Calcium-activated phosphatidylcholine-specific phospholipase C and D in MDCK epithelial MICHAEL Pulmonary University

W. PETERSON

AND MARY

ELIZABETH

Division, Department of Medicine, of Iowa, Iowa City, Iowa 52242

cells

WALTER

College of Medicine,

Refs. 6, 10). The hydrolysis of PC thus provides a more stable and sustained source of DAG in the cell. PC hydrolysis is increased by a number of agonists in different cells. These agonists include purinergic agents, platelet-derived growth factor, vasopressin, thrombin, and bradykinin (2, 13, 18, 20, 22). The nature of the activation pathways used by these agonists is not completely understood, however, and the pathways vary depending on the cells being studied. Several of these agonists also activate PI-specific PLC. PI-PLC leads to increased cell calcium (through inositol trisphosphate generation) and to increased PKC activity (through DAG generation). One explanation is that these agonists increase PC-PLC secondary to PKC activation. This explanation is supported by the finding that activating PKC with phorbol esters in some cells increases PCspecific PLC. Although some agonists do appear to activate PC hydrolysis through PKC, other pathways are also involved (6). Increased cell calcium is a candidate for another potential pathway for increasing PC hydrolysis. Two recent studies in epithelial cells, one in Madin-Darby canine kidney (MDCK) epithelial cells and another in rat type II epithelial cells, reported that calcium ionophore increases cellular DAG through PC hydrolysis (21). Calcium-activated PC-PLC or -PLD has also been reported in polymorphonuclear leukocytes (PMN), endothelial cells, mouse peritoneal macrophages, hepatocytes, and platelets (1, 3,7, 22, 23, 26). Although each of these cells increase PC-PLC or -PLD in response to increased intracellular calcium, the activation pathways differ among cells and among different stimuli. In PMN and endothelial cells, calcium ionophore activates mainly PLD. In PMN, calcium-reguDIGLYCERIDES (DAG) are important second messengers lated PLD activation depends on PKC (23), but similar in epithelial cells. Increased levels of DAG in epithelial data are not yet available in endothelial cells. In mouse cells affect ion transport, alter the barrier integrity of peritoneal macrophages, calcium activates PC-PLC epithelial monolayers, inhibit ciliary action, increase and calcium-activated PC-PLC mitotic indexes, and disrupt actin stress fibers (17, 19, rather than PC-PLD, occurs through a PKC-dependent pathway (26). In con24, 27). Although hydrolysis of phosphatidylinositol trast, calcium ionophore increases PC-PLC in hepato(PI) by phospholipase C (PLC) is a well-described cytes through a PKC-independent pathway (3). It is source of DAG in cells, recent studies have demonPC-PLC strated that phosphatidylcholine (PC) is also a source clear from these data that calcium-activated and -PLD pathways vary significantly among different for generating DAG in epithelial cells (6, 10, 21). Pathways generating DAG from PC are important for two cells. For that reason it is necessary to study the cell of interest to define which pathways are activated in that reasons. First, cells contain considerably more PC than PI; therefore, more DAG can be generated from the cell. Although epithelial cells contain both PC-PLC and hydrolysis of PC than can be generated from the hydrolPC-PLD, the activation pathways are incompletely unysis of PI. Second, DAG generated from the hydrolysis of PI activates protein kinase C (PKC). Activation of derstood. A previous study by Huang and Cabot (12) reported that activating PKC in MDCK cells with phorPKC further downregulates PI-specific PLC (PI-PLC) activity and limits the amount of DAG generated in the bol esters increases cellular DAG mainly through PLD activity. Exposing MDCK cells to calcium ionophore cell through this pathway (15). In contrast to PI-PLC, also increases DAG, but it is not yet known if this repactivating PKC with phorbol esters increases the activity of PC-specific PLC or phospholipase D (PLD; see resents PLC or PLD activity or whether this represents

Peterson, Michael W., and Mary Elizabeth Walter. Calcium-activated phosphatidylcholine-specific phospholipase C and D in MDCK epithelial cells. Am. J. Physiol. 263 (Cell Physiol. 32): C1216C1224 1992.-Calcium ionophore exposure generatesdiglycerides (DAG) from phosphatidylcholine (PC) hydrolysis in Madin-Darby canine kidney (MDCK) epithelial cells.This study comparescalcium ionophore-activated PC hydrolysis with the previously describedphorbol ester-stimulated PC hydrolysis pathway using MDCK cells labeledwith [14C]linoleic acid. Lipid speciesweremeasuredusingthin-layer chromatography. DAG resulted in part from PC hydrolysis because DAG increasedin cellslabeledwith [palmitoyl-2J4C]phosphatidylcholine. Neither protein kinase C (PKC) inhibitors nor PKC depletion affected the ionomycin (IONO)-induced increasein DAG. Ethylene glycol-bis(@-aminoethylether)-N,N,N’,N’-tetraacetic acid prevented the increasedDAG after ION0 but not after phorbol 12,13-dibutyrate (PDBu) exposure. The EGTA effect was reversed by adding excesscalcium but was not reversed by adding excessMg 2+. ION0 exposure also increased phosphatidic acid (PA) production. The PA was produced by phospholipaseD (PLD) becausephosphatidylethanol was produced when ION0 was added to the cells in the presenceof ethanol. Although increasingconcentrations of ethanol resulted in progressivelylessPA, it had no effect on increasedDAG after ION0 exposureat any time point tested. These data are consistent with both increased phospholipaseC (PLC) and increasedPLD activity following ionomycin. In contrast to ION0 exposure,ethanol completely prevented the increase in DAG after PDBu exposure,consistent with DAG produced by PLD activation. These results demonstrate that calcium activates both PC-specific PLC and PLD in MDCK cells and that the calcium-activated pathway is independentof the previously describedPKC activation pathways. protein kinase C; diglyceride; phosphatidic acid

