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Temperature-dependent development of ouabain action in isolated guinea pig heart: Differences in sodium influx?
European Journal of Pharmacology, 104 (1984) 223-233
223
Elsevier
T E M P E R A T U R E - D E P E N D E N T D E V E L O P M E N T OF OUABAIN A C T I O N IN I S O L A T E D GUINEA P I G HEART: D I F F E R E N C E S IN S O D I U M INFLUX? KYOSUKE TEMMA *
Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 48824, U.S.A. Received 8 March 1984, accepted 13 June 1984
K. T E M M A , Temperature-dependent development of ouabain action in isolated guinea pig heart: differences in sodium influx?, European J. Pharmacol. 104 (1984) 223-233. A t 30 o C the development of the positive inotropic action of 0.3/-tM ouabain observed in isolated left atrial muscle preparations of guinea-pig heart was significantly slower and more d e p e n d e n t on the frequency of electrical stimulation than it was at 37 o C. The hypothesis was tested that such differences are due to a variation in the rate of leak sodium influx, and ensuing differences in glycoside binding to sarcolemmal Na,K-ATPase. M o n e n s i n or grayanotoxin, agents which have been shown to increase sodium influx, caused faster development of the inotropic action of ouabain at 30 o C when muscle preparations were stimulated at 0.5 or 1 Hz but their effect was smaller at 2 or 3 Hz stimulation. The contracture caused by toxic concentrations of ouabain in quiescent preparations developed faster at the higher temperature. Ouabain binding to sarcolemmal N a , K - A T P a s e which occurred during exposure of atrial muscle preparations to the glycoside at 25 o C for 30 min was less than the corresponding value observed after a similar exposure at 37 o C; however, electrical stimulation increased glycoside binding to a greater extent at the lower temperature. The ouabain-sensitive 42K+ uptake was lower in quiescent preparations at 3 0 ° C than at 3 7 ° C . Moreover, the e n h a n c e m e n t of the ouabain-sensitive 42 K + uptake caused by electrical stimulation was greater at 30 ° C than at 37 o C. These results are consistent with the hypothesis that, at lower temperatures, the rate of sodium influx is low and is the factor determining the rate of development of the inotropic action of ouabain. At a higher temperature, a relatively high sodium leak influx seems to make the onset of ouabain action less d e p e n d e n t on the frequency of stimulation.
Cardiac glycosides Na,K-ATPase
Ouabain
Beat and temperature dependence of inotropic effect
1. Introduction
The positive inotropic and toxic effects of the cardiac glycosides develop slowly in isolated heart muscle preparations incubated at relatively low temperatures. The rate of development of these effects of the glycosides is substantially increased when the preparations are stimulated at a higher frequency (Moran, 1967; Vincenzi, 1967; Park and Vincenzi, 1975: Bentfeld et al., 1977; Ebner and Reiter, 1977; T e m m a et al., 1982). In contrast, the onset rate of the inotropic and toxic effects of the * Current address: Department of Pharmacology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada-shi, Aomori 034, Japan. 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
Na influx
cardiac glycosides observed at a higher temperature e.g. 37 o C is faster and relatively insensitive to the frequency of electrical stimulation between 0.1 and 1 Hz (Koch-Weser, 1971; Park and Vincenzi, 1973). One possible explanation for the beat-dependent onset of the glycoside action observed at relatively low temperatures is that the binding of the glycoside to sarcolemmal Na,K-ATPase, the putative receptor for the positive inotropic and toxic actions of the glycoside, is regulated by the amount of intracellular sodium ion available to the sodium p u m p (Akera et al., 1977; Li~llmann and Peters, 1979). This concept is supported by the fact that agents and conditions which enhance sodium influx have been shown to increase the
224 onset rate of the positive inotropic action of cardiac glycosides, especially ouabain (Honerj~ger and Reiter, 1975; Akera et al., 1977), concomitant with an enhanced binding of the glycoside to sarcolemmal Na,K-ATPase (Temma and Akera, 1982). Sodium ions enhance glycoside binding to isolated Na,K-ATPase observed in vitro in the presence of Mg 2+ and ATP (Matsui and Schwartz, 1968). Glycoside binding in intact cells is also enhanced by conditions which stimulate turnover of the enzyme (Clausen and Hansen, 1977), except for K + which inhibits glycoside binding. Because intracellular Na + stimulates enzymes activity, it seems likely that it also enhances glycoside binding. Yamamoto et al. (1979) have shown that the sensitivity of the sarcolemmal sodium pump to the inhibitory action of ouabain was elevated by an enhancement of sodium influx. If intracellular sodium available to the sodium pump is the regulator of glycoside binding to sarcolemmal Na,K-ATPase at relatively low temperatures, why is the development of the positive inotropic effect of glycosides less sensitive to the frequency of stimulation at a higher temperature? The study by Yamamoto et al. (1981) suggests that the leak Na + influx of cardiac muscle preparations is substantially higher at 36.5°C than at 30°C. Therefore, we tested the hypothesis that such differences are due to a variation in the rate of sodium influx, and ensuing differences in glycoside binding to sarcolemmal Na, K-ATPase. The glycoside binding to sarcolemmal Na, K-ATPase which occurred during the inotropic study was estimated by homogenizing the tissue and from the reduction of the initial velocity of the [3H]ouabain binding reaction. The rate of sodium influx was estimated indirectly from the ouabain-sensitive 42K+ uptake in preparations which were not sodium-loaded and were presumably in equilibrium with regard to ion movements.
2. Materials and methods
2.1. Force of contraction Guinea pigs of either sex weighing approximately 350 g were stunned by a sharp blow to the
head; their hearts were rapidly excised and perfused via the aorta for 5-10 min at 30°C with freshly oxygenated (95% O 2 and 5% CO2) KrebsHenseleit bicarbonate buffer solution of the following composition: 118 mM NaCI, 27.2 mM N a H C O 3, 4.8 mM KCI, 1.0 mM KHzPO4, 1.2 mM MgSO4, 1.2 mM CaC12 and 11.1 mM glucose (pH 7.4). After visible blood had been removed from the tissue, the left atrial muscle was excised and suspended vertically in a chamber containing 30 ml of the above solution at either 30 or 37°C. The atrial preparations were stimulated at 0.5, 1, 2 or 3 Hz with square wave pulses of 4 ms duration at a voltage approximately 20% above the threshold. Platinum electrodes were used for electrical field stimulation. Resting tension was adjusted to 1.0 g, which produced approximately 90% of the maximal tension developed (Yamamoto et al., 1981). The tension developed was recorded isometrically using a force-displacement transducer and a polygraph recorder (Grass Instrument Company, Quincy, MA; model FT-03C and 7B, respectively). In several experiments, grayanotoxin I or monensin was added to the incubation medium 70 or 30 min, respectively, before the addition of ouabain. In the contracture study, atrial muscle preparations were equilibrated for 60 min under 1.5 Hz stimulation at the temperature indicated and the stimulator was turned off. Subsequently, ouabain was added to the incubation medium and changes in the resting tension were monitored.
2.2. 42K + uptake Left atrial muscle preparations obtained from guinea pig hearts were equilibrated at 30 or 37 °C for 30 rain under 3 Hz electrical stimulation in a chamber containing 30 ml of the Krebs-Henseleit bicarbonate buffer solution described above, except that the K + concentration was reduced to 4 instead of 5.8 mM. Subsequently, tracer amounts of 42K+ were added to the incubation medium. After a 30 min incubation under quiescent conditions or 3 Hz stimulation, the atrial muscle was removed from the incubation bath, rinsed with a tracer-free solution for 15 s, blotted on a filter paper and weighed, the radioactivity in left atrial muscle was estimated using a gamma scintillation
225 spectrometer. The specific (ouabain-sensitive) 42K + uptake is the difference between values observed in the absence and presence of 0.3 mM ouabain (Yamamoto et al., 1979). The addition of 0.3 mM ouabain (final concentration) caused a marked elevation of the resting tension after a transient positive inotropic effect followed by failure of the muscle preparation to follow electrical stimulation within 10 min. Therefore, nonspecific 42 K + uptake was estimated by adding the tracer 10 min after the addition of ouabain and monitoring the 42K+ uptake. With a reduced K + concentration and electrical stimulation at 3 Hz, the ouabain-sensitive 42K+ uptake was significantly elevated over that observed in quiescent preparations, although previous investigators reported that total K + uptake, i.e. ouabain-sensitive plus ouabain-insensitive uptake, was relatively unaffected by electrical stimulation up to 1 Hz (see Goerke and Page, 1965). Assuming that a relationship exists between ouabain-sensitive K + uptake and Na + extrusion by the sodium pump, the rate of N a + pumping can be estimated from the ouabain-sensitive 42K + uptake. In equilibrated, steady state preparations, the rate of Na + influx must be equal to the rate of N a + influx.
2.3. [-~H]Ouabain binding The fractional occupancy of the glycoside binding sites on Na,K-ATPase by ouabain in left atrial muscle preparations was estimated from the reduction in the specific (ATP-dependent) [3H]ouabain binding observed with homogenates from preparations incubated in the presence of unlabelled ouabain (Ku et al., 1974). Left atrial muscle preparations of guinea-pig heart were equilibrated for 60 min at 25 or 3 7 ° C in Krebs-Henseleit bicarbonate buffer solution under 2 Hz stimulation. Subsequently, 0.5 ktM ouabain (final concentration) was added to the medium and preparations were incubated for 30 rain under 0 or 2 Hz stimulation. The muscles were minced with scissors then homogenized in 2.5 ml of an ice-cold solution containing 1 m M E D T A and 10 m M Tris-HC1 buffer (pH 7.5) using a motor-driven Teflon pestle homogenizer with 25 ~ m clearance (3000 r.p.m. for 12 s). A 0.1 ml aliquot of the homogenate
containing approximately 0.1 mg protein was added to 0.9 ml of a prewarmed incubation mixture and incubated at 3 7 ° C for 3 min with 50 nM [3H]ouabain in the presence of 100 m M NaCI, 5 m M MgCI 2, 50 m M Tris-ATP and 50 m M TrisHC1 buffer (pH 7.4). Bound and free [3H]ouabain were separated by filtering the aliquot through a nitrocellulose filter (Millipore Corporation, Bedford, MA; type AA, pore size, 0.8/~m). The filter was washed twice with 6 ml each of an ice-cold solution containing 15 m M KC1 and 50 m M Tris HC1 buffer solution and dissolved in ethylene glycol monomethyl ether (Pierce Chemical Company, Rockford, IL: Piersolve). The radioactivity was estimated with a liquid scintillation spectrometer using a toluene-based counting solution (Temma and Akera, 1982). The specific [3H]ouabain binding is the difference between values observed in the presence and absence of A T P (Ku et al., 1974).
