Strophanthidin-Sensitive Sodium Fluxes in Metabolically Poisoned Frog Skeletal Muscle B R I A N G. K E N N E D Y and P A U L DE W E E R From the Department of Physiology and Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110 ABSTRACT Strophanthidin-sensitive and insensitive unidirectional fluxes of Na were measured in frog sartorius muscles whose internal Na levels were elevated by overnight storage in the cold. ATP levels were lowered, and ADP levels raised, by metabolic poisoning with either 2,4-dinitrofluorobenzene or iodoacetamide. Strophanthidin-sensitive Na efflux and influx both increased after poisoning, while strophanthidin-insensitive fluxes did not. The increase in efflux did not require the presence of external K but was greatly attenuated when Li replaced Na as the major external cation. Membrane potential was not markedly altered by 2,4dinitrofluorobenzene. These observations indicate that the sodium pump of frog skeletal muscle resembles that of squid giant axon and human erythrocyte in its ability to catalyze Na-Na exchange to an extent determined by intracellular ATP/ ADP levels. INTRODUCTION
Caldwell et al. (1960) showed that, in the p r e s e n c e o f cyanide, Na efflux f r o m squid giant axons b e c o m e s insensitive to the r e m o v a l o f external K a n d , instead, requires Na in the b a t h i n g m e d i u m . T h e y suggested that N a - N a e x c h a n g e occurs a f t e r metabolic inhibition. Later, B a k e r et al. (1969) d e m o n s t r a t e d a ouabain-sensitive Na influx in partially p o i s o n e d squid axons. De W e e r (1970, 1974) f u r t h e r characterized this m o d e o f e x c h a n g e a n d showed that p u m p catalyzed N a - N a e x c h a n g e is u n a f f e c t e d by changes in internal Pl, b u t increases with rising intracellular A D P levels. In u n t r e a t e d h u m a n red blood cells, G a r r a han a n d Glynn (1967) f o u n d a sizeable p u m p - m e d i a t e d N a - N a e x c h a n g e , which was inhibited by high levels o f external K. In addition, there was evidence that N a - N a e x c h a n g e increases with decreasing levels o f A T P . Glynn a n d H o f f m a n (1971) f u r t h e r showed that high levels o f ADP also p r o m o t e N a - N a e x c h a n g e . In view o f these findings in squid giant axons a n d h u m a n red blood cells, it s e e m e d i m p o r t a n t to d e t e r m i n e w h e t h e r the sodium p u m p o f muscle will e n g a g e in NaNa e x c h a n g e a f t e r elevation o f intracellular levels o f ADP. An N a - N a e x c h a n g e c o m p o n e n t o f p u m p activity in muscle, t h o u g h small, has b e e n well d o c u m e n t e d . Keynes a n d S t e i n h a r d t (1968) d e m o n s t r a t e d 20% inhibition by o u a b a i n o f b o t h influx a n d efflux o f Na in K-free media. H o r o w i c z et al. (1970) o b s e r v e d a ouabain-sensitive, e x t e r n a l N a - d e p e n d e n t c o m p o n e n t o f Na efflux in f r o g sartorius muscle. More recently, Sjodin a n d Beaug~ (1973), T H E JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 6 8 ,
1976 " pages 405-420
405
406
THE JOURNAL
OF GENERAL
PHYSIOLOGY
• VOLUME
68 •
1976
looking at net m o v e m e n t o f Na as well as unidirectional fluxes, c o n c l u d e d that their findings were consistent with the existence o f a p u m p - m e d i a t e d N a - N a exchange. O n the o t h e r h a n d , r e p o r t s c o n c e r n i n g the effects o f metabolic inhibition on N a fluxes in muscle have b e e n conflicting. Keynes a n d Maisel (1954) r e p o r t e d no m a r k e d changes in Na efflux a f t e r poisoning with cyanide plus iodoacetic acid. Using the s a m e protocol for inhibition, but w o r k i n g in a high K m e d i u m , Frazier a n d Keynes (1959) r e p o r t e d a decrease in efflux a n d a stimulation o f influx. Portela et al. (1974), h o w e v e r , r e p o r t e d decreases in efflux as well as influx after iodoacetic acid poisoning. Beaug~ a n d Sjodin (1975, 1976) m e a s u r e d Na efflux a f t e r t r e a t m e n t with azide, a k n o w n metabolic poison. A ouabain-sensitive increase in efflux, which was a t t e n u a t e d in a m e d i u m free o f Na but not abolished by r e m o v a l o f K, was observed. In the p r e s e n t study two metabolic inhibitors, 2 , 4 - d i n i t r o f l u o r o b e n z e n e (DNFB) a n d i o d o a c e t a m i d e (IAM), were used to lower the A T P / A D P ratio. Portela et al. (1974) have r e p o r t e d that iodoacetic acid, which blocks glycolysis by inhibition o f g l y c e r a l d e h y d e phosp h a t e d e h y d r o g e n a s e , does decrease the A T P / A D P ratio. I n f a n t e a n d Davies (1965) h a v e shown that 2 , 4 - d i n i t r o f l u o r o b e n z e n e inhibits all A T P g e n e r a t i o n in f r o g skeletal muscle: it inactivates creatine p h o s p h o k i n a s e , a n d also interferes with oxidative p h o s p h o r y l a t i o n a n d glycolysis. M o m m a e r t s and Wallner (1967) d e m o n s t r a t e d a decrease in the A T P / A D P ratio a f t e r application o f D N F B . METHODS
Muscle Preparation Northern Rana pipiens were kept at room temperature (approx 23°C) in an aquarium with running water, and fed thrice weekly. Size ranged from 3.5 to 7.5 cm while isolated sartorii ranged in weight from 20 to 80 mg. The muscles were examined for parasites and blood clots before use. For all experiments except those in which membrane potential was to be determined muscles were carefully dissected free and 6-0 surgical thread was tied to both tendons. The muscles were then mounted on wire frames and stored for approximately 24 h (range, 20-30 h) at 3°C in K-free Ringer's solution in order to raise their internal Na levels. Muscles used for membrane potential measurements were taken immediately after dissection and pinned into Petri dishes containing a base of polymer° ized Sylgard 184.
