Down-regulation of protein kinase C attenuates the oxidant hydrogen peroxide-mediated activation of phospholipase A2 in pulmonary vascular smooth muscle cells

Cellular Signalling Vol. 7. No. 1, pp. 75-83. 1995. Elsevier Science Ltd Printed in Great Britain 0898~5568/95 $9.50 + 0,~)

rergamon 0898-6568(94)00061-1

DOWN-REGULATION OF PROTEIN KINASE C ATTENUATES THE OXIDANT HYDROGEN PEROXIDE-MEDIATED ACTIVATION OF PHOSPHOLIPASE A 2 IN PULMONARY VASCULAR SMOOTH MUSCLE CELLS S A J A L C H A K R A B O R T I and T A P A T I C H A K R A B O R T I * Department of Medicine, University of Utah Health Sciences Centre, Salt Lake City, Utah 84132, U.S.A.; and the Department of Biochemistry and Biophysics, University of Kalyani, Kalyani-741235, West Bengal. India (Received 1 Januar3, 1994; and accepted 2 August 1994) Abstract--Exposure of rabbit pulmonary vascular smooth muscle cells to H,O2 dose-dependently stimulates the cell membrane associated protein kinase C (PKC) activity, phospholipase A 2 (PLA2) activity, phospholipase A, (PLA2) activity, and arachidonic acid (AA0) release. Pretreatment of the cells with staurosporine (an inhibitor of PKC) prevents AA release and PLA 2 activity caused by H202. Treatment of the cells with 4~-PMA (an active phorbol ester), or 4ot-PMA (an inactive phorbol ester) does not affect basal AA release. In contrast, 4~-PMA significantly stimulates the cell membrane associated PKC activity. Treatment of the cells with 4[3-PMA for a short time (upregulation of PKC) augments PLA2 activity and AA release caused by a sub-optimal dose of H.,O2 (0.4 mM). Under this condition, staurosporine prevents the stimulatory effects of 4[3-PMA on membrane PLA, activity, and AA release. In contrast to the up-regulation, pretreatment with 4[~-PMA for a longer time (down-regulation of PKC) does not appreciably augment PLA2 activity and AA release caused by 0.4 mM H202. Treatment of the cells with an intracellular Ca 2÷antagonist TBM-8 prevents H202 induced membrane PLA2 activity and AA release without affecting membrane PKC activity. Treatment of the cells with TMB-8 before addition of 4~-PMA (up-regulation of PKC) followed by incubation with 0.4 mM H,O2 does not augment PLA, activity and AA release, although membrane PKC activity increases under this condition. Key words: Oxidant, hydrogen peroxide, protein kinase C, phospholinase A> arachidonic acid, up-regulation, downregulation, smooth muscle cells, signal transduction.

(1,4,5 IP3) and 1,2-Sn-diacylglycerol ( D A G ) act as second messenger molecules which in turn activate the protein kinase C (PKC) enzyme family in membranes [3-5]. This appears to be the primary m e c h a n i s m for initiating P K C - m e d i a t e d cellular effects, although there are alternative p a t h w a y s that may be utilized for generating the necessary Ca 2÷ signal [6]. Physiological activation of P K C by D A G can be m i m i c k e d by tumour-promoting p h o r b o l e s t e r s [7, 8]. P K C can m o d u l a t e the movement o f certain ions into and out of the cytoplasmic compartment o f the cell, and thus can regulate the cellular processes that depend on this. Alteration of the activity of 'exchange' and ' p u m p ' p r o t e i n s b y P K C is w e l l d o c u m e n t e d [9-12]. Recent research indicates that PKC is able to modulate the activity of the numerous pharma-

INTRODUCTION Transmembrane signal transduction systems involve a variety of transducers o f which protein kinase C (PKC) is an important m e m b e r [1, 2]. Receptor-mediated hydrolytic products of inositol 4, 5 biphosphate, i.e. inositol 1,4,5 trisphosphate *To whom correspondence should be addressed at the University of Kalyani. Abbreviations: DMEM--Dulbecco's modified Eagles medium; 4I].----PMA4[~---phorbol 12[~ myristate 13ct acetate; 4ot-PMA-4ct phorbol 1213myristate 13~ acetate; AA--arachidonic acid; PLA,--phospholipase A2; PKC--protein kinase C; PBS--phosphate-buffered saline; HPBS--Hank's buffered physiological saline; HEPES--4-(2-hydroxyethyl)-I-piperazine N-2-ethane sulfonate; FCS--foetal calf serum; H,O2-hydrogen peroxide; TMB-8--8-N.N-diethylamino octyl 3,4,5trimethoxy benzoate; HT--1-(5-isoquinoinesulphonyl)-2methyl piperazine; BSA--bovine serum albumin. 75

