demonstrated that rat type I1 pneumocytes studied at 48 h in culture have the capacity to deacylate esterified arachidonic acid (AA) and subsequently metabolize it, predominantly via the cyclooxygenase pathway, to bioactive eicosanoids [I]. In several cells types, alterations in AA metabolism have been shown to accompany cellular differentiation [2, 31. In vivo, the type I1 pneumocyte, which is known best for its capacity to secrete pulmonary surfactant, serves as the progenitor for the type I cell, the cell that lines the vast majority of the alveolar surface [4]. When cultured for several days on tissue culture grade plastic, type I1 cells assume a phenotype that closely resembles that of a type I cell in vivo with respect to morphologic and biochemical criteria [5-91. We have demonstrated recently that this in vitro model of differentiation is accompanied by marked increases in both the liberation and cyclooxygenase metabolism of AA [lo]. Little information is available, however, regarding the regulation of arachidonate metabolism in alveolar epithelial cells, or whether alterations in regulation might accompany in vitro differentiation. In a variety of cell types, activation of protein kinase C (PKC) augments both the deacylation of AA as well as its metabolism via the cyclooxygenase pathway [ll-141. In the present study, therefore, we have examined the effects of PKC activation on AA metabolism in differentiating alveolar epithelial cells.
METHODS Alveolar Epithelial Cell Isolation and Culture Type I1 alveolar epithelial cells were isolated from male specific pathogenfree Sprague-Dawley rats (Charles River, Portage, MI) weighing 180-200 g by standard techniques [15]. Cells were suspended in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY) containing 10% fetal bovine serum with penicillin G (100 U/mL), streptomycin (100 pg/mL),
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and amphotericin B (0.25 pg/mL) (subsequently referred to as serumcontaining medium) at lo6 cells/mL, then aliquotted to 24-well tissue culture plates (Falcon, Oxnard, CA) at l mL/well and cultured in 5% C0,:air at 37OC. We have demonstrated previously that at 48 h these cultures comprise a confluent monolayer of approximately 3 x lo5 cells/well, of which 92-94'10 are type I1 cells, and contain -70 pg of total protein/well [lo]. Viability exceeded 90% as assessed by trypan blue exclusion. Because culture on tissue culture plastic in serum-containing medium for 7 d permits type I1 cells to assume a type I-like phenotype with respect to a variety of markers [9] and also to maximally up-regulate their capacity for arachidonate liberation and metabolism [lo], cells were studied at 2 and 7 d in culture. Cells to be studied at day 7 had their serum-containing medium changed at days 2 and 4. Cell number, protein content, purity, and viability of cultures have been shown to be stable during the 7-d culture period [lo]. Prelabeling of Epithelial Cell Cultures with [l4C]AA
To radiolabel epithelial cell lipids, serum-containing medium was removed from cultures approximately 18 h prior to experimental incubations and replaced with 1 mL/well of the same medium containing 0.2 pCi (3.6 pM) of [l - 14C]AAhaving a specific activity of 54-57 mCi/mmol (Dupont, New England Nuclear, Boston, MA) [lo]. After labeling for 18 h, medium was removed and monolayers were washed three times with Hanks' balanced salt solution (Gibco) prior to experimental incubations. In parallel with the experimental incubations, prelabeled cells from two additional wells in each experiment were lysed instead in methanol and subjected to scintillation counting to determine the cellular uptake of radiolabel. Uptake of radiolabe1 (mean f. SEM) in day 2 and day 7 cells was 57.2 2 3.1% and 56.6 f. 3.0°/o, respectively, of that added ( n = 11). Experimental Incubations At the indicated experimental time point of 2 or 7 d, prelabeled or unlabeled pneumocyte cultures from a single isolation were washed three times with Hanks' solution and incubated for 1 h with serum-free DMEM alone, DMEM containing the PKC activators phorbol 12-myristate 13-acetate (PMA), or 1-oleoyl-2-acetylglycerol (OAG), or DMEM containing the inactive phorbol ester 4-a-phorbol 12,13-didecanoate (PDD). The ability of PMA or PDD to prime cells for augmented AA metabolism in response to calcium ionophore A23187 was examined by incubating cells for 1 h with 5 pM A23187 [l, 101 in the absence and presence of the phorbol ester (following a 10-min pretreatment with PMA or PDD). The role of PKC activation in mediating PMA-induced responses was assessed by determining the ability of PMA to trigger AA metabolism or prime for ionophore-stimulated
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AA metabolism following either a 10-min pretreatment and subsequent coincubation with the potent PKC inhibitor staurosporine 1161, or an 18-h pretreatment with 1 pM PMA (to down-regulate PKC by its depletion from the cell [17, 181) or PDD. Finally, the effect of PKC activation on cyclooxygenase activity was determined by incubating cells for 1 h with 20 pg/mL AA in the presence or absence of PMA. PMA, OAG, PDD (all from Sigma, St. Louis, MO), A23187 (Calbiochem, San Diego, CA), and staurosporine (Kamiya Biomedical, Thousand Oaks, CA) were prepared in DMSO and freshly diluted into DMEM immediately before use. Final concentrations of DMSO were I0.7%, and had no effect on either cell viability or arachidonate metabolism. Unlabeled AA (Nu-Chek Prep, Elysian, MN) was prepared in sodium hydroxide and diluted prior to use.
Quantitation of Free ["CIAA In experiments designed to assess levels of free [14C]AA, incubations were carried out in the presence of 1 pM indomethacin to inhibit cyclooxygenase metabolism. Indomethacin (Sigma) was prepared in ethanol and diluted to yield a final ethanol concentration of 0.05%. Following experimental incubations, lipids were extracted from cells plus medium with chloroform/methanol (21, v/v), and separated by thin-layer chromatography (TLC) on Silica Gel 60 plates developed with hexane/diethyl ether/acetic acid (70:30:2), as described [19]. Labeled free AA was identified by co-migration with authentic AA, quantitated by scintillation counting, and expressed as a percentage of the incorporated radiolabel. For experiments in which cells were labeled during pretreatment with phorbol esters, free AA levels were expressed relative to uptake determined under the same conditions. For each experiment, the average of duplicate determinations was calculated.
Quantitation of Eicosanoids Eicosanoids were extracted from medium of unlabeled or prelabeled cultures using C,, Sep-Pak cartridges (Waters Associates, Milford, MA) as previously described [20]. Recoveries for AA and prostanoids exceed 90% by this method. Radiolabeled AA and eicosanoids were separated by reverse-phase, high-performance liquid chromatography (HPLC) of the pooled lipid extracts from 3-5 wells using the mobile phase acetonitrile/water/trifluoroacetic acid at a flow rate of 1 mL/min [21]. Products were identified by co-elution with authentic standards, quantitated by scintillation counting, and expressed as a percentage of incorporated label. Prostanoid standards were obtained from the Upjohn Co. (Kalamazoo, MI); 12hydroxyheptadecatrienoic acid (HHT) from Cayman Chemical (Ann Arbor, MI); and lipoxygenase standards from Merck Frosst Inc. (Dorval,
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Quebec, Canada). Prostaglandin E, (PGE,) and 6-keto-prostaglandin F,, (6-kPGF,,), the stable metabolite of prostacyclin, were quantitated in the medium of unlabeled cultures by radioimmunoassay (RIA). Sep-Pak extracts were dried and dissolved in 1 mL of phosphate-buffered saline containing 0.1% gelatin, and 0.l-mL aliquiots were assayed in duplicate. The source, sensitivity, and cross-reactivity of the antibodies employed have been described [I]. Concentrations of immunoreactive prostaglandins were corrected for recovery. In all instances, the average of duplicate determinations was calculated.
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Data Analysis Data were expressed as means k SEM. The significance of differences between the means of more than two groups was assessed by analysis of variance and the Newman-Keuls multiple range test [22]. The significance of differences between the means of identically treated day 2 versus day 7 cultures from a single isolation was assessed by a paired Student’s t test [22]. A P value < .05 was considered significant. RESULTS Effect of PMA on Alveolar Pneumocyte AA Metabolism Since the deacylation of AA from cellular lipids is the initial and probably rate-limiting step in AA metabolism [23], we quantitated levels of the unesterified I4C-labeledfatty acid by TLC in day 2 and day 7 cultures of prelabeled pneumocytes incubated with the potent PKC activator, PMA. Preliminary experiments indicated that at both of these time points, incubation with PMA over the concentration range 5-100 nM resulted in a dosedependent increase in the availability of free AA, which plateaued at a PMA concentration of 50 nM. Therefore, this dose of PMA was selected for subsequent comparisons with unstimulated cultures. Table 1 presents the effects of 50 nM PMA on free AA levels in day 2 and day 7 cultures. Unstimulated cultures contained low levels of free [I4C]AA; as reported previously [lo], quantities of free fatty acid in day 7 cultures exceeded those in day 2 cultures. The presence of 50 nM PMA significantly (P < .02) increased levels of [14C]AA over control levels in both day 2 cells and day 7 cells ( n = 7). The degree of stimulation in day 7 cells (192 k 35% of control) was greater than that in day 2 cells (158 k 18% of control), but not significantly. In contrast to the stimulatory effects of PMA, no dose-dependent increases in free [14C]AAlevels were observed in response to PDD, a phorbol ester that fails to activate PKC [24], at either day 2 (control, 0.28%; 5 nM PDD, 0.23%; 50 nM PDD, 0.28%; and 500 nM PDD, 0.30%) or day 7 (control,
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Table 1 Effect of PMA on [14C]AALevels in Resting and A23187-Simulated Epithelial Cells at 2 and 7 Days in Culture Day 2 [14C]AA
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Control PMA (50 nM) A23187 (5 pM) A23187 + PMA
0.32 f 0.05 0.47 f 0.05" 1.46 f 0.56 1.95 f 0.54'
OO /
(158 f 18)b (164 f 26)d
Day 7 [14C]AA 0.88 1.48 2.82 6.46
0%
f 0.26 f 0.31" f 1.24
(192 f 35)b
f 2.41'
(269 f 40)"'
Data are means & SEM of results from seven separate experiments, each determined in duplicate. [14C]AA levels are expressed as a percentage of total incorporated radioactivity. 'P < .O2 versus appropriate unstimulated control value by paired Student's t test. bValues represent the percentages of the appropriate control values. 'P < .05 versus appropriate A23187 alone value. dValues represent the percentages of the appropriate A23187 alone values. 'When expressed as a percentage of the AA levels observed with A23187 stimulation alone, the degree of priming was significantly greater (P < .05) in day 7 cells than in day 2 cells by paired Student's t test.