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$2.00 Copyright

0 1992 The AmericanPhysiological Society

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CALCIUM-ACTIVATED

PC HYDROLYSIS

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MYR being incorporated into the cells.After 16 h, the medium containing the 14C-labeledfatty acid wasremoved,and the cells were washedtwo times with l-ml volumes of MEM without FBS to remove unincorporated label before the experiments. The washedcells were then exposedto MEM containing the different agonistsand were incubatedat the times noted at 37°C in 95% air-5% CO,. After exposure,the medium wasremoved, and the cellswere covered with ice-cold methanol and immediately placed on ice. Cellsfrom each well were then scrapedinto borosilicate tubes, and the lipids were extracted by the method of Bligh and Dyer (8). In experiments using [ 14C]PC,we removedthe mediumfrom confluent monolayersof MDCK cellsand replacedit with MEM plus 10% FBS containing 0.2 &i/ml [14C]PC. We briefly sonicated the [14C]PC before adding the medium to the cells to evenly dispersethe label in the aqueoussolution, and we incubated the cells for 2 h. We then removed the medium and washedthe cells to remove unincorporated label. In these experiments, 94.8 2 0.6% of the label was incorporated into the cells. The majority of the label was incorporated into PC (80.3 t 0.6%). No label was detected comigrating with PI or phosphatidylserine (PS), and 3.2 * 0.1%of the labelcomigratedwith phosphatidylethanolamine (PE). Neutral lipids contained 2.52 * 0.17% of the label, and the remainder of the label was in sphingomyelin. The labeled cells were exposed to ionomycin, and the lipids were extracted and separatedby thin-layer chromatography (TLC) as described.After we exposedthe labeled cells to IONO, only counts associatedwith PC decreased. Preparation of phosphutidylethanol standard. Phosphatidylethanol (PEtOH) waspreparedaspreviously described(12). In EXPERIMENTAL PROCEDURES brief, 78 mg egg PC was reacted with 2.5 ml cabbagePLD (containing 5.5 mgprotein) in 2.5 ml of a buffer composedof 0.2 Materials. [14C]linoleicacid (50 mCi/mmol) and [palmitoyl2-14C]lysophosphatidylcholine([ 14C]PC, 57 mCi/mmol) were M sodium acetate-acetic acid and 0.08 M CaCl,, pH 5.6, to purchasedfrom Du Pont-New England Nuclear. Cell culture which 200 ~1Triton X-100, 1.5 ml absoluteethanol, and 2.5 ml mediawasobtained from GIBCO, and fetal bovine serum(FBS) diethyl ether were added.The reaction was stirred overnight at waspurchasedfrom Sigma Chemical. PDBu, sphingosine,pro- 25°C. Lipids were extracted by the method of Bligh and Dyer pranolol, egg phosphatidylcholine, cabbage PLD, and ethyl- (8), and PEtOH wasseparatedfrom PC by preparative TLC on ene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid Silica Gel G plates in a solvent system using CHCL,-CH,OH(EGTA) were purchasedfrom Sigma; ionomycin (IONO) was NH,OH (65:30:3vol/vol/vol). The PEtOH was extracted from purchased from Calbiochem; 1-(5isoquinolinesulfonyl) -2-me- the plate again using the method of Bligh and Dyer (8). In thylpiperazine (H-7) was purchasedfrom Seikagaku (LaJolla, subsequentchromatography, the product resolved as a single CA); and staurosporinewas purchasedfrom Kamiya Biochem- band separatefrom PC. The product was dried under N, and ical (Thousand Oaks, CA). Solvents were obtained from Fischer stored at -20°C until resuspendedin CHCl, for use as a stanScientific, and Silica Gel LK5D plates were purchased from dard. Analysis of radiolabeled lipids. Total cellular lipids were exWhatman (Maidstone, UK). Standards for phospholipidsand neutral lipids were obtained from Sigma and Nu Chek Prep tracted as describedby the method of Bligh and Dyer (8). The (Elysian, MN). organic phasewascarefully collectedand dried under a constant Cell culture and radiolabeling. MDCK cells were a clone of stream of N,. The dried lipid wasresuspendedin 20 ~1CHCl,high-resistancecellskindly provided by Barry Gumbiner (Uni- CH30H (2:1 vol/vol) and spotted on LK5D silica gel TLC versity of California at San Francisco, CA). These cells were plates. Neutral lipids (diglyceride, free fatty acid, and triglycerusedin our previous studies of phospholipaseactivation (21). ide) were resolvedusing a solvent system composedof hexanediethyl ether-acetic acid (60:40:1 vol/vol/vol). PA was resolved Cellswere grown in Eagle’sminimal essentialmedium (MEM) supplementedwith 10% FBS, 100 U/ml penicillin, and 100 using a solvent system of CHCl,-pyridine-88% formic acid pg/ml streptomycin. Cellswere maintained on loo-mm culture (50:25:7 vol/vol/vol). PEtOH was resolvedusing a solvent sysplates (Costar) at 37°C in 95% air-5% CO, and passagedweekly tem consistingof CHC&-CH30H-NH40H (65:30:3vol/vol/vol). after exposureto trypsin-EDTA. Cellswere subcultured to six- All chromatography tanks were lined with Whatman paper for well tissue culture plates for experiments. MDCK cells in pas- the chromatography. The distribution of the radioactive label sages45-85 wereusedfor theseexperiments.After the cellshad was determined by scanningthe plate with a Radiomatic TLC achieved confluence, they were labeledwith [14C]linoleic acid plate reader. The area under the curve for each of the peakswas (LIN) or with [14C]myristic acid (MYR). The labeled fatty measured,and the percent of total lipid counts representedby acids were dried under NZ, converted to the sodium salt by each of the peaks was calculated for each sample.Authentic adding2 drops of 1 N NaOH, and resuspended in 1 ml hot water. standardswere run with each of the plates and visualized with This solution was addedto 14 ml MEM plus 10%FBS, and the exposureto I2 vapor after scanningthe plates. pH wasreturned to 7.4 by the dropwiseaddition of 0.5 N HCl. PKC assay. PKC activity was measured in vitro by the The medium, containing 0.2 &i/ml LIN or MYR was addedto method of Yasuda et al. (30) using a commercialkit purchased the cellsat 0.2 &i/well (4 nM) for 16 h in MEM plus 10% FBS from GIBCO. MDCK cellswere grown to confluencein 60-mm (vol/vol). Labeling in this way resulted in 97.5 & 0.5% of the tissue culture plates for these experiments. The medium was [14C]LIN being incorporated and in 94.9 t 0.5% of the [‘“Clremoved, and the cells were incubated with medium alone or

