Alkalosis-induced coronary vasoconstriction: Effects of calcium, diltiazem, nitroglycerin, and propranolol

Alkalosis-induced coronary vasoconstriction: Effects of calcium, diltiazem, nitroglycerin, and propranolol We examined the effects of changes in pH, Ca'÷ concentration, dilUazem, nitroglycerin, and propranolol on the vaBcular tone of the Isolated rabbit coronary artery. Stepwlae Increase in pH of the bath fluid caused pH-dependent Increased vascular tone. Increase In Ca :+ concentration of the bath fluid also resulted In Increased vascular tone, while removal of Ca :+ abollahed the high pH-induced elevated vascular tone. DltUazem and nitroglycerin suppressed the high pH-induced Increased vascular tone. Proprenolol in high concentrations exhibited a direct InhiMlory effect on the high pH-induced Increased vascular tone. We conclude that high pH induces coronary vasoconstriction principally by Increasing transmembrane influx of Ca ~÷ and that dlltlazem and nitroglycerin suppress this action. (AM HEARTJ 102:206, 1981.)

H i r o f u m i Yasue, M.D., Shingo Omote, M.D., Akinori T a k i z a w a , M.D., M a s a o Nagao, M.D., K u n i o N o s a k a , and H i r o m i c h i N a k a j i m a , Ph.D.

Shizuoka City and Saitama, Japan

T h e r e is increasing evidence t h a t c o r o n a r y a r t e r y s p a s m (CAS) p l a y s an i m p o r t a n t role in t h e p a t h o genesis n o t only of v a r i a n t angina, 1-5 b u t also in o t h e r f o r m s of a n g i n a pectoris 6, T a n d p e r h a p s a c u t e m y o c a r d i a l infarction, s, 9 However, t h e m e c h a n i s m b y which CAS occurs requires clarification. W e h a v e demonstrated recently that hyperventilation and T r i s buffer infusion induce CAS a n d anginal a t t a c k s in p a t i e n t s w i t h v a r i a n t angina. 1° T h e s p a s m so produced b y such i n t e r v e n t i o n s was suppressed b y nitroglycerin (NG) or diltiazem (DTZ), a calcium a n t a g o n i s t , b u t n o t b y p r o p r a n o l o l ( P R O P ) , a betaadrenergic blocking agent. I n regard to these clinical findings, we e x a m i n e d t h e effects of a n increase in p H , D T Z , NG, a n d P R O P on t h e v a s c u l a r t o n e of the isolated r a b b i t c o r o n a r y artery. METHODS Experimental preparation. Albino rabbits weighing 2.5 to 3.5 kg were stunned by a head blow and the heart was removed. Large extramural coronary artery segments (outer diameter ranging from 0.5 to 0.8 ram) were dissected from the free wall of the left ventricle and placed in modified Locke solution aerated with 100% 02 at room temperature. The composition of modified Locke solution was (mM):NaCI 136.8, KC1 5.4, CaCl: 1.8, glucose 5.6, and

From the Divisionof Cardiology,Shizuoka City Hospital,and Pharmacological Research Laboratory,Tanabe SeiyakuCo., Ltd. Received for publication Dec. 2, 1980; acceptedApr. 3, 1981. Reprint requests: HirofumiYasue,M.D.,Divisionof Cardiology,Shizuoka City Hospital, 10-93,Ote-Cho,Shizuoka City 420,Japan. 206

Tris-HC1 buffer 5.4. Vessels were stripped of fat and connective tissue using a dissecting microscope, and helical strips, 0.5 to 0.8 mm wide and 5 to 10 mm long, were prepared. The strips were suspended in a tissue bath containing 10 ml of modified Locke solution, aerated with 100% O: at a temperature of 36 + 0.2 ° C. Experimental protocol. After an initial tension of 0.25 gm was applied, the strips were allowed to equilibrate for 1 to 1.5 hours prior to any testing. Isometric contractile tension was recorded on an ink-writing oscillograph (Linearcorder Type WTR 281, Watanabe Instruments, Corp.) utilizing a strain gauge transducer (Type UL-10-120, Shinkoh). In experiments designed to determine the effects of change of pH on vascular tone, each strip was allowed to equilibrate in six changes of Locke solution at different pH for 10 minutes. Ca~÷-free solution was formulated by removal of CaCl2 from the modified Locke solution. Test agents in solution (0.1 ml) were added directly to the tissue bath and the results were expressed in terms of final concentration. RESULTS Effects of elevated pH on coronary tone. As s h o w n in Fig. 1, A stepwise increase in p H f r o m 7.0 to 8.0 of the b a t h fluid caused a p H - d e p e n d e n t increase in vascular t o n e a n d earlier d e v e l o p m e n t of m a x i m u m tension. Fig. 1, B shows t h e relationship b e t w e e n p H in the extracellular b a t h fluid a n d m a x i m u m tension developed during 10-minute periods in seven experim e n t s c o n d u c t e d as depicted in Fig. 1, A. T h e p r e p a r a t i o n was initially i n c u b a t e d at p H 7.2 a n d then increase in v a s c u l a r t o n e was observed b y

0002-8703/81/080206 + 05500.50/0 © 1981 The C. V. Mosby Co.

