H,vpertenston Issue: Volume 30(6), December 1997, pp 1'487-1492 Ccpyrlght: o 1997 American Heart Auadlltbl, Inc. Publication Type: (Sdenttflc Cclltr1buUons] ISSN: 0194-911X Accession: 00004Z6'r 199712Cl00-Cl0an l,Sdentllc Contrtiutfans] Lead-Induced Hypertension: Interplay of Nitric Oxide and Reactive Oxygen Species Gontck, Harwy C.; Ding, Y-'an; Bondy, Steven C.; NI, ZhenrMI; Vllzlrt, Mosratala D.
... Author lnfonnatton Received Man::h 11, 1997; first dedslan April a, 1997; revision llCcepted July 18, 1997. From the Unfwrslty d Cal1fomfa, at Los Angeles t11d lrvlne and Cedars·S'1al Medlcel Center, The Bums and Allen Research "stftute, Los Angeles (H.C..G.) t11d the Unfverslty d California, lrvlne (Y.D., S.C.8., Z.H., M.D.V.). Reprint requests to Hatwy C.. Gonick, MD, DMsm d Nephral~, Becker Buidine., Room ID, Cedars-Sinai Medical Center, 8700 Bewrly Blvd, los Angeles, CA 90048. E· md hganfckSucla.edu ... Abstract
An elevation d meWt blood pressure was found In rm treated with low lead (0.01% lead acetate) for 3 mcnths, as contl'llSted to paired Spr11Ue-Daweey central rm. In these rats, meaturement c( plasma I/Id urine endot.heltls·1 and ·3 re'<'ealed that platma concentration and urlnsy excretion at endothelfn·3 ttcreased stlntflcantly aft:er 3 mcnths (pl•ma: lead 1roup, 31.8 +/· 2.2, venus c«1t.rals, 23.0 •/· 1.7 p1tmL, P < .001; urinaty excretion: lead 1roup, '46.6•11.7, versus controls, 35.6 + 6.7 pi/24 h, P < .05), whereas endot.heltl·1 - unilffect.ed. Plasma illld urinmy nitric Clldde (NO) and cyclfc GMP cmcentratfans were not sflnfflcantly changed. HoweYer, assay of platma and kidney mtex mllondlaldehyde by hfsh·preuure liquid chramatosraphy, as a measure of rew:ttve aicnen species, was elevated In ~·treated rats compared With that tn control rats {Platma: lffld 1roup, 4.74 + 1.27, wrsus controls, 2.14 • .49 micro mol/L, P < .oot; kfdney cortex: lead group, 28.75 + 3.-46, versus CQ'ltrals, 16.38 • 2.37 nmol/t wet ~t, P < .oot). There was ticreased NO synthase actMty ti lead-treated rat brain cortex and cerebellum. In lead-treated rat kidney cortex, the endothelial constftuttw HO synthase protein m•s w• unaffected, whereas the fnducl:lle NO synthase protein ma.u was ticreased. These data. suaest a balance between increased NO synthesis lll'td depatfon (by reactfwo aicnen species) ti lead·treated ms, which results fn norm;sl. l!!'W!ls of HO. Thus, the hypertension may be related to an Increase In the pressure substWJces, endOthelln·l and reacttve QllY!en species, rather than to an absolute dec:re.se fn nitric HD. (~rtenrfon. 1997;30:1'487-1492.)
