the enhancement by wortmannin of protein kinase c-dependent activation of phospholipase d in...

CPL C HEMI S TR Y A N D

Chemistry and Physics of Lipids PHYSICS OF LIPIDS 86 (1997) 65 74 E L S E V I E R

The enhancement by wortmannin of protein kinase C-dependent activation of phospholipase D in vascular endothelial cells

Viswanathan Natarajan *, Suryanarayana Vepa, Mohammed A1-Hassani, William M. Scribner

Department o[ Medicine, Pulmonary Division, Indiana University School of Medicine, 1001 West lOth Street OPW 425, Indianapolis, IN 46202, USA

Received 12 August 1996; received in revised form 20 November 1996; accepted 3 January 1997

Abstract

Phosphatidic acid generation by phospholipase D (PLD) activation has been implicated in agonist- and oxidant- mediated endothelial cell signal transduction. We examined the effect of wortmannin on PLD activation in pulmonary artery endothelial and smooth muscle cells in culture. Pretreatment of bovine pulmonary artery endothelial cells (BPAECs) with wortmannin potentiated TPA- (100 nM), ATP- (100/tM), and bradykinin- (1 /~M) induced [32P]PEt formation, an index of PLD activation. However, wortmannin by itself had no effect on PLD activity. The potentiating effect of wortmannin on TPA-induced PLD activation was dose- (1-10/~M) and time-dependent (5-30 min) and was inhibited by bisindoylmalemide, an inhibitor of protein kinase C (PKC). Furthermore, down-regulation of PKC by prolonged treatment with TPA (100 nM, 18 h) attenuated the wortmannin effect. This effect of wortmannin was specific for TPA- or agonist-induced PLD activation as no potentiation of [32p]PEt formation was observed with H202 (1 mM) or ionomycin (1 ktM). The effect of wortmannin was not due to activation of PKC~ as determined by western blot analysis of PKCct in the cytosol and membrane fractions. Also, genistein, an inhibitor of tyrosine kinases, did not attenuate the wortmannin-mediated potentiation of PLD thereby suggesting non-involve- ment of protein tyrosine phosphorylation. These results indicate that wortmannin potentiates PKC-dependent stimulation of PLD in vascular endothelial cells. © 1997 Elsevier Science Ireland Ltd.

Keywords: Protein kinase C; Phospholipase D; Wortmannin; Phorbol esters; Oxidants; Tyrosine kinases

Abbreviations: BPAECs, bovine pulmonary artery endothelial cells; DAG, diacylglycerol; ECs, endothelial cells; FAK, focal adhesion kinase; LPA, lysophosphatidic acid; PA, phosphatidic acid; PBt, phosphatidylbutanol; PEt, phosphatidylethanol; PI, phosphatidylinositol; PI3K, phosphatidylinositol 3 kinase; PKC, protein kinase C; PLC, phospholipase C; PLD, phospholipase D.

* Corresponding author. Tel.: + 1 317 6306792; fax: + 1 317 6306386.

0009-3084/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. Pll S0009-3084(97)02660-1

66 V. Natarajan et al . / Chemistry and Physics of Lipids 86 (1997) 65 74

1. Introduction

Activation of phospholipase D (PLD) represents an important signaling mechanism in mammalian cells (Billah and Anthes, 1990; Exton, 1994; Liscov- itch and Chalifa, 1994). Stimulation of PLD gener- ates phosphatidic acid (PA) which can be subsequently converted to lyso PA (LPA) or diacyl- glycerol (DAG) by the action of phospholipase A~/A 2 (Billah et al., 1981) or PA phosphatase (PA Pase) (Brindley, 1984), respectively. Earlier studies have clearly established the second-messenger role of DAG as an endogenous activator of protein kinase C (PKC) (Nishizuka, 1992). Phosphatidic acid and lyso PA have been shown to serve as second-messengers (Kroll et al., 1989; Moolenaar, 1995; Boarder, 1994). For instance, exogenous addition of PA activated endothelial cell (EC) PKC in vitro (Stasek et al., 1993; Limatola et al., 1994) and increased protein phosphorylation in the cyto- solic extracts of liver, brain, lung and kidney (Bocckino et al., 1991). Similarly, LPA increased arachidonic acid mobilization in macrophages (Ro- mano et al., 1992) and enhanced protein tyrosine phosphorylation of focal adhesion kinase (FAK) in fibroblasts (Saville et al., 1994).