Volume102

Alkalosis-induced CAS:pH, Ca ~+, diltiazem, nitrate, propranolol effects

Number 2

A

5rain

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207

free

,

pH Z 2

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Fig. 1. Effects of varying extracellular pH on the tone of

isolated rabbit coronary artery. A, Experimental records. Stepwise increase in pH from 7.0 to 8.0 of the bath fluid caused pH-dependent increase in vascular tone and earlier development of maximum tension. B, Relationship between pH in the extracellular bath fluid and maximum tension developed during a 10-minute period in seven experiments conducted as shown in A. Each point on the curve represents the mean of seven experiments; vertical bars represent ___SEM.

pH 7.8

Fig. 2. Experimental records of effects of varying C a =+ concentrations on the tone of coronary artery preparations exposed to Ca=+-free solution at pH 7.2 and pH 7.8.

o~ ®

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i n c r e m e n t a l l y raising p H to 7.8. A t p H 7.8 the contractile response of 415 + 15.6 m g (n = 28, m e a n _+ S E M ) was reversible a n d reproducible in the s a m e p r e p a r a t i o n . Effects of Ca =+ on coronary tone. Fig. 2 shows t h e effect of e x t r a c e l l u l a r Ca 2+ on v a s c u l a r t o n e of t h e p r e p a r a t i o n exposed to Ca2÷-free solution a t either p H 7.2 or p H 7.8. W h e n Ca 2+ was added c u m u l a t i v e ly, v a s c u l a r t o n e increased a t b o t h p H 7.2 a n d 7.8; this response was considerably g r e a t e r a t p H 7.8. Fig. 3 shows t h e results of six e x p e r i m e n t s c o n d u c t e d as depicted in Fig. 2. R e m o v a l of Ca ~+ f r o m Locke solution (Ca 2. 1.8 raM) readily abolished t h e Ca ~÷s u s t a i n e d c o n t r a c t i o n a t p H 7.8. Additional studies showed t h a t t h e contractile response a t p H 7.8 was m i n i m a l l y a p p a r e n t w h e n Ca ~+ was a b s e n t f r o m the b a t h fluid. Effects of DTZ, NG, and PROP on coronary tone. T h e effects of D T Z , NG, a n d P R O P on the p H 7.8induced increase in v a s c u l a r t o n e were examined. Fig. 4 illustrates e x p e r i m e n t a l records a n d Fig. 5 shows dose-response curves for p e a k decrease in vascular t o n e p r o d u c e d b y these drugs. All of these drugs p r o d u c e d a d o s e - d e p e n d e n t decrease in vascular tone; N G was the m o s t active, D T Z the n e x t m o s t active, a n d P R O P t h e least active. N G reduced v a s c u l a r t o n e m o r e rapidly t h a n D T Z or P R O P , b u t

IOmin

0

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3.6 7.2

21.6

Conc. of CoCl=(mM) Fig. :3. Dose-response curves of the effects of Ca 2÷ on the tone of coronary artery preparations exposed to Ca =÷-free solution at pH 7.2 and pH 7.8. Increase in contractile force (C.F.) was expressed as the value relative to the response at pH 7.8 and at 1.8 mM Ca 2÷. Each point on the curves represents the mean of six experiments; vertical bars represent + SEM.

this N G effect was t r a n s i e n t a t the lower concentration (4.4 x 10 -8 M) as s h o w n in Fig. 4, B. D T Z elicited sustained relaxing effect a t b o t h c o n c e n t r a tions (Fig. 4,A). T h e s e drugs also produced inhibitory effects on generation of p H 7.8-induced increase in v a s c u l a r tone. I n Fig. 6, the strips were t r e a t e d with the test drug before a n d during exposure to p H 7.8. T h e inhibitory effect of P R O P was the w e a k e s t (Fig. 6,C) a n d s p o n t a n e o u s r e c o v e r y - S f contractile response was observed in presence of t h e lower N G concentration (Fig. 6, B). T h e inhibitory effects of these drugs were reversible on w a s h o u t a n d the strip regained its original contractile response to p H 7.8. DISCUSSION Alkalosis-lnduced coronary constriction related to enhanced cellular calcium entry. I n t h e present s t u d y

decreased h y d r o g e n ion c o n c e n t r a t i o n

(increased

208

August 1 9 8 1

AmericanHeartJournal

Yasue et al.

