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Can sympathomimetic agents be classified by their action on the force-interval relationship?
Life Sciences, Vol. 32, pp. 329-336 Printed in the U.S.A.
Pergamon Press
CAN SYMPATHOMIMETICAGENTS BE CLASSIFIED BY THEIR ACTION ON THE FORCE-INTERVAL RELATIONSHIP? Andres Manring, Page A. W. Anderson, Rashid Nassar and W. Robins Howe Departments of Physiology, Pediatrics and Surgery, Duke University Medical Center Durham, North Carolina 27710 (Received in final form October 5, 1982)
Summary The use of the f o r c e - i n t e r v a l r e l a t i o n s h i p , the dependence of cardiac c o n t r a c t i l i t y on the rate and pattern of stimulation, has been suggested as an index of cardiac c o n t r a c t i l i t y , e g. i t d i f f e r e n t i a t e s among normal, hypertrophied and f a i l i n g myocardium. In this paper, we describe the e f f e c t s of sympathomimetic agents on the f o r c e - i n t e r v a l r e l a t i o n s h i p and show that this r e l a t i o n s h i p is useful in d i f f e r e n t i a t i n g the actions of these drugs on the myocardium. Rabbit r i g h t v e n t r i c u l a r p a p i l l a r y muscles were exposed to isoproterenol, norepinephrine, methoxamine and phenylephrine during a standard two-stage pacing experiment. The f o r c e - i n t e r v a l r e l a t i o n s h i p is characterized by f o r c e - i n t e r v a l curves which describe how c o n t r a c t i l i t y , i . e . the maximum rate of rise of force (Fmax), changes between contractions. In the control solution the curves were monotonic and the f o r c e - i n t e r v a l r a t i o (FIR), a measure of p o s t - e x t r a s y s t o l i c p o t e n t i a t i o n , was always greater than unity. Isoproterenol and norepinephrine had s i m i l a r actions on the forceinterval r e l a t i o n s h i p : Fmax was potentiated, FIR reduced and the curves became biphasic. Methoxamine had the same e f f e c t on FIR and the curves. Practolol or propr~nolol competitively i n h i b i t e d these changes whereas phentolamine did not i n h i b i t but accentuated them. Phenylephrine increased Fmax, but unlike the other agonists i t increased FIR and the curves remained monophasic. These e f f e c t s were i n h i b i t e d competitively by phentolamine. Based upon the action of the competitive i n h i b i t o r s , these patterns are separable into those produced by ~- and ~-agonists. The results suggest that the forceinterval r e l a t i o n s h i p may be used as a new basis f o r distinguishing the actions of sympathomimetic agents. The f o r c e - i n t e r v a l r e l a t i o n s h i p , the dependence of cardiac c o n t r a c t i l i t y on the rate and pattern of stimulation, has been shown to be a useful tool for exploring changes in cardiac c o n t r a c t i l i t y in the isolated muscle, the i n t a c t animal and the patient (1,2). The r e l a t i o n s h i p is sensitive to a change in inotropy (2) yet is unaltered by a change in muscle length (2), Sympathomimetic agents have been shown to decrease the s e n s i t i v i t y Of isolated v e n t r i c u l a r myocardium to a l t e r a t i o n s in pacing: p o s t - e x t r a s y s t o l i c potentiation was decreased and the steady-state force-frequency curves were f l a t t e n e d (2,3). We recently reported an action of high doses of ~orepinephrine which, at that time, appeared to be unique to norepinephrine: p o s t - e x t r a s y s t o l i c potentiation became poste x t r a s y s t o l i c depression and the time course of c o n t r a c t i l i t y between contrac0024-3205/83/040329-08503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
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tions became biphasic (1). We undertook the present study to determine whether these effects on the force-interval relationship were a specific action of norepinephrine or a general action of either ~- or B-agonists on myocardial cont r a c t i l i t y (4,5).
