~1s.-1611t03.0M) ,.... JouJuv.L 01' PluIlll'OOI01if oU<tI~ ..... TDa.vamaI CopJri&bt 0 191M by The "-rica.a 800et, r ... PhanucoIoc' aJ>d. ~t.al Therropeutica JPET 271:1611- 1616. 1~

Vol. zn. No. 3 l'riIIUd ... U.S.A

Furosemide Enhances the Release of Endothelial Kinins, Nitric Oxide and Prostacyclin

GABAIELE WIEMEA,' EDWIN FINK,2 WOLFGANG UNZ,' MAX HAOPOT,' BEANWARD A. SCHÖLKENS' and PAULUS WOHLFAAT'

Hoechst AG, SBU CardioV83CU1ar Agenls, FrankfurtJMsin, Getmany, Department 01 CJinicaJ Chemie and ClinicaJ BiochemIe, HospItal 01 Suf'9l!llY, UnivefsJty Manchen, Getmeny

Accepted for publlcatlon May 20. 1994

ABSTRACT Oespite a weaIth of date, the mechanism 01 the direct dilator effec:t of furosemlde on the systemlc arteria/ and venous sys­tems Is far from belng satlsfactorily understood. Therefore, we Investlgated whether furosemlde Is capable of stlmulatlng the producUon of the endogenous vasodilators nitric oxide and prostacyclin in primary cultured bovlne aortic endotheliaJ cells by an enhanced synthesis and reIease of endothellum-derived kinins. Nitric oxide production was assessed in terms of intra­cellular guanoslne cyclic-3' ,5' monophosphate accumulatlon; kin!n and prostacyclin reIease were determined by specific radioimmunoassays. Furosemide concentratlon- and lime-de­pendently increased the formation of nitric oxide and prosta-

Earlier investigations in patients suggest that the aeute syatemic: hemodynamie eft'ecta of furoeemide , which appear within a few minutea after intravenous adminiatration be­rare the diuretic response OCCUJ'8, are due to a direct; dilator action on blood vesaels and are independent of its diuretie propemes (Biamino et al., 1974, 1975; Dik.ehit et al., 1973; Schenk. et al., 1975). Nevertheleaa, it haa been ehown that the kidneya playa role in mediating this eft'ed, bec:ause the earIy dilator effed of furoeemide is abolished by nephrectomy in hypervolemie anurie dogs (Bourland et aJ., 1977) and aneph­rie patients (Johnston et aJ., 1983). Furthermore, rat&: given furosernide after bilateral uretalligation ehowed a decrease in arterial blood pressure. In compariaon, bilateralligation of renal blood veesels suppressed this eff'ed, wbich Jed to the conclUlion that the blood pre"UTe-reducing action of furo­aemide requires the integrity of renal vesaels (Sec:h.i et al., 1990). On the basis of the latter studiea, prostaglandins of renal and/or extrarenal origin appear to be involved in the

~ved r.,.. publie,otion J~\IAly 26, 1994 . • "'-tat .cldt..: Ho.ch8t. AG, SBU Caniiovuculat ApDta, H 821.

0.&5926 Frankf'wtJMain, Germany. , J>r.eDt .cIdrM.: DeputmeDt CliDieIU Biocbemiatry. ClinieaI Ceotar City.

LMU, Nupb.1lIUtl'uM 20. D-80336 MUDcben, Germazay.

cytin. Maximal increases 01 both autacoids ware already ob­tained after a 5-min incubation with 3 x 10- 7 10 10- 6 motII of furosemlde. In the same concentratlon range, furosemide lad to an enhanced reIease 01 kinlns Into the SUpem8tant 01 the celis. This observation was supported by the Inhibitory effec:t of the speclfic ~ kinin receptor antagonist lcatlbant (Hoe 140) on the furosemlde-induced Increase of nitric oxide and prostacyclin. Thus lhe hemodynamic effacts, and In particular the direct earty dUator affec:t, 01 furosemlde may be expIaIned In part by an enhanced endotheliaJ synthesis and release 01 bradyklnin and related kin ins, which in tum stimulates endothelial autacold formation via B2 kinin receptor activation.