We and others have demonstrated activation of PLD by a variety of agonists (Billah and Anthes, 1990; Exton, 1994; Liscovitch and Chalifa, 1994; Kiss, 1992; Natarajan and Garcia, 1993; Garcia et al., 1992; Natarajan, 1995) and reactive oxygen species (Bourgoin and Grinstein, 1992; Natarajan et al., 1993a,b; Kiss and Anderson, 1994) in ECs. In addition to accumulation of labelled phos- phatidylalcohol, an index of PLD activation (Kobayashi and Kanfer, 1987; Gustavsson and Alling, 1987), many of the agonists and reactive oxygen species also elevated cellular DAG levels (Leach et al., 1991; Margolis et al., 1989; Taher et al., 1993). At present it is not clear whether the DAG generated in response to an external stimulus is due to phospholipase C (PLC) or PLD stimula- tion followed by conversion of PA to DAG by PA Pase or a combination of both. Recent studies have demonstrated that wortmannin and dimethoxyve- ridin, a structural analog of wortmannin, blocked fMLP-mediated PLD activation in human neu- trophils (Reinhold et al., 1990; Bonser et al., 1991; Naccache et al., 1993). Hence, wortmannin may

serve as a useful pharmacological agent to modu- late PLD activation in mammalian cells. The present study, therefore, was designed to study the effect of wortmannin on agonist-, 12-O-tetrade- canoyl phorbol 13-acetate- (TPA), and oxidant-in- duced PLD activation in bovine pulmonary artery endothelial cells (BPAECs). Our results indicate that wortmannin, instead of inhibiting PLD, aug- ments both agonist- and TPA-induced [32p]phosphatidylethanol (PEt) formation in BPAECs. This potentiating effect of wortmannin was observed only with the agents that activated PLD via a PKC-dependent mechanism while PKC- independent PLD activation was unaffected.

2. Materials and methods

2.1. Mater&&

Wortmannin, 12-O-tetradecanoylphorbol 13-ac- etate (TPA), ionomycin, ATP, bradykinin, minimal essential medium (MEM), fetal bovine serum, trypsin (tissue culture grade), non-essential amino acids, hydrogen peroxide, and antibiotics were purchased from Sigma Chemical Co. (St. Louis, MO). [32p]Orthophosphate (carrier-free) was from DuPont NEN (Boston, MA). Phosphatidylethanol (PEt) and phosphatidylbutanol (PBt) were pur- chased from Avanti Polar Lipids (Alabaster, AL). Bovine pulmonary artery endothelial cells (CCL- 209) were obtained from ATCC (Rockville, MD). Endothelial derived growth factor was purchased from Upstate Biologicals Inc. (Lake Placid, NY). Precoated silica gel plates were procured from Analtech (Newark, DE). Monoclonal antibodies to PKC~ and PKCE were from Transduction Labora- tories (Lexington, KY).

2.2. Cell culture

Bovine pulmonary artery endothelial cells (BPAECs) (passage 16) were grown in T 75-cm 2 tissue culture flasks and maintained in MEM with 10% fetal calf serum, endothelial derived growth factor and antibiotics (Natarajan and Garcia, 1993; Garcia et al., 1992). Confluent cells were seeded in 35-ram dishes and experiments were performed at 85 95% confluency (5 × 105 cells/dish) and at pas- sage 19 or 20.

