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TCM

NV F

Kathy K. Griendling and Masuko Ushio-Fukai

T v a sN A Do xh b s t b t ms oo s u pi t v ew R w h pi n si ni s t ra a ci v ac T e ii n vi b v a ss mm h ya i sf oo i m pe n d o t hr eB ot is ti g ep a ri t p ao h ya a t h e r ot e nt p rr eo sm b i m pd e t eo t c o v d( T rC a rM 1 97 : 30 E S I

Oxidativestresshasbeen the subjectof avast amount of research over the years,initially because of its propensity to in-duce DNA damage. It has now becomeapparent,however, that reactive oxygenspecies may act as intercellular and in-tracellular second messengers in bothnormal and pathophysiologic respon-siveness. In the vascular system, thesourcesof oxidative stresshave not beenclearly established. Recent work hasshown that the traditional sourcesof ox-idative molecules (xanthine oxidase,mi-tochondrial oxidases, arachidonic acid)play a relatively minor role in the pro-duction of reactive oxygen species in thevesselwall and thata nonmitochondrial,membrane-associated NADH/NADPHoxidase is the major source of superox-ide (.02–) in vascular cells (Griendling etal. 1994,Mohazzab-H and Wolin 1994b,Rajagopalan et al. 1996a).

. The NADI-UNADPHOxidaseas a Source of OxidantStressin VascularCells

Vascularcells are exposed to both para-crine and autocrine regulation by reac-

Kathy K. Griendling and Masuko Ushio-Fu-kai are at the Emory University School ofMedicine, Division of Cardiology, Atlanta, GA30322, USA.

tive oxygen species. Endothelial andsmooth muscle cells produce .02– andH202 (Friedl et al. 1989,Pagano et al.1993, Panus et al. 1993, Mohazzab-Hand Wolin 1994b, Rajagopalan et al.1996a),and are exposed to free radicalsreleased by circulating blood cells andinflammatory cells.Cell associated-reac-tive oxygen species appear to derivemainly from an NADH/NADPH oxidase(Mohazzab-H et al. 1994,Rajagopalan etal. 1996a). Acetylcholine-insensitive.02- generation from rabbit aorta wasnoted by Pagano et al. (1993). These in-vestigatorsconcluded that vascular .02–is derived mainly from a nonendothelialsourceand is modulated by endogenoussuperoxide dismutase. Subsequently,a.02--generating NADH oxidase was ob-served in pulmonary arteries by Mo-hazzab-H and Wolin (1994a). This mi-crosomal NADH-dependent productionof .02– is decreased by hypoxia and ap-parently utilizes a cytochrome b~~~elec-tron transport system (see later here).Simultaneously,our laboratory demon-strated (Griendling et al. 1994)that an-giotensin II increases NADH- andNADPH-dependent .02- production incultured vascular smooth muscle cells(VSMC). This activity is membrane as-sociated and is inhibited by diphenyleneiodinium (DPI), an inhibitor of flavin-containing enzymes (Figure la). Paganoet al. (1995) reported an NADPH-depen-