76

S. CHAKRABORTI and T. CHAKRABORT1

cologicaily distinct types of ion channels [13]. Activation of phospholipase A 2 (PLA2) and subsequent release of arachidonic acid (AA) is an important physiological and pathophysiological phenomena [14-16]. W e have previously shown that the P K C inhibitors 1-(5-isoquinoinesulphonyl)-2-methyl piperazine (H7) and sphingosine prevent the oxidant H202-mediated activation of P L A 2 in rabbit pulmonary vascular smooth muscle cells [17]. In view o f this, and to gain an insight into the role of P K C in stimulating membrane PLA2 activity and A A release in rabbit pulmonary vascular smooth muscle cells under exposure to the oxidant H202, the present study was undertaken. Our results show that (i) PKC plays an important role in stimulating membrane P L A 2 activity and subsequent release o f A A under exposure of the smooth muscle cells to H202, and (ii) release o f Ca 2÷ from intracellular storage site(s) along with an increase in membrane P K C activity are equally important and synergistically effective in stimulating membrane PLA2 activity and A A release under exposure of the smooth muscle cells to H~O 2. MATERIALS AND METHODS HBPS, FCS, DMEM, PBS without Ca 2÷ and Mg 2* were obtained from GIBCO Laboratories (Grand Island, N.Y., U.S.A.). Staurosporine, ATP, diolein, fatty acid free bovine serum albumin, HEPES, dithiothreitol, leupeptin, histone type Ills, 4c~-phorbol 1213-myristate 13c~ acetate (4ot-PMA), 413-phorbol 1213 myristate 13or acetate (4~-PMA) and 8,N,N'-diethylamino octyl 3,4,5trimethoxy benzoate (TMB-8) were obtained from Sigma Chemical Co., St. Louis, MO, U.S.A. [)4C] arachidonic acid (sp. activity 54.6 Ci/mol); 1-stearoyl2-[t4C] arachidonyl-L-3-glycero-phosphocholine (sp. activity 58.3 Ci/m mol) and [T-32P] ATP (sp. activity 50-100 cpm/p tool) were obtained from New England Nuclear (Wilmington, DE, U.S.A.). All other chemicals used were of analytical grade.

Cell culture Rabbit pulmonary arterial smooth muscle cells were isolated by following the procedure previously described [18]. Cells were maintained in Dulbecco's modified Eagles medium (DMEM) with 20% FCS, I00 units of streptomycin and penicillin, L-glutamine, non-

essential amino acids and 1.5 g sodium bicarbonate per litre. Cells were subcultured after treatment with 0.25% trypsin. Cells were studied between passages 7 and 12. All experiments were performed using confluent monolayers and in serum free media supplemented with fatty acid free bovine serum albumin (1 mg/ml).

Measurement of [14C] arachidonic (AA) release Cells grown in six-well plates were washed twice with [t4C] AA (sp. activity 54.6 Ci/mol: 2 p_Ci/well). After incubation, the supernatant was removed and the cells were washed twice with phosphate-buffered saline (PBS). AA release was determined as previously described [ 14].

Preparation of cytosol and membrane fraction The smooth muscle cell membrane and cytosol fractions were isolated by following the procedure previously described [ 19]. Briefly, rabbit pulmonary vascular smooth muscle cells grown in T-150 flasks were washed twice with PBS and incubated in a medium containing PBS plus fatty acid free bovine serum albumin (1 mg/ml). After incubation for 15 min, the medium was taken out with a sterile Pasteur pipette then ice-cold homogenizing medium [20 mM HEPES/NaOH (pH 7.4), 3 mM MgC12, 0.1% fatty acid free BSA] was added. The cells were scraped from the flasks and disrupted by sonication with a cell sonicator and centrifuged at 100,000 g for 1 h at 4°C. This supernatant was used as the cytosolic fraction. Its protein concentration was adjusted to 1 mg/ml with the homogenizing medium. The pellet from the first centrifugation was suspended in ice-cold homogenizing buffer containing 0.1% Triton × 100. After sonication, the pellet was kept at 4°C with constant shaking for 1 h then centrifuged at 100,000 g for 1 h at 4°C. This supernatant fraction was used as the membrane fraction. Its protein concentration was adjusted to 1 mg/ml with the homogenizing medium supplemented with Triton X-100.