0.56%; 5 nM PDD, 0.62%; 50 nM PDD, 0.55%; and 500 nM PDD, 0.74%). The effect of 50 nM PMA on ['4C]eicosanoid synthesis by prelabeled epithelial cell cultures was assessed by HPLC (Fig. 1). Unstimulated day 2 cultures (Fig. la) released small amounts of radioactivity corresponding to free AA, PGE,, and 6-k-PGF1,, the stable metabolite of prostacyclin (PGI,). Analysis of day 2 cells incubated with 50 nM PMA (Fig. lb) revealed modest increases in the amounts of radiolabeled free AA, PGE,, and 6-k-PGF1,. As suggested by the TLC data shown in Table 1, and as reported previously [lo], unstimulated day 7 cells (Fig. lc) contained peaks of radioactivity corresponding to free AA, PGE,, and 6-k-PGF1, that were larger than those observed for day 2 cells. Incubation of day 7 cells with 50 nM PMA (Fig. Id) resulted in a marked augmentation over control levels in the quantities of radioactivity corresponding to the cyclooxygenase eicosanoids PGE, and 6k-PGF,,, as well as HHT, a nonenzymatic breakdown product of endoperoxides generated during cyclooxygenation. Of note, a greater proportion of the radioactivity accumulating in the medium in response to PMA stimulation co-eluted with cyclooxygenase products, as opposed to free AA, in day 7 cells than in day 2 cells. Differences in eicosanoid synthesis in response to PMA between day 2 and day 7 cells were confirmed by measuring immunoreactive levels of the major metabolite, PGE,. Once again, 50 nM proved to be an optimal concentration of the phorbol ester (data not shown). As shown in Table 2, incubation with 50 nM PMA resulted in minimal increases in PGE, synthesis over control levels in day 2 cells ( P > .05), but in significant increases over resting levels in day 7 cells ( P < .05).
54 1
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:.I
0 12
k
c l D
al
L
E 0 P
Day 2
a.
I
- Control
0.10 0.08
0 06
0.06
0 0
0.04
.c
0
0.12
6-h-PGFla .
0.00 0 O 02
O
4
1
0.02
$ 0.00 0
20
40
0 12
E a
U
Day 2
b.
80
60
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120
- PMA 0.10
0 10
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0 08
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.-
0 04
0
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.-
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Day 7
80
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- PMA
J -
0.06
O 06 0 o8I
0 0
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JL
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0
6-k-PGF 1 a
/
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HHT I
a-" 0.00
0
40
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0
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Minutes
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Minutes
Figure 1 Representative radioactivity HPLC elution profiles from prelabeled day 2 (u and b) and day 7 (c and d) pneumocytes incubated for 1 h with medium alone (u and c) o r medium containing 50 nM PMA ( b and d). For each condition, medium from triplicate wells was pooled and extracted prior to HPLC analysis. Radioactivity in each fraction was expressed as a percentage of the total radioactivity incorporated into cells prior to stimulation. Peaks were designated on the basis of co-elution with authentic standards.
Table 2 Effect of PMA on Immunoreactive PGE, Synthesis by Resting and A23187-Stimulated Epithelial Cells at 2 and 7 Days in Culture Day 2 ng/mL Control PMA (50 nM) A23187 (5 pM) A23187 + PMA
*
0.78 f 0.13 0.79 f 0.19 1.28 ! I0.28 1.28 f 0.30
OO /
(98 f lo>. (99 f 5)'
Day 7 ng/mL 1.36 f 0.30 2.94 f 0.68 10.40 f 2.10 14.18 f 2.18
YO
(219) f 39Yb (140 f 9)b3c
Data are means SEM of results from three separate experiments, each determined in duplicate. "Values represent the percentages of the appropriate control values. 'I' < .05 versus relevant day 2 value by paired Student's t test. 'Values represent the percentages of the appropriate A23187 alone values.
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Effect of PMA on Ionophore-Stimulated AA Metabolism by Alveolar Pneumocytes We have shown previously that the calcium ionophore A23187 stimulates the liberation and subsequent metabolism of AA in alveolar pneumocytes [I], and that day 7 cells are more responsive to A23187 stimulation than are day 2 cells [lo]. The capacity of PMA to augment A23187-induced AA deacylation was examined by TLC analysis of prelabeled cultures. An ionophore concentration of 5 pM was selected for these experiments, since it has been shown previously to yield maximal responses in cells at both days 2 and 7 [lo]. As also depicted in Table 1, PMA at 50 nM significantly increased levels of free AA over those measured in response to 5 pM ionophore alone in both day 2 and day 7 cells. At both days studied, the free fatty acid levels measured in cultures incubated with A23187 plus PMA exceeded the sum of the responses due to A23187 and 50 nM PMA individually. This synergistic augmentation therefore can be considered true “priming” [25]. Moreover, the degree of priming in day 7 cells (269 k 40% of [14C]AA levels in response to A23187 alone) was significantly greater ( P < .05) than that observed in day 2 cells (164 26% of A23187 alone). The inactive phorbol ester PDD failed to augment ionophore-induced AA deacylation (not shown). Table 2 includes data on the effect of PMA on immunoreactive PGE, levels stimulated by A23187. PMA failed to augment ionophore-stimulated PGE, synthesis in day 2 cells, but did so in day 7 cultures.
*
Dependence of PMA-Induced Responses on PKC Activation The ability of PMA but not PDD to activate AA deacylation and metabolism in alveolar pneumocytes suggests that responses to PMA are a function of the capacity of this agent to activate PKC. We therefore utilized two other approaches to substantiate the PKC dependence of PMA-induced effects on arachidonate metabolism. First, we made use of the fact that in a variety of cell types, prolonged incubation with high doses of PMA ultimately results in down-regulation of PMA-induced responsiveness [ 17, 181. Thus we examined the ability of PMA to trigger AA liberation as well as to augment A23 187-induced liberation in day 7 cells whose PKC was depleted by 18-h pretreatment with 1 pM PMA. The data presented in Table 3 demonstrate that such pretreatment completely abrogated the ability of 50 nM PMA to both trigger and prime AA liberation in prelabeled day 7 cultures. No cytotoxicity was observed following this pretreatment protocol, and uptake of radiolabel was not affected consistently. In contrast, 18-h pretreatment with 1 pM PDD had no such down-regulatory effect on the ability of PMA to increase subsequently the availability of free [I4C]AA in cells incubated with and without A23 187. Interestingly, pretreatment with PMA also
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Table 3 Effect of 18-H Pretreatment with Phorbol Esters on [I4C]AA Levels in Cultures of Day 7 Epithelial Cells ~~~
~~~
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18-h Pretreatment
Incubation
None
1 pM PMA
Control PMA (50 nM) A23187 (5 pM) A23187 + PMA
0.41 f 0.01 0.88 f 0.04' 1.51 f 0.03 3.23 k 0.04"
0.42 0.36 0.99 0.91
f 0.02 k 0.02 f 0.03 f 0.01
1 pM PDD 0.41 2.09 1.77 3.86
f 0.05 f 0.29" k 0.14 f 0.25"
Data represent means f SEM from three separate experiments, each performed in duplicate. [I4C]AA levels are expressed as a percentage of total incorporated radioactivity. "P < .01 versus the non-PMA value immediately above it by analysis of variance.