2.4. Miscellaneous 42K+ was prepared from a K O H solution by the Nuclear Reactor Laboratory of Michigan State University. The irradiated solution was neutralized with HC1 before use. Generally labelled [3H]ouabain (specific activity, 18 C i / m m o l ) was purchased from New England Nuclear, Boston, MA. Monensin sodium was kindly supplied by Lilly Laboratories, Eli Lilly and Company, Indianapolis, IN, and grayanotoxin I by Dr. Junkichi Iwasa, Okayama University, Okayama, Japan. Ouabain octahydrate was obtained from Sigma Chemical Company, St. Louis, MO. Other chemicals were of reagent grade. Statistical analyses were performed with Student's t-test. The criterion for statistical significance was a P value of less than 0.05. Protein concentrations were estimated by the method of Lowry et al. (1951) using bovine serum albumin as the standard.
3. Results
3.1. lnotropic effects of ouabain The degree of beat dependence of the development of the positive inotropic action of ouabain
226
seems to be greatly influenced by the experimental conditions. The dependence of the onset rate of the inotropic effect of ouabain on the frequency of electrical stimulation was therefore examined first to demonstrate beat dependence under the conditions used. Moreover, the effect of the temperature on beat dependence was examined to confirm earlier work. Isolated atrial muscle preparations of guinea-pig heart were equilibrated for 60 min at either 30 or 37°C, under 0.5, 1, 2 or 3 Hz stimulation. At the end of the equilibration period, a greater tension developed at 30 o C than at 37 o C, especially when the preparations were stimulated at a higher frequency (fig. 1). Subsequently, ouabain (final concentration, 0.3 /~M) was added to the medium and caused a gradual increase in developed tension. The development of the positive inotropic effect of ouabain was significantly more rapid at 3 7 ° C than at 3 0 ° C (fig. 1 and also fig. 2, open and filled circles). At 30 o C, the rate of development was clearly dependent on the frequency of stimulation in a range between 0.5 and 2 Hz but the effect of stimulation frequency was less prominent at 37 o C (fig. 2). These results confirm earlier reports (Moran, 1967; Vincenzi, 1967; Koch-
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Fig. 2. Effects of stimulation frequency on the development of the positive inotropic effect of ouabain: Influence of monensin and grayanotoxin 1 (GTX-I). See legend to figs. 1 and 3. The time required for the development of a half-maximal inotropic effect of ouabain was plotted against the frequency of electrical stimulation. The value was calculated for each preparation then averaged. Each point represents the mean of 4 to 6 experiments. Vertical lines indicate S.E.
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Fig. 1. Effects of stimulation frequency on the development of positive inotropic effects of ouabain at 30 or 37 °C. Left atrial muscle preparations of guinea pig heart were electrically stimulated at 0.5 (C)), 1 (O), 2 (A) or 3 Hz (*). After a 60 min equilibration, ouabain (final concentration, 0.3 p,M) was added to the incubation medium at time zero. Each point represents the mean of 4 to 6 experiments.
Weser, 1971; Park and Vincenzi, 1973, 1975; Bentfeld et al., 1977; Ebner and Reiter, 1977; Temma et al., 1982) and indicate that the onset rate of ouabain action is relatively beat-dependent at 3 0 ° C and that the beat dependence is less apparent at 37 °C. The hypothesis that the rate of development ~f the positive inotropic action of ouabain is beat-dependent at 30 o C because the leak sodium influx is relatively low at this temperatures was tested by increasing the rate of the beat-independent sodium influx by means of monensin or grayanotoxin I, agents which have been shown to increase sodium influx in cardiac muscle (Narahashi and Seyama, 1974; Meier et al., 1976). As reported previously (Akera et al., 1977), addition of either monensin or grayanotoxin to the incubation medium caused an increase in the developed tension of isolated atrial
227 m u s c l e p r e p a r a t i o n s ( d a t a n o t shown). We have shown previously that grayanotoxin I c a u s e d a f a s t e r d e v e l o p m e n t of the c o n t r a c t u r e i n d u c e d b y t o x i c c o n c e n t r a t i o n s of o u a b a i n in q u i e s c e n t p r e p a r a t i o n s o n l y w h e n the p r e p a r a t i o n s h a d b e e n p r e v i o u s l y e x p o s e d to this t o x i n u n d e r e l e c t r i c a l s t i m u l a t i o n ( T e m m a a n d A k e r a , 1982). T h i s f i n d i n g raises the p o s s i b i l i t y t h a t the b i n d i n g o f g r a y a n o t o x i n I to its site o f a c t i o n in c a r d i a c m u s c l e m i g h t b e d e p e n d e n t o n t h e f r e q u e n c y of e l e c t r i c a l s t i m u l a t i o n . T h e r e f o r e , left atrial m u s c l e preparations were incubated w i t h 0.2 I~M g r a y a n o t o x i n I for 30 m i n u n d e r 3 H z s t i m u l a t i o n at 30 o C. S u b s e q u e n t l y , the s t i m u l a t o r was reset to the i n d i c a t e d f r e q u e n c y a n d o u a b a i n (final c o n c e n t r a t i o n , 0.3 /~M) was a d d e d 40 rain l a t e r w h e n the d e v e l o p e d t e n s i o n r e a c h e d a s t e a d y state. B o t h 1 ~tM m o n e n s i n o r 0.2 t t M g r a y a n o t o x i n I c a u s e d a m o r e r a p i d d e v e l o p m e n t of t h e p o s i t i v e i n o t r o p i c e f f e c t of o u a b a i n w h e n p r e p a r a t i o n s w e r e s t i m u -
l a t e d at 0.5 o r 1 H z (fig. 3) as r e p o r t e d e a r l i e r ( A k e r a et al., 1977). T h e effect of m o n e n s i n o r g r a y a n o t o x i n I was less p r o m i n e n t w h e n the p r e p a r a t i o n s w e r e s t i m u l a t e d at h i g h e r f r e q u e n c i e s . I n t h e p r e s e n c e o f t h e s e agents, the r a t e o f d e v e l o p m e n t of the p o s i t i v e i n o t r o p i c effect of o u a b a i n ( p l o t t e d as the t i m e to h a l f - m a x i m a l effect) o b s e r v e d at 3 0 ° C , a n d also the p a t t e r n of b e a t d e p e n d e n c e of the o n s e t rate, a p p r o a c h e d t h o s e o b s e r v e d at 37 ° C in the a b s e n c e o f m o n e n s i n or g r a y a n o t o x i n I (fig. 2). T h u s , the d i f f e r e n c e in b e a t d e p e n d e n c e o f the d e v e l o p m e n t of the g l y c o s i d e a c t i o n o b s e r v e d at t w o t e m p e r a t u r e s s e e m e d to r e s u l t f r o m the l o w e r l e a k s o d i u m i n f l u x r a t e at 30 ° C t h a n at 37 o C.
3.2. Ouabain-induced contracture I n c r e a s e s in N a + i n f l u x in t h e q u i e s c e n t m u s c l e h a v e b e e n s h o w n to c a u s e a faster o n s e t of ouabain-induced contracture (Temma and Akera, 1982). T h e r e f o r e , if the l e a k s o d i u m i n f l u x is g r e a t e r at a h i g h e r t e m p e r a t u r e , the d e v e l o p m e n t of c o n t r a c t u r e c a u s e d b y toxic c o n c e n t r a t i o n s of o u a b a i n
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Fig. 3. Effects of monensin or grayanotoxin I (GTX-I) on the development of positive inotropic effects of ouabain at 30 o C. Left panel: Left atrial muscle preparations were equilibrated for 45 min under 0.5 (C)), 1 (o), 2 (zx) or 3 Hz (A) stimulation. Subsequently, monensin (final concentration, 1/LM) was added to the incubation medium. Ouabain (final concentration, 0.3 t~M) was added 30 min later at time zero. Right panel: After equilibration, left atrial muscle preparations were incubated for 30 min in the presence of 0.2 ~M GTX under 3 Hz stimulation. Subsequently, the frequency of electrical stimulation was adjusted to either 0.5 (C)), 1 (O), 2 (zx) or 3 Hz (A), and ouabain (final concentration, 0.3 /~M) was added 40 min later at time zero. Under 3 Hz stimulation, a 30 min exposure to 1 t~M monensin and exposure to 0.2 ~M GTX caused a 33.2 + 6.2 and 43.7 + 8.2% increase in the tension developed. Each point represents the mean of 4 or 5 experiments.
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Fig. 4. Effects of temperature on the ouabain-induced contracture in quiescent atrial muscle preparations. Left atrial muscle preparations were equilibrated for 60 min under 1.5 Hz stimulation at either 30 tO) or 37°C (O). Subsequently, the stimulator was turned off, and ouabain (final concentration, 5 #M) was added to the incubation medium at time zero. Each point represents the mean of 6 (30 ° C) or 4 (37 °C) experiments. Vertical lines indicate S.E. Arrows indicate the mean time to the half-maximal effect of ouabain.
228 in quiescent p r e p a r a t i o n s should be faster at a higher temperature. To test this possibility, quiescent left atrial muscle p r e p a r a t i o n s were exposed to 5 /,M o u a b a i n at either 30 or 3 7 ° C . The addition of 5 /,M o u a b a i n (final c o n c e n t r a t i o n ) to the m e d i u m caused a gradual increase in resting tension at 30 ° C (fig. 4). The d e v e l o p m e n t of contracture due to 5 ~ M o u a b a i n was significantly faster at 37 ° C. The time required for the d e v e l o p m e n t of a h a l f - m a x i m a l response to o u a b a i n was 54.0 + 5.9 m i n at 30 ° C, a n d 28.9 + 4.0 m i n at 37 ° C. These results suggest that o u a b a i n b i n d i n g to the sodium p u m p in atrial muscle p r e p a r a t i o n s is faster at a high temperature a n d that the s o d i u m influx rate in quiescent muscle p r e p a r a t i o n s is higher at 37 ° C t h a n at 30 °C.
