ON VASCULAR SMOOTH MUSCLE

ON VASCULAR SMOOTH MUSCLE

BY

" TINA MARIE MCWILLIAM,

B.Sc .

A thesis submit ted to theSchoolof Graduate Stud ies in pa rtial fulfillme n t otthere q u i re me n t s for the degreeof

Ma ste rof Science

Facul tyof Medicine MemorialUniversityofNewf o und l a nd

August ~990

St.John' s NeWfoundland

•1

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ABSTRACT

The influenceof deu t e r iumoxide (020 ) onaort icvascular smoothmuscle was investigate d. The ef f e cton receptor- and voltage -ope ra tedcalciumcha nne lswas st udied with the use cr phenyl ep hrine and potass i um chlo ride re s pe c ti v e l y . °20 depressed the phenyl e ph r i ne induc ed cont rac tion respons ein a dose dependent manner with sot inh ib i t i on of maximum contraction observed with 60\: °20. The effect of 60t °20 on phenylephrine induced contrac tion wa s reversible and not dependent on an inta c t endoth e lium. Sixty perce n t °20 also reduced potassium chl o ride indu c ed cont r actions by 50%

indicat ingan effect an vo ltage operatedcalcium channels. studies withBay K 8644,all L-typecalciumchanne l activator, and calcium fr e e buffer studies, confirm an ef f e ct on uti li z a tion of extrace llu larcalcium sources. Inhib it i o n of sucrose inducedcontract ion andac e t yl ch ol i ne and iso pre naline ind uc e d relaxatio n wit h 60% °1° sug ges t an effect on intracellular cej.oium mobil ization or on the contractile ele me nts themselves. 010 also affected both the pD1' for nifedipi neandthepAzfor prazosi n.Co lle c t i v ely , the s e data indicate that OzO acts at mUlti p le si tes.

InYi..Y2studies ind icate that chron icoral admi n istration of 25% OzO for 12 days re d uc e s the blood pressure of sponta neousl y hype r t e ns i v e (SHR) andnormote-isdve (WKY) rats.

i i i

However, the lowering of blood pressure did not result in a corresponding change in vascular smooth muscle contractility induced with phenylephrine and potassium chloride. These results support previous evidence that a reductioniJ~blood pressure after hypertension has developed does not reduce va s cularreactivity changes. A further acute study revealed that desensitizationresultsfrom chronic exposure to 020such tha t 60% DzOproduces a significant depressionof contraction onlyin aortic rings obtained from SHR and WKY ratswhichhad not been chronicallytreated with twenty-fivepercentOzO.

Aorta Cal cium channels

Con traction Deuteriumoxide Hyp e rte nsion InY..i.t£g InYi.Y..Q Relaxation

KEY WORDS

Reversibility Vasc ularsmooth muscle

ACKNOWLEDGEMENTS

I would li k e to express deep ap p reciation to my supervisorI Or. An dr e w Rankin for his co nstant su pport and advice. Hi s guidance and genuine in teres t inmy research, al o ng with his light heartedmanner, mad e time spent inthe laboratoryvery enjoyable. Th an ksare extendedto membersof my supervisory commit t e e,Dr.C.R .Triggleand Dr.J.Reynolds forthe ir valuableassistance.

I am grateful for fi na n c i a l support provided by the Facultyof Medicine,the GraduateStudents 'Uni o n,and by Dr.

Rankin through a research gra nt from the Me dical Research Cou nc i l ofCa na d a . Iwouldliketo expresssinceregrat itude to Atomi c Energy of CanadaLimited for their generous supply of deuter ium oxid e thr o ugh Dr. A. Liepins.

Finally , Iwouldli k etoth a nk Harold and myfa mil y for the i rlo v e ,sup p ort, anden cour a gement. Specialthan ksto my sister ElizabethAnn for typingassistance.

T ABLE OF CONTENTS

PAGE '

1.

INTRODUCTION

1.1 Bac kgroun d in f o rma tion ondeuteriu m ox i d e

1. 2 Mechanismsofva s c u l a r smooth muscle contraction 1.3 Mechan i smsofveecur e esmoot h

mu scle relaxation

1.4 Compe t i ti ve andnoncompetitiv e inh i b itio n

13

1.5 Hypertens io n 1.6 Aims ofth e stUdy

15 18

2. MATERIALS ANO METHODS

2.1 Animals 2•2 Dru g s an d reagents 2.3 Preparation of aorticri ngs

2.3.1 Tissueisolation 2.3.2 Experimental apparatus

20 20

22 22

23

30 27 27 26 23 26

2.4. 12 2.4.2 2.4.3

2.4.7 2.4.8 2.4.9 2.4 . 1 0 2.4.11 2.4.4 2.4.5 2.4 .6

2.4 Expe r i me n t al procedures 2.4. 1 Ph e n y l e p h r i n e do se

respo ns e curves Reversibility Mech a ni c a l di s ruption of the endothelium Potassium ch lo r i d e dose respon securve s

Calc i um dose res pon s e curv es BayK 864 4 doseresponse curves

Suc rose ind ucedcon t ract i on 28 Isoprena l i ne inducedrelaxa t ion 28 Ace t ylch o line inducedre te x eeLon 28 DeterminationofpOl ' for ni f e di p ine 29 net.erm fnatLcn of pAlfo r 29 prazo sin

Blo odpressu reee e su r e e e nt-e

2.·Ll~

2.4.14 vii

ChronicinY..1.Y.2 stu d ies us ing twenty-five per cent010 Acute.in.Y..1..t1;:Qst ud ies using sixtypercent010 2.5 Ana l y s i s of data

3. RESULTS

3.1 Effe ct of 010 onao r t icsmooth mu s c l e cont r acti on

3.1 .1 Phen ylephrine Ln dr..ccd co ntrac tion 3.1.2 Rev ersibil i ty 3.1 . 3 Roleofthe endothelium 3.1.4 Potass i um chlorideind uc ed

contract i on

3.1.5 Calciuminduced contract i on 3.1.6 Bay K864 4 ind u ced con t ract ion 3.1.7 Sucrose induc e d con t raction 3.1.8 Syno psis

30 31

31

33 33 38 38 38 43 43 50 50 3.2 Effect of010 on aor ticsmoothmuscle

relaxation

3.2.1 Is op r e nal ine anduced 50 relaxati on

3.2 .2 Ac'tylchollneLnd uced 53

relaxation

3.2.3 synopsis 58

3.3 Effect of 010onthe af f ini ty of an antagonist for itsre c ep t or

3.3. 1 Nonc o mpetitive inhi bition 58 3.3.2 compe t i t i ve inhibition 69

3.3.3 synopsis 69

3.4 Ef f e ct of °1° on aor tic

smoothmuscle from WKY andSHRrats

3.4.1 Bl o od pre s su r e levelsof 78 WKYandSHR ra t s

3.4.2 Chronic.inrlY2effect of twe nty- fiveperce nt D10

3.4.2.1 WKY/ SHR: Potassium 78 chlo r i d e induced

contracti o n

3.4.2.2 WKY/ SHR: Pheny lephri ne 83 ind ucedcont rac tion 3.4.3 Acuteinill.J;:Qeffect of sixty

percent D10

3.4.3 .1 WKY: Pot assium 83

chlor i de indu c e d con t ract ion

3.4.3.2 WRY: Phenylephrine 8B induc edcontraction

3.4.3. 3 SHR: Potassium B8

chlorideinduced contraction

3. 4.3.4 SHR : Phenylephrine 91 inducedcontraction 3. 4. 4

4. DISCUSSION

syno p s is 91

4. 1 Effectof DzOon aorticsmoot h euscn e

cont ractio n ⁹⁷

4.2 Effe ct ofDzOon aortic smoothmusc l e 101 rela x ati on

Effe c t of DzOon the affinity of an 102 antagoni s t fo r its receptor

Chr o nic and acute ef fectsof DzOon 103 aortic smoo t h mus cle fromWKYand SHRrats 4.5 Summar y anddiscu ssion

REFERENCES

107

111

Ta))1e3-1:

Ta))1e 3-2:

ix

LIST OF TABLES

Effect of60\ DzOon the POzI to r nifedi pine.

Effe ctof25% DzOcons ump t i o nrcr 12days on the systolicblood pressuresof spontaneou sly hy pertensive (SHR) and normotensive (WKY ) rats.

Figure 2-1: Experimenta l appar a t us .

Figure3-1: In h i bit i on of lxl O-~ phenylephrine induced aorticsmooth musclecontraction wi t h varying concentra tio ns of

°

2

° .

Fi qure 3-2 : The effect of varyingconcentra tior.sof °2° on phenylephrine induced llor tic smooth muscle contraction.

Fi qur e 3- 3 : Reversal of th e 020 inhi bi to r y effect on phenyleph rine induced aortic smooth muscle contraction.

