Naunyn-Schmiedeberg's
Archivesof
Naunyn-Schmiedeberg's Arch. Pharmacol. 303, 4 7 - 5 3 (1978)
Pharmacology 9 by Springer-Verlag1978
Interactions of Calcium, Dibutyryl Cyclic AMP, Isoprenaline and Aminophylline on the Isometric Contraction of the Isolated Hemidiaphragm of the Rat V. M. Varagi~ and D. Kentera Department of Pharmacology, Faculty of Medicine P,O. Box 662, and Institute for Medical Research, Belgrade, Yugoslavia
Summary. Both di-Na-EDTA and verapamil depressed the tension (T) and the maximum rate of rise of tension (dT/dtm,x) of twitch responses of the isolated hemidiaphragm of the rat to direct electrical stimulation. Depression was preceded by a transient facilitation. The blocking action of di-Na-EDTA was promptly reversed by calcium chloride, whereas the same procedure failed to antagonize the blocking action of veraparail. Isoprenaline and db-cAMP were found to antagonize the blocking action of verapamil. In calcium-free medium verapamil quickly produced block of isometric contractions. The depression of contraction produced by verapamil in calcium-free medium was only slightly or not restored by isoprenaline and db-cAMP. This indicates that the membrane calcium is indispensable for the action of isoprenaline and db-cAMP. The effect of aminophylline on T and dT/dtn,,x depends markedly on calcium in the external medium. In a calcium-free solution, as well as in the presence of verapamil, aminophylline failed to produce any change in the isometric contraction. It is concluded that the actions of isoprenaline, db-cAMP and aminophylline on the isometric contractions of the isolated hemidiaphragm of the rat produced by direct electrical stimulation are possible only in the presence of an optimal concentration of external calcium and of functionally intact calcium channels in the membrane. Key words: Calcium - Verapamil - Cyclic AMP Isoprenaline - Aminophylline.
Introduction It has been postulated that calcium and the cyclic nucleotides, cyclic AMP and cyclic GMP, are the main components of an internal signalling system which Send offprint requests to V, M. Varagi6 at the above address
regulates the activity of most cells (Berridge, 1975). On the other hand, the available information on the interactions between calcium and cyclic nucleotides is very fragmentary. There are indications that cyclic AMP(cAMP) may be important not only in modulating the transmission at neuromuscular junctions, but also in regulating the processes which provide the energy for muscular contraction (Kentera and Varagi6, 1975). It has been known for some time that catecholamines can facilitate neuromuscular transmission (Krnjevi6 and Miledi, 1958) and increase the quantal content of the end-plate potential (Jenkinson et al., 1968). This effect of catecholamines is apparently mediated by cAMP because it can be mimicked by either dibutyryl cyclic AMP(dbcAMP) or theophylline (Breckenridge et al., 1967; Goldberg and Singer, 1969). It was also pointed out that cAMP does not play a direct role in the release of neurotransmitters, but there are indications that it can modulate the calcium signal by increasing the calcium permeability (Miyamoto and Breckenridge, 1974). A similar effect of cAMP on calcium permeability has been noted in heart muscle (Pappano, 1970). It was therefore of interest to study the interactions of calcium, db-cAMP, isoprenaline and aminophylline on the isolated hemidiaphragm of the rat during direct stimulation. All these substances are known to affect the cAMP system.
Materials and Methods The isolated hemidiaphragm of the rat was used essentially as described by Biilbring (1946). The isolated hemidiaphragm from male and female rats was suspended in an isolated organ bath of 15 ml. Tyrode solution was used with double amount of glucose and bubbled with pure oxygen. The composition of the solution was as follows (raM): NaC1 136.7; KCI 2.81; CaC1, 1.8; MgC12 0.105; NaH2PO , 0.417; NaHCO3 11.9; dextrose 11.101. Calcium-free solution was made by omitting calcium chloride, or by adding 0.025 mM di-Na-EDTA to calcium-free solution. The diaphragm was stimulated directly with supramaximal pulses of 0.04-0.Sins
0028-1298/78/0303/0047/$ 1.40
48
Naunyn-Schmiedeberg's Arch. Pharmacol. 303 (1978)
duration. In all experiments with calcium-free medium the pulse duration was regularly 0 . 5 - 1 ms. The isometric contractions were recorded with a microdisplacement myograph transducer (F50, Narco-Bio Systems, Inc.) and displayed on paper (Physiograph IV polygraph). Both tension development (T) and the maximum rate of rise of tension (dT/dtm,,) were recorded simultaneously. The differential of tension with respect to time was recorded by a differentiator Coupler Type 7301 (Narco-Bio Systems, Inc.), which provides the true mathematical derivative of analog signal voltages. The pulses for direct electrical stimulation were delivered via 2 pallador wires, i of which the diaphragm was secured to. The other electrode was placed around the upper part of the diaphragm. In some experiments D-tubocurarine was added to the bath in a concentration sufficient to fully block neuromuscular transmission (41aM). The frequency of stimulation was 9/min. The following substances were used: D-tubocurarine chloride (Burroughs Wellcome), aminophylline (Lek), di-sodium-EDTA (Merck), verapamil (Knoll), adenosine (Serva), N6-2'-O-dibutyrylcyclic-adenosine-3',5'-monophosphate (Boehringer) and (• prenaline hydrochloride.