stimulation. Subsequently, 0.5 ~ M o u a b a i n (final c o n c e n t r a t i o n ) was added to the i n c u b a t i o n m e d i u m a n d the p r e p a r a t i o n s were i n c u b a t e d for an a d d i t i o n 30 m i n u n d e r quiescent c o n d i t i o n s or 2 Hz stimulation. I n c u b a t i o n of atrial muscle p r e p a r a t i o n s in the o u a b a i n - f r e e m e d i u m at different temperatures (25 or 3 7 ° C ) did not alter the specific [3H]ouabain b i n d i n g reaction (fig. 5), indicating that comparison of fractional o c c u p a n c y data at two temperatures is feasible. It should be noted that the assay of [3H]ouabain b i n d i n g with homogenates to assess fractional o c c u p a n c y was performed at 37 oC regardless of the i n c u b a t i o n temperature of atrial muscle p r e p a r a t i o n s prior to homogenization. This
3.3. Ouabain binding to Na, K-A TPase
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The b i n d i n g of u n l a b e l l e d o u a b a i n to glycoside b i n d i n g sites on sarcolemmal N a , K - A T P a s e that occurred d u r i n g the i n c u b a t i o n of isolated atrial muscle p r e p a r a t i o n s was estimated b y homogenizing the tissue a n d assaying the velocity of the specific [3H]ouabain b i n d i n g reaction. The specific [3H]ouabain b i n d i n g reaction was almost linear d u r i n g the 3 m i n i n c u b a t i o n period (data n o t shown), i n d i c a t i n g that the observed value could be used to represent the initial velocity. A reduction in the initial velocity would then indicate previous o c c u p a n c y of the glycoside b i n d i n g site b y u n l a b e l l e d o u a b a i n . Initial experiments showed that the fractional o c c u p a n c y of glycoside b i n d i n g sites on Na, K - A T P a s e which occurred d u r i n g a 30 m i n i n c u b a t i o n of atrial muscle at 3 0 ° C in the presence of 0.3 /,M o u a b a i n was less than the fractional o c c u p a n c y at 37 °C. However, on statistical analysis the difference was f o u n d to be hardly significant, due to the relatively small fractional o c c u p a n c y caused by 0.3 ~ M o u a b a i n a n d to variations in the experimental data. I n order to test the hypothesis that the difference in the inc u b a t i o n t e m p e r a t u r e affects glycoside b i n d i n g to the sodium p u m p , left atrial muscle p r e p a r a t i o n s were i n c u b a t e d at 25 ° C instead of 30 ° C a n d the glycoside c o n c e n t r a t i o n was increased to 0.5 /,M. Left atrial muscle p r e p a r a t i o n s were equilibrated for 60 m i n at either 25 or 3 7 ° C u n d e r 2 Hz
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I I I I 0.5pM Cont ouaoain
Fig. 5. Specific [3H]ouabain binding to homogenates obtained from atrial muscle preparations exposed to unlabelled ouabain. Left atrial muscle preparations were equilibrated for 60 min under 2 Hz stimulation at either 25 or 37 °C. Subsequently, these preparations were exposed to 0.5 p.M unlabelled ouabain, either quiescent or under 2 Hz stimulation. After a 30 min exposure, the muscle preparations were homogenized and immediately added to incubation medium for estimation of the initial velocity of the specific [3Hlouabain binding reaction. Specific [3H]ouabain binding is the difference in values obtained in the absence and presence of 5 mM ATP. Control values were obtained with left atrial muscle preparations incubated for a comparable time period in the absence of unlabelled ouabain under 2 Hz stimulation, homogenized and challenged with [3H]ouabain. a) Significantly different from control values observed at the same temperature, b) Significantly different from the corresponding value observed at 25 o C. Each value represents the mean of 5 experiments. Vertical lines indicate S.E.
229 was done because the p u r p o s e of the [ 3 H ] o u a b a i n b i n d i n g assay was to estimate the fractional occ u p a n c y of the glycoside b i n d i n g sites on N a , K A T P a s e by u n l a b e l l e d o u a b a i n d u r i n g the i n c u b a tion of atrial muscle p r e p a r a t i o n s in the inotropic experiments. The specific b i n d i n g of [3H]ouabain to h o m o g e n a t e s from quiescent left atrial muscle p r e p a r a t i o n s i n c u b a t e d in the absence of unlabelled o u a b a i n was similar to that observed following electrical s t i m u l a t i o n in the absence of o u a b a i n u n d e r the present c o n d i t i o n s (data not shown). Such results were anticipated because 30 m i n i n c u b a t i o n in quiescent c o n d i t i o n s with electrical s t i m u l a t i o n is unlikely to change the n u m b e r of active N a , K - A T P a s e units which b i n d o u a b a i n after h o m o g e n i z a t i o n of the muscle. Therefore, o n l y the values observed with electrically stimulated p r e p a r a t i o n s are shown in fig. 5. Nonspecific b i n d i n g was not affected by either the t e m p e r a t u r e or electrical s t i m u l a t i o n (data n o t shown~ Exposure of quiescent p r e p a r a t i o n s to 0.5 /~M o u a b a i n for 30 rain at either 25 or 3 7 ° C reduced the [3H]ouabain b i n d i n g observed with homogenates o b t a i n e d from these preparations. The red u c t i o n in the specific [3H]ouabain b i n d i n g observed in quiescent p r e p a r a t i o n s exposed to u n labelled o u a b a i n at 37 ° C was significantly greater t h a n that observed after o u a b a i n exposure at 25 o C, i n d i c a t i n g that a greater fractional o c c u p a n c y occurred at 3 7 ° C t h a n at 2 5 ° C in quiescent preparations. Exposure to u n l a b e l l e d o u a b a i n u n d e r electrical s t i m u l a t i o n further reduced the initial velocity of the specific [3H]ouabain b i n d i n g reaction, i n d i c a t i n g that greater b i n d i n g of the unlabelled o u a b a i n occurred d u r i n g the exposure to o u a b a i n at either t e m p e r a t u r e (fig. 5), W i t h electrical stimulation, the difference in fractional occ u p a n c y observed at two t e m p e r a t u r e s was m i n i m a l . These results d e m o n s t r a t e that electrical s t i m u l a t i o n or raising the temperature e n h a n c e d the b i n d i n g of u n l a b e l l e d glycoside to N a , K ATPase, reducing the sites available to [3H]ouabain. The c o m b i n e d effect of electrical s t i m u l a t i o n a n d t e m p e r a t u r e increase however, was not significantly different from that of electrical s t i m u l a t i o n alone.
3.4. Ouabain-sensitive
42K +
uptake
The above results suggest that the leak N a + influx and also the e n h a n c e m e n t of net sodium influx by electrical s t i m u l a t i o n are temperature-dep e n d e n t . T o estimate the rate of sodium influx more directly, tracer a m o u n t s of 42K + were a d d e d to the i n c u b a t i o n m e d i u m a n d the ouabain-sensitive 42K+ uptake was assayed. I n equilibrated heart muscle p r e p a r a t i o n s which are n o t N a +-loaded the o u a b a i n - s e n s i t i v e 42K+ uptake can be used to estimate the rate of s o d i u m influx because the rate of s o d i u m influx must be equivalent to the rate of s o d i u m efflux in steady state preparations, a n d the
28 o
24
30"C r-]; OHz
T 37"C FI; 3Hz a,b I
'q" .~_ ~
12
c
0
Fig. 6. Effects of temperature and electrical stimulation on the ouabain-sensitive 42K+ uptake by isolated atrial muscle preparations. Left atrial muscle preparations were equilibrated for 30 min under 3 Hz stimulation in a Krebs-Henseleit bicarbonate buffer solution containing 4 mM K + at the temperature indicated. Tracer amounts of 42KC1 was added to the incubation medium at this time, and 42K+ uptake was determined after a 30 rain incubation, quiescent or under 3 Hz stimulation. Specific 42K+ uptake is the difference in values obtained in the absence or presence of 0.3 mM ouabain. The values for nonspecific 42K+ uptake were 5.65 _+0.37 (30 o C, 0 Hz), 5.93+0.18 (30°C, 3 Hz), 5.02-+0.39 (37°C, 0 Hz), and 4.87_+0.24 nmol/mg tissue (37°C, 3 Hz). The number in parentheses indicates the number of experiments. Vertical lines indicate S.E. a) Significantly different from the corresponding values observed at 30 o C. b) Significantlydifferent from values observed at same temperature under 0 Hz stimulation, c) Significantly different from the corresponding values observed at 30°C.
230 bulk of Na + efflux seems to be coupled to the ouabain-sensitive K + influx when the extraceilular K + concentration is relatively low (Yamamoto et al., 1979). The ouabain-sensitive 42K+ uptake observed with quiescent preparations incubated at 3 7 ° C was markedly higher than that observed at 30 ° C (fig. 6). Electrical stimulation at 3 Hz caused a significant increase in the specific 42K+ uptake. However, the degree of stimulation-induced increase in 42K+ uptake was relatively small at 37 °C (66 _+ 9% increase) compared to that observed at 3 0 ° C (156 + 22% increase). During 3 Hz stimulation, the ouabain sensitive 42K+ uptake observed at 3 7 ° C was still significantly higher than that observed at 30 ° C but the difference was relatively small. These results indicate that the leak sodium influx at 3 7 ° C was approximately twice that at 30°C, and the effect of electrical stimulation to enhance sodium influx was markedly smaller at the higher temperature.
