Interactions of cryptosin with mammalian cardiac dihydropyridine-specific calcium channels

Life Sciences, Vol. 47, pp. 1667-1676 Printed in the U.S.A.

Pergamon Press

INTERACTIONS OF CRYPTOSIN WITH MAMMALIAN CARDIAC DIHYDROPYRIDINE-SPECIFIC CALCIUM CHANNELS

Venkateswara R. Rao* and J.W. Banning Department of Pharmacology, College of Pharmacy and Allied Health Professions, Wayne State University, Detroit, MI 48202 (Received in final form August 27, 1990)

Summary Cryptosin -a new cardenolide was found to be a potent inhibitor of cardiac Na" and K÷ dependent Adenosinetri-phosphatase (7). In experiments with dog heart ex vivo, development of inotropic and toxic effect correlated with changes in the cardiac dihydropyridine-specific calcium channels as measured by the binding of 3[H]PN 200-110. A significant change in the PN 200-110 binding was observed when guinea pig and dog heart sarcolemmal membranes were pre-incubated with cryptosin in vitro. Binding analysis of 3[H]PN 200-110 (Isradipine) -a 1,4-dihydropyridine analog with very specific calcium channel binding properties -- in both in vitro and ex vivo studies were consistent and indicated a non-specific type of interaction of cryptosin with mammalian cardiac 1,4-dihydropyridine-specific calcium channels.

Cryptosin, a new cardiac glycoside isolated from the leaves of a tropical weed Cryptolepis buchanani Roem & Schult (Asclepiadaceae) (I-3) with a novel structure (4,5) was found to be a potent cardiotonic agent (6). Cryptosin was shown to be an inhibitor of guinea pig and dog cardiac Sodium and Potassium dependent Adenosinetriphosphatase (Na,K-ATPase) (7). Cryptosin displaced 3[H]Ouabain from i t s binding site and demonstrated a significant binding only in the presence of Na÷, Mg"÷ and ATP (8). Although the literature is replete on the role of calcium ion in cardiac glycoside-induced positive inotropy, information is relatively scanty on studies linking calcium channels and cardiac glycosides. However, earlier studies have reported that cardiac glycosides enlarge the size of the exchangeable fraction of tissue calcium (9,10). Electrophysiological studies have implicated involvement of cardiac glycosides in alteration of Na'/Ca÷÷ exchangeability (10,11) and interference with Ca"-Calmodulin dependent systems (12-14). Evidence can also be drawn indirectly from the effects of Calcium Antagonists such as Verapamil, which was found to reduce the positive inotropic effect of cardiac glycosides (15,16,19). Notwithstanding the above salient observations reported in the literature, the effect of cardiac glycosides on calcium channels so far has not received much attention. We, therefore attempted to study the effect of cryptosin on cardiac 1,4-dihydropyridinespecific calcium channels in both in vitro and ex vivo and examine, i f development of cryptosin-induced positive inotropic and toxic effects correlated with changes in dihydropyridine-specific calcium channels in *Corresponding author, Address: Science Applications International Corporation, 8400, Westpark Drive, McLean, VA 22102. 0024-3205/90 $3.00 + .00 Copyright (c) 1990 Pergamon Press plc

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mammalian cardiac tissue. PN 200-110 [Isradipine, (isopropyl 4-(2,1,3benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-pyridine-3,5-dicarboxylic acid methyl 1-methylethyl ester)], a potent calcium antagonist with high specific binding for 1,4-dihydropyridine specific binding sites (17,18) was used as the binding ligand in our studies.

