Hemodynamic vascular forces contribute to impaired endothelium-dependent vasodilation in reperfused canine epicardial coronary arteries

1216

JACC Ve1. 23. No . 5 April 1994:1216-23

EXPERIMENTAL STUDIES Hemodynamic Vascular Forces Contribute to Impaired Endothelium-Dependent Vasodilation in Reperfused Canine Epicardial Coronary Arteries PAMELA OUYANO, MD, FACC, LEWIS C . BECKER, MD, FACC, MARK B . EFFRON . MD, FACC. AHVIE HERSKOWITZ, MD, MYRON L . WEISFELDT, MD, FACC ialdmorr. Maryland

t7¢iealre. We studied eelne area" eel rtd vpareaedrhy alter =man and reperfudon to used= whether redead law a pnmae cehhuted b the ahoesaltlta obenad& iwnyremsdd lac ado and repdidam sitar eddbial and gyecadW New=. Caadve fiselana may laude shard the, mm$ement etvaele or free radical

predicting

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War In-S do 1g%D and datrnad ore pkeylupketoe (10'' mollies 17.6 s 7%1). After reperhdea . A+t~ Caused 133 s 9% van emuaridkoe Mkreprdde and phenyleplerre

reppne was earhayd. Rep f=ad mum eeaaena dm *D,Md bapahvd vowma b rvapum b (0' 6 molllller of aesybbo8ae(1g.6 s 5.1% vs.47.1 s 4.9% b aatrd anal) and

Me*4de. The coronary srMrW of emlhelYad, span clone dogs were subjected to 90-de ocdmion s2 h orrepaide. The

Irnardledbradybiab (4.4 t 6.7% vs 273 x 8% b sachet

egret of reperNWn en arterial response ; he bdaaaaary seyd-

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ddlne, dtropnumlde and idessylephrime was alleged wing be

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Too endothelium appears to playz a major role in the main-

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Several Investigators have reported that after coronary

con-

artery occlusion and repatriation, the vasomotor response of

strictor substances in response to a number of pharmaco-

the epieardial coronary artery may bean= abnormal . Cor-

logic and physical editing (1). Edothellum-derived relaxing

ollary rings taken from segments of comnary artery exposed

factor wall first described by Furchgat and Zawadski (2).

to pehemia and reperNsin dilate normally with nitroptua-

who demonstrated that vascular relaxation was obtained in

side but not with acetylcholine . thrombin, scrotonb or the

response to acetylcholine

in the presence of an intact

calcium lonophore A23187 (6-10). This response pattern is

endothelium (3). Since that time, many receptor-dependent

consistent with loss of normal endothelium dependent vaso-

and receptor-independent stimuli of enddhetium-derived

dilation involving endothelium-derived relaxing factor re-

reining factor have been found (4) . Vasadiletion in response

lam

to these substances (e.g., aectylcholine, brodykinin, seroto-

have been demonstrated in coronary mkravcmls after

nin, thrombin) may be converted to vasoconetdclia after

myocardial isehemia and roperfission (10 .

only

endothelial injury .

from the vascular endothelium . Similar abnormalities

The factors mediating abnormal endothelium-dependent vasodulation after coronary artery occlusion and reperfin on remain unknown . Emlothelial cells arc considered to be

Form the Divides of Cardiology, Jab=s Hopkins Medical lpmmtims .

relatively resistant to hypoxia (12). However, hypoxia and

IWaare. Maryland. This study was supp=ed by Grant 2P MNL1%yf, Ischemic Hears Drsmw . Specilbed Center of Rem". Nations) Head.

aoxygenatin appear to cause molecular changes that in .

Lung, and Rloodlenimm . Nadsd aatame ofHmhh, Betheed ., hesyled .

Mmascrtpt received August b, 1992 ; revhed mnmmpteaaeived November 22,190 . ecoapmd December 1 . 1993. :Dr.pmld.ayang,Dnoim,pfCbedolagy. B-1.5, Frmca Scot Key Medal Center, 4940 Rmmo Boulevard. Bald. a=re, Marybd 21224 .