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Fig. 5. Dose-response curves of the effects of nitroglycerin, diltiazem, and propranolol on the pH 7.8-induced increase in coronary vascular tone. NG = nitroglycerin; DIL = diltiazem; PRO = propranolol. Each point on the curves represents the mean of six experiments; vertical bars represent ___SEM.

Prop IC)s M

Prop 3 x I0"5 M

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Fig. 4. Experimental records of the effects of diltiazem, nitroglycerin, and propranolol on the pH 7.8-induced increase in coronary vascular tone. The pH in the modified Locke solution was increased from 7.2 to 7.8. A, Diltiazem; B, Nitroglycerin (NG); C, Propranolol (Prop).

pH) of the bath fluid consistently increased coronary vascular tone in absence of Pco~ effect. These results are in agreement with those of Fleckenstein et al.', ;~ Increased vascular tone in response to high pH was minimally discernible without Ca 2+ in the bath fluid, and addition of Ca ~+ to bath fluid produced dose-dependent increased vascular tone. These results strongly suggest t h a t extracellular Ca 2÷ is essential for increased vascular tone caused by high pH. Thus it can be inferred t h a t increased extracellular pH principally causes increased membrane permeability to Ca ~+, thereby increasing intracellular free Ca 2+ which is essential for the contraction of vascular smooth muscle./3," This assumption is compatible with the views of Fleckenstein et al.', ~ and van Breemen et al? 5 t h a t hydrogen ions compete with calcium ions for the same active site in the transmembrane calcium transport system. Rel=dlen of p r u e . t i n ~ to ~ i n d u o e d coronary spasm clinically. T h e present s t u d y was performed on isolated rabbit coronary arteries and

extrapolation to humans should be made with caution, since, besides possible species differences,16 our muscle strip investigations focused on individual aspects of the many variables that exist in vivo. However, since increased pH and alkalosis of body fluids caused by hyperventilation and Tris buffer infusion induced CAS in patients with variant angin a / ° it is rational to imply that alkalosis may produce coronary vasoconstriction clinically. Therefore, it may be necessary to avoid hyperventilation, excessive use of diuretics, or any modalities which might result in alkalosis in patients with vasospastic ischemic heart disease. Relief of alkalosis-induced coronary vasospasm by DTZ and NG. Diltiazem, a 1,5-benzothiazepine derivative,17. ~8 is one of the calcium antagonists which have been shown to be specifically effective in the treatment of variant anginaJ °, ~9, ~oNG also promptly relieves attacks of variant angina. In this regard, the present study clearly shows that DTZ and NG dose-dependently suppress increased coronary vascular tone induced by high pH. It may then be concluded that these drugs cause decreased coronary vascular tone (coronary vasodilatation) by interfering with transmembrane influx of Ca ~+ and result in suppression of variant angina episodes. This has indeed been confirmed in clinical studies. ~°, 19 However, in contrast to DTZ, NG in lower concentrations reduced vascular tone only transiently (Figs. 4 and 6). This observation is also in agreement with the report of Fleckenstein et at.," and suggests that NG possesses certain properties different from those of calcium antagonists. Direct coronary vasodilator property of high-dose PROP. The use of PROP in the treatment of variant

Volume102 Number2

Alkalosis-induced CAS:pH, Ca 2+, diltiazem, nitrate, propranolol effects IOmin

A ~

I Diltiozem I0-:'M/,~

~

Diltiazem I0"GM pH Z2"-~' pH~'~-~,8 ' p ~ - 2 "~'"

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Fig. 6. Experimental records of the effects of diltiazem, nitroglycerin, and propranolol on the generation of pH 7.8-induced increase in coronary vascular tone. Preparations were treated with drugs before and during exposure to pH 7.8. A, Diltiazem; B, Nitroglycerin (NG); C, Propranolol (Prop).

angina is controversial.19,21.25in the present study, P R O P in concentration (3 x 10 -~ M or 89 ng/ml) corresponding to its therapeutic dose for betaadrenergic blocking action 53,24 did not exhibit substantive effects on coronary vascular tone in vitro. However, at higher concentrations (3 x 10 -5 M or 8900 ng/ml)the drug decreased coronary vascular tone. This observation is of particular interest in view of some reports that therapeutic doses of P R O P m a y cause coronary vasoconstriction via its beta-adrenergic blocking action in vivo,53,55.5ewith aggravation of episodes of variant angina in some patients.19,55 Whether or not transmembrane movement of Ca 2+ is involved in the direct coronary vasodilatatory action of P R O P is uncertain at the present time, but it m a y be stated that P R O P directly causes coronary vasodilatation at concentrations higher than those required for beta-adrenergic blocking effect.

