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Slow calcium channel blockers and the calcium paradox: Comparative studies in the rat with seven drugs
J Mol
Cell
Cardiol
Slow
(1983)
15, 475-485
Calcium Comparative
Channel Blockers and the Calcium Studies in the Rat with Seven J. E. Baker
and
Paradox: Drugs
D. J. Hearse
The Heart ResearchUnit, The RayneInstitute, St Thomas’sHospital, London,UK (Received30 September 1982, acceptedin revisedform 3 March 1983) J. IX. BAKER ANI) D. J. HEARSE. Slow Calcium Channel Blockers and the Calcium Paradox: Comparative Studies in the Rat with Seven Drugs. Journal of Molecular and Cellular Cardiology (1983) 15, 475-485. In studies of the calcium paradox, the isolated perfused rat heart was used to characterize the relationship between myocardial protein leakage and the concentration of calcium antagonist (verapamil and D600) or calcium in the perfusion fluid during a cycle of calcium depletion and repletion. The results indicated a dose-dependency such that protein leakage could be progressively reduced by decreasing the concentration of calcium during calcium repletion and/or by increasing the concentrations of drug. Detailed doseresponse studies with seven calcium antagonists (verapamil, D600, nifedipine, terodiline, diltiazem, fendiline and prenylamine) and a calcium concentration of 1 .O mmol/l during a 20 min period of calcium repletion following a 10 min period of calcium depletion revealed complex dose-response characteristics for each drug. In the dose range studied (0 to 40 pmol/l) all drugs with the exception of prenylamine were able to reduce protein leakage by up to 25 to 30% Optimal concentrations for verapamil, nifedipine, D600, diltiazem and terodiline were all between 2.0 and 4.0 vmol/l. With the exception of D600, which provided a constant reduction of protein leakage at all concentrations above this optimum, all drugs exhibited hell-shaped dose-response curves with a loss of efficacy at higher concentrations. Fendiline also had a bell-shaped dose-response curve with 23% as a maximal reduction of leakage; however, the optimal concentration for this drug was 21.0 pmol/l. Additional studies with verapamil revealed that the drug acted during the calcium repletion phase and did not influence events during calcium depletion. Simultaneous use of two drugs, verapamil and nifedipine, at their optimal concentrations failed to improve the reduction in protein leakage over and above that observed with one drug alone.
KEY
WORDS:
Protein leakage; Prenylamine.
Calcium Dose
paradox; response;
Slow calcium Verapamil;
channel D600;
Introduction Perfusion of the myocardium with calciumfree, followed by calcium-containing, media results in the immediate occurrence of severe cellular disruption, enzyme and protein leakage and irreversible contracture. The deleterious effects of this phenomenon, originally reported by Zimmerman [27] and termed the calcium paradox, have been attributed to sudden transmembrane calcium movements [7, 15, 23, 26, 27, 281. The extent of damage inflicted upon the myocardium during the calcium paradox can be influenced considerably by a number of Address London,
for reprint UK.
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requests:
J. E. Baker,
11 $03.00/O
The
Heart
blockers; Nifedipine;
Calcium Diltiazem;
antagonists; Terodiline;
Tissue injury; Fendiline;
factors, including the extracellular concentration of ions such as sodium [2&j, magnesium [26] and hydrogen [4], the presence of agents such as taurine [II] and dimethylsulphoxide [25] and the myocardial temperature during the calcium depletion phase [7]. Recently we have reported that the slow channel blockerverapamil can reduce calcium paradox-induced cellular damage by up to one-quarter, as assessed by protein and enzyme leakage [8]. However, some workers have been unable to observe a similar reduction using identical indices of tissue damage [20, 241, while others have reported the
Research
Unit, 0
The 1983
Rayne Academic
Institute, Press
St Thomas’s Inc
(London)
Hospital, Limited
476
J. E. Baker
and D. J. Hearse
ability of this agent [21] and diltiazem [Z] to reduce the leakage of creatine kinase during the calcium paradox. Although the mechanism of this effect is unknown, it may be related to a drug-induced reduction in the rate or extent of intracellular calcium accumulation. In this connection there is some evidence from other studies [I] that verapamil can reduce the initial rate of calcium accumulation during calcium repletion. In addition to verapamil, numerous other compounds have been reported to possess slow calcium channel blocking properties (Table 1). These other drugs, e.g. nifedipine diltiazem and terodiline, have widely differing structures and differing potencies and we were interested to ascertain whether, and to what extent, these agents are able to influence cellular damage associated with the calcium paradox. If similar effects to those seen with verapamil occur, then this may strengthen evidence that calcium movements via the slow calcium channel contribute to the injury associated with the calcium paradox. We have therefore used protein leakage from the isolated rat heart preparation to compare the effects of seven calcium antagonistsIn addition to dose-response studies we have also investigated which phase of the calcium paradox is susceptible to drug modification and also whether the combination of different antagonists produces additive effects.
Materials
and
Experimental
Methods animals
Male CSE rats (250 to 300 g body weight) maintained on a standard diet were used throughout the study. Perfufiion techniques Animals were anaesthetized with diethyl ether, the left femoral vein was exposed and heparin (200 IU) was administered intravenously. After 1 min the chest was opened and the heart was excised and placed in cold (4°C) perfusion medium. After 30 s the aorta was attached to a stainless steel cannula,
the pulmonary artery was incised to permit adequate coronary drainage and the heart was then perfused by the method of Langendorff [I3, 141 at a perfusion pressure equivalent to 10 kPa (100 cm H,O). A side arm on the aortic cannula, located immediately above the heart, was used to facilitate a rapid change between calcium-containing and calcium-free perfusion media. The heart and all perfusion fluids were kept in temperature-controlled chambers in order that the myocardial temperature could be maintained at 37°C. Perfusion media Krebs-Henseleit buffer (pH 7.4 when gassed with 959/o oxygen and 5% carbon dioxide), in which the calcium content was reduced to 1.0 mmol/l, was the standard perfusion fluid [I,?]. To this and all other perfusates was added glucose (11 .l mmol/l). During the period of calcium-free perfusion all calcium was excluded from the perfusion fluid and care was taken to ensure no calcium contamination of constituent reagents or water [ZZ]. Before use all perfusion fluid was filtered through a cellulose acetate membrane of pore size 5.0 pm. In all studies various concentrations of carrier-free dl-verapamil (Knoll AC), dlD600 (Knoll AG), dl-diltiazem (Synthelabo) and dl-terodiline (Kabi) were dissolved in a small quantitity (10 ml) of deionized water before being added to the bulk of the perfusion fluid. Nifedipine (Bayer) and dlfendiline (Dr. Thiemann) were initially dissolved in 10 ml ethanol (19 mol/l) before the inclusion in the perfusate, with the final concentration of ethanol in the perfusate kept constant; dl-prenylamine (Hoechst) in the gluconate form was dissolved in deionized water (10 ml). With the last three drugs appropriate additions of carrier or solvent were made to control solutions to ensure they exerted no effect per se. All experiments with nifedipine were carried out under sodium light to prevent photodegradation of the drug. With the exception of the penultimate series of studies all drugs studied were included in the perfusate throughout both the period of calcium depletion and the period of calcium repletion.