V. Natarajan et al./ Chemistry and Physics of Lipids 86 (1997) 65 74 67

2.3. Measurement of phospholipase D activation 2.5. Immunoprecipitation and PI3 kinase assay

After labeling BPAECs with [32p]orthophos- phate (5 /LCi/dish, carrier free) in DMEM-phos- phate free media for 18 22 h, cells were washed in RPMI-1640 medium and were pretreated with wortmannin (1 /~M 10 /~M) or other agents as indicated. Wortmannin pretreated cells were chal- lenged with RPMI-1640 medium or medium con- taining TPA, bradykinin, ATP, ionomycin or other agents for 15-30 rain in the presence of 0.5% ethanol or 0.05% butanol. Lipids were ex- tracted under acidic conditions and [32PIPEt or PBt was separated by TLC and quantified as described earlier (Natarajan et al., 1993a; 1996a).

2.4. Western blotting and immunodetection of protein kinase C

Confluent BPAECs in T 75-cm 2 flasks were incubated in RPMI-1640 for 60 min at 37°C with or without wortmannin (5 /tM). Thereafter, the cells were washed with medium and challenged with TPA (100 nM) for 15 min. Cells were washed with ice cold PBS and scraped off in 1 ml of homogenization medium (20 mM Tris HCI buffer containing 1 mM EDTA, 1 /,g/ml aprot- inin, 2 / tg /ml leupeptin and 1 mM PMSF) (Taher et al., 1993; Vepa et al., 1997). Cells were soni- cated and subcellular fractions were prepared (Taher et al., 1993). Equal concentrations of protein (10 30 l~g) were fractionated elec- trophoretically using 8"/0 SDS-PAGE. The proteins were transferred to Immobilon P mem- brane and blocked with Tris-buffered saline solu- tion containing 0.1% Tween-20 (TBST) for 3 h at RT. The membranes were incubated with PKC~ (1:5000 dilution) or PKCe (1:10 000 dilution) anti- bodies for 2 4 h at RT. The membranes were subsequently washed in TBST and incubated with a goat-anti-rabbit secondary antibody conjugated to horseradish peroxidase. After incubation, the membranes were extensively washed with TBST and signals were detected with the enhanced chemiluminescence detection system on Hyper au- toradiography film (Amersham Corp).

Confluent BPAECs grown to 90% confluency were rinsed with ice-cold phosphate-buffered sa- line and lysed in 50 mM Tr i s -HCl (pH 7.4) containing 150 mM NaC1, 0.5% Triton X-100, 0.5% Nonidet P-40, 1 mM Na3 VO4, 2 mM EDTA, 2 /~g/ml aprotinin, 2 /~g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride and 1 llM pepstatin. Lysates were sonicated and were cen- trifuged for 15 min at 12000 rev./min in a Brinkman microfuge centrifuge. The superna- rants were adjusted to equal protein concentra- tion and incubated with monoclonal PY-20 antiphosphotyrosine antibody conjugated to Agarose (2 /lg/ml) at 4°C for 3 h. Immunopreci- pates were collected, washed thrice with kinase buffer (10 mM Tris HC1, pH 7.4; 150 mM NaCI, 5 mM EDTA and 20 mM MgC12). PI3 kinase was measured by incubating the im- munoprecipates from control, wortmannin or LY294002 treated cells in 0.2 ml buffer (10 mM Tris HC1, pH 7.4; 150 mM NaC1; 5 mM EDTA: 20 mM MgC12) containing PI (10 itg) and 50 llM ATP, ([7-32p]ATP, 10 /lCi) for 20 min at 26°C with constant shaking (Rahimi et al., 1996). Reactions were terminated by addition of 20 tll of 6 N HC1 and lipids were extracted by the addition of 2 ml of CHC13/CH3OH (2:1, v/v) followed by 0.44 ml of 1 N HCI (Folch et al., 1957). Lipids were spotted on silica gel H plates containing 1% potassium oxalates and developed in chloro- form/methanol/acetone/acetic water/water (7:5: 2:2:2, by vol.). The plates were autoradio- graphed and the labelled PI3 phosphate spot (Rf~0 .6) were scraped and counted by liquid scintillation.

2,6. Statistical analysis

Data are presented as mean + ranges of two independent experiments and mean_+SEM of three independent experiments. Significance of the data were assessed with the use of one- way ANOVA. A P < 0.05 was considered signifi- cant.