Key words nitric c»cicle; endothelils; reactiWI cayeen species; lead
Se&ec:ted Abbrwtatfons and Aaonyms
ecNOS =endothelial CD1stftutf¥e nitric Clldde synthase
EDRF = endot.helt.lm·derfved relmdn9 factor
Er = endothelln
!NOS = lnducl>le nitric oxide synthase
MDA-TBA = malondlaldehyde·thfcbarbfturlc add adduct
MO = nitric Clldde
MOS = nitric Clldde synthase
ROS = reactive aicwen species
Cclltlnucus expc11Ure to low but nGt hlflh levels of lead has been shown to result In '1creased blood pressure In both humans and animals. [1 ·6] Our l.abclratory vertfted that rats treated a:intfnuously With low lead (O.otS lead acetate) had s1tn1ftcantly Increased blood pressure canpared with aae·matched a:introls. Elevation tn blood pressure was observed after 3 months of lead exposure 1nd c:onttlued for 1 year of pemstent lead exposure. The elevatfcn In blood pressure also perststed for 6 months after lead exposure was dfKontlnued. l},6] We sugested that elevattcri In blaod pressure we related to an lll(re•e In an endotheltal-deriwd wscanstr1ttor, endothel'1·3, an tlcrease '1 the Ha·K-ATPase lnhlbttor~tdlfnl hypertenslon-assoctated protetl, and •decrease '1 cyclic auancslne rnonoph01Phate as a second messenter for the vasodilatar ccmpcund, EDRF, which h• been identified as HO. [7-9] Previous 9'1wstiptcn demCll'lstrated beth increased and decreased plasma rem acttvttles In lead~ased rats, (10, 11] with decreased lldMty s"-n fn nits that -re mmt comparable to the present study (100 ppm lead for 6 months). [10]
The purpcses of the present study-re (1) to '1wsttpte the effects of lead expcsure on pl•ma conc:entrat.fon and urinary excretfon of NO, plasma aincentmlons ot ET·1 and ET·3, and urfnary excretion of these hcnnones; (2) to mesu1ate the effects ot lead m pl•ma and kidney MDA·DA, a reflection ot l.,ld peradde fonnatbl (lll'ld thus, ROS); and (3) to detemme whether lead ecpcsure affects NO production by tldudn1 chan,es In NOS acttvtty or protefn mass.
Methods
Animals
Fran the aee of 2 months, male Spraeue·Dlwlev rats (purchased fran Charles R~r. Inc, Wltm9'1ct«i, Mass) ~9'112001 were fed a standard rodent Purina labonltoty chM (ccntafnln1 11 cllcllm) and were also pen 0.011 lead acetate (100 ppm) t'I thri' drTllm'll water for 3 months. The ~rtnental anfmals had aee· matched CU'ltrcls. Six animals each were '1cluded In experimental and control 1roups. Rats were -tshed before entema fnto he study and also at the the of death.
Measurement of Blood ~re
Conscbls nits -re placed ti a restl'Dler and Ill.lowed to rest fnslde the ate for 15 minutes before blood pressure measurements. Rllt tals were placed fnslde a bill cuff, and the cuff was Inflated and released a few times to al'-the anfmal to be condttloned to the procedure. Mean blood pressure lllllues (four conmcutM! readfnp) were taken by a rat tall blCIOd pressure mmltar attached to a student csdllQlraph (Harvard Apparatus Limited) 1nd awraaed for presentation. Th1s apparatus fS caplble ot recordtll beth mean blood pressure and systcltc: blCIOd pressure.
Collection of Blood and Urine
One day before k11Ung. rats were fndtvtclually placed ti metabolic caaes dur1n1 the day, and 24-hour ur1ne samples were collected fn plastic tubes placed on tee. Urine samples were centrifuged and frozen at ·80 [delJ'ee sien] Celsa.is for determ9'1atiCll'I of cGMP, HO, creatil9'1e, and ET-1 and Er·l concentrations. On the day of klli'l1, rats were anesthetized with methmtyflurane nd thefr blood was rerncJYed throulh the heart with ., Ice-cold syringe. NI llliquut (1 ml) ot whole blood - stared llt 4 (degree sllln) Celsfus for detenntlallon of blood lead. Plasma was separated fran the red blood cells by centr1fult'l8 the blood at 3000 rpm for 15 m'1utes at 4 [dearee stfpl] Celsius. Pl•ma allquou (1 ml) were stored at ·80 [degree sign] Celst.is for determtlatfon of cGMP, HO, creat'1'1e, M~·nA, 111d ET-1 Sid ET-3 lewls.
Collec:tlon of Tissues
After re--1 of blocd, kidneys and brains were elldlecl quickly and placed cin tee. Kfdney cortex, brafn cortex, and cerebellum -re sepanted and placed Into lfqufd nttrc11en. All tissues were stclred at -80 [detree sip] Celsius.
Determination of Lead fn Blood
Lead content of whole blocd was measured uma ll'I atomft lbsorptlon spectrophotoneter (Var1an, model «XIZ with graphite furnace). Whcle·blood lead values were expressed as mlcr<11rems per deciliter.