Solutions The composition of normal Ringer's fluid, (Na)Ri, was as follows (in millimoles/liter): NaCI 115; KCI 2.5; CaCI~ 1.8; Na2HPO4 1.08; and NaH2PO4 0.43. In K-free Ringer's, OK(Na)Ri, the KCI was omitted. In Na-free Ringer's, (Li)Ri, LiCI was substituted for NaCI on a mole for mole basis and potassium phosphate was substituted for the sodium phosphate. The Na, K-free, magnesium-substituted Ringer's, OK(Mg)Ri, contained: MgCI2 86.3; CaClz 1.8; and Tris phosphate buffer 1.5. The pH of all solutions was 7.3. DNFB was obtained from Sigma Chemical Co. (St. Louis, Mo.) and dissolved in the appropriate Ringer's solution by vigorous stirring for 1-2 h. DNFB (0.38 mM) and IAM (0.5 mM) solutions were always freshly made on the day of the experiment. Strophanthidin (40 ~M) was added directly to the appropriate Ringer's and dissolved by stirring overnight. Carrier-free 22Na was obtained in a neutral solution from New England Nuclear (Boston, Mass.) and added directly to the appropriate Ringer's (approximately 10 ~Ci/ml).
BRIAN KENNEDY AND PAUL DE WEER
Na-Na Exchange in Muscle
407
Efflux Experiments Muscles, attached to their wire frames, were placed in an OK(2ZNa)Ri labeling solution at 3°C for approximately 6 h (thus the last 6 h of the loading period were spent in labeled Ringer's). T h e paired muscles were then moved through a series of tubes containing 5 ml of unlabeled Ringer's at 23°C. T h e average efflux period in any one tube was 15 min. After an experiment, the muscle was placed in a platinum crucible and ashed at 500°C. T h e ash was dissolved in 1 N nitric acid, and the volume then brought to 5 ml. All tubes were counted in an Auto-gamma scintillation spectrometer (Packard I n s t r u m e n t Co., Inc., Downers Grove, I11.) to approximately 2% counting error. By back calculation, the total counts remaining in the muscle and the instantaneous rate coefficient for efflux could be calculated for each efflux period.
Influx Experiments Influx experiments employed paired muscles from the same frog. T h e control was preincubated in (Na)Ri while the experimental muscle was preincubated either for 30 min in (Na)Ri + DNFB or for 90 min in (Na)Ri + IAM. Muscles were next placed in [Z2Na]Ri for 15 rain. Washout was then followed as described in the previous section, except that muscles were blotted lightly and weighed before ashing. T h e washout solutions consisted of OK(Na)Ri plus 4 × 10-5 M strophanthidin plus 10-7 g/ml tetrodotoxin (TTX). Preliminary experiments had indicated that high-Na muscles occasionally twitched when placed in (Na)Ri + strophanthidin at room temperature. T o circumvent this problem all incubation, labeling, and washout solutions used in the influx experiments contained 10-7 g/ml T T X . Data were analyzed by plotting total counts remaining in the muscle semilogarithmically against time. Total counts gained d u r i n g the 15-min labeling period were d e t e r m i n e d by extrapolating the linear phase of washout to zero time. Influx is expressed as micromoles Na(grams wet weight)-lh -1.
Analysis for Total Na This procedure followed Sjodin and Henderson (1964). Intracellular Na was elevated as described, and muscles were then allowed to recover for 3 h at room temperature in (Na)Ri, rinsed for 15 min in OK(Mg)Ri to remove extracellular Na, lightly blotted on ashless filter paper, weighed, and ashed at 500°C. T h e ash was dissolved in 1 N nitric acid and assayed for total Na by atomic absorption spectrometry.
ATP and ADP Determination Muscles were Na loaded as usual. Muscle pairs were used: the control was incubated in (Na)Ri while the other m e m b e r of the pair was held for 30 rain in (Na)Ri + DNFB or for 90 rain in (Na)Ri + IAM. After incubation the muscles were weighed, frozen in liquid nitrogen, and pulverized with mortar and pestle in liquid N~. 2 vol of 0.1 N HCI in methanol were added to the powder at -20°C, followed by 7 vol of 0.3 N perchloric acid at 0°C. After centrifugation at 4,500 rpm for 20 min, an equal volume of neutralizing solution containing 0.25 M KOH, 0.15 M imidazole, and 0.15 M KCI was added to the supernate. Aliquots of the extract were assayed, in triplicate, for ATP and ADP on the same day they were extracted. T h e fluorometric assay procedures were taken from Lowry and Passonneau (1972).
Membrane Potential Determinations As noted previously, muscles used for m e m b r a n e potential determination were not Na loaded. Paired muscles were dissected and one was p i n n e d directly into the Petri dish; the other remained at room temperature in (Na)Ri until measurements on the first were complete. One muscle served as control, potential measurements being taken in (Na)Ri,
408
T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y " V O L U M E 6 8
"
1976
while measurements in the experimental muscle were taken in (Na)Ri + DNFB. Potentials were recorded as a series of penetrations taken over time, Glass microelectrodes of 10-20 Mlq resistance were used.
