Species difference in myocardial response to actodigin: Correlation with inhibition of myocardial (Na+ + K+)-ATPase

Comp. Biochem. Physiol., Vol. 66C, pp. 153 to 158

0306-4492/80/0701-0153102.00/0

© Pergamon Press Ltd 1980, Printed in Great Britain

SPECIES DIFFERENCE IN MYOCARDIAL RESPONSE TO ACTODIGIN: CORRELATION WITH INHIBITION OF MYOCARDIAL (Na ÷ + K÷)-ATPase J. S. CHARNOCK,W. F. DRYDEN and P. A. LAUZON Department of Pharmacology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada (Received 4 December 1979) Abstract--1. The inotropic response of both atrial and papillary muscle from the active (nonhibernating) ground-squirrel Spermophilus richardsonii is more sensitive to actodigin (AY 22241) than is the response in these muscles from the gninea-pig or the rat. 2. Under constant experimental conditions the magnitude of the positive inotropic response to actodigin in the gninea-pig is greater than that found in the ground-squirrel. 3. Rat cardiac muscles are virtually insensitive to actodigin at any concentration of drug that we could employ in vitro. 4. Actodigin inhibition of myocardial (Na ÷ + K +)-ATPase from these same three species parallels the results from isolated cardiac muscle strips. Enzyme preparations from the active ground-squirrel are more sensitive to inhibition than are those from the guinea-pig, while preparations from the rat are the least sensitive. 5. Comparison of the Iso values for actodigin and ouabain inhibition of myocardial (Na ÷ + K÷)ATPase demonstrates that the semisynthetic glycoside is at least 10-fold less potent than ouabain.

INTRODUCTION

Recent studies in this laboratory have demonstrated large seasonal variations in the positive inotropic response to ouabain by isolated atrial and papillary muscles from hibernating and non-hibernating ground-squirrels. When these animals are awake and active during the early summer months, their body temperature is near 37°C and their heart rate is high (180-200 beats/min). By contrast during the deep torpor of hibernation in mid-winter, their body and organ temperature falls below 5°C and their heart rate decreases to one beat every two or three minutes (Hudson, 1971). We have found that conventional concentrations of ouabain induce a clear positive inotropism in myocardial tissues from non-hibernating animals. Conversely, similar concentrations of this naturally occurring cardiac glycoside only elicited greatly reduced inotropic responses in tissues obtained from hibernating animals. These reduced responses were usually less than 25% of the effect found with their respective controls (Charnock et al., 1980a). However, because of the slow binding constant for ouabain and its pseudo-irreversible action o n cardiac preparations in vitro, our experimental design necessitated the use of only one dose of ouabain per individual tissue, making the determination of conventional concentration-effect relationships subject to considerable experimental error. In the original study, this probably accounts for our observation that the tissues from many hibernating animals failed to demonstrate an unequivocal inotropic response. Recently, a semisynthetic cardiac glycoside has been developed by Deghenghi (1970) from glucose and digitoxigenin. This compound Actodigin (AY 153

22241)3fl-D-glucopyranosyl-14fl,-24 dihydroxyl-21, 23 bisnor- 5fl- chol - 20(22) -ene - 20 - carboxylic~ - lactone, differs from the naturally occurring cardiac glycosides in that the lactone ring is attached to the steroid nucleus via the C-2 atom of the lactone rather than at the C-3 position. Although actodigin retains the qualitative effects of classical cardiac glycosides in that it has been shown to increase the force of contraction in both intact dog heart preparations (Mendez et al., 1974; Zavecz & Dutta, 1977; Cummings & Beaulieu, 1977) and in isolated cardiac muscles from dogs and cats (Pastelin & Mendez, 1972; Gliklich et al., 1975; Cagin et al., 1977; Thomas et al., 1979) it differs from both digitoxin and ouabain in the rapidity of onset of arrhythmia and in the markedly increased ease with which toxicity can be reversed once drug infusion has ceased. On the other hand, in the relatively few studies which have been performed with this drug, actodigin has been shown to be less potent than ouabain in that its concentration-effect curve is shifted to the right by about one order of magnitude (Pastelin & Mendez, 1972; Cummings & Beaulieu, 1977; Cagin et al., 1977; Thomas et al., 1979). In this paper we extend the parallelism between actodigin and the naturally occurring cardiac glycosides by comparing the positive inotropic effect of actodigin upon myocardial preparations from the active (i.e. non-hibernating) ground-squirrel Spermophilus richardosonii, to those found in similar preparations from the glycoside "sensitive" guinea-pig (Carla cobaya) and the glycoside "insensitive" rat (Rattus norvegicus) (Repke, 1963; Repke et al., 1965). In addition, these pharmacological effects have been compared to the inhibition of cardiac (Na ÷ + K+)ATPase by actodigin in these same species.