The study examines the e f f e c t s of isoproterenol, norepinephrine, methoxamine and phenylephrine on the f o r c e - i n t e r v a l r e l a t i o n s h i p . Practolol, propranoloi and phentolamine were used to t e s t whether these effects could be characterized as ~ or B e f f e c t s . Materials and Methods Experiments were performed on hearts from 44 adults New Zealand white rabbits. A r i g h t v e n t r i c u l a r p a p i l l a r y muscle was mounted i s o m e t r i c a l l y and superfused with aerated (95%, 02 , 5% C02) Krebs-Henseleit (K-H) solution containing ascorbic acid, 200 mg/l (38°C). The bath, d e t a i l s of the preparation, the force transducer, and the system used for the data acquisition and analysis have been described previously (1). The method used to analyze the f o r c e - i n t e r v a l r e l a t i o n s h i p has been described elsewhere ( I ) , The muscle was stimulated at a low rate (0.33 Hz = 1/to). Two t e s t stimuli were introduced between every s i x t h and seventh regular beat. The t e s t i n t e r v a l s (the time between the test stimulus and the preceding regular stimulus) were varied systematically in two stages (see Fig. IA): The f i r s t stage determined the way Fmax of a t e s t contraction ( s l ) depended on t 1, the interval between s I and the previous regular contraction (So). Fm~v of s I was plotted against t I (the f i r s t stage curve). The second stage d e t e ~ i n e d the way Fmax of a t e s t contraction (s2) that followed } i depended on t 2, the interval between s o and s 2 ( t I constant, t I always < t 2 ) . Fma~ of s 2 plotted against t 2 formed a d i ~ ferent curve (second stage curve) f o r eacn value of t I. The following procedure was used to determine the e f f e c t s of the agonists, and the a b i l i t y of the antagonists to block those e f f e c t s : The muscle was exposed to a sequence of progressively higher doses of the agonist (isoproterenol, norepinephrine, methoxamine and phenylephrine). Following each exposure the muscle was superfused with K-H u n t i l Fmax reached the control level (usually in 2030 minutes). ~max and the f o r c e - i n t e r v a l r a t i o (FIR:~ma x of s 2, t 2 = 2500 msec,÷ Fmax of So) obtained during e~os-u-r-e~K~HH (termed control) and to the various concentrations of agonist were compared. The f i r s t and second stage curves were also compared. This procedure was repeated a f t e r superfusion with propranolol, practolol or phentolamine f o r 45 minutes. The changes in Fmax and FIR were evaluated using analysis of variance (block design); f a c t o r i a l analysis was used to test the e f f e c t s of the antagonists. The means of all treatments were compared using Duncan's Multiple Range Test (p < 0.05 was considered s i g n i f i c a n t ) . Drugs were purchased in powder forms: (-)-norepinephrine HCl, ( - ) - i s o p r o terenol HCI, (-)-phenylephrine HCl and ascorbic acid (Sigma Corporation); propranolol HCI (Ayerst Laboratory, I n c . ) ; and phentolamine mesylate (CIBA). We thank Burroughs Wellcome Co. for providing methoxamine HCI and Ayerst for practolol. Resul ts Isoproterenol: IO'SM s i g n i f i c a n t l y potentiated Fmax of a l l contractions (Fig. 1C, IF). All curves were monotonic l i k e the control curves, but the f i r s t stage curve was l i f t e d more than any of the second stage curves. FIR declined s i g n i f i c a n t l y (Fig. IG). At IO-TM (Fig. ID, IF) Fmax of all contractions was f u r t h e r potentiated,
Vol. 32, No. 4, 1983
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and FIR was further reduced (Fig, 1G), Both changes were significant. The f i r s t stage curves remained monotonic, but the second stage curves (Fig. 1D) rose for t~ < 700 msec then declined ( i . e , dooped) in a l l muscles. .......... t z . . . . . . . . •
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FIG. 1 Effects of isoproterenol on the force-interval relationship. A. Demonstrates the pacing pattern: see Methods for description. The results from the f i r s t stage (|; solid lines) and the second stage (A; dotted lines, t I = 200 msec) curves of the same muscle prior to (B) and during exposure to 10-8M (C), I0-7M (D), and 10-6M (E) isoproterenol. During exposure to 10-7M and IO-GM isoproterenol, the curves for the second stage of the experiment (dotted lines) were biphasic, i . e . they drooped. Panel F i l l u s t r a t e s the increase in Fmax (± se) of the regular contraction from the control value (KH) to that in isoproterenol (10-8 , 10-7 , 10-6M). Panel G i l l u s t r a t e s the decrease in the force-interval ratio from the control value (KH) to that in isoproterenol (10 -8 , 10-7 , IO-GM). N = 17. At IO-GM (Fig. 1E, 1F) Fmax was further potentiated and FIR was s t i l l further reduced. Both changes were significant. The droop became more pronounced. The second stage curve drooped in a l l muscles, and in 4 muscles even the f i r s t stage curves drooped. The droop in the second stage curves became so severe that in 15 muscles the plateau was lower for the second stage than for the f i r s t stage curve. Consequently, FIR became less than unity, i . e . post-extrasystolic potentiation became post-extrasystolic depression (Fig. 1E, 1G). Norepinephrine: At IO-SM Fmax was potentiated; FIR was s i g n i f i c a n t l y lower, 2.73 ± 0.24 (s.e.) to 1.03 ± 0.05, t I = 225 msec. The second stage curves
332
Cardiac Contractility: ~- and 8-agonists
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Effects of phenylephrine on the force-interval relationship. A. The f i r s t ( c i r cles) and the second (triangles) stage curves from a muscle prior to (KH; 0,~) and during exposure to phenylephrine (~,a; lO-SM: |,A; IO-~M). B. ~max of the regular contraction in KH and in phenylephrine (10 -6, i0 -s, 10-4M). ~. The force-interval ratio (t I = 250 msec) in KH and in phenylephrine (N = 9). Methoxamine: I0-4M did not potentiate Fmax but did cause FIR to f a l l . I0-3M depressed Fmay and FIR f e l l further. The f i r s t and second stage curves drooped (Fig. 2A). - Phenylephrine:
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Vol. 32, No. 4, 1983
Cardiac Contractility:
~-
and B-agonists
333
significant increases at I0 -s and IO-4H (Fig. 3B). At 10-sM FIR was slgnificantly increased over the control (Fig. 3C). At lO-hM an extrasystole (t I = 250 to 500 msec) could not be e l i c i t e d in 4 muscles. Although the mean was increased, FIR was not significantly altered (FIR increased in 3 muscles and f e l l in 2). The force-interval curves remained monophasic in all concentrations of phenylephrine (Fig. 3A).