partially attenuated the response to A23187 alone in day 7 cells. Second, we examined the effect of a potent inhibitor of PKC activation, staurosporine [16], on PMA-stimulated AA release and PGE, synthesis (see Table 4).When prelabeled day 7 cells were pretreated with staurosporine for 10 min and then co-incubated with staurosporine and PMA (50 mM) for 1 h, the increase in [I4C]AAlevels attributable t o PMA was attenuared significantly in a dose-dependent manner by the inhibitor with a half-maximal inhibitory concentration of 5-10 nM. In addition, the capacity of 50 nM PMA to initiate synthesis of immunoreactive PGE, by unlabeled day 7 cultures was inhibited largely by 50 nM staurosporine (control, 1.97 ng/mL; PMA, 4.00 ng/mL; PMA + staurosporine, 2.59 ng/mL). Effect of PMA on Cyclooxygenase Activity of Alveolar Epithelial Cells Although PMA was only slightly more potent a stimulus for the liberation of AA in day 7 cells than in day 2 cells, it was considerably more effective at initiating prostanoid synthesis in day 7 cells (see Table 2). Because PKC activation by PMA has been shown to augment the activity of existing cyclooxygenase enzyme in renal epithelial cells [26], we considered such a mechanism as an explanation for the disproportionate prostanoid synthesis from unesterified AA liberated from endogenous stores in day 7 cells. This possibility was addressed by determining the conversion of exogenously supplied AA to PGE, and 6-k-PGF1,(as an estimate of cyclooxygenase activity independent of a requirement for prior deacylation [lo]) in the presence and absence of 50 nM PMA. RIA (Table 5) was used to quantitate the two prostanoids synthesized from exogenously supplied unlabeled AA (1 and 5 pg/mL), and HPLC with scintillation counting of eluted fractions (Table 6) was used to separate and quantitate radiolabeled prostanoids synthesized from exogenously supplied [14C]AA(0.2 pCi = 1 pg/mL final concentra-
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Table 4 Effect of Staurosporine on [14C]AALevels in Day 7 Epithelial Cells Stimulated with 50 nM PMA Staurosporine (nM)
["C] AA (% of incorp. dpm)
0 5 20 50
0.46 0.02 0.34 f 0.03" 0.29 2 0.09" 0.19 f 0.07"
*
Data represent means k SEM from three separate experiments, each performed in duplicate. The values depicted represent the net quantities of [14C]AA attributable to PMA for each given dose of staurosporine. "P < .05 versus control (0 nM) value by analysis of variance.
tion). Day 7 cells incubated without PMA synthesized greater quantities of the major cyclooxygenase metabolites PGE, and 6-k-PGF,, from exogenous AA than did day 2 cells, in agreement with our previous observations [lo]. This was evident by both RIA (Table 5) and HPLC (Table 6) analyses. In day 2 cells, the addition of PMA resulted in a modest decrease in the conversion of AA to prostanoids as detected by both means of analysis. In day 7 cells, a similar phenomenon was observed for 6-k-PGF,,; however, effects on PGE, levels were inconsistent and noted to vary with the concentration of AA used and the analytical method employed. Total amounts of the two radiolabeled prostanoids and of unmetabolized radiolabeled AA were identical in day 7 cells incubated with and without PMA (Table 6), suggesting no net effect of PMA on cyclooxygenase activity.
Table 5 Effect of PMA on the Conversion of Exogenously Supplied AA to Immunoreactive Prostanoids in Day 2 and Day 7 Epithelial Cell Cultures PGE,
6-k-PGFk
Incubation
Day 2
Day 7
Day 2
1 pg/mL AA PMA + 1 pg/mL AA 5 pg/mL AA PMA + 5 pg/mL AA
2.89 f 0.24 2.54 f 0.43 9.60 0.54 6.72 0.71
19.37 f 2.12 15.43 f 2.10 17.39 2.22 27.56 f 5.05
3.10 2.30 5.92 4.09
* *
*
zk 0.01 f 0.27
f 0.26 f 0.24
Day 7 11.46 6.25 14.58 9.85
f 2.22 f 0.86 f 3.78 f 1.02
Day 2 or day 7 epithelial cell cultures were incubated for 1 h with the indicated concentration of AA in the presence or absence of 50 nM PMA. Data are expressed as ng/mL of immunoreactive eicosanoid, and represent the means k SEM of results from three separate experiments, each performed in duplicate.
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Table 6 Effect of PMA on the Conversion of Exogenously Supplied [14C]AAto ['4C]Prostanoidsin Day 2 and Day 7 Epithelial Cell Cultures Day 7
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Day 2 ['4C]Product
- PMA
+ PMA
- PMA
+ PMA
6-k-PGFP PGE, Free AA
2.88 2.89 85.84
1.28 1.44 93.37
3.22 4.43 86.55
2.37 5.48 86.21
Day 2 or day 7 epithelial cell cultures were incubated for 1 h with 0.2 pCi (- 1 pg/mL) [14C]AA in the presence or absence of 50 nM PMA. Medium was harvested and extracted, and radiolabeled eicosanoids were separated by HPLC and quantitated by scintillation counting. Each value represents the percentage of the total radiolabeled products co-eluting with the appropriate authentic standard. These results are from a single experiment that was representative of a total of three.
DISCUSSION Their diversity of actions implicate the oxygenated eicosanoid metabolites of AA in the modulation of inflammation, immune function, and fibrogenesis [27, 281. We first reported [l] that rat alveolar type I1 cells at 48 h in culture had the capacity to liberate AA and metabolize it predominantly via the cyclooxygenase pathway. Because type I1 cells cultured for several days on tissue culture plastic undergo morphologic and biochemical changes that resemble those observed as they transform into type I cells in vivo [9], and because alterations in arachidonate metabolism have been reported in association with differentiation of other cell types [2, 31, recently we sought to determine if this in vitro model of alveolar type 11 cell differentiation was accompanied similarly by alterations in AA metabolism. Analysis of the products synthesized by epithelial cells at 2, 4,and 7 days in culture revealed a marked increase in the deacylation of AA and in the synthesis of cyclooxygenase metabolites, both constitutively and in response to ionophore A23187, over time in culture [lo]. Evidence that this metabolic change was a function of alteration in phenotype, rather than merely a consequence of time in culture, was provided by demonstrating that cells cultured on fibronectin, which facilitates spreading and differentiation [29, 301, exhibited an even more rapid increase in AA release over time than did cells cultured in standard fashion on plastic. O n the other hand, cells cultured on an extracellular matrix derived from the Engelbreth-Holm-Swarm murine tumor, which preserves many of the phenotypic characteristics of type I1 cells [8], exhibited less of an increase in AA release over time than did cells cultured on plastic. Information about the mechanisms that regulate AA metabolism in alveolar pneumocytes is lacking, however. Therefore, we set out to determine the influence of one important regulatory mechanism, namely the activation of PKC, on the liberation and metabolism of AA in differentiat-
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ing epithelial cells. The approach utilized was to study the effects of PMA, a potent PKC activator, in both resting and A23187-stimulated cells. Responses to PMA were compared between pneumocytes cultured for 2 and 7 days, selected as representing phenotypes with limited and substantial capacities, respectively, for AA liberation and metabolism. Our results indicate that activation of PKC indeed triggered AA deacylation, as well as primed for ionophore-induced deacylation, in alveolar pneumocytes. Moreover, day 7 cells were substantially more responsive than were day 2 cells to the effects of PKC activation. Several lines of evidence lead us to conclude that the capacity of PMA to increase AA metabolism in alveolar epithelial cells was dependent on its capacity to activate PKC. (1) Arachidonate metabolic responses to PMA were observed over the range of concentrations that are typically adequate to activate this calcium- and phospholipid-dependent kinase in a variety of cell types [31]; in fact, the optimally effective concentration, 50 nM, was less than that (80 nM) demonstrated by Sano and co-workers [32] to activate PKC in rat alveolar pneumocytes. (2) Increases in free AA were not initiated by PDD, a phorbol ester that lacks the capacity to activate PKC [24]. (3) Both stimulation and priming of AA deacylation by PMA were abrogated by overnight pretreatment with a high dose of PMA. Similar pretreatment protocols have been shown to deplete both PKC activity and immunoreactivity in a variety of cell types [17, 181, presumably via proteolytic degradation that follows its translocation to the membrane [33]. The fact that an identical pretreatment protocol employing the inactive compound PDD failed to attenuate subsequent PMA-induced responses strongly suggests that the effects of PMA pretreatment were indeed dependent on depletion of PKC. (4) Finally, the potent inhibitor staurosporine inhibited PMA-induced AA liberation and PGE, synthesis at concentrations similar to those reported to inhibit activity of rat brain PKC [16]. However, it should be noted that typically effective concentrations (10-200 pM) of the diacyglycerol analog OAG, which in our hands was capable of triggering AA mobilization in peritoneal macrophages [34], failed to do so in alveolar pneumocytes (data not shown). This is reminiscent of the failure of OAG to translocate PKC from cytosol to membrane in rat alveolar type I1 cells [32], and most likely reflects the rapid degradation of OAG by diglyceride lipases in this particular cell type [32]. We have demonstrated previously that an in vitro model of alveolar epithelial cell differentiation is associated not only with increases in the deacylation of endogenous AA, but also with increases in cyclooxygenase activity, which are identifiable by monitoring the conversion of exogenously supplied fatty acid to prostanoid products [lo]. The result of this latter alteration is that a greater proportion of endogenous AA mobilized by A23187 is metabolized via the cyclooxygenase pathway in day 7 than in day 2 cells. This enhanced capacity for cyclooxygenase metabolism in day 7 cells there-