4. Discussion
Under the conditions used in the present experiments, the rate of development of the positive inotropic effect of ouabain was slow at 30 o C and was dependent on the frequency of electrical stimulation. Elevation of the temperature to 37 o C caused a substantial increase in the rate of development of ouabain action and made the development of the glycoside's action less dependent on stimulation frequency. These results confirm those reported from previous studies: relatively strict beat dependence of the onset of glycoside action at lower temperatures and the lack of the strict beat dependence at higher temperatures (Koch-Weser and Blinks, 1962; Moran, 1967; Vincenzi, 1967; Byrne and Dresel, 1969; Koch-Weser, 1971; Park and Vincenzi, 1973, 1975; Bentfeld et al., 1977; Ebner and Reiter, 1977; Temma et al., 1982). Several hypotheses have been advanced to explain the beat or temperature dependence of the onset of glycoside actions. Park and Vincenzi (1973, 1975) have suggested that the access of the glycoside to its receptor sites requires transport by
a specific carrier system, and proposed that transport might be enhanced by membrane depolarization or at a higher temperature. Alternatively, beat dependence may result from a difference in the magnitude of the inotropic effect of the glycosides at various stimulation frequencies (Wilbrandt et al., 1953), frequency-dependent differences in action potential configuration or in calcium influx (Koch-Weser and Blinks, 1963) or from an additive effect of an increased sodium load by more frequent depolarization and glycoside-induced inhibition of the sodium pump (Reiter, 1981). Another hypothesis, however, appears more attractive: that the amount of intracellular Na t available to the sodium pump is the determinant of glycoside binding to sarcolemmal Na, K-ATPase, and that electrical stimulation or an elevation of the temperature increases the amount of Na + available to the sodium pump by increasing the sodium influx. In isolated Na,K-ATPase preparations, Na + enhances glycoside binding (Matsui and Schwartz, 1968). In intact heart muscle cells, intracellular Na + appears to be an absolute requirement for glycoside binding to sarcolemmal Na,K-ATPase and the development of the inotropic action (Akera et al., 1979; Linden and Brooker, 1980; Wiggins and Bentolila, 1980). Moreover, Na + ionophores or agents which increase sodium influx have been shown to cause a faster development of the positive inotropic action of the cardiac glycoside (Honerj~ger and Reiter, 1975; Akera et al., 1977; Yamamoto et al., 1980), with a concomitant faster binding of the glycoside to sarcolemmal Na,K-ATPase (Temma and Akera, 1982). Bodemann and Hoffman (1976), however, reported that an increase in intracellular Na ~ concentration failed to enhance ouabain binding to Na,K-ATPase in released erythrocyte ghost preparations. Thus, in order to test the above hypothesis, it was essential to examine whether beat dependence of the onset of glycoside action observed at low temperatures and the lack of strict beat dependence at higher temperatures corresponded to the depolarization-induced increases in sodium influx rates and therefore to glycoside binding to sarcolemmal Na,K-ATPase. Our previous study indicated that increasing the
231 amount of intracellular Na t available to the sodium pump, by increasing the frequency of electrical stimulation or adding monensin, grayanotoxin or batrachotoxin, alters glycoside binding to sarcolemmal Na,K-ATPase in isolated heart muscle preparations, in parallel with an enhancement of the rate of development of the positive inotropic action of ouabain (Temma and Akera, 1982). It should be noted that an enhancement of glycoside binding occurs in the absence of an apparent or marked increase in total intracellular Na +, indicating that an increase in turnover rate of the sodium pump is the cause of the enhanced glycoside binding. Conversely, a decrease in extracellular Na + concentration to 27 raM, which causes a marked decrease in the amount of Na + available to the sodium pump, abolished glycoside binding to sarcolemmal Na, K-ATPase and also the development of the positive inotropic effect of ouabain (Temma and Akera, 1983). In the present study, raising the temperature resulted in a greater fractional occupancy of the glycoside binding sites on Na, K-ATPase in atrial muscle preparations exposed to a fixed concentration of ouabain under quiescent conditions. Raising the temperature from 30 to 37 o c also enhanced the development of the contracture caused by toxic concentrations of ouabain, again indicating that the glycoside binds more rapidly to Na,K-ATPase at a higher temperature. Electrical stimulation enhanced glycoside binding irrespective of the temperature. However, the combined effect of electrical stimulation and an elevated temperature was not significantly different from what was observed with electrical stimulation alone. Consistent with this latter finding was the observation that the difference in the time required for the development of a half-maximal inotropic effect of ouabain was relatively unaffected by temperature when the preparations were stimulated at either 2 or 3 Hz. Thus, the effects of incubation temperature and electrical stimulation on glycoside binding to Na,K-ATPase in isolated atrial muscle preparations can explain the beat dependence of the onset of glycoside action at the lower temperature, and the relative lack of the beat dependence at a higher temperature. Glycoside binding to Na,K-ATPase may be en-
hanced by an elevation of temperature per se, independent of changes in sodium influx rates. For example, an elevation of temperature has been shown to increase the association and dissociation rate constants for the interaction between ouabain and Na, K-ATPase observed with isolated enzyme preparations (Erdmann, 1981). This and other possibilities cannot be ruled out. However, the present results indicate that the enhanced glycoside binding to Na,K-ATPase observed with quiescent preparations at a higher temperature and the faster development of the inotropic effect are accompanied by a greater sodium influx. In the present study, sodium influx was estimated indirectly from the ouabain-sensitive 42K+ uptake. Because of the significant back flux of Na +, i.e. substantial portion of the Na + entering the cell during a membrane excitation is pumped out before the next cycle, sodium influx cannot be estimated accurately from the rate of labelled Na + uptake. In isolated heart muscle preparations which are incubated in an equilibrated state without sodium loading, the rate of sodium influx must be equal to the rate of sodium efflux. If the bulk of the sodium efflux is due to N a + / K + transport coupled by the sodium pump, the ouabain-sensirive 42K+ uptake may then be used to estimate the rate of sodium influx (Akera et al., 1981). Several investigators have reported that the total K + flux was relatively insensitive to electrical stimulation up to 1 Hz. However, at a higher frequency and with reduced K + concentration in the medium, the ouabain-sensitive 42K + uptake clearly shows dependence on the frequency of electrical stimulation or the rate of sodium influx (Akera et al., 1981). Yamamoto et al. (1979) have also reported that the ouabain-sensitive 86Rb uptake by cardiac muscle preparations which were not sodium-loaded was greater at a higher temperature. Additionally, in the present study when the rate of sodium influx was increased in isolated atrial muscle preparations incubated at 3 0 ° C by the addition of either monensin or grayanotoxin to the incubation mixture the patterns of beat dependence of the development of the positive inotropic effect of ouabain became similar to those observed at a higher temperature in the absence of these agents.
232 Finally, the increase in sodium influx caused by electrical
stimulation
was
markedly
smaller
References
at
3 7 ° C t h a n a t 3 0 ° C . T h i s f i n d i n g is c o n s i s t e n t with the possibility that the frequency of electrical stimulation has relatively little effect on the rate of development of glycoside action at the higher temperature. The differences in the force of contraction at the time of ouabain addition were not the cause of d i f f e r e n c e s i n t h e r a t e o f d e v e l o p m e n t o f its a c t i o n . Under conditions similar to those of the present s t u d y , a l t e r a t i o n s i n C a 2+ c o n c e n t r a t i o n c a u s e d similar differences in force of contraction but failed to affect the rate of development of glycoside a c t i o n ( A k e r a et al., 1977). T h e d i f f e r e n c e s i n magnitude of the positive inotropic effect of o u a b a i n p e r se a l s o d o n o t s e e m t o b e t h e p r i m a r y c a u s e o f t h e d i f f e r e n c e s i n o n s e t r a t e o f its a c t i o n . When preparations were incubated at 37°C, the positive inotropic action of ouabain at each stimul a t i o n f r e q u e n c y w a s s m a l l e r t h a n t h a t a t 30 ° C. I n contrast, incubation of the atrial muscle preparations in the presence of either monensin or grayanotoxin resulted in a greater inotropic action o f o u a b a i n . I n e i t h e r case, t h e o n s e t o f g l y c o s i d e action was enhanced. I n c o n c l u s i o n , t h e a m o u n t o f N a + a v a i l a b l e to t h e s o d i u m p u m p is r e l a t i v e l y s m a l l a n d is t h e determinant of the rate of ouabain binding to the sodium pump when isolated atrial muscle preparations are incubated at relatively low temperatures and stimulated at a low frequency. At higher temperatures, the beat dependence of onset of the g l y c o s i d e a c t i o n is less p r o m i n e n t , b e c a u s e t h e r e s t i n g s o d i u m i n f l u x is h i g h e r a n d e l e c t r i c a l stimulation has a smaller influence on the rate of sodium influx.
Acknowledgements The author thanks Drs. Tai Akera and Theodore M. Brody for helpful advice and suggestions, and Mr. A.T. Linehan for competent technical assistance. This work was supported by U.S. Public Health Service grant HL-16052 from the National Heart, Lung, and Blood Institute.
Akera, T., M. Hirai and T. Oka, 1979, Sodium ions and the development of the inotropic action of ouabain in guinea-pi~ heart, European J. Pharmacol. 60, 189. Akera, T., M.K. Olgaard, K. Temma and T.M. Brody, 1977, Development of the positive inotropic action of ouabain. Effects of transmembrane sodium movement, J. Pharmacol. Exp. Ther. 203,675. Akera, T., S. Yamamoto, K. Temma, D-H. Kim and T.M. Brody, 1981, Is ouabain-sensitive rubidium or potassium uptake a measure of sodium pump activity in isolated cardiac muscle?, Biochim. Biophys. Acta 640, 779. Bentfeld, M., H. Liallmann, T. Peters and D. Proppe, 1977, Interdependence of ion transport and the action of ouabain in heart muscle, Br. J. Pharmacol. 61, 19. Bodemann, H.H. and J.F. Hoffman, 1976, Comparison of the side-dependent effects of Na and K on orthophosphate-, UTP-, and ATP-promoted ouabain binding to reconstituted human red blood cell ghosts, J. Gen. Physiol. 67, 527. Byrne, J.E and P.E. Dresel, 1969, The effect of temperature and calcium concentration on the action of ouabain in quiescent rabbit atria, J. Pharmacol. Exp. Ther. 166, 354. Clausen, T. and O. Hansen, 1977, Active Na-K transport and the rate of ouabain binding. The effect of insulin and other stimuli on skeletal muscle and adipocytes. J. Physiol. (London) 270, 415. Ebner, F. and M. Reiter, 1977, The dependence on contraction frequency of the positive inotropic effects of dihydroouabain. Naunyn-Schmiedeb. Arch. Pharmacol. 300, 1. Erdmann, E., 1981, Influence of cardiac glycosides on their receptor, in: Handbook of Experimental Pharmacology, 56. Cardiac Glycosides. Part 1, ed. K. Greeff (Springer-Verlag, Heidelberg) p. 337. Goerke, J. and E. Page, 1965, Cat heart muscle in vitro. VI. Potassium exchange in papillary muscles, J. Gen. Physiol. 48, 933. Honerj~tger, P. and M. Reiter, 1975, The relation between the effects of veratridine on action potential and contraction in mammalian ventricular myocardium, Naunyn-Schmiedeb. Arch. Pharmacol. 289, 1. Koch-Weser, J.R., 1971, Myocardial contraction frequency and onset of cardiac glycoside action, Circ. Res. 28, 34. Koch-Weser, J. and J. Blinks, 1962, Analysis of the relation of the positive inotropic action of cardiac glycosides to the frequency of contraction of heart muscle, J. Pharmacol. Exp. Ther. 136, 305. Koch-Weser, J. and J.R. Blinks, 1963, The influence of the interval between beats on myocardial contractility, Pharmacol. Rev. 15, 601. Ku, D., T. Akera, C.L. Pew and T.M. Brody, 1974, Cardiac glycosides: correlations among Na+,K+-ATPase, sodium pump and contractility in the guinea pig heart, NaunynSchmiedeb. Arch. Pharmacol. 285, 185. Linden, J. and G. Brooker, 1980, Sodium requirement for effects of ouabain on contraction of isolated guinea-pig atria, Circ. Res. 46, 553.