Materials and Methods Hartley guinea pigs (400-500 gms) of either sex were obtained from Murphy Laboratories (Plainfield, Inc) housed 2-4 per cage and allowed food and water ad libitum. Dogs (12-20 kgs) of either sex were obtained from the local dog pound and maintained on standard chow and water. All animals were housed under the care and supervision of the Department of Laboratory Animal Resources of Wayne State University. Radiolabeled (+)-(methyl-3H) PN 200-110 (specific activity 82 Ci/mmol) was obtained from Amersham Corp., Arlington Heights IL. Nifedipine, Tris-HCl and other chemicals were obtained from Sigma Chemical Co., St. Louis, MO. Animal preparation and monitoring: Dogs were weighed, anesthetized with 4% sodium thiamylal 17 mg/kg, I.V., intubated and maintained on a respirator (Air Shields, Inc). A tidal volume of 15 ml/kg and respiratory rate of 15 breaths/minute was initiated. Atrial blood gases were monitored to ensure adequate ventilation. Incisional areas were shaved and prepped with Betadine solution. The following monitoring devices were inserted: I. An 18 G.I.V., CATH via the right femoral artery for continuous arterial pressure recording. 2. 7F., flow-directed, balloon-tipped, t r i p l e lumen thermo-dilution catheter via the right jugular vein into the pulmonary artery for recording arterial pressure and temperature. 3. 7F., thermodilution catheter in the l e f t ventricle via the l e f t femoral artery for pressure and dP/dt measurement 4. A five-lead electrocardiograph (EKG), placed in the modified V5 position and continuously recorded the action potential throughout the entire period. EKG, arterial and pulmonary pressures were recorded on a tektronix 414, option 21 dual pressure monitor, model 400 recorder and Cobe transducer and interface module (Cobe Laboratories, Inc.). Left ventricular pressure was recorded and integrated (dP/dtmax) with a Hewlett-Packard F7754A monitor with an externally calibrated, modified 8805C-8811A bio-electric-pressure amplifier system and HP1280 transducer. All lines were equipped with a continuous flush apparatus containing 5 units/ml of heparin solution. A 16G, I.V., CATH was placed in the l e f t femoral vein and balanced electrolyte solution (pH 7.4) infused at 10 ml/kg/hr. Halothane was administered in a 100% oxygen via a Foregger anesthesia machine with calibrated vaporizers. Inspiratory and expiratory anesthetic gas concentrations were verified with an Engstron Multigas Monitor for anesthesia. Cryptosin was dissolved in sterile d i s t i l l e d water U.S.P. and intravenously infused at a rate of 10 ml/kg/hr. The doses of cryptosin studied include 0.01 mg/kg/min, 0.1 mg/kg/min and I mg/kg/min. In order to determine the toxicity in vivo intravenous infusion rate was adjusted in the anesthetized dogs. Cardiac action was monitored by EKG. The end point in the t i t r a t i o n was taken as the time at which action potential cease for over ten seconds. Toxicity was expressed as dose in micrograms per kilogram body weight (12).

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Preparation of Tissue Homogenates: For each experiment the ventricles were removed from two guinea pigs, weighed on a Mettler H34 balance and placed in 30 ml of ice cold 50 mMTris buffer (pH 7.4). In the case of dog, portion of the l e f t ventricles from the apical portion was used. The ventricles were chopped into pieces and homogenized in a polytron homogenizer at a speed setting of 6-7 for 2-3 minutes. The homogenate was centrifuged at 500 g for 10 min. The supernatant of the 500 g spin was again centrifuged at 10,000 g for 15 min. The supernatant was centrifuged at 100,000 g for 30 minutes. The 100,000 g pellet was washed with 50 mM t r i s buffer (pH 7.4) and was taken in the same buffer. The membrane suspension was again centrifuged at 100,000 g for 60 minutes. The final pellet was washed once with 50 mM t r i s buffer (pH 7.4) re-suspended in 50 mM tris-HCl (pH 7.4) to a final protein concentration of 0.4-0.6 mg/ml. All the operations were performed at 4. C. The final preparation was a membrane suspension in 50 mM tris-HCl buffer (pH 7.4) with a final protein concentration of 0.4-0.7 mg/ml. The concentrations of proteins in the membrane preparations were estimated by Bradford's protein-dye binding method (20). Binding Assays: (+)-(methyl-3H) PN 200-110, (Sp.Activity 82 Ci/mmol) was used in the concentration of 0.01 to I nM. The assay was performed in polyethylene vials. A typical binding assay consisted of 3{HI PN 200-110 (0.01 to 5 nM), 50 mM tris-HCl buffer (pH 7.4) containing 20 mM Na÷, 5 mM Mg~÷ and 5 mM ATP and 0.4-0.7 mg/ml of sarcolemmal membrane protein. The assay mixture was incubated with 10 ~M nifedipine to measure the non-specific binding. The value corresponding to each concentration of nifedipine is an average of three runs. Since nifedipine is photo degradable, the vials were enclosed in a dark light-proof box and the contents of the vials were incubated for 60 minutes at room temperature (25- C). At the end of the incubation time, the contents of the vials were quickly f i l t e r e d on a Whatman GF/A membrane f i l t e r s . The f i l t e r s were washed with 6 ml of ice cold tris-HCl buffer and the dried f i l t e r s were counted in a liquid s c i n t i l l a t i o n counter. For in vitro assays sarcolemmal membranes were pre-incubated with cryptosin dissolved in 50 mM tris-HCl buffer (pH 7.4) in the varying concentrations (from I to 100 ~M) for I hour at 4. C. In e_xxvivo experiments with dog heart, cryptosin was administered intermittently at a rate of 10 ml/kg/hr at doses of 0.01, 0.1 and I mg/kg/min. Displacement assays were performed with guinea pig and dog heart membranes. In a final volume of I ml, the heart membrane suspensions, with a final protein concentration of 0.4-0.7 mg/ml, were incubated with a fixed concentration of 3{HI PN 200-110 (1 nM) and t i t r a t e d for competitive binding and displacement of the radioligand with increasing concentrations of cryptosin (from I x 10-~2 to I x 10.4 M). Displacement in binding was expressed as PN 200110 (percent bound) as a function of cryptosin concentrations. Results In order to identify a plausible relationship between cryptosin-induced positive inotropy and Ca~ channel densities, three doses including, a toxic (I mg/kg/min), arrhythmogenic (0.1 mg/kg/min) and positive inotropic (0.01 mg/kg/min) were infused to dog ex vivo and the experiment was terminated at various time-course and the heart was removed and PN 200-110-specific Ca÷~ channel was measured. Cryptosin-induced cardio-toxic effects at toxic (I mg/kg/min) and arrhythmogenic (O.I mg/kg/min) doses was characterized by the development of arrhythmia and cardiac arrest in less than 15 minutes following