Figure 3- 4: The role ofthe endothe lium in the effect 01 sixty pe r c en t D20 on phenylephrine induced aortic smooth muscle contraction.

i'iqu re 3-5: The inhibitoryeffect of sixty percent 0 20 on potassium chloride induced aortic smooth muscle contraction.

Figur e 3-6: Th e inhibitoryeffect of sixtypercent020 on the dose respo nse curve of calcium in the presence of 60roMpotassiumchloride.

Fi gure 3-7: The inhibitoryeffect of sixty percent 020 on Bay I< 8644 ind uced aortic smooth mus cl e contraction.

Figu r e3-8: Th e inhibi toryeffect of sixtypercen t °2°on 250 \tIMsu c r os e induced aortic smooth muscle contraction.

Figure3-9: The inhibitory effect of sixtypercent020on lxlO'^4M isoprena line induced aortic smooth musclerelaxation.

Fi gur e 3- 10: The effect of sixt y percent D20 on th e dose response curve construc ted using lxl O' lOMto lxlO·^4M ac e t yl c hol i ne.

Figure 3- 11: Four consecutive dose response curve s constructed us i ng 9.6ntHto 65.8roMpotassium chlor ide inthe absence of

°

20 .

:rigure3- 12: Four consec utive dose re s po n s e cur-v-as constructedusing 9.6roMto 6!:l.a mMpotassium chlorideIn thepresenceof sixty percentO2°.

xi

Figure 3- 13: Noncompeti t ive in hib i tion of potasl>ium chloride .mdu ced aor t i c smoot h muscle contractioninthe presence of nifedi pineand inthe absenceofD~O.

Figure3-14: Noncompe t i tive inhibitio n of potassium chloride induc ed aortic smooth muscle contra ctionin the presenceof nifedipineand sixtyperc en t 0 20.

Figur e :>- 1 5:

Figu r e 3-16:

Figure 3- 17:

Fi gu r e3-18:

Figure 3-19:

Figur e 3-2 0:

Figure3-2 1:

Figure 3-22 :

Fi gure3-23:

Figure3-2 4:

consecu t ive dose re spons e curves construct ed us ing lxiv·uM to lxlO·sM phenylephrineintheabse nce of O2

°.

Four consecut.Lve dose resp ons e co ns t ructe d us ing lxlO·uM to lxlO-~

phe nylephr ine inthe presence of sixty percent 020.

competitive inhib ition of phenylephrine induced aor t i c smooth muscle contract ion in thepr e-sen c e of prazosin andin the absence of

°2°·

competitive inhibition of phenylephrine induced aortic smooth muscle contract ion in the prese nceof prazosinand sixtypercent°20.

Chronicjnvivoeffectof twenty-fivepercent 020 on potassiumchlorideinducedcontraction of aorticsmoothmusclefrom WKY and SHR rats.

Chronicinyj,yQeerece oft....enty-fivepercent 020 on phenylephrin'~ induced cont r a c tion of aorticsmoot hmusclefroJll WKY and SHRrat s . AcuteinY.i..t1.:Qeffectof si xtypercentD20 on potass ium chloride induced contraction of aortic smoothmusclefrom WKY rats. Acutein:t.i.t.r2effect of si xtypercent 0:20 on phe nyl ep hrine induced contraction of aort ic smoothmuscle fro mWKY r-at.s.

Ac ut einillJ;:gef fectof six typercent020on potassium chloride induc ed contraction of aortic smoothmus c l efro m SHR rats. Ac ut einili1:Qeffe ctof sixtypercent~~Oon pheny lephr i ne induc ed contraction of aortic smoothmuscle fromSHR rats.

ATP

cAMP cecr,

cGMP

D,O EORF BOTA G-protein

"9

IP, K' KCl

mM MgSO.

Na' Nael NaHCOa

S.E . M.

SHR WKY

ABBREVIATIONS

Adenos inetriphosphate Calciumion

cyclic adenos inemonophosphate Ca lciumch loride

cyc lic guanos ine monoph os p hate Deu t e r i umoxi de

End ot he lium deriv edrel axing fac t o r Et hy l e ne d iaminetetraacetic acid Gu a nos inetr ipho s ph a t e bindingprotein Me r cu r y

rncs , to l tripho sphate Potassi umion Potas z i um chloride Pota s s i umdihydrogenphos ph a t e Mo l ar co nce ntra t ion Milli meters

Millimolar conc en t ration Magne s ium sUl p ha te Sodiumion Sodiumchlorid e Sodiumbi l.:~,:,bon ate Sta nda rderrorof th e mean Spo ntaneouslyhypertens iv e ra t s Wistar- Kyoto rats

CHAPTER 1

INTROOUCTION

Background inforaation on4euterlWl1O:ll:ide Deuter iu llI, the heavy stab le is o t o p e or hy d c ogen, discoveredby Urey et a1. in1932 and deuteriumoxide (D:zO) was firstmade available by Ureyin1933. Since thead d i t io n of theextraprotontohydr':lgenpr act i c a l l y doubles th e mass , thereare great differe n ces betwe e nthe or ig ina l element and the is oto pe. Th e ch a n g es include an increaseinthe me l t ing an d boi l ing point by 3.8 2° Cand 1.42 °C resp e c t i v el y , a 25' increase in visco sity , lo we r i n g of vapo r pressure and an increasein the speci f i c gravity to 1.1 074 (Barb o u r, 19 3 7) . There ar ealsoso l ub i l ity changessu ch that carb on di oxid e and ox yg e narebothabout 10perc ent lees so lub l e in ~otha n in HzO (Ka t z:, 1960). Deu t e r i um oxide is fou nd as a natu r al comp o nentofordinary water &t a concent rationof one par t in 50 00 or 0.02 perc e nt (Barbour, 19 3 7 ).

It Isin tere st ingto no t e that the highcost of OzO vas anobs t a c l e forpr o l o n g e djnY1YQst u d i es usi ng OzOduring the th irt y ye a r s followingitsdi s c overy(Tho ms on , 196 0) and even today thehi g h cos t isa limiting fact o r. Si nc e the r e ar e la rg edifferc n e e s between hy dro g enand deuterium,an dhydr o gen is a major co nsti t ue n t of mammal ian ti s s ue s , earlystudi es de a l t withdetennin ingth e effect of DzOon ma mmals . Ba rbour

deuterium is a depressant of biologicalaC'~ivityand thatit s effects can be rtv e r s i ble , or not, depending upon the concentration . Fr om this study it was alsodetermined that mice could not survive replacement of more than one th i r d of bodywater by 020; metabolic rateis in c r e a s e d in the ear ly stages of 020 intoxication~ and 020 has a sympathomi met i c action . A sim ilarstudy was conductedby Thomson (1960)who fou n d that rats died when aboutone third oftheir bodywater wa s replacedby O₂

°.

The effects of O2

°

included impairment of kidney functi on, anaemia, di st ur be d ca r b o hyd r ate metabo lism, central nervous system disturbances , and wide ef fect s onen zy me sy s t e ms. It was conc l u d ed that the toxic action of 020 was due to the additive ef f e c t of the many chan ge s in rate s of enzymaticreactions.

Chemical and biological studiesconductedby Katz (196 0) reinforced the fact that O2

°

^{inhibits many enzymati c}

reactions. He determined th a t although the bindin g rate constantsforHt-and c-ions are similar, the ratecon s t antfor the unbinding of Ot- is much slowe r , demonstrating the increased stability of deuterium bonds and account ing fo r iso t np i c effects. Ex p e r i me n t al rate constants supporting isotopic modifications due toa- and o- exchange have most recently been calculatedby Michael et al. (1990).

Good a ll (19 58 ) su ggestedthat theratelimitingproces s for va scularcon t ract i cnmaybeproto ntransfer . He sh owe d a thr ee foldreduction inthedev e lopment of isollletrictensio n in frog sartoriusmusclewithoutare ducti o ninthe amp litud e of the actionpotentialandconclu d ed that DzO actedon the contrac tile pro tei ns",it.~the involvemen t of poss i blythree pr otons. Thisfi ndingspa rke dinv e stigat ions int othe effe c t s of Dzo on theme chani c al responseof various muscle typ es.

gamine r (1960 ) st udied theeff e c t ofDaO on the is o l a t ed atriaofthefrogandfoun d thatconcent r a tions of hea vy wa t er between 99.aand25pe rc e nt decreasedtheforc eof contr ac tion and the rateof spontane ous beating of the isol at e d atria . Thi s da ta suppo r ts the result found by Barne s and Warren (1935) who vere the first to show that 20t °20 slo ws the frequ e ncyof pUlsationoftheexcis e dhear tof th e frog. In orde r to elucidate thesit e of actionof DzO,Kaminer (1960) perf o rmed subsequentex peri me nts on glycerolextractedpsoas musc l e tibres (wh erecontractile proteins are is ol a t e d ) and found that ~Odoes not af fect ad e n osin e triphos phate (ATP) ind u cedsh o r teni ng of the muscle fibres. Hethen conc luded that it is the exc ita tion -c ontraction coupl i ng whic h is affe cted byDaO andnot theco ntractile prote ins.

