Biochimica et Biophysica Acta, 1093 (1991) 55-64 © 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 ADONIS 0167488991001773

55

BBAMCR 12942

Protein kinase C and cyclic AMP modulate thrombin-induced platelet-activating factor synthesis in human endothelial cells Regine H e l l e r 1.2 F e d e r i c o B u s s o l i n o 2, D a r i o G h i g o 2, G i o v a n n i G a r b a r i n o 3, H e n n i n g SchriSder 4 G i a n p i e r o P e s c a r m o n a 2, U w e Till 1 a n d A m a l i a Bosia 2 I Department of Pathological Biochemistry, Medical Academy, Erfurt (F.R.G.), 2 Department of Genetics, Biology and Medical Chemistry, University of Torino, Torino (Italy), ~ Institute of Internal Medicine, University of Torino, Torino (Italy) and 4 Institute of Pharmacology, University of Diisseidorf, Diisseldorf (F. R. G.)

(Received 14 June 1990) (Revised manuscript received 20 December 1990)

Key words: PAF synthesis; Endothelium; Thrombin; Calcium ion: Protein kinase C; cyclic AMP

Stimulation of human endothelial cells (EC) by thrombin elicits a rapid increase of intracellular free Ca2+ [(Ca2+]i), platelet-activating factor (PAF) production and l.O-alkyl.2-1yso-sn-glycero-3-phosphocholine (lyso-PAF):acetyl-CoA acetyltransferase (EC 2.3.1.67) activity. The treatment of EC with thrombin leads to a 90% decrease in the cytosolic protein kinase C (PKC) activity; this dramatic decline is accompanied by an increase of the enzymatic activity in the particulate fraction. The role of PKC in thrombin-mediated PAF synthesis has been assessed: (1) by the blockade of PKC activity with partially selective inhibitors (palmitoyl-carnitine, sphingosine and H-7); (2) by chronic exposure of EC to phorbol 12-myristate 13-acetate (PMA), which results in down-regulation of PKC. in both cases, a strong inhibition of thrombin-induced PAF production is observed, suggesting obligatory requirement of PKC activity for PAF synthesis. It is suggested that PKC regulates EC phospholipase A 2 (PLA z) activity as thrombin-induced arachidonic acid (AA) release is 90% inhibited in PKC-depleted cells. Brief exposure of EC to PMA strongly inhibits thrombin-induced [Ca2+]i rise, acetyitransferase activation and PAF production, suggesting that, in addition to the positive forward action, PKC provides a negative feedback control over membrane signalling pathways involved in the thrombin effect on EC. Forskolin and iloprost, two agents that increase the level of cellular cAMP in EC, are very effective in inhibiting thrombin-evoked cytosolic Ca2+ rise, acetyltransferase activation and PAF production; this suggests that endogenously generated prostacyclin (PGi 2) may modulate the synthesis of PAF in human endothelial cells.

Introduction

Thrombin-stimulated human endothelial cells (EC) produce platelet-activating factor (PAF) [1,2], that is

Abbreviations: EC, endothelial cells; PAF, platelet-activating factor (1-O.alkyl-2-acetyl-sn-glycero-3-phosphocholine); lyso-PAF, l-O-alkyl-2-1yso-sn-glycero-3-phosphocholine; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; H-7, l-(5-isoquinolinylsuifonyl)-2methylpiperazine; PGI 2, prostacyclin (prostaglandin 12): Ptdlns(4,5) P2, phosphatidylinositol 4,5-bisphosphate; Ins(l,4,5)P3, inositol 1,4,5trisphosphate; Pi, phosphoinositide; DAG, 1,2-diacyiglycerol; BSA, bovine serum albumin: TLC, thin-layer chromatography: [Ca z+ ]i, cytosolic calcium; quin2-AM, quin2-tetraacetoxymethyl ester; IBMX, 3-isobutyl-l-methylxanthine: AA, arachidonic acid; PLA 2, phospholipase A2; PLC, phospholipase C. Correspondence: A. Bosia, Dipartimento di Genetica, Biologia e Chimica Medica, Sezione di Chimica Medica, Via Santena 5/his, 10126 Torino, Italy.

proposed to play an important role in acute inflammatory events. EC appear to synthesize PAF primarily by a remodelling pathway involving a tightly coupled reaction of phospholipase A 2 and 1-O.alkyl-2-1yso-snglycero-3-phosphocholine (lyso-PAF) : acetyl-CoA acetyltransferase [3-5]. Both enzymes are calcium-dependent. The rise in the amount of acetyltransferase active molecules is due to the posttranslational modification of the inactive precursor by a process shown to be phosphorylation in different cell types [6-10]. In human EC, thrombin is known to induce the release of Ca 2+ from intracellular stores and a substantial Ca 2+ influx across the plasma membrane [4,11-15]. We have shown that N a + / H + exchange activation and the resulting intraceilular alkalinization play a direct role in the induction of Ca 2+ influx and PAF synthesis in human EC [4]. It is now established that in human EC thrombin also stimulates the rapid hydrolysis of phosphatidyl-