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—.-DPI +DPI

T

-DPI

**—

+DPI

+Angll

vector p22phox-antiaanaa

+Angll

——vector p22phox-

antisenaa

1

Figure 1. Angiotensin II–induced NADH oxidase activation (a) and hypertrophy (b) areinhibited by diphenylene iodinum, an inhibitor of flavin-containing enzymes (leftpanel) and byantisense p22phox transection (right panel) in vascular smooth muscle cells (VSMC). (a)Initial rate of NADH-dependent .02- production with or without treatment with diphenyleneiodinium (DPI) (100 pM) (left panel) and in vector- and antisense p22phox–transfected cells(rightpanel) stimulated with 100 nM angiotensin II for 4 h, as measured by lucigenin assay. (-)and (+) indicate treatment with vehicle or angiotensin II, respectively. The initial rate ofenzyme activity was calculated over the first 30 to 120 seconds of exposure to NADH and isexpressed as nmol O-/min/mg protein. Statistical analysis was performed by Student’sttest.Values are expressed as mean SEM (n = 15). “ p < 0.05 for increase by angiotensin II inDPI-treated versus untreated cells (leftpanel), and in antisense- versus vector-transfected cells(rightpanel). (b) Bars indicate the percent increase in [3H]-leucine incorporation induced byangiotensin II over the appropriate control. There was no difference in basal incorporation of[3H]-leucine between with and without DPI (left panel), and vector- and antisense p22phox-transfected cells (rightpanel). Each bar represents the mean of four independent experimentsperformed in triplicate. Statistical analysis was performed by one-way analysis of variance.**p <0.01 and *p <0.05 for increase by angiotensin II in DPI-treated versus untreated cells(left panel), and in antisense- versus vector-transfected cells (right panel). Adapted fromGriendling et al. 1994 and Ushio-Fukai et al. 1996.

dent O--generating system in rabbit substrate, this enzyme apparently sharesaorta located in the media andlor adven- many of the characteristics of the oxi-titia but found little evidence that this dase identified by our laboratory and bysystem utilized NADH as a substrate. Mohazzab and Wolin. All of these data

Other than this difference in preferred support the notion that an NADH/

NADPH oxidase is the major source of.02- in vascular cells.

The fate of the NADH/NADPH-derived.02- has not been fully investigated,butcurrentevidenceis consistentwith a rapidconversionof .02– to H202,presumablyvia the action of superoxide dismutase.For example,in VSMC in which agonist-stimulatedNADH/NADPHoxidase activ-ity has been inhibited using antisensetooneof itssubunits,intracellularH202pro-ductionis alsoattenuated(Ushio-Fukaietal. 1996).Additionally,the transientrelax-ationto posthypoxicreoxygenationin calfcoronaryarteriesis apparentlydependenton H202 originating from NADH-depen-dent .02- production(Mohazzab-H et al.1996).H202releasefi-omendothelialcellshas also been demonstrated,althoughitssourcehasnot beendetermined(Panusetal. 1993).Another importantfate of .02 ,particularlythatderived from endothelialcells,is interactionwith nitric oxide (NO.)to form peroxynitnte anion (Beckmanetal. 1990),as discussedlaterhere.

● Structureof the VascularNADWNADPH Oxidase

The endothelial or smooth muscleNADH/NADPH oxidase is a membrane-associated enzyme that generates .02from NADH or NADPH (Griendling et al.1994). It has not been conclusivelyproved that a single enzyme can utilizeboth substrates, although experimentsin antisensecells suggestthat this is thecase (see later here). The complete mo-lecular structure of the vascular oxidaseis unknown; however, it bears some sim-ilarity to the NADPH oxidase that is re-sponsible for the respiratory burst inneutrophils. In vitro studies of the neu-trophil enzyme have indicated that it iscomposed of at least five subunits: twocytosolic components, p47phox andp67phox; a plasma membrane-associ-ated cytochrome b~~~ composed of twosubunits, p22phox and gp91phox; and asmall molecular weight G protein, eitherrac-1 or rac-2 (Rotrosen et al. 1992) (Fig-ure 2). Upon activation, the p47phox andp67phox proteins are translocated to themembrane and associate with the cyto-chrome b~~~,creating the active enzyme.In current models, the full electrontransfer activity of the neutrophilNADPH oxidase resides in the cyto-chrome. Furthermore, the SH3 domainof p47phox interacts directly with the

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cytochrome bS5tl

:*h,pbx

active

Figure2. Molecular structureof the neutrophilNADPHoxidase. The molecular structureof theneutrophiloxidase has been clearlyestablished.The c,ytochromeb~~gresides in the membraneand consists of two subunits, p22phox andgp9tphox. In resting cells, the remaining reW-Iatmy subunits (p47phox, p67phcrx,rac2) [L-side in the cytosol. IJpon stimulation, p47phoxassociates with p22phox via an SH3 domaininteraction, and p67plmx associates withp47phox via a differentSH3 domain, Rac2 alsobecomes part of the active erwymc complex.

