Endothelium-Dependent Vasodilating Effect of Substance P During Flow-Reducing Coronary Stenosis in the Dog

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1 1374 JACC Vol. 15, No.6 May 1990: ndothelium-dependent Vasodilating ffet of Substane P During Flow-Reduing Coronary Stenosis in the Dog AKITO YATANI, MD, MITSUHIRO YOKOYAMA, MD, HOZUKA AKITA, MD, HISASHI FUKUZAKI, MD Kobe, Japan The effets of substane P, a putative entral and peripheral neurotransmitter, on oronary vasulature and its mehanisms were studied in 31 anesthetized open hest dogs. Without oronary stenosis, intraoronary infusion of substane P (0.001 to 1 pmoilkg per min) for 40 s inreased oronary blood flow up to 173 ± 10.7% in dose-dependent fashion. Appliation of oronary stenosis reated by an inflated intraluminal miroballoon that preserved ative vasomotion of the stenosed segment produed a pressure gradient of 34 ± 2 mm Hg, a derease in rest oronary blood flow of 21 ± 1.6% and signifiant depression of the rate of rise in left ventriular pressure (dp/dt). During oronary stenosis, substane P inreased oronary blood flow up to 150 ± 9.4%, lowered mean distal oronary pressure and dereased stenosis resistane in dosedependent fashion. After endothelial denudation of the proximal part of the oronary artery, the substane P indued inrements in oronary blood flow during oronary stenosis were abol- ished. In vitro measurements of isometri tension from both intat and denuded portions of oronary arteries onfirmed a marked inhibition of substane P indued relaxation in the denuded segments. These results show the obligatory role of the endothelium in substane P indued oronary artery dilation. Furthermore, intraoronary infusion of substane P (1 pmoilkg per min) from the site distal to oronary stenosis that preluded the responsiveness of the large oronary artery dereased oronary blood flow by 24 ± 4%, lowered mean distal oronary pressure by 15 ± 1.9 mm Hg and intensified stenosis resistane by 77 ± 7.2%. Thus, substane P exerts a diret potent dilating effet on both large and small oronary arteries. However, beause of its strit endothelium-dependeny, this peptide may play a detrimental role in the regulation of oronary blood flow when an atherosleroti stenoti lesion with endothelial damage or dysfuntion is present in the proximal part of the oronary artery. (J Am Coil CardioI1990;15: ) Substane P is an II-amino aid peptide that was first isolated by von uler and Gaddum (1). xtensive histohemial studies (2-8) in mammals have deteted a widespread distribution of this peptide in different parts of the entral and peripheral nervous systems (2), inluding the heart (3,4) and blood vessels (5-8). Substane P-like immunoreative nerve fibers and variosities are loated extensively in the adventitia and the adventitia-media border of blood vessels (5,7). Substane P, a possible neurotransmitter (9), is From The First Department of Internal Mediine, Kobe University Shool of Mediine, Kobe, Japan. This study was supported in part by Grant-in-Aid for Sientifi Researh No from the Ministry of duation, Siene and Culture, Tokyo. Japan. Manusript reeived August ; revised manusript reeived November 16, 1989, aepted November 29, Address for reprints: Akito Yatani, MD, The First Department of Internal Mediine, Kobe University Shool of Mediine, 7-5-1, Kusunoki-ho, Chuoku, Kobe 650, Japan. thought to partiipate in ardiovasular reflexes and the transmission of ardia pain (6). Systemi infusion of this peptide in animals produed a rapid derease in blood pressure and assoiated tahyardia (10). Loal infusion of the peptide in human forearm vasulature revealed that it was a potent arteriolar vasodilator (11,12). Brum et al. (8) found signifiant onentrations of substane P-like immunoreativity in anine epiardial oronary arteries, and proposed that it might diretly affet oronary vasular tone after its release from peripheral sensory nerve endings. The peptide exerts potent endothelium-dependent dilatory responses in studies using isolated arterial preparations (13) and intat anine femoral arteries (14). These observations have raised the question as to the possible roles of this peptide and the influenes of oronary stenosis on regulating oronary blood flow in an intat animal. Although some investigations desribed the effets of this peptide on oronary irulation in the isolated (15) and intat (16) dog heart, none of these studies were per by the Amerian College of Cardiology /90/$3.50