56 inositol 4,5-bisphosphate (Ptdlns(4,5)P2) [12,16] and the accumulation of inositol 1,4,5-trisphosphate (Ins(I,4,5) P3) [11-13,16], which acts as a second messenger within the cell to stimulate calcium release from the endoplasmic reticulum [17]. The other major product of phospholipase C (PLC)-mediated Ptdlns(4,5)P2 hydrolysis, 1,2-diacylglycerol (DAG), is believed to exert physiological actions by activating protein kinase C (PKC) [18-20], which has been identified as the major cellular receptor for tumor-promoting phorbol esters [21]. Phorbol esters mimic the effect of DAG with respect to activation of PKC. The phospholipid requirement for PKC activity is most likely to be fulfilled by membrane association of cellular enzyme, and agonistdirected changes in subcellular distribution of PKC remains the least ambiguous indicator of its activation. In many cells, the distinctions between Ca 2+- and cAMP-mediated responses are striking. Ca2+-dependent pathways seem invariably to be associated with cellular activation [17], whereas cyclic nucleotides are usually found to be inhibitors. Indeed, agents that increase intracellular cAMP have been shown to decrease Ptdlns(4,5)P2 hydrolysis [22-24], and these results suggest that cAMP may regulate calcium mobilization by inhibiting the generation of Ins(1,4,5)P3 [25]. No evidence till now has been provided of a thrombin-directed change in subcellular distribution of PKC in human endothelial cells. Moreover, the role of PKC and of cAMP in calcium mobilization and in PAF synthesis during thrombin-induced activation remains poorly understood. In the present study, we show: (1) that thrombin elicits a rapid membrane association of PKC in human endothelial cells; (2) that, in thrombin-stimulated EC, PKC activity appears to be required to couple the rise in [Ca2+]i with PAF synthesis, acting presumably at the phospholipase A 2 step; (3) that thrombin-induced calcium flux, acetyltransferase activation and PAF synthesis are inhibited following PKC st,mulation, thus suggesting that the kinase may be invol,.,ed in feedback inhibition of Ptdlns(4,5)P2 hydrolysis; (4) that the elevation of cAMP in EC inhibits the thrombin-evoked Ca 2+ concentration changes and depresses the stimulatory effect of thrombin on acetyitransferase activation and PAF synthesis. Materials and Methods

Reagents. Medium 199 (M199) and fetal calf serum were from Flow Laboratories (McLean, VA); thrombin (human), fatty acid-free bovine serum albumin (BSA), Hepes, 2-mercaptoethanol, phenylmethylsulfonyl fluoride, leupeptin, pepstatin, aprotidin, phosphatidylserine, 1,2-diolein, type III-S histone, palmitoylcarnitine, sphingosine, phorbol 12-myristate 13-acetate (PMA), acetyl-CoA, forskolin and 3-isobutyl-l-methyl xanthine

(IBMX) from Sigma (St. Louis, MO); EDTA and EGTA from Fluka Chemical, top purity grade; DE-52 DEAEcellulose from Whatman (Maidstone, Kent, U.K.); PAF (1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine), and lyso-PAF (1-O-octadecyl-2-1yso-sn-glycero-3-phosphocholine) from Bachem Feinkemikalien (Bubendorf, Switzerland); [3H]PAF (120 mCi/mmol), [3H]acetylCoA (1 mCi/mmol; the specific activity was adjusted by addition of unlabeled acetyl-CoA), [~,-32p]ATP (2 mCi/mmol), [14C]AA (58 mCi/mmol) and quin2-tetraacetoxymethyl ester (quin2-AM) from Amersham (Bucks, U.K.); ionomycin from Behring Diagnostics; iloprost from Schering AG (Berlin, F.R.G.); H-7 from Seikagaku (Miami, FL); thin-layer chromatography (TLC) plates (60F254) and dimethylsulfoxide (Me2SO) from Merck (Darmstadt, F.R.G.). CV-3988 was from Takeda Chemical (Osaka, Japan), and BN52021 was a gift of Dr. P. Braquet (Institut Henri Beaufour, Le Plessis Robinson, France). Other reagents and solvents were analar grade or of the highest purity available. All plastics for the cell culture were from Falcon Labware (Div. Becton-Dickinson, Oxnard, Ca). Stock solutions of PMA, quin2-AM, ionomycin, IBMX and BN52021 were made in Me2SO, forskolin was dissolved in ethanol. Under each experimental condition, control and samples received the same volume addition of solvent, and the final solvent concentration never exceeded 0.1~. M199-BSA: medium 199 supplemented with 0.25% BSA (pH 7.4). Buffer A: 10 mM Hepes, 10 mM 2-mercaptoethanol, 5 mM EDTA (pH 7.5). Buffer B: buffer A containing 0.24 M sucrose, 0.43 mM phenylmethylsulfonyl fluoride, 0.1 mM leupeptin, 0.005 mM pepstatin and 22 U / m l aprotidin. Hepes buffer: 145 mM NaCI, 5 mM KCI, 1 mM MgSO 4, 10 mM Hepes, 10 mM glucose, 1 mM CaCI 2 (pH 7.4). Human endothelial cell cultures. EC were obtained by treating human umbilical cord veins with collagenase, cultured in 75 cm 2 plastic flasks in medium 199 (M199) containing 20~ fetal calf serum and characterized as previously described [1]. Confluent primary cultures were detached by trypsin/EDTA (0.05/0.02~, v/v) and plated on 35-ram diameter wells for the experiments of PAF production, the measurement of acetyltransferase activity and the measurement of cAMP, and on 100-mm diameter dishes for the measurement of PKC activity and AA release from membrane phospholipids. For [Ca2+]i transients measurements, cells were seeded on 12-ram diameter glass coverslips. Cells were used at confluence with a cell number of 5.0 + 0.4.105 per 35-mm dishes, 2.1 + 0.7.106 per 100-mm dishes and 5.0 + 0.3- 104 per glass coverslip (n = 10). No significant variations were observed after manipulations for the different measurements. The viability of EC was determined by trypan blue exclusion and ranged 95-98% after 20 min of incubation under the different conditions described.