In vitro assay of phospholipase A2 (PLA2)activity One to 3 mg of membrane protein suspension (20 ~1) was added to 30 ~tl of reaction mixture which contained (final concentration):Tris-hydroxymethylaminomethane (Tris) buffer (100 ~tM) NaCI (100 mM), deoxycholate (1 mM and the phospholipid L-3-phosphatidylcholine (10 ~tM). PLA2 activity was quantified at pH 9.0 by following the procedure previously described [14].

Measurement of protein kinase C (PKC) activity Protein kinase C activity was quantified by measur-

Regulation of phospholipase A2 by protein kinase C

ing [32p] transfer from [~-32P] ATP into histone [20]. Briefly, 20-~1 aliquots of the cytosolic or membrane fraction were added to 80 ~tl of the mixture containing 25mM HEPES-NaOH pH 7.4, 20 laM ATP, 0.2 mg/ml histone type Ills, 10 ~tM [,~_32p] ATP (50-100 cpm/p mol). Reactions were performed in the presence and absence of 1.5 mM CaCI2, 25 ~tg/ml phosphatidyl serine, and 0.5 ktg/ml diolein. Incubations were performed at 30°C for 10 min. The reaction was terminated with the addition of 1 ml of ice-cold stopping solution containing 10% TCA and 2 mM ATP, followed by the addition of 100DI of 0.5% fatty acid free BSA. After centrifugation at 800 g for 20 min, the supernatant was discarded and the pellet was resuspended in 0.1 ml of 0.1 N NaOH and immediately reprecipitated with 1 ml of ice-cold stopping solution. The precipitated protein was trapped on a Millipore HA filter. The filter was washed five times with 3 ml of cold 5% TCA, dried, and the radioactivity on the filter was measured. PKC activity reflects the difference in activity measured in the presence of calcium, phosphatidyl serine and diolein. Results are expressed as p mol of [32p] incorporated/min/mg protein. Protein concentration was determined by Bradford microassay method [21] using BSA as the standard. Cell viability None of the treatments affected cell viability as assessed by Trypan Blue exclusion. Statistical analysis Data were analysed by the unpaired t-test and one analysis of variance [22]. Statistical significance was assumed when P < 0.05. Experimental protocols Dose effect of H202 on AA release, PLA 2 activity and PKC activity. Cells were exposed to H202 (0-1.2 mM) for 15 min then AA release, membrane PLA2 activity, and membrane PKC activity were determined. Preliminary experiments studying different exposure times (0-20 min) indicated that maximal AA release occurred at 15 min. Effect of staurosporine on H202-induced AA release and PLA z activity. The dose effect of staurosporine on AA release caused by the maximally effective dose of H:O2 was investigated initially. The cells were treated with different concentrations of staurosporine (0-1.25 I.tM) for 20 min before addition of 1 mM H202 for 15 min. To determine the effect of staurosporine on AA

77

release and PLA 2 activity caused by 0.4 mM H202 or 1 mM H202, the cells were treated with 1 laM staurosporine for 20 min before the addition of 0.4 mM or 1 mM H:O2 for 15 min. Effect pretreatment with TMB-8 on H202- induced AA releasemembrane PLA, activi~.', and membrane PKC activity. Cells were treated with TMB-8 (100 ~M) for 20 min in DMEM supplemented with 1 mg/ml fatty acid free bovine serum albumin or in nominal Ca 2+ free PBS before addition of 0.4 mM and 1 mM H,O~ for 15 mins then AA release, membrane PLA2 activity, and membrane PKC activity were determined. 100 ~tM TMB-8 was used because this concentration has been found to be optimal [14]. Effects of combining 4fl-PMA and H20~. The effects of 4[~-PMA dose and incubation time on AA release caused by 0.4 mM H202 were determined. Based upon these results, the effects of treatment of 200 nM 4[3PMA for 10 min before addition of 0.4 mM H_~O2for 15 min on membrane PKC activity and membrane PLA, activity were measured To study the effect of prolonged treatment of 413PMA on H202 induced AA release, membrane PLA, activity and membrane PKC activity, cells were incubated with 200 nM 413-PMA for 2 h before exposure to 0.4 mM H202 for 15 min. To determine whether staurosporine could block the enhanced AA release, membrane PLA 2 activity and membrane PKC activity, ceils were treated with 1 ~tM staurosporine for 20 min before adding 200 laM 413PMA for 10 min followed by 0.4mM H202 for 15 min. To determine the effect of TMB-8, cells were pretreated with TMB-8 (100 ~tM) for 20 min before treatment with 1 mM H.,O2 or 0.4 mM H.~O2 for 15 min. or before adding 413-PMA (200 I.tM) for 10 min followed by 0.4 mM H202 for 15 min. After this treatment, AA release, membrane PLA2 activity, and membrane PKC activity were determined.