Measurements of Urine and Plasma Nitrite and Nitrate (NO (J)"'N03)
The concentration or total NOz"'N03 (NO (x)) fn the test samples was determined by the modtfltatlon of the procedure described by Bnman and Hendrix [12] usfn1 the purse system of a Stew:rs .,struments model 2108 Httrlc Oxide Analyzer (NOA, Sfellers .,struments Inc). Briefty, the urine samples were diluted 10 times ti distilled water before anal~fs. A saturated solution of VCl3 In 1 mol/L HCI was prep.-ed 111d filtered before use. A tatal of 5 ml of the reqent was added to the purp wssel 111d pufled with nttraeen ps for 5 to 10 nmutes before use. The purp 11eSSel WllS equipped with a cold"Wllter condenser and a water jacket to permtt heaUn11 of the reqent to 95 (degree sllln) Celsfus, ust'l1 a drculattia water bath. The hydroc:hlortc: acid vapors were removed by a ps bubbler contaln'11 [nearly=] 15mlof1 mol/L NaOH. The gas flow rate fnto the chemolumlnescence detector was controlled usfng a needle valve adjusted to yteld a cell pressure of [nearly •) 7 mm Hg. The flow rate ot nltroten Into the purse vessel was ad"stecl to prewnt vacuum dfstllattcri of the reapnt.
Simples were fnjected fnto the purte wssel to react with the VC13/Ha reqent, whfch CCll'IW:rted nitrite, n1trate, and S-nltraso ccrnpounds to NO. The HO product was st,..,ped from the reaction chamber (by puf1'111 with nltf08en and vacuum) and detected by mane-Induced chemllumfnescence fn the chemiluminescence detector. The slllnlll aenerated (NO pellk and pellk 11rea) was recorded and processed by a Hewlett-Padwd model 3390 lntevatar. In a typical assay, 5 micro Liter or the test sample was tljected to the purge vessel, and all samples were run fn t,..,lftate. Plasma samples were deprotelnlzed with cdd ethanol and Injected •the urine samples lbove. A standard cumi was constructed usln& YllriDus concentntlons d N03° (5 to 100 micro mcl/L) relalfn& the lum'1escence produced to the pen No,; cmicentmlons d the stlllldard solutfcns. The amount of NC>it In the test sample WllS determ'1ed by fnterpcllltbl d the result tlto the standard cumi.
Determination of cGMP, ET·1, and ET·3
ET'·1 Sid ET'·l fn extracted plasma and urfne were measured by redlotmmunoassay kits purchased fRlm Pentlsula Laboratories. c:GMP t1 plasma and ur1ne we also measured with an radlommunoassay kit supplted by Hew Er11land Nuclear.
Determination of Lfpoperoxtdes in Pluma and Kidney Cortex
~ralddes t1 plasma were detenft'led by hllh·pemrmance llQuld ctiromlt011'1PhY measurement of MOA·TBA acccirdln1 the method ofWon1- [13-14] The whClle procedure, briefly, is as follONS: A volume ol 50 micro Liter plasma WIS milaed with .75 mL 0.44 M H1 PO.., 0.25 mL aqueous 42 mmcl/L TBA, and 0."5 ml H2 0. The mixture was heated In a bcllln1 water bath fir 60 mfnutes. After coolti1 m tee, an equal volume of alkalt'le meth111cl (50 ml meth1nol + 4.5 ml 1 N NIOHJ was added. A volume of 50 micro Uter of tile neutralized reaction mixture WIS tllen injected tlto a 4.6 x 250-mm chromatc1raphlc cd.umn packed with micro Bondapak C18 (5-mtcro meter particle diameter). A guard cclumn, 3.9 x 23 mm, pacbd with Bonda,M Cautl C18 (37· to ~micro meter pllrtlcle dfmneter) was used. Mmle phase was a mixture of SO mmcl/l phosphate buffer (pH 6.8; 600 ml) and methll'lol (o400 ml). The ftow was 1 ml/m'1, and detection was dme at 532 nm. The concentration of plasma Upaperaicldes was determ'1ed trun the callbratlCll'I curw prepared with a tetnmethCKYPropane standard solutb'I (.61 to 4.86 micro mcl/l), processed exactly as the plasma semples.