57

Partial purification of PKC. Confluent EC grown on 100-ram diameter dishes were washed three times with M199-BSA and then stimulated at 37°C for different periods of time in 5 ml of prewarmed M199-BSA (pH 7.4) with the indicated concentrations of thrombin. At the end of the incubation, the cells were scraped in 5 ml of cold buffer B, sedimented and resuspended in I ml of buffer B. After sonication (six pulses of 10 s in ice bath, 100 W, Labsonic 1510, B. Braun Melsungen, F.R.G.), cytosolic and membranous fractions were prepared by ultracentrifugation (100000 x g, 1 h, 4°C). The various cell fractions were applied to a DE-52 DEAE-cellulose (Whatman) column (0.9 x 2.5 cm) equilibrated with buffer A [26]. The kinase activity was eluted by application of 18 ml linear concentration gradient of NaCl (0-0.2 M) in buffer A at a flow rate of 0.2 ml/min. P K C assay. Total PKC activity was quantified by measuring incorporation of 3:p from [y-32p]ATP into type III-S histone [21]. 200 #l of test mixture (10 mM Tris-HC! (pH 7.4), 5 mM MgCl 2, 1.75 mM CaC! 2, 10 mM ATP) contained 10 /~g phosphatidylserine, 10/~g 1,2-diolein, 100 #g III-S histone and 1 /~Ci [32p]ATP. Non specific PKC activity was assayed in the absence of cations and lipids but in the presence of EGTA. Net PKC activity was calculated by difference between total and non specific PKC activity. Production, characterization and measurement of PA F. EC were washed two times with M199-BSA to remove fetal calf serum, then preincubated and stimulated in 1 ml M199-BSA according to the experimental protocol. The reaction was blocked by adding 1 ml of acidified methanol to the incubation medium. Cells were scraped by using a rubber policeman. The cell suspension was transferred into a plastic tube, dishes were rinsed twice with 0.75 ml acidified methanol and the washings were pooled. Lipids were extracted from cells and medium according to a modification of Bligh and Dyer's procedure [27], with acetic acid added to methanol to lower the pH of the aqueous phase to 3.0 [28]. Following this procedure total PAF, i.e., cell-associated and released into the medium, was measured. However, according to former experiments with thrombin-stimulated EC, we could assume that under our conditions more than 9570 of the PAF remained cell-associated. The extracted lipids were submitted to TLC with chloroform/methanol/ water (65:35:6, v/v) as a solvent. The lipid material having an R F from 0.15 to 0.22 was extracted [29] and used for chemical characterization of PAF and for its quantitation. The recovery of 10 nCi of [3H]PAF after extraction and purification was 93-9570. PAF was characterized as previously described [4] by high-performance liquid chromatography (HPLC) [30] and chemical [31] and enzymatic [32] treatments. The PAF concentration was measured by the aggregation of washed rabbit platelets [1], using a calibration curve with synthetic PAF for each series of assays. The specificity of