prolinc-rich region of p22phox (Leto etal. 1994), whereas the C-terminal SH3domain of p67phox associates with aprolinc-rich region of p47phox (Finan etal. 1994). Thus, the cytochrome mole-cule, and in particular the p22phuxsub-unit, is central to the normal functioningof the oxidase because it is responsiblefor electron transfer and serves a dock-ing function for the cytosolic factors.

Many of the characteristicsof the neu-trophil NADPH oxidase are shared bythe VSMC enzyme, most notably stimu-lation by phosphatidic and arachidonicacids, association with the membrane,and sensitivity to the flavin-containingenzyme inhibitor DPI (Griendling et al.1994).The VSMC oxidase differs, how-ever, from that of neutrophils in manyrespects. First and foremost, the timecourse of stimulation of the oxidase dif-fers dramatically in the two cell types.Superoxidegeneration by the neutrophilNADPH oxidase is massive and beginswithin secondsof exposureto hormones(Watson et al. 1991),whereas accumula-tion of .02- in agonist-stimulatedVSMC(about one-tenth the amount in phago-cytes) occursover a period of hours.Sec-ond, the .02– generated in VSMC ap-pears to be mostly intracellular, withonly a limited amount of .02 being re-leased to the exterior (Griendling et al.1994), as occurs in phagocytes. Third,the VSMC enzyme appears

TCM vol. 7, No. 8, 1997

to utilize

NADH to a greater extent than NADPH,which is exactly the opposite 0[ lhe sit-uation in neutrophils. Finally, the phys-iologic function of the .02– that accu-mulates is quite different from that ofphagocytic cells, perhaps serving a sig-naling function for control of vasculartone or growth (Griendling et al. 1994).

On a molecular level, the vascular oxi-dase may share only limited homologyto the neutrophil enzyme. Antibodyscreening and polymerase chain reac-tion (PCR) experiments have shown thatnot all the components of the neutrophiloxidase are expressed in vascular cells.In endothelial cells, gp91phox, p22phox,p47phox, and p67phox are all detectableby reverse transcriptase (RT)-PCR, butonly p22phoxcan be detected by North-ern analysis and only the cytosolic fac-tors are detectable by Western analysis(Jones ct al. 1996).Using hcme spectro-scopy,cytochrome b55xe x p=not bc detected. Expression of a consti-tutively active mutantof rac-1,however,increases the production of reactive ox-ygen species in endothelial cells (SUl-ciner et al. 1996),suggestingthatat leastthe G protein neutrophil NADPH oxi-dase activating factor is functional in

endothelial cells.In smooth muscle cells, p22phox has

beencloned and is relatively abundantatthe mRNA level (Fukui et al. 1995),bulgp91phoxdoes not appear to be present(unpublished data). Functionally, it ap-pears that p22phox may be part of theagonist-sensitive oxidase, as antisenseinhibition of p22phox expression inVSMC leadsto a diminutionof .02- gen-eration (Ushio-Fukai et al. 1996). Inter-estingly,in these cells the spectrophoto-metric peak occurs not at 558 nm, butrather at 553 nm (Ushio-Fukai et al.1996), suggesting that the cytochromeassociated with the vascular enzymemay be different from the neutrophil cy-tochrome and may even be a c-type cy-tochrome. In theneutrophilNADPH oxi-dase, the large subunit of cytochromebSSS(gpglphox) functions as the sub-strate recognition site (Rotrosen et al.1992),the FAD binding site (Sumimotoet al. 1992),and most likely binds heme(Cross et al. 1995).Thus, the unknownlarge subunit of the vascular NADH/NADPH oxidase may be the componentthat confers on smooth muscle itsunique kinetics, substrate specificity,and activation requirements.