01994 by

•

the A-. College of eardioba

duce increased neutrophil adherence to endothelial cells in vitro (13,14) . In vitro studies suggest that hypoxia and endoreoxygenatin may result in free radical release from thelial cells (13) Further endothelial cell injury could be .

caused by activation of serum complement or blood

d

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JACC W1 23. 9ia .5 Ap 1 1990:1216-23

OUYANC E : AL. UOn CURORARY VA5OAC- nVITY

APrER ISLUEMIA

1217

cehvcardaooraplud . One of the knots was kept visible on the image . The maximal cross-sectional diameter of the vessel was recorded during systole and diastole for several cardiac cycles. A small Silaatic catheter (outer diameter 0 .025 in. (0 .063 cm)) was introduced into the proximal coronary artery to allow infusion of phmmacologic agents . The vasoactive drugs were dissolved in 10 ml of saline solution and given at a rate of I mUmin by means of a Harvard pump to achieve the desired concentration in the blood, assuming a coronary blood flow of 100 mllmin. Separate syringes for each drug eonccntrmion were used . The vessel was imaged during infosien of saline solution and during infusion of acetylcholine _° (10-" to 10 -5 malditer), tdnoprusside (l0 to 10-5 mid/liter) 9 and pherylephdne (10_ to 10-s moliliter) administered in random order, Each drug was washed out with a saline infusion before the vessel was exposed to the next vasoactive agent . Images were obtained at baseline and 30 min after Methods reperfusion after a 90-min coronary artery occlusion . The Canine model of myacars 1 ischords and reperfusion . images were recorded on videotape and later reviewed using Male or female adult mongrel dogs were anesthetized with a Hewleet-packerd 7020 AC Ultrasound Imaging Machine . intravenous thiamylal (12 .5 mgikg body weight) mid intrsThe maximal cross-sectional images obtained during diastole mustmMr chloralose (I00 mgikg in urethane) and intubated for each intervention were printed on a paper print-out • and and ventilated with room air using a Harvard respirator. A the cross-sectional intralumen area was obtained by planimthoracotomy was performed in the left fifth intercostal etry. The images were digitized from the paper printout in a space. and the heart was suspended in a pericardial cradle . A blinded manner to obtain the coronary cross-sectional area . peuaisal segment of either the left anterior descending or In vitro evaluation of cvwary reactivity after in vivo left circumflex coronary artery was isolated, and a Mack silk ocrtmlm and reperi'mlon. In another cohort of dogs, coroligature was loosely placed around h . The artery was then nary reactivity was assessed in vitro in vascular segments occluded for 90 min. removed from dogs subjected to in vivo coronary artery Rent nest of intravau,isar oxygen saturation, In three occlusion and repeufirsion. In Group I (n m 16). the hearts dogs a flberoptic oxiuxter (in vivo Combimeter System Were removed at ihx end of the 90•min coronary occlusion, IVS4000 fibpoptic catheter, Schwarzer) was inserted into a and in Group IL (n = 111 hearts were removed after a further distal branch of a left anterior descending diagonal coronary 120-min repeALsion . Ventricular fibrillation occurred either artery and was advanced proximally until it was positioned during mrotmry occlusion or after reperfusion in two Group just distal to the she of the ligature placed around the 11 animals, and resuscitation was performed with cardiac proximal left anterior descending artery . In[mvascular oxymassage and ihirathoraeie defibrillation. No vasoactive gen saturation was measured before occlusion . throughout drugs were given. Two animals could not be resuscitated 90 min of occlusion and during reprtrfusion, from ventricular fibrillation after reperfusion and were not 6ehtnrdlegcaphle evaluating of memory reactivity after included in the dam . lo rlra sedualem ad reperlhxlan . The lumen diameter of the A 4- to 6-cm length of epicaadial coronary artery, at least left anterior descending coronary artery was measured in 2 cm distal to the occlusion, was carefully excised along with four additional open chest dogs by use of a high resolution a strip of surrounding myocardium, The proximal portion of echo transducer placed directly on the anterior surface of the the coronary artery was cannu)ated with a 16-gauge Teflon left anterior descending coronary artery just distal to the catheter, and the vessel was perfitsed with ice cold Hopes takeadof the first diagonal rtery and m least 2 cm distal to buffered crystalloid solution . All side branches were identia silk ligature placed loosely around the proximal portion of fied by gentle perfusion of the coronary artery and were the vessel . The probe was held by hand over the artery, and pressure was adjusted so that the circumferential image Wed . A segment from the unoccluded coronary artery was then removed in a similar manner (Mg. I) . The isolated showed acicularcruss-sectional area with no indentation of vessels were perfused in a recirculating system with Hepes the wait . The image probe had a 12-MHz scanning frebuffered crystalloid plafusnte (sodium chloride, 149 mmoll quency, 2 x 2-cm contact surface, 2- to 3-cm depth of field and a phantom two-point resolution of 0 .1 mm. The imaging liter : magnesium sulfate • 7 water, 1 .0 mmopliter; potassium chloride. 5 mmdAiter; dextrose . 10 mmolAiter, calcium probe was connected to a commercially available recording chloride, 2 .0 mmolAiter; Hepes. 5 mmodlfiter) at pH 7 .4. The instrument (Surgiscan Biosound Corp .) (21) . The position of the transducer was k °!%t c^^..oant by placing a monafilament solution was bobbled with 100% oxygen and maintained at each side of the region of the artery to be 30'C. The vessels were perftesed by a peristaltic pump at a ligature knot at