platelet aggregation was inferred from the inhibitory effect of 5 / t M CV-3988 and 5 #M BN52021 [33], two well-known PAF antagonists. The amount of PAF was expressed in pmol/5 • 105 cells. Acetyltransferase activity. The preparation of cell lysate and the assay condit ons for acetyltransferase have been described in detail L4]. The enzymatic activity was expressed as nmol of [3H]acetate transferred to lyso-PAF/min per mg lysate protein. Measurement of intracellular free calcium ([Ca 2 +] i). Measurements of the time-course of changes in [Ca2+]i were performed using the fluorescent probe qu;-12, as previously detailed [4]. Briefly, confluent cultures of EC grown on coverslips were incubated for 45 min in the presence of 6 #M quin2-AM in culture medium at 37°C and 570 CO 2 atmosphere. The cells were then washed free of the extracellular probe, and allowed to reequilibrate for 10 min at 37 °C in Hepes-buffer solution. Fluorescence of the intracellular quin2 was measured in a Perkin-Elmer LS-5 spectrofluorimeter. For the test, the coverslip (12 mm diameter) was firmly positioned in a 1 cm quartz cuvette at a 45 o angle. The cuvette contained 1 ml Hepes-buffer and was kept at 37°C. Preincubation and stimulation of coverslip-adherent cells were performed under these conditions following the experimental protocol and fluorescence was monitored. The excitation and emission wavelengths were 339 and 492 nm, respectively. The im~acellular free calcium concentration was calculated as described [34]. Measurement of AA release. Confluent EC monolayers in 100 mm diameter dishes were incubated for 24 h with 1.5 ptCi [14C]AA in 7.5 ml complete culture medium [35]. Long-term PMA-pretreatment (1 /~M, 20 h) was performed simultaneously and had no influence on [14C]AA incorporation. Total incorporation was 1 830000 _+ 214000 cpm/dish. The culture medium was aspirated and the cells were washed twice with 6 ml M199 containing 50/~M BSA to remove unincorporated radioactivity. 6 ml M199 containing 50 #M BSA were added and EC were stimulated according to the experimental protocol. During stimulation aliquots of incubation medium were taken and analyzed by liquid scintillation spectrometry to determine the percentage of total cellular radioactivity released in the medium. In each case, agonist-stimulated release was calculated as cpm released in the presence of agonist minus that in control cultures. cAMP determination. Confluent cells in 35-mm dishes were washed twice with 2 ml M199-BSA. The cells were then incubated (37 ° C) in the same medium with forskolin or iloprost at the concentrations and for the indicated time. In some experiments, the cells were preincubated (30 min) in medium containing 0.5 mM 3-isobutyl-l-methylxanthine (IBMX). The final assay volume was 1 ml. After incubation, the medium was

58 aspirated, and 1 ml ice-cold 6% trichloroacetic acid was added to the culture dishes. After freeze-thawing, the cellular extracts were centrifuged at 2000 × g for 10 rain at 4 ° C. The trichloroacetic acid was removed from the supernatant fraction by extracting the samples six times with 2 ml water-saturated diethyl ether. The amount of intracellular cyclic A M P was measured by radioimmunoassay as described [36], and the results expressed as pmol of c A M P per 106 cells. Statistics. The data are presented as means + S.D. of the number of determinations indicated (n). Results

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Effect of thrombin on distribution of PKC In control EC, most (95~) of the PKC activity was found in the cytosol, whereas in thrombin-treated cells PKC activity was found in the particulate fraction extract (Fig. 1). When cells were stimulated with thrombin, a rapid translocation of PKC occurred. The maximal response was observed after 4 - 5 min of stimulation, at which point approx. 80~ of the kinase total activity (cytosolic plus membranous) had translocated to membranes, with a corresponding decrease in cytosolic activity. The response was transient: the translocation persisted with minor loss of PKC for up to 6 min. After 10 min thrombin stimulation, most of the enzyme disappeared from the cells. Fig. 1 shows the time-course and the concentration dependence of thrombin effect on PKC distribution in EC.

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Effect of PKC inhibitors and depletion of PKC on thrombin.induced PA F synthesis The PKC inhibitors palmitoylcarnitine [37], sphingosine [38] and H-7 [39] were used to explore whether thrombin-induced PAF synthesis was a PKC-mediated event. Fig. 2 demonstrates that the inhibitors induced a concentration-dependent decrease in the accumulation of PAF; the synthesis of P A F was 8 0 - 9 0 ~ inhibited by 50 ~tM palmitoylcarnitine and sphingosine and by 100 /tM H-7. This inhibitory effect could be overcome by simultaneous addition of the PKC activator PMA (data not shown). No effect of the inhibitors on basal P A F level was observed, Neither the [Ca2+]i resting level nor the thrombin-stimulated [Ca 2 +]i rise were significantly affected by the inhibitors (not shown). The role of PKC activity in the regulation of thrombin-elicited PAF synthesis was additionally investigated under conditions where the EC enzyme had been down-regulated by chronic exposure to the phorbol ester PMA. Treatment of EC with 1/zM PMA for 20 h resulted in almost complete loss of cellular PKC activity. The kinase C activity of untreated EC was 860 + 126 p m o l / m i n per mg protein (n = 4), while that of PMAtreated EC was 5.6 + 4.9 p m o l / m i n per mg protein (n = 4). Exposure of EC to 1 / t M PMA for 20 h caused