Statistical Analysis Values are reported as means + SEM. Except as noted, all P values were determined from two-tailed t-tests for paired groups. RESULTS
Na Efflux W a s h o u t o f e x t r a c e l l u l a r tuNa r e q u i r e d a b o u t 30 m i n . T h e rate c o n s t a n t t h e n r e m a i n e d stable for o v e r 90 rain, as s e e n in Fig. 1. T h e m e a n rate c o n s t a n t for 56 d e t e r m i n a t i o n s o f N a e f f l u x in (Na)Ri was 0.0160 -+ 0.0006 rain -1. T h i s v a l u e can be c o m p a r e d to 0.016 m i n -1 o b t a i n e d for N a - l o a d e d m u s c l e s by B e a u g ~ a n d Sjodin (1968) a n d 0.014 rain -1 o b t a i n e d by K e y n e s a n d S t e i n h a r d t (1968). A v e r age drift o v e r t i m e was 0.001 m i n -I p e r h. • (Na)Ri
(Na)Ri
o (No)Ri
I 0 K(No)Ri
+ DNFB
OK(No)R
+ DNFB
o
"o
x ;2 .c_ E I,z ¢z I--
o
o
0 o
o
o3
z o (.~
I 0
LU I.,,¢ OC
0
0
0 0
I
I
!
I
2
3
HOURS
FIe;ERE 1. Effects o f 0.38 mM DNFB on the rate constant for efflux of 22Na. The control (O) efflux was followed in (Na)Ri for 145 min, at which point 0.38 mM DNFB was applied. T h e paired muscle (©) displayed an unchanging rate constant in (Na)Ri for 30 min, after which it was exposed to OK(Na)Ri. When the rate constant had again stabilized, OK(Na)Ri + 0.38 mM DNFB was applied. The increase in Na efflux in DNFB did not require external K.
DNFB Effects on Na Efflux T h e effects o f D N F B w e r e d e t e r m i n e d in (Na)Ri, O K ( N a ) R i , a n d (Li)Ri. Fig. 1 s h o w s that w h e n a m u s c l e b a t h e d in ( N a ) R i (solid s y m b o l s ) is e x p o s e d to D N F B , a m a r k e d i n c r e a s e in the rate c o n s t a n t for N a e f f l u x o c c u r s . T h e s t i m u l a t i o n was transient, r e a c h i n g a p e a k value o f 1.5 t i m e s c o n t r o l level. In the paired m u s c l e , the e f f e c t o f D N F B in O K ( N a ) R i can be s e e n . R e m o v a l o f K p r o d u c e d a m a r k e d
BRIAN
KENNEDY
AND PAUL DE
WEER
409
Na-Na Exchange in Muscle
(56%) decrease in the rate constant for efflux. In six normal muscles the magnitude o f the rate constant in OK(Na)Ri relative to that in (Na)Ri was 0.47 + 0.05, comparable to the value of 0.48 reported by Keynes and Steinhardt (1968). T h e significant point for this study is that, even in a K-free medium, a marked DNFB-induced increase in Na efflux, with rate o f rise similar to that in (Na)Ri, is evident: peak response is more than double the base-line level. Clearly, the stimulating effect o f DNFB on Na efflux is larger in the absence o f external K than in its presence. A prompt, transient increase in Na efflux occurred upon replacement of external Na with Li, as expected for a sartorius muscle with elevated internal Na levels (see Horowicz et al. [1970] and Beaug6 and Ortiz [1972] for a discussion of this phenom e non) . In 16 muscles the average Na-free effect (ratio of rate constant in (Li)Ri to that in (Na)Ri) was 1.63 --- 0.045, similar to the ratios of 1.57, • o
{N0)Ri + DNFB
(No)Ri (No)Ri
I (Li)Ri
(U)Ri + DNFB
3"
,N 0
K n
c E
0
2-
0
8
o
• o
I-Z
0 Z 0
~
•°
V
•
•
•
I
o
•
•
~0
0
0 0
i,i p-
i
i
I
I
2
3
HOURS
FIGURE 2. Effect o f 0.38 mM DNFB and Li substitution on the rate constant for efflux of 22Na. DNFB was applied after 150 min of efflux. T h e control muscle (Q) was held in (Na)Ri while the paired muscle (O) was exposed to (Li)Ri after 75 min in (Na)Ri. T h e typical DNFB effect is markedly attenuated in the absence o f external Na.
