Acta Physiol Scand 1990, 139, 289-295

Effects of amrinone on the electromechanical coupling in frog skeletal muscle fibres S. E. J. N. M O R N E R and A. M A N S S O N Department of Pharmacology, University of Lund, Sweden S. E. J. N. & MANSSON, A. 1990.Effects of amrinone on the electromechanical MORNER, coupling in frog skeletal muscle fibres. Acta PhysioI Scand 139, 289-295. Received 24 October 1989, accepted 18 December 1989. ISSN 00014772. Department of Pharmacology, University of Lund, Sweden.

The contractile effects of 1.1 mM amrinone were studied on isolated skeletal muscle fibres of the frog (2-5 "C, slack length). Amrinone potentiated the twitch amplitude and the maximum tetanic tension and also enhanced the maximum contracture response elicited by I 17.5 mM K+ (mean increase 6.3 fI .8 yo,n = 6, P < 0.02). The time to halfrelaxation of the potassium contracture was slightly increased (mean change 13.8 3.5%, n = 7, P < 0.01).Amrinone furthermore shifted the S-shaped curve relating contracture tension to log K+ to the left, and thus reduced the threshold depolarization (the mechanical threshold) needed to elicit a contracture (mean reduction I I f I mV). The duration of the action potential at the -25 mV level was slightly increased by amrinone, whereas the resting membrane potential was unaffected. Key words: action potential, amrinone, mechanical threshold, twitch potentiation.

effect has been considered (e.g. Meisheri et al. 1980) but there is so far no conclusive evidence to support this view (Toda et al. 1984, Morgan et al. 1986). Recently, the contractile effects of amrinone were studied both on single skeletal muscle fibres of the frog (Mlnsson & Edman 1985) and on mammalian skeletal muscle (Minsson et al. 1989). T h e results from these studies suggest that amrinone, in addition to affecting the metabolism of activator calcium, also affects the kinetics of turnover of the myosin cross-bridges. These effects, leading to enhancement of the maximum tetanic tension and reduction of the maximum velocity of shortening, dominate the contractile actions of amrinone in mammalian skeletal muscle. In frog muscle fibres, however, the effect of amrinone on the calcium metabolism in the excitation-contraction coupling was more prominent. This action of the drug led to twitch potentiation and increased rate of rise of force at the onset of contraction, i.e. changes similar to those produced by amrinone in cardiac muscle. T h e aim of the present study was to explore Correspondence : Dr Jonas Morner, Department of Pharmacology, Solvegatan 10, S-223 62 Lund, further the mechanism underlying the rwitchSweden. potentiating effect of amrinone in frog muscle

Amrinone is a bipyridine derivative with cardiotonic (Benotti et al. 1978, 1980, Farah & Alousi 1978, Alousi et al. 1979) and smooth muscle relaxant properties (Meisheri et al. 1980, Maruyama et al. 1981, Mielens & Buck 1982). T h e mechanism underlying the contractile effects is not fully understood, but there is reason to believe that the cardiotonic action is associated with the phosphodiesterase-inhibiting action of the drug (Honerjager et al. 1981, Endoh et al. 1982, Hayes et al. 1984). This effect of amrinone leads to increased intracellular levels of cyclic 3', 5'-adenosine monophosphate (CAMP) and, secondary to this, an enhancement of the intracellular Ca2+stores. I n contrast to the situation in the cardiac muscle, amrinone has been found to reduce the amount of Ca2+ available for contractile activation in smooth muscle (Morgan et al. 1986). This action may account for the amrinone-induced relaxation of this preparation. T h e involvement of increased levels of CAMP in the mediation of the smooth muscle relaxant

289 10

ACT

139

S . E . J . N . Morner and A. Mdnsson

290

fibres. To this e n d action potential parameters and potassium-induced contractures were studied in the presence and absence of amrinone. Whereas amrinone had only minor effects on the time course of the action potential, it shifted the relationship between the external potassium concentration and contracture tension towards lower potassium concentrations. The results demonstrate that the effects of amrinone on the electromechanical coupling exhibit several similarities to the effects of caffeine. Short accounts of the present results have been given earlier (Morner & Minsson 1988, 1989).