PercentRelaxations

o— .!,s

K

— — a,.,

20 \A(T 1

+ Ang II-EL— ~-” U-SOD

T

t— -...m-EL

%

-9- ShAm-SOD

\*

.

T“

I .\-t

60

80

100-9.0 -8.0 -7:0 -6:0

log M [acetylcholine]

Figure 3. Vascular relaxation to acetylcholinein aortic segmentsfrom sham-operatedand an-giotensin II-infused rats treatedwith liposome-encapsulated superoxide dismutase (SOD, 750U/mL) (Sham-SOD and angiotensinII-SOD,re-spectively)or with empty liposomes (Sham-ELand Ang II-EL,respectively).AngiotensinII wasinfused at a rate of 0.7 mg/kg.day for 5 days,which causes increased vasculal oxidativestress.Vesselsfrom both groups were exposedfor 30q5 min to eithe~JjLor SOfJ,and vasol-e-laxationswere examined aftercontraction withphenylepbrinc. ‘ p <0.05 versus sham for per-cent maximal relaxation; + p =: 0.05 versussham for t31350.Reprinkd with permissionfrom Rajagopalan et al. 1996a,

● Role of ReactiveOxygenSpeciesin Endothelial Function

Even before the identification of endo-thelium-derived relaxing factor as NO.,several groups showed that its integritywas compromised by oxygen-derivedfree radicals (Gryglewski et al. 1986,Rubanyi and Vanhoutte 1986) (Figure3). In fact, if the antioxidantdefense sys-tems are inhibited, endothelium-depen-dent relaxations to acetylcholine are im-paired (Mugge et al. 1991a). This isapparently not due to a decrease in theamount of nitrogen oxides released,butrather to the functional destruction ofNO. by .02- (Nlugge et al. 1991a).Super-oxide reacts rapidly with NO. to formperoxynitrite, which itself can alter en-dothelium-dependent relaxations andmay contribute to lipid peroxidation(Radl et al. 1991),or can be dissociatedto an hydroxyllike radical and nitrogendioxide. Nitrogen dioxide is highly reac-tive and may be involved in nitrosationreactions (Pryor and Squandrito 1995).Thus, the balance between ambient lev-els of .02– and releasedNO. is critical tothe normal functioning of the endothe-lium. As noted later here, elevated .02-production is a characteristic of severalvascular diseases,paired endothelial

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suggesting that ir-responsiveness may

303

be a function of the increase in oxygenfree radicals.

Hemodynamic stressalsohasaneffecton the oxidative state of endothelialcells. In a recent paper, Howard et al.(1997) showed that cyclic strain causedan early (2 h), transient increase inNADH/NADPH oxidase activity and asustained increase in H202 in culturedporcine aortic endothelial cells. This in-crease in oxidative stress was reflectedin an increase in lipid peroxidationproducts released from the cells, sug-gesting that reactive oxygen speciesmayserve to transduce mechanical signalsinto biochemical events.

Another effect of reactive oxygen spe-cies on endothelial function is to shiftthe thrombotic or antithrombotic bal-ance to favor adhesionof leukocytes.Forexample, interleukin-l–induced expres-sion of vascular cell adhesion molecule(VCAM-1) is inhibited by treatmentwiththe intracellular antioxidant pyrro-lidinedithiocarbamate (PDTC) (Marui etal. 1993).The redox-sensitive transcrip-tion of VCAM-1 appears to depend, atleast in part, on tandem NFKB-like ele-ments in the promoter region of thisgene. Induction of monocyte chemotac-tic protein (MCP-1) (Cushinget al. 1990)and heme oxygenase-1 (HO-1) (Nath etal. 1992),among others,is alsomediatedby oxidative stress.

Finally, externally applied oxidantstress leads to an upregulation of theantioxidant defense enzymes, includingsuperoxidedismutase,catalase,and glu-tathioneperoxidase (Lu et al. 1993).Thissuggeststhat factors that influence vas-cular oxidantstressare tightly regulated,a requirement if reactive oxygen speciesserve a second messenger function invascular cells.