early vuodilator effed of furoeemide, bec:ause pretreatment with indomethacin aboliahed trua eft'ed. Sulindae, wbieh pre­dominantly aff'ecta the extrarenal ayntheais of proataglan­dina (Wong et 01. , 1986), did not reverae tbe antihyperl.ensive effed of diuretics in patients (Puddey et 01., 1985); this un­deracorea the role of renal pl'08taglandin syntheaia in medi­ating tbe peripheral vascular effecta offuroeemide. However, after furoeemide treatment, plasma ooneentr'atioM of &rac:bi­donic acid (Gerber and Niea 1981) or metabolites of pl'08ta­cyelin (Mac:kay et aJ., 1984; Johnston et aJ., 1983) were too low to produee physiological effecta (Steer et al., 1980). This a.grees with results from Gerkens et al. , (1988) indicating that renal pl'08taglandins are involved in tbe furoaemide­indueed release of an unidentified nonprostanoid hormone from the kidney, whieh producea an endothelium-dependent inhibition of aympathetie vuoconstriction in the in .itu blood-perfused tail artery of the rat.

Few experimental data are available that show a direct; dilatory action offurosemide on i80lated intact blood veseels. In the isolat.ed perfuaed canine lung lobe, furosemide aignif­icantly decreased the mean pulmonary arterial pressure. This eft'ed waa mimieked by pl'08tacyclin and inhibited by indomethacin, whieh suggeats local formation of pl'08tacyclin

A88REVlAl1ON8: BAEC, bovine aortic ~ cella; cycIic GMP, gWlnOSlne cycIic-3',S' monophosphate; HEPES, 4-(2-hydrnxyethyl)-1-pIpef8zIl"IfHJthanesulfonic acId; IBM>:, 3-i8obutyi-1-methyixanthlne; l-NNA, N'l-nitro-L-arglnlne; PG1 2, proatagIandln 12 (prostacycUn); SOD, su­peroxide dlsmutase.

1811

1812

in the presence of furoeemide (Lundergran et al., 1988). In the i801ated rabbit ear artery segments, furoaemide eauaed an inhibition of contraction induced by electric field stimula­tion, which ia endothelium-independent (Tian et ai., 1991).

There ia 00 report of a direct effect of furosemide on the endothelium, wbich ia known to syntheaize and release p0-

tent vasodilatore such as acetycholine, ATP, Bubstance P and kinina (Milner et 01., 1990; Wiemer et aJ., 1994). Kinina, predominanUy bradykinin. set as vasodilators in an au~ crine manner by stimulating endothelial kinin recepton with lIubaequent synthesis and release cf nitrie oxide and proata­cyclin (Schini et 01.,1990; Wiemer et al., 1991). Therefore, we investigated whether cultured endothelial cella from bovine aorta respond to furosemide with an enhanced ayntheais and release of kimns, nitrie oxide and prostacyclin.

Materials and Melhods EDdotbeUaJ ceU culhU'e. BAEC were isolated by digestion with

dUpaae ud were cultured .. previ0\l81y deecribed (Wiemer ud Wirth, 1992). Cella were lleeded on si:r:-well platel (Nunc Intermed, Wiesbaden, Germany), precoated with collagen R) and grown to conßuence. The cultUl'e medium uaed for BAEC waa Dulbeec:o'. mod­ified Eagle'afHam'. F·12 medium (1:1) containin, heat·inactivated fetal calf serum (20%), penicillin (50 lU/mi), .treptomycin (50 ~),

lrllutamine (1 mmoIn), glutathion and L( + ) ucorbie acid (eath 5 I'IImI; Bioteet protection medium). Tbe purity of primlU)' eultured BAEC waa eharact.erit.ed by uptake of ßuoreaeent-labeled low-den· eity lipoprotein (Voyta et cU., 1984) and by negative ataini.ng for Q·.mooth muacle aetin <Abeher et 01., 1989).