Furthe rst udi es on theinhibiti on of enzymes bySvens ma r k (196 1) re ve aled tha t DaOinh i bitedade no s inetriphosph atas e , anenzyme associated wit hmu s cu lar act i vity, by50%. Heth e n

se mite ndino usmusclehe found that the allpl1tudeof theact i o n potential wasunaffected by D:z0 but therewas a reductionof the frequency at which a comp l e t e tetanus cou l d bema i nt ained , sugges ting an eff ect on th e pritRary oxcita tory proce s s es. The r e was also a reduc tion in twi tch force and the rate of forcedevelopment in010 wh ichhe suggestedcould be causedby a directeffect on the contractileelements, Fromthis stUdy itre maine d undecidedwhet h e r the reduction inthe fo r c e of contrac t ion was due to a

° 2°

effect on the co ntractile elements or on excitation-contractio ncoupling. Also, due to ina d equ ate controls, Sve nsma r k ar gue d that no de finite conclusionscouldbe dr a wnfromthe work of Gooda l l (19 5 8) or Kaminer (19 6 0).

The co nflicti ngconc l u s i ons drawnfrollthe sestud i e s le d to increased inte r est in determining thesite of acti on of DzO. Kami ner and Ki mura (1972) supported th eir.previ o us conclusionby determining that the luminesc e nt respo ns e ot aequori n (a calciUm sensitive luminescent prote in) which accompaniescontractionwas abol ishedwhe n H10 was replacedby O2

°.

Howeve r ,itwasagain argue dtha t su i ta b lecont rolswere la cking si nce the possi b l e eff e cts of D10 on calcium sens i tivityandki ne t i c s ofthe aequorinreactionwereunknown (Al l en et aI. , 198 4 ). acudfea cond ucte d by Allen et at. (1984)involveddetermining thein fl ue nc eofthe substitu tion

of OzO for HzO on calciumtrans ients and on contr a cti o n in both intact single skel etalmuscle fi bre s injected with aequorin and in mechanical lyskinned fibres from frogs. These studies revea led that th e calciumsens i tivi ty of the myofibrilsis reduced by Ozo substitu tion but that at sat urati ng calcium conc entratio ns th emyofilamen tsgeneratemorete n s i o n in020 than in H20. Resu ltsalso indicated that cal ciumreleasefrom the sarcoplasmic re t iculum wa s reduc e d in 0 20 and it was concludedthat this reductionin calciumreleasewas e major factor in reducing twitch tensionbut reduced myofibrillar calciumsensitivity also played a role. Also, ti:~ rate of change of aequorin light emission brought about by O2

°

substitution represented a 3 to 5 fold reduction in intracellular calciumconce ntra tionwhich wa s dependent on the concentration of DzO and proportional to the fraction of H20

replaced. This change in li gh t emission, after solvent excha nge , occurredto o fast to be explained by exchange of int r a c e ll ul a r water so it was postulated tha tth e site whereby DzO affected calcium release must be accessible to the extracellu larspace. Itwas conc luded th atthe r e was ti me for O2

°

to diffuse in or out of the T-tubu l e s in tim e to produce the effects oncalcium re l e a s eand therefore i t was proposed tha t the site of action of OzO may be on excitation- contraction coupling in the T- tubules. Earlier studies (Sandowet03.1., 1976 and Yagi and Endo,197 6 ) also support Ozo effectson excitation -con trac tio ncouplingin skeletalmuscle.

Other evidence supporting an ef f e c t of n~o on calcium

al.,1987).

Des p i t e wideinterestin thest udyof th e effects of 020 skeletal muscle, few experiments ha ve been carried out using smooth muscle. The effe c t of OzO on aortic vascular smooth muscle fr omspontaneously hy pe r t e nsiv e rats(Vasdev et al.. 1990a)andDah l salt-s ens itive hy p e r t e n s i v e rats (Vasdev et al., 1990b) ha s beeninves tiga t e d. It was found that°20 inhibitedinilil:2calcium uptake in aortae from these rats.

Since O2

°

has beendemonstratedto reduce calcium uptake in va s cu l a r smooth muscleit might be expected that OzOwill also inh i b i t contractionof vascula rsmooth muscle. The purpose of the present stUdy isto determineif this is so and to try to resolve the previouslypostUlated mechanismsof action of O2

°

on th e contractionprocess.

The thoracic aorta was chosen to st Udy the effect of deuteriumoxide on vascular smoothmuscle. The rat aortawas the vessel of choice since i t is devoid of adrenergic nerves (Pat H et a L,,19 72 ) and Fleisch(1974) reportsthat the rat aortic prepara tionis idealWhe none wishe s to investigate the effects of drugs on vascular smoot hmusclede v o i d of nervous innervation. This conditioniside alsince drugsnot only act at re ce p t o r s , producing aneffect of theirown,but may also actas nerves innervatingtheti s s u eand eU,citingtherele a s e

of endogenous ago nists resul t ing in additional tissue re sponses.

1.2 Kechanis ms ofva s cular smoo t h lIluscle contra ctio n TheIllost importa nt st ep in the contractionor relaxa t i o n of vascular sllloo t h auec Le involves calcium mo bi lization (So ml yoand Somlyo ,1968 ) . An increaseinthe intracel lu l ar calciulQconc entrationat the levelof theco nt rac t i l e prot ei ns re sults in cont ract i on of the muscle , vnexeee removal ot calcium results in relaxat ion (Fleisch, 1974). Vascular smoot hmusclecells may be ac tiva tedeither by agon istswhich activate receptor-operated calcium chann el s or by membran e depola rizati onwhi chact i v a t e sth eopen i ngof voltage-opera t e d calcium cha nnel s.

There are thre e types of vol t age depender rt;calcium ch a nn e l s . The s e are theT. L, and N cha nnelswhic h re f e r to tr an s ient(fa st),lo nglasting(sl ow),and ne urona lCha nn e l s, respect i vel y (Nowyc kyet a1..19 8 51. However, onl y T-andL·

typechannel s are found inva s c u l a r smoothms ucl e (Zel i s and Moore . 1989) .

Inaddition to receptor- andvol t a ge-ope r a ted calcium cha nne ls there is alsoa leakcha nne l which is the calcium ent r y pathway whichallowsacont i nuous ent ry of calciuminto the cell that is not inc r e ase d on stimula tio n by depolarizat ionor agonists (Khalilet a1.. 1987 ).

triphosph ate binding pr o t ein (G-Protein), which in turn eceiva ees enzymes, suc h as phospholipas eC, to generatesecond messengers. Two of these second me s sengers are inositol triphosph ate (I PJ) and ce" which induce the sarcopl asm ic reti culum to inc re ase the release of calcium, there by increas ingthetmrecetnnar co nce nt r a t i o n of ce > ava ilable for bind ing to and ac tiva tio n of calmodulin (Williamson, 1986). Cal modu lin isaub iqu i to us prote i nwhich isthe major re cepto r of ca" and appea rs to media te mos t of the ion's activ i t i es as a sec ond mess e ng er (Wang andWai sman , 1979). The ca lc iu m-ca l modu lin compl e x then activa tes myos i n li g ht chai n kina s e. Myosi nlig ht chai nkina s e isthe enzyme whic h ca t a l ysesth e pho sph orylat i on of myosin; a pr oc e s s important forthe deve l opmentof contr action invascula r smooth mus c l e (Ade l s t ein et a1., 1981 ). Activation of the ki na s ele a dsto rapid cyc lingof the cr os s - br i dg e s betwe en act in and myosin and this int e rac tion of theco nt r ac tile prote i ns re su l t s in con t r act ionof vascular smooth mu s cl e (Kha l ilet aL,, 198 7).

Depo l ar i za tion indu c e d increas es in intra cellul ar calcium conc e nt ra tio n occurby directentry of ca l ciumvia the T- and L-type calciumchannels (Zeli sand Moore, 1989 ).

In thi s stUdy phenylephrine ,an alpha-ad r e nergi c agonist, was cho sentostUdy the DzOeffect on cont ractioninduc e d via

receptor media ted increa s es in int ra cel l u la r calc ium.

Alt hough phenyl e phrine is not compl e tel y sel e ctive for receptor -o pe r ated. calciumchann e ls(Godfrai nd.etat,.;1986 ) it doesact mainly on thI stypeofcha n ne ltoproduc econtra c t i o n of aortic vascular s:lIoot h musc le (Khalil et ea ,.; 1987). Potassiu mchlorideacts mainlyonthe voltage-ope r ate dcatcLue channe l (Meishe riet al.• 1981) and was chos e n tostudy th e ef fect of01

°

onthis channe l type. The effe cts on th i s ty pe of chan nel were also inve st iga te d us ing Bay K 8644 wh ic h enhances calc iumin flux via the voltage sensit ive ca lc iu m channelund er par tialdepo l a r i zingco ndi t ions(Schramm etaL,, 1983). To further ens ure no ef fec t fromreceptor -ope ra t e d calcium channels , theywer e blocked by the al pha-rece ptor antagonis t phe ntolamine. Similarl Y, calc ium fre e buffe r stud i e s were performedinan at tempttocl a ri fytheeff e c t s of Da0on con traction induce d by th e influxof extrace llu l a r calci um.