2 lacc Vol. 15, No.6 YATANI T AL. SUBSTANC P-INDUCD CORONARY VASODILATION 1375 formed under the ondition of oronary stenosis or endothelial dysfuntion, or both, resembling human atherosleroti oronary lesions. We have developed an experimental model of dynami oronary stenosis that preserves ative vasomotion of the stenosed segment, resembling human atherosleroti oronary lesions that ontain some intat smooth musles and maintain stenosis vasomobility (17,18). This model has been proved to be appropriate in investigating the effets of vasoonstriting and vasodilating agents (19-22) and the hanges in myoardial metaboli demand (23) on the oronary vasulature and the therapeuti mehanisms of ardiovasular drugs (20,24,25). In the present study, we evaluated the quantitative dose-response relations of substane P to oronary vasulature in an experimental model of oronary stenosis and larified the mehanisms involved in the observed effets. Methods xperimental preparation. This projet onformed to the "Position of the Amerian Heart Assoiation on Researh Animal Use" adopted by the Amerian Heart Assoiation. Thirty-one mongrel dogs of either gender. weighing II to 18 kg, were pretreated with subutaneous morphine (I mg/kg body weight), anesthetized with intravenous alphahloralose (100 mg/kg) and ventilated by a mehanial respirator with intermittent positive pressure, using room air supplemented with oxygen. Blood gases and aid-base balane were maintained within normal limits (26). A atheter was passed through the right femoral artery and advaned into the aorti arh for aorti pressure monitoring. Another atheter was also plaed in the femoral vein for intravenous infusion. A left thoraotomy was performed at the fifth interostal spae, and the heart was suspended in a periardial radle. In six dogs, a polyethylene tubing (0.6 mm outer diameter) was advaned retrogradely through a small branh of the irumflex oronary artery distal to the proposed olusion site until its tip was just distal to the stenosis for distal oronary infusion of substane P. In the seond distal branh. another polyethylene atheter was plaed to reord distal oronary pressure. A stiff atheter. 10 m long, was inserted into the left ventrile through the apex to reord left ventriular pressure and rate of rise of pressure (dp/do. The left ommon arotid artery was exposed, and a segment approximately 1 m long, of the left irumflex oronary artery was disseted free. After administration of 5,000 U of heparin, the irumflex oronary artery was ligated, promptly annulated just at its distal portion with a thin metal annula (2.4 mm inner diameter) and perfused ontinuously from the arotid artery through bypass perfusion tubing with a minimal internal diameter of 2.4 mm (Fig. I). Heparin (2,000 U) was supplemented every 30 min. Coronary perfusion pressure at the tip of the annula Left Ventriular Pressure from Carotid Artery ::::.: Aorti Pressure Figure 1. xperimental preparation. AO = aorta; ex = left irumflex oronary artery; LAD = left anterior desending oronary artery; PA = pulmonary artery. was measured, and it was onfirmed that mean oronary perfusion and aorti pressures were nearly idential in eah experiment. In addition, the adequay of this perfusion system was onfirmed by preserved autoregulatory reserve (peak reative hyperemi response was>300% of basal flow after a 15 s total oronary artery olusion) in eah dog. Cirumflex oronary blood flow was measured using an extraorporeal eletromagneti flow probe (Nihon Kohden FF-030T, inner diameter 3 mm). Pressures were measured with a Statham P23Db transduer. Heart rate, aorti pressure. mean distal oronary pressure and oronary blood flow were ontinuously monitored. ah preparation was allowed to stabilize for at least 30 min after oronary annulation. Prodution of dynami oronary stenosis. The onept and harateristis of dynami oronary stenosis have been desribed previously (19-25). Briefly, dynami oronary stenosis was produed by inflating a speially made miroballoon oluder, onsisting of a minute rubber balloon attahed to the tip of the polyethylene tubing. This oluder was inserted through the side arm of the perfusion tubing and advaned into the intat proximal portion of the irumflex oronary artery. There was no major branh between the annula and the oluder. It was onfirmed that the plaement of the oluder into the oronary artery did not affet oronary blood flow at rest, its phasi pattern or the peak reative hyperemi response before its operation. The size of the balloon was finely adjusted by expansion with saline solution to obtain a pressure gradient aross the stenosis of approximately 30 mm Hg, and the expansion volume was kept onstant. The severity of the stenosis was evaluated by the mean pressure gradient aross the stenosis; stenosis resistane was alulated by dividing the mean oronary pressure gradient aross the stenosis (that is, subtrating