1670

Cryptosin Affects Cardiac DHP-binding

250

Sites

Vol. 47, No. 18, 1990

1

~ 2O0 z

g t ~ 150 IE

~...,~..f

~ d P/dT mox

I<::)0 I

I

I

30

I

i

I

60

90

TIME (min)

Fig. 1. Cryptosln-induced changes in dP/dt,,, and Left Ventdcular Pressure (LVP) in a time-course in dog heart ex vivo.

400

o

300 ==

i: I00 20@ 13.1 PNZOO -II0BOUN0[ ~~

I

300 / ~ p,o~e,,)

400

Fig. 2. Scatchard analysis of 3[H]PN 200-110 binding to dog ventricle sarcolemmal membranes following exposure to variable dose= of cryptosln.

5OO _ _

--

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• CONTROL O CRYPT0:~N(I M) o CRvpTOS,.(l~p.I

• SLOPE:. 1Z'~ ":~

O"

o 3oo

~

s :

~-07Z

O

I0C

100

200

300

4CO

I 3H ] PN 200- It0 BOUNO( f moles/n~t0rote~n) Fig, 3. Specific binding of =[H]PN 200-110 to guinea pig heart #mrcolemmal membranes following In vitro pre-lncubatlon with cryptosln.

I

Vol. 47, No. 18, 1990

Cryptosin Affects Cardiac DHP-bindlng Sites

"•

400

--

•

• CONTROL CRYPTOSIN(IOOIJM)

~

0

3OO

~ lOO

,

I

100

\

,

I

,

200

I

300

,

"%1

400

[ 3H] PN ~0-110 BOLINO(|t1~les/m9 prote=n)

Fig. 4. Scatchard analysis of 3[H]PN 200-110 binding to dog heart sarcolemmal membranes following in vitro pre-lncubatlon with cryptosin.

o NIFEDEPINE (5x10"4 M IC50] o VERAPAMIL (lx10-8 M [C50} • CRYPTOSIN

O

~

100~

Z n

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ft.

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,

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Competition curves of cryptosin, Nifedipine and VerapamJ binding to guinea pig heart sarcolemmal membranes in the presence of 0.3 nM J[H]PN 200-110.

