mediators of inflammation, 8 we a a possible involvement of ion...

A possible involvement of iontransporter in tumor necrosis factora and cycloheximide-inducedapoptosis of endothelial cells

Hiroshi Fujita1,2,CA, Ikuo Morita2 andSei-itsu Murota2

1Department of Internal Medicine, TokyoMetropolitan Bokutou General Hospital, Sumida-ku,Tokyo; and 2Department of Physiological Chemistry,Graduate School, Tokyo Medical and DentalUniversity, 1–5–45 Yushima, Bunkyo-ku, Tokyo 113,Japan

CACorresponding AuthorPresent Address: Department of Internal Medic ine,Tokyo Metropolitan Bokutou General Hospital, 4–23–15Koutoubashi, Sumida-ku, Tokyo 130–8575, JapanTe l: (+81) 3 3633 6151Fax : (+81) 3 3633 6173

WE ex am in ed the tum or necrosis factor a (TNF a )-in duced apoptosis of vascular endothe lia l cells fromthe standpoin t of ion channe ls. Cultured vascularendothelia l cells from bovin e carotid arte ry wereused. Apoptosis was determ in ed by a propidiumiodide as say. Treatm ent of th e endothelia l cells w ithTNF a and cyclohex im ide for 6 h induced nuclearfr agm entation in a TNF a dose -dependen t m anner(1–10 ng/m l). Concom itan t tr eatm ent of endothe lia lcells w ith TNF a at a dose of 10 ng/m l and cyclohex -im ide at a dose of 10 m g/m l e licited endoth elial cellapoptosis as h igh as 23.4±4.1% at 6 h afte r adm inis tra-tion . However, 10 ng/m l TNF a alone e licited a littleapoptosis at 6 h afte r its adm inis tration (% apopto-s is=4.1±0.8%). Cyclohex im ide (10 m g/m l) did notin duce apoptosis at all. Concom itant tr eatm ent ofendothelia l ce lls w ith 1 m m ol/l of 4,4-diisothiocyana-tostilbe ne -2,2-dis ulfonic acid, w hich is a ch loridebicarbonate ex changer blocker, partially inh ibitedthe TNF a and cyclohex im ide-in duced endothe lia l cellapoptosis . On the other hand, endothelia l cell apop-tosis due to TNF a and cyclohex im ide w as com ple telyin hibited by benzylox ycarbonyl-Asp-CH2OC(O)-2,6-dich lorobenzene (50 m m ol/ l), an in hibitor of cas -pase . Moreover, pyrrolidin e dith iocarbanate , anin hibitor of nuclear factor kappa B (NF- k B), alsosuppres sed endoth elial ce ll apoptosis induced byTNF a and cyclohex im ide com pletely. Th es e findingssuggest th at th e endoth elial ce ll apoptosis induced byTNF a and cyclohex im ide is c losely re lated to not on lychloride ions, but also both NF-k B and caspaseactivation . That is to say, th ere is a poss ibility thatchloride ions or bicarbonate (pH) m ay play anim portan t role in s ign al transduction such as NF-k Band caspase activation in th e apoptosis induced byTNF a and cyclohex im ide .

Key words: Apoptosis , Tumor necrosis factor, Chloridebicarbonate ex changer, Endothelial c ells, Apoptosis

Introduction

Apoptosis (programme d ce ll de ath) is a fundamentalproce ss in embryogenesis, tis sue hemostasis, andimmune system maturation.1 Its deregulation mayhave important implic ations for carc inogene sis andimmune syste m disorde rs. It has been re ported thatapoptos is plays an important role in various patho-physiological conditio ns such as hypox ia/repe rfusioninjury, radiatio n pne umonitis , etc .2,3 Endothelial ce llapoptos is has been observed w hen endothe lial cellsare ex posed to basic fibroblast grow th factor deple-tion.4 Lung injury such as pulmonary edema due toradiation is based on endothelial ce ll apoptosis .3

Moreove r, lupus antic oagulant-induc ed apoptosis ofendothelial c ells w ith recognition of annex in V hasbeen re ported.5 It w as suggested that apoptosis ofendothelial c ells may be re sponsible for the vasculitis

associated w ith syste mic lupus e rythe matosus. Thus,endothelial c ell apoptosis may cause the variouspathologic al conditio ns.