a different pathway from the pathway activated by PKC. We undertook the current study to define the PC-PLC or -PLD activation pathways in MDCK cells after calcium ionophore exposure. On the basis of studies in other cells, calcium-dependent DAG generation in MDCK cells could occur through at least three pathways. First, because some PKCs are calcium activated (l5), one explanation for the calcium-activated pathway is that it represents an alternative pathway for PKC activation with subsequent PC-PLC activation. Second, calcium could directly activate PC-PLC. Third, DAG could be generated by the sequential activity of PC-specific PLD (PC-PLD) followed by phosphatidic acid (PA) phosphohydrolase, as has been described after phorbol exposure (12). To address these questions, we performed studies comparing and contrasting DAG produced in MDCK cells after exposure to calcium ionophore with DAG produced after activating PKC with phorbol 12,13-dibutyrate (PDBu). We did additional studies to investigate the role of calcium and to address the relative activation of PC-PLC compared with PC-PLD. Our results demonstrate that calcium-dependent DAG generation is independent of PKC, depends on extracellular calcium, and is independent of PLD activation in MDCK cells.

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with the PKC inhibitors staurosporine,H-7, or sphingosinefor 30 min. For the prolonged PDBu exposure group, cells were incubated with 500 nM PDBu overnight before the experiment. These inhibitors were then removed, and the cells were incubated with mediumalone, with 500 nM PDBu, or with 500 nM PDBu plus one of the PKC inhibitors for 30 min. After 30 min, the medium wasremoved, and the cellswere rinsed three times with 3 ml phosphate-buffered saline (PBS). The monolayers were then scraped in 1 ml PBS, sonicated for 15 s, and the membraneswere collected by centrifugation at 100,000g for 20 min. The membranepellet wasthen resuspendedin 500 ~1cold extraction buffer containing 20 mM tris(hydroxymethyl)aminomethane(Tris), pH 7.5, 0.5 mM EDTA, 0.5% Triton X-100, 25 pg aprotinin, and 25 pg leupeptin. The membranesuspension wassonicatedon ice for 15s followed by incubation on ice for 30 min. Nonsoluble debris was removed by centrifugation at 100,000g for 20 min. After centrifugation, 100~1of eachsample wassavedfor protein determination by the method of Bradford usingthe commercialBio-Rad kit. Twenty-five microliters from each samplewere placed in each of two tubes. A lipid preparation (5 ~1) containing PS and phorbol ester in Triton X-100 mixed micelleswasaddedto eachtube, 10 ~1water wasaddedto one of each pair of tubes, and 10 ~1 PKC pseudosubstrateinhibitor peptide PKC-( 19-36) was added to the other paired tube. The solutions were incubated for 20 min at room air to allow the inhibitor to bind. [32P]ATP (containing - 1.5 x lo6 counts min- l-10 ~1~l) was added to each tube. The solution wasthen mixed and incubated for 5 min at 30°C. After 5 min, 25 ~1wasremovedand spotted on a phosphocellulosedisc. After eachof the sampleshad beenspotted, the discswerewashedtwo times in phosphoric acid (diluted 5 ml concentrated acid in 500 ml water) followed by two washesin water. Each of the phosphocellulosediscswere then placed in liquid scintillation vials, 5 ml scintillation cocktail was added, and the sampleswere counted in a liquid scintillation counter. [32P]ATP (10 ~1)was alsocounted to determine the specific activity. Specific PKC activity was calculated aspicomoles32Padded to the substrate in the absenceof the inhibitor minus the picomoles32Paddedto the substratein the presenceof the inhibitor. Each of the samples was corrected for the amount of protein present in the sample. SW&al analysis. Groups were analyzed using one-way analysisof variance using the Systat statistical software package. Post hoc testing among the groups was done using the Newman-Keulstest. Differences were consideredto be significant at P < 0.05. l

RESULTS

Role of PKC uctiuation.