Results
Interaction of di-Na-EDTA, Calcium and Isoprenaline on T and dT/dtr,,~ The presence of 2raM di-Na-EDTA in the bath produced a biphasic change in both T and dT/dtm, x : a shortlasting increase in both parameters was followed by a progressive decrease. A 50 ~ depression of T and dT/dtmax was obtained after about 15 rain, the values fell to about 27 ~ of control after about 25 min (Table 1). When this degree of depression was reached, CaC12 was added to the bath to give a final concentration of 1.8raM; it produced a progressive increase of the contraction, which was 50~ of control after about 10 rain. Further increases of the final concentration of CaC12 to 3.6 mM failed to further increase isometric contractions. Even the final concentration of 6.6 mM CaCI2 failed to produce an increase. On the other hand, isoprenaline was found to further counteract the effect of di-Na-EDTA. This
effect was observed when calcium chloride had already been previously added to the bath. Isoprenaline (1 or 2 ~IM) then produced an almost complete abolition of the depressant effect of di-Na-EDTA, T and dT/dt .... reaching 88-97 ~ of the control values (Table I). A typical example of these interactions is shown in Figure 1. Interactions of Verapamil, Calcium and Isoprenaline Verapamil (4.5 ~M) also elicited biphasic changes: a long-lasting increase of the contraction was followed by a progressive decrease, which, eventually, resulted in a complete block of the response to stimulation. A depression to 5 0 ~ of control was seen after about 52rain, whereas di-Na-EDTA caused the same effect within 15 rain. Contrary to the results obtained with diNa-EDTA, increases in the final concentration of calcium (by 1.8 or 6.6raM) did not antagonize the verapamil-induced depression of contraction (Table 2). Isoprenaline (0.24- 1 ~ M) - irrespective of whether calcium chloride had been previously added to the bath or not-antagonized the effect of verapamil (Fig. 2). However, a subsequent increase in the concentration in calcium chloride produced some depression of contraction. Quite opposite results were obtained in experiments in which the interactions of verapamil, isoprenaline and calcium were studied in a calcium-free medium. In this medium the isolated hem• was still able to respond to direct electrical stimulation for the duration of the experiment, though the magnitude of these contraction was smaller, as compared to controls in normal solution. In this series of experiments verapamil regularly produced a quick depression of contraction. Isoprenaline (0.48-1 ~M) only slightly antagonized the effect of varapamil, but the addition of calcium chloride produced a large increase in both T and dT/dtm,x (Fig. 3). In calcium-free solution to which 0.025 mM di-Na-EDTA had been added, and in the
Table 1. Interactions of di-Na-EDTA, calcium and isoprenaline on isometric contraction of the isolated hem• of the rat in response to direct electrical stimulation. The values are given as percentages of control (prior to administration ofdi-Na-EDTA) (Means _+ standard error of the mean). The number of experiments is given in parentheses Maximum of the initial transient increase after 2 mM di-NaEDTA
Time (in rain) for the secondary depression to reach 50 ~
Magnitude o f T and dT/dtmax immediately before addition of CaCI 2
Effect to first addition of CaCI 2 (final conc. 1.8 mM)
Effect of second Effect of isoprenaline addition of CaC12 1 pM 2 laM (final conc. 3.6 mM)
T
144.7 • 7.8 (10)
15.2 • 1.9 (10)
27.5 • 6.6 (10)
42.7 • 3.9 (10)
43.8 • 4.6 (10)
88.5 • 8.2 (9)
94.3 • 8.1 (9)
dT/dtm,x
133.5 • 8.3 (10) 15.2 • 1.9 (10)
26.3 _+ 6.5 (10)
51.2 +_ 3.8 (10)
53.7 • 3 (10)
90
97
_+ 8.7 (9)
• 5.8 (9)
V. M. Varagi6 and D. Kentera: Interactions of Calcium, db-cAMP, Isoprenaline and Aminophylline
49
Fig. 1. The effect of di-Na-EDTA, calcium chloride and isoprenaline on T (upper record) and dT/dtm,x (lower record) of the isolated hemidiaphragm of the rat. Time: 30 s intervals
Table 2. Interactions of verapamil, calcium and isoprenaline on isometric contraction of the isolated hemidiaphragm of the rat in response to direct electrical stimulation. The values are given as percentages of control (prior to administration of verapamiI). (Means _+_standard error of the mean.) The number of experiments is given in parentheses Maximum of the initial transient increase after 4.5 jaM verapamil
Time (in min) for the secondary depression to reach 50 ~
Magnitude of T and dT/dtma • immediately before addition of CaCI2
Effect of first addition of CaC12 (final conc. increased by 1.8 raM)
Effect of second Effect of isoaddition of CaC12 prenaline (finaI conc. increa- (1 gM) sed by a total of 3.6 raM)
T
132.5 +_ 5.6 (6)
52.8 + 2.5 (5)
46.5 _+ 5.3 (6)
35.8 +_ 10.3 (6)
34 + 12.3 (5)
77.2 + 22 (5)
dT/dtm, x
128
52.8 _+ 2.5 (5)
44.8 _+ 6.4 (6)
34.3 _+ 11.8 (6)
30 + 12.5 (5)
75.2 _+ 22.6 (5)
_+ 5.6 (6)
presence o f v e r a p a m i l , i s o p r e n a l i n e a l m o s t c o m p l e t e l y failed to p r o d u c e a n y c h a n g e in c o n t r a c t i o n . Interaction o f Verapamil, d b - c A M P and Adenosine in N o r m a l and Calcium-Free Solution D b - c A M P (0.68 r a M ) a n t a g o n i z e d the d e p r e s s a n t effect o f v e r a p a m i l (4.5 g M ) on i s o m e t r i c c o n t r a c t i o n in n o r m a l s o l u t i o n (Fig. 4, T a b l e 3). H o w e v e r , in a calcium-free m e d i u m d b - c A M P did n o t a n t a g o n i z e the d e p r e s s a n t effect o f v e r a p a m i l (Fig. 4, T a b l e 3). It s h o u l d be a d d e d t h a t T a n d dT/dtm, x values similar to
those shown in T a b l e 3 were o b t a i n e d in p r e p a r a t i o n s either k e p t in calcium-free m e d i u m for 3 - 4 h p r i o r to use in e x p e r i m e n t s with n o r m a l , o r m a i n t a i n e d in calcium-free s o l u t i o n f r o m the very beginning o f experiment. In o r d e r to check the specificity o f the effect o f d b c A M P in a n t a g o n i z i n g the effect o f v e r a p a m i l on the isometric c o n t r a c t i o n , e x p e r i m e n t s were p e r f o r m e d with adenosine. In calcium-free solution a d e n o s i n e ( 0 , 6 8 - 1 . 3 2 m M ) failed to a n t a g o n i z e the d e p r e s s a n t effect o f v e r a p a m i l . H o w e v e r , these c o n c e n t r a t i o n s o f a d e n o s i n e were effective in n o r m a l s o l u t i o n ( T w a s 50
50
Naunyn-Schmiedeberg'sArch. Pharmacol. 303 (1978)
Fig. 2. The effectof verapamil,calciumchlorideand isoprenalineon T (upper record)and dT/dtmax (lowerrecord)of the isolatedhemidiaphragm of the rat. A The initial effect of verapamil. B The final stage of the effect of verapamil. Isoprenaline (ISO, 1 ~M) antagonizedthe effect of verapamil in the presence of previouslyadded calcium chloride. BetweenA and B: 70 min. Time: 30 s intervals
of control after verapamil and 69 _+ 3.7 ~o of control after adenosine; 10 experiments). Aminophylline and Calcium
Doubling the concentration of calcium in the medium produced an increase in response of the isolated hemidiaphragm to aminophylline. However, in a calcium-free solution aminophylline failed to produce any effect (Fig. 5). Reconstitution of Tyrode solution by adding 1.8 m M calcium almost immediately produced a very pronounced increase of contraction. Similarly, be addition of calcium without previous addition of aminophylline to a preparation maintained in a calcium-free medium, produced a very pronounced increase in the isometric contraction. Verapamil completely blocked the response to aminophylline. Even the combination of aminophylline and calcium chloride did not antagonize the blocking action of verapamil. Discussion