~ralddes t1 kidney cortex were measured by h11ti't>erlonnance liquid chn:matography meeurement of the MDA·TBA accordln1 to the metllod of Draper et al. [15] The tilsue samples were mi'lced, and duplicate 0.5~ snples were placed i'I 15 x 1~mm pyrex tubes. A total ol 5 mL of 10X trichloracetic acid plus o.25 ml of !ilOO ppm BHT fn methancl were added. After heatfn1 in a bolling water bath for 30 minutes, the sample WIS collected and centrlfuted at 2!il00 rpm for 10 minutes. A total of 0.25 ml of supemate was cxmblned with 0.25 ml of satunited aqueous DA solutfon and heated fn a boll'19 water bath for 30 minutes. AtWr cool.Ins, tile reactbl mixture was extracted with 1 ml of n·butanol uslna a wrtex mixer. A O.S·ml aliquot of the mllcture was mixed With 0.25 ml metllancll •d 0.25 ml mobile phase. A tot.al al 20 m1cro Liter of the mixture we diluted to 1 ml with mobile phase, and 50 micro Uter of tilts solutb'I was tljectecl tlto the column as lbove. A mobile phase of 1g acetonitrile and O.C tetrahydrofunn i'I 5 mmol/l phmphate buffer, pH 7, WIS used. The flow rate was 1 ml/min, and detection was done at 5J2 nm. The concentration of MDA-TBA In tile supemate was determined from the calfbratlon curw prepared with a tetramethmcy-propane stlllldard solution.
Detection of Ktch!y Corte< NOS Protein Mass by Western Blotting
Kidney ccrtex ecNOS prctein mass was measured by Western btotti't1. [16-18] Briefly, 0.21 kidney cortex was h~ntzed in 0.8 ml of lysls buffer (320 mmol/L sucrase, 0.1 mmcl/L EDTA, 10 mmol/l HEPES pH 7.4, 1 mmcl/l DTT, 10 micro &ram/ml leupeptln, 20 m1cro snm/ml aprotfnt'I). The tis.sue lysates -re centr1fu&ed at 12 000 rpm at 4 [desree slfP'I] Celsius far 20 mtlutes. The supemates -re remo¥ed to fresh tce
Dttennination of NOS ActiVity in Brain Cortex and Cerebellum
NOS activity In tile cytopl11Smic fraction of brDI cortex and cerebellum was determined by tile method or Matsumoto et al, [19) In which the conwrstan ot5 H·ar"Plfne to> H·dtrulllne a determined. control for nonspedlic counts was an incubation tube containi'lg tile NOS inhibitor nttrclfli'lfne. An attempt to measure NOS actMty ti kidney cortex was unsuccetSful beceuw al the ampetttlon be~n NOS and qinase for the meUlballsm d l·arPltle fn the kidney. [2D]
Statistical Treatment of Data
Statistical analysis of the data was performed usfng unplllrad t test and #lf1VA where appniprfllte. Results -re expressed as me• • SD. Stllttstlcll stgnlflcanc:e far the t test was assessed uslns a twi>~led level of P < .05.