233 Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Li~llmann, H. and T. Peters, 1979, Action of cardiac glycosides on the excitation-contraction coupling in heart muscle, Prog. Pharmacol. 2, 1. Matsui, H. and A. Schwartz, 1968, Mechanism of cardiac glycoside inhibition of the ( N a + - K + )-dependent ATPase from cardiac tissue, Biochim. Biophys. Acta 151,655. Meier, C.F., K.C. Lasseter and B.C. Pressman, 1976, Alteration of intracellular sodium activity in canine Purkinje fibers by the ionophore monensin, Pharmacologist 18, 122. Moran, N.C., 1967, Contraction dependency of the positive inotropic action of cardiac glycosides, Circ. Res. 21,727. Narahashi, T. and I. Seyama, 1974, Mechanism of nerve membrane depolarization caused by grayanotoxin 1, J. Physiol. 242, 471. Park, M.K. and F.F. Vincenzi, 1973, Glycoside and aglycone inotropism: influence of temperature on the rate of onset, Life Sci. 13, 743. Park, M.K. and F.F. Vincenzi, 1975, Rate of onset of cardiotonic steroid-induced inotropism: influence of temperature and beat interval, J. Pharmacol. Exp. Ther 195, 140. Reiter, M., 1981, The positive inotropic action of cardioactive steroids on cardiac ventricular muscle, in: Handbook of Experimental Pharmacology 56: Cardiac Glycosides, Part 1, ed. K. Greeff (Springer-Verlag, Heidelberg) p. 187. T e m m a , K. and T. Akera, 1982, Enhancement of cardiac actions of ouabain and its binding to Na +,K+-adenosine triphosphatase by increased sodium influx in isolated guinea-pig heart, J. Pharmacol. Exp. Ther. 223, 490.
T e m m a , K. and T. Akera, 1983, Decreases in active sodium pumping sites and their interaction with ouabain caused by low Na ÷ incubation of isolated guinea-pig atrial muscle, J. Pharmacol. Exp. Ther. 225, 660. Temma, K., T. Akera, T.M. Brody and H. Kondo, 1982, A comparison of inotropic actions of p-chlormercuribenzoate, ouabain, digitoxin and digitoxigenin, European J. Pharmacol. 77, 289. Vincenzi, F.F., 1967, Influence of myocardial activity on the rate of onset of ouabain action, J. Pharmacol. Exp. Ther. 155, 279. Wiggins, J.R. and J.J. Bentolila, 1980, Sodium dependence of the positive inotropic effect of cardiac glycosides, J. Pharmacol. Exp. Ther. 215, 569. Wilbrandt, W., K. Brawand and P.N. Witt, 1953, Die quantitative Abh~mgigkeit der Strophanthosidwirkung auf das Froschherz v o n d e r T~tigkeit des Herzens und v o n d e r Glykosidkonzentration, Naunyn-Schmiedeb. Arch. Exp. Pathol. Pharmakol. 219, 397. Yamamoto, S., T. Akera and T.M. Brody, 1979, Sodium influx rate and ouabain-sensitive rubidium uptake in isolated guinea pig atria, Biochim. Biophys. Acta 555, 270. Yamamoto, S., T. Akera and T.M. Brody, 1980, Positive inotropic action of digitoxigenin and sodium p u m p inhibition: effects of enhanced sodium influx, J. Pharmacol. Exp. Ther. 213, 105. Yamamoto, S., T. Akera, D.-H. Kim and T.M. Brody, 1981, Tissue concentration of N a + ,K+-adenosine triphosphatase and the positive inotropic action of ouabain in guinea-pig heart, J. Pharmacol. Exp. Ther. 217, 701.
223
Elsevier
T E M P E R A T U R E - D E P E N D E N T D E V E L O P M E N T OF OUABAIN A C T I O N IN I S O L A T E D GUINEA P I G HEART: D I F F E R E N C E S IN S O D I U M INFLUX? KYOSUKE TEMMA *
Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 48824, U.S.A. Received 8 March 1984, accepted 13 June 1984
K. T E M M A , Temperature-dependent development of ouabain action in isolated guinea pig heart: differences in sodium influx?, European J. Pharmacol. 104 (1984) 223-233. A t 30 o C the development of the positive inotropic action of 0.3/-tM ouabain observed in isolated left atrial muscle preparations of guinea-pig heart was significantly slower and more d e p e n d e n t on the frequency of electrical stimulation than it was at 37 o C. The hypothesis was tested that such differences are due to a variation in the rate of leak sodium influx, and ensuing differences in glycoside binding to sarcolemmal Na,K-ATPase. M o n e n s i n or grayanotoxin, agents which have been shown to increase sodium influx, caused faster development of the inotropic action of ouabain at 30 o C when muscle preparations were stimulated at 0.5 or 1 Hz but their effect was smaller at 2 or 3 Hz stimulation. The contracture caused by toxic concentrations of ouabain in quiescent preparations developed faster at the higher temperature. Ouabain binding to sarcolemmal N a , K - A T P a s e which occurred during exposure of atrial muscle preparations to the glycoside at 25 o C for 30 min was less than the corresponding value observed after a similar exposure at 37 o C; however, electrical stimulation increased glycoside binding to a greater extent at the lower temperature. The ouabain-sensitive 42K+ uptake was lower in quiescent preparations at 3 0 ° C than at 3 7 ° C . Moreover, the e n h a n c e m e n t of the ouabain-sensitive 42 K + uptake caused by electrical stimulation was greater at 30 ° C than at 37 o C. These results are consistent with the hypothesis that, at lower temperatures, the rate of sodium influx is low and is the factor determining the rate of development of the inotropic action of ouabain. At a higher temperature, a relatively high sodium leak influx seems to make the onset of ouabain action less d e p e n d e n t on the frequency of stimulation.
Cardiac glycosides Na,K-ATPase
Ouabain
Beat and temperature dependence of inotropic effect
1. Introduction
The positive inotropic and toxic effects of the cardiac glycosides develop slowly in isolated heart muscle preparations incubated at relatively low temperatures. The rate of development of these effects of the glycosides is substantially increased when the preparations are stimulated at a higher frequency (Moran, 1967; Vincenzi, 1967; Park and Vincenzi, 1975: Bentfeld et al., 1977; Ebner and Reiter, 1977; T e m m a et al., 1982). In contrast, the onset rate of the inotropic and toxic effects of the * Current address: Department of Pharmacology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada-shi, Aomori 034, Japan. 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
Na influx
cardiac glycosides observed at a higher temperature e.g. 37 o C is faster and relatively insensitive to the frequency of electrical stimulation between 0.1 and 1 Hz (Koch-Weser, 1971; Park and Vincenzi, 1973). One possible explanation for the beat-dependent onset of the glycoside action observed at relatively low temperatures is that the binding of the glycoside to sarcolemmal Na,K-ATPase, the putative receptor for the positive inotropic and toxic actions of the glycoside, is regulated by the amount of intracellular sodium ion available to the sodium p u m p (Akera et al., 1977; Li~llmann and Peters, 1979). This concept is supported by the fact that agents and conditions which enhance sodium influx have been shown to increase the
224 onset rate of the positive inotropic action of cardiac glycosides, especially ouabain (Honerj~ger and Reiter, 1975; Akera et al., 1977), concomitant with an enhanced binding of the glycoside to sarcolemmal Na,K-ATPase (Temma and Akera, 1982). Sodium ions enhance glycoside binding to isolated Na,K-ATPase observed in vitro in the presence of Mg 2+ and ATP (Matsui and Schwartz, 1968). Glycoside binding in intact cells is also enhanced by conditions which stimulate turnover of the enzyme (Clausen and Hansen, 1977), except for K + which inhibits glycoside binding. Because intracellular Na + stimulates enzymes activity, it seems likely that it also enhances glycoside binding. Yamamoto et al. (1979) have shown that the sensitivity of the sarcolemmal sodium pump to the inhibitory action of ouabain was elevated by an enhancement of sodium influx. If intracellular sodium available to the sodium pump is the regulator of glycoside binding to sarcolemmal Na,K-ATPase at relatively low temperatures, why is the development of the positive inotropic effect of glycosides less sensitive to the frequency of stimulation at a higher temperature? The study by Yamamoto et al. (1981) suggests that the leak Na + influx of cardiac muscle preparations is substantially higher at 36.5°C than at 30°C. Therefore, we tested the hypothesis that such differences are due to a variation in the rate of sodium influx, and ensuing differences in glycoside binding to sarcolemmal Na, K-ATPase. The glycoside binding to sarcolemmal Na, K-ATPase which occurred during the inotropic study was estimated by homogenizing the tissue and from the reduction of the initial velocity of the [3H]ouabain binding reaction. The rate of sodium influx was estimated indirectly from the ouabain-sensitive 42K+ uptake in preparations which were not sodium-loaded and were presumably in equilibrium with regard to ion movements.
2. Materials and methods
2.1. Force of contraction Guinea pigs of either sex weighing approximately 350 g were stunned by a sharp blow to the
head; their hearts were rapidly excised and perfused via the aorta for 5-10 min at 30°C with freshly oxygenated (95% O 2 and 5% CO2) KrebsHenseleit bicarbonate buffer solution of the following composition: 118 mM NaCI, 27.2 mM N a H C O 3, 4.8 mM KCI, 1.0 mM KHzPO4, 1.2 mM MgSO4, 1.2 mM CaC12 and 11.1 mM glucose (pH 7.4). After visible blood had been removed from the tissue, the left atrial muscle was excised and suspended vertically in a chamber containing 30 ml of the above solution at either 30 or 37°C. The atrial preparations were stimulated at 0.5, 1, 2 or 3 Hz with square wave pulses of 4 ms duration at a voltage approximately 20% above the threshold. Platinum electrodes were used for electrical field stimulation. Resting tension was adjusted to 1.0 g, which produced approximately 90% of the maximal tension developed (Yamamoto et al., 1981). The tension developed was recorded isometrically using a force-displacement transducer and a polygraph recorder (Grass Instrument Company, Quincy, MA; model FT-03C and 7B, respectively). In several experiments, grayanotoxin I or monensin was added to the incubation medium 70 or 30 min, respectively, before the addition of ouabain. In the contracture study, atrial muscle preparations were equilibrated for 60 min under 1.5 Hz stimulation at the temperature indicated and the stimulator was turned off. Subsequently, ouabain was added to the incubation medium and changes in the resting tension were monitored.
2.2. 42K + uptake Left atrial muscle preparations obtained from guinea pig hearts were equilibrated at 30 or 37 °C for 30 rain under 3 Hz electrical stimulation in a chamber containing 30 ml of the Krebs-Henseleit bicarbonate buffer solution described above, except that the K + concentration was reduced to 4 instead of 5.8 mM. Subsequently, tracer amounts of 42K+ were added to the incubation medium. After a 30 min incubation under quiescent conditions or 3 Hz stimulation, the atrial muscle was removed from the incubation bath, rinsed with a tracer-free solution for 15 s, blotted on a filter paper and weighed, the radioactivity in left atrial muscle was estimated using a gamma scintillation
225 spectrometer. The specific (ouabain-sensitive) 42K + uptake is the difference between values observed in the absence and presence of 0.3 mM ouabain (Yamamoto et al., 1979). The addition of 0.3 mM ouabain (final concentration) caused a marked elevation of the resting tension after a transient positive inotropic effect followed by failure of the muscle preparation to follow electrical stimulation within 10 min. Therefore, nonspecific 42 K + uptake was estimated by adding the tracer 10 min after the addition of ouabain and monitoring the 42K+ uptake. With a reduced K + concentration and electrical stimulation at 3 Hz, the ouabain-sensitive 42K+ uptake was significantly elevated over that observed in quiescent preparations, although previous investigators reported that total K + uptake, i.e. ouabain-sensitive plus ouabain-insensitive uptake, was relatively unaffected by electrical stimulation up to 1 Hz (see Goerke and Page, 1965). Assuming that a relationship exists between ouabain-sensitive K + uptake and Na + extrusion by the sodium pump, the rate of N a + pumping can be estimated from the ouabain-sensitive 42K + uptake. In equilibrated, steady state preparations, the rate of Na + influx must be equal to the rate of N a + influx.