68 V. Natarajan et a l . / Chemistry and Physics c~/' Lipids 86 (1997) 65 74

3. Results

Incubation of BPAECs with TPA (100 nM), ATP (I00 /LM) or Bradykinin (1 /~M) stimulated PLD as measured by the accumulation of [3Ep]PEt in the lipid extracts (Fig. 1). In order to assess the effect of wortmannin on PLD activation, 32p-la- belled BPAECs were preincubated with wortman- nin (5 /~M) for 60 rain before challenging with TPA- or other agonists. As shown in Fig. 1, wortmannin significantly potentiated TPA, ATP and bradykinin-induced [32p]PEt-formation, how- ever, wortmannin by itself had very little effect on basal PLD activity. The potentiating effect of wortmannin on PLD activation was also observed in smooth muscle cells stimulated by endothelin-1 or TPA (Fig. 2). As shown in Fig. 3 and Fig. 4, the potentiating effect of wortmannin on TPA-in- duced PLD activation was dose- and time-depen- dent respectively, with a twofold increase observed at 5 /iM wortmannin. These data fur-

HUVEC RFASMC Vehicle ~ Thrombin~ TPA Vehicle • ET-1 • TPA

(10nM) (100nM) (10nM) (t00nM)

.oo ,00o o "o

3000 3000 .~

E 2000 0ooo

,ooo ,000

0 0

W o r t m a n n i n - - ~ - - ..~ (s pM)

Fig. 2. Effect of wortmannin on agonist-induced [32P]PEt formation in endothelial and smooth muscle cells. Human umbilical vein endothelial cells (HUVECs) and rabbit femoral artery smooth muscle cells (RFASMCs) were labelled with [32p]orthophosphate as described in Fig. 1. Cells were pre- treated with MEM or MEM containing wortmannin (5 l~M) for 30 min and were challenged with thrombin (10 nM) or endothelin-I (10 nM) or TPA (100 nM) in the presence of 0.5% ethanol for 30 min. Lipids were extracted under acidic conditions and [3-~P]PEt formed was separated by TLC and quantified as described under Section 2. Values are m e a n - - ranges of triplicate determination (n = 2).

800

600

| * "

200

F 0

W o r t m a n n i n ( 5 u M )

L ~ (-) I I (+)

cle cells.

. . . . Vehicle - = - - TPA(100 nM)

- , 0 0 0

E 4 0 0 0

~-e" 3000 "~ bide S

(lOOnM) (lOOpM) (1 pM) ~ 2000 I g

Fig. 1. Effect of wortmannin on agonist-induced [32p]PEt formation in BPAECs. BPAECs were labelled with [32p]orthophosphate (5 /~Ci/dish) in DMEM-phospha te free media containing 0.5% fetal calf serum for 18 h. Cells were washed and pretreated with M E M or M E M containing wort- mannin (5 / tM) for 30 min. The medium was aspirated, cells were washed and challenged with TPA (100 nM) or ATP (100 /t M) or bradykinin (1 /IM) in the presence of 0.5% ethanol for 30 min. Lipids were extracted under acidic condition and [3zP]PEt was separated by TLC and quantified as described under Section 2. Values are mean + S E M ( n = 3). *Significantly different (P < 0.01) as compared to TPA treat- ment. **Significantly different (P < 0.05) as compared to ATP or bradykinin (BRK) treatment.

ther provide evidence that wortmannin, an in- hibitor of neutrophil PLD (Bonser et al., 1991), actually enhanced the TPA- and agonist-induced PLD activity in endothelial cells and smooth mus-

": 1000 O.

E" 0 - - ~ " ~ < ' ~ '

2 4 6 0 10 Wortmennln (pM)

Fig. 3. Dose-dependence of wortmannin-mediated potentiation of [32P]PEt formation by TPA. [32P]Orthophosphate-labelled BPAECs, as described in Fig. l, were pretreated with different concentrations of wortmannin for 30 min. Cells were chal- lenged with TPA (100 nM) in the presence of 0.5% ethanol for 30 min and lipids were extracted under acidic conditions. [32PIPEt formed was separated by TLC and quantified as described under Section 2. Values are mean + ranges of tripli- cate determination (n = 2).