M A T E R I A L S AND M E T H O D S Preparation and mounting. Single fibres from the tibialis anterior muscle of Rana temporaria were mounted horizontally in a jacketed Perspex trough between stainless-steel hooks attached to a stationary arm and a force transducer. Clips of aluminium foil (Edman & Reggiani 1984)were used for mounting the fibre. Resting length was adjusted by means of a micrometer screw that controlled the position of the tension transducer. The experiments were carried out at just above slack length of the preparation. Crosssectional area was calculated from the largest and smallest diameters of the fibre assuming an elliptical shape of the cross-section. The fibre diameters were measured at 40 x magnification and the fibre length was measured at 20 x magnification using a Zeiss stereomicroscope. Solutions. The normal Ringer solution had the following composition (mM): NaCl I 15.5, KCI 2.0, CaCI, 1.8, sodium phosphate buffer 2.0, p H 7.0. The composition of the potassium contracture solutions is given in Table I . The ionic strength, osmolality and K x C1 product were kept at constant levels. Amrinone (obtained by courtesy of Sterling-Winthrop Inc.) was

Table

I.

dissolved in 0.5 M lactic acid and added to the normal Ringer solution and to the test potassium contracture solutions. T h e final concentration of the drug was I . I mM (corresponding to 200 mg I-'). Lactic acid was also added to the control solutions to provide the same lactic acid concentration in both test and control. pH was set to 7.0 by addition of appropriate amounts (2.0-2.5 ml I-') of 1.0M NaOH. Muscle chamber and temperature control. Temperaturewascontrolled bycirculatingawater-glycol mixture from a Colora ultrathermostat through the jackets surrounding the plexiglass muscle chamber. The temperature of the bath varied between 2 and 5 "C between different experiments but was keptconstant to ko.3 "C throughout any specific experiment. This applied even during exchange of solutions. The muscle chamber (volume 1 . 2 ml, depth 5 mm, width 6 mm) was continuously perfused with precooled Ringer solution at a rate ofo.6 ml min-'. When potassium contractures were elicited, the continuous perfusion was interrupted. The contracture solution (kept in precooled containers) was flushed into the bath at the force transducer end and removed at the other end by suction. The exchange, of solution was complete within 3 s. Interruption of the contractures was brought about by rapid reintroduction of the normal Ringer solution. A resting period of at least zomin was allowed between contractures. Electrical stimulation. A pair of platinum plate electrodes placed on either side of the muscle fibre were used for electrical stimulation. Either a single rectangular pulse (duration 0.2 ms) or a train of pulses (frequency 15-25 Hz; duration I s) were applied to elicit a twitch or a fused tetanus respectively. The stimulation strength was adjusted to be approximately 1.25 x threshold. The fibre was stimulated at regular 2-min intervals to produce either a twitch or a fused tetanus. Before measurements of twitch and tetanus parameters were carried out the muscle fibre had been paced using this protocol for at least 30 min. A similar

Composition of solutions used for potassium contractures. Concentrations in mM. ~

KCI

NaCl

CaCI,

115.5 10.6 20.2

I .8

2

13.6

I .8

2

11.5

I .8

2

I .8

2

1.8 1.8 I .8 I .03

2

Buffer

NaProp.

I .8

2

-

I .8

2

KMeSO,

CaSO,

~

Ringer KIO K12 KI4 K16 K I ~ Kzo K3o K4o K I r7.5

9.8 8.5 4.47 2.45 ~

2 2 2

96.9 85.4 90.0 90.0 89.7 89.0 83.03 75.05 0.77

-

~

-

1.2

I 2

1.2

'4

I .2

16

1.2

I8

1.2

20

I .2

30 40 "7.5

I .2

NaProp, sodium propionate ; KMeSO,, potassium methyl sulphate.

I .8

1.97

291

Amrinone and electromechanical coupling pacing period was also employed before measurements were resumed after exchange of solutions. Tension recording. Tension was measured by means of a silicon strain gauge transducer AE 801 (Aksjeselskapet Mikroelektronik) similar to that described by Edman (1979). The first derivative of the tension signal was obtained by means of an RC circuit (time constant I ms). In the majority of the experiments the tension signal was recorded on an Elema-Schonander ink jet recorder. In a few experiments, however, tension and the first derivative of tension were displayed on a Tektronix 51 13 storage oscilloscope and photographed on 35-mm orthochromatic film. Membrane potential. Membrane potential was recorded from semitendinosus fibre bundles (5-10 fibres) using conventional glass capillary electrodes (10-20 Mohm) filled with 2 . 5 M KCI. An Ag-AgC1 electrode connected to the bath fluid through an agar-Ringer bridge was used as a reference. The microelectrode and the reference electrode were connected via an electrometer with high-input impedance to a Tektronix 5113 oscilloscope. The displayed voltage signals were photographed on 35-mm orthochromatic film. Measurements of the film records were made on a Nikon model 6C profile projector (Edman 1979). When action potentials were recorded the fibre bundle was stimulated at a single locus 4-5 mm from the position of the niicroelectrode. Statistical analysis. Student's t-test was used for

determination of statistical significance (paired observation). Data are given as mean standard error.