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fore could also explain the greater prostanoid synthesis from unesterified AA mobilized by stimulation with PMA at day 7 than at day 2. One additional possibility, however, was that this greater degree of “coupling” between arachidonate deacylation and cyclooxygenase metabolism in day 7 cells reflected further activation of the cyclooxygenase enzyme resulting from activation of PKC, as has been reported in renal epithelia [26]. However, experiments that directly compared cyclooxygenase activity in the presence and absence of PMA stimulation failed to provide support for such a mechanism. It is worth noting that besides activating existing cyclooxygenase, PKC activation has also been reported to induce de novo synthesis of the enzyme [26, 35, 361. The possibility exists, therefore, that PMA incubations longer than the 1 h utilized in these studies might result in augmentation of cyclooxygenase activity on the basis of new enzyme synthesis; however, such an effect was not examined in the present report. As compared with day 2 cells, day 7 pneumocytes exhibited greater AA metabolic responses to PKC activation. The mechanisms underlying increased responses in this model of epithelial cell differentiation remain to be defined. One possibility is that differentiation is itself associated with increased quantities of cellular PKC, which functions as the receptor for PMA. Precedent for this can be found in reports that increases in PKC activity accompany in vitro differentiation of both HL-60 cells [37] and keratinocytes [38]. Alternatively, pneumocyte differentiation in this model might be characterized by increasing dependence of AA metabolism on enzymatic mechanisms that are regulatable by activation of PKC. Consistent with this possibility is the observation that the marked degree of stimulation of AA liberation by A23187 in day 7 cells also was attenuated substantially by PKC depletion (Table 3). Finally, the mechanisms by which PKC activation increases levels of unesterified AA in alveolar pneumocytes are not known. Levels of free arachidonate reflect a balance between its deacylation from esterified stores within phospholipids as well as its reacylation back into phospholipids 1231. Activation of PKC has been shown in other cell types to both activate phospholipase A, [12], the major enzyme catalyzing AA deacylation [23], and to inhibit arachidonate reacylation enzymes [39, 401. Determining the enzymes that are regulated by PKC activation in differentiating alveolar epithelial cells is the subject for future investigation. Because alveolar pneumocytes comprise the alveolar surface, their capacity for liberation and metabolism of AA is likely to play an important role in the outcome of inflammatory events within the alveolar space. This likelihood is underscored by our recent observation that epithelial-derived AA is available for metabolism not only by epithelial cells themselves, but also by macrophages [41], and potentially by other inflammatory cells in the alveolar space as well. Hence, an understanding of the factors that regulate these metabolic processes is of substantial importance. PKC is a ubiquitous
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enzyme that is involved in receptor-mediated signal transduction, with resultant regulatory effects on a variety of cellular functions [31]. Its activation has been demonstrated previously to play a role in surfactant secretion by isolated pneumocytes [32]. Although the precise enzymatic mechanisms responsible are not yet known, this study establishes for the first time that AA availability and metabolism in alveolar pneumocytes, too, are regulated by activation of PKC. This is true in both resting and agonist-stimulated cells. The potential significance of PKC activation as a model regulatory system is that the actions of a variety of biologically important molecules, including lipopolysaccharide [42], interleukin-1 [43], and interferon, [44], are mediated, at least in part, via activation of PKC. Finally, it is possible that studies of epithelial cells that alter their phenotype in vitro may shed light on their changing metabolic repertoire as they differentiate in vivo in the setting of lung injury. The greater degree to which AA metabolism is regulated by PKC activation as epithelial cells differentiate might signify phenotype-dependent differences in the regulation of other metabolic processes as well. The authors thank Jill Loughney for secretarial assistance, Abdella Feyssa for technical assistance, and Richard Simon for review of the manuscript. This work was supported by Grant HL-01638 from the National Institutes of Health, and by a grant from the American Lung Association. M. Peters-Golden is the recipient of a Career Investigator Award from the American Lung Association.
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9. Dobbs LG: Isolation and culture of alveolar type I1 cells. Am J Physiol 258 (Lung Cell Mol Physiol) 2:L134-L147, 1990. 10. Lipchik RJ, Chauncey JB, Paine R, Simon RH, Peters-Golden M: Arachidonate metabolism increases as rat alveolar type I1 cells differentiate in vitro. Am J Physiol (Lung Cell Mol Physiol. 3) 259:L73-L80, 1990. 11. Parker J, Daniel L, Waite M: Evidence of protein kinase C involvement in phorbol diester-stimulated arachidonic acid release and prostaglandin synthesis. J Biol Chem 262:5385-5393, 1987. 12. Bonventre J, Swidler M: Calcium dependency of prostaglandin E2 production in rat glomerular mesangial cells: evidence that protein kinase C modulates the Ca”-dependent activation of phospholipase A2. J Clin Invest 82:168-176, 1988. 13. Levine L, Xiao D-M: The stimulations of arachidonic acid metabolism by recombinant murine interleukin 1 and tumor promotors or 1-oleoyl-2-acetylglycerol are synergistic. J Immunol 135:3430-3433, 1985. 14. Humes J, Sadowski S, Galavage M, Goldenberg M, Subers E, Bonney R, Kuehl F Jr: Evidence for two sources of arachidonic acid for oxidative metabolism by mouse peritoneal rnacrophages. J Biol Chem 257:1591-1594, 1982. 15. Dobbs L, Gonzalez R, Williams M: An improved method for isolating type I1 cells in high yield and purity. Am Rev Respir Dis 134:141-145, 1986. 16. Tamaoki T, Nomoto H, Takahashi I, Kato Y, Morimoto M, Tomita F: Staurosporine, a potent inhibitor of phospholipid/Ca+ dependent protein kinase. Biochem Biophys Res Commun 135:397-402, 1986. 17. Rodriguez-Pena A, Rozengurt E: Disappearance of Ca2+-sensitive, phospholipid-dependent protein kinase activity in phorbol ester-treated 3T3 cells. Biochem Biophys Res Commun 120:1053-1059, 1984. 18. Ballester R, Rosen OM: Fate of immunoprecipitable protein kinase C in GH3 cells treated with phorbol 12-myristate 13-acetate. J Biol Chem 260:1519415199, 1985. 19. Peters-Golden M, Bathon J, Flores R, Hirata F, Newcombe D: Glucocorticoid inhibition of zymosan-induced arachidonic acid release by rat alveolar macrophages. Am Rev Respir Dis 130:803-809, 1984. 20. Peters-Golden M, Shelley C: Inhibitory effect of exogenous arachidonic acid on alveolar macrophage 5-lipoxygenase metabolism: role of ATP depletion. J Immunol 140:1958-1966, 1988. 21. Peters-Golden M, Thebert P: Inhibition of methylprednisolone of zymosaninduced leukotriene synthesis in alveolar macrophages. Am Rev Respir Dis 135:1020-1026, 1987. 22. Snedecor G, Cochran W: Statistical Methods. Ames, Iowa State UP, 1967. 23. Irvine R: How is the level of free arachidonic acid controlled in mammalian cells? Biochem J 204:3-16, 1982. 24. Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U, Nishizuka Y: Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters J Biol Chem 257:7847-7851, 1982. 25. Cochrane C: The enhancement of inflammatory injury (editorial). Am Rev Respir Dis 136:l-2, 1987. 26. Beaudry GA, Wake M, Daniel LW: Regulation of arachidonic acid metabolism in Madin-Darby canine kidney cells: stimulation of synthesis of the cyclooxy+
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genase system by ~2-O-tetradecanoyl-phorbol-~3-acetate. Arch Biochem Biophys 239:242-247, 1985. 27. Henderson W Jr: Eicosanoids and lung inflammation. Am Rev Respir Dis 135:1176-1185, 1987. 28. Kunkel S: The importance of arachidonate metabolism by immune and nonimmune cells (editorial). Lab Invest 58:119, 1988. 29. Woodcock-Mitchell J, Rannels S, Mitchell J, Rannells D, Low R: Modulation of keratin expression in type I1 pneumocytes by the extracellular matrix. Am Rev Respir Dis 139:343-351, 1989. 30. Clark R, Mason R, Folkvord J, McDonald J: Fibronectin mediates adherence of rat alveolar type I1 epithelial cells via the fibroblastic cell attachment domain. J C h Invest 77:1831-1840, 1986. 31. Nishizuka Y:Studies and perspectives of protein kinase C. Science 233:305-312, 1986. 32. Sano K,Voelker DR, Mason RJ: Involvement of protein kinase C in pulmonary surfactant secretion from alveolar type I1 cells. J Biol Chem 260:12725-12729, 1985. 33. Witters L, Blackshear P: Protein kinase C-mediated phosphorylation in intact cells. Methods Enzymol 141:412-424,1987. 34. Peters-Golden M, McNish RW, Brieland JK, Fantone JC: Diminished protein kinase C-activated arachidonate metabolism accompanies rat macrophage differentiation in the lung. J Immunol 144:4320-4326, 1990. 35. Goerig M, Habenicht AJR, Heitz R, Zeh W, Katus H , Kommerell B, Ziegler R, Glomset JA: sn-1,2-Diacylglycerols and phorbol diesters stimulate thromboxane synthesis by de novo synthesis of prostaglandin H synthase in human promyelocytic leukemia cells. J Clin Invest 79:903-911, 1987. 36. Wu K, Hatzakis H , Lo S, Seong D, Sanduja S: Stimulation of de novo synthesis of prostaglandin G / H synthase in human endothelial cells by phorbol ester. J Biol Chem 263:19043-19047, 1988. 37. Makowske M, Ballester R, Cayre K, Rosen OM: Immunochemical evidence that three protein kinase C isozymes increase in abundance during HL-60 differentiation induced by dimethyl sulfoxide and retinoic acid. J Biol Chem 263:3402-3410, 1988. 38. Jaken S, Yuspa SH: Early signals for keratinocyte differentiation: role of Caz'mediated inositol lipid metabolism in normal and neoplastic epidermal cells. Carcinogenesis 9:1033-1038, 1988. 39. Pfannkuche H-J, Kaever V, Gemsa D, Resch K: Regulation of prostaglandin synthesis by protein kinase C in mouse peritoneal macrophages. Biochem J 260:471-478, 1989. 40. Fuse I, Iwanaga T, Tai H-H: Phorbol ester, 1,2-diacylglycerol, and collagen induce inhibition of arachidonic acid incorporation into phospholipids in human platelets. J Biol Chem 264:3890-3895, 1989. 41. Chauncey JB, Simon RH, Peters-Golden M: Rat alveolar macrophages synthesize leukotriene B4 and 12-hydroxyeicosatetraenoicacid from alveolar epithelial cell-derived arachidonic acid. Am Rev Respir Dis 138:928-935, 1988. 42. Wightman P, Raetz C: The activation of protein kinase C by biologically active lipid moieties of lipopolysaccharide. J Biol Chem 259:10048-10052, 1984.