Results
Blood Pre$sure, Wei9ht, and Blood Lead Concentration
There was a sustained Increase In mean blClod pressure of rats treated With 0.01" lead acetate tl their dml<*11 water. Mell'I blClod pressure elevation was obserwd after 1 month of lead e.xposure (flJUre 1). At. tile end of 3 mcntlls of lead exposure, blood lead concentration fn lead·treated rats was qintftclntly hWier tllan tllat 200 -::cCl> ** E 160 ** §. * ...Cl) = 120 ..."'Cl) 0.. ~ 0 80 0 in c l'IJ 40 Cl) :E 0 0 1 2 2.5 3 Time (months) Figure 1. Ch1nges In mean blood pressure of rats fed O.OI~ lead acetate In their drinking water for Vlt'klus time periods. ~ tilts Figure andthe subsequent fliures, values are expressed as meen +/·SO• ., <.01;.., < .001 compared with c:ontrol. Plasma Concentration and U11nary Excrvtion of NOx and cGMP There were no sipificant differences in plasma NOx lewls when lead-treated rats were compared with CCll'ltrol rats. NOx umary eicicreta. wa.s also not S91ificantly different between the tw'O 1rcups. Pl•m• cGMP concentration 1nd c NO. c GMP Animal Plasma, Urine, Plasma, Urine, Groupings µmol/L µmol/g Cr nmol/L nmol/mg Cr Control 14.3± 4.1 283± 130 11.6± 4.4 1.6= 0.5 Lead 11.8± 3.8 401 ±99 15.2-2.2 1.7= 0.2 NOx indicates nitrite and nitrate; Cr, creatinine. Tilll>le 1. NOx 111d cGMP Plama Cmlcentratlons and Urinary m:retlon ti lead·Treated Rats Plasma Concentrations and Urinary Excretion of £T·1 and ET·3 In lead-treated rats, the plasma concentration of ET'·3 was Tlcreased ~nificantly abow: that al the CCll'ltrol. rats (Table 2), but there was no change in plama concent111tian of ET'·1. Urinary excretion of ET'·l ti lead-treated ms was Ibo stptliCllntly hlllher th111 that of control rats, but tllere was no change ti umary eiccretian of ET'·1 (Table 2). ET-1 ET-3 Animal Plasma, Urine, Plasma, Urine, Groupings pg/ ml pg/24 h pg/ ml pg/24 h Control 7.8.!:2.8 40.3..:. 9.2 23.0z: 1.7 35.6± 6.7 Lead 8.0± 0.7 48.8± 11.0 31.8±2.2· 46.6±. 11.7t ·P< .001 compared with control; tP<.05 compared wrth control. Table 2. EM and ET·3 Plama Concentrations and Ur1nwy eccretton tl Lead·Treated Rats Lfpoperooddes In Plasma and Kidney Cortex Lfpcperaddes In plasma, represented by the Ml».·T8A lewl, were stp'ltt'lcantly hllher In lead-treated rats tllan that In ccntrol rats after lead expc11Ure. Ml».·DA content d kidney Clll'tex also was hllher ti lead-treated rats (Figure 2). c::J Control - Lead 40 8 -5 c: 0 .!c: ** 0 6 §. o- 30 ctJ -1: o_ ~ 0 ~I 20 4 0 ~ g> 0 0 g o~ :E !- 10 2 "'E ,, XI i: 0:: 0 0 Kidney Cortex Plasma Fieure 2. Contents of MDA tL kidney ccrte.x lWld plasma fn lead-treated rats. Kidney Cortex NOS Protein Mass There Mre no ._tftcant dtt'rerences fn kidney ccrtex ecHOS protein mass when lead-treated rats Mre canpared with CCll'ltrol rats, where• IMOS prcteti mass was sttnfficantly elevated (Figure J). A ecNOS iNOS 1 2 3 4 1 2 3 4 140k0a - •••• - ... -- -130k0a B 20 Cl CJ Control -Cl .....Cl )( 15 - Lead =: ••-~ :I! 19 ** -uc co $~ 0 a. ~o Q. • > 5 i GI ~ 0 .__...... _ __ ecNOS iNOS Figure 3•. A, hmunoblot •lllysfs fer ec:NOS and IMOS pratftl under den11tum1 condltfcns. Lanes 1 11nd 2 shaw lyslltes of control rat kidney cortex. Lanes 3 and 4 shaw lysates d lead·treated rat kidney cortex. For ei:HOS, 100 mti:ro grem of lysate protefn was used; fer tHOS, 200 mfcro warn was used. B, Relative aptfi:al densities of the blots fn the study 1roups. NOS ActMty In Brain Cortex MOS actMty In beth brain cortex •d cerebellum was elevated fn lead-treated rats compared with CU'ltrols (l'ltl.lre '4). MOS acttvtty In cortex was 1169 +/· 111 t'I lead· treated rats venus 810 •I· "11 counts per mllll.,am protein fn ccntrds (P < .oot); nnllarly, MOS actMty In cerebellum was 967 +/• 126 fn lead-treated rats venus 737 •I· 103 counts per mtllllram protefn In contrcls (P < .01 ). D Control Lead 1500 - ·~:;: - ti- • m .5 1200 QI~ ma."'e ~ 0) 900 >-E 'O:s c 