It is well established that the physiological contraction of skeletal muscle is elicited by calcium ion released
from the sarcoplasmic reticulum (Ebashi and Endo, 1968; Sandow, 1965; Ebashi, 1975, 1976; Endo, 1977). Coupling of glycogenolysis to muscle contraction is at present also thought to be mediated by calcium ions (Brostrom et al., 1971). The present experiments further demonstrate the importance of the external calcium and the relationship between the external and intracellular calcium in skeletal muscle during direct electrical stimulation. Both diN a - E D T A and verapamil, though probably acting by different mechanisms, reduce isometric contraction of the diaphragm muscle during direct electrical stimulation. Both substances produced an initial increase in force of concentration, followed by a progressive decrease, leading eventually to complete block of contraction. The initial increment in both T and dT/dtm,x was probably due to an alterated ratio between the membrane-located and the intracellular calcium. Verapamil is known to alter the amount of cMcium which is stored at the membrane-located binding sites in heart muscle, hence decreasing the amount of calcium which available for release from these sites during depolarization (Nayler and Szeto, 1972) and displaced inwards into the vicinity of the myofilaments (Nayler and Krikler, 1974). If this is supposed to happen in the skeletal'
V. M. Varagib and D. Kentera: Interactions of Calcium, db-cAMP, Isoprenaline and Aminophyliine
Fig. 3. Interactionsof verapamil,isoprenalineand calciumchloridein calcium-freesolutionon T(upper record) and dT/dtm~ ~ (lowerrecord). Verapamil (4.5 pM) had been added 30min before this record was taken. Note the slight antagonistic action of isoprenaline towards verapamil-induced depression of contraction, and the very marked antagonistic action of calcium chloridein the presenceof previously added isoprenaline. Time: 30 s intervals
muscle as well, then both chelation of calcium in the external fluid and the specific decrease in the membrane-located calcium produce qualitatively identical effects, but with different time characteristics. There is a striking difference between di-Na-EDTA and verapamil-induced depression: the addition of calcium to the external fluid promptly reverses the block of contraction produced by di-Na-EDTA, whereas the same procedure failed to antagonize the blockade by verapamil. It is therefore possible that diNa-EDTA does not change the functional state of the binding sites for calcium within the membrane. On the other hand, verapamil certainly does produce such a change in the membrane (Nayler and Krikler, 1974), more precisely in the calcium channels (Fleckenstein, 1971), so that the action of added calcium becomes impossible. Both isoprenaline and db-cAMP were found to antagonize the blocking action of verapamil on contraction. The catecholamine-induced changes in contraction almost certainly can be accounted for by an
51
increase in calcium which enters the cell during the rising and plateau stage of the action potential (Shigenbou and Sperelakis, 1972). Fleckenstein (1971) has very convincingly shown that isoprenaline increases the calcium influx through the excited cardiac fibre membranes. Under certain conditions also cAMP facilitates the transfer of calcium accross isolated membranes (Kirchberger et al., 1972). In our experiments isoprenaline was also able either to activate the external calcium, or to facilitate its passage across the membrane. It is possible to suppose that isoprenaline activates or opens the same sites on the membrane which have been previously altered or closed by verapamil. In calcium-free medium the isolated hemidiaphragm was still able to respond to direct electrical stimulation, though the responses were small. In this medium verapamil quickly produced block of the isometric contraction, without the preceding enhancement regularly seen in normal solution. This supports the view that the initial enhancement of contraction produced by verapamil in normal solution may be due to an altered ratio of membrane calcium to intracellular calcium. In calcium-free medium db-cAMP failed to antagonize the blocking action of verapamil. On the other hand, isoprenaline was still able to slightly antagonize the blocking action of verapamil even in calcium-free medium. In calcium-free solution to which di-NaEDTA had been added, and in the presence of verapamil, isoprenaline almost completely failed to produce any change in the isometric contraction. This indicates that the membrane calcium is indispensable for the actions of both db-cAMP and isoprenaline. It should be pointed out that the action of db-cAMP does not seem to be specific because adenosine itself also produced an effect similar to that of db-cAMP. The ability of isoprenaline to increase force of contraction even in the absence of external calcium may indicate that either this substance can mobilize the intracellular calcium, or it can increase the energy supply by producing glycogenolysis and lipolysis via cAMP system. The effect of aminophylline on force of contraction is very much dependent on the external calcium. In the calciumXree medium aminophylline failed to produce any change of contraction. The effect of aminophylline was also completely blocked in verapamil-treated preparations. It has already been shown previously that methylxanthines sensitize the calcium release mechanism of the terminal cisternae of the sarcoplasmic reticulum (Bianchi, 1968, 1975). There is no doubt that the action of aminophylline on the skeletal muscle contraction is of complex origin. The results of our experiments indicate that verapamil is able either to affect not only the membrane transfer of calcium, but also its liberation from the sarcoplasmic reticulum, or
52
Naunyn-Schmiedeberg's Arch. Pharmacol. 303 (1978)
Fig. 4. The effect of db-cAMP and verapamil on dT/dtm, x in normal (upper record) and calcium-free solution (lower record). There was a 30 rain interval between the upper records. In calcium-free solution db-cAMP failed to antagonize the depressant effect of verapamil. Time: 30 s intervals
Table 3. Interaction of verapamil and db-cAMP on the isometric contraction of the isolated hem• of the rat in normal and calcium-free medium (Means • standard error of the mean). The values for T and dT/dtm.x are given as percentages of control (prior to administration of verapamil). The number of experiments is given in parentheses Calcium-free solution
Normal solution Depression produced by verapamil (4.5 JAM), before addition of db-cAMP
Time necessary to reach this degree of depression (in rain)
The effect of addition of db-cAMP (0.68 raM)
Depression produced Time necessary to by verapamil reach this degree of (4.5 ~tM), before depression (in rain) addition of rib-cAMP
The effect of addition of db-cAMP (0.68mM)
T 67
67 • 4.7 (17)
96.3 • 10.3 (17)
51.8 • 3.5* (11)
18.4 • 1.9"** (11)
37
67 • 4.7 (17)
81.6 • 10.4 (17)
48.4 _+ 3.1 (11)
18.4 ff 1.9"** (1t)
31.1•
• 4.4 (17)
dT/dtmax 54.5 • 5 (17)
• 11.3"* (11) 9.5** (11)
z"
* = P < 0.05; ** = P < 0.01; *** = P < 0.001. Compared with the corresponding values obtained in normal solution
e v e n to r e d u c e t h e p e n e t r a t i o n o f a m i n o p h y l l i n e through the membrane. These findings might have i m p l i c a t i o n s in t h e r a p y in v i e w o f t h e p o s s i b l e b u t e v i d e n t l y useless c o m b i n a t i o n o f v e r a p a m i l a n d a m i n o p h y l l i n e (e.g., in a n g i n a p e c t o r i s ) .
