Exo-protein kinase release from intact cultured aortic endothelial cells

Biochimica t't BiophDica Acta. I 136 ( 1992) 189-195 [', 1',,'92Elsevier Science Publishers B.V. All rights resetv~:d0167--4889/92/$05.(RI

189

BBAMCR 12,213

Exo-protein kinase release from intact culta:ed aortic endothelial cells Matthias

Hartw.ann

~ and Jiirgen Schrader

Departr:,:':::c,f PhysioloD'. Uni~etstO"of Diisseldo~ , )~tsseldorf (Germany)

K~" ~ords: Exo-protein kinase: Protein kinase; Endot ~elialcell: (Porcine aorta) Extracellular protein kinasc activity is demonstrated in intact cultured porcine aortic endothelial ce!l~ and ig chp."z~:icr.scd. "~hcn cells were i~.cubated with [3,-~2P]ATP (i /zM) a major cell surface protein, corresponding to i 15 kDa. and at least iuur serum proteins (19, 21.55 and 126 kDa) became phosphorylated. Protein kiaase activity is released by intac" endoth:lip~ z.'lls, which is not due to cell damage, as judged by various c¢11 viability parameters (e.g., relc~.se of marker c r ~ , c s , t.nypan blue c,,:.tu~,on). "the activity of the protein kina.se rc|ca.scd amounted to 170 fmol/min per mg endothelial cell protein with phosvitin as substrate, which rcpn.'~cnts 9% ef the to'.al cellular pho~'itin protein kinasc activity. Repetitive incubation of cndothelia' cells substentie'~iy decreased phosvitin-kinasc release. Exo-protein kinase is not influenced by cAMP .-.,,d cGMP but is effc~ively inhibited by heparin (EC~, 0.3 jag/rolL The findings clearly demonstrate: (I) exo-protcin kin=se is released ~:,. iatact porcine aortic endolhelial ceils: (2) substrates of this enzyme are endothelial surface proteins and serum proteins.

Introduction Pro*.ein phospho~-!a.,.[o, has been shown to be an important step of both hormonal and neural signaltransductior, events and various protein kinases responsible .0: these reactiolLs have been described (for review see Ret's. 1 and 2). While most protein kinases are localized intracellularly, there is increasing evidence for a subset of protein kinases, the so called "exo-" and "ecto-proiein kin~Lses', responsible for ex~racel!u!ar p h o s p h o ~ l a t i o n events (for review see Ref. 3). Conccrnit~g the physiological role of these enzymes it has been suggested that ectc~protein kinases may play a role in cell-cell interactionz [4-6]. In the present study we examined w h e t h e r aortic maerovascular endothelial cells exhibit extracellular protein t:inase activity. W e have chosen this cell type, since endothelial cells are known to possess a n u m b e r of very active ¢.ctocnzymcs [7] and have the unique ploperty to be in direct contact w.:th circulating blood components. In this regard platclets are of particular interest since they can release ATP, the substrate of protein kinases. In o r d e r to provide conclusive evi-

Correspondence to: J. Schradcr. Phy~ok~gisch~__Inxtiiut :, M~liziaische Einrichtungen der Un~.'crsit~" Di~eldorf. Mooren:tr. 5. D-4000 Dfisseldofl"L Germany. I Present address: Department ol Anaesth~iology, University of Dfisseldorf, Germany.

dence for ihe r d e a s e of the enzyme by intact cells various control e,'~periments were performed. Methods