RESULTS Twitch and tetanus T h e effects of 1 . 1 mM amrinone on the twitch and tetanus responses of a frog single muscle fibre are shown in Fig. I . Amrinone increased the peak twitch force (Ptw) by 40 + 9 % (n = 12, P < 0.001)and modulated the time course of the isometric twitch. T h e rate of rise of force was increased and there was a prolongation of the time to peak twitch force and the time to halfrelaxation. I n accordance with previous results, amrinone also produced a moderate potentiation of the maximum tetanic output (mean change 9.2+0.8%, n = 12, P < 0 . 0 0 1 ) associated with an increased rate of rise of force at the onset of a tetanus and a slowing of the relaxation phase. T h e effects of amrinone on the earliest phase of tension rise are illustrated in more detail in Fig. 2. T h e derivative of tension is also shown. I t can be seen that the initial rate of rise of tension is higher and that tension starts to rise somewhat earlier in the presence of amrinone.

, , , , , ,

1 1 , 1 , 1 , , 1

U

200 ms

Fig. I. Effects of amrinone on isometric twitch (a) and tetanus (b) of frog single muscle fibre. Myograms I, control solution; myograms 2, 1 . 1 mM amrinone. Cross-sectional area 10.3 x I O - ~mm2. Temperature 3.3 "C.

Table

2.

Effects of

I . I mM

amrinone on resting and action potential Action potential

Amrinone Control ~

~~

Resting potential (mV)

Overshoot (mV)

Maximum rate of rise (V s-l)

Duration at - 2 5 mV level (ms)

89.1 & 0.9 88.4f1.1

38.2 0.8 39.9k1.1

62.3 2.6 63.6f3.1

6.4fo.3 5 . 2 f0.2

~

Frog muscle fibre bundles exposed to amrinone for 3-35 for each measurement given within brackets.

minutes before start of recording. Number of fibres

10-2

292

S . E . J . N . Morner and A . Mdnsson

assium concentration in four different experiments in the presence and absence of amrinone. It is shown that the resting membrane potential is not affected by amrinone at any of the potassium concentrations tested. Thus, a given extracellular potassium concentration corresponds to the same membrane potential in both r test and control. In view of this fact, changes in u 5 ms the threshold depolarization needed to elicit a Fig. 2. Effects of I . I mM amrinone on the initial rate tension response (the mechanical threshold) may of rise of force of a twitch. Superimposed traces of be calculated directly from the S-shaped curve tension (upper), the first derivative of tension (middle) relating contracture tension to log K+. This and stimulus signal (lower) in the presence ( 2 ) and curve was shifted to the left by amrinone, i.e. to absence (I) of I . I mM amrinone. Same fibre as in Fig. lower concentrations of potassium (Fig. 4). The I. threshold concentration of potassium at which a contracture was elicited (17.7 _+ 0.5 mM in the absence of amrinone) was lowered by Time course and magnitude of potassium 6.9k0.6 mM in presence of the drug. As contractures (117.5mM K+) indicated in the inset of Fig. 4, this change The effects of 1 . 1 mM amrinone on the con- corresponds to a reduction of the mechanical tracture tension at 117.5mM K+ are shown in threshold by 11 f I mV. Fig. 3. In accordance with the effects of amrinone on the maximum tetanic output, the maximum Resting and action potentiaj contracture tension was moderately increased by the drug (mean increase 6.3 iI .8 yo, n = 6, The effects of the drug on the action potential P < 0.02). T h e increased tension production were recorded after full development of the was associated with only minor changes in the contractile effects (30 min). As shown in Fig. 5 time course of the spontaneously relaxing potas- amrinone affected neither the maximum rate of sium contracture (Fig. 3). The time to half- rise nor the peak amplitude of the action relaxation, taken as the time from peak tension to potential. On the other hand, the duration of the the half-peak value was increased by 2 . 4 i 0 . 6 s action potential at the - 25 mV level was slightly increased. The change amounted to I. I 0.2 ms (n = 7, P < O.OI), that is by r4+3.5y0. (or 22 +4%, n = 13,P < 0.001).The magnitude of the amrinone effects on the resting potential Mechanical threshold and various action potential parameters are The effects of amrinone on membrane potential summarized in Table 2. were studied by depolarizing the fibre to different degrees by application of various potassium DISCUSSION concentrations. The results are shown in the inset of Fig. 4 where the membrane potential is I t is shown in the present paper that amrinone plotted as a function of the extracellular pot- reduces the threshold depolarization (the mechN

J

i, J

N 0 r

10 s

Fig. 3. Contracture induced by 117.5 mM K'. Myogram I , control solution; myogram 2 , amrinone. Cross-sectional area: 13.4x I O - ~mm'. Temperature 2.9 "C.