Calcium
Antagonists
and
Perfusion sequence Immediately after mounting hearts were perfused at 37°C for a 20 min equilibration period with drug-free calcium-containing buffer. Hearts were then subjected to a 10 min period of calcium-free perfusion. This was followed by a 20 min period of calcium repletion (1 .O mmol calcium/l). During the calcium repletion period total coronary effluent was collected, the volume was recorded and well mixed samples were analysed immediately for total protein content. We have previously shown [8] that protein leakage can be equated to enzyme leakage as an index of tissue injury. At the end of each experiment hearts were taken and heated to 100°C for 24 h in order that dry weights could be determined, Protein determination The total protein content of samples of the coronary effluent collected during the period of calcium repletion was estimated by the method of Bradford [5]. In this assay 50 ~1 of coronary effluent was added to 2500 yl of the protein reagent. The protein reagent was prepared by dissolving 10 mg of Coomassie Brilliant Blue G (Sigma Chemical Co.) in a mixture composed of 5 ml of ethanol ( 19 mol/l) and 10 ml of phosphoric acid (16 mol/l). This was then diluted with distilled water to a final volume of 100 ml and the solution filtered. Following the addition of the coronary effluent to the protein reagent the absorbance of 595 nm was measured after 5 min and compared with appropriate standards (bovine serum albumin, Pentax fraction V) . The protein leakage into the effluent was then calculated and expressed as milligrams released per gram dry weight per 20 min period. Protein leakage has been previously demonstrated to be essentially complete by the end of the 20 min repletion period [8].
Results
Our previous studies [3] have shown that not only are the dose-response characteristics for verapamil concentration versus protein leakage complex, but so too are the characteristics of repletion calcium concentration
the
Calcium
Paradox
477
(at any one verapamil concentration) versus protein leakage. Our primary studies were therefore divided into two parts: first, an assessment of the interrelationship between calcium antagonist concentration, repletion calcium concentration and protein leakage for two calcium antagonists; secondly, an assessment of the relationship between calcium antagonist concentration and protein leakage at a selected repletion calcium concentration for seven calcium antagonists. After these primary studies we carried out some preliminary studies to characteri.ze further these drug effects. Calcium concentration interrelationships For this study equimolar concentrations of D600 and verapamil were studied. Hearts (n = 6 for each group) were subjected to 10 min calcium-free perfusion followed by 20 min calcium repletion at various calcium concentrations (0.1, 0.2, 0.5, 1.0 and 2.4 mmol/l). Verapamil or D600 (0, 1 .O, 4.0 and 20.0 pmol/l) were included in the perfusion fluid during both calcium depletion and calcium repletion. Figure 1 shows the cumulative protein leakage for each condition when D600 was used, Figure 2 shows the corresponding results for verapamil. Examination of the results for D600 (tlhe methoxy derivative of verapamil) shows that at all drug and all calcium concentrations studied the calcium antagonist was able significantly to reduce protein leakage and under some conditions this reduction ‘was very extensive. It is apparent with zero drug or any one drug concentration that there was a dose-dependent increase in protein leakage with increasing calcium repletion concentrations, the higher the repletion concentration the greater was the cumulative leakage. Thus, in the absence of D600, repletion of 0.1, 0.2 0.5, 1.0 and 2.4 mmol calcium/l produced cumulative protein leakage values of 70 f 8, 115+3,132~4,142&5and151 &6mg/g dry weight per 20 min period. Similar stepwise effects were seen with every D600 concentration, thus when 4.0 pmol/l of drug were used the corresponding protein leakage valueswere3&2,66&3,98&4, 120&6 and 132 & 5 mg/g dry weight per 20 min period. In the opposite plane of Figure 1, at
478
$
J. E. Baker
and
D. J. Hearse
14c
FIGURE 1. Calcium-D600-protein leakage doseresponse interrelationships. Dose-response relationships between perfusate D600 and calcium concentration and protein release occurring during calcium repletion. The height of each column represents the mean cumulative protein release from six hearts and the standard error of the mean is indicated by the lightly shaded part.
FIGURE 2. Calcium-verapamil-protein leakage dose-response interrelationships. Dose-response relationship between per&sate verapamil and calcium concentration and protein release occurring during calcium repletion. The height of each column represents the mean cumulative protein release from six hearts and the standard error of the mean is indicated by the lightly shaded part.
all fixed calcium concentrations, D600 caused a dose-dependent decrease in protein leakage. Thus with calcium repletion at 0.2 mmol/l D600 at 0, 1.0, 4.0 and 20.0 pmol/l led to cumulative protein leakage values of 115 & 3, 73 or 5, 66 & 3, and 47 f 5 mg/g dry weight per 20 min period. At the lowest calcium concentration studied (0.1 mmol/l) all D600 doses essentially abolished protein leakage. The observation of progressively reducing protein leakage with increasing drug concentrations or decreasing calcium concentrations was also evident with verapamil (Figure 2), although the results were not quite as sharply resolved. However, it should be noted that the drug was able to achieve a substantial
reduction of protein leakage under all conditions, even at the high calcium concentration of 2.4 mmol/l. In some cases the drug could almost abolish leakage, thus with a calcium repletion concentration of 0.1 mmol/l verapamil (4.0 pmol/l) reduced protein leakage by 99% from its control value of 70 f 8 mg/g dry weight per 20 min period. Drug dose-response curues Having established that two calcium antagonists at a limited number of concentrations are able to reduce protein leakage over a wide range of calcium repletion concentrations, it was decided to select a single calcium
Calcium
Antagonists
and
repletion concentration and investigate detailed dose-response characteristics for seven calcium antagonists over a wide range of concentrations. Although it was tempting to select a low calcium concentration (e.g. 0.1 mmol/l), where dramatic reductions were observed, it was decided, for the present studies, to select a high concentration (1 .O mmol/l) since this represents a physiologically more relevant concentration and also precipitates a full calcium paradox. The drugs to be studied, verapamil, D600, nifedipine, fendiline, terodiline diltiazem, and prenylamine, were selected so as to represent a wide range of structures and potencies. On the basis of our previous studies with verapamil a dose range of 0 to 40.0 pmol/l was selected for investigation.
the
Calcium
Paradox
479
Hearts (a = 6 for each group) were subjected to 10 min calcium-free perfusion followed by 20 min calcium repletioln (I .O mmol/l) . The calcium blocking drugs were included in both perfusion phases at one of the following concentrations: 0, 1 .O, 2.0, 4.0, 10.0, 20.0 and 40.0 pmol/l. Figure 3 shows the extent to which each concentration of each drug was able to reduce protein leakage relative to a paired control. Table 1 details the optimal concentration for each drug and the percentage reduction of protein leakage at that concentration. Verapamil In agreement with our earlier studies [8] verapamil exhibited a multiphasic doseresponse curve. At low concentrations (0 ~to
0 600
FIGURE 3. Dose-response studies. reduction in relation to control) during 10 min period of calcium-free perfusion. the mean is indicated by the bars.