M-.uremea.t of 8-ket.opro-t.".ndln F I ... kinhuI aad c:yeUe GMP. PrimlU)' eulturea of BAEC grown to eonßuence in aiJ:·weU plate. (- 200-250 IA-i protein per welll wera U&ed. After removal of the eulture medium by aapiration, the monolaye", were waahed twiee with 2 ml ofwann (37· C) HEPES-Tyrode', solution, pH 7 .• (in mmolll: KCL 2.7, NaCI 137, CaCl2 1.8, MaCI, 2, NaH,PO. 0.36, alUOOH 6, HEPES 10). Thereafter, the cella were preineubated ror 15 min at 37·C with 1 ml HEPES-Tyrode', solution eontaining IBMX (10-· moIn). Then the drvp or 80Iventa and SOD (3 )( 10- 7 moVl) wera added at the eoneenb'atione and time!! indieated in the reeulte. At the appropriate time, the aupematanta (ineubation medium) were quiekly removed and aaaayed for their eontenta of 6-ketoPl'Oltag· landin F la(6-keto PGF1.), the atable breakdown produet ofpl'Oltaey­cl.iD (PGI,), by a apec:ifie radioimmunoaaaay (Ameraham Buehler, Braunaehweig, Germany). A1tematively, the aupematanta (each 1 mll were direetly tranafeJTed into EDTA solution (final eoneentration 10- · moIn) for determination oflrinina by a radioimmunoaaaay (Fink et cU., 1985). The antibody {f'rom Shimamoto} uaed in thia aaaay did not di.Btinguiah amon, the t.hree mammalian kinina bradykinin, IYllyl.bradykinin and methionyl·IYllyl·braciykinin (almoet 10C1'1> O'OB8·reaetivity relative to bradykinin). Other kinin fragmenta auch aa Dea·Arg'-bradykinin, aa wel1 aa bovine low.moleeuJ.ar-weight ki.ni.nogen, ahowed no aignificant O'OB8oraactivity «0.1%-0.02% rel· ative to bradykinin) (Rönner d cU., 1987; Sbimamoto et cU., 1978). For determination of intracel1u1ar eyclie GMP, the cella weR imme­diatelyextraeted with 0.8 ml of 1 N formie acid/acetone (viv, 15:86) and seraped off with a rubber ac:raper. Tbe cell auspen8ione were tben sonieated for 10 see berore being centrifuged for 10 min at 1000 )( 8. The aupematanta wera lyophilit.ed and reauspended in 0.26 ml aodium acetate buffer (0.05 molll, pR 6 .2) for determination of eydie GMP by a apecifie radioimmunOl.MlY (DuPont NEN, Bad Homburg, Germany). The protein eontenta of the aamplea were meaaured ae­eordin( to the method ofLowJy et cU., (195I>. Cyclie GMP eontent waa upreued aa pioomolea per milligram protein, that of 6-lr.eto PGF I. aa nanograma per milligram protein and that of kinina aa pieomolee per milliliter aupematant.

Val. 271

The valuea wera expresaed ll8 the mean :!: S.E.M. StatiatieaJ. compariaolll were made uaina: Dunnett'. test. Valuee ofP < .06 ware conaidered atatiatiea11y aignil'icant.

Drup. son (bovine erythroeytea; apecifie aetivity, 3300 U/mg) and eol1.n R were p\lJ"Cbued f'rom Serva (Heidelberg, Germany) end DiI·,Ac·LDL f'rom Paeeel + Lorei (Franlr.furtJMain, GermIny). Furoaemide aodium aalt and ieatibant (Hoa 140) were obtained f'rom our department ofpharmaayntheeia. All other eompounda were pur­ehaaed f'rom Sigma Chemie (Deiaenhofen, Germany).

Reoul1s E1fect ot furoaemide OD cyelic GMP aad 1daia pro­

ductioD in BAEC. In primary eultured BAEC, furoaemide induced time- and concentration-dependent increaaeB in in· tracellular eyclie GMP content. Muima..! stimulation of cyclie GMP ayntheaia was reaehed by about 6 min and remained stable for at leut 30 m.in of eontinuoua expoaure of the cells to furosemide (3 x 10- 1 and 10- 6 molll) (fig. 1, A and B). Preincubation (5 min) of the cells with the etereospeci.6e inhibitor of nime oxide synthase L-NNA (10- 6 moVl) totally suppresaed the eff'ect of furosemide without affecting un­stimulated cells (fig. 1 B). An inhibition of the furosemide-­induced cyclie GMP formation was also obaerved in monolay­era that were preincubated (5 mini with 10- 7 molll of the specifie B2 kinin receptor antagoniat icatibant (Hock et 01., 1991) wbereas basal cycüe GMP content was not inßuenced by icatibant (fig. 1 B). Tbe concentration-response relation of

• A

I 1 .. • • , T/-----~.--! • •

5 • ~~ • , .. • , • 0 0-0-0- -0-• 0 0

Q ~ , • " " ,.