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43. Raz A, Wyche A, Needleman P: Temporal and pharmacological division of
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fibroblast cyclooxygenase expression into transcriptional and translational phases. Proc Natl Acad Sci USA 86:1657-1661, 1989. 44. Fan X-D, Goldberg M, Bloom B: Interferon-g-induced transcriptional activation is mediated by protein kinase C. Proc Natl Acad Sci USA 855122-5125, 1988.
ISSN: 0190-2148 (Print) 1521-0499 (Online) Journal homepage: http://www.tandfonline.com/loi/ielu20
Protein Kinase C Activation Modulates Arachidonic Acid Metabolism in Cultured Alveolar Epithelial Cells Marc Peters-Golden, Kathie Coburn & James B. Chauncey To cite this article: Marc Peters-Golden, Kathie Coburn & James B. Chauncey (1992) Protein Kinase C Activation Modulates Arachidonic Acid Metabolism in Cultured Alveolar Epithelial Cells, Experimental Lung Research, 18:4, 535-551, DOI: 10.3109/01902149209064344 To link to this article: http://dx.doi.org/10.3109/01902149209064344
Published online: 02 Jul 2009.
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Date: 21 May 2016, At: 22:50
Protein Kinase C Activation Modulates Arachidonic Acid Metabolism in Cultured Alveolar Epithelial Cells
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Marc Peters-Golden, Kathie Coburn, and James B. Chauncey
ABSTRACT: Cultured alveolar type 11 cells can liberate esterified arachidonic acid (AA)and metabolize it predominantly via the cyclooxygenasepathway, and their capacity to do so increases as they alter their phenotpe over time in culture. Little is known, however, about the regulation of AA metabolism in alveolar pneumocytes. We have examined the effects of protein kinase C (PKC) activation on arachidonate metabolism in primary cultures of rat alveolar epithelial cells studied at 2 and 7 days following isolation. B e potent PKC activator phorbol myristate acetate (PMA) stimulated dose-dependent increases in free A A levels in both day 2 and day 7 cultures, with optimal stimulation at 50 nM. Greater stimulation was demonstrated for day 7 cells, and this was associated with greater prostanoid synthesis in response to PMA by day 7 than by day 2 cells. The capacity of PMA to “prime”epithelial cells for augmented AA liberation and metabolism in response to calcium ionophore A23187 (5 fl)was examined also. Significant priming by PMA was observed i n both day 2 and day 7 cells; once again, augmentation of both free A A levels as well as prostaglandin E, levels was greater for day 7 cells than for day 2 cells. That the capacity of PAL4 to modulate AA metabolism was mediated by activation of PKC was confirmed by demonstrating that (I) phorbol didecanoate, which lacks the ability to activate PKC, failed to activate AA metabolism; (2) pretreatment for 18 h with 1 f l PMA,which depletes cellular PKC, abolished subsequent AA metabolism activated by 50 nM PMA; and (3) the PKC inhibitor staurosporine abrogated increases in the quantities of both fyee AA and prostaglandin E2 in response to PMA. We conclude that activation of PKC increases the availability of AA for prostanoid synthesis in alveolar pneumocytes, and that this ejfict is more evident as t p e 11 cell differentiation is modeled during prolonged cultivation.
From the Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan and Kterans Affairs Medical Centers, Ann Arbor, Michigan. Address all correspondence to Marc Peters-Golden, MD, Division of Pulmonary and Critical Care Medicine, 3916 Taubman Center, University of Michigan Medical Center, Ann Arbor, MI 48109-0360. Received 17 July 1991; accepted 12 November 1991.
Experimental Lung Research 18:535-551 (1992) Copyright 0 1992 by Hemisphere Publishing Corporation
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INTRODUCTION Immune and inflammatory processes occurring within the pulmonary alveolar space are thought to be of primary importance in the initiation and eventual outcome of acute lung injury, as well as of chronic lung disorders such as pulmonary fibrosis. Traditional “effector” cells, including macrophages, lymphocytes, and neutrophils, have been studied extensively in an effort to delineate the cellular mechanisms of alveolar inflammation. However, the participation of the alveolar epithelium itself in such events is poorly understood. This is true despite the fact that alveolar epithelial cells, or pneumocytes, are situated in a position where they have the potential to respond to inhaled agents, as well as to interact with traditional effector cells via the elaboration of mediators. In this regard, previous studies from our laboratory have demonstrated that rat type I1 pneumocytes studied at 48 h in culture have the capacity to deacylate esterified arachidonic acid (AA) and subsequently metabolize it, predominantly via the cyclooxygenase pathway, to bioactive eicosanoids [I]. In several cells types, alterations in AA metabolism have been shown to accompany cellular differentiation [2, 31. In vivo, the type I1 pneumocyte, which is known best for its capacity to secrete pulmonary surfactant, serves as the progenitor for the type I cell, the cell that lines the vast majority of the alveolar surface [4]. When cultured for several days on tissue culture grade plastic, type I1 cells assume a phenotype that closely resembles that of a type I cell in vivo with respect to morphologic and biochemical criteria [5-91. We have demonstrated recently that this in vitro model of differentiation is accompanied by marked increases in both the liberation and cyclooxygenase metabolism of AA [lo]. Little information is available, however, regarding the regulation of arachidonate metabolism in alveolar epithelial cells, or whether alterations in regulation might accompany in vitro differentiation. In a variety of cell types, activation of protein kinase C (PKC) augments both the deacylation of AA as well as its metabolism via the cyclooxygenase pathway [ll-141. In the present study, therefore, we have examined the effects of PKC activation on AA metabolism in differentiating alveolar epithelial cells.
METHODS Alveolar Epithelial Cell Isolation and Culture Type I1 alveolar epithelial cells were isolated from male specific pathogenfree Sprague-Dawley rats (Charles River, Portage, MI) weighing 180-200 g by standard techniques [15]. Cells were suspended in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY) containing 10% fetal bovine serum with penicillin G (100 U/mL), streptomycin (100 pg/mL),
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and amphotericin B (0.25 pg/mL) (subsequently referred to as serumcontaining medium) at lo6 cells/mL, then aliquotted to 24-well tissue culture plates (Falcon, Oxnard, CA) at l mL/well and cultured in 5% C0,:air at 37OC. We have demonstrated previously that at 48 h these cultures comprise a confluent monolayer of approximately 3 x lo5 cells/well, of which 92-94'10 are type I1 cells, and contain -70 pg of total protein/well [lo]. Viability exceeded 90% as assessed by trypan blue exclusion. Because culture on tissue culture plastic in serum-containing medium for 7 d permits type I1 cells to assume a type I-like phenotype with respect to a variety of markers [9] and also to maximally up-regulate their capacity for arachidonate liberation and metabolism [lo], cells were studied at 2 and 7 d in culture. Cells to be studied at day 7 had their serum-containing medium changed at days 2 and 4. Cell number, protein content, purity, and viability of cultures have been shown to be stable during the 7-d culture period [lo]. Prelabeling of Epithelial Cell Cultures with [l4C]AA
To radiolabel epithelial cell lipids, serum-containing medium was removed from cultures approximately 18 h prior to experimental incubations and replaced with 1 mL/well of the same medium containing 0.2 pCi (3.6 pM) of [l - 14C]AAhaving a specific activity of 54-57 mCi/mmol (Dupont, New England Nuclear, Boston, MA) [lo]. After labeling for 18 h, medium was removed and monolayers were washed three times with Hanks' balanced salt solution (Gibco) prior to experimental incubations. In parallel with the experimental incubations, prelabeled cells from two additional wells in each experiment were lysed instead in methanol and subjected to scintillation counting to determine the cellular uptake of radiolabel. Uptake of radiolabe1 (mean f. SEM) in day 2 and day 7 cells was 57.2 2 3.1% and 56.6 f. 3.0°/o, respectively, of that added ( n = 11). Experimental Incubations At the indicated experimental time point of 2 or 7 d, prelabeled or unlabeled pneumocyte cultures from a single isolation were washed three times with Hanks' solution and incubated for 1 h with serum-free DMEM alone, DMEM containing the PKC activators phorbol 12-myristate 13-acetate (PMA), or 1-oleoyl-2-acetylglycerol (OAG), or DMEM containing the inactive phorbol ester 4-a-phorbol 12,13-didecanoate (PDD). The ability of PMA or PDD to prime cells for augmented AA metabolism in response to calcium ionophore A23187 was examined by incubating cells for 1 h with 5 pM A23187 [l, 101 in the absence and presence of the phorbol ester (following a 10-min pretreatment with PMA or PDD). The role of PKC activation in mediating PMA-induced responses was assessed by determining the ability of PMA to trigger AA metabolism or prime for ionophore-stimulated
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AA metabolism following either a 10-min pretreatment and subsequent coincubation with the potent PKC inhibitor staurosporine 1161, or an 18-h pretreatment with 1 pM PMA (to down-regulate PKC by its depletion from the cell [17, 181) or PDD. Finally, the effect of PKC activation on cyclooxygenase activity was determined by incubating cells for 1 h with 20 pg/mL AA in the presence or absence of PMA. PMA, OAG, PDD (all from Sigma, St. Louis, MO), A23187 (Calbiochem, San Diego, CA), and staurosporine (Kamiya Biomedical, Thousand Oaks, CA) were prepared in DMSO and freshly diluted into DMEM immediately before use. Final concentrations of DMSO were I0.7%, and had no effect on either cell viability or arachidonate metabolism. Unlabeled AA (Nu-Chek Prep, Elysian, MN) was prepared in sodium hydroxide and diluted prior to use.