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FIG. 4 Effect of practolol on the isoproterenol-induced changes in the force-interval relationship. Panel A. The f i r s t stage (0; solid lines) and the second stage (A; dotted lines) curves drooped with exposure to IO-?M isoproterenol. In the presence of 10-6M practolol and 10-7M isoproterenol, Fmax was less, and the forceinterval ratio was larger and no droop was present T~,A). Increasing the isoproterenol concentration to IO-GM caused the ratio to f a l l and the curves to droop (O,A) even though practolol was present. Panel B. Fmax of the regular contraction under control conditions (KH) and in isoproterenol (10 -B, 10-7 , ~-GM) are compared in the presence of (hatched bars) and the absence of (open bars) IO-GM practolol. Panel C. The force-interval ratio (t I = 250 msec) under control conditions (KH) and in isoproterenol (10 -8 , 10-7 , IO-GM) are compared in the presence (hatched bars) and absence (open bars) of IO-GM practolol; N = 6. Practolol: 10-6M significantly displaced the concentration-effect curve of isoproterenol for Fmax and FIR to the right (Fig. 4). In IO-GM isoproterenol and practolol, although FIR was lowered, i t did not f a l l below unity as i t had in 10-6M isoproterenol alone. In 10-7M isoproterenol and no practolol the second stage curves drooped in 6 of 6 muscles (Fig. 4). In IO-GM practolol the droop at 10-7M isoproterenol was abolished (Fig. 4). However, the droop at IO-GM isoproterenol was not abolished (Fig. 4).
The potentiation of Fmax, the decrease in FIR and the droop in the forceinterval curves induced by norepinephrine (I0 -~, IO-5M) were shifted to the r i g h t by propranolol or practolol. S i m i l a r l y , propranolol and practolol abolished the droop in the force-interval curves induced by methoxamine (10 -4 ,
334
Cardiac Contractility:
~- and B-agonists
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10-3M; Fig. 2B). However, propranolol did not block the increase in the FIR produced by phenylephrine lO-SM. In 4 muscles the r a t i o increased 20 ± 4% in the presence of phenylephrine alone, and 21 ± 4% in the presence of phenylephrine and propranolol.
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FIG. 5 Effect of phentolamine on the isoproterenol-induced changes in the f o r c e - i n t e r val r e l a t i o n s h i p . Panel A. The f i r s t stage curves ( c i r c l e s , solid lines) and the second stage curves ( t r i a n g l e s , dotted lines) of the same muscle. In IO-~M isoproterenol (O,A) only the second stage curve drooped. In 5 x IO-6M phentolamine and no isoproterenol the curves (~,A} were monotonic. In the presence of 5 x lO-SM phentolamine and IO-6M isoproterenol, the f i r s t stage curve ( | ) drooped and the droop in the second stage curve (A) was accentuated. Panel B. Fmax of the regular contraction under control conditions (KH) and in isoproterenol (10 -8 , 10-7 , IO-GM) are compared in the presence of (hatched bars) and the absence of (open bars) 5 x IO-6M phentolamine. Panel C. The f o r c e - i n t e r v a l r a t i o (t I = 200 msec) under control conditions (KH) and in isoproterenol (10 -B, 10-7 , IO-GM) are compared in the presence (hatched bars) and absence (open bars) of 5 x IO-6M phentolamine; N = 6. Phentolamine: 5 x IO-GM accentuated the e f f e c t of isoproterenol on the f o r c e - i n t e r v a l r e l a t i o n s h i p (Fig. 5). FIR f e l l f u r t h e r and the droop appeared in the f i r s t stage curves and became more pronounced in the second stage curves. The combined e f f e c t of phenylephrine and phentolamine on the r e f r a c t o r y period of the muscles limited the evaluation of the f o r c e - i n t e r v a l r e l a t i o n s h i p as no extrasystoles could be e l i c i t e d at t I = 250 to 500 msec in the majority of muscles in lO-SM and IO-4M phenylephrine and in phentolamine. When an extrasystole could be e l i c i t e d , the increase in the r a t i o induced by phenylephrine was prevented by phentolamine; indeed, the r a t i o f e l l (e.g. f o r t I : 250 msec the r a t i o increased by 33 ± 6.5% in lO-SM phenylephrine but f e l l 27.5 ± 1% in lO-SM phenylephrine and phentolamine).