Cell culture Endothelial cells were isolated from porcine thoracic aorta and cultured in M 199 medium containing 10% fetal calf serum (FCS) and 10% newborn calf ~ r u m (NCS), as recently described in ref. 8. Purity of the preparation was controlled by phas~ Lo~trast microscopy showing a confluent monolayer with contact inhibition. Endothelial cells ingested fluorescentlabelled low-density lipoproteirs. Contamination with fibroblasts or smooth muscle c:lls was studied by antismooth muscle actin ant;bodies. Cells were ~,tbeultured 24 h befo,e phosphorylation experiments on multiwell plates (Falcon 2 crn2). Protein content in all experiments was in the range of 8 0 - 1 0 0 / ~ g / w e l l , as determined by the method of Bradford [9]. Homogcratc$ ;ff endothelial ce!Is were obtained by sonication 0! cells (80-100 p g ) (Branson sonifier, rcodel B-15P, 0°C, Z5 W, 20 bursts) in a Krebs-Ringer solution (400 /zl) leading to a c~mplete release of cellular LDH. Before the phosphorylation experiments ceils were washed twice witb 0.5 mt Krebs-Ringer-sclut.on. Protein phosphor)'latio.'t experiments (a) Cell surface phosphorylation o f in;act endothelf~l ceils. Cells were incubated iw a Hepes-buffered K.-zbs

190 Ringer-solution, consisting of (mM): NaCI 140. KCi 4.0. CaCI, 1.84, MgCI, 1.03, NaH2PO 4 1.0. glucose 5.0, pyruvate Z0, Hepes 10.0 (pH 7.4). Phosphoq/lation reaction was started by addition of [T-32P]ATP (10 p-Ci, 1 a M final concentration) in a final volume o! 400 p-I. Following incub~tlon for 1, 2 and 5 min, supernatant was remceeed li'om the cell monolayer. Immediately tbereaP.er cells ~ere solubilised in 150 p-I SDS/EDTA-buffer 1 consisting of (mMk Tris 30.0, EDTA 3.0, SDS 9%. glycerol 15%. Samples were then boiled. 10 p-I bromophenol blue {ic~) and t3 p-I mercaptoethanol (259[) were added and 55 u.I aliquots were subjected !,) electrophoresis, in a sub+el of experiments the influence of cAMP (10 -'~ M) and cGMP (10 -5 1'4~ on phosphorylation of surface proteins was tested. Furthermore degradation of the exiraceilular o:,clic nucleofides was determined after 5, 10, 30 and 60 min of incubation, using commercially available cAMP and cGMP assays (Amersham).

:he cells. Phosphowlation assay was pe.rformed in a fintl volume of 100 p-i, containing 65 gl aliquots of supernatant and [-,/-3:P]ATP (5 p-Ci, final concentration 1 p-M). To determine the inhibition of the rclc~,cd protein kinase activity by hcparin, the substance was added in various concentrations after the removal of supernatant from cells under otherwise identical conuitions. After a incubation time of 15 min, the reaction was stopped by addition of SDS/EDTA-buffer I! (25/tl) and subsequent boiling for 5 rain. Following addition of bromophenoi biuc (i0 lxl; !%~ and mercaptoethanol (10 p.I; 25~) aliquots v,ere subjected to electrophorcsis.

(b) Phosphorylatio~z of senml ~.:otei,L~ ui presence and absence o f intact endothelial cells. For determination of

For the separat-:.on of proteins, a 5-15% linear gradient gel was used [10]. Gels were stained with Coomassie blue R 250 and dried. Autoradiographs were obtained by exposing gels to Kodak X-Omat AR film and scanning with a laser densitometer (LKB). For quantitative determination of.'['~2P]P,-incorporation into proteins, respective ba~+~'---¢ere cz+'toff and radioac.nvity was determined by Cerenkov-count;ng.

