p. 1 

# OAR Box 1131 Prepped by Ollie Stewart



## Document

Number: 

# 127 ) II-I-40

5 Docket Number: 

# A-83-2 2

Printed 4/21/2008 2:34:12 PM

Sheet 127 of 162, > 

### r* P O ^CJ^JT

- 7 - P. 2 

# (JoS Environmental

Health Perspectives Vol. 78, pp. 113-115,1988 

# Effect s o f Lea d an d Natriureti c Hormon e on Kinetic s o

f Sodium-Potassium Activate d Adenosin e Triphosphatase : Possibl e Relevanc e t o Hypertensio n b y Elma r Weiler, * Farha d Khalil-Manesh, * an d Harve y Gonick ^

Inhibitio n o f

vascula r smoot h muscl e sodium-potassium-activiate d adenosin e triphosphatas e (Na K-ATPase ) ha s bee n postulate d a s a centra l mechanis m i n enhancin g vascula r contractility . I n th e presen tstudy , kinetic s o f inhibitio n o f

Na-K-ATPas e by lead , ouabain , an d natriureti c hormon e (NH ) wa s studie d i n a purifie d ho g cerebra l corte x enzym e preparation . Determinatio n o f I 5 0 value s fo r lead , ouabain , an d NH reveale d tha tN H i s th e mos tpoten tinhibito r o f th e enzym e syste m (0. 8 x IO" 6 M ouabai n equivalents) . Kineti c analyse s indicate d tha tlea d an d NH exhibite d differen t inhibitor y mechanisms . Th e inhibitio n b y lea d wa s noncompetitiv e wit h respec tt o potassiu m an d competitiv e wit h respec tt o sodiu m an d MgATP. Natriureti c hormon e wa s noncompetitiv e wit h respec tt o potassium , uncompetitiv e wit h respec tt o MgATP

, an d exhibite d n o inhibitory effectwit h respec tt o sodium . Synergis m betwee n lea d an d N H i n th e inhibitio n o f Na-K-ATPas e raise s th e possibilit y tha tlea d may b e a contributory facto r i n hypertensio n vi a thi s mechanism . 

## Introductio n Epidemiologica l studie s performe dbot h i n th e Unite dState s (1) an di n Grea t Britai n (2) hav e sug geste dacorrelatio n betwee n bloo dpressur e an dbloo d lea dwithi n th e rang e o

f bloo dlea dlevel s see n in th e normal population . Th e prevalenc e o f hypertensio n in industriall y expose dworker s ha s bee n amatte r o f controversy . Studie s performe db y Crame r an d Dahlber g i

n 196 6 (3) showe dn o differenc e in th e incidenc e o

f hypertensio n i n worker s directl y expose d t o leada s compare dt o age-matche dworker s in th e sam e plan t wh o ha dminima l lea dexposure . Mor e recen t studie s b y d e Kor t

e t al . (4) an dKirb y e t

al . (5 ) demonstrate dapositiv e relationshi p betwee n bloo d lea dan dbloo dpressur e i n lead-expose dpopulations . Thus , th e issu e ha s bee n raise dtha t lea dma y b e apos sibl e causativ e o r

contributor y facto r

in hypertension . Lead , lik e man y othe r

heav y metal s (vanadium , •Trac e Element

Laboratory , Division o

f Nephrology/Departmen t

o f Medicine , Cedars-Sina i Medica l Cente r an dSchoo l o

f Medicine , Universit y o f California , Lo s Angeles , C A 90048 . tAutho r t o who mreprin t

request s shoul db e addressed . mercury , cadmium , uranium ) (6-9) i s apoten t in

hibito r o f th e membran e transpor t enzym e sodium

potassium-activate dadenosin e triphosphatas e (Na-K

ATPase , E . C . 3.6.1.3.) . Thi s effect o f lead , i f exerte do n vascula r smoot h muscle , migh t produc e enhance d vascular contractility . Withi n th e pas t fe w years , ther e ha s bee n considerabl e interes t i n th e possibility tha t