MDCK cells incorporated 95 t over the 16-h incubation. Most of the label was incorporated into PC (53.5 t 0.3%) with 6.3 t 0.3% incorporated into PI, 4.3 t 0.1% into PS, and 13.9 t 0.1% into PE (Table 1). Exposing the cells to the calcium ionophore ION0 or to PDBu resulted in loss of label from PC (Table 1) and to an increase in DAG, as previously reported (Fig. 1). We previously reported that exposing these MDCK cells to the calcium ionophore A23187 did not increase PI hydrolysis (21). To further confirm that the DAG was produced by the hydrolysis of PC, we labeled MDCK cells with [ l*C]PC. When we exposed the MDCK cells labeled with [‘“C]PC to IONO, DAG increased, confirming our results with [ l*C]LIN (Fig. 2). These data, along with our previous studies looking for PI-PLC activity, confirm that some of the DAG produced after ION0 exposure comes from PC hvdrolvsis.

0.7% of the added [‘*C]LIN

PC HYDROLYSIS

Table 1. Distribution of [14C]linoleic acid among cellular lipids before and after exposing to ionomycin or to PDBu (%total counts in lipid species) Control

Ionomycin

PDBu

Phosphatidylinositol 6.3t0.3 7.ltO.l 6.8&O. 1 Phosphatidylserine 4.3tO.l 4.2tO.l 4.1,tO.l Phosphatidylcholine 53.5t0.3 49.5t0.5* 52.6t0.8 Phosphatidylethanolamine 13.9tO.l 13.0t0.7 13.9t0.3 Triglyceride 4.3t0.4 3.9kO.4 4.8t0.5 Free Fatty Acid 0.7to. 1 1.5t0.3* 0.9&O. 1 Values are means t SE for n = 6 experiments. Cells were labeled with [ 14C]linoleic acid for 16 h as described before exposing them to 3 PM ionomycin or to 500 nM phorbol 12,13-dibutyrate (PDBu) for 30 min before lipid analysis. * P < 0.01 compared with control.

To test whether ION0 acted through the previously described PKC pathway, cells were exposed to ION0 or to PDBu after being incubated with the PKC inhibitors H-7 (30 PM; see Ref. 14), staurosporine (100 nM; see Ref. 25), or sphingosine (100 PM; see Ref. 11). Each of these inhibitors stimulated some DAG accumulation themselves and inhibited further increases in DAG after PDBu. However, they had no effect on the IONO-induced increase in DAG (Fig. 1, A-C). These latter results were confirmed using cells labeled with [ ‘*C]PC (Fig. 2). Because each of the PKC inhibitors caused some increase in DAG themselves, we also depleted PKC by incubating the cells with PDBu for 16 h before exposing the cells to IONO. This prolonged incubation with PDBu had no effect on the incorporation of [l*C]LIN into the cells. Prolonged PDBu exposure had no effect on baseline DAG levels in the control cells but completely blocked the DAG increase after additional PDBu exposure. Like the PKC inhibitors, however, prolonged PDBu exposure had no effect on DAG levels produced after adding ION0 (Fig. 1D). The inability of the PKC inhibitors to block IONO-induced DAG generation was not due to incomplete PKC inhibition. Each of these inhibitors completely blocked cell PKC activity when measured by phosphorylation of the artificial PKC substrate Myelin Basic Protein (4-14) (Table 2; see Ref. 30). Role of extracellular cations. These initial data suggested that ION0 stimulated DAG accumulation through a PKC-independent pathway. Our next experiments evaluated the requirement for extracellular calcium in DAG generation after either ION0 or PDBu exposure. When the cells were exposed to ION0 in the presence of 2 mM EGTA (calcium concn of the media 1.2 mM), there was no increase in DAG (Fig. 3A). EGTA itself had no effect on DAG in the cells. Adding 2 mM EGTA to the media before adding PDBu, however, had no effect on the increase in DAG after PDBu exposure (Fig. 3B). The extracellular cation requirement of ION0 was further evaluated by adding excess Ca2+ or Mg2+ to the media containing EGTA or by adding each of the cations separately to media deplete of the cations before exposing the cells to IONO. These studies were done because Wolfe and Gross (28) reported that partially purified canine myocardial PC-PLC activity was increased by excess Ca2+ and by excess Mg 2+. In our experiments, increasing extracellular Ca2+ concentration to 5 mM in the presence

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CALCIUM-ACTIVATED

A

0 Control 3.5

kid PDBu

q

(500 nM)

T

Ionomycin (3 uM)

-I--

PC.01

No Treatment

Cl219

PC HYDROLYSIS

B

pe.01

0 4

Control

q

PDBu

m

Ionomycin

H-7 Treated

T

(3 uM)

No Treatment

n=9 for each bar

T

(500 nM)

px.01

pe.01

Staurosporine Treated

n=6 for each bar

D 4

u

Control PDBu

3.5 -

q q

5 (500 nM)

Ionomycin

px.01 4

PC.01

30 42.5 n : II 2B 3 g 1.5

~1

(3 uM)