V. Natarajan et al. / Chemistry and Physics (~[ Lipids 86 (1997) 65 74 69

~. ,oo,

~ 3 0 0 0

2000

E 1000 L

u

- - : - - Vehicle - - I I - - TPA(100nM)

i i i i i

5 10 10 20 20 30 Pretreetment with Wortmennln(S pM)

(rain)

Fig. 4. Time-dependent potentiation of TPA-induced [32p]PBt formation by wortmannin. [32p]Orthophosphate labelled BPAECs, as described in Fig. 1, were incubated with wort- mannin (5 #M) for varying time periods. Cells were washed and challenged with TPA (100 nM) for 30 min in the presence of 0.05% butanol. Lipids were extracted under acidic condition and [32p]PBt formed was quantified after separation by TLC. Values are mean _+ ranges of triplicate determination (n = 2).

3.1. Effect of PKC inhibitors and down-regulation of PKC on wortmannin-mediated potentiation of TPA-induced PLD activation

3.2. Ejfect of wortmannin on the distribution of PKC~ and PKCE between soluble and particulate fractions

PKC~ (80 kDa) and PKCE (90 kDa) are the major PKC isotypes present in the soluble and membrane fractions, respectively, in BPAECs as determined by immunoblotting (Pottratz et al., 1996). In order to understand whether wortman- nin modulated PKC in BPAECs, we studied the effect of wortmannin (5 /iM) on TPA (100 nM)- induced translocation of PKCT. Preincubation of BPAECs with wortmannin (5 /tM) had no effect on the distribution of PKC7 in unstimulated cells (Fig. 6). Furthermore, translocation of PKCc~ from the cytosol to the particulate-fraction in response to TPA (100 nM) was also not affected by preincubation of BPAECs with wortmannin (Fig. 6). Similarly, wortmannin had no effect on the distribution of PKCE, (predominantly mem- brane associated) as determined by immunoblot- ring (Fig. 6). Together these observations suggest that wortmannin has no direct effect on PKC in BPAECs.

As TPA-, ATP- and bradykinin-induced PLD activation is PKC-dependent in ECs (Natarajan and Garcia, 1993; Garcia et al., 1992; Natarajan, 1995), we investigated the role of PKC inhibitors on the wortmannin-induced potentiation of PLD activity. Preincubation of BPAECs with the PKC inhibitor bisindolylmaleimide (5 /iM/30 min) sig- nificantly attenuated the TPA- and wortmannin plus TPA-induced [32p]PEt accumulation (Fig. 5). Similarly, pretreatment of BPAECs with calphostin C, another inhibitor of PKC, resulted in the inhibition of the TPA and wortmannin plus TPA-induced [32P]PEt accumulation (data not shown). Down-regulation of PKC by prolonged exposure to TPA (100 nM, 18 h) (Natarajan and Garcia, 1993; Garcia et al., 1992) also attenuated the TPA- and wortmannin plus TPA-mediated augmentation of PLD activation in BPAECs (Table 1). These results suggest that the observed effect of wortmannin on agonist-induced PLD activation involves PKC.

! V e h i c l e l TPA(lOO nM)

A

2000 E

1000 L

a - - 0

Woflmannln(5pM) - - -4- - - 4" mN(s pM) - - - - + +

Fig. 5. Effect o f b is indo lymale imide on T P A - and wor tmmnnin plus TPA-induced [32p]PBt formation. BPAECs were labelled with [32p]orthophosphate as described in Fig. 1. Cells were treated with bisindolylmaleimide (5 /tM) for 30 min before addition of wortmannin (5 /tM). Incubations were continued for an additional 30 min before challenging with TPA (100 nM) for 30 min in the presence of 0.05% butanol. Lipids were extracted under acidic conditions and [32P]PBt were separated by TLC and quantified. Values are mean + SEM (n = 3). *Significantly increased (P < 0.01) as compared to TPA treat- ment. **Significantly decreased (P<0 .01) as compared to TPA treatment.