a circulatin g transpor t

enzym e inhibito r

[th e putativ e hypothalamic-rena l natriureti c hormon e (NH) ] migh t b e amajo r

caus e o f essentia l hypertensio n vi a th e sam e mechanis m(10-12). A hypothesi s advance d b y Blaustei n e t

al . (11) i s tha t

inhibitio n o

f th e vas cula r smoot h muscl e sodiu mpump by . N H lead s t o increase dintracellula r

calciu mconcentrations , thu s enhancin g vascula r

responsivenes s t o th e vasocon strictiv e hormone s norepinephrin e an dangiotensi n II . I f lea dan dN

H coul db e show n t o inhibi t Na-K

ATPas e b y different

mechanisms , the n th e possibility exist s tha t

lea dmigh t

ac t t o increas e vascula r

con tractilit y b y synergizin g with N H i n inhibitin g th e vascula r smoot h muscl e sodiu mpump . Th e goal s o f th e presen t

stud y wer e first

t o contras t

th e kinetic s o

f inhibitio n o

f highl y purifie dNa-K-ATPas e b y lea d 

# JP. 3

11 4 WEILER, KHAULMANESH, AND GONICK

an dasemi-purifie dplasm aNa-K-ATPas e inhibitor , (NH) , with th e know n specifi c inhibito r o f Na-K

ATPase , ouabain , an dsecon dt o explor e th e possibility o f synergisti c inhibitio n o f th e enzym e b y lea dan d NH . 

## Method s Purificatio n o

f Natriureti c Hormon e fromHuma n Plasm a Separatio n o

f lo w molecular

weigh t N H fro mhig h molecula r weigh t materia l wa s achieve db y passin g th e plasm asampl e fro mahypertensiv e patien t throug h a

n Amico n Y

M 2 ( 1 kilodalto n cutoff

) mem bran e at 20 psi . Fo r

furthe r purificatio n o

f th e lo w molecula r

weigh t material , th e filtrat e wa s passe d throug h aSEP-PA K

cartridg e an delute dwit h acetonitril e (ACN

) an d0.1 % trifluoroaceti c aci d(TFA) a s describe di

n a n earlie r publicatio n (13). Th e fractio n elutin g betwee n 0

% AC N an d20 % AC N wa s lyophilize d an dstore dfo r

kineti c studies . Concentratio n o f inhibito r wa s expresse da s ouabain-equivalent s (13). Lea dacetat e use dfo

r th e kineti c studie s wa s purchase d fro mFishe r

Scientifi c (Fai r

Lawn , NJ) . 

## Na-K-ATPas e Inhibitio n Assa y I n thes e experiment s apurifie dNa-K-ATPas e fro m ho g cerebra l corte x wa s employed , purchase dfro m Sigm aChemica l (St . Louis , MO) . Th e incubatio n tube s containe dasubstrat e solutio n providin g final concentration s o

f 1 m M ATP ; 1 m

M Mg ; 1 0 m

M imidazole-HC l buffer , p H 7.2 ; 100 m M Na ; 2 0 m

M K ; an d1 m M ethylen e glycol-bis-((3-aminoethy l ether ) NyN' tetraaceti c aci d(EGTA) . Th e tube s wer e incubate d a t 37° C for 5 min . Th e reactio n wa s starte db y addin g 0.02 5 m L o f enzym e preparatio n (2 5 mg/mL) . Th e incubatio n wa s stoppe dafter 1 0 mi n b y addin g ice-col d trichloraceti c aci d(TCA) . Afte r centriiugatio n (HOOgx 5 min) , 0. 5 mL o

f supernatan t

wa s assaye dfo r inorgani c phosphat e b y th e metho do f Fisk e an d Subbaro w (14). I n al l instances , enzym e inhibitio n wa s expresse da s jimol e Pi/m g protein/hr . Fo r determinatio n of