The influence of drug concentration upon protein leakage (percentage a 20 min period.of calcium repletion (I’.0 mmol calcium/l) following: Each point represents the mean of six hearts and the standard error
a of
281
415
315
329
486
Terodiline (‘Bicor’)
Diltiazem (‘Herbesser’)
Fendiline (‘Sensit’)
Prenylamine (‘Segontin’)
D600
++
+++
++
+++
++++
++++
Slow channel inhibition
+
Cardiac
+++
++
+++
+
?
?
?
AV node conduction decrease
++
+++
if
+
f ?
7
Ability to induce coronary vasodilation
sites of action”
+++
+++
No
+
No
Alphaadrenoceptor binding
Subsidiary
?
?
i
+
f
t
I
No
++
?
Fast channel inhibition
pharmacological
-t
-+++-I
?
?
?
Max ?
Possible
-
Yes
Yes
Yes
-
Yes
Yes
Coronary heart diasease
conditions.
3.0
0.006-0.03
0.1-0.3
0.03-0.16
0.06-0.2
0.08-0.06
(PM)
Therapeutic Plasma levels
to in viva or clinical
++
++
+++
Peripheral vascular relaxation
propertiesb
? No information available. a Note: Cardiac sites of action referred to above are based upon in vitro experimental studies and may not apply b Plasma levels not corrected for protein binding, all drug actions referred to are concentration dependent.
346
Nifedipine (‘Adalat’)
Molecular weight
to be investigated
455
1. Drugs
Verapamil (‘Isoptin’)
Name
TABLE
uses
-
Yes
Yes
Yes
Yes
Supraventricular arrhythmias
clinical
Calcium TABLE
2. Optimal
drug Drug
Verapamil Nifedipine Terodiline Diltiazem Fendiline Prenylamine D600
Antagonists
concentration
and
and protein
Optimal drug concentration (wolil)
the
Calcium
4,8 1
Paradox
leakage Maximum percentage reduction in protein leakage
4 4 4 2 20 4+&o
26 31 31 29 23
f & * & f 0 24 &
the mean for six hearts; the variation leakage relative to a paired drug-free
in reduction control.
P
3 2 3 3 3
3
N.S.
Each result represents the reduction in protein
4.0 pmol/l) there was a steep and linear increase in the ability of the drug to reduce protein leakage, which could be reduced maximally by 26 f 3% with a verapamil concentration of 4.0 pmol/l. In comparison with control this reduction was significant (P.< 0.01). At higher drug concentrations (10.0 to 40.0 pmol/l) the reduction of leakage declined to a plateau level (13 -& 3%) but remained significantly different from control values (P < 0.05). Nifedipine, terodiline and diltiazem Figure 3 reveals that for these three drugs comparable results to those for verapamil were obtained, thus rapid increases in drug efficacy were observed in the range 0 to 4.0 ymol/l, with similar maximal reductions of protein leakage of between one-quarter and one-third at an optimal concentration of 2.0 to 4.0 pmol/l. As with verapamil each of these three drugs had reduced effects in the higher concentration range, but in general the plateau values were considerably lower and at 40.0 pmol/l the reduction in protein leakage did not achieve a level of statistical significance when compared with drug-free controls. In the cases of terodiline and nifedipine bell-shaped curves were obtained, the drug appearing to be active only over a very narrow concentration range (2.0 to 6.0 pmol/l). Fendiline Fendiline response
also exhibited a bell-shaped dosecurve with an optimal reduction of
is indicated
as is the significance
of
protein leakage of 23 f 3:/,. However, in contrast to the previously mentioned drugs, the optimal concentration was much higher (20.0 pmol/l). This effect fell rapidly to zero at higher concentrations. Prenylamine This drug did not reduce leakage at any of the. concentrations studied; in view of the close similarity in structure between prenylamine and fendiline this observation was particularly surprising. However, the movement, relative to other calcium antagonists, of the optimal dose of fendiline to higher concentrations may be further continued in its molecular conversion to prenylamine and in these studies the possibility could not be ruled out that prenylamine may be effective at concentrations beyond the range studied. 0600 In contrast to the other active ca1ciu.m antagonists D600 did not exhibit a bellshaped dose-response curve but inhibitled protein leakage maximally at all higher concentrations. However, like the other drugs, maximal reduction of protein leakage was about one-quarter and this could be achived at concentrations between 4.0 and 40.0 pmol/l. Characteristics of drug @ects In a preliminary attempt to investigate possible mechanisms of action for the effects observed in these studies three additional investigations were carried out: one was to
482
J. E. Baker
and
eliminate the possibility that the reduction of protein leakage was merely a delay in the kinetics of injury, the second was to ascertain whether the drugs acted during the period of calcium depletion or calcium repletion and the third was to see whether the combination of two structurally unrelated drugs could have any additive effect upon the reduction of protein leakage. Reduction or delay of leakage? To eliminate the possibility that the drugs were acting to extend the duration of calcium-free perfusion required to predispose the myocardium to a calcium paradox, hearts (n = 4 in each group) were subjected to 10, 20 and 30 min of calcium depletion followed by 20 min of calcium repletion. In the control series verapamil was not used, in the test series it was included (4.0 pmol/l) during both calcium depletion and repletion phases. Figure 4 shows that for each of the three depletion times a similar and significant reduction of protein leakage could be achieved through the use of verapamil, thus effectively
D. J. Hearse
excluding the possibility that the effect was via a delay in the induction process. Phase of action The interpretation of the preceding results was further reinforced when studies were carried out to ascertain whether the drug was active during the calcium depletion or the calcium repletion phase. In these studies, hearts (n = 4 for each group) were subjected to 10 min of calcium-free perfusion followed by 20 min of calcium repletion. In control hearts there was no verapamil in any phase; in the test hearts verapamil (4.0 pmol/l) was included in either the calcium depletion phase alone or during the calcium repletion phase alone. The results (Figure 5) indicate that the drug had no effect when used only during calcium depletion but was effective when included during the entire calcium repletion phase alone or during both phases, although there was no significant difference (P > 0.01) between the last two groups studied, indicating that verapamil exerts its effect at some stage of calcium repletion.
.E 14c R k
120
i ‘p
100
,. 6 p
60
‘I-
E j
Duration
of calcium
depletion
(min)
FIGURE 4. Duration of depletion study. The effect of various durations of calcium depletion upon the ability of verapamil (4.0 ~mol/l) to reduce protein leakage in the calcium paradox. The unshaded histograms represent the cumulative protein leakage in control drug-free hearts; the shaded histograms represent the verapamil-treated hearts. Each group represents the mean of four hearts and the bars represent the standard error of the mean.
B .c al ‘i 9 B .6 2 (3
6o 40
20 0
FIGURE 5. Verapamil phase of action. The effect upon cumulative protein leakage during calcium repletion of the inclusion of verapamil (4.0 pmol/l) in the calcium depletion phase alone, the calcium repletion phase alone or during both phases. Each group represents the mean of four hearts and the bars represent the standard error of the mean.