It is c o n c l u d e d t h a t the a c t i o n s o f i s o p r e n a l i n e , d b c A M P a n d a m i n o p h y l l i n e o n the i s o m e t r i c c o n t r a c tions of the hem• d u r i n g direct electrical s t i m u l a t i o n are o n l y p o s s i b l e w i t h o p t i m a l c o n c e n t r a t i o n s o f c a l c i u m in the e x t e r n a l m e d i u m , as well as w i t h
V. M. Varagi6 and D. Kentera: Interactions of Calcium, db-cAMP, Isopreanaline and Aminophylline
150
T
~ .~o~~ + + l
130
+ "~
g 90
50 30
~
~.~/
D: C~+-free
~,
10 - - e
/
C~.+ oddedl []
,,
D
"'~,1
0:20:L, 0~,6
I],8 110 112 I',/-, 116 118 2,0 Cone. of ctminophyLline(in mM)
Fig. 5. The effect of aminophylline on T of the isolated hemidiaphragm of the rat and its dependence on calcium. Note the slight potentiation of the response to aminophylline in the solution with double amount of calcium and the complete failure to produce any effect in the calcium-free solution. Reconstitution of Tyrode solution by adding calcium chloride produced a very pronounced potentiation of the response to aminophylline. * = P < 0.05; ** = P < 0.01
functionally operating calcium channels in the membrane. Acknowledgements. One of the authors (V.M.V.) wishes to express his gratitude to the Wellcome Trust for providing a grant which partly covered the expenses for the equipment. Thanks are also due to Miss Ljubica Nikoli6 for skilful technical assistance. This work was financed by ZMNU of SR Serbia.
References Berridge, M. J. : The interaction of cyclic nucleotides and calcium in the control of cellular activity. Adb. Cyclic. Nucleotide Res. 6, 1--98 (1975) Bianchi, C. P. : Pharmacological actions on excitation-contraction coupling in striated muscle. Fed. Proc. Am. Soc. Exp. Biol. 27, 126-131 (1968) Bianchi, C. P. : Cellular pharmacology of contraction of skeletal muscle. In: Cellular pharmacology of excitable tissues. Springfield: C. C. Thomas 1975 Breckenridge, B. McL., Burn, J. H., Matschinsky, F. M. : Theophylline, epinephrine and neostigmine facilitation of neuromuscular transmission. Proc. Natl. Acad. Sci. U.S.A. 57, 1893-1897 (1967) Brostrom, C. O., Hunkeler, F. L., Krebs, E. G.: The regulation of skeletal muscle phosphorylase kinase by calcium. J. Biol. Chem. 246, 1961--1967 (1971)
53
Btilbring, E. : Observations on the isolated phrenic nerve diaphragm preparation of the rat. Br. J. Pharmacol. 1, 3 8 - 4 9 (1946) Ebashi, S.: Regulatory mechanisms of muscle contraction with special reference to the Ca-troponin-tropomyosin system. Essays Biochem. 10, 1 - 3 6 (1975) Ebashi, S. : Excitation-contraction coupling. Ann. Rev. Physiol. 38, 293-313 (1976) Ebashi, S., Endo, M. : Ca ion and muscle contraction. Prog. Biophys. Mol. Biol. 18, 123-183 (1968) Endo, M. : Calcium release from the sarcoplasmic reticulum. Physiol. Rev. 57, 71-108(1977) Fleckenstein, A. : Specific inhibitors and promoters of calcium action in the excitation-contraction coupling of heart muscle and their role in the prevention and production of myocardial lesions. In: Calcium and the heart, pp. 135-188. London-New York: Academic Press 1971 Goldberg, A. L., Singer, J. J. : Evidence for the role of cyclic AMP in neuromuscular transmission. Proc. Natl. Acad. Sci. U.S.A. 64, 134-141 (1969) Jenkinson, D. H., Stamenovi6, B. A., Whitaker, B. D. L. : The effect of noradrenaline on the end-plate potential in twitch fibres of the frog. J. Physiol. (Lond.) 195, 743-754 (1968) Kentera, D., Varagi6, V. M. : The effect of cyclic N-2-0-dibutyryl adenosine Y,5'-monophosphate, adrenaline and aminophylline on the isometric contractility of the isolated hemidiaphragm of the rat. Br. J. Pharmacol. 54, 375-381 (1975) Kirchberger, M. A., Tada, M., Repke, D. I., Katz, A. M.: Cyclic 3',5'-adenosine monophosphate-dependent protein kinase stimulation of calcium uptake by canine cardiac microsomes. J. Mol. Cell. Cardiol. 4, 673-685 (1972) Krnjevi6, K., Miledi, R. : Some effects produced by adrenaline upon neuromuscular propagation in rats. J. Physiol. (Lond.) 141, 291-304 (1958) Miyamoto, M. D., Breckenridge, B. McL.: A cyclic adenosine monophosphate link in the catecholamine enhancement of transmitter release at the neuromuscular junction. J. Gen. Physiol. 63, 609-624 (1974) Nayler, W. G., Krikler, D.: Verapamil and the myocardium. Postgrad. Med. J. 50, 441-446 (1974) Nayler, W. G., Szeto, J. : Effect of verapamil on contractility, oxygen utilization and calcium exchangeability in mammalian heart muscle. Cardiovasc. Res. 6, 120-128 (1972) Pappano, A. J.: Calcium-dependent action potentials produced by catecholamines in guinea pig atrial muscle fibres depolarized by potassium. Circ. Res. 27, 379-390 (1970) Sandow, A.: Excitation-contraction coupling in skeletal muscle. Pharmacol. Rev. 17, 265-320 (1965) Shigenbou, K., Sperelakis, N. : Calcium current channels induced by catecholamines in chick embryonic hearts whose fast sodium channels are blocked by tetrodotoxin or elevated potassium. Circ. Res. 31, 932-945 (1972)
Received August 28, 1977/Accepted January 24, 1978
Archivesof
Naunyn-Schmiedeberg's Arch. Pharmacol. 303, 4 7 - 5 3 (1978)
Pharmacology 9 by Springer-Verlag1978
Interactions of Calcium, Dibutyryl Cyclic AMP, Isoprenaline and Aminophylline on the Isometric Contraction of the Isolated Hemidiaphragm of the Rat V. M. Varagi~ and D. Kentera Department of Pharmacology, Faculty of Medicine P,O. Box 662, and Institute for Medical Research, Belgrade, Yugoslavia
Summary. Both di-Na-EDTA and verapamil depressed the tension (T) and the maximum rate of rise of tension (dT/dtm,x) of twitch responses of the isolated hemidiaphragm of the rat to direct electrical stimulation. Depression was preceded by a transient facilitation. The blocking action of di-Na-EDTA was promptly reversed by calcium chloride, whereas the same procedure failed to antagonize the blocking action of veraparail. Isoprenaline and db-cAMP were found to antagonize the blocking action of verapamil. In calcium-free medium verapamil quickly produced block of isometric contractions. The depression of contraction produced by verapamil in calcium-free medium was only slightly or not restored by isoprenaline and db-cAMP. This indicates that the membrane calcium is indispensable for the action of isoprenaline and db-cAMP. The effect of aminophylline on T and dT/dtn,,x depends markedly on calcium in the external medium. In a calcium-free solution, as well as in the presence of verapamil, aminophylline failed to produce any change in the isometric contraction. It is concluded that the actions of isoprenaline, db-cAMP and aminophylline on the isometric contractions of the isolated hemidiaphragm of the rat produced by direct electrical stimulation are possible only in the presence of an optimal concentration of external calcium and of functionally intact calcium channels in the membrane. Key words: Calcium - Verapamil - Cyclic AMP Isoprenaline - Aminophylline.