Finally. the effect on sucrose induced contr actionwas stud i ed to deterJIine the effec t of Ca0 on int racellular ca a cIue mobil iza tlon since it oc cur s independe nt l y of the le ve l of m~mbranepotenti a l and inthe absenc eof externa l calcium so urce s (Anders s on et a L;, 1974).

1. 3 Me c h a n I sms Of vas cular smoothmusc lerelaxa t ion The two mai n proposed mecha nisms of va scul a r smooth musc le relaxa t i on inv ol ve an inc r ea s e in either cyclic

ad eno sine monophosphate (c AMP) cyclic guanos in e lDon oph o sphate (cGKP) leve ls. In th isstu d y a representat i v e wa s chosenfrom each group: is op re na line, which is a be t a - ago ni s t an d acts via an incr ease in cAMP le vels; an d ace t y l ch oline , Whi chre s ultsin relaxat i o nvi a anincre asein cGMP lev el s.

va r iouse.t rre r e n e aech ansfee are tho ught to resul t in va s cu larsmoot h mu s cl ere l axatio nthrough aninc reasein cAMP;

inclu ding beta-adre n e rgic stimUlati o n, inhibit ion of phosp h odiesterase, and regu latio n of myos in ligh t chai n ki nas e.

Althoug h var ious mech anisms have beenpropos e d for beta- adrenergi c re c epto r mediated rela xation of vascula r smooth auacLe , al lthe orie s suggest tne Ln votveee nc of c~p as the secon dmes s e nger (Muel lerandvan Bxe ee en , 1979). Rel"xatio n of vas c ula r smoot h musc le in res po n s e to adrener-gic st i mu l at ion is ae di ated by 1J1, 13" or both 1J1 and fJ, ad r e nocept ors(Lucchesi,19 89) . The be ta- a dre nergicre c e p to r is coup l e d to adenylatecyclas e by a stim ula tory G-pro t ein . Act iva ti on ofad enylatecyclase conv erts ATPto cAMPand th e cAM P in turn activa t e s a pro te in ki na s e wh i ch ca ta lyses a protein phosphory lation and le ads to a reduct i on in intra cell ular freecal cium (Muel l e r and va n nr -eemen, 19 79).

To date , it has not beendetermined howthe intrace llul a r ca lcium concent ra t i on isredu c e d. The pro po sedme chanismsby

11

which cAMP may decrease the calcium io n co n c e n t ra tion avail a b l e to the contractileproteinsinclude : inc reased Ca²+ sequestration into intracellular st o r a ge sites such as the sarcoplasmic reticulum (Muelle.\; and va n areeaen, 1979 ) ; inhibitionof calciuminflux (Meisher! and van Breaman, 19 8 2 ) ; andincreased calciumextrusionviaincreased ac t i v i tyofthe na'-K+pump (Scheid an d Fay, 1984) . At pres e ntitisthought thatcal c i umsequestrationby increased ca" uptake int o the sarcoplasmic ret icul um proba bly plays the pr i ma r y role in de c rea s i ng free intracellular ca lciu m (Bu l b ri ng and Tomi t l\, 19B7 ).

Th e r eisal soevidencetosup por t a roleforincrea ses in cAMPvi a inh i bit ionof phosphodiesterase. Kal!:l:fma n et al.

(1987 ) showedthataser i e sof dihydropyridaz one shadva s cul ar smoo t h mus cle rel ax a nt potencies which co r rel a te d directl y with theirability toin hi b i t an isozyme ofcycl i c nuc ler:-tide phosphodiesterase loc ated inthe sa r c op la smic reti..:ul u m of car d i a cmus c le. Theysuggestedthat the vascularrelaxatio n pr oduced bythese agents,whichproduced signif icant increases incAM P, was related to their ability to inh i bita vascular en zyme si mil a r or identicalto this isozyme .

It is also suggested thatincreasedcAMP level s maycause rel a x a t i o n by the requ LatrLon of myo sin light ch ai n kina s e.

The ac t i vity of myosi n light chain kinase is regUla te d by cAM P- d e p e nd e n t prote i n ki nase which diphosphor y lates th e

enzy me and weakens its abi l i t y to bind toandbeactivatedby calmodulin. Evidencesuppo rtingdipho s phory lationof myos in light cha i nkinas e hasbe e n found by Valletet a1. (1981) in vascularsmoot hmuscle .

Inrecentycaee th e reisinc r e a s i ngevide ncein support of a role tor cGM P as a mediato r of vascularsmoothmuscle relaxation. cyclicGMP is though t tome d i ate vasorelaxation by endothel ium derived relax i ng fa c t or (EDRF ) , atrial na triur et i cfa c t or , and thenitr ov asodi lato rs(Khalil at al., 19 87). The inc r easedcGMPle v el s are attainedby activation of guany latecycla seandthe inc reased le ve l s of cGMP le a d to decrease dintracellularcalciumleve l s.

In 1980, Furchgott and Zawadzki di s c ove r e d that the relaxationby acetylcholi neofpr e c ontra c t e dis ol a t e dva s c ul a r prepa rat ions is dependent on the presence of intact en dothe lial cells. It ha s bee n de mons t r a t e d that EDRF directly activates the soluble Laoayme form of guanylate cycla se (Fos terrnan et al., 1986) andit is th ou gh t that EDRF is a reactive oxidiz ed material , analogous to ni tricoxide, thatcangive rise to atre eradical\oih ichactivatesguanyla te cyclase (Murad , 1986).

Unlike EDRF, atrial natriure tic fact or selectivel y acti vates the par t iculate form of guan yl a t e cyclase in vascular smoothmuscle (Winquistet a L,, 19 64 ) and has no

13

effect on theso l u b l e form of the enzyme. Theendothelium independentrelaxant effect of at11al na t r i u r e t i c factor is alsothought to be me d i a t e d via inc r e a s e d ti s s u e level s of cGHP (Wi nqu ist et al., 1984). Ho....ever, Naka tsuand Diamo nd (1989) believethat cGHPturn o v e r in theti s s ue may be more iUlpo r t a nt thancGHPconc e ntra t i o n .

Nitrovasodilators are also though t to mediate smooth muscl e relaxation by activati ng the par t iculat e form of guanylatecyc laseand incre a s i ngintrace l l u larconc e nt ra tions of cGMP (Waldman and Murad, 198 7 ). Thes-xe ct;me c ha n ismOf enzyme activation rema i ns unknownbu t it ha s beensuggested that the seagentsareallca pabl e of gene ra tingni t ricoxide and that this free rad i c al is the activator of guanyl ate cyclase.

1.-4 Comp e ti t ive and noncompetitive in h i b i t i o n

Thisstudyinv o l v e d determining th e effectof DzOon th e pAzvalue ofth e alpha-adr enocep torantagonistprazosin whic h competitive lyblocks th e res p on s e to the alpha-adrenocepto r agonist phenylephri ne.Schild (1947)"',-·fined thepAzva l ueas the neg ative logarithm of the molllr concentration of II comp e ti tive antagonist wh ich red u c e s the effec t of a double conc e nt r a t ionof ago n ist to thatof a single one_ The pAz value shouldals obe an accu rate imlicatio noftheaff i n i ty of an an tagonist fo r itsreceptor(Muramatsuct aI., 19 90 ).

An ag o n i s t is adr ugwhi chWhen coupled wi th the rece p tor in an agonist-receptorcomplex will elicit a respons e.

antagonililtonthe otherhand,may forman antagon is t- recepto r co mp l e x but i t does not produce IS cellular response . compet itive antagonis t is one which wil l sh i f t the do s e response curve of an agonist to th e right in a mann e r prop or t i on alto theantago nis t conce nt r ati o n. However, th e maximal respons e remai ns the same since the er rec e ot the antagon ist can be overcome by raising the. agonist co nc e n t ra t i o n. ApA~val u e maybe determ i nedby usingafi x ed co nce n t ra tio n of an t a go n i s t along wi thgrad ed concentrations of an agonist whi chacts at the same re c eptor.

Al o ng wit h deter mini ng th e O~O effect on compe t i tiv e inhibi t ionthe ef f ecton noncompetitiv e inhi b it i o nw~ seisc st u d i e d. Noncompetitive in h i bi t i o n is a type ofant aqon i s m whi chis not su rmo u n t abl e andthe r efo r e cannot beove r c o me by in c r e a s i n g the concentrationof agon i s t or depola r i z i ng agen t.