3 1376 YATANI T AL. SUBSTANC P-INDUCD CORONARY VASODILATION JACC Vol. IS, No.6 mean distal oronary pressure from mean aorti pressure) by mean oronary blood flow. ffets of intraoronary substane P administration. Dose-response relations of intraoronary infusion of substane P (0.001, 0.01, 0.1 and 1 pmol/kg per min) were examined in 16 dogs. The dogs were lassified into two subgroups: eight dogs without and eight dogs with oronary stenosis. In the latter group, oronary stenosis was produed after reordings under rest onditions. The peptide was dissolved in a saline solution ontaining 0.1 % bovine serum albumin, diluted in an appropriate onentration and infused through the bypass tubing into the irumflex oronary artery (proximal infusion) for 40 s at a rate of 0.2 ml/min. The response to the peptide was always apparent within this time period. Beause the hemodynami hanges in response to this peptide were relatively transient, maximal responses of eah hemodynami variable were observed in eah perturbation. In the preliminary study, it was onfirmed that this perfusion rate of substane P vehile did not affet oronary hemodynami variables and that the transit time between injetion site and annula tip was less than a few seonds in the presene and absene of oronary stenosis. The sequene of varying doses of the peptide was seleted randomly. Under eah ondition, at least 20 min were interposed between eah perturbation to onfirm the full reovery of all hemodynami variables to the ontrol state and exlude the possible development of tahyphylaxis. ffets of endothelial denudation in vivo. To examine the role of the endothelium in response to intraoronary substane P administration, endothelial denudation was performed around the site where the miroballoon oluder was plaed in eight dogs. After the effets of substane P (0.1 and 1 pmol/kg per min) infusion for 40 s were examined without and with oronary stenosis as ontrol studies, endothelial denudation was performed. A 2F Fogarty emboletomy atheter (Amerian dwards Laboratories) was inserted through the side arm of the perfusion tubing and plaed in the proximal portion of the irumflex artery. The intima of this portion was mehanially deendothelialized by balloon inflation and drawing of the atheter. The balloon size of the emboletomy atheter was previously adjusted so as not to distend the artery. The miroballoon oluder was then plaed in the portion of the artery where mehanial rubbing had been applied. The effets of intraoronary substane P (0.1 and 1 pmol/kg per min) were again examined in the absene and presene of partial oronary obstrution. To exlude the possibility that endothelial damage might alter the responsiveness of the vasular smooth musle, the responses to a 40 s infusion of nitroglyerin (1 p,g/kg per min) were also ompared before and after the proedure. Organ hamber experiment on oronary artery segments. At the end of the denudation experiment in vivo, oronary arteries from six dogs were immediately isolated and leaned of surrounding tissue. Helial strips approximately 2 mm wide and 20 mm long were obtained from both the endothelium-denuded portion of the irumflex oronary artery and the intat proximal portion of the left anterior desending artery. For reording isometri tension, strips were suspended in 30 ml organ baths ontaining buffer of the following omposition (mm): sodium hloride, 118; potassium hloride, 4.0; alium hloride, 1.5; magnesium sulfate, 1.2; sodium phosphate, monobasi, 1.2; sodium biarbonate, 25; and gluose, 5 (27), and equilibrated at 37 C with a 95% oxygen-5% arbon dioxide gas mixture. Final ph was approximately An initial preload of 1.5 g was applied to eah strip. The equilibration time before the relaxation experiment was 1.5 to 2 h. The tension of the oronary artery at rest was approximately I g. A test ontration was indued by adding 20 mm of potassium hloride. When developed tension attained its peak value, the strips were relaxed by rinsing with the buffer. They were then preontrated by 2 pm of prostaglandin F 2u and subsequently relaxed by the umulative addition of substane P (l pmol to 100 nmol). Dose-response relations were determined for eah portion of oronary artery segments. Histologi examination. To assess the morphologi harateristis of the denudation site, the vasular samples from the intat and denuded oronary arteries were fixed in a 10% formaldehyde solution. The speimens were stained with hematoxylin-eosin and examined mirosopially. In some dogs. vital staining was performed to onfirm the vasular area of denudation. vans blue dye (400 mg dissolved in 20 ml of isotoni sodium hloride solution) was intravenously administered at the end of the experiment. Thirty minutes later, the dog was killed and the heart was promptly reseted. The oronary tree was flushed with the buffer, and the irumflex oronary artery was opened longitudinally and inspeted for blue staining, whih signified endothelial trauma (28). ffets of distal substane P infusion. In six dogs, the effets of distal oronary infusion of substane P were ompared with those of proximal infusion through the perfusion iruit in both stenosis-absent and stenosis-present onditions. All hemodynami measurements were made ontinuously during rest onditions, oronary stenosis, drug infusion and for 5 min after essation of the infusion. Materials. Syntheti substane P (Sigma Chemial Co.) was dissolved in normal saline solution ontaining 0.1% bovine serum albumin to give a dose of 0.1 mg/ml, and stored at -80 C before use. Nitroglyerin (Millisrol, Nihonkayaku Co., Tokyo, Japan) was dissolved in distilled water; prostaglandin F 2u was obtained from Sigma Chemial Co. ah agent was prepared in an appropriate dose eah time before use.