Introduction It has been postulated that calcium and the cyclic nucleotides, cyclic AMP and cyclic GMP, are the main components of an internal signalling system which Send offprint requests to V, M. Varagi6 at the above address
regulates the activity of most cells (Berridge, 1975). On the other hand, the available information on the interactions between calcium and cyclic nucleotides is very fragmentary. There are indications that cyclic AMP(cAMP) may be important not only in modulating the transmission at neuromuscular junctions, but also in regulating the processes which provide the energy for muscular contraction (Kentera and Varagi6, 1975). It has been known for some time that catecholamines can facilitate neuromuscular transmission (Krnjevi6 and Miledi, 1958) and increase the quantal content of the end-plate potential (Jenkinson et al., 1968). This effect of catecholamines is apparently mediated by cAMP because it can be mimicked by either dibutyryl cyclic AMP(dbcAMP) or theophylline (Breckenridge et al., 1967; Goldberg and Singer, 1969). It was also pointed out that cAMP does not play a direct role in the release of neurotransmitters, but there are indications that it can modulate the calcium signal by increasing the calcium permeability (Miyamoto and Breckenridge, 1974). A similar effect of cAMP on calcium permeability has been noted in heart muscle (Pappano, 1970). It was therefore of interest to study the interactions of calcium, db-cAMP, isoprenaline and aminophylline on the isolated hemidiaphragm of the rat during direct stimulation. All these substances are known to affect the cAMP system.
Materials and Methods The isolated hemidiaphragm of the rat was used essentially as described by Biilbring (1946). The isolated hemidiaphragm from male and female rats was suspended in an isolated organ bath of 15 ml. Tyrode solution was used with double amount of glucose and bubbled with pure oxygen. The composition of the solution was as follows (raM): NaC1 136.7; KCI 2.81; CaC1, 1.8; MgC12 0.105; NaH2PO , 0.417; NaHCO3 11.9; dextrose 11.101. Calcium-free solution was made by omitting calcium chloride, or by adding 0.025 mM di-Na-EDTA to calcium-free solution. The diaphragm was stimulated directly with supramaximal pulses of 0.04-0.Sins
0028-1298/78/0303/0047/$ 1.40
48
Naunyn-Schmiedeberg's Arch. Pharmacol. 303 (1978)
duration. In all experiments with calcium-free medium the pulse duration was regularly 0 . 5 - 1 ms. The isometric contractions were recorded with a microdisplacement myograph transducer (F50, Narco-Bio Systems, Inc.) and displayed on paper (Physiograph IV polygraph). Both tension development (T) and the maximum rate of rise of tension (dT/dtm,,) were recorded simultaneously. The differential of tension with respect to time was recorded by a differentiator Coupler Type 7301 (Narco-Bio Systems, Inc.), which provides the true mathematical derivative of analog signal voltages. The pulses for direct electrical stimulation were delivered via 2 pallador wires, i of which the diaphragm was secured to. The other electrode was placed around the upper part of the diaphragm. In some experiments D-tubocurarine was added to the bath in a concentration sufficient to fully block neuromuscular transmission (41aM). The frequency of stimulation was 9/min. The following substances were used: D-tubocurarine chloride (Burroughs Wellcome), aminophylline (Lek), di-sodium-EDTA (Merck), verapamil (Knoll), adenosine (Serva), N6-2'-O-dibutyrylcyclic-adenosine-3',5'-monophosphate (Boehringer) and (• prenaline hydrochloride.