2.3. [-~H]Ouabain binding The fractional occupancy of the glycoside binding sites on Na,K-ATPase by ouabain in left atrial muscle preparations was estimated from the reduction in the specific (ATP-dependent) [3H]ouabain binding observed with homogenates from preparations incubated in the presence of unlabelled ouabain (Ku et al., 1974). Left atrial muscle preparations of guinea-pig heart were equilibrated for 60 min at 25 or 3 7 ° C in Krebs-Henseleit bicarbonate buffer solution under 2 Hz stimulation. Subsequently, 0.5 ktM ouabain (final concentration) was added to the medium and preparations were incubated for 30 rain under 0 or 2 Hz stimulation. The muscles were minced with scissors then homogenized in 2.5 ml of an ice-cold solution containing 1 m M E D T A and 10 m M Tris-HC1 buffer (pH 7.5) using a motor-driven Teflon pestle homogenizer with 25 ~ m clearance (3000 r.p.m. for 12 s). A 0.1 ml aliquot of the homogenate
containing approximately 0.1 mg protein was added to 0.9 ml of a prewarmed incubation mixture and incubated at 3 7 ° C for 3 min with 50 nM [3H]ouabain in the presence of 100 m M NaCI, 5 m M MgCI 2, 50 m M Tris-ATP and 50 m M TrisHC1 buffer (pH 7.4). Bound and free [3H]ouabain were separated by filtering the aliquot through a nitrocellulose filter (Millipore Corporation, Bedford, MA; type AA, pore size, 0.8/~m). The filter was washed twice with 6 ml each of an ice-cold solution containing 15 m M KC1 and 50 m M Tris HC1 buffer solution and dissolved in ethylene glycol monomethyl ether (Pierce Chemical Company, Rockford, IL: Piersolve). The radioactivity was estimated with a liquid scintillation spectrometer using a toluene-based counting solution (Temma and Akera, 1982). The specific [3H]ouabain binding is the difference between values observed in the presence and absence of A T P (Ku et al., 1974).
2.4. Miscellaneous 42K+ was prepared from a K O H solution by the Nuclear Reactor Laboratory of Michigan State University. The irradiated solution was neutralized with HC1 before use. Generally labelled [3H]ouabain (specific activity, 18 C i / m m o l ) was purchased from New England Nuclear, Boston, MA. Monensin sodium was kindly supplied by Lilly Laboratories, Eli Lilly and Company, Indianapolis, IN, and grayanotoxin I by Dr. Junkichi Iwasa, Okayama University, Okayama, Japan. Ouabain octahydrate was obtained from Sigma Chemical Company, St. Louis, MO. Other chemicals were of reagent grade. Statistical analyses were performed with Student's t-test. The criterion for statistical significance was a P value of less than 0.05. Protein concentrations were estimated by the method of Lowry et al. (1951) using bovine serum albumin as the standard.
3. Results
3.1. lnotropic effects of ouabain The degree of beat dependence of the development of the positive inotropic action of ouabain
226
seems to be greatly influenced by the experimental conditions. The dependence of the onset rate of the inotropic effect of ouabain on the frequency of electrical stimulation was therefore examined first to demonstrate beat dependence under the conditions used. Moreover, the effect of the temperature on beat dependence was examined to confirm earlier work. Isolated atrial muscle preparations of guinea-pig heart were equilibrated for 60 min at either 30 or 37°C, under 0.5, 1, 2 or 3 Hz stimulation. At the end of the equilibration period, a greater tension developed at 30 o C than at 37 o C, especially when the preparations were stimulated at a higher frequency (fig. 1). Subsequently, ouabain (final concentration, 0.3 /~M) was added to the medium and caused a gradual increase in developed tension. The development of the positive inotropic effect of ouabain was significantly more rapid at 3 7 ° C than at 3 0 ° C (fig. 1 and also fig. 2, open and filled circles). At 30 o C, the rate of development was clearly dependent on the frequency of stimulation in a range between 0.5 and 2 Hz but the effect of stimulation frequency was less prominent at 37 o C (fig. 2). These results confirm earlier reports (Moran, 1967; Vincenzi, 1967; Koch-
3
!
!
i
=
=
!
.i i 0 0
2O
4O
Time
6O
80
(min)
lO0
120
i
i
i
i
O ; 3TC .E
• ; 30"C 30
._E x
~ ~
Lx:
~
A: 3 0 C
N
30~
1pM monensin
20
E e-
E_ I--
0
I O.S
1 l Stimulation
I 2 frequency
1 3 (Hz)
Fig. 2. Effects of stimulation frequency on the development of the positive inotropic effect of ouabain: Influence of monensin and grayanotoxin 1 (GTX-I). See legend to figs. 1 and 3. The time required for the development of a half-maximal inotropic effect of ouabain was plotted against the frequency of electrical stimulation. The value was calculated for each preparation then averaged. Each point represents the mean of 4 to 6 experiments. Vertical lines indicate S.E.
!
37 "C
30 "C
40
0
20
4O
6O
Time (min)
Fig. 1. Effects of stimulation frequency on the development of positive inotropic effects of ouabain at 30 or 37 °C. Left atrial muscle preparations of guinea pig heart were electrically stimulated at 0.5 (C)), 1 (O), 2 (A) or 3 Hz (*). After a 60 min equilibration, ouabain (final concentration, 0.3 p,M) was added to the incubation medium at time zero. Each point represents the mean of 4 to 6 experiments.
Weser, 1971; Park and Vincenzi, 1973, 1975; Bentfeld et al., 1977; Ebner and Reiter, 1977; Temma et al., 1982) and indicate that the onset rate of ouabain action is relatively beat-dependent at 3 0 ° C and that the beat dependence is less apparent at 37 °C. The hypothesis that the rate of development ~f the positive inotropic action of ouabain is beat-dependent at 30 o C because the leak sodium influx is relatively low at this temperatures was tested by increasing the rate of the beat-independent sodium influx by means of monensin or grayanotoxin I, agents which have been shown to increase sodium influx in cardiac muscle (Narahashi and Seyama, 1974; Meier et al., 1976). As reported previously (Akera et al., 1977), addition of either monensin or grayanotoxin to the incubation medium caused an increase in the developed tension of isolated atrial
227 m u s c l e p r e p a r a t i o n s ( d a t a n o t shown). We have shown previously that grayanotoxin I c a u s e d a f a s t e r d e v e l o p m e n t of the c o n t r a c t u r e i n d u c e d b y t o x i c c o n c e n t r a t i o n s of o u a b a i n in q u i e s c e n t p r e p a r a t i o n s o n l y w h e n the p r e p a r a t i o n s h a d b e e n p r e v i o u s l y e x p o s e d to this t o x i n u n d e r e l e c t r i c a l s t i m u l a t i o n ( T e m m a a n d A k e r a , 1982). T h i s f i n d i n g raises the p o s s i b i l i t y t h a t the b i n d i n g o f g r a y a n o t o x i n I to its site o f a c t i o n in c a r d i a c m u s c l e m i g h t b e d e p e n d e n t o n t h e f r e q u e n c y of e l e c t r i c a l s t i m u l a t i o n . T h e r e f o r e , left atrial m u s c l e preparations were incubated w i t h 0.2 I~M g r a y a n o t o x i n I for 30 m i n u n d e r 3 H z s t i m u l a t i o n at 30 o C. S u b s e q u e n t l y , the s t i m u l a t o r was reset to the i n d i c a t e d f r e q u e n c y a n d o u a b a i n (final c o n c e n t r a t i o n , 0.3 /~M) was a d d e d 40 rain l a t e r w h e n the d e v e l o p e d t e n s i o n r e a c h e d a s t e a d y state. B o t h 1 ~tM m o n e n s i n o r 0.2 t t M g r a y a n o t o x i n I c a u s e d a m o r e r a p i d d e v e l o p m e n t of t h e p o s i t i v e i n o t r o p i c e f f e c t of o u a b a i n w h e n p r e p a r a t i o n s w e r e s t i m u -
l a t e d at 0.5 o r 1 H z (fig. 3) as r e p o r t e d e a r l i e r ( A k e r a et al., 1977). T h e effect of m o n e n s i n o r g r a y a n o t o x i n I was less p r o m i n e n t w h e n the p r e p a r a t i o n s w e r e s t i m u l a t e d at h i g h e r f r e q u e n c i e s . I n t h e p r e s e n c e o f t h e s e agents, the r a t e o f d e v e l o p m e n t of the p o s i t i v e i n o t r o p i c effect of o u a b a i n ( p l o t t e d as the t i m e to h a l f - m a x i m a l effect) o b s e r v e d at 3 0 ° C , a n d also the p a t t e r n of b e a t d e p e n d e n c e of the o n s e t rate, a p p r o a c h e d t h o s e o b s e r v e d at 37 ° C in the a b s e n c e o f m o n e n s i n or g r a y a n o t o x i n I (fig. 2). T h u s , the d i f f e r e n c e in b e a t d e p e n d e n c e o f the d e v e l o p m e n t of the g l y c o s i d e a c t i o n o b s e r v e d at t w o t e m p e r a t u r e s s e e m e d to r e s u l t f r o m the l o w e r l e a k s o d i u m i n f l u x r a t e at 30 ° C t h a n at 37 o C.