I 50 , i.e. , th e amoun t o f inhibito r necessar y for

50 % enzym e inhibition , seria l dilution s o

f th e purifie dlo w molecular weigh t plasm a N H wer e prepared . Fo r

comparison , ouabai n an dlea d wer e dissolve di n wate r t o resul t

i n final concen tration s rangin g from4 x IO" 8 M t o 4 x 10" 3 M ouabai n an d5 x IO- 5 M t o 5 x 10 ^ M Pb . T o furthe r

characteriz e th e inhibitor y activitie s o f lead , ouabain , an dNH , kineti c studie s wer e performe d wit h concentration s o

f potassiu mrangin g fro m0.12 5 m M t o 5. 0 mM , sodiu mrangin g fro m5 m M t o 12 0 mM , an dMgAT P rangin g fro m0.2 5 m M t o 3. 0 mM , in th e presenc e o

f constan t amount s o

f lead , ouabain , an dNH . Fo r

th e MgATP studies , th e rati o betwee n M g an dATP molar

concentration s wa s maintaine da t 1:1 . Michaelis-Mente n parameter s wer e calculate dfromth e conventiona l Lineweaver-Bur k plot . 

## Synerg y I n orde r t o determin e whethe r

lea dan d N H were synergisti c inhibitor s o

f Na-K-ATPase , alea ddose

respons e curv e wa s first established . Th e combine d effect s o f lea dan dN

H wer e assaye di n th e presenc e o f tw o different

dose s o f NH . Th e lo w dos e o

f N H produce d 2 % inhibitio n o

f th e enzym e system , wherea s th e hig h dos e yielde d12

% inhibition . 

## Result s Lead , ouabain , an dN

H wer e foun dt o b e poten t inhibitor s o

f th e Na-K-ATPas e enzym e system . Th e I5 0 value s fo r eac h substanc e ar e show n i n Tabl e 1 . 

## Kineti c Studie s Potassium. Th e effect s o f varyin g concentration s o f potassiu mo n Na-K-ATPas e activit y i n th e presence o f lea d( 5 x 10- 5 M) , ouabai n ( 4 x 10" 9 M) , an dN

H (6. 9 x IO" 9 M ) ar e show n i

n Figur e 1 . Th e inhibitio n o f Na

K-ATPas e b y ouabai n wa s foun dt o b e competitiv e wit h respec t t o potassium , wherea s th e inhibitory effect s o f N H an dlea dwer e foun dt o b e noncompetitive . Sodium. Wit

h sodiu ma s th e variabl e sustrate , th e kineti c analysi s reveale dtha t lea d(2. 5 x IO - 5 M ) i s a competitiv e inhibito r

(Fig . 2) , wherea s N

H exhibite d n o inhibitory effect . 

### Magnesium Adenosine Triphosphate (MgATP). Wit

h MgAT P a s th e variabl e substrate , ki

neti c analysi s reveale dtha t lea di s acompetitiv e in

hibitor ; N

H (1. 3 x IO -7 ) exhibite duncompetitiv e inhi

bitio n (Fig . 3) . Th e K m an dV

mix value s ar e liste d in Table s 2 and3 . 

## Synerg y Figur e 4 display s th e dose-relate dinhibitio n o f Na

K-ATPas e i

n th e presenc e o

f increasin g lea dconcen tration s rangin g fro m0. 1 x 10- " M t o 5 x 10" 4 M . In the presenc e o

f lo w dos e NH , th e inhibitio n curv e i s shifte d t o th e left , i.e. , inhibitio n i s increased . In th e presenc e o f hig h dos e NH , afurthe r leftwar dshif

t o f th e Na-K

ATPas e inhibitio n curv e wa s observed . I n bot h in

stances , th e incremen t

o f inhibitio n produce d b

y add

Tabl e I . Determinatio n o f I 5 0 value s fo r inhibitor s o f Na-K-ATPase . Substanc e I(so.M ' Ouabai n Lea d N H 2X10- 6 80x10- * 0. 8 xlO- 6 a Th e substrat e concentration s fo r leadan douabai n ar e expresse da M , wherea s th e concentratio n fo r N H i s expresse da s M ouaba ' equivalent .