11m. (mln)

• B < • , • • • 5 • , .. , • • o •

" ' 0-0'-----0

QL-__ ~ __ ~ __________ _

• • " tlma (mln)

Ag. 1. A) Basal (0)- and furoaemide (3 X 10- 7 moVr}-induced (e) accumulation of cGMP In primary cultured BAEC as a function of time. B) lcatlbant (10- 7 moII1;. and l-NNA (10-& moVI; 6) wen! added 5 mIn before the addItion of furosemIde (10- & moVI). Eact1 vaIue repreeenta the rnean :!: $ .E.M. of sill: dlshes performed on two d~ cell batehes.

1994

furosemide ie shown in figure 2. After a lO-min incubation, maximal increases in cyclic GMP were obtained byabout 3 X 10 7 to 10- 8 molll offurosemide with a threshold concentra­bon of about 3 x lO- e molll. Preincubation of the ceils with either L-NNA (10-6 molll) or icatibant (10- 7 molll) totally prevented tbe furosemide-induced mcrease8 in cyclic GMP. In compari80D, exogenously added bradykinin (whieh we al­ways used as a "standard'" for the reepective cell batches) led. at an optimal concentration of 10- 8 moV! after 1 min ineu­bation (Wiemer et al" 1991), to a little lower content of cyclic GMP (3.70 :t 0.61 pmollmg protein V8. motrol incubation 0.61 :!: 0.12 pmollmg protein; n = 11) than did the optimal efl'ective concentrations offuroeemide (3 X 10-'_10- 8 mol/l).

In parallel to the concentration-dependent increa.se in in­tracellular cyclic GMP by furosemide, the compound en­hanced the concentration ofkinine in the 8888Y media (fig. 3). Already at 3 x 10- 8 molll of furoaemide (after a lo.min incubation), an approximately 8.S-fold higher concentration of kinin.s (-60 pmoVm1 8upernatant) /J8. control incubation was obtained. Cyelie GMP production occurring at th.is con­centration of kinins in the asaay media ia approximately comparable with changes in cyclie GMP and PGI2 induced by bradykinin UO- a molll).

Effect of turo.emide on proAaeyclin <PG"') aynthe­Iris in BAEC. The time course ofthe furoeemide (3 x 10- 7

molll)..sti.mulated release of PGl.J: is depicted in figure 4. A plateau that remained stable for at leaat 30 Drin waa reached after a 6-Drin incubation. The concentration-reaponse rela­tion of furosemide revealed marimal increases in PGI2 re­lease at about 3 x 10-7 molll, with a threshold cencentration ofabout 3 X 10- 9 molll (fig. 5). Preincubation with icatibant UO- 7 molll) significantly inhibited trus effect without affect­ing the baaal release of PGl.J:. Thua the time- and cencentra­tion-dependent eft'ect of furoeemide on PGI2 release was quite parallel to that observed for cyclie GMP accumulation (see figs. 1 and 2). In compariaon, bradykinin at a cencentra­tion of 10- s molll (l-min incubation) cauaed the release of an

7

o • ~ o E 3 ~ .. 2

" 'il

• .. . o \0" 10" 10" 10"

,

10.1 10'"

fUfosemlde (molfl)

Ag. 2. Effect of fumsemide (e) on the accumuIatIon of Intracellular cGMP In primary cuttured BAEC as • function of COf'ICeI .batiorl (1O-m1n Iocubatlon). lcatlbant (10·' moVI; IIJ and l-NNA (10- 5 moIII; 6) W9f8

added 5 min before the additIon of the respectIve concentratlons of turosemlde. Reaults are axpressed as the rnean :!: S.E.M. ExperIments (n - 7-11, performed on sIx dI1'fera'rt cell batches) were done on tripllcated dishe8. - P < .05 vs. controI (abscissa. zero point).

1613

8 T " ? • 7 T T '\T

70 , , e 8 80 ~

0 5 50 ~

E

• -I 40 " • • e c

" , v· '0 e .. • "

, 2. ? 0 • 0 , 10

• 0 -I' , , 0 0 10< 10" ,..

luro.amlde (moi/l)

Ag. 3. RelatIonship between the accumulation 01 Intracellular cGMP Oeft ordinate) and that 01 kinins (fight ordinate) in the supematant of primary eultured BAEC Incubatecl wlth furosemlde (10-mln Incubation). cGMP Md kinin eoncentratlons wenII determlned concomltantty In the same dish. Each valoe represents the mean :!: S.E.M. of 91)( dishes performed on two different cell batches.

1400 c T T • 1200

J.-...........~.-. e ~ 1000 0

.!;: 0 800

I -1 600 ~

0 400 • ..