[3:P]P~-incorporation into serum-proteins, 1.5% FCS/NCS (I : 1) was added to the Krebs-Ringcr solulion iLscd for incubation of endothelial cells. In a further series of experiments this medium was incubated with [-I,-3-'P]ATP ,,~ititout cells, while all other incubation parameters wexe identical to t h e e reported above, in addition, serum-f:ee medium was incubated with endothelial cells and [T-3-'P]ATP (see also ~cli )n (a) above) to evalua~'c a rossiblc rclea.se of phosphoproteins from the cells. Incubation was terminated by removing supernatant aqd adding 100 pA S D S / E D T A buffer II. consisting of tmM): Tris 30. EDTA 15, SDS 9% and glycerol 15'7~. Alter boiling the .samples for 15 min and addition o~ bromophenol blue (20 p-i. 1%) and mercaptoethanol ~,21) gL "~%), 2(I0 /~I aliquots were subjected to clcctrophoresi+,.

go) Serum-protein phosphotylation by endothelial cell supertzatant. Following 5 rain of incubation with endothelial cells, supernatar,t containing I-';% FCS/NCS was incubated with ['I,-3: P]ATP (see section (b) a~'~ove). (d) PhosphorHation f~attem of damaged cells. Experimeres were pcrfornteti according to section (b) with the exception that endothelial cell integrity was destroyed by sonieation and serum was omitted.

(e) Phosphorylation o f endothehal cells by /32P/P,. Cells were incubated 'ruder condition', identical to section (a), except that: (I) I"q']P, (lit gCi) instead of [y-3:P]ATF' was used and: (2) buffer was phosphatcfrcc in a sebs::t of experiments.

(f) Phos~'itin-phosl;f~orylmion by er~.a'o~helial cell snpen~atant. Endothelial cells were ir.eubated for 5 min with Krebs-Ringer buffer, which, in .,,dine experiments, additionally contained I mg/ml phosvitin. When cells were incubated in t'le ab.~nce of phosvitin this substrate was added to the supernatant aher removal from

(g) Phoscitin-phosphoo'lation by homogenates. Experiments were performed according to section (e), except tha" different amounts of cell homogcnate instead of supcrnatant were added.

SDS gel electrophor~os

Eik'3'me assays For determination of ecto-ATPase activity, cells were im:ubated with [y-32p]ATP (1 p-Ci, I p-M) in 200 p-i final volume. During ir.cubation 5 ~1 aliquots were remo,,ed after 0.5, !. 2. 5, 7.5 and 10 rain and were collected in 2 ml ice-cotd buffer consisting of (M): 0.0!1 tetrabutyl ammonium hydrogen sulfate, 0.061 KH:PO.+ (pH 5.8). For subsequent separation of [y32PIATP from [32p]p/ SFO.Pak C 18 cartridges were used, which had previously been equilibrated with 10 ml of the above buffer. Ft:llt~ing sample application 5 ml of buffer wits passel.~ over the column and the combined eluat~ of both these stens contained the [~:P]P+-fraction, free of '[y-32p]ATP. For elution of retained [-),-32p]ATP, columns were washed with 5 mi buffer-acetonitrile (1 : I). The .separation of [-),-32P]ATP from [32p]p+ as well as the recovery, were about 99%. Even addition of I mM ATP to the .'+amples did not cause any interference, as could be shown by use of tracer amounts of IS,-~:P]ATP and [:'+'P]Pi. For determination of radioactivity by_ Cerenkov counting, H 2 0 was added to lhe column c'uate to a final volume of 20 ml. in :i::pcrnatant (5 rain of cell contact) and soni~ates of ~.r,dothchal cells lactate dehy'Jrogenase activity was determined according to Ref. 11. Within 5 min of incubation 0.4-P,,.6% of cellular lactate debydrogenase, amounting to 2.4 ± 0.3 U / m g prot(:ir (.~ ± S.E.), was released (n = 6).