# I F ""V r f w "" r ' w ^iW 1 f""°'r "*-"

" • P. 4

LEAD, NATRIURETIC

HORMONE, AND NA-K-ATPASE 11 5 

### : n 

### g N H wa s mor e tha n woul db e predicte db

y asimple additio n effect.

2. 4 -

2. 0 •

1. 6 -

1 / V 1. 2 •

0. 8 •

0. 4 f la

/ / • Enzym e T Ouabai n o N H P b

8. 0 FIGUR E 1 . Lineweaver-Bur

k plot o f kinetic s o f inhibitio n o

f Na-K-ATPas e b y lea d(5 x IO" 5 M) , ouabai n (

4 x IO" 9 M) , andN

H (0.69 x 10-

9 M ouabai n equivalents ) wit

h potassiu ma s th e variabl e substrate . 

## Discussio n Th e primar

y functio n o f th e membrane-boundNa

K-ATPas e enzym e systemi s th e active transpor t o f sodiu man dpotassiu m(15,16). Thi s enzym

e syste mi s als o involve

di n salt an dwate r homeostasi s (17), th e transpor t o f nonelectrolyte s (18), an

dth e secretio n o

f potassiuman

dhydrogen b

y kidne y tubule s (18). Th e activit y o f th e Na-K-ATPas

e enzym e syste mi s affecte

d b y avariet y o f hormones , includin g mineralo

- and glucocorticoid s (19,20), thyroi dhormon e (21), insulin (22,23), catecholamine s (24), an dN H (25,26), a s wel l a s trac e metal s [e.g. , lead , vanadium , mercury , uranium , cadmium , silve

r (8,9,27)]. Thi s interactio n

c P b o N H • Enzym e

2. 0 3. 0 1/[MgATP ] FIGUR E 3 . Lineweaver-Bur

k plot o f kinetic s o f inhibitio n o

f Na-K-ATPase by lea d(5 x IO - 5 M

) andN H (1. 3 x IO - 7 M ouabai n equivalents ) wit h MgAT P a s th e variabl e substrate . 1. 4 - 1. 2 - 1. 0 - 1 / V 0. 8 • 0. 6 • 0. 4 - 0. 2 - /

*s"* / i jS"^* m

• Enzym e o p b

-0. 1 0. 1 0. 4 0. 5 0. 2 0. 3 l/(Na ] FIGUR E 2 . Lineweaver-Bur

k plot o f kinetic s o f inhibitio n o

f Na-K-ATPase by lea

d(25 x IO - 6 M

) wit h sodiu ma s th e variabl e substrate .P. 5

11 6 WEILER, KHAUL-MANESH, AND GONICK' Tabl e 2 Substrat e Enzym e Lea d N H Ouabain Tabl e 3 . Substrat e Enzym e Lea d N H Ouabai n * m

^ma > value s fo r inhibitor s o f Na-K-ATPase . K 0.4 8 0.4 4 1.6 1 0.5 0 tf

m ,mM Na 34. 9 96. 8 MgAT

P 2.69 6.6 4

value s fo r inhibitor s o f

Na-K-ATPase . v r ma x K 4.6 5 3.4 8 2.3 0 4.7 7 limol e Pi/m g protein/h r

Na 17.54 17.3 0 MgAT

P 20.1 6 18.4 2 10.1 3

10 0

- 80 - 60 - o P b + N H hig h dos e • P b + N H lo

wdos e cP b

0. 3 0. 5 0. 7 0. 9 1. 3 5. 0 x 1 0 M

P b concentratio n FIGUR E 4 . Inhibitio n o

f Na-K-ATPas e by lea di

n th e presenc e an d absenc e o f lo w dos e andhig h dos e NH . Th e inhibitor y effec t o f lo w dos e N H alon e wa s 2% ; th e inhibitor y effec t o f hig h dos e N