1.1

mM

Amrinone and electromechanical coupling

293

z120 3 E .rl

;loo E

- 80 H

C 0 .rl

(D

60

C a, c,

a,

40

L

3

;20

+ J

L

c, C

0

u

0 i

t

~

,

,

,

,

,

, ,

I

1

10 K+ c o n c e n t r a t i o n

100

(mM)

Fig. 4. Relation between peak contracture tension and extracellular K' concentration in the presence ( 0 )and absence (0) of I. I mM amrinone. Data (mean f SE) normalized with respect to maximum contracture tension induced by 117.5 mM [K']. Data from eight experiments or as given within parentheses. Inset : Relation between the membrane potential and the extracellular potassium concentration in the presence ( 0 )and absence (0) of amrinone. Mean from four experiments (SE smaller than the size of symbols). Straight lines fitted by means of linear regression to data obtained at K+ 2 10 mM. The intercepts of the dotted line on the abscissa represent the lowest K' concentration at which contractures are elicited. The intercepts on the ordinate give the mechanical threshold. I , control solution; 2, 1.1 mM amrinone.

ation and the increased rate of rise of force in the presence of amrinone may be accounted for on this basis. The enhancing effect of the drug on the maximum tetanic tension however is not explainable by increased calcium release. This may be inferred from evidence suggesting that the contractile system is saturated with calcium during the plateau of a tetanus (cf. Mlnsson & I Edman 1985). The amrinone-induced potenti10 m s Fig. 5. Effects of 1 . 1 mM amrinone on intracellularly ation of the maximum tetanic output may instead recorded action potential of a frog single muscle fibre. be attributed to changes in the kinetics of turnover of the myosin cross-bridges (Mlnsson I , control solution; 2, amrinone. Temperature 3.3 "C. & Edman 1985). It is reasonable to assume that the potentiating effect of amrinone on the anical threshold) needed to elicit a contracture. maximum contracture response to I 17.5 mM K+ As further discussed below, this change may is also attributable to this change. reflect a facilitation of the Ca2+release in response A potentiation of the twitch amplitude is a to membrane excitation. Both the twitch potenti- characteristic contractile effect of agents that are

294

5'. E . J . N . Morner and A . Mdnsson

known to increase the calcium release in response to membrane excitation (Lopez et al. 1981). These twitch-potentiating agents may, on the basis of their main mechanism of action, be classified into two distinct groups (Sandow et al. 1965, Taylor et al. 1969, 1972): first, those that prolong the period of calcium release by increasing the duration of the action potential (e.g. Zn2+, +aminopyridine), and second those that enhance the mechanical response to a given membrane depolarization (e.g. NO;, caffeine, diethylstilboesterol). The finding that amrinone reduced the threshold depolarization that was required for a mechanical response suggests that this drug has properties characteristic of the second group. The small increase of the action potential duration in the presence of amrinone (20%) may contribute to the twitch-potentiating effect. However, the change was of considerably smaller magnitude than that seen in response to substances whose main mechanism of action is at the level of the action potential. Thus, twitchpotentiating concentrations of Zn2+ (Edman & Grieve 1961, Edman et al. 1966, Taylor et al. 1972) and 4-aminopyridine (Khan & Edman 1979) increase the duration of the action potential by more than rooyo. The effects of 1.1 mM amrinone are, on the other hand, similar to the effects of 0.5-1 mM caffeine. The degree of potentiation of the isometric twitch was about the same for the two agents (Minsson & Edman 198j), and both substances reduce the mechanical threshold and induce a small prolongation of the action potential (present paper and Taylor et al. 1969). Furthermore, amrinone (see Results), like caffeine (Sandow et al. 196j), shortened the latent period slightly and increased the rate of tension rise during the first milliseconds of an isometric contraction. The latter change has also been demonstrated in response to other agents (e.g. anions like NO-,; Taylor et al. 1969) that reduce the mechanical threshold but not in response to substances that act mainly by prolonging the action potential (Taylor et al. 1972). Neither has this change been demonstrated in response to diethylstilboestrol, an agent that has been shown to reduce the rate of re-uptake of calcium by the sarcoplasmic reticulum (Khan 1979). The existence of the similarities between amrinone and caffeine points to the possibility that both drugs affect the electromechanical coupling in a similar way. That is, amrinone might, like caffeine (Lopez ei af. 1981, Kovacs &