r e por t ed by Keynes and Steinhardt (1968), and 1.52, obtained by Beaug6 and Sjodin (1968). Fig. 2 illustrates one experiment in which DNFB was applied to a muscle that had been exposed to (Li)Ri for 75 min. T h e DNFB effect is greatly attenuated compared to that observed in the paired control kept in (Na)Ri (solid symbols). Since a muscle in Na-free medium will be losing internal Na, the attenuation of the DNFB effect in (Li)Ri could conceivably be due to decreased levels o f internal Na rather than to the absence o f external Na. T o test this point five experiments were perform ed, one o f which is shown in Fig. 3. Paired muscles were used and both were exposed to (Li)Ri for approximately 90 min. One muscle, indicated by the filled symbols, was then ret urned to (Na)Ri just long enough to establish a stable efflux, whereupon both muscles were exposed
410
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
68
•
1976
to D N F B . A l t h o u g h b o t h muscles were d e p l e t e d o f internal Na, only the muscle b a t h e d in Na containing Ringer's exhibited the characteristic stimulation u p o n application o f D N F B . Finally, the s t r o p h a n t h i d i n sensitivity o f the D N F B - s t i m u l a t e d Na efflux was studied. Fig. 4 shows virtually no effect on the rate constant for Na efflux when D N F B is applied in the p r e s e n c e o f s t r o p h a n t h i d i n . ( T h e paired muscle, indicated with filled symbols, shows a n o r m a l D N F B effect in (Na)Ri.) Pooled data for all e x p e r i m e n t s c o n c e r n i n g the effects o f D N F B on the rate constant for 22Na efflux are p r e s e n t e d in Fig. 5. In these c o m p a r i s o n s , each muscle served as its own control, base-line efflux being d e t e r m i n e d i m m e d i a t e l y b e f o r e d r u g application. T h e rate constant in D N F B was taken as the p e a k value r e a c h e d a f t e r application o f the inhibitor. 23 d e t e r m i n a t i o n s were m a d e in o
No(Ri) N,"(Ri)
I
Li (Ri) (Li)Ri
I ( L i ) R i + DNFB I (No)Ri I (No)RI + DNFB
3"
I-z
0 2"
o'o
o~o
•
•
Z O ~: ~T"
~~ he-
:
•
I o o
I I
I 2 HOURS
o
o
°°
o
I 3
! 4
FIGURE 3. Effect of 0.38 mM DNFB and Li substitution on the rate constant for efflux of 22Na for low Na muscles. Both muscles were depleted of internal Na by exposure to (Li)Ri-the control muscle (C)) for 120 min, the paired experimental muscle (0) for 90 min. The experimental muscle was then re-exposed to (Na)Ri for 30 min before addition of 0.38 mM DNFB. Attenuation of the DNFB effect in the absence of external Na is not due to depletion of internal Na levels. (Na)Ri. T h e a v e r a g e effect o f D N F B application was a significant 50% increase in the rate constant f o r Na efflux (P < 0.001). In O K ( N a ) R i , D N F B also p r o d u c e d a significant 150% stimulation (P < 0.001). Also shown in Fig. 5 are pooled data for the D N F B effect in (Li)Ri. T h e effect, t h o u g h small, is significant at the 0.02 level. H o w e v e r , it should be n o t e d that the rate constant in (Li)Ri was still falling w h e n the inhibitor was applied. T h e control rate constant was thus estimated by e x t r a p o l a t i o n o f a falling base line; this p r o c e d u r e m a y i n t r o d u c e s o m e e r r o r . T h e results f r o m five control e x p e r i m e n t s p e r f o r m e d in N a - d e p l e t e d muscles are also shown. T h e D N F B stimulation o f Na efflux into (Na)Ri for these low Na fibers was similar to that o b s e r v e d in loaded fibers; the m e a n rate constants in the p r e s e n c e o f D N F B were not significantly d i f f e r e n t for the two g r o u p s . ( T h e P value, as d e t e r m i n e d by a t-test for i n d e p e n d e n t g r o u p s ,
BRIAN KENNEDY AND PAUL DE WEER
411
Na-Na Exchange in Muscle
was g r e a t e r t h a n 0.2). Finally, Fig. 5 also presents the p o o l e d data f o r six e x p e r i m e n t s on the effect o f D N F B on Na efflux in the p r e s e n c e o f s t r o p h a n t h i din. T h e m e a n rate constants are virtually identical ( P > 0.2). It should be n o t e d that the base-line efflux was still d e c r e a s i n g slightly w h e n the inhibitor was applied (as can be seen in Fig. 4); again, this m a d e evaluation o f small inhibitor i n d u c e d effects difficult. H e n c e , it is possible that D N F B did p r o d u c e a small decrease in strophanthidin-insensitive Na efflux. T h e conclusion is that the m a r k e d D N F B - i n d u c e d stimulation o f N a efflux is greatly r e d u c e d in the absence o f e x t e r n a l N a a n d abolished in the p r e s e n c e o f s t r o p h a n t h i d i n . Iodoacetamide Effects on N a Efflux
M a r k e d transient stimulation o f the rate constant f o r Na efflux was o b s e r v e d a f t e r application o f 0.5 m M I A M . I n the e x p e r i m e n t s h o w n in Fig. 6, stimulation (Na)Ri o (Na)Ri
(No)Ri + DNFB I(No)Ri +](No)RJ + DNFB + STROPH STROPH
•
:5 °
o ,'-'~ 2 E Iz IZ
O
I°
o 0
tel
In,-
o• o
0
o
o
I
I
I
i
2
3
HOURS
FIGURE 4. Effect of 0.38 mM DNFB and 4 x 10-5 M strophanthidin on the rate constant for efflux of 22Na. The control muscle (0) was held in (Na)Ri for 120 min and then exposed to (Na)Ri + DNFB. The experimental muscle (©) was held in (Na)Ri for 90 min and then exposed to 4 x 10-5 M strophanthidin for 30 min. DNFB was added in the presence of strophanthidin. The DNFB effect is strophanthidin sensitive. was a b o u t 60%. C o m p a r e d to D N F B , I A M acted with a m u c h longer delay, a v e r a g e time to p e a k b e i n g a b o u t 90 min. T h e effect o f I A M was less pron o u n c e d a n d m o r e variable than that o b s e r v e d with D N F B . Fig. 7 s u m m a r i z e s the results for efflux d e t e r m i n a t i o n s in I A M . 15 d e t e r m i n a t i o n s in (Na)Ri indicated an a v e r a g e 40% increase (P < 0.001) in rate constant d u e to I A M . Similar m e a s u r e m e n t s m a d e in K-free Ringer's show a twofold increase (P < 0.001); thus, as for D N F B t r e a t m e n t , the stimulation p r o d u c e d by I A M is not blocked by r e m o v a l o f K. T h e effect o f I A M is greatly a t t e n u a t e d , however, in an N a - f r e e m e d i u m . Finally, w h e n applied in the p r e s e n c e o f s t r o p h a n t h i d i n , I A M had no significant effect on the rate constant for Na efflux, which suggests that the e x t r a efflux is p u m p m e d i a t e d .