RESULTS H202 caused a d o s e - d e p e n d e n t increase in A A release, m e m b r a n e PLA2 activity, and m e m b r a n e P K C activity reaching a m a x i m u m at 1 m M (Figs l, 2). Staurosporine has been demonstrated to be a potent inhibitor of P K C in different systems [23]. To investigate whether P K C activity in the m e m brane plays a role in the H202 response, the effect pf pretreatment with the P K C inhibitor staurosporine has been studied. At 1 I.tM concentration, staurosporine elicited m a x i m u m inhibitory

78

S. CHAKRABORTI and T. CHAKRABORTI

1400

14

_

/I--2~;

_

.>, •; T

1200

12

"d Qi ~Z

tooo

10 ~

o ~

800

"~ ~'

600

~ ~

400

"7--

l r ~

t/

6

20O

.~'~

o-.,(

2 I

l

I

I

3.75

3.50

3.25

3.00

¢

~

HzO 2 ( - l o g M )

Fig. 1. Hydrogen peroxide (H,O,) dose-dependently stimulates [~4C] AA release (-@-) and membrane PLA~ activity (-A-) in rabbit pulmonary arterial smooth muscle cells [Control:AA release, 402 _+32 (cpm/10 -scells); PLA_~'activity, 4.4 _+0.42 (p mol AA/min/mg protein)]. *P < 0.05 vs basal (control) condition. 5"P < 0.001 vs basal (control) condition. Results are expressed as mean _+S.E. (n = 4).

->- "~

~

~

1400

-

14

1200

-

12

soo

~ 6oo •"

~:

~

";

- l0

~

- 8

~,.~

-6

~.~_ ~.~

--2

~'~

400

200 o

I

I

I

I

3."/5

3.50

3.25

3.00

0

H z O 2 (-IogM) Fig. 2. D o s e - r e s p o n s e c u r v e for h y d r o g e n peroxide (H202)-stimulated m e m b r a n e P K C activity and PLA2 activity in

rabbit pulmonary arterial smooth muscle cells [Control:PKC activity, 180 __ 26 (p mol{32P} incorporated/min/mg protein]; PLA 2 activity 4.4 __ 0.42 (p tool AA/min/mg protein]. -tP 0.001 vs basal (control) condition. Results are mean _+S.E. (n = 4). effect on A A release caused by H202 (Fig 3). Staurosporine also prevents P L A 2 activity caused by H_~O2 (Fig 4). To investigate further the potential role o f PKC in activating PLAz activity, the effects o f an active phorbol ester 4[~-PMA and an

inactive phorbol ester 4c~-PMA by themselves or combined with a suboptical dose of H202 (0.4 mM) was studied. Neither 4 a - P M A nor 4 ~ - P M A increases basal A A release (control, 406 _+ 25 cpm/105 cells; 200 nM 4[3-PMA, 402 ___ 32

Regulation of phospholipase A2 by protein kinase C

1200

--

1000

--

o a ¢J

~ = ID LI

.<

800

--

600

--

400

--

200

--

79

~t

U

v

(J

0

I

I

I

I

I

6.75

6.50

6.25

6.00

5.75

Staurosporin (-logM) Fig. 3. Dose-response profile of staurosporine to prevent (H302)-caused stimulation of AA release in rabbit pulmonary arterial smooth muscle cells [AA release: control 402 _+32 cpm/l@ cells vs 1308 -+ 84 cpm/105 cells with 1 mM H202]. t P < 0.001 compared with 1 mM H:O.~ treatment. 1600

--

1400

--

(a)

t200 ..~ ,~ ~

. T

12 ....