1671

1672

Cryptos±n Affects Cardiac DHP-blndlng Sites

Vol. 47, No. 18, 1990

infusion. However, at inotropic dose (0.01 mg/ kg/min with a total infusion time of 30 minutes followed by intermittent re-perfusion with saline, cryptosin was found to increase both Left Ventricular Pressure (LVP) and dP/dtmax (Fig.l). LVP and dP/dtmax stabilized by 35 minutes at 100% above control levels and the steady state was maintained for up to 65 minutes (at 350 ~g/kg dose of cryptosin). 3[H] PN 200-110 binding to dog ventricles pre-treated with cryptosin e__xx vivo demonstrated an interesting effect. At the toxic dose (I mg/kg/min) cardiac arrest was observed in less than 15 minutes. The calcium channel densities was reduced by about 80% and was 45.11 (± 18) f moles/mg, protein (Tab. I). At the arrhythmogenic dose (0.1 mg/kg/min) a reduction of up to 70% in the PN 200-110 specific calcium channel density [B~o~ 99.67 (, 40) f moles/ mg. protein] was observed. No significant alteration in the binding site densities was observed at 0.01 mg/kg/min (corres-ponding to inotropic dose); the Bmx , was 318.09 (, 110) f moles/mg, protein. The results of the scatchard analysis is illustrated in Fig.2. It is interesting to note that although the Bm°x was significantly altered at both toxic and arrhythmogenic doses, apparently no significant changes in the KD was observed in all the groups. Binding constants obtained from Hill plot analysis and scatchard analysis were internally consistent. TABLE I Scatchard and Hill plot of 3[H]PN200-110 Binding to the Sarcolemmal Membranes of Dog Ventricles Treated with Cryptosin ex vivo

Ko

Control Cryptosin

(1 mg/kg/

0.988

(,0.08) 0.186

Bmox

393.86

(,53.76)

Hill

coeffcient 1.107

(,0.02)

(,18.22)

45.11"

0.963

0.442 (,0.06)

99.67* (,40.18)

0.994

318.09

1.342

K'D

nl

0.94

40

0.47

16

0.49 (,0.03)

16

1.22

16

(,0.04)

(,0.01)

min) Cryptosin (0.1 mg/ kg/min) Cryptosin

(0.01 mg/ kg/min)

1.064

(,0.11)

(,110.26)

(,0.04)

* S t a t i s t i c a l l y significant decrease from controls. nl = number of data points used in linear least square regression analysis. 3[H] PN 200-110 (0.01 to I nM) bindng and saturation isotherms to sarcolemmal calcium channels is dose-dependent. Incubation of guinea pig sarcolemmal membranes with 100 ~M cryptosin showed significant differences in the receptor density specific to PN 200-110. There was almost a 45% reduction

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Cryptosin Affects Cardiac DHP-binding Sites

L673

in the receptor population (Fig.3 and Tab. II) following pre-incubation with 100 ~M Cryptosin [Bm, X ]96.97 (,36) f moles/mg, protein]. Similarly, a nonsignificant reduction in the a f f i n i t y of the ligand to its binding site (KD) was observed. Correspondingly the slopes of the Hill plots also changed from 1.054 (control) to 0.679 (100 ~M cryptosin pre-treated membranes). Binding constants determined by Hill and scatchard plots are mutually consistent (Tab. I I ) . In the presence of lower concentration of cryptosin (I ~M), no significant changes in the calcium channel densities were observed. The B,.. was 365.79 (,85) f moles/mg, protein. Similarly the Kodid not change significantly in comparison to controls. Pre-incubation of dog heart sarcolemmal membranes with 100 ~M cryptosin reduced the calcium channel density of the sarcolemmal membranes by almost 66% (Tab. I l l ) . Bm°X was 133.85 (,53) f moles/mg, protein for 100 ~M cryptosin pre treated membranes [controls had a Bm., of 393.87 (, 110) f moles/mg, protein]. However, KD did not change significantly, which was 0.683 (,0.43) and 0.988 (±0.067) nM respectively (Fig 4.). However, a nonsignificant change in h i l l co-efficient was observed between control (0.973) and 100 ~M cryptosin pretreated membranes (0.656). TABLE II Scatchard and Hill Plot Analysis of 3[H]-PN200-110 Binding to Guinea Pig Heart Sarcolemmal Membranes in the Presence of Cryptosin in vitro ...................

KD

Control

1.099 (,0.24)

Bm, ,

Hill coefficient (slope)

371.89 (,74.48)

1.054

Cryptosin (I ~M)

0.784 365.79 ( ~ 0 . 1 4 ) (,85.92)

0.679

Cryptosin (100 ~M)

1.388 (,0.45)

1.056

196.97" (,39.42)

mean , sem . . . . . . . . . . . . . .