Calcium
Antagonists
and
Additive ejkts In order to investigate the possibility that two slow channel blockers used together could improve the reduction in protein leakage induced during the calcium paradox, verapamil (4.0 lJ,rnol/l) and nifedipine (4.0 ymol/l) were included simultaneously in both the calcium depletion and calcium repletion phases. Hearts (n = 6) were subjected to 10 min of calcium-free perfusion followed by a 20 min period of calcium repletion during which time coronary effluent was collected and assayed for total protein. The results (Figure 6) indicate that there was no further reduction in protein leakage when nifedipine and verapamil were combined. This may indicate a common site of action for these two structurally unrelated compounds,
-1
FIGURE 6. Combination of verapamil and nifedipine. The effect upon cumulative protein leakage during calcium repletion of the inclusion in both the calcium depletion and repletion phases of verapamil (4.0 wol/l) alone, nifedipine (4.0 pmol/l) alone and both agents combined. Each group represents the mean of four hearts and the bars represent the standard error of the mean. Discussion
The results of this study have shown that a broad spectrum of highly diverse calcium antagonists was able to reduce protein leakage associated with the calcium paradox. The extent of this reduction of leakage was dependent upon the concentration of calcium used during the calcium repletion phase and
the
Calcium
Paradox
~I.83
upon the concentration of the calcium antagonist. At low calcium repletion ccmcentrations (0.1 mmol/l) protein leakage could be reduced by as much as 9994. At any single calcium concentration the drugs exhibited complex dose-response characteristics. These varied between drugs but certain important similarities were apparent. Thus, with the exception of prenylamine, which had no effect in the dose range studied, ,a11 drugs were able to reduce leakage by onequarter to one-third at their optimal concentration when calcium repletion was carried out at a concentration of 1.0 mmol/l. All of the active drugs except fendiline exerted a maximal effect at an identical concentration range of 2.0 to 4.0 pmol/l. With fendiline the concentration optimal was 20.0 pmoll/l. Fendiline and all other active drugs except D600 exhibited bell-shaped dose-response curves with a significant loss of activity at higher concentrations, in some instances activity being completely lost. Our investigations have further shown that this ability of the drugs to reduce protein leakage refle’cts some mechanism which is operative during the phase of calcium repletion and it is tempting to speculate that this effect is a blockade of the slow calcium channel at a time when massive influxes of calcium are known to be occurring. However, a number of aspects of this study would make us cautious in attributing our results to such a specific effect as blockade of the slow calcium channel. In particular, we refer to the almost identical optimal molar concentration for most drugs and the almost identical maximal reduction in leakage achieved by each. In view of the known wide range in slow calcium channel blocking potencies of these drugs [6] such a result is perhaps surprising and may argue :for a much more non-specific membrane effect. This is all the more 1ikel.y in view of the high concentrations relative to clinically effective doses (Table l), needed to achieve the results reported in this study. It should, however, be noted that the concentrations used in this study, and particularly the optimal concentrations observed, are very similar to those used in many experimental studies [16, l7, 181 and as such might lead one to question the specificity of calcium antagonists in other such studies in the literature.
J. E. Baker
484
and D. J. Hearse
In attempting to determine in which phase of the calcium paradox the drugs exert their effect we observed that verapamil acted when included during calcium repletion alone or during both calcium depletion and calcium repletion. It would appear therefore that this agent is active primarily during the phase of calcium repletion. In support of this possibility Ohhara [21] has demonstrated that the massive release of creatine kinase that occurs during the first 2 min of calcium repletion can be substantially reduced if verapamil is present during this period. Alto and Dhalla [I] have also shown that verapamil is able to reduce the rate of calcium accumulation during the induction of the calcium paradox in that after 2 min of calcium repletion significantly less (P < 0.05) calcium is present in the myocardium in hearts perfused with verapamil. The precise mechanism for the effects seen in these investigations remains obscure. However, recent studies have implicated other possible sites of action in addition to the slow channel. In this connection Nayler [19] has demonstrated that verapamil, D600, diltiazem, but not nifedipine, inhibit the binding of u-agonists to varying degrees in rat myocardium. In addition, Karliner [IO] has shown that in membranes prepared from rat heart ventricles both verapamil and D600 competitively inhibited binding to a-adrenergic receptors (at a dose of 10 pmol/l) and to muscarinic receptors (at a dose of 30 pmol/l).
Thus it may well be that calcium antagonists act as ‘promiscuous molecules’ [ 101, influencing in a non-specific yet dose-dependent manner a number of membrane channels and receptors which in turn, either directly or indirectly act to reduce protein leakage and tissue injury. It should be noted that throughout this article we have deliberately avoided the use of the word ‘protection’ when discussing the reduction of tissue injury as evidenced by a reduction in protein leakage. As we have stressed before [9], in this severe form of calcium-mediated injury, even with major reductions of enzyme or protein leakage, sufficient injury remains to render the tissue irreversibly injured; as such the term ‘tissue protection’ would not seem appropriate. However, in less severe models of calciummediated injury, e.g. incomplete calcium depletion or calcium repletion at low calcium levels, it is possible that the slow calcium channel blocking drugs may be able to reduce injury to such an extent that a return to normal function may be feasible. Acknowledgements This work was supported by grants from the Wellcome Trust, the British Heart Foundation and St Thomas’s Hospital Research Endowments Fund. The authors gratefully acknowledge the assistance of C. Boles.