meats. such as platelets or neutrophils (b •3 7) . Diminished Dow and pressure accompany coronary artery occlusion followed by rapid increases during reperfusion that may lead to alterations in vascular endothelium through changes in shear stress. Shear stress and mechanical forces have been shown to influence several membrane functions of endaha dial cells, including transmembrane ionic currents (19,19) and intracellular pH (20). The goal of this study was to evaluate the role of altered vascular hemodynamic forces in the development of abnormalities of endothelium-dependent vasodilation after ischemia . By studying the responses of petfused canine coronary artery segments ex vivo, we were able to separate the contribution of flow- and pressurerelated forces from the complicating effects of blood proteins and cells.



OUYANG Er AL DOG CORONARY vASOACRVRY AFea ISCHEMIA

JAM Vol . 23. No. 5 Apil 1994 :1216-23

obtained from Sigma Chemical Company. All drugs were freshly prepared on the day of the experiment. Potassium chloride, uk opmsside and acetylcholine were prepared in dishiled wafer . Bradykioin was dissolved in phosphatebuffered saline solution containing 0.151 gelatin as a 10' muifliter stock solution. ludomethacin was dissolved in a minimal amount of methanol and brought up to volume with phmphate-buffered saline solution to achieve a stuck solution of 10" 2 molRtmr. An aliquot was added to the pelfosate to achieve a final concentration of 10 - ' moitliter of indomethaein. Solutions of potassium chloride, nhropmsside, acetylcholine, bradyPinin and irKloincthocin were freshly prepared for each experiment, and concentrations quoted are the final concentrations in the bath perfusate . Statistical analysis . Dane-response curves for potassium chloride were plotted for each vascular segment and normalized for the maximal perfusion pressure developed . The vasodilator response to a (bug dose was quantitated using the observed decrease in perfusion pressure as a percent of the increase in perfusion pressure obtained after partial contraction with 25 mmelditer of potassium chloride . Because low was maintained constant throughout the expon meat, changes in perfusion pressure correlated with charges in vascular resistance . Vessels exposed to in vivo coronary occlusion or reperfusion were compared with central vessels from the same animals . The in vivo coronary artery responses were analyzed using the maximal diastolic diameter during saline infusion as the baseline measurement . The dAtdioes or cvoauiction produced by the I0' moVliter dose of the vasmetive drugs studied was expressed as a percent change compared with the baseline diameter. Dam me presented as mean value ± SEh4. Comparison between groups were made by analysis of variance with the Sche fe test for multiple comparison . Significance was considered at p < 0.05 .