0

Control

q q

PDBu Ionomycin

PC.01 PC.01

_ -

l1 0.5 O-

No Treatment n=9 for each

.bar Sphingosine Treated

0

No Pretreatment n=9

Fig. 1. Madin-Darby canine kidney (MDCK) cells were grown to confluence and labeled with [14C]linoleic acid ([‘“ClLIN) for 16 h. For experimental groups depicted in A, B, and C, unincorporated label was removed, and cells were incubated with media alone (no treatment) or with PKC inhibitor. Later (30 min), 500 nM phorbol l&13-dibutyrate (PDBu), 3 PM ionomycin (IONO), or vehicle (control) was added, and incubation was continued for additional 30 min. l-(5-isoquinolinesolfonyl)-2-methylpiperazine (H-7) was used at 30 PM (A), staurosporine was used at 100 nM (B), and sphingosine was used at 100 PM (C). Cell lipids were then extracted, as described in EXPERIMENTAL PROCEDURES, and analyzed by thin-layer chromatography. Each experiment was done two or three times with an n = 3 in each group. Both PDBu and ION0 resulted in increased diacylglycerol (DAG). PKC inhibitors H-7, staurosporine, and sphingosine generated increased DAG themselves and completely blocked further increase after exposure to PDBu. They did not, however, have any effect on increased DAG after ION0 exposure. When cells were incubated with PDBu (500 nM) for 24 h to deplete protein kinase C (PKC; D), phorbol-stimulated phospholipase activation was blocked, but IONOstimulated hydrolysis was unaffected. Each bar represents mean t SE. NS, not significant.

of 2 mM EGTA partially restored the increase in DAG after IO-NO, but increasing Mg2+ concentration to 5 mM in the presence of 2 mM EGTA had no effect on the DAG levels after ION0 exposure (Fig. 4A). Similar results were obtained when cells were exposed to ION0 in incomplete Hank’s buffered saline (IHBSS) containing no added Ca2+ or Mg2+ (Fig. 4B). Exposing the cells to ION0 in IHBSS blunted the increase in DAG, and adding 1.2 mM Mg2+ to the media did not restore the response. Adding 1.2 mM Ca2+ to the media, however, restored the DAG response to ION0 addition. Adding both Mg2+ and Ca2+

to the media had no more effect than adding Ca2+ alone. Role of PLD activity. DAG can be generated through the action of a PLC or can be generated through the sequential actions of PLD and PA phosphohydrolase. Directly relevant to our work, Huang and Cabot (12) recently reported that phorbol esters increased DAG in MDCK cells by increasing PLD activity (12). To determine whether ION0 increased PLD activity, we analyzed the extracted lipids for both PA and DAG and compared the time course of their appearance. As shown in Fig. 5, exposing the MDCK cells to ION0 resulted in increased

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q q

3.5

PC HYDROLYSIS

Control Ionomycin

2 3 n 2 2.5 2 3 8

2 1.5

-II&

1 0.5 1/////A

0

Control

H-7 n=6 for each bar

Staurosporine

Fig. ‘2. To confirm that DAG generated after ionomycin was from phosphatidylcholine (PC) hydrolysis, cells were labeled with [palmitoyl-2 14C]lysophosphatidylcholine ( [ 14C]PC), which preferentially labels the PC pool. DAG increased after ION0 exposure, and neither H-7 nor staurosporine prevented the increase. (P < 0.01 for ionomycin-treated groups compared with control for each of paired bars.) Control

DAG and in increased PA. The increase in PA and DAG followed a similar time course. Just as DAG can be generated from PA, PA can also be generated from DAG, and an increase in PA may occur without PLD activation. We therefore sought evidence for PLD activity by measuring the appearance of PEtOH when the cells were exposed to ION0 in the presence of ethanol (29). PLD catalyzes a trans-phosphatidylation reaction, producing a phosphatidylalcohol when it is activated in the presence of an alcohol (29). Figure 6 demonstrates that exposing the cells to ION0 in the presence of increasing concentrations of ethanol resulted in increasing concentrations of PEtOH. This result supports the conclusion that ION0 activates PLD. Although these data confirm that ION0 increases the activity of PLD in MDCK cells, they do not answer the question of whether this PLD activity is the source of the increased DAG. To address this question, we measured DAG in IONO-exposed cells in the presence of increasing concentrations of ethanol. If the DAG is produced from PA, the increasing ethanol concentration will generate more PEtOH at the expense of PA production, and less DAG will be produced. Increasing concentrations of ethanol did result in decreased levels of PA after exposure to ION0 (Fig. 7B). The ethanol did not affect the activity of PLD because the combined production of PA and PEtOH Table 2. Protein kinase C activity in MDCK membranes after addition of inhibitors Condition

pmoles

s2P added.

min-l

-100

pg protein-l

1.70t0.81 Control PDBu (500 nM) 4.21kO.68 + Staurosporine (100 nM) 1.22kO.06 2.06kO.61 + H-7 (30 PM) + Sphingosine (100 PM) 0.20t0.25 0.30t0.12 + PDBu Pretreatment Values are means & SE. MDCK, Madin-Darby canine kidney cells. Cells were exposed to PDBu for 30 min before measuring protein kinase C activity. Cells exposed to staurosporine, 1- (5-isoquinolinesulfonyl) -2 methylpiperazine (H-7), or sphingosine were incubated with inhibitor for 30 min before and during incubation with PDBu. Cells pretreated with PDBu were incubated with PDBu (500 nM) for 24 h before adding fresh PDBu.