70 V. Natarajan et al . / Chemistry and Physics of Lipids 86 (1997) 65 74

Table 1 Effect of PKC down regulation on TPA and TPA plus wort- mannin induced [32p]PET formation

Pretreatment Treatment [32p]PEt formed (dpm/ dish)

Vehicle Vehicle 235 + 58 Vehicle TPA 2643 + 159 Wortmannin Vehicle 276 _+ 71 Wortmannin TPA 3962 _+ 270 TPA (18 h) Vehicle 170 _+ 38 TPA (18 h) TPA 346+ 126 TPA (18 h )+wor t - Vehicle 196_ 74

mannin TPA (18 h )+wor t - TPA 376_+ 94

mannin

BPAECs in 35-mm dishes were labelled with [32p]orthophosphate as indicated in Fig. 1. Cells were washed and pretreated with TPA (100 nM) for 18 h to down-regulate PKC. At the end of 18 h, cells were incubated with wortman- nin (5 / tM) for 30 min and were challenged with MEM or MEM containing TPA (100 nM), in the presence of 0.5% ethanol, for an additional 30 min. Lipids were extracted under acidic condition and [32p] PEt was separated by TLC and quantified as described under Section 2. Values are mean ___ ranges of triplicate determination (n = 2).

3.3. Specificity of wortmannin augmentation of PLD activation

To further investigate the specificity of wort- mannin-induced augmentation of agonist-induced PLD activation, BPAECs were challenged with TPA (100 nM), H202 (1 mM) or ionomycin (1

C M C M C M C M PKC(x 8 0 k D a ¢ID - ~ l -..

I I I I I I I I

T P A ( 1 0 0 n M ) ~ .4- ~ -[-

W o r t m a n n l n (5 ~ M ) ~ ~ -I- -!-

~ 1 I I I I I PKC~ 9 0 K D a ~ ~ I I I ~1)

Fig. 6. Effect of wortmannin on TPA-mediated translocation of PKCct from cytosol to membrane. BPAECs in T 75-cm 2 flasks were pretreated with MEM or MEM containing wort- mannin (5 ,uM) for 30 min. Cells were washed and challenged with TPA (100 nM) for 15 min and cell lysates were prepared as described under Section 2. The cytosol (C) and membrane (M) fractions were subjected to SDS-PAGE, transferred to membrane and incubated with PKC~ antibody (1:5000 dilu- tion) for 24 h. Signals were detected with ECL system.

W o r t m a n n l n (5pM) i . ( . ) s l im ( . )

~---~ 0 0 0 0

E 0 O O O ]

4 0 0 0 , t .,O =

2 0 0 0

o .

o TPA (100 f tM) ~ ~ ~ "~"

H , L ~ 2 ( l m M ) - - .4- ~ - - I o n o m y c l n ( l p M ) - - ~ 4 " - -

Fig. 7. Effect of wortmannin on TPA-, H202 or ionomycin- induced [32pIPEt fomaation. BPAECs labelled with [32P]orthophosphate, as described in Fig. 1, were pretreated with wortmannin (5 /~M) for 30 rain. Cells were washed and were challenged with TPA (100 nM) or H202 (1 mM) or ionomycin (1 IBM), in the presence of 0.5"/,, ethanol, for an additional 30 rain. Lipids were extracted under acidic condi- tion and [32P]PEt was separated by TLC and quantified as described under Section 2. Values are mean +_ ranges of tripli- cate determination (n = 2).

/~M). Under these experimental conditions all three agents activated endothelial cell PLD. In contrast to the effect on TPA-induced potentia- tion of PLD activation, wortmannin had no effect o n H2O 2- or ionomycin mediated PLD stimula- tion (Fig. 7). We have recently demonstrated that H202-induced PLD activation in ECs is PKC-in- dependent and may involve protein tyrosine phos- phorylation (Natarajan et al., 1996a,b). These data suggest that the effect of wortmannin was specific for agents that activated PLD through PKC-dependent mechanism.