Results
Interaction of di-Na-EDTA, Calcium and Isoprenaline on T and dT/dtr,,~ The presence of 2raM di-Na-EDTA in the bath produced a biphasic change in both T and dT/dtm, x : a shortlasting increase in both parameters was followed by a progressive decrease. A 50 ~ depression of T and dT/dtmax was obtained after about 15 rain, the values fell to about 27 ~ of control after about 25 min (Table 1). When this degree of depression was reached, CaC12 was added to the bath to give a final concentration of 1.8raM; it produced a progressive increase of the contraction, which was 50~ of control after about 10 rain. Further increases of the final concentration of CaC12 to 3.6 mM failed to further increase isometric contractions. Even the final concentration of 6.6 mM CaCI2 failed to produce an increase. On the other hand, isoprenaline was found to further counteract the effect of di-Na-EDTA. This
effect was observed when calcium chloride had already been previously added to the bath. Isoprenaline (1 or 2 ~IM) then produced an almost complete abolition of the depressant effect of di-Na-EDTA, T and dT/dt .... reaching 88-97 ~ of the control values (Table I). A typical example of these interactions is shown in Figure 1. Interactions of Verapamil, Calcium and Isoprenaline Verapamil (4.5 ~M) also elicited biphasic changes: a long-lasting increase of the contraction was followed by a progressive decrease, which, eventually, resulted in a complete block of the response to stimulation. A depression to 5 0 ~ of control was seen after about 52rain, whereas di-Na-EDTA caused the same effect within 15 rain. Contrary to the results obtained with diNa-EDTA, increases in the final concentration of calcium (by 1.8 or 6.6raM) did not antagonize the verapamil-induced depression of contraction (Table 2). Isoprenaline (0.24- 1 ~ M) - irrespective of whether calcium chloride had been previously added to the bath or not-antagonized the effect of verapamil (Fig. 2). However, a subsequent increase in the concentration in calcium chloride produced some depression of contraction. Quite opposite results were obtained in experiments in which the interactions of verapamil, isoprenaline and calcium were studied in a calcium-free medium. In this medium the isolated hem• was still able to respond to direct electrical stimulation for the duration of the experiment, though the magnitude of these contraction was smaller, as compared to controls in normal solution. In this series of experiments verapamil regularly produced a quick depression of contraction. Isoprenaline (0.48-1 ~M) only slightly antagonized the effect of varapamil, but the addition of calcium chloride produced a large increase in both T and dT/dtm,x (Fig. 3). In calcium-free solution to which 0.025 mM di-Na-EDTA had been added, and in the
Table 1. Interactions of di-Na-EDTA, calcium and isoprenaline on isometric contraction of the isolated hem• of the rat in response to direct electrical stimulation. The values are given as percentages of control (prior to administration ofdi-Na-EDTA) (Means _+ standard error of the mean). The number of experiments is given in parentheses Maximum of the initial transient increase after 2 mM di-NaEDTA
Time (in rain) for the secondary depression to reach 50 ~
Magnitude o f T and dT/dtmax immediately before addition of CaCI 2
Effect to first addition of CaCI 2 (final conc. 1.8 mM)
Effect of second Effect of isoprenaline addition of CaC12 1 pM 2 laM (final conc. 3.6 mM)
T
144.7 • 7.8 (10)
15.2 • 1.9 (10)
27.5 • 6.6 (10)
42.7 • 3.9 (10)
43.8 • 4.6 (10)
88.5 • 8.2 (9)
94.3 • 8.1 (9)
dT/dtm,x
133.5 • 8.3 (10) 15.2 • 1.9 (10)
26.3 _+ 6.5 (10)
51.2 +_ 3.8 (10)
53.7 • 3 (10)
90
97
_+ 8.7 (9)
• 5.8 (9)
V. M. Varagi6 and D. Kentera: Interactions of Calcium, db-cAMP, Isoprenaline and Aminophylline
49
Fig. 1. The effect of di-Na-EDTA, calcium chloride and isoprenaline on T (upper record) and dT/dtm,x (lower record) of the isolated hemidiaphragm of the rat. Time: 30 s intervals
Table 2. Interactions of verapamil, calcium and isoprenaline on isometric contraction of the isolated hemidiaphragm of the rat in response to direct electrical stimulation. The values are given as percentages of control (prior to administration of verapamiI). (Means _+_standard error of the mean.) The number of experiments is given in parentheses Maximum of the initial transient increase after 4.5 jaM verapamil
Time (in min) for the secondary depression to reach 50 ~
Magnitude of T and dT/dtma • immediately before addition of CaCI2
Effect of first addition of CaC12 (final conc. increased by 1.8 raM)
Effect of second Effect of isoaddition of CaC12 prenaline (finaI conc. increa- (1 gM) sed by a total of 3.6 raM)
T
132.5 +_ 5.6 (6)
52.8 + 2.5 (5)
46.5 _+ 5.3 (6)
35.8 +_ 10.3 (6)
34 + 12.3 (5)
77.2 + 22 (5)
dT/dtm, x
128
52.8 _+ 2.5 (5)
44.8 _+ 6.4 (6)
34.3 _+ 11.8 (6)
30 + 12.5 (5)
75.2 _+ 22.6 (5)
_+ 5.6 (6)
presence o f v e r a p a m i l , i s o p r e n a l i n e a l m o s t c o m p l e t e l y failed to p r o d u c e a n y c h a n g e in c o n t r a c t i o n . Interaction o f Verapamil, d b - c A M P and Adenosine in N o r m a l and Calcium-Free Solution D b - c A M P (0.68 r a M ) a n t a g o n i z e d the d e p r e s s a n t effect o f v e r a p a m i l (4.5 g M ) on i s o m e t r i c c o n t r a c t i o n in n o r m a l s o l u t i o n (Fig. 4, T a b l e 3). H o w e v e r , in a calcium-free m e d i u m d b - c A M P did n o t a n t a g o n i z e the d e p r e s s a n t effect o f v e r a p a m i l (Fig. 4, T a b l e 3). It s h o u l d be a d d e d t h a t T a n d dT/dtm, x values similar to
those shown in T a b l e 3 were o b t a i n e d in p r e p a r a t i o n s either k e p t in calcium-free m e d i u m for 3 - 4 h p r i o r to use in e x p e r i m e n t s with n o r m a l , o r m a i n t a i n e d in calcium-free s o l u t i o n f r o m the very beginning o f experiment. In o r d e r to check the specificity o f the effect o f d b c A M P in a n t a g o n i z i n g the effect o f v e r a p a m i l on the isometric c o n t r a c t i o n , e x p e r i m e n t s were p e r f o r m e d with adenosine. In calcium-free solution a d e n o s i n e ( 0 , 6 8 - 1 . 3 2 m M ) failed to a n t a g o n i z e the d e p r e s s a n t effect o f v e r a p a m i l . H o w e v e r , these c o n c e n t r a t i o n s o f a d e n o s i n e were effective in n o r m a l s o l u t i o n ( T w a s 50
50
Naunyn-Schmiedeberg'sArch. Pharmacol. 303 (1978)
Fig. 2. The effectof verapamil,calciumchlorideand isoprenalineon T (upper record)and dT/dtmax (lowerrecord)of the isolatedhemidiaphragm of the rat. A The initial effect of verapamil. B The final stage of the effect of verapamil. Isoprenaline (ISO, 1 ~M) antagonizedthe effect of verapamil in the presence of previouslyadded calcium chloride. BetweenA and B: 70 min. Time: 30 s intervals
of control after verapamil and 69 _+ 3.7 ~o of control after adenosine; 10 experiments). Aminophylline and Calcium
Doubling the concentration of calcium in the medium produced an increase in response of the isolated hemidiaphragm to aminophylline. However, in a calcium-free solution aminophylline failed to produce any effect (Fig. 5). Reconstitution of Tyrode solution by adding 1.8 m M calcium almost immediately produced a very pronounced increase of contraction. Similarly, be addition of calcium without previous addition of aminophylline to a preparation maintained in a calcium-free medium, produced a very pronounced increase in the isometric contraction. Verapamil completely blocked the response to aminophylline. Even the combination of aminophylline and calcium chloride did not antagonize the blocking action of verapamil. Discussion