t000

~

10

~E ~

A

B

~

C

2 0

D

T

T

~

A

B

~

C

D

Fig. 4. Effect of pretreatment with staurosporine on AA release (a) and membrane PLA2 activity (b). Rabbit pulmonary arterial smooth muscle cells are pretreated with I ~M staurosporine for 20 min then exposed to one of the four conditions. (A) control (D) vs control + staurosporine (II). (B) 0.4 mM H202 (V]) vs I ~M staurosporine + 0.4 mM H_~O2(II). (C) 200 nM 413-PMA + 0.4 mM HzO: ([]) vs staurosporine + 200 nM 4I~-PMA + 0.4 mM HzO_~(IlL (D) l mM H20: (rq) vs l IktM.~taurosporine + I mM H202 (III). *P < 0.001 compared to basal condition. "IP < 0.001 compared to the response produced by 4~-PMA + 0.4 mM H20_~. ~P < 0.001 compared to the response produced by l mM H202. cpm/105 cells; 200 nM 4tx-PMA, 410 _+ 28 cpm/105 cells; not significant). 4[3-PMA does not affect basal PLA2 activity (control 4.25 _+ 0.29 p mol A A / m i n / m g protein vs 4.20 -4- 32 pmol A A / m i n / m g protein with 200 nM 4 ~ - P M A ; not significant). 4ct-PMA treatment did not augment 0.4 mM HzOz-induced A A release (710 _+ 42 cpm/105 cells with 0.4 mM H20_~ vs 702 _+ 38 with 200 nM 4o~-PMA and 0.4 mM H202; not signifi-

cant). Treatment with 413-PMA, however, augments the increase in A A release and PLA~ activity caused by a sub-optimal dose 0.4 mM H_~O, (Figs 5, 6). Consequently, adding 4[3-PMA caused dose-dependent increase in membrane PKC activity (Fig. 7) which accounts for the stimulatory effect as indicated by the ability of staurosporine to prevent the synergistic effect of 413-PMA on membrane P L A 2 activity and A A release (Fig. 4).

80

S. CHAKRABORTI and T. CHAKRABORTI

~E

t~ ~

120

"~ "~ 100 N

go

~

60

- -

20

•-~

o 0

2.5

5.0

7.5

10.0 12.5

30

60

90

120

Time (rain) Fig. 5. Time and dose effect of treatment with 4~-PMA on AA release caused by 0.4 mM H_,O~. After treatment, all cells were exposed to 0.4 mM H202. The 413-PMA doses studied were 100 nM (©), 200 nM (O) and 300 nM (U]). Results are mean _ S.E. (n = 4). 1400

o

14

(a)

(b)

I200

.o

000 o

~

800

0 "~ ~

600

~

400

rJ

200

0

A

B

C

A

B

C

Fig. 6. Effect of treatment of rabbit pulmonary arterial smooth muscle cells with 4[3-PAM for 10 min or 2 h on AA release (a) and PLA_, activity (b). Three conditions were studied. (A) control ([]) vs 0.4 mM H202 (m). (B) 200 nM 4~-PMA treatment for 10 min (U]) vs 200 nM 4[3-PMA treatment for 10 min followed by 0.4 mM H_,O2 ( I ) . (C) 200 nM 4 I3-PMA treatment for 2 h (IS]) vs 4~-PMA (200 nM) for 2 h followed by 0.4 mM H202 (m). *P < 0.001 compared to control, t P < 0.001 compared to the response produced by treatment with 413-PMA for 10 min. The effect of 413-PMA dose and incubation time on A A release caused by 0.4 m M H202 has also been studied (Fig 5). In a concentration dependent manner, 413-PMA augments A A release caused by 0.4 mM H202 (Fig 5). Interestingly, treatment with 200 nM 413-PMA enhances A A release more than treatment with 300 nM 413P M A (Fig. 5). The ability o f a given 413-PMA dose to increase H202-medicated A A release corresponds with its ability to increase PKC activity in membrane (Figs 5, 6, 7). Increasing 413-PMA dose from 100 nM to 200 nM augments membrane PKC activity but a dose of 300 nM

decreases membrane PKC activity compared to the effect of 200 nM (Fig. 7). The time of pretreatment with 413-PMA also significantly influences the stimulatory effect on H202 -induced A A release (Fig 5). Treatment with 413-PMA for 10 min augments A A release and PLA2 activity caused by 0.4 m M H202 due to up-regulation of PKC whereas treatment with 413-PMA for longer periods attenuates the augmenting effect o f 413P M A on A A release caused by 0.4 m M H20,. (Figs 5, 6) due to down-regulation o f PKC. Because of the previous observation that oxidant-mediated release of intracellular Ca 2÷ may