4 lacc Vol. 15, No.6 May 1990: YATANI T AL. SUBSTANC P-INDUCD CORONARY VASODILATION 1377 Table 1. Systemi Hemodynami ffets of Substane P in ight Dogs Without Coronary Stenosis Mean Aorti LV Pressure (mm Hg) HR Pressure LV dp/dt (beats/min) (mm Hg) Systoli nd-diastoli (mm Hg/s) pmol/kg per min Preinjetion 137 ± ± ± 6.2 5,1 ± 0.5 2,224 ± 137 During injetion 137 ± ± ± ±0.5 2,167 ± 142 nd of injetion 136 ± ± ± ± 0.5 2,161 ± min after injetion 136 ± ± ± ± 0.5 2,162 ± pmol/kg per min Preinjetion 132 ± ± ± ± 0.5 2,275 ± 110 During injetion 133 ± ± ± ± 0.5 2,249 ± 134 nd of injetion 135 ± ± ± ± 0.5 2,269 ± min after injetion 133 ± 9,3 106 ± ± ± 0.5 2,248 ± pmol/kg per min Preinjetion 132± ± ± ± OJ 2328 ± 155 During injetion 132 ± ± 6. I 131±7,3 5.1 ± ± 160 nd of injetion 132 ± ± ± ± OJ ± min after injetion 133 ± ± ± ± OJ 2,364 ± 152 I pmol/kg per min Preinjetion 140 ± ± ± 6,5 503 ± 0.4 2,246 ± 165 During injetion 139 ± ± ± ± ± 165 nd of injetion 142± ± ± ± 0.4 2,286 ± min after injetion 141 ± ± 6,0 130 ± ± 0.4 2,251 ± 164 Values are mean values ± SM. HR = heart rate: dp/dt = rate of rise of pressure: LV = left ventriular. Statistial analysis. The results of in vivo experiments were expressed as mean values ± SM. Data for a single response in the same dog were analyzed by Student's t test for paired omparison. Differenes among the different doses of eah group were analyzed by analysis of variane and Tukey's test. In in vitro experiments, the responses to the peptide were expressed as mean values ± SM perent relaxation of the prostaglandin F 2u -indued ontration, and the signifiane of the differene between group means was assessed with Student's t test for Table 2. Systemi Hemodynami ffets of Substane P in ight Dogs With Coronary Stenosis Mean Aorti LV Pressure (mm Hg) HR Pressure LV dp/dt (beats/min) (mm Hg) Systoli nd-diastoli (mm Hgls) pmol/kg per min Preobstrution 137 ± ± ± ± 0.4 2,137 ± J13 Preinjetion 135 ± ± ± ± OJ' 2,009 ± 126' During injetion 133 ± ± ± ± OJ ± pmol/kg per min Preobstrution 140 ± ± ± ± 0.4 2,164 ± 130 Preinjetion 142 ± ± ± ± OJ' 1,998 ± 97' During injetion 143 ± ± ± ± OJ 2,002 ± pmol/kg per min Preobstrution 140 ± ± ± ± OJ 2,281 ± 132 Preinjetion 145 ± ± ± ± 0.2' ± Ill' During injetion 144 ± ± ± ± 0.2 2,253 ± 134 I pmo1/kg per min Preobstrution 137 ± ± ± ± OJ 2,273 ± 128 Preinjetion 145 ± ± ± ± OJ' 2,140 ± 121' During injetion 145 ± ± ± ± 0.2 2,149 ± 130 'Compared with above, p < Values are mean values ± SM. Abbreviations as in Table 1.

5 1378 YATANI T AL. SUBSTANC P-INDUCD CORONARY VASODILATION lacc Vol. 15. No.6 Table 3. Coronary Hemodynami ffets of Substane P in Dogs Without and With Coronary Stenosis Without Coronary Stenosis With Coronary Stenosis (n = 8) (n = 8) SR CBF Mean DCP CBF Mean DCP (mm Hg/ml (ml/min) (mm Hg) (ml/min) (mmhg) per min) pmol/kg per min Preobstrution 23.5 ± ± 3.2 Preinjetion 25A ± ± 6J 18.8 ± 1.9* 70 ± 3.4* 1.9 ± OA During injetion 27.9 ± 2.7* 100 ± ± ± 3A 1.8 ± OA 0.01 pmol/kg per min Preobstrution 23.9 ± ± 3.8 Preinjetion 25.1±2A 101 ± ± 1.8* 67 ± 4.1* 1.9 ± OA During injetion 29J ± 3.0* 102 ± ± ± ± OA 0.1 pmol/kg per min Preobstrution 23J ± 2J 99 ± 2.9 Preinjetion 25.0 ± ± 6J 18.6 ± 2.0* 67 ± 3.0* 2.2 ± OJ During injetion 37.0 ± 4.5* 100 ± ± 2.2t 62 ± 4At 1.8 ± 0.2t I pmol/kg per min Preobstrution 23.5 ± ± 2.9 Preinjetion 24.6 ± 2A 102 ± ± 2.4* 66 ± 4.2* 2.0 ± OA During injetion 43.8 ± 7.0* 98 ± 6.7t 27.9 ± 4.5* 60 ± 4.2* 1.7 ± OAt Compared with above: *p < tp < Values are mean ± SM. CBF = oronary blood flow; DCP = distal oronary pressure: SR = stenosis resistane. unpaired samples. Signifiane was aepted at the p value <0.05. Results ffets of intraoronary substane P administration. Systemi and oronary hemodynami variables in dogs without and with oronary stenosis are shown in Tables 1 to 3. Without oronary stenosis. intraoronary administration of substane P (0.001 to 1 pmol/kg per min) signifiantly inreased oronary blood flow in a dose-dependent manner. Maximal inreases in oronary blood flow were 10 ± 2.5% (p < 0.01). 16 ± 3.8% (p < 0.01). 48 ± 11.4% (p < 0.01) and 73 ± 10.7% (p < 0.01) at the doses of 0.001, , and 1 pmol/kg per min. respetively (Fig. 2). Systemi hemodynami variables did not hange during the infusion of any doses of substane P (Table I). ah dose of the peptide produed transient but signifiant inreases in oronary blood flow that were observed several seonds after the start of the infusion and began to deline during the infusion period. The oronary effets of repeated administration of any dose of substane P were onfirmed to be reproduible at 20 min intervals. Appliation of oronary stenosis (Table 2), produed a mean pressure gradient of 34 ± 2 mm Hg (p < 0.01) and a derease in oronary blood flow of 21 ± 1.6% (p < 0.01). Left ventriular dp/dt dereased by 127 ± 21 mm Hg/s (p < 0.01). During oronary stenosis. the dose-dependent inreases in oronary blood flow and dereases in mean distal oronary pressure in response to intraoronary substane P infusion were signifiant at doses >0.1 pmol/kg per min (Fig. 2). Maximal inreases in oronary blood flow were 8 ± 4.7% (p = NS), 10 ± 5.3% (p = NS). 