3.2. Ouabain-induced contracture I n c r e a s e s in N a + i n f l u x in t h e q u i e s c e n t m u s c l e h a v e b e e n s h o w n to c a u s e a faster o n s e t of ouabain-induced contracture (Temma and Akera, 1982). T h e r e f o r e , if the l e a k s o d i u m i n f l u x is g r e a t e r at a h i g h e r t e m p e r a t u r e , the d e v e l o p m e n t of c o n t r a c t u r e c a u s e d b y toxic c o n c e n t r a t i o n s of o u a b a i n
--AA
~
2 2.6
I
24
lu
l_o'
2.2 c* 2.0 0
i 0
i 20
40 60
i 80 100 120 0
Time (min)
t i i i 20 40 60 80 100 120 Time (min)
~
I
/
o~ g
1.s
3o~
14
Fig. 3. Effects of monensin or grayanotoxin I (GTX-I) on the development of positive inotropic effects of ouabain at 30 o C. Left panel: Left atrial muscle preparations were equilibrated for 45 min under 0.5 (C)), 1 (o), 2 (zx) or 3 Hz (A) stimulation. Subsequently, monensin (final concentration, 1/LM) was added to the incubation medium. Ouabain (final concentration, 0.3 t~M) was added 30 min later at time zero. Right panel: After equilibration, left atrial muscle preparations were incubated for 30 min in the presence of 0.2 ~M GTX under 3 Hz stimulation. Subsequently, the frequency of electrical stimulation was adjusted to either 0.5 (C)), 1 (O), 2 (zx) or 3 Hz (A), and ouabain (final concentration, 0.3 /~M) was added 40 min later at time zero. Under 3 Hz stimulation, a 30 min exposure to 1 t~M monensin and exposure to 0.2 ~M GTX caused a 33.2 + 6.2 and 43.7 + 8.2% increase in the tension developed. Each point represents the mean of 4 or 5 experiments.
"
1.2 1.(1
oT
I
0
20
i
40
60
Time
( rnin )
80
100
120
Fig. 4. Effects of temperature on the ouabain-induced contracture in quiescent atrial muscle preparations. Left atrial muscle preparations were equilibrated for 60 min under 1.5 Hz stimulation at either 30 tO) or 37°C (O). Subsequently, the stimulator was turned off, and ouabain (final concentration, 5 #M) was added to the incubation medium at time zero. Each point represents the mean of 6 (30 ° C) or 4 (37 °C) experiments. Vertical lines indicate S.E. Arrows indicate the mean time to the half-maximal effect of ouabain.
228 in quiescent p r e p a r a t i o n s should be faster at a higher temperature. To test this possibility, quiescent left atrial muscle p r e p a r a t i o n s were exposed to 5 /,M o u a b a i n at either 30 or 3 7 ° C . The addition of 5 /,M o u a b a i n (final c o n c e n t r a t i o n ) to the m e d i u m caused a gradual increase in resting tension at 30 ° C (fig. 4). The d e v e l o p m e n t of contracture due to 5 ~ M o u a b a i n was significantly faster at 37 ° C. The time required for the d e v e l o p m e n t of a h a l f - m a x i m a l response to o u a b a i n was 54.0 + 5.9 m i n at 30 ° C, a n d 28.9 + 4.0 m i n at 37 ° C. These results suggest that o u a b a i n b i n d i n g to the sodium p u m p in atrial muscle p r e p a r a t i o n s is faster at a high temperature a n d that the s o d i u m influx rate in quiescent muscle p r e p a r a t i o n s is higher at 37 ° C t h a n at 30 °C.
stimulation. Subsequently, 0.5 ~ M o u a b a i n (final c o n c e n t r a t i o n ) was added to the i n c u b a t i o n m e d i u m a n d the p r e p a r a t i o n s were i n c u b a t e d for an a d d i t i o n 30 m i n u n d e r quiescent c o n d i t i o n s or 2 Hz stimulation. I n c u b a t i o n of atrial muscle p r e p a r a t i o n s in the o u a b a i n - f r e e m e d i u m at different temperatures (25 or 3 7 ° C ) did not alter the specific [3H]ouabain b i n d i n g reaction (fig. 5), indicating that comparison of fractional o c c u p a n c y data at two temperatures is feasible. It should be noted that the assay of [3H]ouabain b i n d i n g with homogenates to assess fractional o c c u p a n c y was performed at 37 oC regardless of the i n c u b a t i o n temperature of atrial muscle p r e p a r a t i o n s prior to homogenization. This
3.3. Ouabain binding to Na, K-A TPase
1.0 ! r-
The b i n d i n g of u n l a b e l l e d o u a b a i n to glycoside b i n d i n g sites on sarcolemmal N a , K - A T P a s e that occurred d u r i n g the i n c u b a t i o n of isolated atrial muscle p r e p a r a t i o n s was estimated b y homogenizing the tissue a n d assaying the velocity of the specific [3H]ouabain b i n d i n g reaction. The specific [3H]ouabain b i n d i n g reaction was almost linear d u r i n g the 3 m i n i n c u b a t i o n period (data n o t shown), i n d i c a t i n g that the observed value could be used to represent the initial velocity. A reduction in the initial velocity would then indicate previous o c c u p a n c y of the glycoside b i n d i n g site b y u n l a b e l l e d o u a b a i n . Initial experiments showed that the fractional o c c u p a n c y of glycoside b i n d i n g sites on Na, K - A T P a s e which occurred d u r i n g a 30 m i n i n c u b a t i o n of atrial muscle at 3 0 ° C in the presence of 0.3 /,M o u a b a i n was less than the fractional o c c u p a n c y at 37 °C. However, on statistical analysis the difference was f o u n d to be hardly significant, due to the relatively small fractional o c c u p a n c y caused by 0.3 ~ M o u a b a i n a n d to variations in the experimental data. I n order to test the hypothesis that the difference in the inc u b a t i o n t e m p e r a t u r e affects glycoside b i n d i n g to the sodium p u m p , left atrial muscle p r e p a r a t i o n s were i n c u b a t e d at 25 ° C instead of 30 ° C a n d the glycoside c o n c e n t r a t i o n was increased to 0.5 /,M. Left atrial muscle p r e p a r a t i o n s were equilibrated for 60 m i n at either 25 or 3 7 ° C u n d e r 2 Hz
a~ •" o
~.
.c:: .j= .E_ f..O
25"C
37"C
-i-i 0.9a,b
~0.8
~
. ~
O
v 0~
O
E
v
0.7.~
0Tl
..
I
I
05pM Cont ouaoain
2Hz~
I I I I 0.5pM Cont ouaoain
Fig. 5. Specific [3H]ouabain binding to homogenates obtained from atrial muscle preparations exposed to unlabelled ouabain. Left atrial muscle preparations were equilibrated for 60 min under 2 Hz stimulation at either 25 or 37 °C. Subsequently, these preparations were exposed to 0.5 p.M unlabelled ouabain, either quiescent or under 2 Hz stimulation. After a 30 min exposure, the muscle preparations were homogenized and immediately added to incubation medium for estimation of the initial velocity of the specific [3Hlouabain binding reaction. Specific [3H]ouabain binding is the difference in values obtained in the absence and presence of 5 mM ATP. Control values were obtained with left atrial muscle preparations incubated for a comparable time period in the absence of unlabelled ouabain under 2 Hz stimulation, homogenized and challenged with [3H]ouabain. a) Significantly different from control values observed at the same temperature, b) Significantly different from the corresponding value observed at 25 o C. Each value represents the mean of 5 experiments. Vertical lines indicate S.E.
229 was done because the p u r p o s e of the [ 3 H ] o u a b a i n b i n d i n g assay was to estimate the fractional occ u p a n c y of the glycoside b i n d i n g sites on N a , K A T P a s e by u n l a b e l l e d o u a b a i n d u r i n g the i n c u b a tion of atrial muscle p r e p a r a t i o n s in the inotropic experiments. The specific b i n d i n g of [3H]ouabain to h o m o g e n a t e s from quiescent left atrial muscle p r e p a r a t i o n s i n c u b a t e d in the absence of unlabelled o u a b a i n was similar to that observed following electrical s t i m u l a t i o n in the absence of o u a b a i n u n d e r the present c o n d i t i o n s (data not shown). Such results were anticipated because 30 m i n i n c u b a t i o n in quiescent c o n d i t i o n s with electrical s t i m u l a t i o n is unlikely to change the n u m b e r of active N a , K - A T P a s e units which b i n d o u a b a i n after h o m o g e n i z a t i o n of the muscle. Therefore, o n l y the values observed with electrically stimulated p r e p a r a t i o n s are shown in fig. 5. Nonspecific b i n d i n g was not affected by either the t e m p e r a t u r e or electrical s t i m u l a t i o n (data n o t shown~ Exposure of quiescent p r e p a r a t i o n s to 0.5 /~M o u a b a i n for 30 rain at either 25 or 3 7 ° C reduced the [3H]ouabain b i n d i n g observed with homogenates o b t a i n e d from these preparations. The red u c t i o n in the specific [3H]ouabain b i n d i n g observed in quiescent p r e p a r a t i o n s exposed to u n labelled o u a b a i n at 37 ° C was significantly greater t h a n that observed after o u a b a i n exposure at 25 o C, i n d i c a t i n g that a greater fractional o c c u p a n c y occurred at 3 7 ° C t h a n at 2 5 ° C in quiescent preparations. Exposure to u n l a b e l l e d o u a b a i n u n d e r electrical s t i m u l a t i o n further reduced the initial velocity of the specific [3H]ouabain b i n d i n g reaction, i n d i c a t i n g that greater b i n d i n g of the unlabelled o u a b a i n occurred d u r i n g the exposure to o u a b a i n at either t e m p e r a t u r e (fig. 5), W i t h electrical stimulation, the difference in fractional occ u p a n c y observed at two t e m p e r a t u r e s was m i n i m a l . These results d e m o n s t r a t e that electrical s t i m u l a t i o n or raising the temperature e n h a n c e d the b i n d i n g of u n l a b e l l e d glycoside to N a , K ATPase, reducing the sites available to [3H]ouabain. The c o m b i n e d effect of electrical s t i m u l a t i o n a n d t e m p e r a t u r e increase however, was not significantly different from that of electrical s t i m u l a t i o n alone.
3.4. Ouabain-sensitive
42K +
uptake
The above results suggest that the leak N a + influx and also the e n h a n c e m e n t of net sodium influx by electrical s t i m u l a t i o n are temperature-dep e n d e n t . T o estimate the rate of sodium influx more directly, tracer a m o u n t s of 42K + were a d d e d to the i n c u b a t i o n m e d i u m a n d the ouabain-sensitive 42K+ uptake was assayed. I n equilibrated heart muscle p r e p a r a t i o n s which are n o t N a +-loaded the o u a b a i n - s e n s i t i v e 42K+ uptake can be used to estimate the rate of s o d i u m influx because the rate of s o d i u m influx must be equivalent to the rate of s o d i u m efflux in steady state preparations, a n d the
28 o
24
30"C r-]; OHz
T 37"C FI; 3Hz a,b I
'q" .~_ ~
12
c
0
Fig. 6. Effects of temperature and electrical stimulation on the ouabain-sensitive 42K+ uptake by isolated atrial muscle preparations. Left atrial muscle preparations were equilibrated for 30 min under 3 Hz stimulation in a Krebs-Henseleit bicarbonate buffer solution containing 4 mM K + at the temperature indicated. Tracer amounts of 42KC1 was added to the incubation medium at this time, and 42K+ uptake was determined after a 30 rain incubation, quiescent or under 3 Hz stimulation. Specific 42K+ uptake is the difference in values obtained in the absence or presence of 0.3 mM ouabain. The values for nonspecific 42K+ uptake were 5.65 _+0.37 (30 o C, 0 Hz), 5.93+0.18 (30°C, 3 Hz), 5.02-+0.39 (37°C, 0 Hz), and 4.87_+0.24 nmol/mg tissue (37°C, 3 Hz). The number in parentheses indicates the number of experiments. Vertical lines indicate S.E. a) Significantly different from the corresponding values observed at 30 o C. b) Significantlydifferent from values observed at same temperature under 0 Hz stimulation, c) Significantly different from the corresponding values observed at 30°C.