H alon e wa s 12 % (plotte dadjacen

t to y-axis) . o f metal s wit

h Na-K-ATPas e ma

y hav e physiologica l a s wel l a s toxicological significance . Na-K-ATPas e ha s bee n classifie da s asulfhydry l (SH) enzyme , i n par t

becaus e o

f it s inhibitio n b y mercu rial s (28). Lead , a s ameta l tha t react s wit

h S H groups , woul dals o b e predicte dt o ac t a s aNa-K-ATPas e in

hibitor . Krame r

e t al , (9) foun dtha t

in rena l cortica l tissu e homogenates , Na-K-ATPas e wa s inhibite db y 50 % a t alea dconcentratio n o f 7 x IO" 5 M . Simila r result s wer e reporte db y Necha y an dSaunder s (7). Thes e investigator s showe dtha t th e concentratio n for 50 % inhibitio n o

f Na-K-ATPas e range dfro m6 t o 8 x IO- 5 M in bot h microsome s an dtissu e homogenates . Th e homogenat e di dno t contai n an y protectiv e sub

stance s tha t

ameliorate dNa-K-ATPas e inhibition , a s ha dbee n reporte dfor

mercur y (8). Both studie s demonstrate dtha t

lea dcause dareversibl e inhibitio n o f th e enzym e system . Ethylenediaminetetraaceti c aci d (EDTA ) prevent s o r

reverse s lead-induce dinhibitio n o f Na-K-ATPas e (7). Krame r e t al . (9) demonstrate d tha t lea di s acompetitiv e inhibito r wit h respec t t o it s substrat e ATP . Fro mearlie r studie s b y Tic e (29), i t woul dappear tha t lea dcompete s with magnesiu mfo r chelatio n b y ATP . Leadi s als o know n t o inhibi t Na-K

ATPas e at

it s sodium-sensitiv e sit e (7). In th e presen t study , whic h employe dapurifie dho g cerebra l corte x Na-K-ATPas e enzym e preparation , w e als o foun d tha t 50 % inhibitio n o

f th e Na-K-ATPas e enzym e syste mwa s achieve da t

alea dconcentratio n o

f 8 x Iff

5 M . Th e inhibitio n o

f Na-K-ATPas e b y lea dwa s foun dt o b e noncompetitiv e wit

h respec t t o potassium , bu t

com petitiv e wit

h respec t t o sodiu man dMgATP . Endogenou s inhibitor s o f Na-K-ATPas e hav e bee n isolate dfro mbot h plasm aan durin e b y differin g methodologies . Thus , th e N H studie db y variou s lab

oratorie s may consis t

o f simila r o r differen t

sub

stances . Krame r

e t al . (30), studyin g asemi-purifie d urinar y inhibitor , describe dth e N H inhibitio n o f Na

K-ATPas e a s noncompetitiv e wit h respec t t o potassium . Crabo s e t al . (31) reporte dtha t

NH , als o isolate dfrom urine , inhibite dNa-K-ATPas e noncompetitivel y wit h respec t t o ATP . Inhibitio n o

f th e enzym e syste mwa s facilitate db y sodium . Ouabain , adigitali s glycosid e an daspecifi c inhibito r

o f th e Na-K-ATPas e system , ha s bee n show n t o b e competitiv e wit h respec t t o potassiu m (32) an dnoncompetitiv e wit h respec t t o AT P (31). I

n th e presen t

study , w e employe dasemi-purifie d N H preparatio n derive dfro mplasm ao f apatien t

wit h essentia l hypertensio n t o performkineti c studies . Thi s N H inhibit s Na-K-ATPas e in anoncompetitiv e man ne r with respec t

t o potassiu man duncompetitivel y wit h respec t t o magnesium . Th e semi-purifie dmate rial exerte dn o effect

o n th e sodiu msit e o

f th e enzym

e system . Thes e result s clearl y demonstrat e tha t N H an d lea ddiffe r fro meac h othe r

an dals o fro mouabai n i n thei r Na-K-ATPas e inhibitor y characteristics . A t present , th e rol e o

f lea da s acontributor y factor i n hypertensio n remain s speculative ; however , ther e ar e in vitro dat atha t