31 ± 7.2% (p < 0.01) and 50 ± 9.4% (p <0.01) for eah inremental dose between and 1pmol/kg per min. and maximal dereases in mean distal oronary pressure were 5 ± 1.8 (p < 0.05) and 7 ± 2.2 mm Hg (p < 0.01) for the doses of 0.1 and 1pmol/kg per min (Fig. Figure 2. Perent hange in oronary blood flow (IlCBF) in response to various doses of intraoronary substane P (SP) (0.001 to 1 pmol/kg per min) without (open bars) and with (hathed bars) oronary stenosis. Signifiantly different from preinjetion values: **p < u:: 40 CD u <l 10 ' 0

6 lacc Vol. 15. No.6 YATANI T AL. SUBSTANC P-INDUCD CORONARY VASODILATION 1379 A SP(I Opmllkglmln, ) I I Coronary Blood Flow (mllmln) Dostal Coronary Pressure 100 (mmhg) 0 Aorti Pressure (mmhg) 100 o LV Pressure (mmhg) LVdPldt (mmhgls) B Coronary Blood Flow (mllm,n) SPl1 Opmollkglmln Ie l j I 50[. -...r-j,',. '-.J".U6iW.._4iii". o Distal Coronary Pressure (mmhg) AortiC Pressure (mmhg) loo[_.r--... o LV Pressure (mmhgl LV dpldt (mmhgls) L oss-l lmln 3). Stenosis resistane dereased signifiantly by administration of the two higher doses; however, hanges in left ventriular dp/dt did not reah statistial signifiane. ffets of endothelial denudation in vivo (Table 4). There were no signifiant differenes in basal systemi and oronary variables before and 15 min after endothelial removal. Coronary blood flow inrement in response to intraoronary Figure 3. ffets of intraoronary (i.e.) substane P (SP) (I pmol/kg per min). A. without oronary stenosis, it an be seen that substane P inreased oronary blood flow without any hanges in systemi hemodynami variables. B, With oronary stenosis, substane P inreased oronary blood flow, with a onomitant derease in distal oronary pressure. However, no signifiant hanges in left ventriular (LV) end-diastoli pressure or rate of rise of pressure (dp/dt) were seen during the experimental period.

7 1380 YATANI T AL. SUBSTANC P-INDUCD CORONARY VASODILATION JACC Vol. 15, No.6 May 1990: Table 4. Hemodynami Data Before and After ndothelial Denudation Proedure in ight Dogs LV Pressure Mean Aorti (mmhg) HR Pressure LV dp/dt (beats/min) (mmhg) Systoli nd-diastoli (mm Hg/s) Before 144 ± ± ± ± ± 277 After 138 ± ± ± ± ± 264 Values are mean values ± SM. Abbreviations as in Tables I and 3. CBF (ml/min) 24.6 ± ± 2.9 substane P infusion without oronary stenosis (Fig. 4, right) was not altered signifiantly before and after endothelial denudation (before: 30 ± 4.4%; after: 35 ± 5.7% for 0.1 pmol/kgpermin, and before: 50 ± 4.7%; after: 51 ± 4.8% for 1 pmol/kg per min). However, oronary blood flow inrement indued by this peptide during oronary stenosis (Fig. 4, right and Fig. 5A) was almost ompletely abolished after this proedure (before, 25 ± 4.4%; after, 8 ± 2.2% [p < 0.01] for 0.1 pmol/kg per min; and before, 40 ± 3.2%; after, 10 ± 3.8% [p < 0.01] for 1pmol/kg per min). Mean distal oronary pressure dereased further, and the derement of stenosis resistane was ompletely abolished (Fig. 5A). Intraoronary infusion ofnitroglyerin (1 J.Lg/kg per min) was performed in seven dogs with oronary stenosis before and after endothelial denudation. There was no signifiant differene in preinjetion values of systemi and oronary hemodynami variables before and after the proedure. Nitroglyerin signifiantly inreased oronary blood flow and mean distal oronary pressure and dereased stenosis resistane after endothelial denudation to the same extent as before the proedure (oronary blood flow inrement: before, 35 ± 11.0%; after, 28 ± 16.0%; mean distal oronary pressure inrement: before, 13 ± 3.7 mm Hg; after, 8 ± 1.5 mm Hg; stenosis resistane derement: before, 48 ± 3.7%; Figure 4. Perent hange in oronary blood flow (CBF) in response to intraoronary substane P (SP) before (open bars) and after (hathed bars) endothelial denudation. Left, without oronary stenosis; right, during oronary stenosis. Whereas an inrease in oronary blood flow is unhanged in the absene of oronary stenosis after removal of the endothelium, an inrease in oronary blood flow is almost abolished in the presene of oronary stenosis after the denudation. Vertial bars indiate standard error of the mean. Signifiantly different from values before endothelial denudation: **p < '00 '00 after, 37 ± 6.7%) (Fig. 5B). No signifiant systemi hemodynami hanges were seen with intraoronary nitroglyerin infusion before and after the proedure. Organ hamber experiment on oronary segments. Figure 6 shows the