The noncompe t i t ive antag o n ist used was ni f ed ip i n e whi ch is referrecl toas a cal ciumantagon ist and blockscalc i u m entry via L-type calcium channels only (Zeli s and Mo ore , 198 9 ). Potass iu m chlo r i d e wa s us e d asth e depolarizing ag ent .

Go df.cai n d et a L, (1986)define a calcium arrt e q onist;as a drug that alters the cellul ar function of calcium by inhibiting its en t ry or release or :"y interfe rin g wi t h it s in trac e l l u la r action. Al t ho u g h Ch urc h and Zs o t e r (1980)

15

suggest an intracel l u lar action fo r nifed i pine, Godfraind (198 3) argu es that theac t i o n of nifedipine is related to blockade of calciumentry.

To determi ne the effect of D20 on the action of nifedipine,a pDz' valuewa s determ ined. The pDz' is defined as the neqat.Lvc. logarithm of the molar conce n tra tion of a noncompeti tiveantagonistwhLchreduces the maximaleffec t of an agonist to50%of itsinit i alvalue (Ariens and van Ros su m, 1957)•

1. 5 Hypert en sio n

In order to gain knowledge in understanding the development of hypertensionin manand tofindmethodsfo rit s control and treatment, studies have been carried out on variousanimal mo dels, the most popular sp ecies ofwhi c h is the rat. Hypertension is mducee in animal modelsby a number of methods including deoxycorticosterone ace t a te administrationandrenal arterycons t ricti o n withor withou t removalof the contralateral kidney(Fieldet a L,, 197 2) but a number of gen etic models of hypertensio n have also been developed. The most commonly us ed strains include the spo nta ne ous ly hypertensive rat (SHR) whichwas de ve l ope d by brother-sister inbreeding and the Da hl salt-se nsitive rat whichdevelops hypertensiondueto a sensitivityto Lno rea ees in dietary sodium chloride (Tr iggle, 1969 ). One pr-cb'lern associatedwi th usingthese genetic models ofhyp e r t e n s ion is

that th e r e is no sing l e ade qu a t e contro l for the models (Clineschmidt et aI., 1970) and often the re s ultsde p end on thetyp eof normo tensivecont ro lwh ichis used for the study. Clineschmidtet ar. (1970) suggested the us e of morethanone st r a in af ra t as a con trol sin ce they found th a t al t hough aortae from SHR did not show a reduced co ntract i leresponse to noradrenal ine in comparisonto National Instituteof Health wistar rats there was a depression of contraction in compar i sonto Carworth FarmWi sta r rats.

A further problem associatedwith the use of geneticall y hypertensiverats arises fromthe fact that different stra in s showvarying resu lts. For example , Massingham and Shev de (1971) showed that aortae from the New Zealand strain of genet ica lly hyp ert e ns i v e rat exh ibitedreducedreac tivityto norepinephrineWhile Clineschmidt et a1. (1970 ) showedtha t the sensitivity of SHR rats was the same as contro ls.

Furthermore, the results obt a i ne d for any part icularst ra i n varydepending onwhether the work is perforllled.inYiY..9.orin vitro. However, generallyinY.i.t.r2 stud iesshow a decrease in the sensitivity of tissues from SHRratsto e-ont.r-ac ttIe agents incompa rison to normotensivecontrols(Tr l gg l e,1989).

Although many problemsexistwith the use of theseanimal models in f o rmation has been qa med contributing to the knowledgeofthedi s e a s e processof hy pe rte n sioninmanand to the developmentof dru gsfor treat me nt.

17

It iswidelyaccepted that the ge ne ti c a llyhype r te nsive ra t deno nstrate svascular res pon s e swhi c har edifferent!roll those of normotensive control s. Mos t re s e arch is geared toward investigati ngwhatdeceradnes the s e vascular reacti vity changes and if they are a cause or a consequence of the hype rt e n sion. In th isstudyan asse s s me ntwas made asto the effect of ~o on the blood pr e s sure of spontaneousl y hy pertens i ve ratsand wi star Kyoto normotensive rats (WKY) , and on the contrac t ile resp onse to bot hpheny lephrine and potassiumchloride .

Previ ou s studiesbyVa s dev et a1. (199 0a ) usi ngSHRrat IO showtha t 020re duc e s the blood.pressure and highin Yi.W.

calcium upt a ke inducedbyphenylephrine viare c e ptor-ope r ated calcium channe ls and by potaS!l;ium chl oride via voltage- ope r ated channels in aorta e. Similarstudiesusing Dahlsalt~

sensitiveratsenevecthat~Onormalizesbothelevatedblood pres su re andin~aorticcalciumuptake (Vas devet al.• 1990b). Since~Ohasbe en shown to normalize cafc Iua uptake in SHR ra t.s andreducebl oodpress ur e itlIa y be expected that the phe ny l e phrineandpotassiumchloride induced contractions of SHR aortae fro m ra t str e a t ed withDzO will not di f f e r from those of normotensive contro ls . In cons ideri ng this hypot hesis, however, it is wo rth noting that va s c ula r reactivity cha nge s using othe r hypoten s i ve agents were not reve r sed once hype rtension had developed (TriggIe, 1989 ). Sinc e this study inv olve s the useof aortae from rats which

ha ve al readyde veloped hype rtens i o n , thi s latte r observation should be takeninto consi de rat ion.

1.6 Aims of the study

1. Todetermineif020has aneffect onvascu la rsmooth mus cle contra ctioninduced by pheny lephri n ewhichstimu l a tes calciumentry viathe re c e ptor-op e r ate d calci umchannel.

2. Ifthe r e is a DzOef fect on phenyle p hrine indu c e d vascular smoothmuscle contraction,to determi neifthe ef fect is reversible.

3. Since vascular end o thel ium re leases EDRF when stimulatedbya range of pharmacologica l agents (Furchgottet a1., 19 8 3 ) , to invest igatewhether the end o t h eliu m plays a role in th e 020 mediated effect on vascular smoothmus cle contraction.

4. To determineifO2

°

^hasan ef fe c t on vascu larsmooth muscle contraction induced by pot a s s i um chloride which stimulates ca lcium entry vi a vol tage-operated calci um channels.

5. Toinvestigatetheeffect of 020on vascularsmooth muscle contrac tion induced by Bay K 6644 which enh a nc e s calcium influxvia voltage-oper a ted cake-tum cha n n e l s under parti a l depolarizing conditions.

6. To determine the effect of 020onvascula rsmooth musc lecontrac tio ninduced by extracellular ca l c i um influx via th e voltage-operated ca lc ium ch a nn el after deplet ion of in tracellularcalcium.

19

7. To assess the effect of 020 on sucrose induced vascular smooth musclecontraction....hichoccurs independently of membrane potential and inthe absence of extracellu lar calcium.

8. To determine whet her 020 wi l l have a select ive ef f ectonvas c u la rsmoo thmusclerelaxationinduced by either is o p r e na l i n e or acetylcholi ne which act by in c r eas i ng intra c e l l u l arconcentrations of cAMP andcGMP, respec t i ve ly.

9. To investigate whether D20will have a sel e ctive effect on either noncompetitive inhibi t ion of potassium ch l o r ide ind uc e d cont raction by nifedip ine or comp etit ive inhibition of phenyle ph r i ne induced contract ionbyprazos i n. 10. To exa mine whether0:0produces a hypoten sive ef f ec t inSHR and WKY rats and, if so, to determine whether there will be corresponding va s c ula r reactivitychang e s.

11. To attemp t to cla r i f y the previ ous l y postul ate d mechani sms of acti on of 020.

CHAPTER 2

MATERIALSAND METHODS

2.1 Anima ls

For experimental procedures 2.4.1 to 2.4.11, male sprague-D awley rats weighing between 100-150 grams purchased from Canadian Hybrid Farms, Nova Scotia, Canada. WKY and SHR rats (100-150grams), for experimentalprocedures 2.4.12 to 2.4.14, were purchased from Charles River Inc., Montreal, Canada. At the time of sacrifice the rats weighed 300-400grams.

The rats were maintained under standard light and temperature conditions (twelvehour light/darkperiod, 20-22

°C, 40-60% humidity). Housi ng was provided by rectangular shoe box cages, made af polycarbonate plastic, with a detachable metal rod lid (three rats per cage). The cages contained Beta-Chipwood chip bedding purchased from Charles River Inc. Dietcone Lst.ed of free access to Purina Rat Chow flT-50l2 (Ralston Purina Company) and ta p water unless otherwise specifiedin the experimental procedures.

2.2 Dru g s andreag e nt s

The list of drugs and chemicals us e d throughout the experimental procedures is:

21

Ac etylcho line chl orid e

Bay K 8644

Calciulllchloride

Deuteriullloxi d e

Dext ros e

Et h yle n e d iami ne t etr a llce t i c ac id

Is o p r e nal i n e

Ma g n es i um su l p hate Nifedipine

Phentolami ne

Phenylephrine

Potas sium chloride

Potassiumdihydrogenphosp ha te

Sigma Chemical Comp a n y , MO,USA

Research Biochemica l s Inc••KA, USA si gma Chemical Comp a ny, HO,USA

sigma Chemical Company, MO,USA

Atomic Ene rgy of Canad a Ltd•• Ont., Can.