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Fig. I. Time-course and dose-r,=~ponse curve of protein kinase C (PKC) redistribution in human enciothelial cells (EC~ :t:,mulated with thrombin. Upper panel: EC (2..~06/dish) were stimulated with 0.2 U/ml thrombin in 5 ml of M199-gSA at 37 °C for the indicated time. The reaction was stopped by cooling ~e dishes in an ice bath. Each value was obtained by the addition of net PKC activity measured in the fractions of DEAE-cellulose column eluted with 0.06-0.09 M NaCI. Total (cytosolic plus membranous) PKC activity in unstimulated cells was 8604-126 pmol 32p incorporated/min per mg protein (n---4). The figure shows a typical experiment out of four done with similar results. Each point represents the mean of a determination performed in duplicate. The difference between the two determinations never exceeded 5~. Lower panel: EC (2.106/dish) were stimulated for 3 rain at 37°C in 5 ml of M199-BSA with different concentrations of thrombin and processed as described above. Total (cytosolic plus membranous) PKC activity in unstimulated cells was 7584-135 pmol 32p incorporated/rain per mg protein (n--4). The figure shows a typical experiment out of four done with similar results. Each point represents the mean of a determination performed in duplicate. The difference between the two determinations never exceeded 5~. Thrombin concentration is plotted on a logarithmic scale.

59 Thrombin

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Fig. 2. Palmitoylcarnitine (PC), sphingosine (SF), H-7 and PKC depletion inhibit PAF production by thrombin-stimulated human endothelial cells. EC (5.10S/dish) were incubated with the following concentrations of inhibitor (/~M): a: 1; b: 10; c: 50; d: 5; e: 100 for 10 rain prior to the addition of 0.5 U/ml thrombin. After an additional 2 min period, the amount of PAF generated was quantitated as described under Materials and Methods. No effect of the inhibitors on PAF level in unstimulated cells (C) was observed. PKC depletion was obtained by treating EC with 1 tam PMA for 20 h. The PKC activity of PMA-treated EC was 5.6 4-4.9 pmol 32p incorporated/rain per mg protein (untreated cells: 8604.126) (n = 4). Results are expressed as the mean±S.D, from four experiments each done in duplicate. S = stimulated.

97% inhibition of thrombin-induced PAF synthesis (Fig. 2). Loss of responsiveness to thrombin was not due to cell death, because cells remained viable by trypan blue exclusion. Moreover, [ C a 2 + ] i rise in response to thrombin (0.5 U / m l ) was even greater and much more persistent in PKC-depleted cells than in control cells (peak [Ca24]i (/~M): PMA-treated cells: 0.769 _+ 0.208 ( n - 4); control cells: 0.647 +_ 0.170 (n = 10). These results show that in thrombin-stimulated EC, besides the rise in intraceilular C a 2+, PKC activity is required to express full synthesis of PAF. It is suggested that kinase inhibition a n d / o r depletion interfere with a different step, presumably the activation of PLA 2.

Cells were prelabelled with [14CIAA as described under Materials and Methods. Long-term pretreatment with PMA (1 #M, 20 h) was performed simultaneously to get PKC-depleted cells. Short-term PMA pretreatment (250 nM, 2 and 20 win) was performed after removing unincorporated radioactivity in M199 containing 50 /zM BSA. 0.5 U/ml thrombin was added to contro! or PMA-treated cells and after indicated times aliquots were taken to determine the percentage of total cellular radioactivity released into the medium. Agonist-stimulated release was calculated as cpm released in the presence of agonist minus that in non-stimulated EC with or without PMA. PMA itself did not influence AA release. Total incorporation was 1830000_+ 214000 cpm. Values are means_+S.D, from three different experiments. Radioactivity released (~) after 0.5 U/ml thrombin

Non-treated EC PMA 250 nM 2 min PMA 250 nM 20 min PKC-depleted EC

T o test t h e h y p o t h e s i s w h e t h e r P K C a c t i v i t y c o u p l e s t h e rise in i n t r a c e l l u l a r C a 2+ w i t h P A F s y n t h e s i s via a c t i n g o n P L A 2 step, w e m e a s u r e d the t h r o m b i n - i n d u c e d r e l e a s e o f A A f r o m m e m b r a n e p h o s p h o l i p i d s in P K C - d e p l e t e d cells as c o m p a r e d to c o n t r o l cells. T a b l e I s h o w s t h a t t h e A A release is 9 0 ~ i n h i b i t e d in P K C - d e p l e t e d cells a t all m e a s u r e d times.

Effect of a short-term pretreatment with PMA on thrombin-induced [Ca2+]~ rise, AA release, acetyltransferase activation and PAF synthesis F i g . 3 s h o w s r e p r e s e n t a t i v e t r a c i n g s o f t h r o m b i n (0.5 U / m l ) - i n d u c e d [Ca2+]i rise i n q u i n 2 - 1 o a d e d E C follow-

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ing brief (2-20 min) treatment with the PKC activator PMA (0.025-1.0 pM). Although PMA had no effect on the basal [Ca2+]i (basal [ C a 2 + ] i level (/~M): 0.154_+ 0.025, n = 10), pretreatment with PMA strongly inhibited the thrombin-evoked [ C a 2 + ] i peak. The inhibition was concentration- and time-dependent. The thrombin-stimulated release of AA was significantly decreased after 250 nM PMA pretreatment (2 and 20 min). The effect was present both after short-time thrombin stimulation (2 min) and after longer stimulation (10 rain) (Table I). PMA itself did not induce AA release from EC (data not shown). Whereas the effect of [C°++ ]i ~ M ] 0.8. •