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Cardiac Contractility: ~- and B-agonists
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Discussion The action of sympathomimetic drugs used in this study f e l l into two dist i n c t classes (A and B) according to t h e i r e f f e c t on the f o r c e - i n t e r v a l r e l a tionship. Class A (phenylephrine) increased the FIR and class B (isoproterenol, norepinephrine and methoxamine) decreased i t . High doses of a class B drug produced e f f e c t s which appear to be unique among inotropic agents ( I ) : the FIR curves which are normally monophasic become biphasic; and the FIR which is normally greater than unity ( i . e , p o s t - e x t r a s y s t o l i c p o t e n t i a t i o n ) became smaller than unity ( i . e . p o s t - e x t r a s y s t o l i c depression). The present c l a s s i f i c a t i o n of the sympathomimetic agents coincides approximately with the t r a d i t i o n a l c l a s s i f i c a t i o n as alpha and beta agonists. However, one important exception was methoxamine, which is a recognized alpha agonist (6) but whose action on the FIR f e l l into class B along with the beta agonists. This exception could be related to the phylogenetic and ontogenetic differences in receptor number and a c t i vation (7). Alpha and beta adrenergic blocking agents were used to explore these effects f u r t h e r . The beta blockers shifted the concentration-ratio curves to the r i g h t and eliminated or g r e a t l y reduced the droop in the FIR curves induced by the class B drugs, including methoxamine. This indicates competitive antagonism. The alpha blocker abolished the increase in the FIR produced by phenylephrine. The anomalous c l a s s i f i c a t i o n of methoxamine suggests that two d i s t i n c t beta actions may e x i s t in the heart: one is potentiation of force and the acceleration of r e l a x a t i o n ; and the other is the action on the f o r c e - i n t e r v a l r e l a t i o n ship. Isoproterenol and norepinephrine produced both actions and practolol and propranolol blocked both. Methoxamine altered only the f o r c e - i n t e r v a l r e l a t i o n ship. What is needed to explore this f u r t h e r are beta blockers that s e l e c t i v e l y a f f e c t e i t h e r the p o t e n t i a t i o n of force or the droop. The mechanism f o r class A and B action on the FIR is not known. Elevation of CAMP l e v e l s , which is widely believed to be involved in the p o s i t i v e inotropic action of beta agonists, is probably not responsible for the action of class B drugs on this r e l a t i o n s h i p because d i b u t r y l cyclic-AMP increases rather than decreases the FIR (unpublished observation). An increase in a slow inward calcium current by the beta agonists might explain the f a l l in the r a t i o , for this is s i m i l a r to the e f f e c t of elevating e x t r a c e l l u l a r calcium concentration on the f o r c e - i n t e r v a l r e l a t i o n s h i p ( I ) , but the droop in the curve and the transformation from p o s t - e x t r a s y s t o l i c p o t e n t i a t i o n to p o s t - e x t r a s y s t o l i c depression is unlike the e f f e c t of elevated calcium or of any other inotropic intervention of which we are aware. Acknowledgements The forming part by cipient
authors would l i k e to thank Mrs. Annette Oakeley for her help in perthe experiments and analyzing the r e s u l t s . This work was supported in NIH grants: HL 20749, HL 11307 and HL 18270. Dr. Anderson was the reof Research Career Development Award HL 00500. References
1. P.A,W. ANDERSON, A. MANRING, and E.A. JOHNSON, J. Mol. Cell Cardiol. 2, 131-150 (1977). 2. A. MANRING and P.A.W. ANDERSON, C r i t . Rev. Bioeng. 4, 165-201 (1980). 3. J. KOCH-WESER, C.M. BERLIN, JR. and J.R. BLINKS, Pharmacology of Cardiac Function, pp. 63-72, The MacMillan Co., New York (1964). 4. J.T. STULL and S.E. MAYER, Handbook of Physio199y, pp. 741-774, American Physiological Society, Bethesda (1979).
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5. B.G. BENFY, Br. J. Pharmac. 48, 132-138 (1977). 6. B. RABINOWITZ, L. CHUCH, M. KLINGERMAN, and W.W. PARMLEY, Am. J. Physiol. 229, 582-585 (1975). 7. R.---~-.-.-.-.-.-.~.FELDER, P.L. CALGAGNO, G.M. EISNER, and P.A. JOSE, Pediat. Res. 16, 340-342 (1982), 8. J.B. OSNERS, H. REFSUM, T. SKIMEDAL, and I. BYE, Acta. Pharm. Tox. 42, 235-247 (1978). 9. A.M. KATZ, Adv. Cycl. Nucl. Res. 11, 303-343 (1979). 10. H. SCHOLZ, Handbook of Experimental Pharmacology, 5__4, 651-778 (1980).