● ReactiveOxygenSpeciesand VascularSmooth Muscle

In addition to their influenceon vasculartone via alterations in endothelium-de-pendentrelaxations,reactive oxygen spe-cies also directly affect vascular smoothmuscle.Mohazzab-Handcolleagues(Mo-hazzab-Het al. 1996)haveshownthatthetransient pulmonary vasorelaxation oc-curringduringposthypoxicreoxygenationis a direct result of H202 derived fromNADH-dependent superoxide formation.This effect is apparentlydue to the stimu-lation of guanylate cyclase via catalase-

aBlood

Pressure(nmnlig)

120~1 2 3450

b(mmHg)

100

0

D o Infusion

*

PIIX4SUE

onmd/midmg

120001I

10

5

042- production

moo

4(XIO

o

‘

❑ Sham❑ Angll❑ Angll+l-lydmlazine

*

Figure 4. Role of .02- in hypertension induced by high circulating levels of angiotensin II. (a)Changes in systolic blood pressure (Zefipanel)and .02- production from aortic segments (rightpanel) in angiotensin II- and norepinephrine (NE)-infused rats. Angiotensin II was infused ata rate of 0.7 mg/kg.day, whereas NE was infused at 2.8 mg/kg.day. Systolic pressure and .02–production were measured by tail cuff sphygmomanometry and by lucigenin chemilumines-cence, respectively.Measurement of .02– production was performed 5 days after drug infusion,when blood pressure levels plateaued at similar values in both groups. (b) Effects of hydrala-zine, an antihypertensive drug, on systolic blood pressure (left panel), the increases in .02–production (middle panel), and p22phox mRNA expression (right panel) in angiotensin II-infused rats. Rats were killed 5 days after angiotensin II infusion, and hydralazine (15mg/kg.day) was given in the drinking water. ‘<p < 0.05 versus sham-operated rats. Adaptedfrom Rajagopalan et al. 1996a and Fukui et al. 1997.

dependent H202 metabolism. These in-vestigators have described a similarmechanismfor photorelaxationof pulmo-nary arteries denuded of endothelium(Wolin et al. 1991).

Reactive oxygenspecieshavealsobeenimplicated in growth of vascularsmoothmuscle.AngiotensinII–inducedhypertro-phy is mediatedin partby NADH oxidase

activation because inhibition of this en-zyme systemwith diphenylene iodiniumor transection with antisensep22phoxat-tenuatesprotein synthesis(Griendling etal. 1994,Ushio-Fukaiet al. 1996)(Figurelb). Furthermore, treatment of VSMCwith antioxidantssuch as PDTC and N-acetylcysteineleadsto apoptosis,suggest-ing that some level of oxidant stress is

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required for normal growth (Tsai et al.1996).Tncfeed,H202and 02- activatem-merous growth-related signaling path-ways in VSMC, including activation of

kinases (BaasandBerk 1995);induc-tion of’ the protooncogencsand c-imyc (Rao and Berk 1992,Rao et al.1993aand b); and activationof the AP-1transcriptionfactor (Puri et al. 1995).Arecent study(Traniet al. 1997)on the roleof supcmxide in mitogenic signaling infibroblasts afso implicates ras, a smallguanosinetrisphosphatebinding proteinknown LObe important in proliferationand differentiation. Fibroblasts h-ans-formedwith a constitutivelyactivemutantof ras produce large amountsof .O1–viaactivationof the NADPH oxidasecompo-nent rac-1,and thesecells have a greatlyincreased DNA turnovm If a similarmechanismholdsin VSMC, thisstudynotonlyprovidessupportfor a role of rac-1 inthe nonphagocyticoxidasc but also sug-gestsa new mitogenic signalingpathwayby which oxidantsexert their effects.