References ALTO, L. E., DHALLA,
N. S. Myocardial cation contents during induction of calcium paradox. Am J Physiol H-713-H-719 (1979). ASHRAF, M., ONDA, M., BENEDICT, J. B., MILLARD, R. W. Prevention of calcium paradox-related myocardial cell injury with Diltiazem, a calcium channel blocking agent. Am J Cardiol 49, 1675-l 68 1 ( 1982). BAKER, J. E., HEARSE, D. J. A comparison of the ability of slow calcium blockers to reduce tissue damage during the calcium paradox in the rat heart. .J Mol Cell Cardiol 13, Suppl. 1, 6 (1981) (abstr.). BIELE&I, K. The inkuence of changes in pH of the perfusion fluid on the occurrence of the calcium paradox in the isolated rat heart. Cardiovasc Res 3, 268-271 (1969). BRADFORD, M. M. A rapid and sensitive ‘method for the’quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254 (1976). FLEKENSTEIN, A. Specific pharmacology of calcium in myocardium, cardiac pacemakers and vascular smooth muscle. Ann Rev Pharmacol Toxic01 17, 149-166 (1977). HEARSE, D. J., HUMPHREY, S. M., BULLOCK, G. R. The oxygen paradox and the calcium paradox: two facets of the same problem? J Mol Cell Cardiol 10, 641-668 (1978). HEARSE, D. J., BAKER, J. E., HUMPHREY, S. M. Verapamil and the calcium paradox. J Mol Cell Cardiol 12, 733-739 (1980). HEARSE, D. J., BAKER, J. E. Verapamil and the calcium paradox: a reaffirmation. J Mol Cell Cardiol 13, 1087-1090 (1981). 237,
Cell
Cardiol
Slow
(1983)
15, 475-485
Calcium Comparative
Channel Blockers and the Calcium Studies in the Rat with Seven J. E. Baker
and
Paradox: Drugs
D. J. Hearse
The Heart ResearchUnit, The RayneInstitute, St Thomas’sHospital, London,UK (Received30 September 1982, acceptedin revisedform 3 March 1983) J. IX. BAKER ANI) D. J. HEARSE. Slow Calcium Channel Blockers and the Calcium Paradox: Comparative Studies in the Rat with Seven Drugs. Journal of Molecular and Cellular Cardiology (1983) 15, 475-485. In studies of the calcium paradox, the isolated perfused rat heart was used to characterize the relationship between myocardial protein leakage and the concentration of calcium antagonist (verapamil and D600) or calcium in the perfusion fluid during a cycle of calcium depletion and repletion. The results indicated a dose-dependency such that protein leakage could be progressively reduced by decreasing the concentration of calcium during calcium repletion and/or by increasing the concentrations of drug. Detailed doseresponse studies with seven calcium antagonists (verapamil, D600, nifedipine, terodiline, diltiazem, fendiline and prenylamine) and a calcium concentration of 1 .O mmol/l during a 20 min period of calcium repletion following a 10 min period of calcium depletion revealed complex dose-response characteristics for each drug. In the dose range studied (0 to 40 pmol/l) all drugs with the exception of prenylamine were able to reduce protein leakage by up to 25 to 30% Optimal concentrations for verapamil, nifedipine, D600, diltiazem and terodiline were all between 2.0 and 4.0 vmol/l. With the exception of D600, which provided a constant reduction of protein leakage at all concentrations above this optimum, all drugs exhibited hell-shaped dose-response curves with a loss of efficacy at higher concentrations. Fendiline also had a bell-shaped dose-response curve with 23% as a maximal reduction of leakage; however, the optimal concentration for this drug was 21.0 pmol/l. Additional studies with verapamil revealed that the drug acted during the calcium repletion phase and did not influence events during calcium depletion. Simultaneous use of two drugs, verapamil and nifedipine, at their optimal concentrations failed to improve the reduction in protein leakage over and above that observed with one drug alone.
KEY
WORDS:
Protein leakage; Prenylamine.
Calcium Dose
paradox; response;
Slow calcium Verapamil;
channel D600;
Introduction Perfusion of the myocardium with calciumfree, followed by calcium-containing, media results in the immediate occurrence of severe cellular disruption, enzyme and protein leakage and irreversible contracture. The deleterious effects of this phenomenon, originally reported by Zimmerman [27] and termed the calcium paradox, have been attributed to sudden transmembrane calcium movements [7, 15, 23, 26, 27, 281. The extent of damage inflicted upon the myocardium during the calcium paradox can be influenced considerably by a number of Address London,
for reprint UK.
0022-2828/83/070475+
requests:
J. E. Baker,
11 $03.00/O
The
Heart
blockers; Nifedipine;
Calcium Diltiazem;
antagonists; Terodiline;
Tissue injury; Fendiline;
factors, including the extracellular concentration of ions such as sodium [2&j, magnesium [26] and hydrogen [4], the presence of agents such as taurine [II] and dimethylsulphoxide [25] and the myocardial temperature during the calcium depletion phase [7]. Recently we have reported that the slow channel blockerverapamil can reduce calcium paradox-induced cellular damage by up to one-quarter, as assessed by protein and enzyme leakage [8]. However, some workers have been unable to observe a similar reduction using identical indices of tissue damage [20, 241, while others have reported the
Research
Unit, 0
The 1983
Rayne Academic
Institute, Press
St Thomas’s Inc
(London)
Hospital, Limited
476
J. E. Baker
and D. J. Hearse
ability of this agent [21] and diltiazem [Z] to reduce the leakage of creatine kinase during the calcium paradox. Although the mechanism of this effect is unknown, it may be related to a drug-induced reduction in the rate or extent of intracellular calcium accumulation. In this connection there is some evidence from other studies [I] that verapamil can reduce the initial rate of calcium accumulation during calcium repletion. In addition to verapamil, numerous other compounds have been reported to possess slow calcium channel blocking properties (Table 1). These other drugs, e.g. nifedipine diltiazem and terodiline, have widely differing structures and differing potencies and we were interested to ascertain whether, and to what extent, these agents are able to influence cellular damage associated with the calcium paradox. If similar effects to those seen with verapamil occur, then this may strengthen evidence that calcium movements via the slow calcium channel contribute to the injury associated with the calcium paradox. We have therefore used protein leakage from the isolated rat heart preparation to compare the effects of seven calcium antagonistsIn addition to dose-response studies we have also investigated which phase of the calcium paradox is susceptible to drug modification and also whether the combination of different antagonists produces additive effects.
Materials
and
Experimental
Methods animals
Male CSE rats (250 to 300 g body weight) maintained on a standard diet were used throughout the study. Perfufiion techniques Animals were anaesthetized with diethyl ether, the left femoral vein was exposed and heparin (200 IU) was administered intravenously. After 1 min the chest was opened and the heart was excised and placed in cold (4°C) perfusion medium. After 30 s the aorta was attached to a stainless steel cannula,
the pulmonary artery was incised to permit adequate coronary drainage and the heart was then perfused by the method of Langendorff [I3, 141 at a perfusion pressure equivalent to 10 kPa (100 cm H,O). A side arm on the aortic cannula, located immediately above the heart, was used to facilitate a rapid change between calcium-containing and calcium-free perfusion media. The heart and all perfusion fluids were kept in temperature-controlled chambers in order that the myocardial temperature could be maintained at 37°C. Perfusion media Krebs-Henseleit buffer (pH 7.4 when gassed with 959/o oxygen and 5% carbon dioxide), in which the calcium content was reduced to 1.0 mmol/l, was the standard perfusion fluid [I,?]. To this and all other perfusates was added glucose (11 .l mmol/l). During the period of calcium-free perfusion all calcium was excluded from the perfusion fluid and care was taken to ensure no calcium contamination of constituent reagents or water [ZZ]. Before use all perfusion fluid was filtered through a cellulose acetate membrane of pore size 5.0 pm. In all studies various concentrations of carrier-free dl-verapamil (Knoll AC), dlD600 (Knoll AG), dl-diltiazem (Synthelabo) and dl-terodiline (Kabi) were dissolved in a small quantitity (10 ml) of deionized water before being added to the bulk of the perfusion fluid. Nifedipine (Bayer) and dlfendiline (Dr. Thiemann) were initially dissolved in 10 ml ethanol (19 mol/l) before the inclusion in the perfusate, with the final concentration of ethanol in the perfusate kept constant; dl-prenylamine (Hoechst) in the gluconate form was dissolved in deionized water (10 ml). With the last three drugs appropriate additions of carrier or solvent were made to control solutions to ensure they exerted no effect per se. All experiments with nifedipine were carried out under sodium light to prevent photodegradation of the drug. With the exception of the penultimate series of studies all drugs studied were included in the perfusate throughout both the period of calcium depletion and the period of calcium repletion.