effets of substane P on vasular tones of isolated oronary arteries obtained from both intat and denuded portions of the endothelium. Dose-response urves for substane P-eliited hanges in isometri tension during preontration by prostaglandin F 2a were ompared. Substane P (0.1 to 100 nmol) indued dose-dependent relaxation in endothelium-intat arteries. Almost maximal relaxation was ahieved by appliation of 10 nmol of the peptide (67 ± 9.0%). However, substane P-indued relaxation of Figure 5. ffets of intraoronary substane P (SP) (A) and nitroglyerin (TNG) (B) on oronary hemodynami variables during oronary stenosis before (open irles) and after (solid irles) endothelial denudation. Signifiantly different from preinjetion values: *p < 0.05, **p < 0.01; signifiantly different from nondenudation values: tp < The oronary hemodynami response to intraoronary nitroglyerin was not signifiantly different before and after endothelial denudation. CBF = oronary blood flow; DCP = distal oronary pressure; SR = stenosis resistane. A t "- 10 "- CD CD '0 u U q4 -; " 0 1 B :I: a- u 0 * "- CD 40 U <J t "- CD 40 U <J 20 3 If 2 2 t hi N r, a: I1'J I1'J * 0 ' '.0 SPlpmol/kg/min) P P P 0 ',{) ' 0 SP(pmol/kglmin) TNG lg/kg/min)

8 lacc Vol. 15, No.6 YATANI T AL. SUBSTANC P-INDUCD CORONARY VASODILATION ;;; 60 )( ro BO " 10 SP (-logm) 8 Figure 6. ffets of substane P (SP) on helial strips obtained from both intat (open irles) (left anterior desending) and denuded (solid irles) (left irumflex) portion of the oronary arteries preontrated by prostaglandin F 2a (211M). Relaxation is expressed as a perent of the ontration to prostaglandin F 2a and shown as mean values ± standard error of the mean, indiated by vertial lines. Substane P indued a dose-dependent relaxation in the endothelium-intat artery, but the relaxation was almost ompletely abolished in the endothelium-denuded artery. Signifiantly different from values of the endothelium-denuded portion: *p < 0.05, **p < B the endothelium-denuded arteries was almost ompletely abolished (7 ± 3.7% by 10 nmol). Histologi examination. Light mirosopi study revealed the presene of an endothelial lining in intat oronary artery segments (Fig. 7A). Denuded segments showed the absene of endothelial ells, with a few adherent platelets attahed to the luminal side and preserved internal elasti lamina and media (Fig. 7B). In addition, the denuded area in the irumflex artery in whih the vital staining was performed was learly tinted blue by vans blue dye. The blue-stained area was approximately 2 m in length and was present in the entire irumferene. ffets of distal oronary infusion of substane P. Without oronary stenosis, inreases in oronary blood flow indued by substane P were similar between the proximal and distal oronary infusions. The perent inrements in oronary blood flow in response to substane P infusion were 34 ± 6.9% and 27 ± 6.7% (p = NS) at 0.1 pmol/kg per min and 74 ± 16.9% and 70 ± 15.1% (p = NS) at I pmol/kg per min, respetively. With oronary stenosis, distal infusion of substane P at 0.1 pmol/kg per min did not inrease oronary blood flow signifiantly in ontrast with proximal infusion (23 ± 4.4% and 3 ± 4.8% for proximal and distal infusion, respetively), and distal infusion of the peptide at its highest dose exhibited a substantial derease in oronary blood flow (50 ± 12.8% inrease and 24 ± 4.0% derease for proximal and distal infusion, respetively, p < 0.05). This derement in oronary blood flow was aompanied by an aentuated derease in mean distal oronary pressure (8 ± 1.2 versus IS ± 1.9 mm Hg) and an intensifiation of stenosis resistane Figure 7. Photomirographs of setions of anine oronary arteries before and after endothelial denudation. A, Transverse setion of the ontrol oronary artery with an intat endothelial ell layer. B, Transverse setion of the denuded segment of oronary artery showing an absent endothelial layer with normal internal elasti lamina and media with a few adherent platelets (hematoxylin-eosin stain; original magnifiation x100, redued by 29%). (20 ± 6.6% derease versus 77 ± 7.2% inrease, p < 0.01) (Fig. 8). Disussion This study demonstrates that substane P, an amino aid peptide, is a potent dilator in both large and small oronary arteries and that this peptide produes a benefiial effet on oronary vasulature during dynami oronary stenosis. The dilating ation of this peptide on epiardial oronary arteries is present only in the presene of intat endothelium in vivo and in vitro. ffets of substane P without oronary stenosis. In intat oronary irulation, intraoronary infusion of substane P produed dose-dependent inreases in oronary blood flow without hanging systemi hemodynami variables, indiating that it has a diret vasodilatory ation on the small oronary artery. Whether this peptide has a diret ation on