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Fig. 4, Eff~t of PMA pretreatment on thrombin- and ionomycin-induced PAF production and acetyltransferase activation in human endothelial cells, EC (5,10S/dish) were exposed to PMA (laM: a: 0.1: h: 0.25: c: I) for the indicated times (2 -- 2 mira 20 -- 20 min) prior to the addition of 0.5 U / m l thrombin (left} or I/~M ionomycin (right), After an additional 2 rain period, the amount of PAF generated and acetyltransferase activity were measured as described under Materials and Methods. Results are expressed as the mean :t: S. D, from four experiments each done in duplicate, C = unstimulated control cells. S -- stimulated. AT = acetyitransferase.

PMA alone on acetyltransferase was negligible, an enhancement of PAF synthesis after PMA could be seen (data not shown). This effect was apparently not via the remodeling pathway and will be investigated further. However, compared with adequate controls (PMAtreated, non-stimulated), brief PMA exposure (2-20 min) markedly inhibited thrombin-mediated acetyltransferase activation and PAF synthesis (Fig. 4, left), the inhibition was time- and concentration-depender~. In contrast with its inhibition of receptor-mediated response, PMA had little or no influence on the increase in [Ca .'+ ]~ (not shown), and on acetyitransferase activation and PAF production induced by the Ca -'+ ionophore ionomycin (1/~M) (Fig. 4, right). These results provide evidence that PKC activation by PMA can inhibit early membrane signaling pathways involved in thrombin-induced acetyltransferase activation and PAF synthesis in EC.

Effect of agents that increase cAMP concentration on thrombin-induced [Ca" +], rise, acetyltransferase activation and PA F synthesis Cellular cAMP levels were measured in EC following treatment with forskolin, a diterpene that directly activates EC adenylate cyclase [40], and with iloprost, a stable synthetic analogue of prostacyclin [41]. The addition of forskolin (10-50 /~M) elicited a rapid and dose-dependent increase in cAMP content (Fig. 5, upper panel), even in the absence of cyclic nucleotide phosphodiesterase inhibition. The maximal accumulation (50 FM forskolin), reached by 10 min, corresponded to a 10-fold increase in cAMP concentration above baseline. 1 laM iloprost induced a 2.5-fold in-

crease after 10 min incubation (Fig. 5, lower panel). A large amplification of iloprost-elicited cAMP production was observed in IBMX-treated cells (Fig. 5, lower panel), but the inhibitor of cyclic nucleotide phosphodiesterase was omitted in further experiments, as it was shown that the compound inhibits the activities of both p h o s p h o l i p a s e A 2 and C in cultured EC [42,43]. Fig. 6 shows that forskolin (10-50 #M) was very effective in inhibiting thrombin-evoked cytosolic [Ca2+]i rise. A similar impairment of the C a 2+ response was obtained when the EC cAMP was increased by the addition of iloprost (0.1-1 /~M) (Fig. 6). When thrombin-elicited PAF synthesis was measured in cells preincubated with forskolin or iloprost, a dose-dependent inhibition was observed, that reached 100% with 50/~M forskolin (Fig. 7, left, c) and 90% with 1 #M iloprost 280

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Fig. 7. Effect of forskolin and iloprost pretreatment on thrombin- and ionomycin-induced PAF production and acetyltransferase activation in human endothelial cells. EC (5.105/dish) were exposed to forskolin (vM: a: 10; b: 20: c: 50) and iloprost (#M: d: 0.1; e: 1) for 10 rain prior to the addition of 0.5 U / m i thrombin (left) or 1 #M ionomycin (right). After an additional 2 min period, the amount of PAF getterated and acetyltransferase activity were measured as described under Materials and Methods. Results are expressed as the mean:LS.D. from four experiments each done in duplicate. C = unstimulated control cells. S = stimulated. AT = acetyltransferase.