Pergamon Press
CAN SYMPATHOMIMETICAGENTS BE CLASSIFIED BY THEIR ACTION ON THE FORCE-INTERVAL RELATIONSHIP? Andres Manring, Page A. W. Anderson, Rashid Nassar and W. Robins Howe Departments of Physiology, Pediatrics and Surgery, Duke University Medical Center Durham, North Carolina 27710 (Received in final form October 5, 1982)
Summary The use of the f o r c e - i n t e r v a l r e l a t i o n s h i p , the dependence of cardiac c o n t r a c t i l i t y on the rate and pattern of stimulation, has been suggested as an index of cardiac c o n t r a c t i l i t y , e g. i t d i f f e r e n t i a t e s among normal, hypertrophied and f a i l i n g myocardium. In this paper, we describe the e f f e c t s of sympathomimetic agents on the f o r c e - i n t e r v a l r e l a t i o n s h i p and show that this r e l a t i o n s h i p is useful in d i f f e r e n t i a t i n g the actions of these drugs on the myocardium. Rabbit r i g h t v e n t r i c u l a r p a p i l l a r y muscles were exposed to isoproterenol, norepinephrine, methoxamine and phenylephrine during a standard two-stage pacing experiment. The f o r c e - i n t e r v a l r e l a t i o n s h i p is characterized by f o r c e - i n t e r v a l curves which describe how c o n t r a c t i l i t y , i . e . the maximum rate of rise of force (Fmax), changes between contractions. In the control solution the curves were monotonic and the f o r c e - i n t e r v a l r a t i o (FIR), a measure of p o s t - e x t r a s y s t o l i c p o t e n t i a t i o n , was always greater than unity. Isoproterenol and norepinephrine had s i m i l a r actions on the forceinterval r e l a t i o n s h i p : Fmax was potentiated, FIR reduced and the curves became biphasic. Methoxamine had the same e f f e c t on FIR and the curves. Practolol or propr~nolol competitively i n h i b i t e d these changes whereas phentolamine did not i n h i b i t but accentuated them. Phenylephrine increased Fmax, but unlike the other agonists i t increased FIR and the curves remained monophasic. These e f f e c t s were i n h i b i t e d competitively by phentolamine. Based upon the action of the competitive i n h i b i t o r s , these patterns are separable into those produced by ~- and ~-agonists. The results suggest that the forceinterval r e l a t i o n s h i p may be used as a new basis f o r distinguishing the actions of sympathomimetic agents. The f o r c e - i n t e r v a l r e l a t i o n s h i p , the dependence of cardiac c o n t r a c t i l i t y on the rate and pattern of stimulation, has been shown to be a useful tool for exploring changes in cardiac c o n t r a c t i l i t y in the isolated muscle, the i n t a c t animal and the patient (1,2). The r e l a t i o n s h i p is sensitive to a change in inotropy (2) yet is unaltered by a change in muscle length (2), Sympathomimetic agents have been shown to decrease the s e n s i t i v i t y Of isolated v e n t r i c u l a r myocardium to a l t e r a t i o n s in pacing: p o s t - e x t r a s y s t o l i c potentiation was decreased and the steady-state force-frequency curves were f l a t t e n e d (2,3). We recently reported an action of high doses of ~orepinephrine which, at that time, appeared to be unique to norepinephrine: p o s t - e x t r a s y s t o l i c potentiation became poste x t r a s y s t o l i c depression and the time course of c o n t r a c t i l i t y between contrac0024-3205/83/040329-08503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
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tions became biphasic (1). We undertook the present study to determine whether these effects on the force-interval relationship were a specific action of norepinephrine or a general action of either ~- or B-agonists on myocardial cont r a c t i l i t y (4,5).