600 ·->< :::s0 0 u u- z"C- 300 0 Cortex Cerebellum Flaure 4. NOS activity in brVt cortex and cerebellum. Discussion ~ure to low but not "WI lewis of lead has been reported to cause hypertensb'I fn hum1ns and antnels. [1-61 NI fncrease fn blood pressure of rats oc:airs at lciw lead e.Jq*ure lewis of appraiclmately 0.1 to 100 ppm, which is siTlilar to the e.Jq*ure lewis seen in the envll'Cll'lment. ~r. rats eJcpased to ca1centrattons greater than 500 ppm of lead (equlwllent to lndustrllll lewl opasure) do nat develop hypertension. Our previous studies suaestettlon of Na, K, ATPase by oulblfn causes lmpSlllent of ace~cholt'le-fnduced relantlon tl human resistance arteries, conststent with an eft'ect on EllRF synthesis or release and therefcre affording a passible link between these two effects.) we shcMed also that ION lead feedfng increased rather than decreased glomeruler flltnltlon rate end that no morphcloglcel or renal funct1cllal abnormalities -re associated. [22] In the study presented here, blood lead concentratiCllls at 3 months ti lead-treated rats l').01S lead acetate 1n drlnldn9 water) -re much hlllher than those In contrcl rats but lOllll"er than thclff in low lead·treated rats studied previously (12.4 +/· U wrsus 29.4 +/· 4.1 micro 1rem/dL). The epparent reason for the difference was that a stendard chaw was used In place of the S)Tlthettc dt.t used previously. The latter ts known to tlcrease susceptlb111ty to lead tmclclty, possl>ly through a le7ft1er calcium content (4n of labcntcty chow). (23) Despite the lower blood lead lew:ls, an elevation of blood pressure produced by ...-ure to lead was ccnfinned by the present lnvestlptbl. There W11S a slpfflcant tlcrease In plasma concentration and In ur1nary ellCretbl d ET·3 but no chlll'lge ti plmma ccncentratfon or ur1nary eiacret1cl1 d ET·1. The plasma results -re compatt>le with the results of the prior study, [5] although the absolute values for plasma concentrations af ET·l and ET·1 -re lclwer because of the use of extracted rather th1n unextrected plasma. It was notev.athy that ur1nary lewis of ET'·l were also elevated, tlasmuch as sewral studtes have shown that ur1nary endotheltl ts a better reftectlon of lntrarenal events. [2+26] 18 lead deposition ts localtzed prlmal1ly to the practnal tubule rA the ktdney [27] 1nd as ET·l ts produced within thts seF1ent of the nephron, (2B] this suggests that the effect al lead on enclothel In production may occur via Inc remed renal tubular rather than ' EDRF was also thoullht to play a alttclll role In lead·tlduced hypertension In our e11rller study, as bath plasma and urinary lewls al cGMP, the second messen9er for EllRF, -re reduced by lead adnmtstratlon. 1.5] In the present experiment, by contrast, EDRF, as measured by plasma and urine cGMP and more directly by NO, was unatrected by lead. Howewir, HO lew:ls reflect the bal1nce between HO synthesis Via NOS •d NO dearadatton, a process affected by e.icposure to ROS. (32,33] Levels of clrculatcty NO may be increased, normal, ot decreased when l' Measurement d NOS activity fn brain wes bind to be Increased after lead eicpc11Ure, whereas In kidney cortex INOS, but not ecNOS, pnitetl mass was Increased, 1ncltcatt11 that HO synthesis was uprepleted. Of Interest ts the 1n Vitro observation that lead fnhtllts rather than stimulates bra. constltuttw NOS activity [35,361 while hlllint no effect on cytdme-i'lducl>le brain NOS actiYity, [36] therefore nplyint that 9'1 the present study the increased NOS activity is a compensatory response to NO degradlltlan by ROS or seccndary to 11nother biochemical chanp Induced by lead oposure. An tlcreased production of ROS In response to lead, previously suggested by Hennes-Lima et al, [37] was confirmed by the elewted lewis of MDA·TBA '1 plasma 11nd kidney cortex of lead-treated rats. 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