230 bulk of Na + efflux seems to be coupled to the ouabain-sensitive K + influx when the extraceilular K + concentration is relatively low (Yamamoto et al., 1979). The ouabain-sensitive 42K+ uptake observed with quiescent preparations incubated at 3 7 ° C was markedly higher than that observed at 30 ° C (fig. 6). Electrical stimulation at 3 Hz caused a significant increase in the specific 42K+ uptake. However, the degree of stimulation-induced increase in 42K+ uptake was relatively small at 37 °C (66 _+ 9% increase) compared to that observed at 3 0 ° C (156 + 22% increase). During 3 Hz stimulation, the ouabain sensitive 42K+ uptake observed at 3 7 ° C was still significantly higher than that observed at 30 ° C but the difference was relatively small. These results indicate that the leak sodium influx at 3 7 ° C was approximately twice that at 30°C, and the effect of electrical stimulation to enhance sodium influx was markedly smaller at the higher temperature.
4. Discussion
Under the conditions used in the present experiments, the rate of development of the positive inotropic effect of ouabain was slow at 30 o C and was dependent on the frequency of electrical stimulation. Elevation of the temperature to 37 o C caused a substantial increase in the rate of development of ouabain action and made the development of the glycoside's action less dependent on stimulation frequency. These results confirm those reported from previous studies: relatively strict beat dependence of the onset of glycoside action at lower temperatures and the lack of the strict beat dependence at higher temperatures (Koch-Weser and Blinks, 1962; Moran, 1967; Vincenzi, 1967; Byrne and Dresel, 1969; Koch-Weser, 1971; Park and Vincenzi, 1973, 1975; Bentfeld et al., 1977; Ebner and Reiter, 1977; Temma et al., 1982). Several hypotheses have been advanced to explain the beat or temperature dependence of the onset of glycoside actions. Park and Vincenzi (1973, 1975) have suggested that the access of the glycoside to its receptor sites requires transport by
a specific carrier system, and proposed that transport might be enhanced by membrane depolarization or at a higher temperature. Alternatively, beat dependence may result from a difference in the magnitude of the inotropic effect of the glycosides at various stimulation frequencies (Wilbrandt et al., 1953), frequency-dependent differences in action potential configuration or in calcium influx (Koch-Weser and Blinks, 1963) or from an additive effect of an increased sodium load by more frequent depolarization and glycoside-induced inhibition of the sodium pump (Reiter, 1981). Another hypothesis, however, appears more attractive: that the amount of intracellular Na t available to the sodium pump is the determinant of glycoside binding to sarcolemmal Na, K-ATPase, and that electrical stimulation or an elevation of the temperature increases the amount of Na + available to the sodium pump by increasing the sodium influx. In isolated Na,K-ATPase preparations, Na + enhances glycoside binding (Matsui and Schwartz, 1968). In intact heart muscle cells, intracellular Na + appears to be an absolute requirement for glycoside binding to sarcolemmal Na,K-ATPase and the development of the inotropic action (Akera et al., 1979; Linden and Brooker, 1980; Wiggins and Bentolila, 1980). Moreover, Na + ionophores or agents which increase sodium influx have been shown to cause a faster development of the positive inotropic action of the cardiac glycoside (Honerj~ger and Reiter, 1975; Akera et al., 1977; Yamamoto et al., 1980), with a concomitant faster binding of the glycoside to sarcolemmal Na,K-ATPase (Temma and Akera, 1982). Bodemann and Hoffman (1976), however, reported that an increase in intracellular Na ~ concentration failed to enhance ouabain binding to Na,K-ATPase in released erythrocyte ghost preparations. Thus, in order to test the above hypothesis, it was essential to examine whether beat dependence of the onset of glycoside action observed at low temperatures and the lack of strict beat dependence at higher temperatures corresponded to the depolarization-induced increases in sodium influx rates and therefore to glycoside binding to sarcolemmal Na,K-ATPase. Our previous study indicated that increasing the
231 amount of intracellular Na t available to the sodium pump, by increasing the frequency of electrical stimulation or adding monensin, grayanotoxin or batrachotoxin, alters glycoside binding to sarcolemmal Na,K-ATPase in isolated heart muscle preparations, in parallel with an enhancement of the rate of development of the positive inotropic action of ouabain (Temma and Akera, 1982). It should be noted that an enhancement of glycoside binding occurs in the absence of an apparent or marked increase in total intracellular Na +, indicating that an increase in turnover rate of the sodium pump is the cause of the enhanced glycoside binding. Conversely, a decrease in extracellular Na + concentration to 27 raM, which causes a marked decrease in the amount of Na + available to the sodium pump, abolished glycoside binding to sarcolemmal Na, K-ATPase and also the development of the positive inotropic effect of ouabain (Temma and Akera, 1983). In the present study, raising the temperature resulted in a greater fractional occupancy of the glycoside binding sites on Na, K-ATPase in atrial muscle preparations exposed to a fixed concentration of ouabain under quiescent conditions. Raising the temperature from 30 to 37 o c also enhanced the development of the contracture caused by toxic concentrations of ouabain, again indicating that the glycoside binds more rapidly to Na,K-ATPase at a higher temperature. Electrical stimulation enhanced glycoside binding irrespective of the temperature. However, the combined effect of electrical stimulation and an elevated temperature was not significantly different from what was observed with electrical stimulation alone. Consistent with this latter finding was the observation that the difference in the time required for the development of a half-maximal inotropic effect of ouabain was relatively unaffected by temperature when the preparations were stimulated at either 2 or 3 Hz. Thus, the effects of incubation temperature and electrical stimulation on glycoside binding to Na,K-ATPase in isolated atrial muscle preparations can explain the beat dependence of the onset of glycoside action at the lower temperature, and the relative lack of the beat dependence at a higher temperature. Glycoside binding to Na,K-ATPase may be en-
hanced by an elevation of temperature per se, independent of changes in sodium influx rates. For example, an elevation of temperature has been shown to increase the association and dissociation rate constants for the interaction between ouabain and Na, K-ATPase observed with isolated enzyme preparations (Erdmann, 1981). This and other possibilities cannot be ruled out. However, the present results indicate that the enhanced glycoside binding to Na,K-ATPase observed with quiescent preparations at a higher temperature and the faster development of the inotropic effect are accompanied by a greater sodium influx. In the present study, sodium influx was estimated indirectly from the ouabain-sensitive 42K+ uptake. Because of the significant back flux of Na +, i.e. substantial portion of the Na + entering the cell during a membrane excitation is pumped out before the next cycle, sodium influx cannot be estimated accurately from the rate of labelled Na + uptake. In isolated heart muscle preparations which are incubated in an equilibrated state without sodium loading, the rate of sodium influx must be equal to the rate of sodium efflux. If the bulk of the sodium efflux is due to N a + / K + transport coupled by the sodium pump, the ouabain-sensirive 42K+ uptake may then be used to estimate the rate of sodium influx (Akera et al., 1981). Several investigators have reported that the total K + flux was relatively insensitive to electrical stimulation up to 1 Hz. However, at a higher frequency and with reduced K + concentration in the medium, the ouabain-sensitive 42K + uptake clearly shows dependence on the frequency of electrical stimulation or the rate of sodium influx (Akera et al., 1981). Yamamoto et al. (1979) have also reported that the ouabain-sensitive 86Rb uptake by cardiac muscle preparations which were not sodium-loaded was greater at a higher temperature. Additionally, in the present study when the rate of sodium influx was increased in isolated atrial muscle preparations incubated at 3 0 ° C by the addition of either monensin or grayanotoxin to the incubation mixture the patterns of beat dependence of the development of the positive inotropic effect of ouabain became similar to those observed at a higher temperature in the absence of these agents.
232 Finally, the increase in sodium influx caused by electrical
stimulation
was
markedly
smaller
References
at
3 7 ° C t h a n a t 3 0 ° C . T h i s f i n d i n g is c o n s i s t e n t with the possibility that the frequency of electrical stimulation has relatively little effect on the rate of development of glycoside action at the higher temperature. The differences in the force of contraction at the time of ouabain addition were not the cause of d i f f e r e n c e s i n t h e r a t e o f d e v e l o p m e n t o f its a c t i o n . Under conditions similar to those of the present s t u d y , a l t e r a t i o n s i n C a 2+ c o n c e n t r a t i o n c a u s e d similar differences in force of contraction but failed to affect the rate of development of glycoside a c t i o n ( A k e r a et al., 1977). T h e d i f f e r e n c e s i n magnitude of the positive inotropic effect of o u a b a i n p e r se a l s o d o n o t s e e m t o b e t h e p r i m a r y c a u s e o f t h e d i f f e r e n c e s i n o n s e t r a t e o f its a c t i o n . When preparations were incubated at 37°C, the positive inotropic action of ouabain at each stimul a t i o n f r e q u e n c y w a s s m a l l e r t h a n t h a t a t 30 ° C. I n contrast, incubation of the atrial muscle preparations in the presence of either monensin or grayanotoxin resulted in a greater inotropic action o f o u a b a i n . I n e i t h e r case, t h e o n s e t o f g l y c o s i d e action was enhanced. I n c o n c l u s i o n , t h e a m o u n t o f N a + a v a i l a b l e to t h e s o d i u m p u m p is r e l a t i v e l y s m a l l a n d is t h e determinant of the rate of ouabain binding to the sodium pump when isolated atrial muscle preparations are incubated at relatively low temperatures and stimulated at a low frequency. At higher temperatures, the beat dependence of onset of the g l y c o s i d e a c t i o n is less p r o m i n e n t , b e c a u s e t h e r e s t i n g s o d i u m i n f l u x is h i g h e r a n d e l e c t r i c a l stimulation has a smaller influence on the rate of sodium influx.
Acknowledgements The author thanks Drs. Tai Akera and Theodore M. Brody for helpful advice and suggestions, and Mr. A.T. Linehan for competent technical assistance. This work was supported by U.S. Public Health Service grant HL-16052 from the National Heart, Lung, and Blood Institute.
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