Results ®ten of mommy mchtslar and repeflnlon on in vies rapine a of eploedrl =many aAeits Is vmauilve agents . Before occlusion, thediesmlic mms-sectiond area of the left anterior descending coronary artery, determined by high frequency ultrasound. was 2.7 ± 0.2 mm2 during infusion of saline solution (Fig. 2). The cross-sectional area increased by 10.5 ± 9% during inltslm of 10' mobliter of acetylcholine and 22 .5 ± 10% during itdusion of 10-5 mol/iter of aitropmaside and decreased by 7.6 ± 7% during infusion of 10-5 mdlliter of pheeylephrine . During proximal occlusion, the epicm6d coronary artery collapsed, and only a very small intralumen echo-lee space was visible. After reperfumon after 90-sain occlusion . cross-sectional area was 2 .2 0.2 min" during saline infusion p = (III compared with cross-sectional befre occlusion). Instead of increasing, the area now decreased by 13 .5 t 9% during acetylcholine infusion (p < 0.05 compared with before occlusion) . In contrast, the responses to nitropeusside and phenylephrine

1219

ripae 2 . Top, Percent change in maximal diastolic cross-sectional errs of the kit aaeerine descending coronary artery in response to tmmaomeary admidsnalon of 10 -' =Miter of acetylcholine IACItI, nitrop oadde (NPI and pheaylephrine (PHS) . Mean tospome Y SEM before and after 90-min coronary occlusion and reperfasion are shown (n = 4). set=a, Diastolic blood incisors at baseline (OL1 and during iniaion of acetylcholine, mtro(amside and phenykphrise before and after 9g-mho coronary occlusion and repetfustar.

were unchanged (increase of 50 .6 ± 34.8% with nitroprusside, decrease of 14.1 ± 7% with pbenylephrine) . As shown in Figure 2. the difference in acetylcholne response between preocdusion and occlusion and reperltsion could not be attributed to a diftrence in blood pressure . Elect of in vivo cereonry oedusfna and repelmion on vascular smamh muwte responsiveness . Exposure of perfused coronary artery segments to increasing concentrations of potassium chloride resulted fn a curvilinear increase in perfusion pressure consistent with an increase in coronary artery resistance (Fig. 3). No differences were fond in the dose-response curves ammng control vessels, vessels subjeeted to occlusion only (Group 1) and vessels subjected to occlusion and reperfusion (Group Il) . The vasodilator respoons to nitropmsside are shown in Figure 3 as a percent of the contraction obtained after preconstriction with 35 mmoNiter of potassium chloride . There were no significant differences in the dine-response curves in response to cumulative addition of nitropeusside among control and