3.4. Effect of wortmannin and L Y294002 on TPA-induced PLD activation

Wortmannin is not only a potent inhibitor of myosin light chain kinase activity (Nakanishi et al., 1992) but also inhibits PI3 kinase in mam- malian cells (Arcaro et al., 1993). To further understand the mechanism of action of wortman- nin, its effect on TPA-induced PLD activation was compared to LY29002, a specific PI3 kinase inhibitor (Vlahos et al., 1994). Immunoprecipi- tates obtained with antiphosphotyrosine antibody treated BPAECs from wortmannin and LY294002 inhibited PI3 kinase activity (Table 2). Also

V. Natarajan et al . / Chemistry and Physics of Lipids 86 (1997) 65 74 71

Table 2 Effect of myosin light chain kinase and phosphatidylinositol-3-kinase inhibitors on TPA-induced PLD activation

Treatment Inhibitor PI3 kinase activity TPA [32p]PEt formed (dpm/dish) % Control

Vehicle - - 100% - 255 _+ 35 100 + 1855 + 287 727

Wor tmannin M L C K and PI3K 25% - 242 _+ 35 95 + 3372 _+ 152 1322

LY294002 PI3K 18% -- 248 _+ 8 97 + 1835 +_ 65 720

BPAECs labelled with [32P]orthophosphate (5/~Ci/dish in DMEM-phospha te free media) for 18 h were washed and pretreated with wor tmannin ( 5 / t M ) for 30 min. Cells were washed and challenged with M E M or M E M containing TPA (100 nM) in the presence of 0.5% ethanol for an additional 30 min. Lipids were extracted under acidic condition and [32p]PEt was separated by TLC. PI3 kinase activity was determined in the ant iphosphotyrosine antibody immunoprecipitates using [732p]ATP and P! as substrate. Radioactivity in PI3-phosphate was determined as described under Section 2. Radioactivity associated with PI3-phosphate in control cells ~ 1500 dpm/mg of protein in cell lysates. Values are mean + ranges of triplicate determinations (n = 2).

shown in Table 2, pretreatment of BPAECs with LY294002 did not alter TPA-induced [32p]PEt formation as compared to wortmannin. These data suggest that the effect of wortmannin on TPA-induced PLD activation does not involve PI3 kinase.

3.5. Effect of genistein on TPA- and wortmannin plus TPA-induced [32p]PEt formation

In order to determine whether the effect of wortmannin on TPA-induced PLD activation was through protein tyrosine phosphorylation, we ex- amined the role of genistein, an inhibitor of protein tyrosine kinases (Akiyama et al., 1987). Genistein (100 /tM), by itself or in wortmannin- treated cells, had no effect on TPA-induced [32p]PEt formation (Table 3). However, genistein attenuated the H202-induced [32p]PEt formation (Natarajan et al., 1996a), and the addition of wortmannin had no effect on H202-induced PLD activation. Furthermore, wortmannin had no ef- fect on H2Oz-induced protein tyrosine phosphory- lation in ECs (data not shown). These data suggest that wortmannin-induced augmentation of TPA-induced PLD activation does not involve tyrosine kinases.

4. Discussion

We have previously reported that agonist- and oxidant-mediated activation of PLD in ECs is

regulated by different mechanisms (Natarajan et al., 1996a,b). Stimulation of PLD by thrombin or bradykinin or ATP required Ca 2 ÷ and was PKC- dependent (Natarajan and Garcia, 1993; Garcia et al., 1992), while H202 and 4-hydroxynonenal-in- duced PLD activation was independent of Ca 2 ÷ and PKC (Natarajan et al., 1993a,b). However, the oxidant-mediated PLD activation was attenu- ated by tyrosine kinase inhibitors (Natarajan et al., 1996a) and potentiated by phosphatase in- hibitors suggesting regulation by protein tyrosine

Table 3 Effect of genistein on wortmannin-induced potentiation of [32p]PEt formation (% control)

Pretreatment Treatment

Vehicle TPA H202

Vehicle 100 326 260 Wor tmannin 81 526 289 Genistein 118 321 168 Genistein + wortmannin 91 572 162

BPAECs were labelled with [32P]orthophosphate (5 /~Ci/dish) in DMEM-phospha te free media for 18 h. Cells were washed and pretreated with wor tmannin (5/~M) or genistein (100 ~M) or wor tmannin (5/~M) plus genistein (100/~M) for 60 min in MEM. The cells were washed and were challenged with TPA (100 nM) or H20 2 (1 mM) for 30 min in MEM containing 0.5% ethanol. Lipids were extracted under acidic condition and [32p]PEt was separated by TLC and quantified as de- ~r ibed under Section 2. Values are expressed as 0% control and calculated from two independent experiments. Radioactivity associated with [32P]PEt: vehicle 532 ± 33.