Regulation of phospholipase A: by protein kinase C

...q

~o •~ ~

1600

--

1400

--

1200 --

o ~ ~: ".~

tooo

~

800

oe ~ 8 o . _~

600

~

o

~. -

tf'

-

400 -

I 7.00

I 6.75

I 6.50

81

appear to contribute to an increase in AA release and PLA 2 activity [14], studies have been carried out to investigate the effect of an intracellular Ca -,+ antagonist TMB-8 on H202-induced AA release and PLA 2 activity. Treatment of the cells with TMB-8 before the addition of H202 prevents AA release and PLA: activity without affecting membrane PKC activity (Table 1). In addition, treatment with TMB-8 before adding 413-PMA (upregulation of PKC) followed by 0.4 mM H:O, prevents AA release and PLA2 activity without causing an appreciable alteration of membrane PKC activity (Table 1).

41~-PMA ( - l o g M )

DISCUSSION

Fig. 7. D o s e effect o f 413-PMA o n m e m b r a n e P K C activity in rabbit p u l m o n a r y v a s c u l a r s m o o t h m u s c l e cells. 200 n M 413-PMA increase P K C activity m o r e t h a n does 300 n M (basal P K C activity, 192 _+ 28 p m o l [3.,p] i n c o r p o r a t e d / m i n / m g protein), t P < 0.001 c o m p a r e d to basal condition. R e s u l t s are m e a n _+ S.E. (n = 4).

The oxidant H202 dose-dependently stimulates AA release and membrane PLA2 activity in rabbit pulmonary vascular smooth muscle cells (Fig. 1). The mechanism by which H_,O_~stimulates PLA 2 activity and subsequent increase in AA release appears to be via involvement of membrane PKC (Fig. 2). Several lines of evidence suggest that PKC plays an important role in increasing AA

Table 1. Effect of different treatments on H_,O:-mediated AA release, membrane PLA_, activity and membrane PKC activity in rabbit pulmonary arterial smooth muscle cells

AA release (cpm/105 cells)

Condition Basal H20~ (1 mM) TMB-8 ( 100 p.M) TMB-8 (100 MM) + H20:(1 mM) H,O.~ (0.4 mM) TMB-8 ( 100 HM) + H202 (0.4 raM) 4~-PMA (200 nM) (up-regulation of PKC) TMB-8 (100 MM) + 413-PMA (200 nM) (up-regulation of PKC) 4 ~I-PMA (200 nM) (up-regulation of PKCt + H202 (0.4 mM) TMB-8 (100 p_M) + 413-PMA (200 nM) (up-regulation of PKC) + H.,O_, (0.4 mMI 413-PMA (200 nm) (down-regulation of PKC) 4[3-PMA (200 nM) (down-regulation of PKC) + H202 (0.4 mM) Results are *P < 0.001 t P < 0.001 :I:P < 0.001 §P < 0.001

PLA_, activity (p mol AA/min/mg protein)

PKC activity (p mol [~-'P] incorporated/ mirdmg protein)

392 ± 38 1308 -+ 84* 378 ± 34 592 ± 4 4 t 682 ± 42 424 ± 38+ 386 ± 28 374 ± 32

3.94 ± 0.42 10.96 ± 0.82* 3.76 ± 0.40 4.82 ± 0.44t 6.22 -* 0.48 3.98 ± 0.36:~ 3.78 ± 0.42 3.72 ± 0.44

192 +_28 1096 ± 48* 186 +_.24 1116 ± 62* 324 -+ 34 312 ± 26 1416 ± 64 1402 ± 56

1156 ± 78~:

9.82 ± 0.66:{:

1484 ± 72+

482 ± 36§

4.62 ± 0.38§

1420 -+ 68

378 +- 28 762 _+64§

3.82 - 0.36 • 6.78 ± 0.48§

418 ± 32 448 ± 36§

mean ± S.E. (n = 4). compared with control (basal) condition. compared to the response produced by 1 mM H20 :. compared to the response produced by 0.4 mM H,O2. compared to the response produced by 413-PMA (200 nM) (up-regulation of PKC) followed by 0.4 mM H_,O2.