suppor t th e possibility tha t

lea d enhance s vascular

reactivity . Web b e t

al . (33) foun dtha

t tai l arterie s fromhypertensiv e mal e rat s expose dt o lea d at ados e o f 10 0 pp mshowe da

n increas e in maxima

l contractil e forc e whe n teste dwit h th e a-adrenergi c agents , norephinephrin e an dmethoxamine . Thi s findin g wa s attribute dt o a n increas e i n th e intra

cellula r poo l o f activato r

calciu mi n th e vascula r smooth muscl e cell . Piccin i e t al . (34) an dFavalli e t

al . (35) foun dtha t

calciu mwa s necessar y i n th e perfusio n solutio n for

lea dt o exer t

an effect o n vascula

r contractilit y o

f th e isolate dra t

tai l artery . Further

more , tissu e calciu mwa s increase din th e presenc e o f lead , a n effect tha t coul db e explaine db y th e inhibitor y effect o f lea don th e vascular

smooth muscl e membrane Na-K-ATPas e system , a s predicte db y th e Blaustein hypothesi s (11). A s lea dari d N H hav e asynergisti c effect on thi s enzym e system , on e possibl e actio n o f lea d ma y b e t o potentiat e vascula r reactivity in geneticall

y susceptibl e individuals . Suc h ahypothesi s l S supporte db y observation s o f Cloi x e t al . (36) tha t

som e normotensiv e individual s with afamil y histor y ° hypertensio n hav e elevate dlevel s o f NH . A s hypertensio n i s amultifactoria l comple x di s 

# ALEAD, NATRIURETIC HORMONE, AND NA-K-ATPASE 11 7 

### order , i t i s unlikel y tha t asingl e hormone o

r toxi n ca n accoun t fo r th e entire pathogenesis . A mor e plausibl e outcom e i s tha t man y substance s wil

l b e foun dt o b e contributor y factor s an

dth e interpla

y between thes e substance s wil

l determin e whether

agive n individua

l develops asustaine dincreas e i n bloodpressure . Th e author s than k

Rub y McCart y an dBeverl y Gil for thei r se cretaria l assistance . Thi s stud y wa s supporte db y Endocrin e Science s Laboratories , th e Louis e an dGustavu s Pfeiffe r Foundation , a n d th e International Lea

dZin c Researc h Organization . REFERENCE S 1 . Pirkle , J . L. , Schwartz , J. , Landis , J . R. , an dHarlan , W . R . Th e rela

tionship betwee n bloo dleadan dbloo dpressur e an dit s cardio

vascula r ris k implications . Am . J . Epidemiol . 121 : 246-25 8 (1985) . 2. Pocock , S . J. , Schwartz , A . G. , Ashby , D. , Delves , T. , Whitehead , T P . Bloodlea

dconcentration , bloo dpressure , an drenal function . Br . Med . J . 289 : 872-87 4 (1984) . 3 . Cramer , K. , an dDahlberg , L . Incidenc e o f hypertensio n amon g lea dworkers : A follow-u

p study base do n regular

contro l ove

r 20 years . Br . J . Ind . Med . 23:101-10 4 (1966) . 4 . d e Kort , W . L , Verschoor , M . A. , Wibowo , A . A , va n Hemmen , J . J . Occupationa

l exposur e to leadandbloo dpressure . A study o f 10 5 workers . Am . J . Ind . Med . 11:145-15 6 (1987) . 5 . Kirby , H. , an dGyntelberg , F . Bloo dpressur e an dothe r cardio

vascula r ris k factor s o f long-ter mexposur e to lead . Scand . J . Wor k Environ . Healt

h 11:15-1 9 (1985) . 6 . Nechay , B . R. , an dSaunders , P . J . Inhibitio n b y vanadiu mo f sodium