REFERENCES 1. Oliva PB, Potts DE, Plum RG: Coronary arterialspasm in Prinzmetars angina. Documentation by coronary arteriography. N Engl J M e d 288:745, 1973. 2. MacAlpin RN, Kattus AA, Alvaro AB: Angina pectoris at rest with preservation of exercise capacity: Prinzmetars variant angina. Circulation 47:946, 1973.

209

3. Yasue H, Touyama M, Kato H, Tanaka S, Akiyama F: Prinzmetars variant form of angina as a manifestation of alpha-adrenergic receptor-mediated coronary artery spasm. Documentation by coronary arteriography. AM HEART J 91:148, 1976. 4. Endo M, Hirosawa K, Kaneko N, Hase K, Inoue Y, Konno S: Prinzmetars variant angina. Coronary arteriogram and left ventriculogram during attack induced by methacholine. N Engl J Med 294:252, 1976. 5. Meller J, Pichard A, Dacks S: Coronary arterial spasm in Prinzmetars angina: A proved hypothesis. Am J Cardiol 37:938, 1976. 6. Maseri A, L'Abbate A, Pesola A, Ballestra AM, Marzilli M, Maltinti G, Severi S, DeNes, DM, Parodi O, Biagini A: Coronary vasospasm in angina pectoris. Lancet 1:713, 1977. 7. Yasue H, Omote S, Takizawa A, Nagao M, Miwa K, Tanaka S: Exertional angina caused by coronary arterial spasm: Effects of various drugs. Am J Cardiol 43:647, 1979. 8. Oliva PB, Breckenridge JC: Arteriographic evidence of coronary arterial spasm in acute myocardial infarction. Circulation 56:366, 1977. 9. Maseri A, L'Abbate A, Baroldi G, Chierchia S, Marzilli M, Ballestra AM, Severi S, Parodi O, Biagini A, Distante A, Pesola A: Coronary vasospasm as a possible cuase of "preinfarction" angina. N Engl J Med 299:1271, 1978. 10. Yasue H, Nagao M, Omote S, Takizawa A, Miwa K, Tanaka S: Coronary arterial spasm and Prinzmetal's variant form of angina induced by hyperventilation and Tris-buffer infusion. Circulation 58:56, 1978. 11. Fleckenstein A, Nakayarna K, Fleckenstein-Griin G, Byon YK: Interactions of vasoactive ions .and drugs with Cadependent excitation-contraction coupling of vascular smooth muscle. In Carafoli E, Clementi F, Drabikowski W, Margreth A, editors: Calcium transport in contraction and secretion. Amsterdam, 1975, North-Holland Publishing Co, p 555. 12. Nakayama K, Fleckenstein A, Byon YK, Fleckenstein-Griin G: Fundamental physiology of coronary smooth musculature from extramural stem arteries of pig and rabbit. Eur J Cardiol 3:319,1978. 13. Somlyo AP, Somlyo AV: Vascular smooth muscle. II. Pharmacology of normal and hypertensive vessels. Pharmacol Rev 22:249, 1970. 14. Bohr DF: Vascular smooth muscle updated. Circ Res 32:665, 1973. 15. van Breemen C, Farinas BR, Gerba P, McNaughton ED: Excitation-contraction coupling in rabbit aorta studied by the lanthanum method for measuring cellular calcium influx. Circ Res 30:44, 1972. 16. Ginsburg R, Brisow MR, Harrison DC, Stinson ED: Studies with isolated human coronary arteries: Some general observations, potential mediators of spasm, role of calcium antagonists. Chest 78(Suppl):180, 1980. 17. Sato M, Nagao T, Yamaguchi I, Nakajima H, Kiyomoto A: Pharmacological studies on a new 1,5-benzothiazepine derivative (CRD). Arzneim Forsch 21:1338, 1971. 18. Nakajima H, Hoshiyama M, Yamashita K, Kiyomoto A: Electrical and mechanical responses to diltiazem in potassium depolarized myocardium of the guinea pig. Jpn J Pharmacol 26:571, 1976. 19. Yasue H, Omote S, Takizawa A, Nagao M, Miwa K, Tanaka S: Pathogenesis and treatment of angina at rest as seen from its response to various drugs. Jpn Circ J 42:1, 1978. 20. Schroeder JS, Rosenthal S, Ginsburg R, Lamb I: Medical therapy of Prinzmetal's variant angina. Chest 78(Suppl):231, 1980. 21. Guazzi M, Fiorentini C, Polese A, Margrini F, Olivari MT: Treatment of spontaneous angina pectoris with beta-adrenergic blocking agents. Br Heart J 37:1235, 1975. 22. Yasue H: Beta-adrenergic blockade and coronary arterial spasm. In Sandoe E, Julian DG, Bell JW, editors: Manage-