Calcium
Antagonists
and
Perfusion sequence Immediately after mounting hearts were perfused at 37°C for a 20 min equilibration period with drug-free calcium-containing buffer. Hearts were then subjected to a 10 min period of calcium-free perfusion. This was followed by a 20 min period of calcium repletion (1 .O mmol calcium/l). During the calcium repletion period total coronary effluent was collected, the volume was recorded and well mixed samples were analysed immediately for total protein content. We have previously shown [8] that protein leakage can be equated to enzyme leakage as an index of tissue injury. At the end of each experiment hearts were taken and heated to 100°C for 24 h in order that dry weights could be determined, Protein determination The total protein content of samples of the coronary effluent collected during the period of calcium repletion was estimated by the method of Bradford [5]. In this assay 50 ~1 of coronary effluent was added to 2500 yl of the protein reagent. The protein reagent was prepared by dissolving 10 mg of Coomassie Brilliant Blue G (Sigma Chemical Co.) in a mixture composed of 5 ml of ethanol ( 19 mol/l) and 10 ml of phosphoric acid (16 mol/l). This was then diluted with distilled water to a final volume of 100 ml and the solution filtered. Following the addition of the coronary effluent to the protein reagent the absorbance of 595 nm was measured after 5 min and compared with appropriate standards (bovine serum albumin, Pentax fraction V) . The protein leakage into the effluent was then calculated and expressed as milligrams released per gram dry weight per 20 min period. Protein leakage has been previously demonstrated to be essentially complete by the end of the 20 min repletion period [8].
Results
Our previous studies [3] have shown that not only are the dose-response characteristics for verapamil concentration versus protein leakage complex, but so too are the characteristics of repletion calcium concentration
the
Calcium
Paradox
477
(at any one verapamil concentration) versus protein leakage. Our primary studies were therefore divided into two parts: first, an assessment of the interrelationship between calcium antagonist concentration, repletion calcium concentration and protein leakage for two calcium antagonists; secondly, an assessment of the relationship between calcium antagonist concentration and protein leakage at a selected repletion calcium concentration for seven calcium antagonists. After these primary studies we carried out some preliminary studies to characteri.ze further these drug effects. Calcium concentration interrelationships For this study equimolar concentrations of D600 and verapamil were studied. Hearts (n = 6 for each group) were subjected to 10 min calcium-free perfusion followed by 20 min calcium repletion at various calcium concentrations (0.1, 0.2, 0.5, 1.0 and 2.4 mmol/l). Verapamil or D600 (0, 1 .O, 4.0 and 20.0 pmol/l) were included in the perfusion fluid during both calcium depletion and calcium repletion. Figure 1 shows the cumulative protein leakage for each condition when D600 was used, Figure 2 shows the corresponding results for verapamil. Examination of the results for D600 (tlhe methoxy derivative of verapamil) shows that at all drug and all calcium concentrations studied the calcium antagonist was able significantly to reduce protein leakage and under some conditions this reduction ‘was very extensive. It is apparent with zero drug or any one drug concentration that there was a dose-dependent increase in protein leakage with increasing calcium repletion concentrations, the higher the repletion concentration the greater was the cumulative leakage. Thus, in the absence of D600, repletion of 0.1, 0.2 0.5, 1.0 and 2.4 mmol calcium/l produced cumulative protein leakage values of 70 f 8, 115+3,132~4,142&5and151 &6mg/g dry weight per 20 min period. Similar stepwise effects were seen with every D600 concentration, thus when 4.0 pmol/l of drug were used the corresponding protein leakage valueswere3&2,66&3,98&4, 120&6 and 132 & 5 mg/g dry weight per 20 min period. In the opposite plane of Figure 1, at
478
$
J. E. Baker
and
D. J. Hearse
14c
FIGURE 1. Calcium-D600-protein leakage doseresponse interrelationships. Dose-response relationships between perfusate D600 and calcium concentration and protein release occurring during calcium repletion. The height of each column represents the mean cumulative protein release from six hearts and the standard error of the mean is indicated by the lightly shaded part.
FIGURE 2. Calcium-verapamil-protein leakage dose-response interrelationships. Dose-response relationship between per&sate verapamil and calcium concentration and protein release occurring during calcium repletion. The height of each column represents the mean cumulative protein release from six hearts and the standard error of the mean is indicated by the lightly shaded part.
all fixed calcium concentrations, D600 caused a dose-dependent decrease in protein leakage. Thus with calcium repletion at 0.2 mmol/l D600 at 0, 1.0, 4.0 and 20.0 pmol/l led to cumulative protein leakage values of 115 & 3, 73 or 5, 66 & 3, and 47 f 5 mg/g dry weight per 20 min period. At the lowest calcium concentration studied (0.1 mmol/l) all D600 doses essentially abolished protein leakage. The observation of progressively reducing protein leakage with increasing drug concentrations or decreasing calcium concentrations was also evident with verapamil (Figure 2), although the results were not quite as sharply resolved. However, it should be noted that the drug was able to achieve a substantial
reduction of protein leakage under all conditions, even at the high calcium concentration of 2.4 mmol/l. In some cases the drug could almost abolish leakage, thus with a calcium repletion concentration of 0.1 mmol/l verapamil (4.0 pmol/l) reduced protein leakage by 99% from its control value of 70 f 8 mg/g dry weight per 20 min period. Drug dose-response curues Having established that two calcium antagonists at a limited number of concentrations are able to reduce protein leakage over a wide range of calcium repletion concentrations, it was decided to select a single calcium
Calcium
Antagonists
and
repletion concentration and investigate detailed dose-response characteristics for seven calcium antagonists over a wide range of concentrations. Although it was tempting to select a low calcium concentration (e.g. 0.1 mmol/l), where dramatic reductions were observed, it was decided, for the present studies, to select a high concentration (1 .O mmol/l) since this represents a physiologically more relevant concentration and also precipitates a full calcium paradox. The drugs to be studied, verapamil, D600, nifedipine, fendiline, terodiline diltiazem, and prenylamine, were selected so as to represent a wide range of structures and potencies. On the basis of our previous studies with verapamil a dose range of 0 to 40.0 pmol/l was selected for investigation.
the
Calcium
Paradox
479
Hearts (a = 6 for each group) were subjected to 10 min calcium-free perfusion followed by 20 min calcium repletioln (I .O mmol/l) . The calcium blocking drugs were included in both perfusion phases at one of the following concentrations: 0, 1 .O, 2.0, 4.0, 10.0, 20.0 and 40.0 pmol/l. Figure 3 shows the extent to which each concentration of each drug was able to reduce protein leakage relative to a paired control. Table 1 details the optimal concentration for each drug and the percentage reduction of protein leakage at that concentration. Verapamil In agreement with our earlier studies [8] verapamil exhibited a multiphasic doseresponse curve. At low concentrations (0 ~to
0 600
FIGURE 3. Dose-response studies. reduction in relation to control) during 10 min period of calcium-free perfusion. the mean is indicated by the bars.