412
T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y ' V O L U M E
68
• 1976
Inhibitor Effects on Na Influx T h e a p p e a r a n c e o f a N a - d e p e n d e n t Na efflux in response to metabolic poisoning, as described above, leads one to expect the c o n c o m i t a n t induction o f a strophanthidin-sensitive Na influx. Increased Na influx into metabolically poisoned muscles was indeed f o u n d . Fig. 8 A illustrates a typical washout experim e n t after a 15-min period o f e x p o s u r e to ZZNa. A slow phase o f washout is evident in both muscles after 30 min, and extrapolation o f this phase to zero time EFFLUX RATE C O N S T A N T min-lxlO
-2
3
f~x
(Na)Ri n=23 P
Caldwell et al. (1960) showed that, in the p r e s e n c e o f cyanide, Na efflux f r o m squid giant axons b e c o m e s insensitive to the r e m o v a l o f external K a n d , instead, requires Na in the b a t h i n g m e d i u m . T h e y suggested that N a - N a e x c h a n g e occurs a f t e r metabolic inhibition. Later, B a k e r et al. (1969) d e m o n s t r a t e d a ouabain-sensitive Na influx in partially p o i s o n e d squid axons. De W e e r (1970, 1974) f u r t h e r characterized this m o d e o f e x c h a n g e a n d showed that p u m p catalyzed N a - N a e x c h a n g e is u n a f f e c t e d by changes in internal Pl, b u t increases with rising intracellular A D P levels. In u n t r e a t e d h u m a n red blood cells, G a r r a han a n d Glynn (1967) f o u n d a sizeable p u m p - m e d i a t e d N a - N a e x c h a n g e , which was inhibited by high levels o f external K. In addition, there was evidence that N a - N a e x c h a n g e increases with decreasing levels o f A T P . Glynn a n d H o f f m a n (1971) f u r t h e r showed that high levels o f ADP also p r o m o t e N a - N a e x c h a n g e . In view o f these findings in squid giant axons a n d h u m a n red blood cells, it s e e m e d i m p o r t a n t to d e t e r m i n e w h e t h e r the sodium p u m p o f muscle will e n g a g e in NaNa e x c h a n g e a f t e r elevation o f intracellular levels o f ADP. An N a - N a e x c h a n g e c o m p o n e n t o f p u m p activity in muscle, t h o u g h small, has b e e n well d o c u m e n t e d . Keynes a n d S t e i n h a r d t (1968) d e m o n s t r a t e d 20% inhibition by o u a b a i n o f b o t h influx a n d efflux o f Na in K-free media. H o r o w i c z et al. (1970) o b s e r v e d a ouabain-sensitive, e x t e r n a l N a - d e p e n d e n t c o m p o n e n t o f Na efflux in f r o g sartorius muscle. More recently, Sjodin a n d Beaug~ (1973), T H E JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 6 8 ,
1976 " pages 405-420
405
406
THE JOURNAL
OF GENERAL
PHYSIOLOGY
• VOLUME
68 •
1976
looking at net m o v e m e n t o f Na as well as unidirectional fluxes, c o n c l u d e d that their findings were consistent with the existence o f a p u m p - m e d i a t e d N a - N a exchange. O n the o t h e r h a n d , r e p o r t s c o n c e r n i n g the effects o f metabolic inhibition on N a fluxes in muscle have b e e n conflicting. Keynes a n d Maisel (1954) r e p o r t e d no m a r k e d changes in Na efflux a f t e r poisoning with cyanide plus iodoacetic acid. Using the s a m e protocol for inhibition, but w o r k i n g in a high K m e d i u m , Frazier a n d Keynes (1959) r e p o r t e d a decrease in efflux a n d a stimulation o f influx. Portela et al. (1974), h o w e v e r , r e p o r t e d decreases in efflux as well as influx after iodoacetic acid poisoning. Beaug~ a n d Sjodin (1975, 1976) m e a s u r e d Na efflux a f t e r t r e a t m e n t with azide, a k n o w n metabolic poison. A ouabain-sensitive increase in efflux, which was a t t e n u a t e d in a m e d i u m free o f Na but not abolished by r e m o v a l o f K, was observed. In the p r e s e n t study two metabolic inhibitors, 2 , 4 - d i n i t r o f l u o r o b e n z e n e (DNFB) a n d i o d o a c e t a m i d e (IAM), were used to lower the A T P / A D P ratio. Portela et al. (1974) have r e p o r t e d that iodoacetic acid, which blocks glycolysis by inhibition o f g l y c e r a l d e h y d e phosp h a t e d e h y d r o g e n a s e , does decrease the A T P / A D P ratio. I n f a n t e a n d Davies (1965) h a v e shown that 2 , 4 - d i n i t r o f l u o r o b e n z e n e inhibits all A T P g e n e r a t i o n in f r o g skeletal muscle: it inactivates creatine p h o s p h o k i n a s e , a n d also interferes with oxidative p h o s p h o r y l a t i o n a n d glycolysis. M o m m a e r t s and Wallner (1967) d e m o n s t r a t e d a decrease in the A T P / A D P ratio a f t e r application o f D N F B . METHODS
Muscle Preparation Northern Rana pipiens were kept at room temperature (approx 23°C) in an aquarium with running water, and fed thrice weekly. Size ranged from 3.5 to 7.5 cm while isolated sartorii ranged in weight from 20 to 80 mg. The muscles were examined for parasites and blood clots before use. For all experiments except those in which membrane potential was to be determined muscles were carefully dissected free and 6-0 surgical thread was tied to both tendons. The muscles were then mounted on wire frames and stored for approximately 24 h (range, 20-30 h) at 3°C in K-free Ringer's solution in order to raise their internal Na levels. Muscles used for membrane potential measurements were taken immediately after dissection and pinned into Petri dishes containing a base of polymer° ized Sylgard 184.