3

Ionomycin

EGTA (2 mM)

Ionomycin + EGTA

n=6 for each bar

T 2.5 -

2 2n 3 1.5 z 9 A # l-

0.5 -

O-

Control

PDBu

EGTA

PDBu

t EGTA

Fig. 3. After labeling MDCK cells with [ 14C]LIN, cells were exposed to 3 PM ION0 or to 500 nM PDBu for 30 min in presence or absence of 2 mM EGTA. Lipids were then extracted and analyzed as described. A: EGTA completely prevented increased DAG after IONO. Each bar represents an n = 9, and only ION0 group differed from control with P < 0.01. B: in contrast, EGTA had no effect on increased DAG after PDBu exposure. Each bar again represents n = 9. Both PDBu and PDBu + EGTA differed from control, with P < 0.01.

remained constant among the five different concentrations of ethanol. Total PA plus PEtOH at 0% is equal to 6.62% of the label; at 0.5%, it is equal to 6.72%; at l.O%, it is equal to 6.4%; at 1.5%, it is equal to 6.27%; and, at 2.0%, it is equal to 6.63%. However, increasing the concentration of ethanol from 0.5 to 2.0% had no effect on the increase in DAG (Fig. 7A). In similar studies using phorbol esters, however, we found that ethanol completely prevented the increase in DAG after phorbol exposure (Fig. 8). These data suggest that ION0 stimulates both PLC and PLD activity in MDCK cells and that the increased DAG is not due to PLD activity. To further test this

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CALCIUM-ACTIVATED

Two lines of evidence support a role for PKC stimulating PC hydrolysis. First, activation of PKC with phorbol esters increases PC hydrolysis in a number of cells (6,10). Second, depletion of PKC by prolonged phorbol exposure before agonist exposure partially blocks PC hydrolysis in some systems (6). This block by PKC depletion is neither universal nor always complete, however, suggesting that other activation pathways exist. One potential amplification pathway is through increased intracellular Ca2+. Calcium-activated PC hydrolysis has been reported in a number of cells, including MDCK cells (1, 3, 18, 23, 26). However, the PKC- and calcium-activated pathways have not been compared in many of these cells, including MDCK cells and rat type II epithelial cells. Because PKC

n=6 for each bar

Control

EGTA

EGTA

Ionomycin

EGTA tea + Ionomycin

5

0

Without

Cl221

PC HYDROLYSIS

EGTA +Mg

6-

(3 uM)

Ionomycin

Diglyceride rlu

n=9 for each point

PhosphatidicI I

0 0

I

I

1

I

I

20

40

60

80

100

Acid

I

120

140

Time of Exposure (in minutes) IHBSS + Ca

IHBSS

IHBSS + Ca + Mg

n=9 for each bar

Fig. 4. Two different experiments were done to evaluate role of extracellular Ca2+ or extracellular Mg2+ in IONO-induced increase in DAG. A: after labeling cells, they were exposed to 3 PM ION0 in presence of 2 mM EGTA, and either Ca2+ or Mg2+ was added to media to total concentration of 5 mM. Adding excess Ca2+ restored response to IONO, but adding excess Mg 2+ had no effect on DAG generation after IONO. Each bar represents n = 6. Only ION0 in the absence of EGTA and ION0 + EGTA + 5 mM Ca2+ differed from control, with P < 0.01. B: similarly, exposing cells to 3 PM ION0 in media containing no added Ca2+ or Mg2+ blunted increase in DAG. Adding Ca2+ to 1.2 mM restored response to IONO, but adding Mg 2+ to 1.2 mM did not. Adding both Ca2+ and Mg2+ was no different than adding Ca2+ alone. Each bar represents n = 9.

Fig. 5. ION0 exposure (3 PM) resulted in both increased DAG and in increased phosphatidic acid (PA) in similar time course. After labeling cells with [14C]LIN and after exposing cells to ION0 for times noted (expressed in min), cell lipids were extracted. Lipid fraction from each monolayer was split and analyzed separately for DAG or PA, as described in EXPERIMENTAL METHODS. Each data point represents n = 9.

r -6

aa

’

---B-

Control

-+-

Ionomycin

postulate, we exposed the cells to ION0 in the presence of 100 PM propranolol to block PA phosphohydrolase, the second enzyme in the PLD pathway for DAG generation (7). Propranolol had no effect on the increase in DAG after ION0 exposure. This observation is consistent with our results using ethanol that ION0 activates PLC. DISCUSSION

PC hydrolysis by PLC or PLD is being increasingly recognized as an important source of DAG in cells (6,10, 21). PC hydrolysis is associated with PI hydrolysis after some stimuli (9) but is independent of PI hydrolysis after other stimuli (18,21). PI-PLC generates two intracellular signals, PKC activation through increased DAG and increased intracellular Ca2+ from inositol trisphosphate (5).

5% EtOH

1.0% EtOH

15% EtOH

2.0% EtOH

Fig. 6. To confirm that PA produced after ION0 exposure is due to PLD activity, labeled cells were exposed to ION0 in presence of increasing concentrations of ethanol (EtOH). Extracted cell lipids were then analyzed for phosphatidylethanol (PEtOH). There was marked increase in PEtOH after ION0 exposure, and quantity of PEtOH increased, as expected by increasing ethanol from 0.5 to 2.0%. Each data point represents n = 9, and each IONO-exposed point differs from cntrol, with P < 0.01.