Tumor ne crosis factor a (TNFa ) is an inducer ofapoptos is in various cells as we ll as an inf lammatorycytokine . TNFa ac tivate d the Fas-associate d prote inw ith death domain (FADD) through the receptor ofTNFa , subsequently caused the ac tivation of thecaspase , re sulting in apoptosis.6 TNFa receptor bind-ing prote in, TRAF2, e lic ited nuclear factor kappa B(NF-k B) ac tivation, w hich has an inhibitory action oncaspase ac tivity.7 On the other hand, ce ramide , w hichis produced from sphigomye lin due to activate dsphingomyelinase by TNFa , can also induc e both NF-k B and apoptos is.8

In the pre sent study, w e show that TNFa caninduc e apoptosis of endothe lial c ells concomitantlyw ith cyclohex imide . This apoptosis w as inhibited by

ISSN 0962-9351 print/ISSN 1466-1861 online/99/040211-08 © 1999 Taylor & Francis Ltd 211

Research Paper

Mediators of Inflammation, 8, 211–218 (1999)

an NF-k B inhibitor such as pyrrolidine dithioc arbo-nate (PDTC) or a caspase inhibitor. More over, w e w illdemonstrate that 4,4-diisothiocyanato stilbene-2,2-dis-ulfonic ac id (DIDS), a blocker of chloride bicarbonateex change r, c an pre vent TNFa and cyclohex imide -induc ed apoptosis of endothe lial c ells.

Materials and methods

Reagents

Human re combinant TNFa w as purchased from Endo-gen Inc . (NY) DIDS, 5-N, N-dime thyl amiloride ,propidium iodide (PI), PDTC, N-acetyl cyste ine (NAC)and anthrac ene carbox ylic ac id w ere purchased fromSigma Co. Ltd (St. Louis , MO). Benzylox ycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene (zD-dcb) w aspurifie d as described previously.9

Endothelial cell culture

Endothe lial ce lls we re isolated from fre shly ex cisedbovine carotid arte rie s as described pre viously.10

Brie fly, endothelial c ells w ere obtaine d by lightlysc raping the intimal surface of longitudinally opene dvessels. The ce lls we re then seeded into 60 mm dishes(Falcon Labw are , Divis ion of Bec ton Dickinson andCompany, Lincoln Park, NJ) in a grow th mediumcontaining minimum essential medium (MEM) (GibcoLaboratorie s, Grand Is land, NY) supplemented w ith20% heat-inac tivate d fetal calf se rum (FCS) (Inte rgenCo. Purchase , NY), penic illin, and streptomycin (2%total medium volume, Gibco Laboratorie s) and sub-cultured in a 10% FCS-containing MEM. Culture s w eremaintaine d in a humidifie d incubator at 37°C under95% air/5% CO2, and w ere ide ntifie d as endothelialce lls from their typical cobblestone appearanc e andtheir incorporation activity of ac etylated low -densitylipoprote in. For ex pe rime ntal use, the endothelialce lls (passage leve ls unde r 20) were plated in 24-we lldishe s (Corning Glass Works , Corning , NY). Cellsw ere plated in e ight-w ell slide chambers (Nunc Inc .,Herlev, Denmark) for PI staining s.

Apoptosis

Apoptosis w as dete rmined from the presenc e ofnuclear fragmentatio n on PI staining .11 Cells w erefix ed w ith 70% alcohol for 10 min at 4°C. Afte r beingw ashed w ith phosphate -buffere d saline , the cellsw ere treated w ith PI solution (0.01 g/l) containingRNAse (100 mg/l) for 30 min at 37°C. Follow ing theinc ubation, the nuclear fragmentation (ove r 2) w asobserved using a fluorescenc e mic roscope (Nikonmic rophoto-FX, Nikon Japan Co., Tokyo, Japan), andce lls (endothelial ce lls , 100 cells) w ere counted in 10diffe rent fie lds chosen at random. The percentage ofce lls that unde rw ent apoptosis w as calculate d using

the formula: % apoptosis= (apoptotic ce lls/total coun-te d cells) 3 100.

Viability assay

Cell viability w as evaluate d using a 3–4,5-dime thylth-iazol-2-yl-2,5-diphenylte trazolium bromide (MTT)assay.12 Brie fly, endothelial c ells we re seeded into96-w ell dishes (Corning ). The ce lls w ere tre ated w ithTNFa (1–10 ng/ml) and cyclohex imide (10 m g/ml) for6 h, and the MTT solution (200 mg/l) w as then added.Thirty minute s late r, dime thylsulfox ide w as adde d todissolve the cells. The fluorescenc e at 570 nm w asmeasured. The ce ll survival rate w as ex pre ssedaccording to the formula: % survival rate = [(test value– blank value)/(max imal value – blank value )] 3 100,w here the blank value repre sents the ce ll-free dish,and the max imal value repre sents the fluorescenc efrom the total cells.