● ReactiveOxygenSpeciesand Microphage Function

Reactive oxygen species are both pro-duced by,and alter,the function of mac-rophages and leukocytes. In leukocytes,.02- and H202 promote CD1lb andCD18 expression and increased adhe-sion to endothelial cells (Fraticelli et al.1996),similar to their effect on endothe-lial cell adhesion molecule expression.Furthermore,microphage-derived foamcells from hypercholesterolemic rabbitsproduce .02– and H202 (Rajagopalan etal. 1996b).These reactive oxygen speciesregulate the activity of smooth muscle-derived matrix metalloproteinasesMMP-2 and MMP-9 (Rajagopalan et al.1996b), suggesting that oxidant stressmay contribute to vascular remodeling.

. The NADI-UNADPHOxidasein Hypertension

Recently,there has been intense interestin the role of .02– in hypertension in-ducedby high circulating levels of angio-tensin II. Rajagopalan et al. (1996a)demonstrated that in rats made hyper-tensive by prolonged infusion of angio-tensin II, vascular NADH/NADPH oxi-dase activity is selectively increased andcontributes to impaired endothelium-dependent vasodilation (Figure 3). This

elevated enzymatic activity is accompa-nied by an increase expression0[p22phoxmRNA (Fukui et al. 1997)(Fig-ure 4), suggesting that the amount ofoxidase present in the vessel wall mightbc upregulated in this model of hyper-tension. Notably, infusion of anothervasoconsLrictol;nolepincphrine, had noeffect on oxidase activity even though itcaused an equivalent in bloodpressure (Rajagopalan et al. 1996a).Both oxidase activity and p22phox ex-pression were blocked by the antihypcr-tensive agent hydralazine (Figure 4) andthe angiotensin ATI receptor blockerlosartan, raising the possibility thatblood pressureitself might contribute tooxidase upregulation in the setting ofelevated angiotensin H (Fukui et al.1997).A possiblealternativeexplanationarises from the recent obsemation thathydralazine has antioxidant proper-ties(Mtinzel et al. 1996). These findingswould thusalsobe consistentwith a rolefor oxidant stressin blood pressurereg-ulation. In fact, administrationof super-oxide dismutasebeginning 3 daysbeforeangiotensin II infusion significantly at-tenuates the subsequent rise in bloodpressure (Bech-Laursen et al. 1997).Consistent with the lack of effect ofnorepinephrine on oxidase activity, su-peroxide dismutasehas no effect on thenorepinephrine-induced increase inblood pressure.

● OxidativeStressin Atherosclerosis

There is accumulatingevidence thatath-erosclerosis is accompanied by, or isconsequentupon,oxidative stresswithinthe vessel wall. Oxygen free radical pro-duction is higher in aortas from rabbitsfed a high-cholesterol diet for severalweeks than in aortas from animals onnormal diets (Harrison et al. 1987,Ohara et al. 1993). Hypercholester-olemic animals exhibit impaired endo-thelial-dependentrelaxation, which canbe restored with antioxidants (Miigge etal. 1991b,Keaney et al. 1995).Further-more, it hasbeen demonstratedthataor-tic atheroscleroticlesion formation is as-sociated with the accumulation of lipidperoxidation products and induction ofinflammatory genesby NFKB-like(redoxsensitive) transcription factors (Liao etal. 1994). In vitro data suggest that ex-pression of endothelial-leukocyteadhe-

sion molecules,an early event in athero-genesis that mediates migration ofmonocytes into the vessel wall, is alsoredox sensitive (Marui et al. 1993,Khanet al. 1995). The increased free radicalproduction may thushave multiple con-sequences, including monocyte recruit-ment into the vessel wall (Alexander1995), inactivation of NO resulting inendothelial dysfunction (Miigge et al.1991b), and increased smooth musclecell growth (Griendling et al. 1994).To-gether,these data suggestthaLincreasedoxidative stress in the arterial wall maybe a fundamental feature of atheroscle-rosis (Afexander 1995).