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CALCIUM-ACTIVATED

PC HYDROLYSIS

A n=6 for each

0% EtOH

EtOH

1.0% EtOH

1.5% EtOH

2.0% EtOH

Ionomycin (3 u”)

+I-

Phoephatidic

-.-

Phosphtidykthmol

Control

0% EtOH

3% EtOH + Ionomycin

1% EtOH

1% EtOH

PDBu (500 nM) Fig. 8. Unlike results of experiments with ION0 exposure, exposing labeled cells to 500 nM PDBu for 30 min in presence of 1 or 2% ethanol completely prevented increase in DAG. Each bar represents n = 6, and only PDBu exposure in 0% ethanol differed from control at P < 0.01.

Acid

1 0

0% EtOH

1.5% EtOH

2% EtOH

(3 uM)

Fig. 7. A: increasing concentration of ethanol from 0 to 2.0% had no effect on generation of DAG after 30 min of 3 PM ION0 exposure. Each bar represents n = 9, and each IONO-exposed group differed from control at P < 0.01. B: increasing concentration of ethanol from 0 to 2.0% did result in progressive increase in PEtOH and progressive decrease in PA. Each data point represents n = 9. Progressive decrease in PA production without decrease in DAG production after ION0 exposure supports hypothesis that ION0 activates both PLC and PLD enzymes.

is a calcium-regulated enzyme, one potential explanation for the calcium-activated PC hydrolysis pathway reported in these epithelial cells is that increased intracellular calcium directly activates PKC. Our current study addresses this question by comparing phorbol-activated PC hydrolysis with calcium ionophore-activated PC hydrolysis in MDCK cells. This cell line is well suited to these studies because our previous work demonstrated that calcium ionophore activates PCspecific phospholipolysis without increasing the hydrolysis of phosphoinositide lipid species (21). We compared phorbol-activated DAG accumulation with IONO-activated DAG accumulation in the following three ways: by comparing the effect of PKC inhibition on the two pathways, by comparing the requirement for extracellular calcium on each of the pathways, and by comparing the relative contributions of PLC compared with PLD activ-

ity in the generation of DAG after each of the stimuli. The cumulative data from these experiments support the hypothes 1s that the calcium-activated PC hydrolysis pathway is different from the PC hydrolysis pathway activated by phorbol esters. The first set of experiments evaluated the ability of different PKC inhibitors to block DAG accumulation after either PDBu or ION0 exposure. We used three different PKC inhibitors because each of the inhibitors acts differently, because each of them has different abilities to inhibit other kinases, and because different PKC isozymes may be variably sensitive to the different inhibitors (9, 15, 16). In spite of these differences, each of the inhib iitors generated a sim .ilar response. DA .G increased after incubation with each of the inhibitors, and each of the inhibitors blocked any further increase after adding PDBu. Of importance to the current study, however, none of the inhibitors prevented increased DAG after ION0 exposure. In spite of using multiple inhibitors, it is possible that none of them blocked all of the PKC in the cells. For that reason, we also depleted the cells of PKC by prolonged exposure to PDBu (4). Unlike the inhibitors, this treatment did not itself increase DAG but completely prevented further DAG generation after adding PDBu. Like each of the PKC inhibitors, however, prolonged PDBu exposure had no effect on the IONO-induced increase in DAG. The universal finding in these experiments is that blocking PKC activity has no effect on DAG generation after IONO. This result cannot be explained by incomplete PKC inhibition because each of these steps blocked PKC activity measured with an in vitro assay system. These data together support the hypothesis that ION0 increases PC hydrolysis through a PKC-independent pathway. Our next set of experiments was designed to determine the role of extracellular Ca2+ or extracellular Mg2+ in IONO-induced DAG generation. The rationale for this set of experiments comes from the work of Wolf and

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CALCIUM-ACTIVATED

Gross (28). Those investigators reported that both Ca2+ and Mg2+ increased the activity of a partially purified PC-specific PLC isolated from canine myocardium (28). Consistent with their report, we found that EGTA prevented increased DAG after IONO, but, unlike their report, only Ca2+ was important in our system. Mg2+ did not affect the activation of the enzyme in our studies. In contrast to the effect of EGTA on ION0 stimulation, EGTA had no effect on DAG generation after PDBu exposure. The results of the experiments with EGTA and PDBu are consistent with the recent report by Huang and Cabot (12). They similarly found that phorbol-activated PLD activity was not affected by Ca2+ chelation with EGTA (12). These data suggest that IONO-stimulated DAG production depends on extracellular calcium but that PDBu-stimulated DAG production is independent of extracellular calcium. Although calcium contributes to PLC and PLD activation, our results do not allow us to speculate further about the mechanism through which Ca2+ increases the activity of the enzymes. The final set of experiments examined the relative roles of PLC and PLD in IONO-generated DAG. Huang and Cabot (12) demonstrated that the relative activities of PLC and PLD after phorbol stimulation depended on the cell studied. MDCK cells stimulated with phorbol esters generated virtually all of their DAG by increasing PLD activity. Our data with PDBu and increasing concentrations of ethanol support their conclusions. IONO, in contrast, stimulates both PLD and PLC activity. Our data measuring the generation of PA and measuring the production of PEtOH in the presence of ethanol are consistent with the hypothesis that ION0 increases PLD activity in the cells. Unlike phorbol stimulation, however, increasing concentrations of ethanol had no effect on DAG accumulation after ION0 exposure even though PA production was blunted. This result demonstrates that decreasing the substrate available for PA phosphohydrolase had no effect on IONO-induced DAG production. In addition, inhibiting PA phosphohydrolase with propran0101 had no effect on DAG accumulation. These data support the conclusion that ION0 activates both PLC and PLD but that DAG is generated mainly by PLC. An increasing body of data now identifies PC hydrolysis as an important source of increased DAG in a number of different tissues. Our report confirms that DAG can be generated in MDCK cells through a calcium-activated PC-specific PLC and compares this pathway with the PKC-dependent pathway. The calcium ionophoreactivated pathway differs from the previously described phorbol-activated pathway in three ways. First, calcium ionophore activates both PC-specific PLC and PC-specific PLD in MDCK cells, whereas phorbol activates mainly PC-specific PLD. Second, calcium ionophore activation depends on extracellular calcium, whereas phorbol activation is independent of extracellular calcium. Finally, phorbol activation depends on PKC activity, whereas calcium ionophore activation occurs through a PKC-independent pathway. These data suggest that cells may contain at least three different pathways for increasing PC hydrolysis. PC hydrolysis may be increased through increased PKC activitv. mav be increased di-