Statistical analysis

Data are ex pressed as the mean ± standard error ofthe mean (SEM). For comparis ons of the mean valuesbetw een tw o subsets of data, Stude nt’s t-te st w asused, employing the standard c rite rion of P<0.01 orP<0.05 to indic ate statis tically signific antdiffe rences.

Results

TNF a -induced apoptosis of endothelial cellsconcomitantly with cycloheximide

We ex amined the effe ct of TNFa (1–10 ng/ml) onendothelial ce lls . Contro l endothe lial c ells displayed atypic al cobblestone morphology, but the ir treatme ntw ith 1–10 ng/ml of TNFa for 6 h caused a little ce llshrinkage . Concomitant tre atment w ith TNFa(1–10 ng/ml) and cyc lohex imide (10 m g/ml) causedsevere ce ll shrinkage for 6 h. To de termine w hetherthe cell damage w as due to apoptosis or not, w estaine d the cells w ith propidium iodide . The nuclei ofthe contro l endothe lial c ells w ere round, w here as theTNFa (10 ng/ml) and cyclohex imide (10 m g/ml) tre a-te d ce lls revealed nuclei that w e re fragmented. Thedose dependency of the TNFa effec t on the apoptosisis illustrate d in Fig. 1. Cyclohex imide (10 m g/ml)given alone affected ne ither the morphology nor theapoptos is induc tion (Fig. 1).

Prevention by the NF- k B inhibitor, PDTC, ofTNF a and cycloheximide-induced apoptosis ofendothelial cells

Concomitant treatme nt of endothe lial c ells w ithPDTC (1 mmol/l) complete ly inhibited the TNFa andcyclohex imide-induced morphologic al changes, and

H. Fu jita et al.

212 Mediators of Inflammation · Vol 8 · 1999

Io n tra ns po rte r in apo pto s is o f endo the lia l ce lls

Mediators of Inflammation · Vol 8 · 1999 213

FIG. 1. Apoptosis of endothelial cells treated with TNF a and cycloheximide. Endothelial cell monolayers were treated with1–10 ng/ml TNF a in the presence or absence of cycloheximide (CHX) (10 m g/ml) for 6 h. After the incubation treatment, the cellswere stained with PI, as described in Materials and methods. The percentage of apoptotic endothelial cells represents thenumber of apoptotic cells in the counted 100 cells (apoptotic cells + viable cells). The data show the mean±SEM for 10 wells.* P<0.05 versus TNFa -treated group. Each experiment was repeated three times.

FIG. 2. PDTC-inhibited TNF a and cycloheximide-induced apoptosis of endothelial cells. (A) Endothelial cell monolayers weretreated with TNFa (10 ng/ml) and cycloheximide (10 m g/ml) in various concentrations of PDTC (0–1 mmol/l) for 6 h. After theincubation treatment, the cells were stained with propidium iodide, as described in Materials and methods. ** P<0.01 versusTNF a and cycloheximide-treated group. Each experiment was repeated three times. (B) Endothelial cell monolayers weretreated with TNF a (10 ng/ml) and cycloheximide (10 m g/ml) in the presence or absence of PDTC (1 mmol/l) for 6 h. After theincubation treatment, the cells were stained with MTT, as described in Materials and methods. The data show the mean±SEMfor eight wells. #P<0.05 versus TNF a and cycloheximide-treated group. Each experiment was repeated three times.

nuclear fragmentation of the cells, as demonstrate d inFig. 2A. PDTC (1 mmol/l) given alone affe cted ne itherthe morphology nor the apoptosis induc tion. PDTCsuppressed the TNFa and cyclohex imide -inducedendothelial c ell apoptos is in a dose -dependent man-ne r, as show n in Fig. 2A. PDTC also re ve rsed thedecre ased survival rate caused by TNFa and cyclohex -imide in a dose-depende nt manne r (Fig. 2B). PDTCgiven alone did not inf luence the cell survival rate .

NAC, anothe r inhibitor of NF-k B, did not suppressthe TNFa and cyclohex imide -induced apoptos is (NAC1 mmol/l; % inhibition= 0.3±4.0%).