9 1382 YATANI T AL. SUBSTANC P INDUCD CORONARY VASODILATION lacc Vol. 15. No o ontrol o ontrol distal infusion distal infusion 40.< ;;! ai... U ai 20 <I 40 U '" 100 I 80 p p P SP (pmol/kg/min) H-+1 P SP (pmol/kg/min) P SP (pmollkg/min) Figure 8. ffets of distal substane P (SP) infusion on oronary hemodynami variables during oronary stenosis as ompared with proximal substane P infusion (ontrol). Distal infusion of the highest dose of substane P eliited a derease in oronary blood flow (CBF), an augmented deline in mean distal oronary pressure (DCP) and an intensifiation of stenosis resistane (SR). The data are expressed as mean ± standard error of the mean during preinjetion (P) and infusion (I) periods. Compared with preinjetion: *p < 0.05, **p < 0.01, respetively; ompared with orresponding values in ontrol: tp < 0.05, ttp < respetively p<o.oi SP (pmol/kg/min) 4 60 Q u I a a: 20 If)." the heart is ontroversial, although the loalization of substane P-like immunoreative nerves in the atrium and within the ventriular myoardium is well observed (3,4). Relatively high doses of the peptide, whih aused systemi hypotension, reflex tahyardia and assoiated inrease in ardia output, were used in studies (16,29-31) favoring positive ardiotropi ation of this peptide. At a onentration as low as that used in other studies (15,32-34) and in ours, this peptide may not exert a ardiotropi ation. An inrease in oronary blood flow in response to substane P infusion was rapid, transient and began to disappear within a minute, even during ontinued infusion. This result is in aordane with that in other studies (11,30) in whih the peptide was infused systemially in dogs and in humans and showed a rapid ahievement of its maximal effet. Mwan et al. (12) demonstrated that the biologi half-time of substane P in a human forearm blood vessel is about 15 s. Desensitization to the peptide is also demonstrated (13) in isolated arterial preparations of various speies, whih is usually omplete within 10 min and readily disappears after the washout. Perhaps suh shortness of biologi ativity would aount for insignifiant hanges in left ventriular dp/dt during the oronary flow inrement eliited by the peptide. In this experiment, tahyphylaxis was avoided beause the repeated infusion of the same dose of the peptide after a 20 min interval aused similar hemodynami responses, whih is in aordane with previous human observations (31). ffets of substane P with oronary stenosis. The effets of substane P were evaluated under the ondition of flowlimiting oronary stenosis. In this situation, the autoregulatory reserve is almost exhausted in the subendoardium, whereas some reserve is still present in the subepiardium (20) (although regional blood flow was not measured in this study). Intraoronary infusion of substane P during oronary stenosis inreased oronary blood flow together with a derease in mean distal oronary pressure and stenosis resistane. This an be explained by the preferential dilation of the large oronary artery and the onomitant dilation of the small oronary artery. Both endothelial denudation and distal infusion experiments were performed to further larify the role of this peptide in regulating large oronary artery tone. After endothelial denudation of the proximal oronary artely. the oronary blood flow inrement in response to intraoronary substane P infusion without oronary stenosis was not signifiantly altered, showing that the dilating apaity of the small oronary artery was well preserved by this intervention. Careful attention was paid to establishing an inflation volume of the balloon atheter to produe only endothelial denudation without disernible overdistension of the artery wall and smooth musle damage. The adequay of this proedure was onfirmed by the response to nitroglyerin, a diret vasular smooth musle dilator, and histologi studies and the organ hamber experiment on oronary segments. Benefiial effets of nitroglyerin during oronary stenosis were a result of seletive dilating ation of the large oronary artery (24), and this response was similar before and after endothelial denudation. In ontrast, the oronary blood flow inrement in response to the peptide during oronary stenosis was almost ompletely abolished after endothelial denudation. These results suggest that vasodilator ativity of substane P depends on endothelial integrity. Distal oronary infusion of substane P inreased oronary blood flow to the same extent as proximal infusion without oronary obstrution, indiating almost the same dilatory responsiveness of the small oronary artery. In ontrast, the deleterious response of the oronary vasulature to the highest dose of this peptide during oronary stenosis an be explained by the mehanism of passive narrowing of the stenosed segment. Briefly, potent dilation of the small oronary artery in response to this peptide auses a derease in intraluminal-distending pressure distal