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OOYANG ET AL. MG CORONARY VASOACr7VITY AFTER 1SCHEMLA

the right for Group 11 vessels . Higher concentrations of bradykinia resulted in an increase in perfusion pressure . signifying vasoconstriction . This effect was more marked in Group I and TL vessels than in control vessels . Emmluellsn of fades during occlusion and reperfusiou that n 5y attar subsequent eodathdium-dependent rmometor :oapossivenma. Byporia . Oxygen saturation in coronary artery blood, measured by catheter oximeterdownstreans, from the snare oceluder, was X8.5% before, dur)ng and after reperfusion or the coronary artery . This absence of hypoxemia in the occluded vascular segment most likely reflects inflow of oxygenated arterial blood from collateral vessels and low metabolic activity of the coronary artery . Regnfeemrnr for blood components . Coronary artery segments were harvested from eight normal dogs and perfused in vitro with buffer solution in the absence of blood calls cc proteins . In vessels in which flow was stopped for 90 nun and followed for 2 h, acetylcholine perfusion (10-6 molfliter) resulted in relaxation of only 11 .9 *_ 8.2% compared with 35 .3 d: 7.1% in control vessels in which flow had not been stopped (p < 0.03) . The dose-response curve was signifnautly shifted down and to the right for no Ilow/reIow vessels compared with control vessels (p < 0 .05 for difference in area under curves) . The response to bmdykinia was not affected by temporary cessation of perfusion . At a concentration of 10'1 mol/liter. beadykinia caused a mean decrease of 8 .8 ± 9 .5% in relaxation in the no bw/reflow vessels compared with 11 .6 ± 17% in the control vessels (p = NS). (Fig. 5). Vascular collapse. To separate the effects of loss of decreased inaralumen pressure with vascular collapse . norcoal vessels from 10 dogs were perfused with buffer solution in vitro and during a 90-mtn flow iatermption . The distal vessel was clamped to prevent vascular collapse and to produce wmtioued vessel disterlson . After reflow, the vasodilator responses of these vessels to acetylcholine and lmrdykinin remained normal (Fig . 6).

Dlactmion There is a growing body of experimental evidence that coronary artery occlusion and repetfusion may result in abnorma0ties of eplcardlal coronary eadmhelium to stimulators of andolhdml-derived relaxing factor release . Wish use area vivo online coronary rings (5.101, previous studies reported marked structural abnormalities of the endothelium in repedused vessels and both reduclion of acetylcholine induced vasodOmion and evidence of abnormality of amoosh vessel response to potassium chloride (51 . Coronary roperAnion has also been shown to impair the vasoditator response ro acetyleholin a and bsadykinin in intact dots (7,10) . &dalkdW dyrlbnatlun after seclusion and reperlmlna. Our study also demonstrated that occlusion of canine epieardW coronary arteries with or without repedusion impairs endothelium-dependent vasodilation . This was demonstrated in excised perfused coronary artery segments and in

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Figures. Respousetoacetylchuke (tool and bradykinin (bottom) in normal vessels to = 8) compmedwkb vessels subjected to 90 min of Now cessation (no fowkelow) and in control vessels in which flow was maintained continuously . Results are shown as a decrease in perfusienpressute expressed as a percent ofpreconstricted pressure obtained afler irethuien of 25 mmeldiur of potassium ch olide. The response to 10-6 mdnterefscasykhdineis signillcamly dmdnislnd in the ao flrwlrefew vemels coo mod with conbol vessels (p < U03) .

in vivo perfusion by use of high resolution epicardiul ultrasound imaging. Our study shows that the response of the coronary artery to different mediators of endatheliumderived relaxing factor release may not be altered uniformly after occlusion and reperf3siou. The vasodilator response to acetylchoine and bradykinin were impaired after occlusion alone, but when the vessel was then subjected to reperf s%ion, there appeared to be further impairment of coronary vasodrlation with aceylcholine but not bradykinin. In addition, when nor ml coronary arteries were subjected to no flow/reflow ex vivo, abnormal responses to acetylcholine were found but hoof to b eadykinin . These observations suggcst that the endothelial acetylcholine receptor may be more sensitive to injury than the hredykinn receptor . This study used a model of perfused segments (4 to 6 cm long) of coronary artery rather than a stretched coronary ring. The advantage of the perfused vascular segment model is that it allows in vitro modeling of changes in intravascular few . The disadvantage is that it does not readily lend itself to removal of the endothelium to allow study of the possible

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JACC Vol. 23. No. 5 April 19ae1216-23

OUYANG ET AL . DOG CORONARY VASOACrIVI Y AFTER ISCHEMIA

complement or white blood cells can contribute to the impaired endothelium-dependent vasodilation seen after reperfasion . To determine whether the altered hemodynamic vascular forces contribute in a significant way to the abnormalities of endothelial function seen after coronary occlusion and reperfusian will require further study . v

aknmvlaip the leebniwl ondtanee d Deborah F . Senders . the torrid technical assistance of Aatbooy DiPada and the seaRlwial espeftise of Wendy Wightmm.