191 To'pan blue excht, ion test Endothelial cells attached to culture dishes were iucul:,atcd in Hopes-buffered Krcbs-Ringcr solution for 20 rain. Then cells were stained with 9.2% trypan blue dissolved in the same buffer. After washing the plates twice with buffer, cell viability was determined by light microscopy, counting about 5tl00 cells for each dish. The trypan blue exclusion test revealed cell damage in a range of 0.5-0.7% (n = 5). Furthermore. no detachment of endothelial cells v~thin the time of incubation u ~ d in our experiments could be observed by phase contrast microseopy. Materials The sou=ce of the substances was as follows: [7~2P]ATP (3000 Ci/mmol), [~-'P]P~ (carrier free) (Amersham, Brauoschweig, Germany); acrylamide, bisacrylamide, TEMED, ammonium persulfate, EDTA, bromophenol blue, Coomassie blue R 250, molecular weight standards (Sewa. H~ideiberg, Germany); heparin, phowitin (Si~.g*n~.Mr.mchen, Germany); fetal calf serum (Gibco, Eggenstein, Germany); newborn calf serum (Biochrom, E.,zrlin, Germany). All other substanc~ were ot highest ¢iuality available. Results In a first series of experiments, phosphorylation of both e~dothelial cell surface proteins and serum-containing culture medium caused by endothelial extracellular acting p[otein kinases was investigated. For this purpose, endothelial cells were incubatt:d with [732P]ATP (I v M ) for different times (1, 2 and 5 rain). [32P]P, conte,it of cellular '~roteins separated by SDSgel electrophoresis was d :t*:,~a~ined autoradiographically. Fig. i shows rcpres:ntative de.-.si,ometHe scans from a sc0ies of four experiments. Incubation of intact •adotho.!ia| cells with |7-32P]ATP for 5 rain in saline supernatant caused the phosphorylation of a major surface protein, exhibiting a molecular weight of !15 kDa (lane 1. Fig. 1). This main substrate did not detach into serum-free medium as evidenced by the analysis of the cellular supernatant (Fig. I, lane 2). Destruction of endothelial cells by sonication prior to the addliio=i of [7-~:P]ATP resulted in a completely different phosphorylation pattern (Fig. I, lane 3) demonstrating the existence of intra- and extracellular compartmenL~ of protein phosphorylation, in ordcr to exclude the possibility, that protein phosphory!ation was caused by [-~:P]P, incorporation into the mtracellular ATP pool following cxtracellular hydrolysis of [ y- 3~,P]ATP, phospho.-ylation experiments were performed with [32P]Pi in a phosphate-containing (I mM) and phosphate-free medium. Although the absolute amount of radioactiviq, was the same in both sets of cxpcrimcnts [3_,pip, did not cause significant labelling of cellular proteins i:~ the

1

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19 21

55

t15

YOLECULAR WEIGHTtkDg) Fig. I. Densitometric scans of phospho~+lation patterns of endothelial cell surface,saline supernatanland cell homogenalc.Lane I. cell

surface phosphorylationof intact endothelial cells incubated in a phosphate-containing s.dine buffer b~,'[7-~'PJATP (Ill #Cil. The arrow indicates the mainsurface substrate.Lane 2. lack of phosphorytatcd proteins in the ~ l u m free supcrnatant of cndolhelial cells from the ¢xl~riment of Lane I. Lane 3. phospho~'latlon of endothelial cell homogenatc by [y-~"P]ATP (10 pCi). Lane 4. lack of phospho~lation of endothelial cells by [:*2pip, (tO pCO in a phosphate containing medium. Protein content of endothel,al cells and

homogenatesamountedto ~0- liP, pg. Th(. markon the rightside of the figure~mbolizes a cnange in 2ptJcal absorban*:.eof (I.5 for lanes I. 2 and 4 end 1.0 f.,r lane 3. respecti.cl).