The study examines the e f f e c t s of isoproterenol, norepinephrine, methoxamine and phenylephrine on the f o r c e - i n t e r v a l r e l a t i o n s h i p . Practolol, propranoloi and phentolamine were used to t e s t whether these effects could be characterized as ~ or B e f f e c t s . Materials and Methods Experiments were performed on hearts from 44 adults New Zealand white rabbits. A r i g h t v e n t r i c u l a r p a p i l l a r y muscle was mounted i s o m e t r i c a l l y and superfused with aerated (95%, 02 , 5% C02) Krebs-Henseleit (K-H) solution containing ascorbic acid, 200 mg/l (38°C). The bath, d e t a i l s of the preparation, the force transducer, and the system used for the data acquisition and analysis have been described previously (1). The method used to analyze the f o r c e - i n t e r v a l r e l a t i o n s h i p has been described elsewhere ( I ) , The muscle was stimulated at a low rate (0.33 Hz = 1/to). Two t e s t stimuli were introduced between every s i x t h and seventh regular beat. The t e s t i n t e r v a l s (the time between the test stimulus and the preceding regular stimulus) were varied systematically in two stages (see Fig. IA): The f i r s t stage determined the way Fmax of a t e s t contraction ( s l ) depended on t 1, the interval between s I and the previous regular contraction (So). Fm~v of s I was plotted against t I (the f i r s t stage curve). The second stage d e t e ~ i n e d the way Fmax of a t e s t contraction (s2) that followed } i depended on t 2, the interval between s o and s 2 ( t I constant, t I always < t 2 ) . Fma~ of s 2 plotted against t 2 formed a d i ~ ferent curve (second stage curve) f o r eacn value of t I. The following procedure was used to determine the e f f e c t s of the agonists, and the a b i l i t y of the antagonists to block those e f f e c t s : The muscle was exposed to a sequence of progressively higher doses of the agonist (isoproterenol, norepinephrine, methoxamine and phenylephrine). Following each exposure the muscle was superfused with K-H u n t i l Fmax reached the control level (usually in 2030 minutes). ~max and the f o r c e - i n t e r v a l r a t i o (FIR:~ma x of s 2, t 2 = 2500 msec,÷ Fmax of So) obtained during e~os-u-r-e~K~HH (termed control) and to the various concentrations of agonist were compared. The f i r s t and second stage curves were also compared. This procedure was repeated a f t e r superfusion with propranolol, practolol or phentolamine f o r 45 minutes. The changes in Fmax and FIR were evaluated using analysis of variance (block design); f a c t o r i a l analysis was used to test the e f f e c t s of the antagonists. The means of all treatments were compared using Duncan's Multiple Range Test (p < 0.05 was considered s i g n i f i c a n t ) . Drugs were purchased in powder forms: (-)-norepinephrine HCl, ( - ) - i s o p r o terenol HCI, (-)-phenylephrine HCl and ascorbic acid (Sigma Corporation); propranolol HCI (Ayerst Laboratory, I n c . ) ; and phentolamine mesylate (CIBA). We thank Burroughs Wellcome Co. for providing methoxamine HCI and Ayerst for practolol. Resul ts Isoproterenol: IO'SM s i g n i f i c a n t l y potentiated Fmax of a l l contractions (Fig. 1C, IF). All curves were monotonic l i k e the control curves, but the f i r s t stage curve was l i f t e d more than any of the second stage curves. FIR declined s i g n i f i c a n t l y (Fig. IG). At IO-TM (Fig. ID, IF) Fmax of all contractions was f u r t h e r potentiated,
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and FIR was further reduced (Fig, 1G), Both changes were significant. The f i r s t stage curves remained monotonic, but the second stage curves (Fig. 1D) rose for t~ < 700 msec then declined ( i . e , dooped) in a l l muscles. .......... t z . . . . . . . . •
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FIG. 1 Effects of isoproterenol on the force-interval relationship. A. Demonstrates the pacing pattern: see Methods for description. The results from the f i r s t stage (|; solid lines) and the second stage (A; dotted lines, t I = 200 msec) curves of the same muscle prior to (B) and during exposure to 10-8M (C), I0-7M (D), and 10-6M (E) isoproterenol. During exposure to 10-7M and IO-GM isoproterenol, the curves for the second stage of the experiment (dotted lines) were biphasic, i . e . they drooped. Panel F i l l u s t r a t e s the increase in Fmax (± se) of the regular contraction from the control value (KH) to that in isoproterenol (10-8 , 10-7 , 10-6M). Panel G i l l u s t r a t e s the decrease in the force-interval ratio from the control value (KH) to that in isoproterenol (10 -8 , 10-7 , IO-GM). N = 17. At IO-GM (Fig. 1E, 1F) Fmax was further potentiated and FIR was s t i l l further reduced. Both changes were significant. The droop became more pronounced. The second stage curve drooped in a l l muscles, and in 4 muscles even the f i r s t stage curves drooped. The droop in the second stage curves became so severe that in 15 muscles the plateau was lower for the second stage than for the f i r s t stage curve. Consequently, FIR became less than unity, i . e . post-extrasystolic potentiation became post-extrasystolic depression (Fig. 1E, 1G). Norepinephrine: At IO-SM Fmax was potentiated; FIR was s i g n i f i c a n t l y lower, 2.73 ± 0.24 (s.e.) to 1.03 ± 0.05, t I = 225 msec. The second stage curves
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Effects of phenylephrine on the force-interval relationship. A. The f i r s t ( c i r cles) and the second (triangles) stage curves from a muscle prior to (KH; 0,~) and during exposure to phenylephrine (~,a; lO-SM: |,A; IO-~M). B. ~max of the regular contraction in KH and in phenylephrine (10 -6, i0 -s, 10-4M). ~. The force-interval ratio (t I = 250 msec) in KH and in phenylephrine (N = 9). Methoxamine: I0-4M did not potentiate Fmax but did cause FIR to f a l l . I0-3M depressed Fmay and FIR f e l l further. The f i r s t and second stage curves drooped (Fig. 2A). - Phenylephrine:
Fmax was significantly potentiated at I0-6M with further
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Cardiac Contractility:
~-
and B-agonists
333
significant increases at I0 -s and IO-4H (Fig. 3B). At 10-sM FIR was slgnificantly increased over the control (Fig. 3C). At lO-hM an extrasystole (t I = 250 to 500 msec) could not be e l i c i t e d in 4 muscles. Although the mean was increased, FIR was not significantly altered (FIR increased in 3 muscles and f e l l in 2). The force-interval curves remained monophasic in all concentrations of phenylephrine (Fig. 3A).