The influence of drug concentration upon protein leakage (percentage a 20 min period.of calcium repletion (I’.0 mmol calcium/l) following: Each point represents the mean of six hearts and the standard error
a of
281
415
315
329
486
Terodiline (‘Bicor’)
Diltiazem (‘Herbesser’)
Fendiline (‘Sensit’)
Prenylamine (‘Segontin’)
D600
++
+++
++
+++
++++
++++
Slow channel inhibition
+
Cardiac
+++
++
+++
+
?
?
?
AV node conduction decrease
++
+++
if
+
f ?
7
Ability to induce coronary vasodilation
sites of action”
+++
+++
No
+
No
Alphaadrenoceptor binding
Subsidiary
?
?
i
+
f
t
I
No
++
?
Fast channel inhibition
pharmacological
-t
-+++-I
?
?
?
Max ?
Possible
-
Yes
Yes
Yes
-
Yes
Yes
Coronary heart diasease
conditions.
3.0
0.006-0.03
0.1-0.3
0.03-0.16
0.06-0.2
0.08-0.06
(PM)
Therapeutic Plasma levels
to in viva or clinical
++
++
+++
Peripheral vascular relaxation
propertiesb
? No information available. a Note: Cardiac sites of action referred to above are based upon in vitro experimental studies and may not apply b Plasma levels not corrected for protein binding, all drug actions referred to are concentration dependent.
346
Nifedipine (‘Adalat’)
Molecular weight
to be investigated
455
1. Drugs
Verapamil (‘Isoptin’)
Name
TABLE
uses
-
Yes
Yes
Yes
Yes
Supraventricular arrhythmias
clinical
Calcium TABLE
2. Optimal
drug Drug
Verapamil Nifedipine Terodiline Diltiazem Fendiline Prenylamine D600
Antagonists
concentration
and
and protein
Optimal drug concentration (wolil)
the
Calcium
4,8 1
Paradox
leakage Maximum percentage reduction in protein leakage
4 4 4 2 20 4+&o
26 31 31 29 23
f & * & f 0 24 &
the mean for six hearts; the variation leakage relative to a paired drug-free
in reduction control.
P
3 2 3 3 3
3
N.S.
Each result represents the reduction in protein
4.0 pmol/l) there was a steep and linear increase in the ability of the drug to reduce protein leakage, which could be reduced maximally by 26 f 3% with a verapamil concentration of 4.0 pmol/l. In comparison with control this reduction was significant (P.< 0.01). At higher drug concentrations (10.0 to 40.0 pmol/l) the reduction of leakage declined to a plateau level (13 -& 3%) but remained significantly different from control values (P < 0.05). Nifedipine, terodiline and diltiazem Figure 3 reveals that for these three drugs comparable results to those for verapamil were obtained, thus rapid increases in drug efficacy were observed in the range 0 to 4.0 ymol/l, with similar maximal reductions of protein leakage of between one-quarter and one-third at an optimal concentration of 2.0 to 4.0 pmol/l. As with verapamil each of these three drugs had reduced effects in the higher concentration range, but in general the plateau values were considerably lower and at 40.0 pmol/l the reduction in protein leakage did not achieve a level of statistical significance when compared with drug-free controls. In the cases of terodiline and nifedipine bell-shaped curves were obtained, the drug appearing to be active only over a very narrow concentration range (2.0 to 6.0 pmol/l). Fendiline Fendiline response
also exhibited a bell-shaped dosecurve with an optimal reduction of
is indicated
as is the significance
of
protein leakage of 23 f 3:/,. However, in contrast to the previously mentioned drugs, the optimal concentration was much higher (20.0 pmol/l). This effect fell rapidly to zero at higher concentrations. Prenylamine This drug did not reduce leakage at any of the. concentrations studied; in view of the close similarity in structure between prenylamine and fendiline this observation was particularly surprising. However, the movement, relative to other calcium antagonists, of the optimal dose of fendiline to higher concentrations may be further continued in its molecular conversion to prenylamine and in these studies the possibility could not be ruled out that prenylamine may be effective at concentrations beyond the range studied. 0600 In contrast to the other active ca1ciu.m antagonists D600 did not exhibit a bellshaped dose-response curve but inhibitled protein leakage maximally at all higher concentrations. However, like the other drugs, maximal reduction of protein leakage was about one-quarter and this could be achived at concentrations between 4.0 and 40.0 pmol/l. Characteristics of drug @ects In a preliminary attempt to investigate possible mechanisms of action for the effects observed in these studies three additional investigations were carried out: one was to
482
J. E. Baker
and
eliminate the possibility that the reduction of protein leakage was merely a delay in the kinetics of injury, the second was to ascertain whether the drugs acted during the period of calcium depletion or calcium repletion and the third was to see whether the combination of two structurally unrelated drugs could have any additive effect upon the reduction of protein leakage. Reduction or delay of leakage? To eliminate the possibility that the drugs were acting to extend the duration of calcium-free perfusion required to predispose the myocardium to a calcium paradox, hearts (n = 4 in each group) were subjected to 10, 20 and 30 min of calcium depletion followed by 20 min of calcium repletion. In the control series verapamil was not used, in the test series it was included (4.0 pmol/l) during both calcium depletion and repletion phases. Figure 4 shows that for each of the three depletion times a similar and significant reduction of protein leakage could be achieved through the use of verapamil, thus effectively
D. J. Hearse
excluding the possibility that the effect was via a delay in the induction process. Phase of action The interpretation of the preceding results was further reinforced when studies were carried out to ascertain whether the drug was active during the calcium depletion or the calcium repletion phase. In these studies, hearts (n = 4 for each group) were subjected to 10 min of calcium-free perfusion followed by 20 min of calcium repletion. In control hearts there was no verapamil in any phase; in the test hearts verapamil (4.0 pmol/l) was included in either the calcium depletion phase alone or during the calcium repletion phase alone. The results (Figure 5) indicate that the drug had no effect when used only during calcium depletion but was effective when included during the entire calcium repletion phase alone or during both phases, although there was no significant difference (P > 0.01) between the last two groups studied, indicating that verapamil exerts its effect at some stage of calcium repletion.
.E 14c R k
120
i ‘p
100
,. 6 p
60
‘I-
E j
Duration
of calcium
depletion
(min)
FIGURE 4. Duration of depletion study. The effect of various durations of calcium depletion upon the ability of verapamil (4.0 ~mol/l) to reduce protein leakage in the calcium paradox. The unshaded histograms represent the cumulative protein leakage in control drug-free hearts; the shaded histograms represent the verapamil-treated hearts. Each group represents the mean of four hearts and the bars represent the standard error of the mean.
B .c al ‘i 9 B .6 2 (3
6o 40
20 0
FIGURE 5. Verapamil phase of action. The effect upon cumulative protein leakage during calcium repletion of the inclusion of verapamil (4.0 pmol/l) in the calcium depletion phase alone, the calcium repletion phase alone or during both phases. Each group represents the mean of four hearts and the bars represent the standard error of the mean.