Effect of the sphigomyelinase inhibitor,L-cycloserine, and the tyrosine phosphataseinhibitor, vanadate, on endothelial cellapoptosis induced by TNF a and cycloheximide

Concomitant treatme nt of endothe lial c ells w ithL-cyclose rine (an inhibitor of sphingomyelinase,1–10 mmol/l) did not inhibit the TNFa and cyclohex -imide -induced nuclear fragmentatio n of the cells, asdemonstrate d in Fig. 3A. The phosphatas e inhibitor,sodium vanadate (1 mmol/l), complete ly inhibited the

TNFa and cyclohex imide -induc ed apoptosis , asshow n in Fig. 3B.

Protective effect of the caspase inhibitor,zD-dcb, on endothelial cell apoptosis inducedby TNF a and cycloheximide

Concomitant tre atment of endothe lial ce lls w ith zD-dcb (a caspase inhibitor, 50 m mol/l) comple te ly inhib-ite d the TNFa and cyclohex imide -induced nuclearfragmentatio n of the ce lls, as demonstrate d in Fig. 4A.zD-dcb suppressed the TNFa and cyclohex imide -induc ed endothe lial c ell apoptosis in a dose-depend-ent manne r, as show n in Fig. 4A. zD-dcb also re ve rsedthe decre ased survival rate caused by the TNFa andcyclohex imide in a dose-dependent manne r (Fig.4B).

Effects of ion transport inhibitors onendothelial cell apoptosis induced by TNF a andcycloheximide

Concomitant treatme nt of endothe lial c ells w ithDIDS (a chloride bicarbonate ex changer blocker,

H. Fu jita et al.


FIG. 3. Effects of L-cycloserine and vanadate on TNF a and cycloheximide-induced apoptosis of endothelial cells. (A) Endothelialcell monolayers were treated with TNF a (10 ng/ml) and cycloheximide (10 m g/ml) in the presence or absence of L-cycloserine(1–10mmol/l) for 6 h. After the incubation treatment, the cells were stained with propidium iodide, as described in Materialsand methods. The percentage of apoptotic endothelial cells represents the number of apoptotic cells in the counted 100 cells(apoptotic cells+viable cells). The data show the mean±SEM for 10 wells. (B) Endothelial cell monolayers were treated withTNF a (10 ng/ml) and cycloheximide (10 m g/ml) in the presence or absence of sodium vanadate (0.01–1 mmol/l) for 6 h. After theincubation treatment, the cells were stained with propidium iodide, as described in Materials and methods. The percentage ofapoptotic endothelial cells represents the number of apoptotic cells in the counted 100 cells (apoptotic cells+viable cells). Thedata show the mean±SEM for 10 wells. ** P<0.01 versus TNFa and cycloheximide-treated value. Each experiment wasrepeated three times.

1 mmol/l), in part, inhibited the TNFa and cyclohex -imide -induced nuclear fragmentatio n of the cells, asdemonstrate d in Fig. 5A. DIDS (1 mmol/l) givenalone affec te d ne ithe r the morphology nor theapoptos is induc tion. DIDS suppressed the TNFa andcyclohex imide-induced endothe lial cell apoptos is ina dose-depende nt manne r, as show n in Fig. 5A. Onthe othe r hand, the sodium proton antiporte rblocker, dimethyl amiloride (0.1 mmol/l), ex erte d noeffect on the TNFa and cyclohex imide -inducedapoptos is (Fig. 5B). The chloride ion pump inhibitor,anthrac ene carbox ylic ac id (1 mmol/l), did notinhibit the TNFa and cyclohex imide -induced apopto-sis of endothe lial c ells (Fig. 5C).

Discussion

TNF a and cycloheximide-induced apoptosis ofendothelial cells was due to NF- k B activationand was different from TNF a -inducedapoptosis

TNFa has been reported to be an induc er of apoptosisin some kinds of ce lls . This substance induc ed DNAladdering in tumor cells, and the cells displaye d a