DIFCO Laborato r ies, HI, USA

Sigma Chemical Company , MO,USA

Sigma Chemical Compa ny, HO,USA

BDH cneedcars,Ont., Can.

Sigm a Chemical Compa ny, HO,USA

sigma Chemical Comp a ny, MO, USA

Sigma Chemical Company, MO, USA

Fi s her Scie n t i f ic Co., Ont .,Can .

Fisher scient i fic Co., one•• Can.

pxa aoe Ln

Sodium bicarb onate

Sodium chloride

Suc r o s e

2.3 Preparation ofao r t i c rings

Sigma Chemical Company, MO,USA

Sigma Chemical Company. MO,USA

Fisher Scientific co- ,

Ont., Can.

Sigma Chem ical Company, MO, USA

2.3.1 Tiusu e is olat i on

Rats were killed by a blow to the head and e:<sanguinat ed. The chest cavity was then surgicallyopened and the thorac ic ao r t a was removed and placed in Krebs sol uti on of the following compos!tiLc n (inmM): NaCl 118,KCI 4.7. cacr, ^{2.5, KH2PO ~ 1.2, MgSO~ 1.2. NaHCO, 12.5, dextros e}

11.1, and EDTA0.01. These soluteswere diluted in either deionized water or deuter ium oxide, as speci fied in the

ex pe r i me n t a l procedures , and aeratedwith 95%" oxygen and 5%

carbon dioxide. The aorta was then cleanedofall fat, blood and connective tissue and cut into sectional rings of approximatelythree mmin length.

2.3 .2 Experimentalapparatus

Aortic rings were stationed on metal hooks (made from insectpins) and attached to force transducers, by 5-0

23

sil k thread, at one end ....hil e the other end wa s held st at ionary. Pai r s of ring s were lowere d into 25 mL baths hea tedat 38°e, conta ining aerated Kreb s solution. Tens i o n measuredby the for c etr ansduc e r s was re corde d on a Bec k man R611 pol yg r aph. A diagramatic rep r e s e ntat i on of the expe rime ntalapparatusmay bese e n inFigure2- 1 . On egra m of tensio nwas app l iedto the ringsand the y wereequilibr at e d in the Krebs solut i on for at ime specif iedin the expe rime nta l procedures.

2. '" Experimenta l pro c ed ure s

2."'. 1 Phenyle ph rinedoserespo ns e curves

Aortic rings were al lowed to equilibrate for two hourr in Krebssolut ion(ma de upin0, 20,40,60,or 80%DzO by volume). the soluti onbei ngchanged everythirtyminutes.

A dose response curve was the n constructed by adding consecutive doses of phen ylephrine (lxlO-uM tolxl O'~ ) tothe bath containing the aortic rings once contraction to the previousconcent ra tio n ha dre ach ed aplateau . Followingthe con tra ct i o n wit hpheny lephr ine, lxlO-5Macetylcholine wa s added anda relaxati o nnoted toconfirmtha t theendo t hel iumof the tissue wasinta c t (alsoperformedinprotocols2.4.2-2.4.4).

2.4.2 Reversibili ty

Ringswer e allowedto equil ibrateinsixty pe r c e nt D20 Krebs for one hour; Krebswit h zero percent D20was us e d

, .

9~%Oxygen :i %Corbon

Dioxide 25

Fore. Transducer

AortIc Ring

asa cont r ol. A phenylephr inedos e resp on se curve was then constructed us ing lxlO-12M to lX10-~M phen yle ph r i ne. Rings whichha d been equilibrated insixtyperce n t 020Kre bs were then re -equilibrated in re gular Kr eb s for two hou rs. The secondequilibrationtime wa sdoUble thatofth e firstbecause al thoughthe bind i ng rat e con s ta n ts forH+ and 0+ ions ar e similar, the rate constan for theunbi ndi ngofD+ is slower by a factorof about 2.5 (Prod1 hom at a1.. 19 8 7 ). A sec ond phenylephrine contraction dose response curve was then constructed.

2.4 . 3 Mechanicaldisruption of the end o t h eli um Afterthe aorta had been removed, it was cut into two sections. One section had the endothelium mechanica lly disrupted by gently scrapi ngthe insideof the aortawitha tube of soft plastic. Thre e rnmring s were th e ncut anda ring with an intact endotheliumwa s equilib ra tedin the same bath as a ringwitha disrupted end ot h e l ium. Ti s s ueswere setup in organbaths containing sixtypercent020Krebsor regula r Krebs as the contro l. The tio:;sues....ereallowedto equilibrate fortwo hours andth e n a phenylephrinedose response curvewas performed. Acetylcholinein duc e d relaxationwas demonstrated to be absent toconfirm that the endothelium scraping was effective .

2.4.4 Potas siumchloride doseresponsecurves Ringswere allowedto equi l ibrateinsi xtypercent

27

020 for two hours (regular Krebs for controls) . Krebs solutions were thenmade up withvaryi ng concentrationsof potassium chloridefrom 9.6mM tl)65.8 rnM. The concentration ofsodium chloride added was also simul taneouslychangedso as to maintainan isotonic sol ution. A dose responsecurve was then constructed by changing the bat h solutions and SUbstituting Krebs solution containing increasing concentrationsof potassium chloride.

2.4.5 Ca l c i umdoseresponse cu rve s

Aortic rings were permitted to equilibratein either sixty percent DzO Krebs or r-equf ar-Krebs for two nours.

lxlO-~Mphenylephrineinducedcontraction was then measured to confirm the presence of the 020inhibitoryeffect. The rings were then washed in calcium free Krebs solution,containing 60 mMKCl, at fifteen minute intervals untiI no contr-a-it.Lon reep on e e to KCl couldbe elicited (at least one ho u r) ; th e ca lc i um free Krebs was alsodiluted with either sixty percent OzO or H20. A.calciumdose response curve was then constructed by adding increasingconcentrationsof cecr,fr om 0.5mMto5 roM.

2.4.6 BayK8644dose re s p o n s e cu rves

Aortic rings were allowedto equilibrate in sixty percent020 Krebsor regUlarKrebs for two hours. The Krebs solutionwasthe neub stLt.uted forKr e bs solution containi ng10 mM KCl; lx l O-⁶M phentolamine was added to block receptor

oper a tedcal c i um channr,ls. A Bay K 8644 doseresponsecurve wasthe" cons tructed usinglXlO·~tolxlO-~Bay K 8644.

2•••7 sucroseinduc e d contrac tion

Aortic ring s wer e permittedtoequi l ibratein ei t h er sixty perc e nt 0,.0 Kr eb s or regula r Kreb s tor two hours. A sucr os econt r ac t ion respo nsewas then perforwled using250mM

2^0••9 Is oprenalineinduced relaxati on

Ringswerepermitted to equilibratein eit herc;ixt y percent 020 Kreb s or regular Krebs for one hou r. phe nyle ph ri ne indu c edcontractionrespons ewasthen mea sur ed ....ithlx l0'~phen yLe ph r-Lneto confirmthepr e s e nc e of the Dzo inhi b i t oryeffect. Aft erwashingtor one fur ther hour th e ti s s ues equ.li bra t edinregularKr e bswereexposed to lxl O-'H phe nto l a mi neto bring thecontracti on levelst."thesame level astis s ue s incx;,ated in sixtypercent U20 Krebs. Asecond lxlO-'i1; phen ylephrine inducedcontracti onwa s then perf o rme d and the precontra cted ti s s ues we r e relaxed with lxl0-~M is opre nal ine .

2.4. 9 Acet.ylcholi n e induce d re l a xatio n

Following the fina l phenylephrine control dos e responsecurves in the absence of D:.Oand inthe presence of si xty per c ent D20 in exp e r i me nt a l pr oc e d ur e 2.4.11 an acet y l c ho li ne dos e re sponse curve wa s const ruc t ed using

2.4. 1 0

29

lxlO-!CMtolX10·~Macetylcho l i ne.

Determinationof pDll fornifedipine

Tissues were equilibratedin ei ther sixty peecent, 020 Krebs or regula r Krebs fo r two hours . A potassium chlori dedose responsecurvewa sthen const ructe daccordingto the metl,od statedinex perimentalprocedure2.4.4. Subsequent re spons e curves were cons t ru cte d on the same ti s sue s pre- incubated withthreedifferentconcentrations ofnife d i p i ne: Controls were performed

2. 4.11

simultaneouslyin which four dose response curves topotassium chloridewere constructedwithoutthe additionof nifedipine . The pOl' wascalculated according to the following equatio n derivedbyBickerton (1963):

pDz'

=

pDX' + log ( Earn/ Eabm - 1]

where: pOx'= the negative logarithm of the mol ar co nc e nt r a tion of nifedipine

Earn = the maximal cont raction height inthe absence of nifedipine

Eabm

=

the maximalcont rac tion height in thepresence of nifedipine

Determ inationof pAzfor prazosi n

Tissueswere incuba ted ineitt.""rsixtypercent 010 Kr e b s or regUlar Krebs for two hour s. Aphenylephrine dose

2....12

response curve was then constructed using IxIO· 12M to lXlO'~

phenylephrine. SUbsequentresponse curveswere constructedon the same ti ssu e s pre-incube bed with three different concentrations of prazosin: lXlO-9M, lX l O-aM, and lxlO-1M.