- an dpotassium-dependen t adenosinetriphosphatas e derive dfro manima l an dhuma n tissues . J . Environ . Pathol . Toxicol . 2 : 247-26 2 (1978) . 7 . Nechay , B . R. , an dSaunders , J . P . Inhibitor y characteristic s o

f lea d chlorid e in sodium

- an dpotassium-dependen t adenosinetri

phosphatas e preparation s derive dfro mkidney , brain , an dhear t o f severa l species . J . Toxicol . Environ . Healt h 4 : 147-15 9 (1978) . 8 . Nechay , B . R. , an dSaunders , J . P . Inhibitory characteristic s o f cadmium , lead , an dmercur y i n huma n sodiu man dpotassiu m dependen t

adenosinetriphosphatas e preparations . J . Environ . Pathol . Toxicol . 2 : 283-29 0 (1978) . 9 . Kramer, H . J. , Gonick , H . C , an dLu , E . I n vitro inhibitio n o

f Na-K

ATPas e b y trac e metals : Relatio n t o rena l an dcardiovascula r damage . Nephro n 44 : 329-33 6 (1986) . 10 . d e Wardener , H . E. , an dMacGregor , G . A . Th e relationshi p o f a circulatin g sodiu mtranspor t inhibito r (th e natriureti c hormone? ) t o hypertension . Medicin e 62 : 310-32 6 (1983) . 11 . Blaustein , M . P . Sodiu mions , bloo dpressur e regulation , an d hypertension : A reassessmen t an dahypothesis . Am . J . Physiol . 232 : C165-C16 9 (1977) . 12 . Haddy , F . J . Sodium-potassiu mpum p in low reni n hypertension . Ann . Intern . Med . 98 : 781-78 4 (1983) . 13 . Weiler , E „ Tuck , M. , andGonick , H . C . Observation s on th e "cas

cade " o f Na-K-ATPas e inhibitor

y an ddigoxin-lik

e immunoreactiv e material i n human urine : Possibl e relevanc e t o essentia l hyper

tension . Clin . Exp . Hypertens . Par t A Theor y Pract . A7(5,6) : 809-83 6 (1985) . 14 . Fiske , C . H. , an dSubbarow , J . Th e colorimetri c determinatio n o f phosphorous . J . Biol . Chem . 66 : 375-38 1 (1925) . 15 . Skou , J . C . Enzymati c basi s fo r activ e transport

o f N aandK acros s cel l membrane . Physiol . Rev . 45 : 596-61 7 (1965) . 16 . Skou , J . C . Th e (Na + -K + ) activate denzym e syste man dit s relationshi p t o transpor t

o f sodiu man dpotassium . Rev . Biophys . 7 : 401-43 4 (1975) . 17 . Nechay , B . R . Biochemica l basi s o f diureti c action . J . Cli n Pharmacol . 17:626-64 1 (1977) . 18 . Jorgensen , P . L . Sodiu man dpotassiu mio n pum p in kidne y tubules . Physiol . Rev . 60 : 864-91 6 (1980) . 19 . Westenfelder , C . G. J. , Arevalo , R . L. , Baranowski , N . A , Kurtzman , N . A. , an dKatz , A . I . Relationship betwee n mineralocorticoid s and rena l Na-K-ATPase : Sodiu mreabsorption . Am . J . Physiol . 233 : F593-F59 9 (1981) . 20 . Mujais , S . K. , Chekai , M . A , Jones , W . J. , Hayslett , J . P. , an dKatz , A . I . Regulatio n o

f renal Na-K-ATPase i

n th e rat . J . Clin . Invest . 73 : 13-1 9 (1984) . 21 . Katz , A . I. , andLindheimer , M . D . Rena l sodium