Quantitation of Free ["CIAA In experiments designed to assess levels of free [14C]AA, incubations were carried out in the presence of 1 pM indomethacin to inhibit cyclooxygenase metabolism. Indomethacin (Sigma) was prepared in ethanol and diluted to yield a final ethanol concentration of 0.05%. Following experimental incubations, lipids were extracted from cells plus medium with chloroform/methanol (21, v/v), and separated by thin-layer chromatography (TLC) on Silica Gel 60 plates developed with hexane/diethyl ether/acetic acid (70:30:2), as described [19]. Labeled free AA was identified by co-migration with authentic AA, quantitated by scintillation counting, and expressed as a percentage of the incorporated radiolabel. For experiments in which cells were labeled during pretreatment with phorbol esters, free AA levels were expressed relative to uptake determined under the same conditions. For each experiment, the average of duplicate determinations was calculated.
Quantitation of Eicosanoids Eicosanoids were extracted from medium of unlabeled or prelabeled cultures using C,, Sep-Pak cartridges (Waters Associates, Milford, MA) as previously described [20]. Recoveries for AA and prostanoids exceed 90% by this method. Radiolabeled AA and eicosanoids were separated by reverse-phase, high-performance liquid chromatography (HPLC) of the pooled lipid extracts from 3-5 wells using the mobile phase acetonitrile/water/trifluoroacetic acid at a flow rate of 1 mL/min [21]. Products were identified by co-elution with authentic standards, quantitated by scintillation counting, and expressed as a percentage of incorporated label. Prostanoid standards were obtained from the Upjohn Co. (Kalamazoo, MI); 12hydroxyheptadecatrienoic acid (HHT) from Cayman Chemical (Ann Arbor, MI); and lipoxygenase standards from Merck Frosst Inc. (Dorval,
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Quebec, Canada). Prostaglandin E, (PGE,) and 6-keto-prostaglandin F,, (6-kPGF,,), the stable metabolite of prostacyclin, were quantitated in the medium of unlabeled cultures by radioimmunoassay (RIA). Sep-Pak extracts were dried and dissolved in 1 mL of phosphate-buffered saline containing 0.1% gelatin, and 0.l-mL aliquiots were assayed in duplicate. The source, sensitivity, and cross-reactivity of the antibodies employed have been described [I]. Concentrations of immunoreactive prostaglandins were corrected for recovery. In all instances, the average of duplicate determinations was calculated.
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Data Analysis Data were expressed as means k SEM. The significance of differences between the means of more than two groups was assessed by analysis of variance and the Newman-Keuls multiple range test [22]. The significance of differences between the means of identically treated day 2 versus day 7 cultures from a single isolation was assessed by a paired Student’s t test [22]. A P value < .05 was considered significant. RESULTS Effect of PMA on Alveolar Pneumocyte AA Metabolism Since the deacylation of AA from cellular lipids is the initial and probably rate-limiting step in AA metabolism [23], we quantitated levels of the unesterified I4C-labeledfatty acid by TLC in day 2 and day 7 cultures of prelabeled pneumocytes incubated with the potent PKC activator, PMA. Preliminary experiments indicated that at both of these time points, incubation with PMA over the concentration range 5-100 nM resulted in a dosedependent increase in the availability of free AA, which plateaued at a PMA concentration of 50 nM. Therefore, this dose of PMA was selected for subsequent comparisons with unstimulated cultures. Table 1 presents the effects of 50 nM PMA on free AA levels in day 2 and day 7 cultures. Unstimulated cultures contained low levels of free [I4C]AA; as reported previously [lo], quantities of free fatty acid in day 7 cultures exceeded those in day 2 cultures. The presence of 50 nM PMA significantly (P < .02) increased levels of [14C]AA over control levels in both day 2 cells and day 7 cells ( n = 7). The degree of stimulation in day 7 cells (192 k 35% of control) was greater than that in day 2 cells (158 k 18% of control), but not significantly. In contrast to the stimulatory effects of PMA, no dose-dependent increases in free [14C]AAlevels were observed in response to PDD, a phorbol ester that fails to activate PKC [24], at either day 2 (control, 0.28%; 5 nM PDD, 0.23%; 50 nM PDD, 0.28%; and 500 nM PDD, 0.30%) or day 7 (control,
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Table 1 Effect of PMA on [14C]AALevels in Resting and A23187-Simulated Epithelial Cells at 2 and 7 Days in Culture Day 2 [14C]AA
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Control PMA (50 nM) A23187 (5 pM) A23187 + PMA
0.32 f 0.05 0.47 f 0.05" 1.46 f 0.56 1.95 f 0.54'
OO /
(158 f 18)b (164 f 26)d
Day 7 [14C]AA 0.88 1.48 2.82 6.46
0%
f 0.26 f 0.31" f 1.24
(192 f 35)b
f 2.41'
(269 f 40)"'
Data are means & SEM of results from seven separate experiments, each determined in duplicate. [14C]AA levels are expressed as a percentage of total incorporated radioactivity. 'P < .O2 versus appropriate unstimulated control value by paired Student's t test. bValues represent the percentages of the appropriate control values. 'P < .05 versus appropriate A23187 alone value. dValues represent the percentages of the appropriate A23187 alone values. 'When expressed as a percentage of the AA levels observed with A23187 stimulation alone, the degree of priming was significantly greater (P < .05) in day 7 cells than in day 2 cells by paired Student's t test.
0.56%; 5 nM PDD, 0.62%; 50 nM PDD, 0.55%; and 500 nM PDD, 0.74%). The effect of 50 nM PMA on ['4C]eicosanoid synthesis by prelabeled epithelial cell cultures was assessed by HPLC (Fig. 1). Unstimulated day 2 cultures (Fig. la) released small amounts of radioactivity corresponding to free AA, PGE,, and 6-k-PGF1,, the stable metabolite of prostacyclin (PGI,). Analysis of day 2 cells incubated with 50 nM PMA (Fig. lb) revealed modest increases in the amounts of radiolabeled free AA, PGE,, and 6-k-PGF1,. As suggested by the TLC data shown in Table 1, and as reported previously [lo], unstimulated day 7 cells (Fig. lc) contained peaks of radioactivity corresponding to free AA, PGE,, and 6-k-PGF1, that were larger than those observed for day 2 cells. Incubation of day 7 cells with 50 nM PMA (Fig. Id) resulted in a marked augmentation over control levels in the quantities of radioactivity corresponding to the cyclooxygenase eicosanoids PGE, and 6k-PGF,,, as well as HHT, a nonenzymatic breakdown product of endoperoxides generated during cyclooxygenation. Of note, a greater proportion of the radioactivity accumulating in the medium in response to PMA stimulation co-eluted with cyclooxygenase products, as opposed to free AA, in day 7 cells than in day 2 cells. Differences in eicosanoid synthesis in response to PMA between day 2 and day 7 cells were confirmed by measuring immunoreactive levels of the major metabolite, PGE,. Once again, 50 nM proved to be an optimal concentration of the phorbol ester (data not shown). As shown in Table 2, incubation with 50 nM PMA resulted in minimal increases in PGE, synthesis over control levels in day 2 cells ( P > .05), but in significant increases over resting levels in day 7 cells ( P < .05).
54 1
Pneumocyte Arachidonate Metabolism and PKC
:.I
0 12
k
c l D
al
L
E 0 P
Day 2
a.
I
- Control
0.10 0.08
0 06
0.06
0 0
0.04
.c
0
0.12
6-h-PGFla .
0.00 0 O 02
O
4
1
0.02
$ 0.00 0
20
40
0 12
E a
U
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b.