82

S. CHAKRABORTIandT.CHAKRABORTI

release and PLA2 activity caused by the oxidant H202. First the PKC inhibitor staurosporine prevents the increase in AA release and PLA 2 activity (Fig. 4). Secondly, H202 stimulates membrane PLA 2 activity and membrane PKC activity in a dose-dependent manner (Figs 1, 2). Third, activating membrane PKC by 4[3-PMA for a short time (up-regulation of PKC) significantly augments AA release and PLA2 activity caused by a suboptimal dose of 0.4 mM H202 (Fig. 6). Under this condition membrane PKC activity also corresponds with membrane PLA2 activity and AA release (Fig. 6, Table 1). Fourthly, the augmenting effect of 413-PMA on AA release and PLA~ activity caused by 0.4 mM H202 was prevented upon pretreatment of the cells with staurosporine (Fig. 4). Finally, prolonged treatment of the cells with 4]3-PMA (down-regulation of PKC) attenuates the increase in AA release and PLA2 activity caused by 0.4 mM H202 (Fig. 6). Under this condition membrane PKC activity also corresponds with membrane PLA2 activity and AA release (Fig. 6, Table 1). Previous studies showed that oxygen-derived free radicals may be involved in the development of Ca 2÷ overload [24]. Reperfusion and reoxygenation after prolonged ischemia and hypoxia which produces aggressive free radicals in situ have been shown to cause an increase in cytosolic Ca 2+ content [25-27]. The rise of cytosolic Ca -,+ may subsequently activate a variety of Ca -~* dependent enzymes, for example, phospholipase A 2 directly or indirectly via intracellular regulators, for instance, PKC [18, 28-31]. In hepatocytes, oxidant-mediated release of Ca ,-+ from both mitochondrial and extra-mitochondrial stores have been indicated by previous researchers [31, 32]. Pretreatment of bovine pulmonary vascular endothelial cells with the intracellular calcium antagonist TMB-8 has been shown to prevent the oxidant t-buOOH stimulated AA release [14], which indicates that the oxidant may release stored intracellular Ca 2÷ and this could play an important role in stimulating membrane PLA~ activity and AA release. The present study indicates that 4]3-PMA by itself stimulates membrane PKC activity without stimulating AA release and

PLA_~ activity iFigs 6, 7). Pretreatment with TMB8 prevented H20_, stimulated membrane PLA, activity and AA release without causing an appreciable alteration of membrane PKC activity (Table 1). Additionally, treatment with TMB-8 prevented 413-PMA induced augmentation of AA release and PLA2 activity caused by a sub-optimal dose of 0.4 mM H202 without affecting membrane PKC activity (Table 1). Thus, in the pulmonary vascular smooth muscle cells, PKC activation by itself is an insufficient stimulus for activating PLA~ activity and triggering AA release. Treatment with 4I~-PMA, however, stimulates membrane PKC activity and causes a synergistic increase in PLA, activity and AA release produced by a sub-optimal dose of 0.4 mM H202 (Figs 6, 7). In platelets, human umbilical endothelial cells and bovine pulmonary vascular endothelial cells, a similar effect of PKC activation on AA release and PLA2 activity has been observed. These activators do not affect basal A A release, but combined with a small rise in intracellular Ca 2+ concentration they produce a synergistic increase in AA release and PLA2 activity [18, 33, 34]. Conceivably, a rise in intracellular Ca ,-+ concentration and an increase in membrane PKC activity are equally important and synergistically effective in stimulating membrane PLA_~ activity and the subsequent increase in AA release in rabbit pulmonary vascular smooth muscle cells under exposure to the oxidant H_~O2. Acknowledgement Thanks are due to Dr John R. Michael (Department of Medicine, University of Utah Health Sciences Center, Salt Lake City, Utah), Professor Prasanta K. Ray (Director, Bose Institute, Calcutta) and Dr W. Selvamurthy (Director, Defence Institute of Physiology and Allied Sciences, Delhi).

REFERENCES 1. Nishizuka Y. (1986) Science (Washington D.C.) 233, 305-312. 2. Kikkawa U. and Nishizuka Y. (1986)A. Rev. Cell Biol. 2, 149-178. 3. Berridge M. J. (1987) A. Rev. Biochem. 56, 15993. 4. Nishizuka Y. (1984) Nature (Lond.). 308, 693698.