154

J.S. CrIARNOCK,W. F. DRYDENand P. A. LAUZON MATERIALS AND METHODS

RESULTS

The ground-squirrels used in these experiments were obtained from the colony of Spermophilus richardsonii maintained in our department. Full details relating to the trapping and maintenance of these animals have been published previously (Charnock et al.; 1980a,b). The guineapigs and the Wistar white rats were obtained from commercial suppliers. The method of atrial and papillary muscle preparation and the determination of the mechanical effect of the cardiac glycoside in vitro have also been published recently (Charnock et al., 1980a). Because of the limited solubility of actodigin in aqueous solution (about 10 mg%), the drug was first dissolved in dimethylsulfoxide (DMSO) after paired experiments had shown that the final concentration of DMSO that we employed in these experiments (1.5%) was without effect upon the contractile properties of the myocardial muscle preparations (Hughes & Charnock, unpublished data). Even so, the maximal concentration of actodigin that could be reliably attained in our experiments was 5 x 10-4M. The preparations of myocardial (Na + + K +)-ATPase and its assay by the coupled spectrophotometric method utilizing the regeneration of ATP and the oxidation of NADH at 340 m#, has also been described in full in a recent publication from this laboratory (Charnock et al., 1980b). Actodigin (AY 22241) was the gift of Ayerst Laboratories, Montreal, Canada; ouabain (octahydrate) and dimethylsulfoxide(DMSO) were obtained from the Sigma Chemical Co, St Louis, Missouri. The structural formulae of actodigln and ouabain are compared with those of their related genins in Fig. 1.

Effects of actodioin on twitch tension of isolated cardiac muscle preparations The positive inotropic effects of actodigin were compared on the isolated atrial and papillary muscles of three different species of small mammals (the rat, the guinea-pig, and the ground-squirrel), chosen because of their reported difference in response to the naturally occurring cardiac glycosides (Gersmeyer & Holland, 1962; Repke et al., 1965; Charnock et al., 1980a). The dose-dependent relationships obtained with atrial muscle preparations from all three species are shown in Fig. 2. Whereas the greatest maonitude of response is clearly obtained with the tissue of the guinea-pig (more than 700% increase in twitch tension above the control level was observed with actodigln concentrations > 10-4M under constant experimental conditions), maximal responses could not be obtained in these tissues because of the limited solubility of actodigin even in DMSO. However, the atria of the ground-squirrel was the most sensitive in the sense that the maximal positive inotropic effect (350% increase in twitch height above the control) was obtained with 3.3 x 10 -6 M actodigin. The EDso for ground-squirrel atrial muscle was 6.6 x 10-TM, which was about two orders of magnitude less than that which could be estimated for the guinea-pig muscle from our results. By contrast, the atria from

OU/~BAGENIN MW 439

OUABAIN MW 5 8 4

029 H44 O12

o

C23 H34 08

oH

H

~

o

oH

O-

OH

OH OH

rhamnose ACTODIGIN MW 537'

C29 H45 09

o 0

!

i

1 °"

OlGITOXlGENIN MW 375

C23 H34 04

o

1,z 3

HO~

OH

H OH

glucose Fig. 1. Comparison of the structural formulae of ouabain and actodigin, and the genins from which they are derived.

800

Species difference in response to aetodigin

155

ATRIA /

/~ Rg

600

4OO

10-'

10-'

10-'

10°'

~-'

Molar Concentration ACTODIGIN

Fig. 2. Species difference in sensitivity of atrial muscle to various concentrations of actocligin. Increases in twitch tension are the means of at least five separate experiments on tissues from different animals, given as % increases above control levels (see Methods). The order of sensitivity is ground squirrel (I) > guinea-pig (4)) > rat (O). The values for EDso calculated from this data are given in the text.

the rat were virtually isensitive to actodisin at the concentrations examined; the maximum increase in twitch tension we observed being only 30?/o above the control level at 1.1 x 10- 3 M. Again, the limited solubility of actodigin (even in DMSO) prevented examination of the effect of even higher drug concentrations. Qualitatively similar results were obtained with the papillary muscle preparations from the same three species of mammals. Our results are given in Fig. 3, where again the tissue of the ground-squirrel was found to be the most sensitive, displaying a measurable positive inotropic effect at 1.1 x 10 -6 M; and a maximum effect at 8.0 x 10 -6 M where an increase of 1707/o above the control level was obtained. The EDs0 value for ground-squirrel was 1.5 x 10 -6 M. How-

ever, the greatest magnitude of the inotropic effect was again found with tissue from the guinea-pig where more than 400?/o increase in twitch tension above the control level was observed with actodigin at 3.3 x 1 0 - ' M . As found previously with the preparations of atrial muscle from the guinea-pig, the limited solubility of the drug (even in DMSO) again prevented the demonstration of maximal effects in this tissue. Papillary tissue from the rat was virtually insensitive (maximum increase of 30?/o above control level at 4 x 10 -4 M) at any concentration of actodigin we were able to examine. In general, these results with the semisynthetic cardiac glycoside actodigin, support the earlier work of