phosphate-containing medium (Fig. i, lane 4). In phosphate-free medium, however, phosphorylation of cellular protei,s could be observed (data not shown). Phosphoxytation of sentm protein.~ caused by intact endothelial cells shows a pattern quite different from cell surface phosphorylation (compare Fig. 2, lane ! with Fig. 1, lane i). The time-course of radioactivity incorporation into both surface and .serum proteins was linear for up to 5 rain (data not shown). Addition of serum (I.5% FCS/NCS) to the supernatant did not alter the phosphorylation of cellular surface proteins under otherwise identical experimental conditions. Serum incubated with [T--~2P]ATP in the absence of cudothclial eel':, but under otherwise identical conditions, exhibited significant protein kinase activity (Fig. Z lane 2). When comparing the phosphorylation pattern with that obtained in the presence of endothelial cells (Fig. 2, lane 1) it is evident that at least four additional serum proteins (19, 21, 55 and i26 kDa) are plnosphorylated when cndothelial ceils are present. A separate series of experiments was performed to determine whether the observed end~thelium-derived protein kinasc ac:ivity is associated with the plasma membrane of endothelial cells or was released in:to the supernatant. For th~ purpose, endothelial cells were

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.'40LEGULaR WEICHT13UO] Fig. ") Dcnsitometric ~¢=r~ of [7-3:P]ATP - (10 pC!) in6uced ph(~,phoq;|ation patterns of serum prot~,;.n.~ pht~phoq,'latud in the plc~,-=nceo absence and foliowiog a 5 m~;, incubation wifi~ endothelial cells. L~ne I, phosphorylalion of serum proteins in the pre~cnee of

endothelial cells. Lane ~ phosphoq,'lation of serum in the absence of endoth¢liai ct:l|~. La;:.c 3. ~...-~'z,hal.~.'!.~llO,patte.-'n of serum, which ~,~.- inct;~a;c,:]~,izh ea,h)zl',,|ial ccll~ before addition of [7- ~:P~.'~TP. Serum proteins which are exclusivelyphosphor~'l=ted [%' the endothelial cxo-orotein kinas¢ arc marked ~," arrows. Incubat on was performed ":;ith ['r-~:P/ATIP (10 pCi)" for 5 rain. Protein con:cnz amounted to 80-lO0/zg ifl all c0_ses. "1he change in optical densi|.~. as illustrated I:q.'the mark on the : ght side of the figure, was 0.5. im:uba!ed w i t h a s e r u m - c o n t a i n i n g m e d i u m for 5 min in the a b s e n c e o f [ T J Z P [ A T P . The s u p e r n a t a n ! was t h e n r e m o v e d a n d i n c u b a t J d with [ 7 - 3 : P ] A T P for a n additional 5 min. A s shown in Fig. 2, lane 3, the p h o s p h o r y l a t i o n p a t t e r n was similar to t h a t d e p i c t e d in Fig. ~ l a n e l, indicating that t h e e n d o t h c i , u m - d c r h . c d p r o t e i n kinase activity, at least in part, is released into the s u p c r n a t a n t . I n c u b a t i o n u f cndothe!;~! ,ct~ ""::b c A M P (lO - s M ) a n d c G M P G 0 - - M) did not c h a n g e the p h o s p h o r y l a !ion p a t t e r n o f s u r f a c e a n d s e r u m p r o t e i n s ~n f c u r e x p e r i m e n t s ( d a t a not shown). Control c x p : r i m e n : ~ revealed @,a' this result is not d u e to a r a p i d d e g r a d a tion o r u p t a k e o f cyc!ic nucleotides, since t h e i r c o n c e n trations in the s u p c r n a t a n t r e m a i n e d c o n s t a n t within 60 rain o f i n c u b a t i o n with endothelial cells (n = J.; d a t a not sho~at ). In the a t t e m p t to fltrther explore t h e ,,ubstrate specifity o f e p d o t h e l i u m - d e r i v e d protein k i n a ~ , experim e n t s with the artificial substrate phosvitin [12] were p e r f o r m e d . E n d o t h e l i a l cells w e r e i n c u b a t e d will| saline m e d i u m in the a b s e n c e o r presence o t phosvitin ( f i n d c o n c e n t r a t i o n ! m g / r d ) at~d the s u p e r n a t a n t w a s c h a n g e d f o u r times with time intervals o f 5 min. A f t e r removing the s u p e r n a t a n t , phosvifin was s u p p l e m e n t e d to those sa,nples c o n t a i n i n g n o artificial s u b s | r a t e . A s c a n be seen f r o m Fig. 3, phosvitin protein kinase activity w a s released it; the presenze, as well as in the