typic al morphologic al appearanc e of apoptosis .13 Inthe pre sent study, w e first demonstrate d apoptosisdue to TNFa in endothelial ce lls from the view pointof ion transport. Apoptosis of vascular endothelialce lls may play an important role in the deve lopmentof inc reased vascular permeability and capillary leaksyndrome during systemic inf lammatory responsesyndrome. Irradiatio n can also e lic it a vascular pe rme-ability inc re ase in rat lungs , re sulting from apoptosisof the pulmonary endothe lial ce lls as judged from thepathologic al finding s.3 Recently, lupus antic oagulant-induc ed apoptosis of endothe lial c ells w ith recogni-tion of annex in V has been reported.5 It w assuggested that apoptosis of endothe lial c ells may berespons ible for the vasculitis assoc iate d w ith syste miclupus erythe matosus. The mechanism w here by TNFainduc es apoptos is w as not fully unde rstood. Clarify-ing the mechanism w ill reach the goal to tre at thevascular disease such as vasculitis . In general, it isknow n that TNFa elic ited apoptosis through itsreceptor,6 subsequently stimulating the deathdomain, FADD, and activating caspase activ ity, re sult-ing in apoptos is .6 On the other hand, TNFa alsoactivate s the sphyngomyelinase and produces thece ramide , w hich is capable of ac tivating the kinas e.



FIG. 4. Effect of an inhibitor of caspase on TNF a and cycloheximide-induced apoptosis of endothelial cells. (A) Endothelial cellmonolayers were treated with TNF a (10ng/ml) and cycloheximide (10 m g/ml) in the presence of various concentration of zD-dcb, an inhibitor of ICE (0–50 m mol/l), for 6 h. After the incubation treatment, the cells were stained with PI, as described inMaterials and methods. * P<0.05 versus TNF a and cycloheximide-treated group; ** P<0.01 versus TNF a and cycloheximide-treated group. Each experiment was repeated three times. (B) Endothelial cell monolayers were treated with TNF a (10 ng/ml)and cycloheximide (10 m g/ml) in the presence of various concentration of zD-dcb, an inhibitor of caspase (0–50 m mol/l), for 6 h.After the incubation treatment, the cells were stained with MTT, as described in Materials and methods. The data show themean±SEM for eight wells. ** P<0.01 versus TNFa and cycloheximide-treated group. Each experiment was repeated threetimes.

(A) (B)

The ce ramide -activating kinas e stimulate s NF-k B ac ti-vation.8 TNFa -induced apoptosis through the FADD isindepende nt on NF-k B activatio n.7 Howeve r, TNFacan induc e apoptos is through the NF-k B ac tivation inthe concomitant pre sence of cyclohex imide .14 Wechecked the invo lvement of NF-k B in TNFa andcyclohex imide-induced apoptosis (Fig. 2). In theendothelial ce lls from bovine carotid arte ry, TNFa andcyclohex imide induc ed apoptos is through PDTC-sensitive NF-k B (Fig. 2). How eve r, anothe r inhibitor ofNF-k B, NAC, did not suppre ss the apoptosis inducedby TNFa and cyclohex imide . Bessho e t a l. alsoreporte d that etoposide-induced apoptosis of HL-60ce lls and thymocytes w as inhibite d by PDTC, but notNAC.15 In our pre liminary data, hydrogen perox ideand cyclohex imide -induced apoptosis of endothelialce lls w as inhibited by e ithe r PDTC or NAC (data notshow n). Thus, TNFa and cyclohex imide -inducedapoptos is is diffe re nt from TNFa -induc ed apoptosisfrom the point of the invo lvement of NF-k B. Never-theless, endothelial c ell apoptos is induced by TNFaand cyclohex imide as w ell as TNFa alone , is re sponsi-ble for the caspase activatio n (Fig. 4).

On the othe r hand, the reason w hy cyclohex imideenhance s the TNFa -induced apoptos is remainsunclear. While TNFa dire ctly cause s apoptosis oftumor ce lls , normal ce lls are generally res istant.However, most resis tant cells, inc luding vascularendothelial cells, can be re ndered susceptible toTNFa by inhibiting RNA and prote in synthe sis.16 Thissuggests that TNFa provide s a ce ll survival signal inadditio n to a death signal. Ce ll survival factorsinduc ed by TNFa are BCl-2 analogue , A1, FGF-1 andnitric ox ide (induc ible nitric ox ide synthase).17,18

Cyclohex imide may inhibit the prote in synthe sis oftheir antiap optotic fac tors and be able to easilyenhance the TNFa -induc ed apoptosis.