It is well established that the physiological contraction of skeletal muscle is elicited by calcium ion released
from the sarcoplasmic reticulum (Ebashi and Endo, 1968; Sandow, 1965; Ebashi, 1975, 1976; Endo, 1977). Coupling of glycogenolysis to muscle contraction is at present also thought to be mediated by calcium ions (Brostrom et al., 1971). The present experiments further demonstrate the importance of the external calcium and the relationship between the external and intracellular calcium in skeletal muscle during direct electrical stimulation. Both diN a - E D T A and verapamil, though probably acting by different mechanisms, reduce isometric contraction of the diaphragm muscle during direct electrical stimulation. Both substances produced an initial increase in force of concentration, followed by a progressive decrease, leading eventually to complete block of contraction. The initial increment in both T and dT/dtm,x was probably due to an alterated ratio between the membrane-located and the intracellular calcium. Verapamil is known to alter the amount of cMcium which is stored at the membrane-located binding sites in heart muscle, hence decreasing the amount of calcium which available for release from these sites during depolarization (Nayler and Szeto, 1972) and displaced inwards into the vicinity of the myofilaments (Nayler and Krikler, 1974). If this is supposed to happen in the skeletal'
V. M. Varagib and D. Kentera: Interactions of Calcium, db-cAMP, Isoprenaline and Aminophyliine
Fig. 3. Interactionsof verapamil,isoprenalineand calciumchloridein calcium-freesolutionon T(upper record) and dT/dtm~ ~ (lowerrecord). Verapamil (4.5 pM) had been added 30min before this record was taken. Note the slight antagonistic action of isoprenaline towards verapamil-induced depression of contraction, and the very marked antagonistic action of calcium chloridein the presenceof previously added isoprenaline. Time: 30 s intervals
muscle as well, then both chelation of calcium in the external fluid and the specific decrease in the membrane-located calcium produce qualitatively identical effects, but with different time characteristics. There is a striking difference between di-Na-EDTA and verapamil-induced depression: the addition of calcium to the external fluid promptly reverses the block of contraction produced by di-Na-EDTA, whereas the same procedure failed to antagonize the blockade by verapamil. It is therefore possible that diNa-EDTA does not change the functional state of the binding sites for calcium within the membrane. On the other hand, verapamil certainly does produce such a change in the membrane (Nayler and Krikler, 1974), more precisely in the calcium channels (Fleckenstein, 1971), so that the action of added calcium becomes impossible. Both isoprenaline and db-cAMP were found to antagonize the blocking action of verapamil on contraction. The catecholamine-induced changes in contraction almost certainly can be accounted for by an
51
increase in calcium which enters the cell during the rising and plateau stage of the action potential (Shigenbou and Sperelakis, 1972). Fleckenstein (1971) has very convincingly shown that isoprenaline increases the calcium influx through the excited cardiac fibre membranes. Under certain conditions also cAMP facilitates the transfer of calcium accross isolated membranes (Kirchberger et al., 1972). In our experiments isoprenaline was also able either to activate the external calcium, or to facilitate its passage across the membrane. It is possible to suppose that isoprenaline activates or opens the same sites on the membrane which have been previously altered or closed by verapamil. In calcium-free medium the isolated hemidiaphragm was still able to respond to direct electrical stimulation, though the responses were small. In this medium verapamil quickly produced block of the isometric contraction, without the preceding enhancement regularly seen in normal solution. This supports the view that the initial enhancement of contraction produced by verapamil in normal solution may be due to an altered ratio of membrane calcium to intracellular calcium. In calcium-free medium db-cAMP failed to antagonize the blocking action of verapamil. On the other hand, isoprenaline was still able to slightly antagonize the blocking action of verapamil even in calcium-free medium. In calcium-free solution to which di-NaEDTA had been added, and in the presence of verapamil, isoprenaline almost completely failed to produce any change in the isometric contraction. This indicates that the membrane calcium is indispensable for the actions of both db-cAMP and isoprenaline. It should be pointed out that the action of db-cAMP does not seem to be specific because adenosine itself also produced an effect similar to that of db-cAMP. The ability of isoprenaline to increase force of contraction even in the absence of external calcium may indicate that either this substance can mobilize the intracellular calcium, or it can increase the energy supply by producing glycogenolysis and lipolysis via cAMP system. The effect of aminophylline on force of contraction is very much dependent on the external calcium. In the calciumXree medium aminophylline failed to produce any change of contraction. The effect of aminophylline was also completely blocked in verapamil-treated preparations. It has already been shown previously that methylxanthines sensitize the calcium release mechanism of the terminal cisternae of the sarcoplasmic reticulum (Bianchi, 1968, 1975). There is no doubt that the action of aminophylline on the skeletal muscle contraction is of complex origin. The results of our experiments indicate that verapamil is able either to affect not only the membrane transfer of calcium, but also its liberation from the sarcoplasmic reticulum, or
52
Naunyn-Schmiedeberg's Arch. Pharmacol. 303 (1978)
Fig. 4. The effect of db-cAMP and verapamil on dT/dtm, x in normal (upper record) and calcium-free solution (lower record). There was a 30 rain interval between the upper records. In calcium-free solution db-cAMP failed to antagonize the depressant effect of verapamil. Time: 30 s intervals
Table 3. Interaction of verapamil and db-cAMP on the isometric contraction of the isolated hem• of the rat in normal and calcium-free medium (Means • standard error of the mean). The values for T and dT/dtm.x are given as percentages of control (prior to administration of verapamil). The number of experiments is given in parentheses Calcium-free solution
Normal solution Depression produced by verapamil (4.5 JAM), before addition of db-cAMP
Time necessary to reach this degree of depression (in rain)
The effect of addition of db-cAMP (0.68 raM)
Depression produced Time necessary to by verapamil reach this degree of (4.5 ~tM), before depression (in rain) addition of rib-cAMP
The effect of addition of db-cAMP (0.68mM)
T 67
67 • 4.7 (17)
96.3 • 10.3 (17)
51.8 • 3.5* (11)
18.4 • 1.9"** (11)
37
67 • 4.7 (17)
81.6 • 10.4 (17)
48.4 _+ 3.1 (11)
18.4 ff 1.9"** (1t)
31.1•
• 4.4 (17)
dT/dtmax 54.5 • 5 (17)
• 11.3"* (11) 9.5** (11)
z"
* = P < 0.05; ** = P < 0.01; *** = P < 0.001. Compared with the corresponding values obtained in normal solution
e v e n to r e d u c e t h e p e n e t r a t i o n o f a m i n o p h y l l i n e through the membrane. These findings might have i m p l i c a t i o n s in t h e r a p y in v i e w o f t h e p o s s i b l e b u t e v i d e n t l y useless c o m b i n a t i o n o f v e r a p a m i l a n d a m i n o p h y l l i n e (e.g., in a n g i n a p e c t o r i s ) .