80
60
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- PMA 0.10
0 10
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0 08
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AA
.-
0 04
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.-
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J -
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O 06 0 o8I
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a-" 0.00
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40
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0
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Figure 1 Representative radioactivity HPLC elution profiles from prelabeled day 2 (u and b) and day 7 (c and d) pneumocytes incubated for 1 h with medium alone (u and c) o r medium containing 50 nM PMA ( b and d). For each condition, medium from triplicate wells was pooled and extracted prior to HPLC analysis. Radioactivity in each fraction was expressed as a percentage of the total radioactivity incorporated into cells prior to stimulation. Peaks were designated on the basis of co-elution with authentic standards.
Table 2 Effect of PMA on Immunoreactive PGE, Synthesis by Resting and A23187-Stimulated Epithelial Cells at 2 and 7 Days in Culture Day 2 ng/mL Control PMA (50 nM) A23187 (5 pM) A23187 + PMA
*
0.78 f 0.13 0.79 f 0.19 1.28 ! I0.28 1.28 f 0.30
OO /
(98 f lo>. (99 f 5)'
Day 7 ng/mL 1.36 f 0.30 2.94 f 0.68 10.40 f 2.10 14.18 f 2.18
YO
(219) f 39Yb (140 f 9)b3c
Data are means SEM of results from three separate experiments, each determined in duplicate. "Values represent the percentages of the appropriate control values. 'I' < .05 versus relevant day 2 value by paired Student's t test. 'Values represent the percentages of the appropriate A23187 alone values.
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Effect of PMA on Ionophore-Stimulated AA Metabolism by Alveolar Pneumocytes We have shown previously that the calcium ionophore A23187 stimulates the liberation and subsequent metabolism of AA in alveolar pneumocytes [I], and that day 7 cells are more responsive to A23187 stimulation than are day 2 cells [lo]. The capacity of PMA to augment A23187-induced AA deacylation was examined by TLC analysis of prelabeled cultures. An ionophore concentration of 5 pM was selected for these experiments, since it has been shown previously to yield maximal responses in cells at both days 2 and 7 [lo]. As also depicted in Table 1, PMA at 50 nM significantly increased levels of free AA over those measured in response to 5 pM ionophore alone in both day 2 and day 7 cells. At both days studied, the free fatty acid levels measured in cultures incubated with A23187 plus PMA exceeded the sum of the responses due to A23187 and 50 nM PMA individually. This synergistic augmentation therefore can be considered true “priming” [25]. Moreover, the degree of priming in day 7 cells (269 k 40% of [14C]AA levels in response to A23187 alone) was significantly greater ( P < .05) than that observed in day 2 cells (164 26% of A23187 alone). The inactive phorbol ester PDD failed to augment ionophore-induced AA deacylation (not shown). Table 2 includes data on the effect of PMA on immunoreactive PGE, levels stimulated by A23187. PMA failed to augment ionophore-stimulated PGE, synthesis in day 2 cells, but did so in day 7 cultures.
*
Dependence of PMA-Induced Responses on PKC Activation The ability of PMA but not PDD to activate AA deacylation and metabolism in alveolar pneumocytes suggests that responses to PMA are a function of the capacity of this agent to activate PKC. We therefore utilized two other approaches to substantiate the PKC dependence of PMA-induced effects on arachidonate metabolism. First, we made use of the fact that in a variety of cell types, prolonged incubation with high doses of PMA ultimately results in down-regulation of PMA-induced responsiveness [ 17, 181. Thus we examined the ability of PMA to trigger AA liberation as well as to augment A23 187-induced liberation in day 7 cells whose PKC was depleted by 18-h pretreatment with 1 pM PMA. The data presented in Table 3 demonstrate that such pretreatment completely abrogated the ability of 50 nM PMA to both trigger and prime AA liberation in prelabeled day 7 cultures. No cytotoxicity was observed following this pretreatment protocol, and uptake of radiolabel was not affected consistently. In contrast, 18-h pretreatment with 1 pM PDD had no such down-regulatory effect on the ability of PMA to increase subsequently the availability of free [I4C]AA in cells incubated with and without A23 187. Interestingly, pretreatment with PMA also
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Table 3 Effect of 18-H Pretreatment with Phorbol Esters on [I4C]AA Levels in Cultures of Day 7 Epithelial Cells ~~~
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18-h Pretreatment
Incubation
None
1 pM PMA
Control PMA (50 nM) A23187 (5 pM) A23187 + PMA
0.41 f 0.01 0.88 f 0.04' 1.51 f 0.03 3.23 k 0.04"
0.42 0.36 0.99 0.91
f 0.02 k 0.02 f 0.03 f 0.01
1 pM PDD 0.41 2.09 1.77 3.86
f 0.05 f 0.29" k 0.14 f 0.25"
Data represent means f SEM from three separate experiments, each performed in duplicate. [I4C]AA levels are expressed as a percentage of total incorporated radioactivity. "P < .01 versus the non-PMA value immediately above it by analysis of variance.
partially attenuated the response to A23187 alone in day 7 cells. Second, we examined the effect of a potent inhibitor of PKC activation, staurosporine [16], on PMA-stimulated AA release and PGE, synthesis (see Table 4).When prelabeled day 7 cells were pretreated with staurosporine for 10 min and then co-incubated with staurosporine and PMA (50 mM) for 1 h, the increase in [I4C]AAlevels attributable t o PMA was attenuared significantly in a dose-dependent manner by the inhibitor with a half-maximal inhibitory concentration of 5-10 nM. In addition, the capacity of 50 nM PMA to initiate synthesis of immunoreactive PGE, by unlabeled day 7 cultures was inhibited largely by 50 nM staurosporine (control, 1.97 ng/mL; PMA, 4.00 ng/mL; PMA + staurosporine, 2.59 ng/mL). Effect of PMA on Cyclooxygenase Activity of Alveolar Epithelial Cells Although PMA was only slightly more potent a stimulus for the liberation of AA in day 7 cells than in day 2 cells, it was considerably more effective at initiating prostanoid synthesis in day 7 cells (see Table 2). Because PKC activation by PMA has been shown to augment the activity of existing cyclooxygenase enzyme in renal epithelial cells [26], we considered such a mechanism as an explanation for the disproportionate prostanoid synthesis from unesterified AA liberated from endogenous stores in day 7 cells. This possibility was addressed by determining the conversion of exogenously supplied AA to PGE, and 6-k-PGF1,(as an estimate of cyclooxygenase activity independent of a requirement for prior deacylation [lo]) in the presence and absence of 50 nM PMA. RIA (Table 5) was used to quantitate the two prostanoids synthesized from exogenously supplied unlabeled AA (1 and 5 pg/mL), and HPLC with scintillation counting of eluted fractions (Table 6) was used to separate and quantitate radiolabeled prostanoids synthesized from exogenously supplied [14C]AA(0.2 pCi = 1 pg/mL final concentra-
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Table 4 Effect of Staurosporine on [14C]AALevels in Day 7 Epithelial Cells Stimulated with 50 nM PMA Staurosporine (nM)
["C] AA (% of incorp. dpm)
0 5 20 50
0.46 0.02 0.34 f 0.03" 0.29 2 0.09" 0.19 f 0.07"
*
Data represent means k SEM from three separate experiments, each performed in duplicate. The values depicted represent the net quantities of [14C]AA attributable to PMA for each given dose of staurosporine. "P < .05 versus control (0 nM) value by analysis of variance.
tion). Day 7 cells incubated without PMA synthesized greater quantities of the major cyclooxygenase metabolites PGE, and 6-k-PGF,, from exogenous AA than did day 2 cells, in agreement with our previous observations [lo]. This was evident by both RIA (Table 5) and HPLC (Table 6) analyses. In day 2 cells, the addition of PMA resulted in a modest decrease in the conversion of AA to prostanoids as detected by both means of analysis. In day 7 cells, a similar phenomenon was observed for 6-k-PGF,,; however, effects on PGE, levels were inconsistent and noted to vary with the concentration of AA used and the analytical method employed. Total amounts of the two radiolabeled prostanoids and of unmetabolized radiolabeled AA were identical in day 7 cells incubated with and without PMA (Table 6), suggesting no net effect of PMA on cyclooxygenase activity.
Table 5 Effect of PMA on the Conversion of Exogenously Supplied AA to Immunoreactive Prostanoids in Day 2 and Day 7 Epithelial Cell Cultures PGE,
6-k-PGFk
Incubation
Day 2
Day 7
Day 2
1 pg/mL AA PMA + 1 pg/mL AA 5 pg/mL AA PMA + 5 pg/mL AA
2.89 f 0.24 2.54 f 0.43 9.60 0.54 6.72 0.71
19.37 f 2.12 15.43 f 2.10 17.39 2.22 27.56 f 5.05
3.10 2.30 5.92 4.09
* *
*
zk 0.01 f 0.27
f 0.26 f 0.24
Day 7 11.46 6.25 14.58 9.85
f 2.22 f 0.86 f 3.78 f 1.02
Day 2 or day 7 epithelial cell cultures were incubated for 1 h with the indicated concentration of AA in the presence or absence of 50 nM PMA. Data are expressed as ng/mL of immunoreactive eicosanoid, and represent the means k SEM of results from three separate experiments, each performed in duplicate.