TNF a and cycloheximide-induced apoptosiswas related to the tyrosine phosphatase, butnot to the sphyngomyelinase

TNFa also activate s the sphyngomyelinase and pro-duces the ceramide , w hich is capable of ac tivating thekinas e. The ce ramide -activating kinase stimulate s NF-k B ac tivation.8 Neve rthe less , TNFa and cyclohex -

H. Fu jita et al.


FIG. 5. Effects of various ion transporter inhibitors on TNFa and cycloheximide-induced apoptosis of endothelial cells. (A)Endothelial cells were treated with TNF a (10 ng/ml) and cycloheximide (10 m g/ml) in the presence of various concentrations ofDIDS (0.1–1 mmol/l) for 6 h. After the incubation treatment, the cells were stained with propidium iodide, as described inMaterials and methods. The percentage of apoptotic endothelial cells represents the number of apoptotic cells in the counted100 cells (viable cells+apoptotic cells). The data show the mean±SEM for 10 wells. * P<0.05 versus TNF a and cycloheximide-treated group. Each experiment was repeated three times. (B) Endothelial cell monolayers were treated with TNF a (10 ng/ml)and cycloheximide (10 m g/ml) in the presence of dimethyl amiloride (0.1 m mol/l) for 6 h. After the incubation treatment, the cellswere stained with PI, as described in Materials and methods. Each experiment was repeated three times. (C) Endothelial cellmonolayers were treated with TNFa (10 ng/ml) and cycloheximide (10 m g/ml) in the presence of anthracene carboxylic acid(1 m mol/l) for 6 h. After the incubation treatment, the cells were stained with PI, as described in Materials and methods. Eachexperiment was repeated three times.

(A) (B) (C)

imide -induced apoptosis w as not inhibited by aninhibitor of sphyngomyelinase (Fig. 3A), show ing thatthe apoptosis w as inde pendent of the ac tivation ofsphingomyelinase through the TNFa receptor in ourassay. Slow ik e t a l. al3o re ported that TNFa -inducedapoptos is w as not re lated to the ce ramide through thesphingomyelinase ac tivation.19,20 In additio n, Modure t a l. reporte d that ceramide production in theendothelial ce lls treate d w ith TNFa did not ac tivateNF-k B.21 These re ports may suggest the agreement onour data.

Tyros ine kinase ac tivation, as w ell as a grow th factor,is a survival fac tor. Tyrosine kinase inhibitors such ashe rbimysin or geniste in caused the apoptosis ofleukemic ce lls, w hich w as rescued by the tyros inephosphatas e inhibitor, sodium vanadate .22,23 TNFaand cyclohex imide -induced apoptosis w as comple te lyinhibited by sodium vanadate , as show n in Fig. 3B. Themechanis m by w hich vanadate pre vente d endothelialce lls from TNFa and cyclohex imide -induc ed apoptosisis unclear. We speculate tw o possibilitie s: (1) vanadatemay have an action on activatio n of tyrosine kinas e,resulting in ce ll survival; (2) vanadate may cause amarked dec rease in p53, the endogenous apoptoticinduc er, as described by the pre vious report.24

TNF a and cycloheximide-induced apoptosiswas involved in the chloride bicarbonateexchanger

In the present study, w e attempte d to use a blocker ofchloride bic arbonate ex changer to inhibit TNFa andcyclohex imide-induced apoptosis. In orde r to main-tain cell shape, it is nece ssary to re gulate the osmoticpressure and ion inf lux . We first cons ide red thechloride channe l, since this channe l is re sponsible forregulation of the anionic prope rtie s and intrac ellularpH. Moreove r, chloride ion inf lux inf luenc es theshrinkage of ce lls.15 A chloride bicarbonate ex change ris pre sent in various kinds of c ells inc luding ven-tric ular myocytes , gastric ce lls , and renal tubularce lls .26–28 DIDS, one of the chloride bicarbonateex change r blockers, prevented TNFa and cyclohex -imide -induced apoptos is in endothelial c ells. In addi-tion, anthrac ene carbox ylic ac id, w hich inhibits thechloride inf lux via the blockade of chloride channe l,did not affec t the TNFa and cyclohex imide -inducedapoptos is (Fig. 5C). There is one possibility that TNFaand cyclohex imide affe ct the function of the chloridebicarbonate ex change r, the reby e lic iting a change inthe intrac e llular chloride ion leve l as w ell as theintrac e llular bicarbonate leve l. Apoptosis could thenbe induc ed by chloride ion e fflux , followed by achange in the intrac ellular pH. In our unpublisheddata, DIDS inhibited the endothelial ce ll apoptosisand an inc re ase in pH, induced by staurosporine ,anothe r apoptos is induce r. Staurosporine changed theintrac e llular pH from 6.9 to 7.4 through a chloride