10 JACC Vol. 15, No.6 YATANI T AL. SUBSTANC P-INDUCD CORONARY VASODILATION 1383 to the stenosis and results in a paradoxi intensifiation of stenosis severity (35,36). The result also minimizes any role of flow-dependent dilation of the large oronary artery (37) in the inrease in oronary blood flow during oronary stenosis beause the mehanism of passive narrowing easily outstripped the flow-dependent dilator effet during oronary stenosis. In addition, the possibility of "oronary steal" and diversion of myoardial blood flow from subendoardium to subepiardium should also be onsidered in this situation (38). Role of endothelial denudation. If the endothelial denudation proedure nullifies the epiardial arterial vasodilator response, this intervention would, theoretially, produe the same effet on oronary vasulature as that of distal infusion experiments. However, the inrement in oronary blood flow during oronary stenosis after the denudation was not perfetly inhibited. The reason for this small inrease in oronary blood flow may be explained by inomplete deendothelialization in situ or release of a relaxing fator from the intat endothelial ells adjaent to the damaged site, whih was ompletely preluded by the distal oronary infusion of this peptide. The ontribution of released prostaylin on endothelium-mediated vasorelaxation ould be negleted beause relaxation by substane P is not influened by ylooxygenase inhibitors (39). Thus, the responses to proximal infusion of the higher doses of substane P during oronary stenosis an be explained by its opposing effets on oronary vasulature (namely, a redution in stenosis severity due to ative dilation of the intat large oronary artery and a passive narrowing of the stenosed segment aused by small oronary artery dilation). Net effets of the two mehanisms are manifest in the proximal infusion experiment, but the former effet is ompletely preluded in the distal oronary infusion experiment and is markedly attenuated in the denudation experiment. Clinial impliations. It has been suggested (40) that endothelial damage an playa role in the pathophysiology of oronary vasospasm. Furthermore, the relation between atheroslerosis and vasospasm raises the possibility that atheroslerosis-indued endothelial damage would alter vasoative responsiveness to neurohumoral and pharmaologi stimuli. Impairment of the endothelium-dependent relaxation in response to agents suh as aetylholine, substane P and bradykinin has been noted in atherosleroti arteries of human and animals (39,41,42). This lak of vasodilating ability in atherosleroti arteries appears to be important as the ause of myoardial ishemia. The preise mehanism whereby substane P peptidergi nerves in oronary arteries are ativated remains to be eluidated, as well as the extent to whih they influene oronary vasodilator tone under normal and pathophysiologi onditions in humans. Conlusions. We have demonstrated that substane P indued dilation of the anine epiardial oronary artery is endothelium-dependent in vivo and in vitro. Substane P an indue a diret potent dilating effet on the large and small oronary arteries and exhibits a benefiial effet in restoring oronary blood flow during oronary stenosis. However. in view of its strit endothelium dependene, this peptide ould playa detrimental role in the regulation of myoardial blood flow supplied by an atherosleroti stenoti artery with endothelial dysfuntion or damage. We express our gratitude to Yasunori Ihikawa, MD, Takashi Fujii, MD. Seiihiro Usuki, MD, Ken-ihi Hirata, MD and Yuihi Matsuda, MD for their valuable help and suggestion. We also aknowledge the skillful seretarial assistane of Noriko Hamana. Referenes I. von uler US. Gaddum JH. An unidentified depressor substane in ertain tissue extrats. J Physiol (Lond) 1931 ;72: Pernow B. Substane P. Pharmaol Rev 1983;35: Rehardt L. Aalto-Setala K, PUQeranta' M, Pelto-Huikko M, Kyosola K. Peptidergi innervation of human atrial myoardium: an eletron mirosopial and immunoytohemial study. J Autonom Nerv Sys 1986;17: Dalsgaard CJ. Frano-Cereeda A. Saria A, Lundberg JM. Theodorsson Norheim, Hokfelt T. Distribution and origin of substane P- and neuropeptide Y-immunoreative nerves in the guinea-pig heart. Cell Tissue Res 1986;243: Reineke M, Weihe. Forssmann WG. Substane P-immunoreative nerve fibers in the heart. Neurosi Lett 1980;20: Wharton J. Polak JM, MGregor GP, Bishop A, Bloom SR. The distribution of substane P-like immunoreative nerves in the guinea-pig heart. Neurosiene 1981 ;6: Barja F. Mathison R, Huggel H. 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