Regulationof phospholipase A: by proteinkinase C 5. Nishizuka Y. (1988) Nature (Londl) 334, 661-665. 6. Berridge M. J. (1993) Nature (Lond.) 361, 315-325. 7. Ashendel C. L., Staller J. M. and Boutweli R. K. (1983) Biochem. biophys. Res. Commun. 111. 340-345. 8. Castagna M., Takai Y., Kaibuchi K., Sano K., Kikkawa U. and Nishizuka Y. (1982) J. biol. Chem. 257, 7847-7851. 9. Grinstein S., Smith J. D., Onizuka R., Cheung R., Gelfand E. W. and Benedict S. (1988) J biol. Chem. 263, 8658-8665. 10. Parker J., Daniel L. W. and Waite M. (1987) J. biol Chem. 265, 2091-2097. 11. Wang K. K. W., Wright C. C., Machan C. L., Allen B. G., Conigrave A. D. and Roufogalis B. D. (1991 ) J. biol Chem. 266, 9078-9085. 12. Neyses L., Reinlib L. and Carafoli E. (1985) J. biol Chem. 260, 10,283-10,287. 13. Harris K. M., Kongsamut S. and Miller R. (1986) Biochem. biophys. Res. Commun. 134, 1298-1305. 14. Chakraborti S., Gurtner G. and Michael J. R. (1989) Am J. Physiol. 257, L430-L437. 15. Clark M. A., Littlejohn D., Mong L. and Crooke S. T. (1986) Prostaglandins 31, 157-166. 16. Farukh I. S., Michael J. R., Peters S. P., Scuito A. M., Adkinson N. F. Jr, Freeland H. S., Paky A., Spannhake E. W., Summer W. R, and Gurtner G. H. (1988)Am. Rev. Respir. Dis. 137, 1343-1349. 17. Chakraborti S. and Michael J. R. (1993) Molec. Cell Biochem. 122, 9-15. 18. Chakraborti S., Michael J. R. and Patra S. (1991) FEBS Lett. 285, 104-107. 19. Chakraborti S., Michael J. R. and Sanyal T. (1992) Eur. J. Biochem. 206, 965-972. 20. Kitano T., Go M., Kikkawa U. and Nishizuka Y. (1986) Methods Enzymol. 124, 349-353.

83

21. Bradford M. M. (1976) Analvt. Biochem. 72, 248-254. 22. Snedecor G. M. and Cochran W. G. (1980) Statistical Methods. (7th edition), Iowa State Univ. Press, Ames, Iowa, U.S.A. 23. Vagesna R.V.K., Wu H., Mong S. and Crooke S.T. (1988) Molec. Pharmac. 33, 537-542. 24. Hess M. L. and Manson N. H. (1984) J.Molec. Cell. Cardiol 16, 969-985. 25. Bourdillion P. D.,Poole-Wilson P. A. (1982) Circ. Res. 50, 360-368. 26. Neyler W. G., Papagiotopoulos S., Elz J. S.. Daly M. J. (1988) J. Molee. Cell. Cardiol. 20 (Suppl. II), 41-54 27. Poole-Wilson P. A., Harding D. P., Bourdillion P. D. V. and Tones M.A. (1984) J. Molec. Cell. Cardiol. 16, 175-187. 28. Wong P. Y. K. and Wong W. Y. (1979) Biochent. biophys. Res. Commun. 90, 473--480. 29. Hirata F., Schiffman E., Venkatasubramonium K., Salomom D. and Axelrod J. (1980) Proc. natn. Acad. Sci. U.S.A. 77, 2533-2536. 30. Clark M. A., Chen M. J., Crooke S. T. and Bomalaski J. S. (1988) Biochem. J. 250, 125-132. 31. Baumhuter S. and Christoph R. (1982) FEBS Lett. 148, 271-275. 32. Lewis M. S., Whatley R. E., Cain P., Mclntyre T. M., Prescott S.M. amd Zimmerman G.A. (1988) J. clin. Invest. 82, 2045-2055. 33. Touqui L., Rothhut B., Shaw A. M., Vargaftig B. B. and Russo-Marie F. (1986) Nature (l_xmd.) 321, 177-180. 34. Takayama H., Krou M. H. Gibrone M. A. J. and Schafer A. I. (1989) Biochem. J. 258, 427--434.