It is c o n c l u d e d t h a t the a c t i o n s o f i s o p r e n a l i n e , d b c A M P a n d a m i n o p h y l l i n e o n the i s o m e t r i c c o n t r a c tions of the hem• d u r i n g direct electrical s t i m u l a t i o n are o n l y p o s s i b l e w i t h o p t i m a l c o n c e n t r a t i o n s o f c a l c i u m in the e x t e r n a l m e d i u m , as well as w i t h
V. M. Varagi6 and D. Kentera: Interactions of Calcium, db-cAMP, Isopreanaline and Aminophylline
150
T
~ .~o~~ + + l
130
+ "~
g 90
50 30
~
~.~/
D: C~+-free
~,
10 - - e
/
C~.+ oddedl []
,,
D
"'~,1
0:20:L, 0~,6
I],8 110 112 I',/-, 116 118 2,0 Cone. of ctminophyLline(in mM)
Fig. 5. The effect of aminophylline on T of the isolated hemidiaphragm of the rat and its dependence on calcium. Note the slight potentiation of the response to aminophylline in the solution with double amount of calcium and the complete failure to produce any effect in the calcium-free solution. Reconstitution of Tyrode solution by adding calcium chloride produced a very pronounced potentiation of the response to aminophylline. * = P < 0.05; ** = P < 0.01
functionally operating calcium channels in the membrane. Acknowledgements. One of the authors (V.M.V.) wishes to express his gratitude to the Wellcome Trust for providing a grant which partly covered the expenses for the equipment. Thanks are also due to Miss Ljubica Nikoli6 for skilful technical assistance. This work was financed by ZMNU of SR Serbia.
References Berridge, M. J. : The interaction of cyclic nucleotides and calcium in the control of cellular activity. Adb. Cyclic. Nucleotide Res. 6, 1--98 (1975) Bianchi, C. P. : Pharmacological actions on excitation-contraction coupling in striated muscle. Fed. Proc. Am. Soc. Exp. Biol. 27, 126-131 (1968) Bianchi, C. P. : Cellular pharmacology of contraction of skeletal muscle. In: Cellular pharmacology of excitable tissues. Springfield: C. C. Thomas 1975 Breckenridge, B. McL., Burn, J. H., Matschinsky, F. M. : Theophylline, epinephrine and neostigmine facilitation of neuromuscular transmission. Proc. Natl. Acad. Sci. U.S.A. 57, 1893-1897 (1967) Brostrom, C. O., Hunkeler, F. L., Krebs, E. G.: The regulation of skeletal muscle phosphorylase kinase by calcium. J. Biol. Chem. 246, 1961--1967 (1971)
53
Btilbring, E. : Observations on the isolated phrenic nerve diaphragm preparation of the rat. Br. J. Pharmacol. 1, 3 8 - 4 9 (1946) Ebashi, S.: Regulatory mechanisms of muscle contraction with special reference to the Ca-troponin-tropomyosin system. Essays Biochem. 10, 1 - 3 6 (1975) Ebashi, S. : Excitation-contraction coupling. Ann. Rev. Physiol. 38, 293-313 (1976) Ebashi, S., Endo, M. : Ca ion and muscle contraction. Prog. Biophys. Mol. Biol. 18, 123-183 (1968) Endo, M. : Calcium release from the sarcoplasmic reticulum. Physiol. Rev. 57, 71-108(1977) Fleckenstein, A. : Specific inhibitors and promoters of calcium action in the excitation-contraction coupling of heart muscle and their role in the prevention and production of myocardial lesions. In: Calcium and the heart, pp. 135-188. London-New York: Academic Press 1971 Goldberg, A. L., Singer, J. J. : Evidence for the role of cyclic AMP in neuromuscular transmission. Proc. Natl. Acad. Sci. U.S.A. 64, 134-141 (1969) Jenkinson, D. H., Stamenovi6, B. A., Whitaker, B. D. L. : The effect of noradrenaline on the end-plate potential in twitch fibres of the frog. J. Physiol. (Lond.) 195, 743-754 (1968) Kentera, D., Varagi6, V. M. : The effect of cyclic N-2-0-dibutyryl adenosine Y,5'-monophosphate, adrenaline and aminophylline on the isometric contractility of the isolated hemidiaphragm of the rat. Br. J. Pharmacol. 54, 375-381 (1975) Kirchberger, M. A., Tada, M., Repke, D. I., Katz, A. M.: Cyclic 3',5'-adenosine monophosphate-dependent protein kinase stimulation of calcium uptake by canine cardiac microsomes. J. Mol. Cell. Cardiol. 4, 673-685 (1972) Krnjevi6, K., Miledi, R. : Some effects produced by adrenaline upon neuromuscular propagation in rats. J. Physiol. (Lond.) 141, 291-304 (1958) Miyamoto, M. D., Breckenridge, B. McL.: A cyclic adenosine monophosphate link in the catecholamine enhancement of transmitter release at the neuromuscular junction. J. Gen. Physiol. 63, 609-624 (1974) Nayler, W. G., Krikler, D.: Verapamil and the myocardium. Postgrad. Med. J. 50, 441-446 (1974) Nayler, W. G., Szeto, J. : Effect of verapamil on contractility, oxygen utilization and calcium exchangeability in mammalian heart muscle. Cardiovasc. Res. 6, 120-128 (1972) Pappano, A. J.: Calcium-dependent action potentials produced by catecholamines in guinea pig atrial muscle fibres depolarized by potassium. Circ. Res. 27, 379-390 (1970) Sandow, A.: Excitation-contraction coupling in skeletal muscle. Pharmacol. Rev. 17, 265-320 (1965) Shigenbou, K., Sperelakis, N. : Calcium current channels induced by catecholamines in chick embryonic hearts whose fast sodium channels are blocked by tetrodotoxin or elevated potassium. Circ. Res. 31, 932-945 (1972)
Received August 28, 1977/Accepted January 24, 1978