bicarbonate ex change r (unpublished data). Thechange in pH doe s occur prior to the caspaseactivatio n (unpublished data). We speculate thatendothelial cell apoptosis induc ed by TNFa andcyclohex imide as w ell as staurosporine may be due tothe activatio n of a chloride bic arbonate ex changer;chloride efflux and bicarbonate inf lux (an inc re ase inpH). We consider that pH change due to a chloridebicarbonate ex changer may be invo lved not in NF-k Bactivatio n, but caspase activatio n, although w e haveno dire c t proof. On other hand, Li and Eastman29

demonstrate d that inte rleukin-2-stimulate d lympho-cytes induced apoptosis of targe t ce lls via intrac ellularac idosis through the sodium proton transporte r. Wespeculate , there fore , that one of the common path-w ays of apoptosis may be via a sodium protontransporte r. How eve r, the sodium proton trans porte rblocker, dimethyl amiloride , did not affe ct the TNFaand cyclohex imide -induc ed apoptos is (Fig. 5B). Inour assay syste m, a sodium proton transporte r w asevide ntly not re late d to TNFa and cyclohex imide -induc ed apoptosis .

Currently, w e are attempting to e luc idate moreprec ise ly the mechanism re sponsible for the apopto-sis , w hich is re late d to various ion pumps, inc ludingan ex aminatio n of the role of chloride ion andintrac e llular pH.

References1. Raff MC. Social controls on c ell survival and ce ll death. Scien ce 1992;

356: 397–400.2. Gottlieb RA, Burle son KO, Kloner RA, Babior BM, Engler RL. Reperfusion

injury induces apoptosis in rabbit cardiomyoc ytes . J Clin Inve s t 1994;94: 1621–1628.

3. Fuks Z, Persaud RS, Alfieri A e t a l. Basic fibroblast grow th factor prote ctsendothelial ce lls against radiation-induce d programme d ce ll de ath invitro and in vivo . Ca nc er Re s 1994; 54: 2582–2590.

4. Araki S, Shimada Y, Kaji K, Hayashi H. Role of prote in kinase C in theinhibition by fibroblast grow th factor of apoptosis in serum-de pletedendothelial ce lls. Bio che m Bio phy s Re s Co m m un 1990; 172:1081–1085.

5. Nakamura N, Shidara Y, Kaw aguchi N e t a l. Lupus anticoagulantautoantibody induce s apoptosis in umbilical ve in endothelial ce lls.Bio che m Bio phy s Re s Co m m un 1994; 205: 1488–1493.

6. Boldin MP, Goncharov TM, Goltsev YV, Wallach D. Involve ment of MACH,a novel MOLT-1/FADD-interacting protease, in Fas/Apo-1 and TNFre ceptor-induced apoptos is. Cell 1996; 85: 803–815.

7. Liu ZG, Hsu H, Goedde l DV, Karin M. Dissection of TNF rece ptor 1effecte r functions: JNK activation is not linked to apoptosis w hile NF-kappa B activation prevents ce ll death. Ce ll 1996; 87: 565–576.

8. Kitajima I, Soe jima Y, Takasaki I, Beppu H, Tokioka T, Maruyama I.Ceramide-induced nucle ar translocation of NF-kappa B is a potentialmediator of the apoptotic re sponse to TNF-alpha in murine clonalosteoblasts. Bo ne 1996; 19: 263–270.

9. Mashima T, Naito M, Kataoka S, Kaw ai H, Hayashi H. Identification ofactin as a substrate of ICE and an ICE-like protease and involveme nt of anICE-like protease but not ICE in VP-16-induced U937 apoptos is. Bio ch emBiophy s Re s Co m m un 1995; 217: 1185–1192.

10. Fujita H, Morita I, Murota S. A possible mechanism for vasc ularendothelial ce ll injury elicited by activate d leukocyte s. Arch Bio ch emBiophy s 1994; 309: 62–69.

11. We i YQ, Zhao X, Kariya Y, Fukuta H, Teshigaw ara K, Uchida A. Induc tionof apoptosis by querce tin. Ca nce r Re s 1994; 54: 4952–4957.

12. Mosmann T. Rapid calorime tric assay for ce llular grow th and survival. JIm m uno l Methods 1983; 65: 55–63.

13. Wang CY, Mayo NW, Baldw in AS. TNF and cancer therap y-inducedapoptosis. Scien ce 1996; 274: 784–787.

14. Polunovsky VA, Wendt CH, Ingbar DH, Pete rson MS, Bitte rman PB.Induction of endothelial ce ll apoptos is by TNF alpha: modulation byinhibitors of prote in synthesis. Exp Ce ll Re s 1994; 214: 584–594.