PAPILLARY

8001 ~4oo4

lo-,

I

Guinea~

~b-, Mokmr ~-"

~

Concentration ACTOOIGIN

~b-,

Fig. 3. Species difference in sensitivity of papillary muscle to various concentrations of actodigin. Increases in twitch tension are the means of at least five separate experiments on tissues from different animals, given at % increases above control levels (see Methods). The order of sensitivity is ground squirrel (I) > guinea-pig (e) > rat (O). The values for EDso calculated from this data are given in the text. C,B,P, 66/2C--C

156

J. S. CnARNOCK, W. F. DRYDEN and P. A. LAUZON

Gersmeyer & Holland (1962) and Repke et al. (1965) with the naturally occurring cardiac glycosides. However, we are not aware that the extreme sensitivity of the ground-squirrel (Spermophilus richardsonii) cardiac muscles to either naturally occurring or semisynthetic cardiac glycosides when compared to other more common laboratory animals has been reported previously. In this respect they appear to be very similar to the responses to Actodigin which were reported by Thomas et al. (1979) while this work was in progress. Effect o f actodigin (Na + + K+)-ATPase

and

ouabain

on

myocardial

Dose response curves for both actodigin and ouabain inhibition of myocardial (Na + + K+)-ATPase were obtained by the coupled optical assay procedure we have employed in several previous studies (Charn o c k e t al., 1977; Charnock et al., 1980b) which allows us to compare glycoside-sensitive ATPase with "cation-stimulated" ( N a + + K+)-activation of the enzyme, thus establishing a reliable value for either maximal activation or 100% inhibition of the enzyme. The results for actodigin inhibition of myocardial ATPase from the three different species (guinea-pig, ground-squirrel and rat) are given in Fig. 4). As one might expect from the previous results with isolated strips of cardiac tissue, the myocardial (Na + + K+)-ATPase from the ground-squirrel is very sensitive to inhibition by actodigin. Although from the Iso values given in Table 1, actodigin is less potent than ouabain, measurable inhibition can be seen at about 5 x 10-7 M and more than 90% of the maximal activity in the presence of N a + + K + is inhibited by 2 x 10-5 M actodigin. This drug concentration is the greatest we could achieve in our enzyme assay system before D M S O must be used to solubilize actodigin (Flasch & Heinz, 1978). However, it is clear that

the 15o for actodigin inhibition of ground-squirrel myocardial (Na + + K+)-ATPase is near 5 x 10 -6 M, which is about an order of magnitude greater than the value we obtained for ouabain with these groundsquirrel preparations (see Table 1). Guinea-pig myocardial (Na + + K + ) - A T P a s e is also sensitive to inhibition by actodigin. Measurable effects were found at 1 x 1 0 - S M and 20?/0 of the cation activated enzyme activity was inhibited by 2 x 10 -5 actodigin. Our estimated value for Iso for actodigln inhibition of the guinea-pig enzyme is 7 x 10 -5 M which is more than an order of magnitude higher than that for ouabain for this tissue. Only partial inhibition of rat myocardial (Na + + K+)-ATPase could be demonstrated at the highest concentration of actodigm that we were able to employ. N o reduction in activity was obtained at 2 x 10-5 M. This result compares with a reduction of 33% found with the same concentration of ouabain (not shown in Fig. 3). These latter values for ouabain inhibition are very similar to those previously reported for rat heart preparations by Allen & Schwartz (1969). The values for 15o for actodigin and ouabain we obtained with myocardial (Na ÷ + K+)-ATPase for some other tissues or species are also given in Table 1 where they are compared with values obtained from the literature. DISCUSSION Since the work of Vulpian in 1855, the marked species difference in the sensitivity of animals to cardiac glycosides has become well established. The detailed study of this phenomenon by Repke (1963) and Repke et al. (1965) led to the hypothesis that the characteristic positive inotropic response of these drugs was due to inhibition of the ( N a + + K+) ATPase of the myocardium.