Controls were per formed simu l taneously in which four dose response curvesto phenylephrinewereconstructedwit houtthe addition of prazosin. The pA2was calcula tedaccordingto the Schild Plot method as descr ibed by Schild (1947 ).The Schild Plotisa plotof log (X'iX)-1versus-log (A) where:

x'/x= the agonistdose rat i o A=theantago nist concentration

For a competitive antagonist, theli ne has a slopeof -1 and the intercepton the abscissa corresponds topAz.

Bl oo d pressure me a s ure men t s

Sy s t olic bl o od pressure was measured by tail plethysmog raphy according to the method describedby Byrom (1969); signals we r e amplified using a mode l SAamplifier (ITTCLi f e Science Instruments,california,USA). Fourbl ood pressure measurements we r e recorded beforethe animals were sacrificed.

2.4.13 Chronic!!! vivo st u di es usingtwenty-rivepercent

D ,.

WKY and SHRra t s we r e treated as in section 2.1

31

un tilseven weeksof age and at th atti methe drinking water was rep l a c e d by eit he rdi s t il l e d wa te ror twenty-fivepe r c e nt 020in distil led waterfortwe lvedays. Theanimal s were then sacr i f icedand thetho rac i c aorta remov ed. Aort ic ringswe r e equilibrated in regularKreb s for two ho u r s follow ingwhi ch a dosere s pon se curve was const ructedusing either lx l O·^{1 2M} to lx l O - ~Mphenylephrineor 9.6 rnMto 65.8mMpo t a s sium chloride by the me t hod s sta ted in expe r ime ntal procedur es 2.4.1and 2.4.4 (only regul ar Krebs solut ionwas used).

2.4.14 Acute!nVit1'Ostudies usingsixty percent020 WKY and SHRrats weretr e ate d asin section 2.4.13.

Experimental procedures 2.4.1 and 2.4.4 were the n performed (both r-e quLa z- Krebs so lution and sixty percent Dzo Kre bs solutionwereused).

2. 5 Analysisof data

Resultsare expressed as mean±standard er r or of the mean. Whe r eno S.E .M.is indicated thelev e l oferrorwastoo small to bede t e cte d wi t h the means ofrecor d ingandanalysis employed. Tissue re s pons e s are expre s sed as pe r c e nt age maximum contraction or relaxa t ion , whe r e th e maximum cont rac tion or relaxat i o n (lOOt) corresponds tothatof the controltissue in each group.

Maximumcontractio ns be twee ngroups were compa re d us i ng anunpaired st ude nt 'st- t est. Significanc e....as acceptedat a

lev e l of p<0.05.

Plots were -:onst ru cte d usingsigmaPlot, scientifi cGraph System. The pA2 and EC50values vet-e ca l c ulat e d using Ph a rma c ol og i c Calculat ionSyste ro, version4.1byTal l a ridaand Murra y (Sp ri n g e r-Ve rl a g New York Inc.,19 87 ).

33

CHAPTER 3

RESULTS

3. 1 Effectof020 onao r ticsmootb mU9c l e contra c t ion

3.1.1 Pheny lephrine induc e d con t ra ction

In nonnal Krebs solution contractionof aor ticrings using l)CIO'~M phenylephri ne result ed in a maximum control contractionof1.08±0.11grams. Aorticri ngs incuba tedwith inc r e a s i ng concentrations of 010 ha d increasinglydepressed phenylephrine induced maximal con tractio ns (Figure 3-1) . TwentyI forty, sixty, and eighty percent D10 depressed the phenylephrine induced maximum con tractionto 86.9%±12 . 6%, 74.3%±5.0%, 51.9% ±6.5%, and 42.4%±3.7%of the control value re s pe c t i vely. The contractions were significantly depre ssedfrom forty percentto ei g htypercent 0₁₀.

Figure 3-2 shows theeffe c t of varyingconc e ntr ati ons of 020 on the entiredose response cu rv e cons tructedusing i.xtn"

l2M to lxlO-'M pheny lephrine. Twenty, sixty, and eighty percent D20uni f o rml y depress thedosere s po ns e curve compared to the control curve. Howev er ,forty percent 020re sulte d in enhanced contrac t ion fro m lxlO,uM to lXlO'IMpheny l e phr ine.

The dose response cuz-ve wa s de pres s e d from lx l O-7Mtolxlo-~

phenylephrine.

with varyingconcentrati o n s ofD2.0.

Columns represe nt mean percent maximum contraction and bars S.E.M. for eight tissues. An asterix represents a si g ni f i c a nt difference from the contral contraction (p<0. 01 ).

35

~

Ow

CXl O

~C X

CD::;;:

::J

~~O~

'¢::J W

~O 0 1 - N Z W 1-• • • • • • • • • •

~

^o

^{o ~}

0 0 0 0 0 0 0

N 0 CXl CD '¢ N

NOI1::>'v'~lNO::> V'm~lx'v'~ IN3::>~3d

smoo t hmu s c l eoontraction.

Dose re s pon s e curves were constructed using lxl O-lZM to lxl O,sMphenylephrine.

Points repre s e nt mean percent max i mum contract ion and bars S.E.M. for eight tissues.

J7

'<I"

I

U1

I

2:

<0 W

I D:

Z :I:

0...

r--. w

1 --'

>-

ZW

eo

:I:0...

0 0 0 0 I _u,

ONNNN 0

NClOClO

0<ll!~<ll! ~ Z

Ol 0

~o o o o I

~

ONv<OlIJ

00<1<1. ....

III II

⁰

^{z w}

0

00<1<1. z

0

~ 0

~

N

~

0 0 0 0 0 0 0

I

N 0 <Xl

'"

^{V N}

NOIIO~lNOO

rln rl lXVY'llN30H3d

3.1.2 Reversibility

Re ve rsa l of the ao r tic smooth musc le inhibit o r y effect of sixty percent 020 on phenylephr ine induced contra ction is shown in Figure 3-3 . The firstcontr a cti on dosere s pon s e curves denoted byDiOa and HzOaare signficantly different(p <0.05). HzOacorre spondsto the firstco n t r o l curveconstructed in the absence ofO2

°.

ozOa correspondsto thefi r s t dose re s pons e curveconstr ucted inthepre s e nc e of sixtypercen t O2

°.

^'l'he020ha s reduce d themaxi mum contra ction to64.9\±9.6\ of th e cont r ol value. However, the second phenylephrinedose responsecurvetwohour s followi ngremoval ofO₂

°

^{(OzOb ;} Fi gure 3-3) isnotsignifican tlydiffere n t from the second contro lcurve(RzOb; Figure)-3).

3.1.3 Roleof the endothe l ium

Me chan ica l disruption of the endothe liumhad no effect on th e cont ro l phenylephrine dos e response curves (Figure 3-4~H~Ointact/scraped )or onthe depressed curves of tissuesincubated in sixty percent D~OKr e b s solution (Figur e 3- 4~ D~O intact/scraped). A le vel of aortic contrac tion similar to that previ ouslyobs erved was demonstra tedin all tissu esinc u ba ted with either regula r Kre bsor sixtypercent D~OKr e bs solution.

3.1.4 pot assium chlorideinduc ed contrac tio n Six ty perc entD~Odepressed thedo s ere s pons e curve constructedus ing9.6tuMto 65. 8mMpotassiu1l' chl oride(Figure

Figure3-3: Reversalof the 0₂₀inh i b i t or y effect on phenylephrine induced aortic smoo t h mus cle cont r a ction.

H20 a/b represent th e first and second control curves respectively. D20a represents the first curve of tissues exp os e d to sixty percen t ~O Krebs solution; D_20b rep res e n ts th e second curve after removal of O2

°.

^Points

represent percent max Imux

contraction and ba r s S.E.M. for eight tissues.

'<t I

---.

...

^~

... ^{...0 ....}

^It)

_I C _w

\ \ \

^<0

^1'2

^:I:

^z

...

^~...-

...0 ....