August1981

210 Yasue et al. ment of ventricular tachycardia-Role of mexiletine. Amsterdam-Oxford, 1978, Excerpta Medica, p 305. 23. Nies AS, Shand DG: Clinical pharmacology of propranolol. Circulation 52:6, 1975. 24. Walle T, Conradi EC, Walle UK, Fagan TC, Gaffney TE: The predictable relationship between plasma levels and dose during chronic propranolol therapy. Clin Pharmacol Ther 24:668, 1978.

AmericanHeadJournal 25.

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Whitsitt LS, Lucchesi BR: Effects of propranolol and its stereoisomers upon coronary vascular resistance. Circ Res 21:305, 1967. Wolfson S, Gorlin R: Cardiovascular pharmacology of propranolol in man. Circulation 40:501, 1969.

Ventricular dysfunction and necrosis produced by adren hrome metabolite of epin rine: Relation to p ~ - - h o ~ ~ L j S of catecholamine cardiomyopathy We have examined the effects of adrenochrome and other metabolites of epinephrine on the uitrastructure and contractile activity of isolated rat hearts pertused under c o n d ~ in which the heart rate and coronary flow were controlled. Perfusinn of heads with epinephrine or metanephrine significantly increased contractile force; vanillyimandelic acid and dihydroxymandelic acid did not alter contractile force development, whereas adrenochrome (SO mg/L) declined contractile force with complete disappearance of contractile activity by 30 minutes, increased contractile force with epinephrine (50 mg/L) was associated with increased resting tension and maximum rates of force development and relaxation, and decreased time for peak tension development and 1/: relaxation. On the other hand, hearts per/used with adrenochrome showed early decline followed by steady increase in resting tension; maximum rates of force development and relaxation were reduced and times for peak tension development and r/= relaxation were increased. Heads per/used for 10 minutes or more with adrenochrome (50 mg/L), but not epinephrine, metanephrlne, dlhydroxymandelic acid or vanillylmandelic acid, showed ultrastructural damage. Adrenochrome concentrations of 10 or 25 mg/L altered the appearance of mitochondria alter 30 minutes of perfusion, infusion of epinephrine (1 rag/L) during perfuslon with adrenochrome partially maintained contractile force during the first 15 minutes of pertuslon but did not alter the severity of ultrestrucinral changes due to adrenochrome. These results are consistent with the concept that oxidation products of catechoiamines such as adronochrome are partly rasponsibl9 for inducing myocardial necrosis and failure following massive catacholamine injections in intact animals. (AM HEART J 102:210, 1981.)

John C. Yates, Robert E. Beamish, and Naranjan S. Dhalla.

Winnipeg, Manitoba, Canada It is well recognized t hat catecholamines in large doses can produce necrotic lesions in the myocardium. 1-3 Catecholamines also produce a number of From the Division of Experimental Cardiology, Departments of Physiology and Medicine, Faculty of Medicine, University of Manitoba. This study was supported by grants from The Great West Life Assurance Company, Winnipeg. Received for publication Feb. 7, 1981; accepted March 27, 1981. Reprint requests: Dr. N. S. Dhalla, Division of Experimental Cardiology, Dept. of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3.

profound cardiovascular pharmacologic effects which include changes in peripheral resistance, arterial blood pressure, cardiac o u t p u t and venous return; increases in heart rate and cardiac work causing increased myocardial oxygen demand; release of further catecholamines within adrenergic nerve endings; and alterations in lipid and carbohydrate metabolism resulting in accumulation of exogenous lipids in the heart. As a consequence, it has been difficult to determine whether catecholamines exert a direct toxic influence on the myocardium, or 0002-8703/81/080210 + 12501.20/0 © 1981 The C. V. Mosby Co.