• 0_0_0 __ 0_0 0 • 200 ~ ~

0 , 1 , 5 10 15 30

IIme (mln)

FIQ.4. Basal (0) and furosemlde (3 x 10- T molll)-induced (e ) accumu­lalion 01 6·keto PGF1a In the supema1ant 01 primary eultured BAEC aa a lunctlon 01 time. Each value represents the mean :!: S.E.M. of sIx dishes performed on two different cell batehes.

approximately sirnilar &mount of PGI2 (752 :: 70 nglmg pro­tein V8. centrol incubation 292 :: 30 nglmg protein; n = 7).

Discusslon This study has shown that furoaemide stimulates the for·

mation of cyclie GMP and PGI2 in primary cultured BAEe. Similar findings to those with BAEC were obtained with primary cultured endothelial cella from cardiae microveaaels of the rat (unpubliahed data). An enhanced aynthesis of en­dothelial cyelie GMP had been weil documented as an index of endothelial Ditrie oxide syntheais and release in response to bradykinin, ATP, ADP and calcium ionophore (Boulanger et aJ., 1990; Martin et 01., 1988; Schini et 01., 1990). In theae inveatigationa it was ahown that the stimu1ated increase in endothelia1 eyclie GMP was inhibited by methylene blue, an inhibitor ofthe activation of soluble guanylyl cyclase and by hemoglobin, a scavenger of Ditrie oxide. Increasea in endo­thelial cyelic GMP generation by stimu1ation of a soluble guanylyl cyclase through Ditrie oxide were also demonatrated by the inhibitory effect of the atereoapecific inhibitor of Ditrie oxide synthase vNNA (Mülach and Busae 1990) on bradyk,i-

1814

c

li • ~ 0

~ 0

E-· ~

~ ~

• • ~ ~

WlMneret ...

1200

'000

800

600

<00

200

0 I'

01~1 1~ 10" 10~ 10~ 10~

lurosemld. (mol/l)

Ag. 5. Effect cf furosemide tel on the accumolatlon of 6·kelo PGF, .. 1n the supematant 01 primary cuhured SAfe as a functlon of concentra· tion (10-mln incubation). lcatibant (10" maUl; ., was added 5 mIn before the addition 01 the respec1ive conceotrations of furosemide. Resutts 8(8 expressed as the mean :!: S.E.M, Experiments (n " 7-11, pecfooned on six different cell batche$) -. dolle on triplica1ed dishes. • P < .05 vs, controI (abscissa zero point).

nin-induced cyclic GMP generation (Duhbin et al., 1990; Hecker et al., 1993; Wiemer et ol" 1991; Wiemer and Wirth, 1992). Thus the obaerved prevention of the furosemide-in­duced increase in endothelial cyclic GMP indicates an eo­hanced generation of nitric oxide by thi8 compound.

The increase in cyeUc GMP by furoaemide ia associated with an augmented release of endothelial kinins. This is support.ed by the observation that preincuhation of the cella with the specific 8 2 k.ini.n receptor antagonist icatihant aOOI­isbed the furoaemide·induced increase in nitric oride synthe­eis aB weil as the release of PGI2• It also indicates Ule 000' tribution of endogenous bradykinin in this response. The stight dissociation of tbe concentration-response relation of furoaemide on eyelic GMP and kinins U1g. 3) eannot be sat.­isfactorily explained. 1t may be tbat furosemide at higher concentrations (>3 X 10- 8 mol/l) leads to an enhanced gen­eration and release of kinins (e.g., De8-~-bradykinin or other kinin fragments) tbat are not detected by tbe radioim­munoassay used (see "Materials and Methods") but are prob­ably due in part to the production of cyelie GMP. This as­sumption is supported by the finding that in eultured BAEC. an increase in eyelie GMP could be indueed to the same extent by eitber bradykinin or Des-&-g9-bradykinin and, fur­thennore, that the effect of both compounds was prevented through icatibant (Wiemer and Wirth, 1992). Thua furo.. semide seems to cause an enhanced synthesis and release of endothelium-derived kinins. whieh in turn triggers the for­mation ofthe vasodilators nitrie ozide and PGI2 via enhance­ment of cytosolie calcium (Lüekhoff et al .• 1988).