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Fic. 3. Rclea_~ of exo-p=ah:io I.;ina~¢ acl,vil~ in the SUl~zrnatam of ir,,Jct cn.lo!he!ial cells in the prc~cncc and al~zncc of phos,,itin ( I rag/roll. Cc[k v,ere repctilkcK- iocufiatcd (4 times (A-DE 5 mine Pho~',-illn ~as used as the subSlrale. SignifiGmt differences in the rel~;~ of ex~pmtein kina_~ in the pre~',ence and absence of sl:bslralc, rcspccln'el)', were c~tc.:tablc in ~roap C and D ( P < t t . ( ~ ; Student's r-I.~l [or pai;'cd sampl=..'sE .~ _+S.E.. n = 4.

absence, o f phos~Itin in the s u p e r n a t a n t . With subseq u e n t m e d i u m c h a n g e s e n z y m e activi~ rapidly declined. I n t h e pr~:scncc o f p h ~ V i i l l l , ~i:lw¢:vcr, li|oFE

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HEPAR IN l u 3 / m [ I Fig- J. llepariv= inhibition of ¢xo-pro;cin kina.~ a~k'ily re!casetl t~ cndothclisl c~[Is. Different concuntralk is .'ff hcpann and the sl;bstrafe ph~.b~,'itio (I mg/l~d) ~ere adde£ to the saline supcrnatant. ~hich had heen incubated ~ith end~t ¢lial ceils before t5 rain). Radioactivity incorporation into pho~dtl,1 ¢,~LSdetermined after 15 rain of incubation ~ith [7-:~-'PJATP (I p.M) [%" Cerenkov counting of the c.lcclrophoretically-separated st ~ ,Irate. ~ 4-_S.E.: n = 3.

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centration within certain limit~. This may be difficult in view of the presence of highly active endothelial ectoATPase [7]. We, therefore, have studied ATP-degradation caused by intact and sonicated endotheE,,~ cells, as w,:ll ~ ~ul~ernat.ant exposed to cndothelium. As shown in Fig. 6, the half-life of ATP (1 p.M) was 6.5 rain and ! min. when incubated with intact and Ilomogeni~d el~dotbelial cells, respectively. ATP-dcgra:lation in the supcruatant was only 15% within 180 min. This is not due to release of ATPases from endothelial cells since the same extent of ATP hydrolysis also occurs in incubation buffer without cell contact.

Fng. 5. Relationship hemcen the amounl of ~.n~,thelial cell Ilo-

mogenatcand phos~-/tir,proiein kinaseactivity. Cell homogenatcwas prepared from endothelial oells equivalent to ~Q0-100/~g protein (100~,c). n ==2. protein kinase activity is rcl~:ased (Fig. 3, C,D). A.g shown in Fig. 4, heparin inh~ited extracellular protein kinase activity released ~ endothelial cells dose dependently up to 98%. The ECso was about 0.3/zg/ml. Control experiments, in which serum containing medium (I.5% FCS/NCS) was incubated with [y32p]A-I'-P and phosvitin (! mg/'ml) for 15 rain in the absen,:e of endothelial cells revealed that the serum protein kinase activity does not phospimrylate phosvitin (data not shown). To estimate the degree of cell damage, which could have elicited the observed protein kinase activity in t_he superuata~k a cell homogena'.e was prepared from endothelia~ cells and examined for phosvitin protein kinase activity (Fig. 5). Using the same assay conditions as before it is now evident that a protein kinasc activity of 170 fmol/min per mg protein which is relca~d into the supernatant by intact endothelial cells (see Fig. 3) corresponds to 9% sonicated cells. For a precise estim.ate of ecto-protein kinase ~ctivity it is important to maintain thc extrace!!alar A'lP-con-

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0-1 TJME tmml Fig. 6. Time-courseol de.gradatFonof ATP (1 ~aM)in the presenceof in:act and homogenizedendothelial cells(ECL respectively,endothelial cell supernatant~5rain incubation)and buffer. £ ± S.E.: n = 4.