K'D

1.013 (,0.11)

nl

n2

48

8

0.835 (,0.04)

16

4

3.847 (,0.33)

16

4

* S t a t i s t i c a l l y significant decrease from controls (P
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Cryptosin Affects Cardiac DHP-binding Sites

Vol. 47, No. 18, 1990

Discussion Our studies on the effect of cryptosin on calcium channels demonstrated that pre-incubation of the guinea pig heart sarcolemmal membranes with cryptosin significantly reduce the Ca++ channel density specific to PN 200110. There was a 45% reduction in the Bma, in guinea pig membranes in vitro. Dog heart sarcolemmal membranes demonstrated greater sensitivity to cryptosin pre-incubation. There was a 66% reduction in the calcium channel density corresponding to PN 200-110 binding sites. The results on the effect of cryptosin on calcium channel densities obtained by Ex vivo studies on dog corroborated with the results from in vitro studies from guinea pig and dog heart membrane preparations. In general, there was l i t t l e evidence of any significant changes in the a f f i n i t y of PN 200-110 to its binding site following

TABLE I l l Scatchard and Hill plot Aalysis of 3[H]PN200-110 Binding to Dog Heart Sarcolemmal Membranes in the Presence of Cryptosin in vitro . . . . . . . . . . . . mean , sem . . . . . . . . . . . . . . . . . . . . . . . . . . KD B.ax Hill K' o n, coefficient Control

Cryptosin (100 ~M)

(±0.067)

0.988

(±110.68)

393.87

0.973

1.08

40

0.683 (±0.432)

133.85" (~53.11)

0.656

1.41 (±0.91)

16

(,0.05)

* S t a t i s t i c a l l y significant decrease from controls. nl = number of data points used in linear square regression analysis. a pre-exposure to cryptosin. However, toxic concentrations of cryptosin was found to reduce the a f f i n i t y constants, albeit, insignificantly in both _ i_n_n vitro and ex vivo experiments, indicating probable involvement of Ca÷" channels in cryptosin-induced cardio-toxic effects. Although the possible involvement of Ca÷÷ channels in cryptosin-induced cardio-toxicity needs further confirmation, we presently have evidence from dog and guinea pig studies with different experimental strategies (ex vivo and in vitro) that exposure to higher concentrations of cryptosin reduces calcium channel densities. I t is plausible that cryptosin-induced reduction in the calcium channel densities at 100 ~M may involve a non-specific interaction with PN 200-110 binding sites. This is supported by our observation that at inotropic dose (0.01 mg/kg/min) ex vivo and a corresponding low dose (1 ~M) i n vitro, cryptosin did not significantly change the calcium channel densities. Whereas, at arrhythmogenic and toxic doses a 70 to 80% reduction in channel densities was observed. The non-specificity of interaction with PN 200-110 site

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Cryptosin Affects Cardiac DHP-binding Sites

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was further evident from competitive binding where cryptosin did not demonstrate a significant competition for PN 200-110 binding sites even at higher concentrations. However, the apparent discrepancy of toxic dose cryptosin-induced changes in KD ex vivo and its low a f f i n i t y to PN 200-110 binding sites in competitive binding experiments r e f l e c t the i n t r i n s i c differences in the experimental design. While the former may directly involve a hitherto unknown cryptosin-induced toxicological effect(s) or indirectly due to some as yet unknown compensatory mechanism; the l a t t e r is purely a physicochemical phenomenon of binding and displacement. Cryptosin was shown to inhibit the cardiac Na,K-ATPase (7). Both dog and guinea pig enzymes were found to be responsive to cryptosin-induced inhibition of the enzyme. Binding studies with guinea pig heart membranes revealed that cryptosin has high a f f i n i t y to its binding site on the Na,K-ATPase only in the presence of Nat , Mg++and ATP (7)° Further, studies by means of circular dichroism spectroscopy, i t was shown that p a r t i a l l y purified guinea pig Na,KATPase was more susceptible to cryptosin-induced secondary structural change than the purified enzyme (8). There is a general agreement that Ca'* ions largely contribute to the slow inward current, which flows during the plateau phase of the cardiac action potential (21,22)o Presently there are many observations which links the positive inotropic effect of cardiac glycosides with their effects on ion exchangeability (10,11). Although, studies indicate that the positive inotropic effect of the cardiac glycosides can be dissociated from the effect on Na+ and K÷ movement (23,24), the possibility of an involvement of Ca** ion remains strong. In order to explain the non-specific effect of cryptosin on calcium channels i t is necessary to invoke the concept that cryptosin-induced positive inotropic effect and the changes in calcium channel densities at arrhythmogenic and toxic concentrations of cryptosin may involve the alteration of Ca** binding and Ca+" release characteristics of cardiac plasmalemma. Evidence exist on the involvement of secondary mechanisms in cardiac glycoside-induced toxic effects (25-27). Earlier studies have indicated the alteration of Ca'" binding and release characteristics of cardiac plasmalemma as a plausible mechanism of ouabain and digoxin-induced toxic effects (25-27). A reduction in PN 200-110 binding sites at toxic concentrations of cryptosin may essentially reflect reduction in the recycling efficiencies of Na,K-ATPase. The resulting ionic imbalances may probably enhance Na'/Ca" or less favored K'/Ca"÷ exchange. Enhanced Ca+" levels compensatorily reduce available channel densities. To our knowledge this is the for the f i r s t time an attempt has been made to measure Ca÷*-channel densities in cardiac tissues both in vitro and ex vivo following an exposure to cardiac glycoside° Also we have demonstrated for the f i r s t time a relationship between the calcium channel densities and a cardiac glycosideinduced positive inotropic, and toxic effects° References I. 2o 3.