About the mechanism of action leading to an enhanced synthesis and release of kinins by furosemide, one can spec­ulate that the compound decreases intracellular pH in endo­tbelial cells. In peripheral (Wilkens et al., 1970) and card.iae blood vessels (Noda et al., 1993) under acid conditions, an enhanced release ofkinins was observed to occur presumably by the activ:ity of an acid optimum protease (M08hi et al., 1992; Zeitlin et 0/., 1989), which cleaves kinins from kinino­gens. The presence of kininogens in endothelial cella was shown by van Iwaarden et oi. (1988).

Maximal endothelial synthesi.s ofkinins. as weil as of cyelie

Val. 271

GMP and PGI!!, was rea.ched within a 5-min incubation ofthe cells with furosemide at eoncentrations of 3 X 10- 7 to 10- 8

mol/l, which were also obaerved in human plasma within several minutes after oral administration of 40 mg of furo.. semide (Rakhit et 0/., 1987). In contrast, such concentrations of furosemide are insufficient to produce a significant diure­sis. However, in the kidney, circulating plasma concentra­tions of furosemide become concentrated in the loop of the Henle (at least to 10- 6 molll) because of an active secretion of furosemide into the tubular lumen and subsequent fluid re... absorption (Gutsche et 01., 1982). Thus the initial vasodila­tory effect offurosemide, which a1ready occurs in tbe absence of extracellular fluid volume 1088 by diureais, seems to be in part mediated locally by the stimulation of endothelial Ditrie oxide and PGI2 production in resistance and/or capacitance veesels.

The elevation of endothelial nime oxide and POl!! (produc­tion) by furosemide migbt bave further relevant biological implications. It bas been shown that both autacoids can inhibit platelet aggregation and adhesion (Thiemermann, 1991). In agreement with tbis bypothesis. an inhibition of ADP-induced platelet aggregation by furosemide bas been docum.ented in the human (Kribben d ai .• 1988). Besides these effects, the elevation of endothelial Ditrie oxide and PGI:!; production by furosemide might help to prevent the initiation of atherosclerosis. Nitrie oxide prevents the adhe­sion of .monocytes to the endothelium. whereas POl:!; inhibits their chemotactie responae (Bath d al., 1991). Additionally. antiatherosclerotie effects ofDitrie oxide and POI2 may result from tbeir respective cyclie nucleotide second-messenger sys­tems, which may inhibit smootb muscle mitogenesis and proliferation (Garg and Hasaid, 1989; Thiemennann, 1991).

In coneluaion, our findinga suggeet that the aeute hemody­namie effects of furosemide are mediated directly by an en­banced synthesis and release of endothelium-derived kinins. wbicb aet as a potent stimulus for tbe endotbelial formation of nitrie oxide and PGI!!. Whetber tbe direct effect of furo­semide on endothelial cells is significant for the treatment of cardiovascular diseases remains 10 be detennined.

kknowledp:leau

We thank. Anke Hullmann and Marlon Jorge for theiT exoellent technical aasiatance.

.... ~ AIsIWl, M., Wooooocx-Mrrcmu., J ., MITCHW., J., 8.w:JoR. L., 1.0 ...... , R .um

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BIAlllHO, G., W&1IHL, H. J., SoriiuK, K. P., lWmoHa, B., NÖllINO, J . .um Sootöou, R : HImod.}'Il&IDi8cbe Auawirkun&en von ~ i. v . • Aue­drudt einee VUIOdilatoriacben Mechaaiemue. Z. Kardiol. a: 61, 1974.

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Bouu./'IoQ, C., Sc/lUll, V. B., MONCUlA, S. AM) V A1fflOl.ITft, P. M.: StimubotioD of cyclic GMP produeüon in tultured eadotbeüal celll of the pir by bradykinm, edenoÄ!lt! dipboephate, wo ...... iOl"lopbo .... A23187 and Ditrit: ozide. Br. J. ~1. 101:162-166, 1990.

8ooa1.vfo, W. A., DAY, D. K..um WlWAX9OH, H. E.: 1'be role ofthe kidDey in the early nondilll"ltic aetion of fwoMmide to reduce elevated Iett. arterial

preMIIN ifI tbe ~ q . J . I'harm.mI. Exp. 'I'bu. JOt: 221-229. 1977.

I>otaoT. K.. "VTDDI. J . K.. Fousnu, J. S., ClLl.TftIUD, K.. Pa.t.nsH, R. A1fO

SwAK. H. J . C.: Renal utdatrveGaI ~etrect. offuroMmide in COJIP'tive heart failllN after acuta ~ Wardion. N. Enci. J. Weil. ... 1087_1090. 1973.

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