References 1- Vaahaete PM, Rabanyi GM, Miller VM, Housmn 1151- MWuhliap of vascular smooth muscle contraction by the endothelium. Anna Rev FlOaid ioio1e:J67-70 . 2 . Pnrcb oliRF,ZawadrkiJV,Theobligatoryroleofesdothelialcellsinthe relmauun of amuial smooth meek by nayklwaoe . Nature 1996288: 373-6 . 3. Fanigpntt RE Role of endothelium in response of ocular rmteA tomato. Cic Roe 199353357-734. Vahmae PM . The esdmhelum: omdutotor of r.weukr samolb muscle tape. N End I Med 199d319e512 .3 . 5. Van Bentnaymn KM. McMaty IF. Harwitz LD. Repertusion aferacale summary oeclasim in dogs impairs endadhalium-dependent relaxation to aoetytebolihe and augments oonuaclile reactivity in vitro. I Ctin Invest 1957:79:265-74. . Pmtson PJ, Sclf IIV . Vatdlonlle PM. Acute impabmea of codotheR6 am-0epeadem retaliation to amrnptiag pomlds fdbwing repefustrn igjmy in wrdae wramry armies. Cue Red 1090A7AM-M. 7. Mdtm IL, Nichols WW. Dangly WH. Irwson DL, Sateen TGP . Inlpdred ennee cauumy vaaodiotar response to ocetylcho8m and bradykhbt after occhdoarepedsian . Chc Re, 1959;64:41-54. 5 . Nucleic WW, Mehh JL Deadly Wit. la-son D . Thompson L. Tee Riot M. Reduction in contrary smoothest reserve tallovdng coronary . aedaioe and repel nnim h aeeabeleel dogs : rote of endothelium derived ralauhe factor myocardial teuROphil inttaion and prostsglmdia I Mel CA Cordial 1955 ;28943-54. . Ku IJD. Camaey vaaatar raativhy near acme anyasdhl iotucdos 9 Sdeece 1992218576-8. 10 . Salary CO, D nneiog GJ, Consonant HJ . Woodman OL . Impaired vstdii don d epkmdW c ounary angles and resistance manila ralowlng myocardial ischeeia and tepcrfmion is ar"belized dogs . Car Art Dim 1990a:363-74. 11. Qatike 1& SNSe FW, Brooks LA . Hannsm DG. Isebemia-repedusien lapis makshidmsdepn dint relwtoa of coronary mknwestah but does ant afoul hose stories . Cucabiioo 199092 :586-90 . 12. KImerRA . Rude RE . Carkm N . Matdw PR. DeBam LWV. &amwatd R . Ulbakmlurd evdence dmicrorancds damn, ad myawdal coil irdety after contrary artery oeclutoa which wow tint' Circulation 19".4L"2. 13. Oliver MO . Spoon RD Pony MA . Oramer DN. Morphologic assesMM of kxdacyiesndothead all interactions in .mrrneeric retailer n lliamd to bohemia and nepnfction . InBmananion 1991115:331-46. It &mtM M. houses W. Kvieiys PR. et al. Sapemslde meeaw repetnt-