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FIG. 4 Effect of practolol on the isoproterenol-induced changes in the force-interval relationship. Panel A. The f i r s t stage (0; solid lines) and the second stage (A; dotted lines) curves drooped with exposure to IO-?M isoproterenol. In the presence of 10-6M practolol and 10-7M isoproterenol, Fmax was less, and the forceinterval ratio was larger and no droop was present T~,A). Increasing the isoproterenol concentration to IO-GM caused the ratio to f a l l and the curves to droop (O,A) even though practolol was present. Panel B. Fmax of the regular contraction under control conditions (KH) and in isoproterenol (10 -B, 10-7 , ~-GM) are compared in the presence of (hatched bars) and the absence of (open bars) IO-GM practolol. Panel C. The force-interval ratio (t I = 250 msec) under control conditions (KH) and in isoproterenol (10 -8 , 10-7 , IO-GM) are compared in the presence (hatched bars) and absence (open bars) of IO-GM practolol; N = 6. Practolol: 10-6M significantly displaced the concentration-effect curve of isoproterenol for Fmax and FIR to the right (Fig. 4). In IO-GM isoproterenol and practolol, although FIR was lowered, i t did not f a l l below unity as i t had in 10-6M isoproterenol alone. In 10-7M isoproterenol and no practolol the second stage curves drooped in 6 of 6 muscles (Fig. 4). In IO-GM practolol the droop at 10-7M isoproterenol was abolished (Fig. 4). However, the droop at IO-GM isoproterenol was not abolished (Fig. 4).
The potentiation of Fmax, the decrease in FIR and the droop in the forceinterval curves induced by norepinephrine (I0 -~, IO-5M) were shifted to the r i g h t by propranolol or practolol. S i m i l a r l y , propranolol and practolol abolished the droop in the force-interval curves induced by methoxamine (10 -4 ,
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~- and B-agonists
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10-3M; Fig. 2B). However, propranolol did not block the increase in the FIR produced by phenylephrine lO-SM. In 4 muscles the r a t i o increased 20 ± 4% in the presence of phenylephrine alone, and 21 ± 4% in the presence of phenylephrine and propranolol.
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FIG. 5 Effect of phentolamine on the isoproterenol-induced changes in the f o r c e - i n t e r val r e l a t i o n s h i p . Panel A. The f i r s t stage curves ( c i r c l e s , solid lines) and the second stage curves ( t r i a n g l e s , dotted lines) of the same muscle. In IO-~M isoproterenol (O,A) only the second stage curve drooped. In 5 x IO-6M phentolamine and no isoproterenol the curves (~,A} were monotonic. In the presence of 5 x lO-SM phentolamine and IO-6M isoproterenol, the f i r s t stage curve ( | ) drooped and the droop in the second stage curve (A) was accentuated. Panel B. Fmax of the regular contraction under control conditions (KH) and in isoproterenol (10 -8 , 10-7 , IO-GM) are compared in the presence of (hatched bars) and the absence of (open bars) 5 x IO-6M phentolamine. Panel C. The f o r c e - i n t e r v a l r a t i o (t I = 200 msec) under control conditions (KH) and in isoproterenol (10 -B, 10-7 , IO-GM) are compared in the presence (hatched bars) and absence (open bars) of 5 x IO-6M phentolamine; N = 6. Phentolamine: 5 x IO-GM accentuated the e f f e c t of isoproterenol on the f o r c e - i n t e r v a l r e l a t i o n s h i p (Fig. 5). FIR f e l l f u r t h e r and the droop appeared in the f i r s t stage curves and became more pronounced in the second stage curves. The combined e f f e c t of phenylephrine and phentolamine on the r e f r a c t o r y period of the muscles limited the evaluation of the f o r c e - i n t e r v a l r e l a t i o n s h i p as no extrasystoles could be e l i c i t e d at t I = 250 to 500 msec in the majority of muscles in lO-SM and IO-4M phenylephrine and in phentolamine. When an extrasystole could be e l i c i t e d , the increase in the r a t i o induced by phenylephrine was prevented by phentolamine; indeed, the r a t i o f e l l (e.g. f o r t I : 250 msec the r a t i o increased by 33 ± 6.5% in lO-SM phenylephrine but f e l l 27.5 ± 1% in lO-SM phenylephrine and phentolamine).