R. VENKATESWARA, 'Isolation, Characterization and Mode of Action of a new Cardiac Glycoside, Cryptosin'o Ph.D Thesis, Indian Institute of Science, Bangalore, India (1987)o R. VENKATESWARARAO. K. SANKARARAO and C.S. VAIDYANATHAN, ICSU Short Reports, 6:310-311 (1986). Ro VENKATESWARARAO, K. SANKARARAO and C.S. VAIDYANATHAN, Plant Cell Reports, 6:291-293 (1987)o

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R. VENKATESWARARAO, N.NARENDRA, M.A. VISWAMITHRA and C.S. VAIDYANATHAN, Phytochemistry, 28:1203-1205 (1989). N. NARENDRA, R. VENKATESWARA, M.A. VISWAMITHRA, K. SANKARARAO and C.S. VAIDYANATHAN, Acta Crystallographica, C43:1562-1564 (1986). R. VENKATESWARAPJ~O, J.W. BANNING, and C.S. VAIDYANATHAN ICSU Short Reports, 6:312-313 (1986). R. VENKATESWARARAO, DIPAK DASGUPTA., and C.S. VAIDYANATHAN Biochemical Pharmacology, 38:2039-2041 (1989). R. VENKATESWARARAO, DIPAK DASGUPTA, J.W. BANNINGand C.S. VAIDYANATHAN, Biochemical International (In Press), (1990). H. HEINEM and E. NOACK, Arch. Expt. Pathol. Pharmacol., 275:359-371 (1972). W.G. NAYLER, Br. Heart J., 35:561-566 (1973). H. REUTER, Circ. Res., 34:599-605 (1974). W.G.NAYLER, Adv. Drug Res., ]__2:39-51 (1977). P. COHEN, A. BARACHELL, J.G. FOULKEO, T.W. COHEN, T.C. VANAMANand A.C. NAIM, FEBS Lett., 9__22:287-293 (1978). M.P. WALSH, C.J. LePEUCH, B. VALLET, J.C. CAVADOREand J.C. DEMAILLE, J. Mol. Cell. Cardiol., I__22:1091-1101 (1980). S. SHIBATA, T. IZUMI, D.G. SERIGUCHI and T.R. NORTON,J. Pharmacol. Expt. Therp., 205:683-692 (1978). B.N. SINGH and E.M. VAYGHAN-WILLIAMS, Cardiovasc. Res., 6:109-119 (1972). H.R. LEE, W.R. ROESKE and H.I. YAMAMURA, Life Sciences, 35: 721-732, (1984). J. PTASIENKI, K.K. McMAHONand M.M. HOSEY, Biochem. Biophys. Res. Commn., 129: 910-917, (1985). J.S. KARLINER, P. BARNES, M. BROWNand C. DOLLERY, Eur.J. Pharmacol., 6_!:115-118 (1980). M.M. BRADFORD, Anal.Biochem., 7_22:248-254 (1976). H. REUTER, Progress in Biophysics & Molecular Biology; J.A.V. Butler and D. Noble (eds) Vo126, 1-43, Pergamon Press, Oxford (1973). H. REUTERand H. SCHOLZ, J. Physiol., 264:17-47 (1977). H.R. BESCH and A.M. WATANABE, J. Pharmacol. Expt. Therp., 207:958-965 (1978). P.A. POOLE-WILSON and G.A. LANGER, Cir. Res., 34:599-601 (1975). W. NAYLERand E. NOACK, Cardiac Glycosides, K. Greeff, (ed.) Part I, 407 -436, Springer Verlag, Berlin, (1982). F.R.A. ZWAAL, B. ROELOFSENand C.M. COLLEY, Biochem.Biophys. Acta., 300:159 -182 (1973).