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en-ioduced leukocyte eodothetial cell otmaelions . Am I Physid 1969; 257 :H174a-45. 15 . Zweier IL . Kupposamy P . Laity GA . Measurement of eDdothelial tell free radical generation : evidence for a central mechanism of free radical irnury in pmtischunictimers . P,oc Nad Acrid Sci USA 198895 :4046-56. 16. Sacks T. Motdew CF . Craddock PR, Raveren TK . Jacob BE. Oxygen as d. area ate neddnstiat cell domeW by coat hums-adadated nnaaulocyles . I Clin Invest 19iK61 :1161-7. 17. Sherida FM . Dauber IM, McMmvy IF, Leaefsky El, Horwitx I .D. Roleofk,tkagtesramao nyvmwrmreMm eltoinpnymremedren® and repmibdon, Cite Res 199tt69d366 74. 1K Oleaen S-P. Clapham DR, Davits PP . Hmmdnmoic char siren andvale' a K' cmrem to vascular eodolhefal cells . Nolure 1968:33k16617a. 19. L,nsnrania .HaltamTJ,RinkTJ.Sagtnnkald livatediwsdrgmdniu noontime modmlhl malls as neebtemmnsdueers . Nature 1907925: ell-2 . 20. Zksehteia RC. Clean L Capagosi MC. Flaw-0ependmt cymsolc acidification of vascular cWothztio ratio . Science 1992258 :666-9, r-1 . Hirutzka LF. McPheson DD . Immbcdh WC Jr. d al. Inlneperaave evaluation of corollary artery bypass graft anastonoses with high, frrvaueay eoleardial ograpby : experimental validation and ion Sal potion studies' Circulation 1986;17:1199-265. ,,. Weirs HR. Effeel of coronary artery allusion an regional anotia mad ate O0. r*ootim. O. eshadien, blood ft and Oc cemurrption in the dog bent. Cho Rat 199R47NOF7 . 13. Werber AIL Reload KK. DMI19atd support of arteries . Am I "to] 1945:241:11901-6 . 24. lintel. Fushkni S. AboT. Argmmlcd contractile mspoma locndotnelin and blared endothetium-depeadent relaxation In post-ischemic rrperfused coranay armies. Jpn Ciec 3 1992:56:657-70. 25 . Rubonyi GM, Vmnoutte Phil . Hypoxia retentts a vaseconteictor sub stance furor me wane vaswkrendothelum . J Physiol London 1995;364: 45-56. 16. Harder DR. Solder-Fever C . Sorer K. Seklel WJ. Rubmyl GM. Pressure tetanus tnnsfenble endolhetid wntracdk factor in cat cerebral aeerta. Cite Ran 19a9 ;6k193-6. 27. Hultz F. Fomentation U. Pohl U. Gabber M. Stowage R . Flowdepudem-mdolhnfaa lwdiaadd8aipndepicaedidternarynteies conscious dogs: effects of cycooxygeonse Inhibition. I Cardiavase Phamaol 1984o116l-9. 29. Smiwko V . Rook J. Ddozel 5 . Role of endmhel ram in the contra or arkrinl diameter by blood Row . Blood Vouch 191160 :Z" I . 2% Rumnyi GM. Romero IC . Vmenme PM. Pow.induced mime of endmke8umderived rehxhg faaa . Am I Physo119M;256:H1145-9. 30 . HaroorOR .Pmeaae-iaducedmysrgenkm7ivatieeofemamhmlanede is dependen on beet mdothe8um . Cln Res 1987:66:166-7 . 31 . Rrtbmnyi GM. Rnddldkundepwdem prcmum-i duced commotion of Worst canine mmlid weaken . An I Physid 19Ba;255:H783-8. 32 . Rubanyi GM, Fiery AD. Know K. taken A . Harder DR. Meelsmonceptnn by the admktiumt medktmn ad meehanhaa of ptenae-and bow-induced vaswler testamsa . Blood Vessels 194027:246-7. 33 . Doothubei N& Kim D. Bencc TA . Intracellular alkallobadon leads to Ca" mobilization from epelsl-semMve pools In bovine aenlic eadolhe SW cells. J Blot Chnu 1999269.1907-6. 34. Lenin DA, Loomu JL, Seed SS . Preservation or ednhelol cells in excised rat cemtld marks. Ell of Intentional presents Rod saymem length . Cite Re, 19910.997-1002.