Discussion I'he present study clearly demonstrates the presence of an extracellular protein kinase activity of intact porcine aortic endothelial cells which phosphorylates proteins present at the cell surface, the major phosphoprotein exhibiting a molecular mass corresponding to 115 kDa. Interestingly, the 115 kDa major substrate in our experiments corresponds well to the major surtaoe suhstrates of ecto-protein kinases in HeLa cells (116 kDa; Ref. 13) and neural cells (120 kDa; Ref. 4). The functional importance of this suhstrate is unknown at present. In addition to the phosplnorylation of surface proteins, endothelia~ cells were also able to phosphorylate serum proteins. The fact that serum itself possessed some protein kinase activity is in accordance with the literature [14,15]. However, this finding did not complicate the interpretation of our results since at least four serum proteins (19, 21, 5.~ and 126 kDa) were exclusively phosphorylated by endothelial cells ( ~ e Fig. 2). In addition, our study provides evidence that the extracellular protein kinase of endothelial cells is released into the superuatant. A release of nrotein kinases, induced by phosvitin, has also been described in HeLa ~,cnns [i3]. Aithougil pzo[ein kinas¢ in lh¢ latter study was associated with the plasma membrane under normal conditions, addition of substrate led to a nearly complete release of the enzyme. Similarly, in the present study pho,~'itln facilitated the release of the endothelial protein kinase. The enzyme, however, was released even in the absence of the substrate. Therefore, the extracellular acting protein kinases of endothelial cells and HeLa cells share proly:-"~es of both exo- and ectoenzymes. While some of the studies on ecto-protein kinases describe an activation of enz~ne activity by cAMP [16-18], others could not detect any de~endence on cyclic nuc!eotides [4,13,19-23]. In our experiments neither cAMP nor cGMP (1O ~.M) changed the phosphorylation pattern of surface and .serum proteins. Degradation of cyclic nucleotides by endothelial cells gould be excluded, confirming that the exo-prote: ". kinase of