Calcium
Antagonists
and
Additive ejkts In order to investigate the possibility that two slow channel blockers used together could improve the reduction in protein leakage induced during the calcium paradox, verapamil (4.0 lJ,rnol/l) and nifedipine (4.0 ymol/l) were included simultaneously in both the calcium depletion and calcium repletion phases. Hearts (n = 6) were subjected to 10 min of calcium-free perfusion followed by a 20 min period of calcium repletion during which time coronary effluent was collected and assayed for total protein. The results (Figure 6) indicate that there was no further reduction in protein leakage when nifedipine and verapamil were combined. This may indicate a common site of action for these two structurally unrelated compounds,
-1
FIGURE 6. Combination of verapamil and nifedipine. The effect upon cumulative protein leakage during calcium repletion of the inclusion in both the calcium depletion and repletion phases of verapamil (4.0 wol/l) alone, nifedipine (4.0 pmol/l) alone and both agents combined. Each group represents the mean of four hearts and the bars represent the standard error of the mean. Discussion
The results of this study have shown that a broad spectrum of highly diverse calcium antagonists was able to reduce protein leakage associated with the calcium paradox. The extent of this reduction of leakage was dependent upon the concentration of calcium used during the calcium repletion phase and
the
Calcium
Paradox
~I.83
upon the concentration of the calcium antagonist. At low calcium repletion ccmcentrations (0.1 mmol/l) protein leakage could be reduced by as much as 9994. At any single calcium concentration the drugs exhibited complex dose-response characteristics. These varied between drugs but certain important similarities were apparent. Thus, with the exception of prenylamine, which had no effect in the dose range studied, ,a11 drugs were able to reduce leakage by onequarter to one-third at their optimal concentration when calcium repletion was carried out at a concentration of 1.0 mmol/l. All of the active drugs except fendiline exerted a maximal effect at an identical concentration range of 2.0 to 4.0 pmol/l. With fendiline the concentration optimal was 20.0 pmoll/l. Fendiline and all other active drugs except D600 exhibited bell-shaped dose-response curves with a significant loss of activity at higher concentrations, in some instances activity being completely lost. Our investigations have further shown that this ability of the drugs to reduce protein leakage refle’cts some mechanism which is operative during the phase of calcium repletion and it is tempting to speculate that this effect is a blockade of the slow calcium channel at a time when massive influxes of calcium are known to be occurring. However, a number of aspects of this study would make us cautious in attributing our results to such a specific effect as blockade of the slow calcium channel. In particular, we refer to the almost identical optimal molar concentration for most drugs and the almost identical maximal reduction in leakage achieved by each. In view of the known wide range in slow calcium channel blocking potencies of these drugs [6] such a result is perhaps surprising and may argue :for a much more non-specific membrane effect. This is all the more 1ikel.y in view of the high concentrations relative to clinically effective doses (Table l), needed to achieve the results reported in this study. It should, however, be noted that the concentrations used in this study, and particularly the optimal concentrations observed, are very similar to those used in many experimental studies [16, l7, 181 and as such might lead one to question the specificity of calcium antagonists in other such studies in the literature.
J. E. Baker
484
and D. J. Hearse
In attempting to determine in which phase of the calcium paradox the drugs exert their effect we observed that verapamil acted when included during calcium repletion alone or during both calcium depletion and calcium repletion. It would appear therefore that this agent is active primarily during the phase of calcium repletion. In support of this possibility Ohhara [21] has demonstrated that the massive release of creatine kinase that occurs during the first 2 min of calcium repletion can be substantially reduced if verapamil is present during this period. Alto and Dhalla [I] have also shown that verapamil is able to reduce the rate of calcium accumulation during the induction of the calcium paradox in that after 2 min of calcium repletion significantly less (P < 0.05) calcium is present in the myocardium in hearts perfused with verapamil. The precise mechanism for the effects seen in these investigations remains obscure. However, recent studies have implicated other possible sites of action in addition to the slow channel. In this connection Nayler [19] has demonstrated that verapamil, D600, diltiazem, but not nifedipine, inhibit the binding of u-agonists to varying degrees in rat myocardium. In addition, Karliner [IO] has shown that in membranes prepared from rat heart ventricles both verapamil and D600 competitively inhibited binding to a-adrenergic receptors (at a dose of 10 pmol/l) and to muscarinic receptors (at a dose of 30 pmol/l).
Thus it may well be that calcium antagonists act as ‘promiscuous molecules’ [ 101, influencing in a non-specific yet dose-dependent manner a number of membrane channels and receptors which in turn, either directly or indirectly act to reduce protein leakage and tissue injury. It should be noted that throughout this article we have deliberately avoided the use of the word ‘protection’ when discussing the reduction of tissue injury as evidenced by a reduction in protein leakage. As we have stressed before [9], in this severe form of calcium-mediated injury, even with major reductions of enzyme or protein leakage, sufficient injury remains to render the tissue irreversibly injured; as such the term ‘tissue protection’ would not seem appropriate. However, in less severe models of calciummediated injury, e.g. incomplete calcium depletion or calcium repletion at low calcium levels, it is possible that the slow calcium channel blocking drugs may be able to reduce injury to such an extent that a return to normal function may be feasible. Acknowledgements This work was supported by grants from the Wellcome Trust, the British Heart Foundation and St Thomas’s Hospital Research Endowments Fund. The authors gratefully acknowledge the assistance of C. Boles.
References ALTO, L. E., DHALLA,
N. S. Myocardial cation contents during induction of calcium paradox. Am J Physiol H-713-H-719 (1979). ASHRAF, M., ONDA, M., BENEDICT, J. B., MILLARD, R. W. Prevention of calcium paradox-related myocardial cell injury with Diltiazem, a calcium channel blocking agent. Am J Cardiol 49, 1675-l 68 1 ( 1982). BAKER, J. E., HEARSE, D. J. A comparison of the ability of slow calcium blockers to reduce tissue damage during the calcium paradox in the rat heart. .J Mol Cell Cardiol 13, Suppl. 1, 6 (1981) (abstr.). BIELE&I, K. The inkuence of changes in pH of the perfusion fluid on the occurrence of the calcium paradox in the isolated rat heart. Cardiovasc Res 3, 268-271 (1969). BRADFORD, M. M. A rapid and sensitive ‘method for the’quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254 (1976). FLEKENSTEIN, A. Specific pharmacology of calcium in myocardium, cardiac pacemakers and vascular smooth muscle. Ann Rev Pharmacol Toxic01 17, 149-166 (1977). HEARSE, D. J., HUMPHREY, S. M., BULLOCK, G. R. The oxygen paradox and the calcium paradox: two facets of the same problem? J Mol Cell Cardiol 10, 641-668 (1978). HEARSE, D. J., BAKER, J. E., HUMPHREY, S. M. Verapamil and the calcium paradox. J Mol Cell Cardiol 12, 733-739 (1980). HEARSE, D. J., BAKER, J. E. Verapamil and the calcium paradox: a reaffirmation. J Mol Cell Cardiol 13, 1087-1090 (1981). 237,