sis is strongly dependent on thrombin-induced elevation of [Ca2+]i and the subsequent stimulation of the calcium-dependent acetyltransferase activity [4,5]. Thrombin acts via stimulation of PLC-mediated Ptdlns(4,5)P 2 hydrolysis [11-13,16] and induces formation of Ins(1A,5)P3 which releases calcium from the endoplasmic reticulum [17] thereby eliciting intracellular calcium rise. The second major product of PLC-mediated Ptdlns(4,5)P2 hydrolysis, DAG, in concert with membrane phospholipids and Ca "-+, promotes translocation to the plasma membrane and activation of protein kinase C [18-20]. The tumor-promoting phorbol esters mimic the effects of endogenously produced DAG in activating PKC [19,21] and, thus, have provided a powerful tool to study the role of PKC in cell function. The results of the present study show that translocation of PKC can be added to the list of biochemical events that occur in human endothelial cells upon activation by thrombin (Fig. 1). Fig. 1 shows that the treatment of EC with thrombin (0.2 U/ml) for 5 rain leads to a 90% decrease in the cytosolic PKC activity. This dramatic decline is accompanied by an increase of the enzymatic activity in the particulate fraction. Only approx. 80% of the activity apparently lost at the cytosolic level upon thrombin treatment is recovered at the particulate level, suggesting that the translocation of the PKC from the cytosol to the membrane compartment is not complete, or alternatively that the translocated enzyme is very rapidly down-regulated. Such a phenomenon may be attributed to modification of the catalytic properties of the PKC [44], or it may be the consequence of proteolytic degradation [45,46]. Indeed, thrombin acts very rapidly with an almost maximal effect observable already after 5 min, then the enzymatic activity translocated from cytosol to the particulate fraction is immediately decreasing, and after 10 rain the recovery is only 16% (139 pmol/min per mg), The kinetics of translocation are consistent with the rapid initiation of PI turnover, and both PI turnover and translocation can occur in the absence of extracellular calcium, Therefore, we can assume that thrombin-induced translocation of PKC is a result of phosphoinositide (PI) turnover in EC. The question arises as to whether the activation of PKC characterized in this study has a role in transducing the thrombin-evoked signal for initiating PAF synthesis. The role of PKC in thrombin-mediated PAF synthesis, which occurs primarily by a remodelling pathway, has been assessed: (1) by the blockade of kinase activity with partially selective inhibitors (palmitoylcarnitine, sphingosine, H-7) [37-39]; (2) by chronic exposure of EC to phorbol esters, which results in down-regulauon of PKC and a concomitant loss of phorbol ester responsiveness [19,45]. The chronic exposure of EC to phorbol esters was used as a well

62 established method of down-regulation of PKC despite we found also profound disappearance of PKC already after 10 min thrombin (0.6~ of total activity after long-term PMA treatment versus 16% after thrombin). Our data show that thrombin-mediated PAF synthesis is strongly inhibited by the three inhibitors, whereas the same inhibitors concentrations do not affect significantly thrombin-elicited [Ca2+]i rise. Exposure of EC to 1 pM PMA for 20 h causes complete loss of cellular PKC activity, and a concomitant 97% inhibition of thrombin-induced PAF synthesis. Loss of thrombinmediated response in PKC-depleted cells is not due to impairment of early receptor function, as Ca 2+ mobilization by thrombin is maintained, and many reports are available on sensitization of the Pl signalling pathway to ag,onists after long-term phorbol ester treatment [47,48], PKC is apparently regulating the EC PLAn which has been assessed by measuring the thrombinstimulated release of AA in PKC-depleted cells (90% inhibition, Table 1). These results strongly suggest that PKC activity is required for the thrombin-mediated PAF synthesis in human endothelial cells and that the regulatory role of the kinase is not via alterations in calcium concentrations, but through regulation of PLA 2 activity. Our data are in accordance with recent data, which showed that PKC inhibitors block A23187-induced PAF synthesis in human neutrophils [49] and bradykinin-stimulated PAF production in bovine EC [3], and thrombin-stimulated PGI 2 and PAF synthesis in HUVEC [50]. The mechanisms accounting for this synergistic interaction between Ca 2+ and PKC activation are incompletely understood at present, but may involve: (1) the phosphorylation of phospholipase A 2 [51,52] or endogenous PLA 2 modulatory proteins thus lowering calcium sensitivity of PLA2 [53-55]; (2) the activation of a N a + / H + exchange process [56] that we have identified in EC [4]. This may activate PLA 2 by a PLC-independent mechanism as has been reported in platelets [57]. It is important to point to the fact that PKC and Ca 2+ act synergistically in thrombin-mediated PAF synthesis. PKC activation alone by brief PMA treatment of EC did neither induce AA release from membrane phospholipids nor stimulate acetyltransferase activity, thus confirming that PMA is not able to mimic agonist-induced, PLC-mediated effects. Our results show that, in addition to the positive action in mediating thrombin effect which has been assessed by PKC inhibition or depletion, PKC provides negative feedback control over membrane signalling pathways involved in thrombin effect on EC PAF synthesis. Brief PMA exposure (2 min up to 20 rain) strongly inhibits thrombin-induced [Ca 2+]i rise, AA release, acetyltransferase activation and PAF synthesis in a time- and concentration-dependent fashion. It is well known that short-term activation of PKC with