194 endothelial cells is not u n d e r control o f these second messengers. Phosvitin proved to be a g o o d substratc for e n d o t h e lial cell exo-protein kinase, revealing a n activity of 170 f m o l / m i n p e r mg protein. Phosvitin ecto-protein kinase activity was shown to be present al,;o in a m a c r o p h a g e - l i k e cell line [19]. H e L a cells [13] a n d c u l t u r e d 3T3 a n d S V 4 0 - t r a n s f o r m e d 3T3 ccl[-~ [24]. T h e enzymatic activity of endothelial exo-protein kinasc (170 f m o l / m g p e r min) is similar to that described for H e L a cell s (21)7 f m o l / m g p e r min; ReL 13). C o m p a r i .son with the results o f o t h e r a u t h o r s , however, is difficult, since r e p o r t e d e n z y m e ac.'i~ity is related to cell n u m b e r . T h e p r o t e o g l y c a n h e p a r i n was shown to be a very p o t e n t inhibitor o f phosvitin protein kina:;c released by endothelial cells. A c o n c e n t r a t i o n o f only 0.3 p , g / m l resulted in a 5(l c~ inhibition (Fig. 4). T h e heparin-ind u c e d inhibition o f ecto-protein kinasc activity has also b e e n descr+bed for H e L a cells, a l t h o u g h the EC~. was slightly h i g h e r (ECsn. i t a g / m l ; Ref. 3). it is interesting to note. f u r t h e r m o r e , that the intracellularly local!seal casein kinase II is significantly inhibited by heparin (EC~. 0.15 p , g / m l ; Ref. 25) suggesting that similarities m a y exist between these different classes o f kinases. V a r i o u s control experiments were p e r f o r m e d to d e m o n s t r a t e t h a t the observed protein kinase activity trnly is a n exo-prntein kinase released by intact cells: T h e s e e x p e r i m e n t s were: ( ! ) Er:dothcliel cehs, which a r e c h a r a c t e r i s e d by a highly active e c t o - A T P a s e [7]. might have critically lowered the A T P in the m e d i u m d u r i n g incubation. D e t e r m i n a t i o n of A T P - c o n c c n t r a tions in the assay medium, however, revealed that 6 0 ~ of the A T P a d d e d was still present at the e n d o f the incubation time. F u r t h e r m o r e , labelling o f surface proreins was linear with time. (2) U p t a k e of [y-~2P]ATP in a m o u n t s sufficient to p r o d u c e d e t e c t a b l e intraeellular p r o t e i n phosphorylations, is highly unlikely because: (i) there is a c o n s i d e r a b l e c o n c e n t r a t i o n g r a d i e n t b e t w e e n intracellular a n d extracellular A T P (mill!molar r a n g e intraccllular [26] vs. I p.M extrat, cllular) a n d a n active uphill t r a n s p o r t o f A T P has not b e e n de,~ribed and: (ii) intra- a n d extraeellular p h o s p h o r y l a t i o n patterns. o b t a i n e d by incubation of intact a n d sonicated cells with [ 7-3," P]ATP, are differ greatly. (3) Endothelial cell p h o s p h o r y l a t i o n p a t t e r n with [y-~2P]A'['P as a substratc is not d u e to u p t a k e of the [~2P]Pi f o r m e d by d e g r a d a t i o n Gf ['y-~-'P]ATP by c c t o - A T P a s c s . Control e x p e r i m e n t s with 1 m M extraccllular p h o s p h a t e revealed t h a t intraccllular p h o s p h o r y l a t i o n is completely suppressed. (4) S e r n m - c o n t a i n i n g culture m e d i u m is not the origin o f endothelial exo-protein kinase since .serum p r o t e i n kinase activity was neither capable o f p h o s p h o r y l a t i n g certain surface proteins (19, 21, 55, a n d 126 k D a ) n o r phosvitin. F u r t h e r m o r e , the p a t t e r n o f surface protein p h o s p h o r y l a t i o n was u n a f f e c t e d w h e n

0he saline supernatant was supplemented with serum. ~51 Endothelial cells rclea.',,c protein kinase at a rate of 170 f m o l / m i n per mg protein (Fig. 3L which would be equivalent to 9% o f damaged endothelial cells (Fig. 5). This, however, is highly unlikely since only 0.4-0.6% of cellular L D H is rclea.sed within 5 rain and cell damage, asses.sed by trypan blue, ranged from 0.5-0.7%. Furthermorc, a release of cellular ATPases into the medium did not occur within 180 rain of incubation. Taken together, the rcsulLs of thi~ study clearly demonstrate the release of an exo-protein kinasc from intact porcine aortic endothelial cells capable of phnsphoD'lating ~ r u m proteins, as well as phosvitin. The m a j o r substrate is a 115 k D a surface protein, the p h o s p h o r y l a t i o n o1" which is not r e g u l a t e d by the cyclic nucleotides c A M P a n d c G M P . but is effectively inhibited by h e p a r i n . T h e endothelial cxo-protein kina,se share~ i m p o r t a n t p r o p e r t i e s with the e c t o - p r o t e i n kinasc o n H e L a cells a n d with the intracellularly Iocaliscd casein k i n a ~ II. T h e functional significance of the endothelial exo-protcin kinase is not presently known, it is t e m p t i n g to speculate, however, that A T P released from a g g r e g a t i n g platelcts in sufficiently high c o u c c n t r a t i o n s [27], c a n directly activate this enzyme. References

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