Solutions The composition of normal Ringer's fluid, (Na)Ri, was as follows (in millimoles/liter): NaCI 115; KCI 2.5; CaCI~ 1.8; Na2HPO4 1.08; and NaH2PO4 0.43. In K-free Ringer's, OK(Na)Ri, the KCI was omitted. In Na-free Ringer's, (Li)Ri, LiCI was substituted for NaCI on a mole for mole basis and potassium phosphate was substituted for the sodium phosphate. The Na, K-free, magnesium-substituted Ringer's, OK(Mg)Ri, contained: MgCI2 86.3; CaClz 1.8; and Tris phosphate buffer 1.5. The pH of all solutions was 7.3. DNFB was obtained from Sigma Chemical Co. (St. Louis, Mo.) and dissolved in the appropriate Ringer's solution by vigorous stirring for 1-2 h. DNFB (0.38 mM) and IAM (0.5 mM) solutions were always freshly made on the day of the experiment. Strophanthidin (40 ~M) was added directly to the appropriate Ringer's and dissolved by stirring overnight. Carrier-free 22Na was obtained in a neutral solution from New England Nuclear (Boston, Mass.) and added directly to the appropriate Ringer's (approximately 10 ~Ci/ml).
BRIAN KENNEDY AND PAUL DE WEER
Na-Na Exchange in Muscle
407
Efflux Experiments Muscles, attached to their wire frames, were placed in an OK(2ZNa)Ri labeling solution at 3°C for approximately 6 h (thus the last 6 h of the loading period were spent in labeled Ringer's). T h e paired muscles were then moved through a series of tubes containing 5 ml of unlabeled Ringer's at 23°C. T h e average efflux period in any one tube was 15 min. After an experiment, the muscle was placed in a platinum crucible and ashed at 500°C. T h e ash was dissolved in 1 N nitric acid, and the volume then brought to 5 ml. All tubes were counted in an Auto-gamma scintillation spectrometer (Packard I n s t r u m e n t Co., Inc., Downers Grove, I11.) to approximately 2% counting error. By back calculation, the total counts remaining in the muscle and the instantaneous rate coefficient for efflux could be calculated for each efflux period.
Influx Experiments Influx experiments employed paired muscles from the same frog. T h e control was preincubated in (Na)Ri while the experimental muscle was preincubated either for 30 min in (Na)Ri + DNFB or for 90 min in (Na)Ri + IAM. Muscles were next placed in [Z2Na]Ri for 15 rain. Washout was then followed as described in the previous section, except that muscles were blotted lightly and weighed before ashing. T h e washout solutions consisted of OK(Na)Ri plus 4 × 10-5 M strophanthidin plus 10-7 g/ml tetrodotoxin (TTX). Preliminary experiments had indicated that high-Na muscles occasionally twitched when placed in (Na)Ri + strophanthidin at room temperature. T o circumvent this problem all incubation, labeling, and washout solutions used in the influx experiments contained 10-7 g/ml T T X . Data were analyzed by plotting total counts remaining in the muscle semilogarithmically against time. Total counts gained d u r i n g the 15-min labeling period were d e t e r m i n e d by extrapolating the linear phase of washout to zero time. Influx is expressed as micromoles Na(grams wet weight)-lh -1.
Analysis for Total Na This procedure followed Sjodin and Henderson (1964). Intracellular Na was elevated as described, and muscles were then allowed to recover for 3 h at room temperature in (Na)Ri, rinsed for 15 min in OK(Mg)Ri to remove extracellular Na, lightly blotted on ashless filter paper, weighed, and ashed at 500°C. T h e ash was dissolved in 1 N nitric acid and assayed for total Na by atomic absorption spectrometry.
ATP and ADP Determination Muscles were Na loaded as usual. Muscle pairs were used: the control was incubated in (Na)Ri while the other m e m b e r of the pair was held for 30 rain in (Na)Ri + DNFB or for 90 rain in (Na)Ri + IAM. After incubation the muscles were weighed, frozen in liquid nitrogen, and pulverized with mortar and pestle in liquid N~. 2 vol of 0.1 N HCI in methanol were added to the powder at -20°C, followed by 7 vol of 0.3 N perchloric acid at 0°C. After centrifugation at 4,500 rpm for 20 min, an equal volume of neutralizing solution containing 0.25 M KOH, 0.15 M imidazole, and 0.15 M KCI was added to the supernate. Aliquots of the extract were assayed, in triplicate, for ATP and ADP on the same day they were extracted. T h e fluorometric assay procedures were taken from Lowry and Passonneau (1972).
Membrane Potential Determinations As noted previously, muscles used for m e m b r a n e potential determination were not Na loaded. Paired muscles were dissected and one was p i n n e d directly into the Petri dish; the other remained at room temperature in (Na)Ri until measurements on the first were complete. One muscle served as control, potential measurements being taken in (Na)Ri,
408
T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y " V O L U M E 6 8
"
1976
while measurements in the experimental muscle were taken in (Na)Ri + DNFB. Potentials were recorded as a series of penetrations taken over time, Glass microelectrodes of 10-20 Mlq resistance were used.
Statistical Analysis Values are reported as means + SEM. Except as noted, all P values were determined from two-tailed t-tests for paired groups. RESULTS
Na Efflux W a s h o u t o f e x t r a c e l l u l a r tuNa r e q u i r e d a b o u t 30 m i n . T h e rate c o n s t a n t t h e n r e m a i n e d stable for o v e r 90 rain, as s e e n in Fig. 1. T h e m e a n rate c o n s t a n t for 56 d e t e r m i n a t i o n s o f N a e f f l u x in (Na)Ri was 0.0160 -+ 0.0006 rain -1. T h i s v a l u e can be c o m p a r e d to 0.016 m i n -1 o b t a i n e d for N a - l o a d e d m u s c l e s by B e a u g ~ a n d Sjodin (1968) a n d 0.014 rain -1 o b t a i n e d by K e y n e s a n d S t e i n h a r d t (1968). A v e r age drift o v e r t i m e was 0.001 m i n -I p e r h. • (Na)Ri
(Na)Ri
o (No)Ri
I 0 K(No)Ri
+ DNFB
OK(No)R
+ DNFB
o
"o
x ;2 .c_ E I,z ¢z I--
o
o
0 o
o
o3
z o (.~
I 0
LU I.,,¢ OC
0
0
0 0
I
I
!