72 v. Natarajan et al. ~Chemistry and Physics of Lipids 86 (1997) 65 74

phosphorylat ion (Natarajan et al., 1997). In the present study, we investigated the effect of wort- mannin, a fungal metabolite, on agonist-induced PLD activation. The findings presented in this paper demonstrate that (i) wortmannin treatment of ECs did not inhibit agonist- or oxidant-medi- ated [32p]PEt formation, an index of PLD activa- tion, (ii) wortmannin treatment enhanced agonist-mediated and not oxidant-mediated [32p]PEt !formation and (iii) the effect of wortman- nin on agonist-induced [32p]PEt formation is not linked to the PI3 kinase pathway. Also, the data presented here suggest that the enhancement by wortmannin was restricted to agents that activated PLD through the PKC-dependent pathway.

Although earlier studies have demonstrated that wortmannin actually enhanced TPA- or agonist- induced PLD activity (Kanoh et al., 1992; Kozawa et al., 1995a; Kiss and Tomono, 1995), the possible mechanism(s) of wortmannin action was not inves- tigated. To address this, the effect of wortmannin on PKC was determined. While TPA (100 nM) treatment of BPAECs resulted in translocation of P K C e from the cytosol to the membrane fraction (Fig. 6), wortmannin (5/2 M) showed no effect on the distribution of PKCc¢ in the absence or pres- ence of TPA. Also, wortmannin did not alter the total PKC activity as determined by in vitro his- tone 1 phosphorylat ion (Taher et al., 1993) or bradykinin-induced intracellular calcium release (data not shown). It is possible that the site of action of wortmannin in ECs is upstream or downstream to PKC. Interestingly, the precise mechanism of PLD activation by PKC itself is unclear. Earlier studies suggest that there is no direct role for ATP-dependent protein phosphory- lation in TPA-mediated PLD activation (Conri- code et al., 1992). However, recent experiments in neutrophils indicate that PKC phosphorylates a target protein in the plasma membrane after A T P H s / T P A treatment (Lopez et al., 1995).

While PKC activation is critical to TPA- o~ agonist-induced PLD stimulation, recent studies suggest that TPA may also activate PLD via tyrosine kinase activation (Kozawa et al., 1995b). Furthermore, wortmannin inhibited fMLP-medi- ated protein tyrosine phosphorylat ion in human neutrophils (Reinhold et al., 1990) and genistein

attenuated the effect of wortmannin on TPA-in- duced PLD activation (Kozawa et al., 1995a). These studies suggest that in neutrophils and os- teoblast-like cells, wortmannin was modulating tyrosine kinase-dependent PLD activation (Rein- hold et al., 1990; Kozawa et al., 1995a). However, in ECs, wortmannin did not alter basal or H2Oz-in- duced protein tyrosine phosphorylation (data not shown). Genistein, an inhibitor of tyrosine kinases (Akiyama et a l , 1987), also showed no effect on wortmannin plus TPA-induced [32p]PEt formation (Table 3).

In summary, the results of the present study demonstrate that wortmannin, an inhibitor of fMLP-induced PLD activation in neutrophils, ac- tually enhances TPA- or agonist-induced and not oxidant-mediated PLD activation in ECs. In ECs, the enhancement by wortmannin in PLD activa- tion seems to involve agents that activate PLD via PKC.

Acknowledgements

We are grateful to Mrs. Beverly Clark for secre- tarial assistance and Rita Shamlal for technical assistance. This work was supported by N I H grants HL47671, KO4 HL03095 and American Lung Association Career Investigator Award to VN.

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