Szucz 1983, Delay et al. 1986), facilitate the release of Ca2+from the sarcoplasmic reticulum. However, the possibility also exists that the similarities between the two drugs are merely fortuitous and that amrinone mimics the effects of caffeine on contractility by exerting a combination of different effects. For example, a facilitation of the Caz+ release from the sarcoplasmic reticulum, an increased Ca2+sensitivity of the contractile proteins and a slowing of the Ca2+sequestration by the sarcoplasmic reticulum may be consistent with the reduction of the mechanical threshold. Irrespective of which is the precise mechanism of action of amrinone however, it is likely that the substance acts at an intracellular site. This is suggested by the findings of Minsson & Edman (1985) that the twitch-potentiating effect developed slowly upon immersion in drug-containing solution and that the effect was difficult to reverse. Amrinone, in the concentrations used here, is known to increase the cAMP levels in both cardiac (Honerjager et al. 1981) and smooth muscle (Meisheri et al. 1980). The possibility may therefore be considered that the present effects of amrinone are related to the phosphodiesterase-inhibiting activity of the drug. The resulting increase of CAMP would be expected to give effects consistent with some of those seen experimentally in response to amrinone. Thus, Gonzales-Serratos et al. (1981) demonstrated that preincubation of skinned muscle fibres of the frog in CAMP-containing solution increased the amount of calcium that is available for release from the sarcoplasmic reticulum. Furthermore, these authors showed that adrenaline, presumably by increasing the intracellular cAMP concentrations, produced twitch-potentiation in intact fibre bundles of the frog semitendinosus muscle. In conclusion, available evidence (present paper and Minsson & Edman 1985) suggests that amrinone acts at an intracellular site in frog skeletal muscle. T h e effects of amrinone on the electromechanical coupling are caffeine-like. This points to the possibility that, like caffeine, amrinone facilitates the release of calcium from the sarcoplasmic reticulum, thus explaining the twitch-potentiating effect of the drug. The authors wish to thank Professor K. A. P. Edman for support and helpful discussions and Mrs Britta Kronborg, Mrs Gunilla Prins and Mrs Marianne Ekman for their excellent technical assistance.