I
2
3
HOURS
FIe;ERE 1. Effects o f 0.38 mM DNFB on the rate constant for efflux of 22Na. The control (O) efflux was followed in (Na)Ri for 145 min, at which point 0.38 mM DNFB was applied. T h e paired muscle (©) displayed an unchanging rate constant in (Na)Ri for 30 min, after which it was exposed to OK(Na)Ri. When the rate constant had again stabilized, OK(Na)Ri + 0.38 mM DNFB was applied. The increase in Na efflux in DNFB did not require external K.
DNFB Effects on Na Efflux T h e effects o f D N F B w e r e d e t e r m i n e d in (Na)Ri, O K ( N a ) R i , a n d (Li)Ri. Fig. 1 s h o w s that w h e n a m u s c l e b a t h e d in ( N a ) R i (solid s y m b o l s ) is e x p o s e d to D N F B , a m a r k e d i n c r e a s e in the rate c o n s t a n t for N a e f f l u x o c c u r s . T h e s t i m u l a t i o n was transient, r e a c h i n g a p e a k value o f 1.5 t i m e s c o n t r o l level. In the paired m u s c l e , the e f f e c t o f D N F B in O K ( N a ) R i can be s e e n . R e m o v a l o f K p r o d u c e d a m a r k e d
BRIAN
KENNEDY
AND PAUL DE
WEER
409
Na-Na Exchange in Muscle
(56%) decrease in the rate constant for efflux. In six normal muscles the magnitude o f the rate constant in OK(Na)Ri relative to that in (Na)Ri was 0.47 + 0.05, comparable to the value of 0.48 reported by Keynes and Steinhardt (1968). T h e significant point for this study is that, even in a K-free medium, a marked DNFB-induced increase in Na efflux, with rate o f rise similar to that in (Na)Ri, is evident: peak response is more than double the base-line level. Clearly, the stimulating effect o f DNFB on Na efflux is larger in the absence o f external K than in its presence. A prompt, transient increase in Na efflux occurred upon replacement of external Na with Li, as expected for a sartorius muscle with elevated internal Na levels (see Horowicz et al. [1970] and Beaug6 and Ortiz [1972] for a discussion of this phenom e non) . In 16 muscles the average Na-free effect (ratio of rate constant in (Li)Ri to that in (Na)Ri) was 1.63 --- 0.045, similar to the ratios of 1.57, • o
{N0)Ri + DNFB
(No)Ri (No)Ri
I (Li)Ri
(U)Ri + DNFB
3"
,N 0
K n
c E
0
2-
0
8
o
• o
I-Z
0 Z 0
~
•°
V
•
•
•
I
o
•
•
~0
0
0 0
i,i p-
i
i
I
I
2
3
HOURS
FIGURE 2. Effect o f 0.38 mM DNFB and Li substitution on the rate constant for efflux of 22Na. DNFB was applied after 150 min of efflux. T h e control muscle (Q) was held in (Na)Ri while the paired muscle (O) was exposed to (Li)Ri after 75 min in (Na)Ri. T h e typical DNFB effect is markedly attenuated in the absence o f external Na.
r e por t ed by Keynes and Steinhardt (1968), and 1.52, obtained by Beaug6 and Sjodin (1968). Fig. 2 illustrates one experiment in which DNFB was applied to a muscle that had been exposed to (Li)Ri for 75 min. T h e DNFB effect is greatly attenuated compared to that observed in the paired control kept in (Na)Ri (solid symbols). Since a muscle in Na-free medium will be losing internal Na, the attenuation of the DNFB effect in (Li)Ri could conceivably be due to decreased levels o f internal Na rather than to the absence o f external Na. T o test this point five experiments were perform ed, one o f which is shown in Fig. 3. Paired muscles were used and both were exposed to (Li)Ri for approximately 90 min. One muscle, indicated by the filled symbols, was then ret urned to (Na)Ri just long enough to establish a stable efflux, whereupon both muscles were exposed
410
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
68
•
1976
to D N F B . A l t h o u g h b o t h muscles were d e p l e t e d o f internal Na, only the muscle b a t h e d in Na containing Ringer's exhibited the characteristic stimulation u p o n application o f D N F B . Finally, the s t r o p h a n t h i d i n sensitivity o f the D N F B - s t i m u l a t e d Na efflux was studied. Fig. 4 shows virtually no effect on the rate constant for Na efflux when D N F B is applied in the p r e s e n c e o f s t r o p h a n t h i d i n . ( T h e paired muscle, indicated with filled symbols, shows a n o r m a l D N F B effect in (Na)Ri.) Pooled data for all e x p e r i m e n t s c o n c e r n i n g the effects o f D N F B on the rate constant for 22Na efflux are p r e s e n t e d in Fig. 5. In these c o m p a r i s o n s , each muscle served as its own control, base-line efflux being d e t e r m i n e d i m m e d i a t e l y b e f o r e d r u g application. T h e rate constant in D N F B was taken as the p e a k value r e a c h e d a f t e r application o f the inhibitor. 23 d e t e r m i n a t i o n s were m a d e in o
No(Ri) N,"(Ri)
I
Li (Ri) (Li)Ri
I ( L i ) R i + DNFB I (No)Ri I (No)RI + DNFB
3"
I-z
0 2"
o'o
o~o
•
•
Z O ~: ~T"
~~ he-
:
•
I o o
I I
I 2 HOURS
o
o
°°
o
I 3
! 4