Cl223

PC HYDROLYSIS

rectly, or may be increased through lar calcium.

increased intracellu-

We thank Dr. Michael J. Welsh for critically reviewing the manuscript. This work was supported by National Heart, Lung, and Blood Institute Grant HL-42358, a Program Project Grant on Cystic Fibrosis, and by grants from the American Heart Association, Iowa Affiliate and the American Lung Association of Iowa. M. W. Peterson is a recipient of a Clinical Investigator Award from the National Institutes of Health and a Career Investigator of the American Lung Association. Address for reprint requests: M. W. Peterson, C33H General Hospital, University of Iowa, Iowa City, IA 52242. Received 20 April 1992; accepted in final form 17 June 1992. REFERENCES 1. Agwu, D. E., L. C. McPhail, M. C. Chabot, L. W. Daniel, R. L. Wykle, and C. E. McCall. Choline-linked phosphoglycerides. A source of phosphatidic acid and diglycerides in stimulated neutrophils. J. Biol. Chem. 264: 1405-1413, 1989. 2. Anderson, M. P., and M. J. Welsh. Isoproteronol, CAMP, and bradykinin stimulate diacylglycerol production in airway epithelium. Am. J. Physiol. 258 (Lung Mol. PhysioZ. 6): L294-L300,1990. 3. Auger, G., S. B. Bocckino, P. F. Blackmore, and J. H. Exton. Hormonal stimulation of diacylglycerol formation in hepatocytes: evidence for phosphatidylcholine breakdown. J. BioZ. Chem. 264: 21689-21698, 1989. 4. Ballester, R., and 0. M. Rosen. Fate of immunoprecipitable protein kinase C in GH3 cells treated with phorbol 12-myristate 13-acetate. J. BioZ. Chem. 260: 7295-7303, 1985. 5. Berridge, M. J., and R. F. Irvine. Inositol trisphosphate and diacylglycerol as second messengers. Nature Lond. 312: 315-321, 1984. 6. Besterman, J. M., V. Duronio, and P. Cuatrecasas. Rapid formation of diacylglycerol from phosphatidylcholine: a pathway for generation of a second messenger. Proc. NatZ. Acad. Sci. USA 83: 6785-6789, 1986. 7. Billah, M. M., S. Eckel, T. J. Mullman, R. W. Egan, and M. I. Siegel. Phosphatidylcholine hydrolysis by phospholipase D determines phosphatidate and diglyceride levels in chemotactic peptide-stimulated human neutrophils. Involvement of phosphatidate phosphohydrolase in signal transduction. J. BioZ. Chem. 264: 17069-17077, 1989. 8. Bligh, E. G., and W. J. Dyer. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: 91 l-917, 1959. 9. Castagna, M., Y. Takai, K. Kaibuchi, K. Sano, U. Kikkawa, and Y. Nishizuka. Direct activation of calciumactivated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J. BioZ. Chem. 257: 7847-7851, 1982. 10. Daniel, L. W., M. Waite, and R. L. Wykle. A novel mechanism of diglyceride formation. J. BioZ. Chem. 261: 9128-9132, 1986. 11. Hannun, Y. A., C. R. Loomis, A. H. Merrill, and R. M. Bell. Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets. J. BioZ. Chem. 261: 12604-12609, 1986. 12. Huang, C., and M. C. Cabot. Phorbol diesters stimulate the accumulation of phosphatidate, phosphatidylethanol, and diacylglycerol in three cell types. Evidence for the indirect formation of phosphatidylcholine-derived diacylglycerol by a phospholipase D pathway. J. BioZ. Chem. 265: 14858-14863, 1990. 13. Hughes, B. P., K.-A. Rye, L. B. Pickford, G. J. Barritt, and A. H. Chalmers. A transient increase in diacylglycerols is associated with the action of vasopressin on hepatocytes. Biothem. J. 222: 535-540, 1984. 14. Kawamoto, S., and H. Hidaka. 1-(5-Isoquinolinesulfonyl)-2methylpiperazine (H-7) is a selective inhibitor of protein kinase C in rabbit platelets. Biochem. Biophys. Res. Commun. 125: 258-262, 1984. 15. Kikkawa, U., A. Kishimoto, and Y. Nishizuka. The protein kinase C family: heterogeneity and its implications. Annu. Rev. Biochem. 58: 31-44, 1989.

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