Table 1. Comparison of the inhibitory effect of actodigin and ouabain on the (Na ÷ + K÷)-ATPase from various tissues and species Species Groundsquirrel Guinea pig Rat

Dog Beef Cat Porcine

Tissue

[M] Actodigin 15o

heart brain heart heart heart heart heart heart heart heart heart heart heart heart heart heart heart brain

4.4 x 1 0 - 6 5.8 x 1 0 - 6 7.0 x 10 -s --1.9 x 10-3:~ ---3.4 x 10 -6 ---1.8 x 10 -6 --2.8 x 1 0 - 6 3.6 × 10 -6

[M] Ouabain I50 4.7 4.4 1.7 6 1 5.7 6 1 1 1.7 6 6 3

Reference*

x 10 -7 x 10 -7

t t

X 10 -6

~"

X 10 -7

Repke et al. (1965) Flasch & Heinz (1978) 5" Repke et al. (1965) Allen & Schwartz (1964) Flasch & Heinz (1978) 1" Repke et al (1969) Schwartz et al (1969) Schwartz et al (1969) Thomas et al. (1979) Repke et al. (1965) Flasch & Heinz (1978) Thomas et al. (1979) Ross & Pessah (1975)

x

10 -6

x 10 -s x 10 -s x 10 -4 x 10 -4 x 10 -7 x 10 -8 × 10- 7 x 10 -7 -1.3 x 10 -7 1.9 x 10 -7 ---

* Cited in full in the Reference section of this paper. 3"This publication. An estimated value since actodigin precipitates at concentrations greater than 2 × 10 -3 M.

Species difference in response to actodigin

157

100" Ground

Squ|rreil~lll --i

/

GuineaPig

/

"r.l.J

~40-

C)

0

a~)-a

lb-7

lb-S

10-s

1()-4

1()-3

Molar Concentrath)n ACTODIGIN

Fig. 4. Species difference in actodigin inhibition of myocardial (Na + + K+)-ATPase from ground squirrel, guinea-pig and rat. All data expressed at percentage inhibition of system fully inhibited by ouabain. The order of sensitivity is ground squirrel ( I ) > guinea-pig (e) > rat (o). The values for lso from this data are compared in Table 1 to the I50 for ouabain we obtained on the same enzyme preparation.

Several accounts of close parallelism between instropic concentrations of naturally occurring cardiac glycosides and their inhibitory action on mammalian heart (Na + + K+)-ATPase have supported this view (Akera et al., 1973; Erdmann & Schoner, 1974a,b; Schwartz et al., 1974; Fricke, 1976; Charnock et al., 1980a). Very recently, Flasch & Heinz (1978) have extended this parallelism to a large series of polar cardenolide-conjugates, nonpolar cardenolides and cardanolides and concluded "that the obvious correlation between the cardenolide-induced inhibition of (Na + + K +)-stimulated membrane ATPase and the positive inotropic cardiac activity strongly supports Repke's hypothesis". Associated findings of Allen & Schwartz (1969) with the glycoside-insensitive rat heart add support to this general view. In the present study we have extended Repke's hypothesis to the new semisynthetic glycoside, actodigin (AY 22241) which has qualitatively similar pharmacological properties to the naturally occurring glycosides. It is clear from our results that while the guinea-pig and the rat continue to represent glycoside "sensitive" and glycoside "insensitive" species respectively, the myocardium of the ground-squirrel Spermophilus richardsonii is the most sensitive of the species examined here. When these animals are awake and active, the sensitivity of their myocardium to actodigin is similar to that reported recently for the cat (Thomas et al., 1979). However, it should be recalled that the sensitivity of the ground-squirrel myocardium to cardic glycosides is subject to very considerable seasonal variation (Charnock et al., 1980a, b). It should also be noted that in confirmation of the few pharmacological studies performed to date with actodigin, this semisynthetic cardiac glycoside is less potent than ouabain, digitoxin or digitoxigenin but is similar to digitoxigenin to which it is also closely related (Flasch & Heinz, 1978). The parallelism between the EDso values of actodigin which induce half maximal positive inotropic

effects and the I50 values for half maximal inhibition of myocardial (Na + + K+)-ATPase in a variety of species, adds further support to the hypothesis that the (Na + + K+)-ATPase macromolecule is itself the drug-receptor for cardiac glycosides (Repke et al., 1965; Schwartz et al., 1975, Akera, 1977). As actodigin has already been shown to inhibit sodium transport (Erlij & Elizalde, 1974) it is an ideal tool for the further investigation of the parallelism between the pharmacological action of this group of clinically important drugs and their biochemical mechanism of action. For example, studies of the seasonal variation in the myocardial response to actodigin in the hibernating and non-hibernating groundsquirrel are currently in progress in our laboratory. Acknowledgements--The authors are most grateful to Mr L. P. Simonson, M.Sc. for his skilled assistance in the preparation and assay of myocardial ( N a + + K+)-ATPase. We also wish to thank Ayerst Laboratories of Montreal for supplying the Actodigin used in this study, which was supported by a grant from the Alberta Heart Foundation. REFERENCES

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J.S. CHARNOCK,W. F. DRYDENand P. A. LAUZON

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