_I

^o,

~\ \ ^{r-- w}

^...J

^>-

^Z

^w

~+ ~~

^{I :I:c,}

^z

00 0

,

^Ol

^I 1 ^~

^0:::I-Z

^w o

~

^Z

0..00.0 0

0 00 0 0 U

NNNN

o

ClCl:C :I:

I

I 0

O.<I~ ...J

III I

^~~

O.<I~

I

^N

0 If) 0 It) 0 It) 0

1

It) N 0 r-, It) N

NOJIO~lNOO

rl n ViIXVVi

IN30 ~3d

Fiqure 3-4: Theroleof the ondotheliumin theeffe ct of sixty percent Dp on phenylephrine inducedaortic smoothmusclecontr action.

Points represent mean percent maximum contraction and bars S.E. M. for eight ti s sue s.

. - . 020 SCRAPED

i=

D-D H20 INTA CT T

~ ¹⁰⁰ _{. - .H20 SCRA PED} ~~::::::::::,

a::

I-

z

0

(J

75 J ' ' ^e

::;: • __T ::::'

:::>

::;:

~ ⁵⁰ T/9

::;: »> ?

I-

z

"J

25

(J

a::

w

a.

a -12 -11 -10 -9 - 8 - 7 -6 -5 -4

CONCENTRATION OF PHENYLEPHRINE (M)

43

3-5 ). The controlJIlaxima l co nt ract i on ind uced by 65.8 DIM potass iUlllchlor idewas1.15±0.10gr amsandsixty percent~O depressed thepotassium ch l orideinduced mll.xilllWi contraction to 51.9\ ± 6.5' as compa red to the cont ro l va l ue. The difference in the potassiun chlori de dose respo ns e curve, constructedin D10Krebs , froDthe contro l curve,co n s tructed in reg ula r Krebs, wasstatistica llysi g n i fica nt (p<0.02).

3. 1.5 Ca laium. ind u c edco ntraot ion

A calcium indu ce d dos e respons e curve was co ns t ruc t ed after the co nt r a cti o n re spons eto 60roMKCl had be en elimina t ed by wash i n g aortic rings with ca lci umfree Kre bs solut i on . The levelof theco ntrol maximum contra c t i on at 5.0mMcalciumchlorid e was 0.93±0.07 grams andsixty percent D:O significantly lp < 0.05) reduced the maximull calcium contraction to 76.n ± 5.9 ' as compared to control values (FigureJ-6).

3.1 .6 Bay It864411t:IC!ucedcon traction

The dosere s po n securve con structed usin g1xl0-8Mto lXI0-~Bay K 8644 wassignifica n t l ydep r essedby si x typer-cent;

0:0 (p<0.05). The maximum control re s pons e inth eabsence of DzOwas 1.16±0.11 grams. Sixtyperc entDlQdepresse dthi s co n t r a c tio n re spons e to 65.8% ± 11.2' as comp a r e d to the max imu m co nt r ol val ue (Figure3-7 ) .

smo o t h muscle co n t r a c t i o n.

Dose response curves were constructed usinq 9.6 ltIM to 65.8 mM potassium chloride. Points representmeanpe r c e nt maximum cont ractionand bars S.E .M. for eight tissues.

45

0 I'-

... -<

- 0 -

\ ^\ \ ^a

^to

...-<

- 0 -

\ ^\

^0L()

_~

... 1-0-

\ ^\

^0'!"

^s

^-lU

... >-0-

::£

~\

^l.L.0

0 0 ⁰ Z

N N ...-0-< ^I") 0

ClCl

<, ^~

ClIt Cllt

~O

0 ^f-Z

o.

^{N W}

~ ^o

I I ^z

⁰

O.

⁰

^o

0

L() 0 L() 0 L() 0

N 0 I'- L() N

~

NOI1:)'v'~lNO:)

rinV'llx'v'V'I

IN3::J~3d

ill the pres~:!e!J of 60 mM potassium chloride .

Dose re s p o nse curves were constructed using1.0mMto5.0mMcalciumchloride . Points represe nt mean percent ma.ximum contraction and bars S.E.M. for eight tissues.

47

to

....0-< ~.~ Il1

I ^{I ~}

1-0 ... ....

-4

.r -5.

\ \ ^{-c o}

^u,

^{+ +}

....0-< ~. ~

n

₀

\ \ ^z

⁰

0 0

~

_et:

NN ^~O... ... ... N I-

0 0

\\ ^{z w}

~~

o

0 0to Z0

o.

⁰⁾

II ^1-0...

^{1 - .-4}

"''''

o. _~O~.

I I I

I ^~.O

Ll) 0 Il1 0 Il1 0

N 0 r-, Il1 N

~ ~

NOll::J\I~lNO::J ~nrm(\I~ IN3::J~3d

muso l e oontraot ion.

Dose re s pon se curves were constructed using lxlO-9M to lx l O-~M Ba y K 8644. Poin ts rep r e sent mea n perc en t maximum contraction and ba r s S.E.M. for ei g ht tis sues.

49

.r I

>-...

1--0-

^l()

\ ^I

^<0I~

^{2- .r}

^'<t

^'"

⁰⁰

".~

^I~IIIZ0>.

0

I'-~

0 0

~ ^10::

^l-

N N Z

0 0

\\ ^w

<R<R

^U_Z

0 0

000

to

. 0 1--0"- _I

^U

I I o ^o

^--l

. 0

en

l() 0 l() 0 l() 0

1

N 0 r-, tn N

NOll:)V~lNO:)V'lnV'lIXV~~ IN3:)~3d

:1.1 . 7 Sucrose induc e d contraction

Following incubation or aortic ri ng s in regular Krebs a contraction response to 250^mMsucrosewas performed.

The amount of contraction of the control tissues to the sucrose was 0.23 ± 0.0 1 grams. Sixty percent DzO significa ntly (p <0.01) re d u c e d thecont ract ion to 5S.6%± 0.0\ofthe control value (Figure3-8).

:1.1 . 8 syn o psis

DzOinhibited pheny lephrine induced aorti c smooth muscle contractio n in a dose dependent manner with approximately50% inhibition using60\ 020. This inhibitory effect was endothelium independent and reversib le. sixty percent DzO alsosignificantly inh i b i t e d potassium chloride, calcium, Bay K 8644.and sucrose ind uc e d aortic smoot hmuscle cont.zact rc n.

3.2 Effect of DzOon aortic smooth muscl e rel axation

J.2 .1 rs oprenal ine induced rel axation

Ri ng s equilibrated insixty percent

°

2°Kr e bs and regu lar Krebs were precontracted wit h IXI 0·~M phenylephrine before relaxatio n with IxlO -'M isoprenaline. The level of contractionof tissues incubated in regular Krebs was 0.69± 0.01grams while th a t of tissues incubated in sixtypercent D20 Krebs was 0.46± 0.01 grams. Treatment of the rings equilibrated in regular Krebs with Ixl0·~fo1 phentolamine

Fi gure3-8: The inhibitory effect of si x t y pe rc e nt DzO on 250 DIM sucrose ind.uc ed aortic smoo t hmu s c le contraction .

Columns re p r e s e nt mean percent maximum cont rac t i on and bars S.E.M. for four tissues . asterix indicates significant differe nce fr om control values (p<0.01).

w a o X :::;;

~

i2 w ...

e a

!z _w

~ ~ _w

n,

o o o

lXl

o

10

o ..,. ^o

_N

^o

NOI10V~lNOO Y'lnY'lIXVY'llN30~3d

53

rCdulted in a second contraction response to lxlO'flM phenylephrine of 0.69±0.01grams. Thesecondcontrac tio n responsefortissues equilibra tedinsixty percent OzOKrebs was 0. 6 6±0.02grams and this le ve l of contraction was not signi f i c ant l ydi f f e r e nt fr om the second contractionresponse of tissuesequilibrated inregularKrebs and phentolamine.

Si xt y percent DzO significantly (p <0.01) re d u ce d the re laxat ionrespons eind ucedusinglxl O-'M isoprenalineto50.3%

±5.7%of themax imumcontrolresponse (Figure 3-9 ) .

3.2.2 Acet y lcho l i n e indu ce drelaxat i on

Priortorelaxationwith acetylcholine,aorticrings were precontracted with lxlO' 3Mphenylephrine. For rings equ i l ib ra te d in regular Krebs solution the level of cont rac t i o n was 1.39 ±0.0 8 grams :the level of contraction for ri ng s equilibrated insi x t y percentDzO Krebsso l u t i o n was 1.06±0.07grams . Sixty percent DzOsignificantly(p <0.05 ) re duce d the maxi mumrelaxationto 76.7%±7.7%as comp are dto the control va l u e of ti s s u e s incubated in regUlar Kr ebs so l u t i o n (Figure 3-10). Sixty percent DzOalsoshifted the dos e response curve of acetylcholine to tole right. For tissues incuba ted in r-eqirLar- Krebs solution the EC~o was 9.6 2 xl O·bM while for ti ssues incubated in sixty percent DzO Kr e b s th e EC}Owas 5.64xlO-1M.

smoo t h mus clerelaxation .

Columns represent mean percent maximum relaxation and bars S.E.M. for eight tissues. An accexLx indicates significant differencefrom the control value (p<0.01).