- an dpotassiu m activate dadenosin e triphosphatas e andsodiu mreabsorptio n i n th e hypothyroi drat . J . Clin . Invest . 52 : 796-80 4 (1973) . 22 . Rogus , E. , Price , T. , an dZierler , K . L . Sodiu mplu s potassiu m activate douabain-inhibite dadenosin e triphosphatas e fro ma fraction o f rat skeletal muscle , an dlack o f insuli n effec

t on it . J . Gen . Physiol . 54 : 188-20 2 (1969) . 23 . Garvyck

, W . A. , Moore , R

. D. , an dThompson , R

. C . Effec t o f insuli n upo n membran e boun d(Na-K)-ATPas e extracte dfro mfro g skeletal muscle . J . Physiol . 252 : 43-5 8 (1975) . 24 . Herrera , F . C , Whittemburry , G. , an dPlanchart , A . Effec t o f insuli n o n short-circui t

curren t acros s isolate dfro g ski n i n th e presenc e o

f calciuman dmagnesium . Biochem . Biophys . Act a66 : 170-17 2 (1963) . 25 . Haddy , J . F. , Pamnani , M . B. , andClough , D . L . Humora l factor s an dth e sodium-potassiu mpum p i n volum e expande d hypertension . Lif

e Sci . 24 : 2105-211 8 (1979) . 26 . Buckalew , V . M. , an dNelson , D . B . Natriureti c an dsodiu m transpor t inhibitor y activit y i n plasm ao

f volum e expande ddogs . Kidne y Int . 5:12-22(1974) . 27 . Nechay , B . R. , andSaunders , P . J . Inhibitio n o f adenosin e triphos

phate s i n vitr o by silve r nitrat e andsilve

r sulfadiazine . J . Environ . Pathol . Toxicol . Oncol . 54/5:119-12 6 (1984) . 28 . Har t W . M. , an dTitus , E . O . Sulfhydryl group s o f sodium

- potas siu mtranspor t

adenosin e triphosphatase . J . Biol . Chem . 248 : 4674-468 1 (1973) . 29 . Tice , L

. W . Lead-adenosin e triphosphat e complexe s i n adenosin e triphosphatas e histochemistry . J . Histochem . Cytochem . 17 : 85-9 1 (1969) . 30 . Kramer , H . J. , Baecker , A . Weiler, E . J , andLiddiard , C . Studie s o n ouabain-lik

e natriureti c factor s i

n huma n urine . Inhibitio n o

f Na-K

ATPas e and3H-ouabain binding . Klin . Wochenschr . 64 : 760-76 6 (1986) . 31 . Crabos , M. , Grichois , M-L. , Guicheney , P. , Wainer, I . W. , andCloix , J-F . Furthe r

biochemica

l characterizatio n o

f a n Na+ pum

p inhibito r purifiedfro

mhuma n urine . Eur . J . Biochem . 162:129-13 5 (1987) . 32 . Nechay , B . R. , Nelson , J . A. , Contreras , R . R. , Sarles , H . E. . Remmers , A . R. , Beathard , G. . A . Fish . J . C , Lindley , J . D. , Brady , J . M. , an dLerman , M . J . Ouabain-sensitiv e adenosin e triphosphatas e fro

mhuman kidneys . J . Pharmacol . Exper . Ther . 192:303-309(1975) . 33 . Webb , R

. C , Winquist , R

. J. , Victery , W. , an

dVander, A . J . I

n viv o an din vitr o effect s o

f lea do n vascula

r reactivit y in rats . Am . J . Physiol . 241 : H211-H21 6 (1981) . 34 . Piccini , F. , Favalli , L. , an dChiari , M . C . Experimenta l investigation s on th e contractio n induce db y lea din arteria l smoot h muscle . Toxicolog y 8 : 43-5 1 (1977) . 35 . Favalli , L , Chiari , M . C , Piccinini , F. , an dRozza, A . Experimental investigation s on th e contractio n induce db y lea din arteria l smoot h muscle . Act

aPharmacol . Toxicol . 41 : 412-42 0 (1977) . 36 . Cloix , J . F. , Devynck , M-A. , Elghozi , J-L. , Kamal , L. A. , Lacerda

Jacomini , L . C , Meyer , P. , Pernollet , M-G. , Rosenfeld , J . B. , andd e The , H . Plasm aendogenou s sodiu mpum p inhibito r

i n essentia l hypertension . J . Hypertensio n 1 (suppl . 2) : 11—1 4 (1983) .