FIGURE 3. Effect of 0.38 mM DNFB and Li substitution on the rate constant for efflux of 22Na for low Na muscles. Both muscles were depleted of internal Na by exposure to (Li)Ri-the control muscle (C)) for 120 min, the paired experimental muscle (0) for 90 min. The experimental muscle was then re-exposed to (Na)Ri for 30 min before addition of 0.38 mM DNFB. Attenuation of the DNFB effect in the absence of external Na is not due to depletion of internal Na levels. (Na)Ri. T h e a v e r a g e effect o f D N F B application was a significant 50% increase in the rate constant f o r Na efflux (P < 0.001). In O K ( N a ) R i , D N F B also p r o d u c e d a significant 150% stimulation (P < 0.001). Also shown in Fig. 5 are pooled data for the D N F B effect in (Li)Ri. T h e effect, t h o u g h small, is significant at the 0.02 level. H o w e v e r , it should be n o t e d that the rate constant in (Li)Ri was still falling w h e n the inhibitor was applied. T h e control rate constant was thus estimated by e x t r a p o l a t i o n o f a falling base line; this p r o c e d u r e m a y i n t r o d u c e s o m e e r r o r . T h e results f r o m five control e x p e r i m e n t s p e r f o r m e d in N a - d e p l e t e d muscles are also shown. T h e D N F B stimulation o f Na efflux into (Na)Ri for these low Na fibers was similar to that o b s e r v e d in loaded fibers; the m e a n rate constants in the p r e s e n c e o f D N F B were not significantly d i f f e r e n t for the two g r o u p s . ( T h e P value, as d e t e r m i n e d by a t-test for i n d e p e n d e n t g r o u p s ,
BRIAN KENNEDY AND PAUL DE WEER
411
Na-Na Exchange in Muscle
was g r e a t e r t h a n 0.2). Finally, Fig. 5 also presents the p o o l e d data f o r six e x p e r i m e n t s on the effect o f D N F B on Na efflux in the p r e s e n c e o f s t r o p h a n t h i din. T h e m e a n rate constants are virtually identical ( P > 0.2). It should be n o t e d that the base-line efflux was still d e c r e a s i n g slightly w h e n the inhibitor was applied (as can be seen in Fig. 4); again, this m a d e evaluation o f small inhibitor i n d u c e d effects difficult. H e n c e , it is possible that D N F B did p r o d u c e a small decrease in strophanthidin-insensitive Na efflux. T h e conclusion is that the m a r k e d D N F B - i n d u c e d stimulation o f N a efflux is greatly r e d u c e d in the absence o f e x t e r n a l N a a n d abolished in the p r e s e n c e o f s t r o p h a n t h i d i n . Iodoacetamide Effects on N a Efflux
M a r k e d transient stimulation o f the rate constant f o r Na efflux was o b s e r v e d a f t e r application o f 0.5 m M I A M . I n the e x p e r i m e n t s h o w n in Fig. 6, stimulation (Na)Ri o (Na)Ri
(No)Ri + DNFB I(No)Ri +](No)RJ + DNFB + STROPH STROPH
•
:5 °
o ,'-'~ 2 E Iz IZ
O
I°
o 0
tel
In,-
o• o
0
o
o
I
I
I
i
2
3
HOURS
FIGURE 4. Effect of 0.38 mM DNFB and 4 x 10-5 M strophanthidin on the rate constant for efflux of 22Na. The control muscle (0) was held in (Na)Ri for 120 min and then exposed to (Na)Ri + DNFB. The experimental muscle (©) was held in (Na)Ri for 90 min and then exposed to 4 x 10-5 M strophanthidin for 30 min. DNFB was added in the presence of strophanthidin. The DNFB effect is strophanthidin sensitive. was a b o u t 60%. C o m p a r e d to D N F B , I A M acted with a m u c h longer delay, a v e r a g e time to p e a k b e i n g a b o u t 90 min. T h e effect o f I A M was less pron o u n c e d a n d m o r e variable than that o b s e r v e d with D N F B . Fig. 7 s u m m a r i z e s the results for efflux d e t e r m i n a t i o n s in I A M . 15 d e t e r m i n a t i o n s in (Na)Ri indicated an a v e r a g e 40% increase (P < 0.001) in rate constant d u e to I A M . Similar m e a s u r e m e n t s m a d e in K-free Ringer's show a twofold increase (P < 0.001); thus, as for D N F B t r e a t m e n t , the stimulation p r o d u c e d by I A M is not blocked by r e m o v a l o f K. T h e effect o f I A M is greatly a t t e n u a t e d , however, in an N a - f r e e m e d i u m . Finally, w h e n applied in the p r e s e n c e o f s t r o p h a n t h i d i n , I A M had no significant effect on the rate constant for Na efflux, which suggests that the e x t r a efflux is p u m p m e d i a t e d .
412
T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y ' V O L U M E
68
• 1976
Inhibitor Effects on Na Influx T h e a p p e a r a n c e o f a N a - d e p e n d e n t Na efflux in response to metabolic poisoning, as described above, leads one to expect the c o n c o m i t a n t induction o f a strophanthidin-sensitive Na influx. Increased Na influx into metabolically poisoned muscles was indeed f o u n d . Fig. 8 A illustrates a typical washout experim e n t after a 15-min period o f e x p o s u r e to ZZNa. A slow phase o f washout is evident in both muscles after 30 min, and extrapolation o f this phase to zero time EFFLUX RATE C O N S T A N T min-lxlO
-2
3
f~x
(Na)Ri n=23 P