phorbol esters results in the inhibition of agonist-induced Ptdlns(4,5)P2 hydrolysis [47,48,58-61], and Ca 2+ mobilization [58,62-64] in a variety of tissues, including human endothelial cells [12,65]. The decrease of Ins(1,4,5)P3-induced elevation of intracellular Ca 2+ levels elicited by PMA may alone explain the inhibition of AA release, acetyltransferase activation and PAF synthesis. PKC may inhibit calcium mobilization by blocking the receptor-mediated hydrolysis of inositol phospholipids, or by stimulating the hydrolysis of Ins(1,4,5)P3 by activating an Ins(1,4,5)P3 phosphatase [66]. Alternatively, PKC may stimulate the removal of intracellular Ca 2+ by activation of the Ca2+-transport ATPase and the N a + / C a 2+ exchange protein (reviewed in Ref. 18). Our findings do not agree completely with recent data showing that, under some experimental conditions, PMA enhances bradykinin-induced [3H]AA release and PAF synthesis in bovine aortic EC [3], thrombin-stimulated PAF production in human umbilical vein EC [50] and thrombin-mediated PGI 2 production in human umbilical vein EC [55,65]. As far as bovine aortic EC are concerned, PMA has no effect on bradykinin-induced Ca 2+ influx [3]: so far the negative effect on signal transduction, which is evident in human umbilical vein EC with thrombin as an agonist [12,55,65], is lacking in bovine aortic EC, where only up-regulation of PLA 2 is present. As far as human umbilical vein EC are concerned, PMA effect on agonist-induced PLA 2 activation and PAF (or PGI2) production depends in a complex manner on the time of preincubation and the concentrations of both PMA and agonist, as most authors agree [50,55,65]. Depending on experimental conditions, the positive effect exerted by PKC activation on PLA 2 activity (which includes the enhancing of PLA 2 sensitivity to Ca 2+, Ref. 55) can be counterbalanced by the PKC-dependent negative feedback mechanism on thrombin-induced cell activation. Indeed, PMA reduces the production of PGI 2 elicited by !Gw (0.1 U / m l ) thrombin concentrations independe,~tly of duration of treatment, while with higher dose of thrombin (1 U/ml) the duration of treatment is critical [65]. It is likely that, under our experimental conditions, the concentrations and the duration of treatment with PMA decrease the thrombin-evoked Ca 2+ transient to a level which is below the threshold for PLA 2 and acetyltransferase full activation, even if this threshold had been lowered by PMA pretreatment. The observation that PKC activation inhibits PLC-mediated events, suggests that endogenous DAG formation may be involved in feedback inhibition of phosphoinositide hydrolysis. This phenomenon may modulate the physiological responsiveness of EC to vasoactive mediators that act via Ca 2+ and induce PAF synthesis in endothelium. We finally looked to see whether agents that are known to increase levels of cellular cAMP exerted any

63 effect on thrombin-m¢:liated [Ca2+]i rise, acetyltransferase activation and P A F synthesis. In neutrophils, platelets and lymphocytes, agents that increase intracellular c A M P have been shown to decrease Ptdlns (4,5)P2 hydrolysis [22-24] and these results sugges,' that c A M P may regulate calcium mobilization by inhibiting the generation of inositol phosphates [25]. We used forskolin, a diterpene which directly stimulates adenylate cyclase activity in EC m e m b r a n e [40], and iloprost, a stable synthetic analogue of PGI 2 [41], in order to increase cellular c A M P level. O u r results show that both forskolin and iloprost are very effective in inhibiting t h r o m b i n - e v o k e d c y t o s o l i c [Ca 2+] rise, W h e n thrombin-dependent acetyltransferase activation and P A F synthesis are measured in cells preincubated with forskolin or iloprost, a strong inhibition of both responses is observed. By contrast, nearly no inhibition of acetyltransferase activation and P A F production occurs in cells pre-treated with forskolin and stimulated with ionomycin, pointing to Ca 2+ mobilization block as the major cause of forskolin and iloprost effect. By analogy with other cAMP-sensitive regulatory systems, the inhibition m a y possibly involve the phosphorylation, by a cAMP-dependent protein kinase, of one or more components of the thrombin signal transduction pathway. It has been recently reported that c A M P inhibits AIF4--induced activation of phospholipase C [67-69], and that cAMP-induced suppression of GTP~,S-mediated PLC activation is caused by the cAMP-dependent phosphorylation of the G protein [70]. O u r findings do not agree with those of a prior study [11] in which preincubation of quin2-1oaded EC with 8-Br-cAMP did not suppress the thrombin-induced [Ca2+]i response. We cannot account for this discrepancy: one relevant methodologic difference is the 4-fold thrombin concentration used in the previous study (2 U / m l vs. 0.5 U / m l used in our study), as the inhibitory effect of c A M P on PLC activation is much more pronounced at low thrombin concentrations [67]. Our data are in accordance with previous studies [1,71] which showed that the addition of exogenous PGI 2 caused inhibition of agonist-stimulated production of PAF. The demonstration of an effect of c A M P concentration on the PLC signal transduction p a t h w a y in human endothelial cells can be added to a growing list of examples of 'cross-talk' between different second messengers. Moreover, these results suggest that endogenously generated PGI 2 may modulate the synthesis of PAF.

Acknowledgements This work was supported by grants from Ministero della Pubblica Istruzione (Rome) to A.B. and G.P. (60% and 409~), by a grant from C.N.R. (Rome, No. 90.01261.CT14) to A.B., by a grant from Associazione Italiana per la Ricerca sul Cancro to F.B., and by grants

from I.S.I. (Institute for Scientific Interchange, Torino) and from University of Torino to R.H. and U.T.

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