15. Bessho R, Matsubara K, Kubota M, Lin YW, Okuda A, Nishimori R.Pyrrolidine dithiocarbamate , a pote nt of nuclear factor kappa Bactivation, pre vents apoptosis in human promye locytic leukemia HL-60 c ells and thymocyte s. Bio ch em Pharm a co l 1994; 48:1883–1889.

16. Kars an A, Yee E, Harlan JM. Endothelial ce ll death induced by tumornec rosis fac tor-alpha is inhibited by the Bcl-2 family me mber, A1. J Bio lChe m 1996; 271: 27201–27204.

17. Maier JA, Morelli D, Menard S, Colnaghi MI, Bals ari A. Tumornec rosis factor-induced fibroblast grow th factor-1 acts as a survivalfac tor in a transformed endothelial ce ll line. Am J Pa tho l 1996; 149:945–952.

18. Dimmeler S, Hae ndele r J, Nehls M, Ze iher AM. Suppre ssion of apoptosisby nitric ox ide via inhibition of interle ukin 1 beta converting enzymelike and cys teine prote ase prote in 32 like proteases. J Exp Med 1997;185: 601–607.

19. Slow ik MR, De Luca LG, Min W, Pober JS. Ceramide is not a signal fortumor necrosis factor-induced gene expre ssion but does cause pro-gramme d cell death in human vas cular endothelial ce lls. Circ Re s 1996;79: 736–747.

20. Slow ik MR, Min W, Ardito T, Kars an A, Kashgariam M, Pober JS. Evidencethat tumor necrosis factor trigge rs apoptosis in human endothelial ce llsby interleukin 1 converting enzyme like prote ase-depe ndent andindependent pathw ays . Lab Inve s t 1997; 77: 257–267.

21. Modur V, Zimme rmann GA, Prescott SM, McIntyre TM. Endothelial ce llinflammatory responses to tumor nec rosis factor alpha. Ceramide-dependent and indepe ndent mitogen-activate d prote in kinase casc ade s.J Bio l Chem 1996; 271: 13094–13102.

22. Bergamas chi G, Rosti V, Danova M, Ponchio L, Lucotti C, Cazzola M.Inhibitors of tyrosine phosphorylation induce apoptosis in humanleukemic ce ll lines. Leukem ia 1993; 7: 2012–2018.

23. Min A, Hasuma T, Yano Y, Matsui-Yussa I, Otani S. Regulation of apoptos isof interleukin 2-dependent mouse T-cell line by prote in tyros inephosphorylation and polyamine s. J Ce ll Phy s io l 1995; 165: 615–623.

24. Chen ZP, Ye ung DC. Re gulation of p53 expre ssion in HeLa ce lls. Bio ch emMo l Bio l In t 1996; 38: 607–616.

25. Widdas WF, Baker GK. The pH volume changes of human red ce lls invitro due to the exchange of chloride and hydrox yl anions. Cy to bio1992; 72: 139–152.

26. Xu P, Spitzer KW. Na-independent Cl––HCO3– exchanger mediates

re cove ry of pHi from alkalosis in guinea pig ve ntricular myocyte s. Am JPhy s io l 1993; 267: H85–H91.

27. Horie S, Yano S, Watanabe K. Inhibition of gastric acid secre tion in vivoand in vitro by an inhibitor of Cl––HCO3

– exchange r, 4,4 9 -diisothiocya-nostilbene-2,2 9 -disulfonic acid. J Pha rm a co l Exp Theu r 1993; 265:1313–1318.

28. We iner ID, Wingo CS, Hamm LL. Re gulation if intrace llular pH in tw o c ellpopulations of inner stripe of rabbit outer me dullary colle cting duc t. AmJ Phy s io l 1993; 265: 1313–1318.

29. Li J, Eastman A. Apoptosis in an interleukin-2-dependent cytotox ic Tlymphocyte line is assoc iated w ith intrace llular acidification. J Bio lChe m 1995; 270: 3203–3211.

Received 2 August 1999;accepted 24 August 1999

H. Fu jita et al.


Submit your manuscripts athttp://www.hindawi.com

Stem CellsInternational

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014

Immunology ResearchHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinson’s Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com

mediators of inflammation, 8 we a a possible involvement of ion...

Documents