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Discussion The action of sympathomimetic drugs used in this study f e l l into two dist i n c t classes (A and B) according to t h e i r e f f e c t on the f o r c e - i n t e r v a l r e l a tionship. Class A (phenylephrine) increased the FIR and class B (isoproterenol, norepinephrine and methoxamine) decreased i t . High doses of a class B drug produced e f f e c t s which appear to be unique among inotropic agents ( I ) : the FIR curves which are normally monophasic become biphasic; and the FIR which is normally greater than unity ( i . e , p o s t - e x t r a s y s t o l i c p o t e n t i a t i o n ) became smaller than unity ( i . e . p o s t - e x t r a s y s t o l i c depression). The present c l a s s i f i c a t i o n of the sympathomimetic agents coincides approximately with the t r a d i t i o n a l c l a s s i f i c a t i o n as alpha and beta agonists. However, one important exception was methoxamine, which is a recognized alpha agonist (6) but whose action on the FIR f e l l into class B along with the beta agonists. This exception could be related to the phylogenetic and ontogenetic differences in receptor number and a c t i vation (7). Alpha and beta adrenergic blocking agents were used to explore these effects f u r t h e r . The beta blockers shifted the concentration-ratio curves to the r i g h t and eliminated or g r e a t l y reduced the droop in the FIR curves induced by the class B drugs, including methoxamine. This indicates competitive antagonism. The alpha blocker abolished the increase in the FIR produced by phenylephrine. The anomalous c l a s s i f i c a t i o n of methoxamine suggests that two d i s t i n c t beta actions may e x i s t in the heart: one is potentiation of force and the acceleration of r e l a x a t i o n ; and the other is the action on the f o r c e - i n t e r v a l r e l a t i o n ship. Isoproterenol and norepinephrine produced both actions and practolol and propranolol blocked both. Methoxamine altered only the f o r c e - i n t e r v a l r e l a t i o n ship. What is needed to explore this f u r t h e r are beta blockers that s e l e c t i v e l y a f f e c t e i t h e r the p o t e n t i a t i o n of force or the droop. The mechanism f o r class A and B action on the FIR is not known. Elevation of CAMP l e v e l s , which is widely believed to be involved in the p o s i t i v e inotropic action of beta agonists, is probably not responsible for the action of class B drugs on this r e l a t i o n s h i p because d i b u t r y l cyclic-AMP increases rather than decreases the FIR (unpublished observation). An increase in a slow inward calcium current by the beta agonists might explain the f a l l in the r a t i o , for this is s i m i l a r to the e f f e c t of elevating e x t r a c e l l u l a r calcium concentration on the f o r c e - i n t e r v a l r e l a t i o n s h i p ( I ) , but the droop in the curve and the transformation from p o s t - e x t r a s y s t o l i c p o t e n t i a t i o n to p o s t - e x t r a s y s t o l i c depression is unlike the e f f e c t of elevated calcium or of any other inotropic intervention of which we are aware. Acknowledgements The forming part by cipient
authors would l i k e to thank Mrs. Annette Oakeley for her help in perthe experiments and analyzing the r e s u l t s . This work was supported in NIH grants: HL 20749, HL 11307 and HL 18270. Dr. Anderson was the reof Research Career Development Award HL 00500. References
1. P.A,W. ANDERSON, A. MANRING, and E.A. JOHNSON, J. Mol. Cell Cardiol. 2, 131-150 (1977). 2. A. MANRING and P.A.W. ANDERSON, C r i t . Rev. Bioeng. 4, 165-201 (1980). 3. J. KOCH-WESER, C.M. BERLIN, JR. and J.R. BLINKS, Pharmacology of Cardiac Function, pp. 63-72, The MacMillan Co., New York (1964). 4. J.T. STULL and S.E. MAYER, Handbook of Physio199y, pp. 741-774, American Physiological Society, Bethesda (1979).
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5. B.G. BENFY, Br. J. Pharmac. 48, 132-138 (1977). 6. B. RABINOWITZ, L. CHUCH, M. KLINGERMAN, and W.W. PARMLEY, Am. J. Physiol. 229, 582-585 (1975). 7. R.---~-.-.-.-.-.-.~.FELDER, P.L. CALGAGNO, G.M. EISNER, and P.A. JOSE, Pediat. Res. 16, 340-342 (1982), 8. J.B. OSNERS, H. REFSUM, T. SKIMEDAL, and I. BYE, Acta. Pharm. Tox. 42, 235-247 (1978). 9. A.M. KATZ, Adv. Cycl. Nucl. Res. 11, 303-343 (1979). 10. H. SCHOLZ, Handbook of Experimental Pharmacology, 5__4, 651-778 (1980).