● The NADH Oxidasein NitrateTolerance

The clinical problem of the developmentof tolerance to nitrates has receivedenormous attention, but the underlyingmechanisms remain elusive. A recentstudy by Miinzel et al. (1996) providesevidence for a role for the vascularNADH oxidase in nitrate tolerance,basedon ananalysisof the drug hydrala-zinc. In rabbits exposed to nitroglycerincontinuously for 3 days, vascular .02production is increased, and this in-crease is completely abolished by hy-dralazine. In vitro studies indicate thatthis drug apparently exerts its effect byinhibiting the NADH oxidase. Impor-tantly, concomitant administration ofhydralazine and nitroglycerin preventsthe development of tolerance to acutenitroglycerin-induced endothelium-de-pendent relaxation, suggesting thatNADH-derived .02- may contribute tothe vascular dysfunction that is pathog-nomonic for nitrate tolerance.

. Future Directions

The recent work summarized here sug-geststhat the proteins and enzymes thatproduce reactive oxygen speciesor serveas the antioxidant defense system areimportant determinantsof the course ofvascular disease. A corollary of thispremise is that the mechanismscontrol-ling activity or expression of these pro-teins or enzymes are also critical. Fur-ther investigation is necessary todetermine the subunit structure of thevascularNADH/NADPHoxidase,the sig-nals that regulate oxidase activity, thefate of the .02- generated, and the func-

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tional consequences of increased .02–production in both endothelial andsmooth muscle cells (Rajagopalan et al.1996b). Of particular interest will beidentification of redox-sensitive genesthat play a role in both normal vascularfunction and the pathogenesis of vascu-lar disease.

● Acknowledgments

This work was supported by NationalInstitutes of Health grant HL38206.Wethank Dr. David Harrison for a criticalreading of the manuscript and BarbaraMerchant-Bailey for editorial assistance.

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K PRT a S G D SJulie Chao and Lee Chao

K a l l if id i sa a h k a l lp it c i r cs hh h ow o p s pt e ii n h i( s eI f a c ol c wt ik a l la i nk a la cS ueh a c c ui r ey i nt k am p ar oi b lp r er e gi n do i i nwt ik a l lI n ti no k ai r a mr e si a r aa t r ar eo b p i ad o s e -m aF u na ni t rm oe x p rr k a l l i kp ra a no h kl i s tr e vt t hm h s i gl b pc o mw c ol i t tA d e nt d o th uk a l lg c c s i gb p rf 4 w ei s p o nh y pr F k aci nv a s o ri i sr a r a r rp e r -p r ei t i sp ek T tf i ns u ga d ir f k a li r eb pa r at p o s sf t d e vo n p ht r e af h y p e( TC aM 1 73 1 0 1E l sS cI

. Discoveryof Kzdlistatinas aTissueKallikrein-BindingProtein

Tissue kallikrein activity was first iden-tified in humanurine in 1909as a resultof its profound but transientblood pres-sure lowering effect on dogs (Abelousand Bardier 1909).In 1928,a tissuekal-

Julie Chao and Lee Chao are in the Depart-ment of Biochemistry and Molecular Biology,Medical Universityof South Carolina, Charle-ston, SC 29425, USA.

likrein-binding protein (KBP), whichrapidly inhibited kallikrein’s activity,was first observed in human plasma(Frey and Kraut 1928), but its identityremained unclear. Geiger et al. (1981)reported that human cxl-antitrypsin (cil-AT) binds to and inhibits human tissuekallikrein. Although al-AT is present in anenormous quantity in the circulation, theformation of the complex between al-ATand kallikrein is a very slow process. Thesignificance of its physiological functionin regulating tissue kallikrein’s function

TCM vol. 7, No. 8, 1997 @ 1997,ElsevierScienceInc., 1050-1738/97/$17.00PIIS105O-1738(97)OOO89-3 307