Amrinone and electromechanical coupling

295

KHAN,A.R. 1979. Effects of diethylstilboestrol on single fibres of frog skeletal muscle. Acta Physiol Scand 106, 69-73. KHAN,A.R. & EDMAN,K.A.P. 1979. Effects of 4aminopyridine on the excitation-contraction coupREFERENCES ling in frog and rat skeletal muscle. Acta Physiol ALOUSI,A.A., FARAH, A.E., LESHER, G.Y. & OPALKA, Scand 105, 443-452. C.J. 1979. Cardiotonic activity of amrinone - WIN KOVACZ, L. & Szucz, G. 1983. Effects of caffeine on 40680 (5-amino-3,4’-bipyridine-6( IH)-one). Circ intramembrane charge movement and calcium transients in cut skeletal muscle fibres of the frog. 3 Res 45, 6 6 6 6 7 7 . BENOTTI,J.R., GROSSMAN, W., BRAUNWALD, E., Physiol 341, 559-578. DAVOLOS,D.D. & ALOUSI,A.A. 1978. Hemo- LOPEZ,J.R., WANEK,L.A. & TAYLOR,S.R. 1981. dynamic assessment of amrinone: a new inoSkeletal muscle : Length-dependent effects of tropic agent. New Engl3 Med 299, 1373-1377. potentiating agents. Science 214, 79-82. BENOTTI,J.R., GROSSMAN, W., BRAUNWALD, E. & MANSSON,A. & EDMAN,K.A.P. 1985. Effects of CARABELLO, B.A. 1980. Effects of amrinone on amrinone on the contractile behaviour of striated myocardial energy metabolism and hemodynamics muscle fibres. Acta Physinl Scand 1 2 5 , 481-493. in patients with severe congestive heart failure due MANSSON,A,, MORNER,J. & EDMAN,K.A.P. 1989. to coronary arterial disease. Circulation 62, 28-34. Effects of amrinone on twitch, tetanus and shortening kinetics in mammalian skeletal muscle. Acta DELAY, M., RIBALET, B. & VERGARA, J. 1986.Caffeine potentiation of calcium release in frog skeletal Physiol Scand 136, 37-45. MARUYAMA, M., SATOH, K. & TAIRA, N. 1981.Effects muscle fibres. 3 Ph.ysiol 375, 535-559. of amrinone on the tracheal musculature and EDMAN,K.A.P. 1979. The velocity of unloaded vasculature of the dog., 3ap 3 Pharmacol 31, shortening and its relation to sarcomere length and isometric force in vertebrate muscle fibres. 3Physiol 1095-1097. MEISHERI,K.D., PALMER, R.F. & VANBREEMEN, C. 291, 143-159. EDMAN,K.A.P. & GRIEVE, D.W. 1961. The role of 1980. The effects of amrinone on contractility, Ca2+ calcium and zinc in the electrical and mechanical uptake and CAMP in smooth muscle. Eur 3 Pharmacol 61, 159-165. responses of frog sartorius muscle. Experzentia 17, MIELENS, Z.E. &BUCK,D.C. 1982. Relaxant effects of 557. amrinone upon pulmonary smooth muscle. PharEDMAN,K.A.P., GRIEVE, D.W. & NILSSON,E. 1966. macology 25, 262-271. Studies of the excitation-contraction mechanisms in the skeletal muscle and the myocardium. F‘j’zigers MORGAN,J.P., GWATHMEY, J.K., DEFEO,T.T. & Arch Ges Physiol 290, 320-334. MORGAN, K.G. 1986. The effects of amrinone and related drugs on intracellular calcium in isolated EDMAN, K.A.P. & REGGIANI, C. 1984. Redistribution mammalian cardiac and vascular smooth muscle. of sarcomere length dhring isometric contraction of frog muscle fibres and its relation to tension creep. Circulation 73 (Suppl. 111), 65-77. MORNER,J. & MANSSON, A. 1988. Amrinone affects 3 Ph.ysio1 351, 169-198. the mechanical threshold in frog skeletal muscle ENDOH,M., YAMASHITA, S. & TAIRA, N. 1982. Positive inotropic effects of amrinone in relation to fibres. In: L.C. Sellin, R. Libelius & S. Thesleff (eds.) Neuromuscular Junction, p. 608A. Elsevier, cyclic nucleotide metabolism in the canine ventricular muscle.3Pharmacol Exp Ther 221,775-783. Amsterdam. FARAH, A.E. & ALOUSI,A.A. 1978. New cardiotonic MORNER, J. & MANSSON, A. 1989. Effects of amrinone agents: a search for a digitalis substitute. Life Sci on the excitation-contraction coupling in frog skeletal muscle fibres. 3 Muscle Res Cell Mot 10, 22, 1139-1 148. GONZALEZ-SERRATOS, H., HILL, L. & VALLE‘73A. A., TAYLOR, S.R. & PREISER, H. 1965. Role AGUILERA, R. 1981. Effects of catecholamines and SANDOW, cyclic AMP on excitation-contraction coupling in of the action potential in the excitation-contraction coupling. Fed Proc 24, I I 1 6 - 1 123. isolated skeletal muscle fibres of the frog. 3Physiol TAYLOR, S.R., PREISER,H. & SANDOW, A. 1969. 315, 267-282. HAYES, J.S., BOWLING, N., BODER,G.B. & KAUFFMAN, Mechanical threshold as a factor in excitationcontraction coupling. 3 Gen Physiol 54, 352-368. R. 1984. Molecular basis for the cardiovascular S.R., PREISER,H. & SANDOW, A. 1972. activities of amrinone and AR-Ls7. 3 Pharmacol TAYLOR, Action potential parameters affecting excitationExp Ther 230, 124-132. contraction coupling. 3 Gen Physiol 59, 421-434. HONERJAGER, P., SCHAFER-KORTING, M. & REITER, M. 1981. Involvement of cyclic AMP in the direct TODA, N., NAKAJIMA, M., NISHIMURA, K. &MIYAZAKI, M. 1984. Responses of isolated dog arteries to inotropic action of amrinone. Naunyn amrinone. Cardiooasc Res 18, 174-182. Schmiedeberg’s Arch Pharmakol 318, I 12-120. This study was supported by grants from the Swedish Medical Research Council (project no. 14X184) and the Medical Faculty, University of Lund.