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Table 6 Effect of PMA on the Conversion of Exogenously Supplied [14C]AAto ['4C]Prostanoidsin Day 2 and Day 7 Epithelial Cell Cultures Day 7
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Day 2 ['4C]Product
- PMA
+ PMA
- PMA
+ PMA
6-k-PGFP PGE, Free AA
2.88 2.89 85.84
1.28 1.44 93.37
3.22 4.43 86.55
2.37 5.48 86.21
Day 2 or day 7 epithelial cell cultures were incubated for 1 h with 0.2 pCi (- 1 pg/mL) [14C]AA in the presence or absence of 50 nM PMA. Medium was harvested and extracted, and radiolabeled eicosanoids were separated by HPLC and quantitated by scintillation counting. Each value represents the percentage of the total radiolabeled products co-eluting with the appropriate authentic standard. These results are from a single experiment that was representative of a total of three.
DISCUSSION Their diversity of actions implicate the oxygenated eicosanoid metabolites of AA in the modulation of inflammation, immune function, and fibrogenesis [27, 281. We first reported [l] that rat alveolar type I1 cells at 48 h in culture had the capacity to liberate AA and metabolize it predominantly via the cyclooxygenase pathway. Because type I1 cells cultured for several days on tissue culture plastic undergo morphologic and biochemical changes that resemble those observed as they transform into type I cells in vivo [9], and because alterations in arachidonate metabolism have been reported in association with differentiation of other cell types [2, 31, recently we sought to determine if this in vitro model of alveolar type 11 cell differentiation was accompanied similarly by alterations in AA metabolism. Analysis of the products synthesized by epithelial cells at 2, 4,and 7 days in culture revealed a marked increase in the deacylation of AA and in the synthesis of cyclooxygenase metabolites, both constitutively and in response to ionophore A23187, over time in culture [lo]. Evidence that this metabolic change was a function of alteration in phenotype, rather than merely a consequence of time in culture, was provided by demonstrating that cells cultured on fibronectin, which facilitates spreading and differentiation [29, 301, exhibited an even more rapid increase in AA release over time than did cells cultured in standard fashion on plastic. O n the other hand, cells cultured on an extracellular matrix derived from the Engelbreth-Holm-Swarm murine tumor, which preserves many of the phenotypic characteristics of type I1 cells [8], exhibited less of an increase in AA release over time than did cells cultured on plastic. Information about the mechanisms that regulate AA metabolism in alveolar pneumocytes is lacking, however. Therefore, we set out to determine the influence of one important regulatory mechanism, namely the activation of PKC, on the liberation and metabolism of AA in differentiat-
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ing epithelial cells. The approach utilized was to study the effects of PMA, a potent PKC activator, in both resting and A23187-stimulated cells. Responses to PMA were compared between pneumocytes cultured for 2 and 7 days, selected as representing phenotypes with limited and substantial capacities, respectively, for AA liberation and metabolism. Our results indicate that activation of PKC indeed triggered AA deacylation, as well as primed for ionophore-induced deacylation, in alveolar pneumocytes. Moreover, day 7 cells were substantially more responsive than were day 2 cells to the effects of PKC activation. Several lines of evidence lead us to conclude that the capacity of PMA to increase AA metabolism in alveolar epithelial cells was dependent on its capacity to activate PKC. (1) Arachidonate metabolic responses to PMA were observed over the range of concentrations that are typically adequate to activate this calcium- and phospholipid-dependent kinase in a variety of cell types [31]; in fact, the optimally effective concentration, 50 nM, was less than that (80 nM) demonstrated by Sano and co-workers [32] to activate PKC in rat alveolar pneumocytes. (2) Increases in free AA were not initiated by PDD, a phorbol ester that lacks the capacity to activate PKC [24]. (3) Both stimulation and priming of AA deacylation by PMA were abrogated by overnight pretreatment with a high dose of PMA. Similar pretreatment protocols have been shown to deplete both PKC activity and immunoreactivity in a variety of cell types [17, 181, presumably via proteolytic degradation that follows its translocation to the membrane [33]. The fact that an identical pretreatment protocol employing the inactive compound PDD failed to attenuate subsequent PMA-induced responses strongly suggests that the effects of PMA pretreatment were indeed dependent on depletion of PKC. (4) Finally, the potent inhibitor staurosporine inhibited PMA-induced AA liberation and PGE, synthesis at concentrations similar to those reported to inhibit activity of rat brain PKC [16]. However, it should be noted that typically effective concentrations (10-200 pM) of the diacyglycerol analog OAG, which in our hands was capable of triggering AA mobilization in peritoneal macrophages [34], failed to do so in alveolar pneumocytes (data not shown). This is reminiscent of the failure of OAG to translocate PKC from cytosol to membrane in rat alveolar type I1 cells [32], and most likely reflects the rapid degradation of OAG by diglyceride lipases in this particular cell type [32]. We have demonstrated previously that an in vitro model of alveolar epithelial cell differentiation is associated not only with increases in the deacylation of endogenous AA, but also with increases in cyclooxygenase activity, which are identifiable by monitoring the conversion of exogenously supplied fatty acid to prostanoid products [lo]. The result of this latter alteration is that a greater proportion of endogenous AA mobilized by A23187 is metabolized via the cyclooxygenase pathway in day 7 than in day 2 cells. This enhanced capacity for cyclooxygenase metabolism in day 7 cells there-
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fore could also explain the greater prostanoid synthesis from unesterified AA mobilized by stimulation with PMA at day 7 than at day 2. One additional possibility, however, was that this greater degree of “coupling” between arachidonate deacylation and cyclooxygenase metabolism in day 7 cells reflected further activation of the cyclooxygenase enzyme resulting from activation of PKC, as has been reported in renal epithelia [26]. However, experiments that directly compared cyclooxygenase activity in the presence and absence of PMA stimulation failed to provide support for such a mechanism. It is worth noting that besides activating existing cyclooxygenase, PKC activation has also been reported to induce de novo synthesis of the enzyme [26, 35, 361. The possibility exists, therefore, that PMA incubations longer than the 1 h utilized in these studies might result in augmentation of cyclooxygenase activity on the basis of new enzyme synthesis; however, such an effect was not examined in the present report. As compared with day 2 cells, day 7 pneumocytes exhibited greater AA metabolic responses to PKC activation. The mechanisms underlying increased responses in this model of epithelial cell differentiation remain to be defined. One possibility is that differentiation is itself associated with increased quantities of cellular PKC, which functions as the receptor for PMA. Precedent for this can be found in reports that increases in PKC activity accompany in vitro differentiation of both HL-60 cells [37] and keratinocytes [38]. Alternatively, pneumocyte differentiation in this model might be characterized by increasing dependence of AA metabolism on enzymatic mechanisms that are regulatable by activation of PKC. Consistent with this possibility is the observation that the marked degree of stimulation of AA liberation by A23187 in day 7 cells also was attenuated substantially by PKC depletion (Table 3). Finally, the mechanisms by which PKC activation increases levels of unesterified AA in alveolar pneumocytes are not known. Levels of free arachidonate reflect a balance between its deacylation from esterified stores within phospholipids as well as its reacylation back into phospholipids 1231. Activation of PKC has been shown in other cell types to both activate phospholipase A, [12], the major enzyme catalyzing AA deacylation [23], and to inhibit arachidonate reacylation enzymes [39, 401. Determining the enzymes that are regulated by PKC activation in differentiating alveolar epithelial cells is the subject for future investigation. Because alveolar pneumocytes comprise the alveolar surface, their capacity for liberation and metabolism of AA is likely to play an important role in the outcome of inflammatory events within the alveolar space. This likelihood is underscored by our recent observation that epithelial-derived AA is available for metabolism not only by epithelial cells themselves, but also by macrophages [41], and potentially by other inflammatory cells in the alveolar space as well. Hence, an understanding of the factors that regulate these metabolic processes is of substantial importance. PKC is a ubiquitous
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enzyme that is involved in receptor-mediated signal transduction, with resultant regulatory effects on a variety of cellular functions [31]. Its activation has been demonstrated previously to play a role in surfactant secretion by isolated pneumocytes [32]. Although the precise enzymatic mechanisms responsible are not yet known, this study establishes for the first time that AA availability and metabolism in alveolar pneumocytes, too, are regulated by activation of PKC. This is true in both resting and agonist-stimulated cells. The potential significance of PKC activation as a model regulatory system is that the actions of a variety of biologically important molecules, including lipopolysaccharide [42], interleukin-1 [43], and interferon, [44], are mediated, at least in part, via activation of PKC. Finally, it is possible that studies of epithelial cells that alter their phenotype in vitro may shed light on their changing metabolic repertoire as they differentiate in vivo in the setting of lung injury. The greater degree to which AA metabolism is regulated by PKC activation as epithelial cells differentiate might signify phenotype-dependent differences in the regulation of other metabolic processes as well. The authors thank Jill Loughney for secretarial assistance, Abdella Feyssa for technical assistance, and Richard Simon for review of the manuscript. This work was supported by Grant HL-01638 from the National Institutes of Health, and by a grant from the American Lung Association. M. Peters-Golden is the recipient of a Career Investigator Award from the American Lung Association.
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43. Raz A, Wyche A, Needleman P: Temporal and pharmacological division of
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