Action Potential Duration in Frog Heart

On the Effects of Divalent Cations

Ethylene Glycol-bis-(/3-Aminoethyl

N,N,N',N'-Tetraacetate

on

Action Potential Duration in Frog Heart

and

Ether)

D A V I D J. M I L L E R and A L F R E D M C ) R C H E N

From the Department of Cell Physiology, Ruhr University, 4630 Bochum, Federal Republic of Germany. Dr. Miller's present address is the Department of Physiology, University of Glasgow, Glasgow G12 8QQ, Scotland.

A B $ T R A C T Resting and action potentials were recorded from superfused strips of frog ventricle. Reducing the bathing calcium concentration ([Ca2+]0) with or

without ethylene glycol-bis(~-aminoethyl

ether)N,N,N',N'-tetraacetate

prolongs the action potential (AP). T h e change in the duration of the AP extends over many minutes, but is rapidly reversed by restoring calcium ions. Other changes (e.g., in resting potential and overshoot) are, however, only more slowly reversed. Reducing [Ca2+]0 with 0.2, 2, or 5 mM EGTA produces progressively greater prolongation of AP; maximum values were well in excess of 1 rain. This prolongation can be reversed by other divalent cations in EGTA (Mg 2+, Sr 2+) or Ca-free (Mn 2+) solutions, or by acetylcholine. Barium ions increase AP duration in keeping with their known effect on potassium conductance. D600, which blocks the slow inward current in cardiac muscle, is without effect on the action potentials recorded in EGTA solutions, or on the time course and extent of the recovery to normal duration upon restoring calcium ions. It is concluded that divalent cations exert an influence on membrane potassium conductance extracellularly in frog heart. T h e cell membrane does not become excessively "leaky" in EGTA solutions.

I N T R O D U C T I O N

It is c o m m o n l y o b s e r v e d that the d u r a t i o n o f the c a r d i a c action p o t e n t i a l (AP) is i n f l u e n c e d by t h e b a t h i n g calcium c o n c e n t r a t i o n . G e n e r a l l y , a r e d u c t i o n in calcium p r o l o n g s t h e d u r a t i o n o f the AP, a l t h o u g h this e f f e c t is, f o r e x a m p l e , d e p e n d e n t u p o n stimulus f r e q u e n c y (Bassingthwaighte et al., 1976). A n u m b e r o f a u t h o r s h a v e r e p o r t e d t h a t calcium r e d u c t i o n by the u s e o f c h e l a t i n g a g e n t s , e.g., e t h y l e n e d i a m i n e tetraacetic acid, a n d e t h y l e n e glycol-bis-(fl-aminoethyl

ether)N,N,N',N'-tetraacetate

tentials ( T r i t t h a r t et al., 1973), s o m e t i m e s o f several s e c o n d s ' d u r a t i o n ( H o f f m a n a n d Suckling, 1956; R o u g i e r et al., 1969). T h i s effect is o f i m m e d i a t e interest b e c a u s e b o t h intracellular a n d e x t r a c e l l u l a r calcium levels h a v e b e e n f o u n d to affect p o t a s s i u m c o n d u c t a n c e in a n u m b e r o f cell types ( M e e c h , 1974; M e e c h a n d S t a n d e n , 1975; I s e n b e r g , 1975; Kass a n d T s i e n , 1975, 1976; Lew a n d THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 71. 1978 " pages 47-67 47

4 8 T H E J O U RN A L OF GENERAL PHYSIOLOGY • VOLUME 7 1 • 1 9 7 8

F e r r e i r a , 1976), quite a p a r t f r o m the d e p e n d e n c e o f t h e "slow i n w a r d c u r r e n t " o f cardiac muscle o n t h e b a t h i n g calcium level (see R e u t e r , 1973).

T h e effect o f e x t r e m e calcium r e d u c t i o n o n h e a r t m u s c l e is also i n t e r e s t i n g since, especially in E D T A solutions, the m e m b r a n e is r e p o r t e d to b e c o m e highly p e r m e a b l e , allowing f r e e r m o v e m e n t o f ions a n d l a r g e r molecules into the cell ( W i n e g r a d , 1971). Until n o w t h e r e has b e e n n o detailed o b s e r v a t i o n o f tlae time c o u r s e o f t h e effects o f E G T A o n the h e a r t o r o f the possible influence o f divalent cations o t h e r t h a n calcium a n d m a g n e s i u m o n the r e s p o n s e s .

T h e p r e s e n t e x p e r i m e n t s o n f r o g ventricle fibers w e r e u n d e r t a k e n to p r o v i d e i n f o r m a t i o n a b o u t the effects o f E G T A - c o n t a i n i n g solutions o n the h e a r t u n d e r precisely d e f i n e d conditions (e.g., e x t r a c e l l u l a r e x c h a n g e , p H , e x t r a c e l l u l a r divalent cation c o n c e n t r a t i o n , IX2+]0). Special a t t e n t i o n was p a i d to o b t a i n i n g c o n t i n u o u s r e c o r d i n g s f r o m individual cells t h r o u g h o u t a s e q u e n c e o f solution c h a n g e s . T h e results show that r e s t i n g a n d action potentials a r e well m a i n t a i n e d in divalent c a t i o n - p o o r m e d i a , a l t h o u g h the d u r a t i o n o f the AP is d r a m a t i c a l l y increased. T h e o b s e r v a t i o n s indicate t h a t the e x t r a c e l l u l a r divalent cation c o n c e n t r a t i o n IX2+]0 affects the t i m e o f onset o f r e p o l a r i z a t i o n in f r o g ventricle. C h a n g e s in [Ca~+]t a r e c o n c l u d e d to be o f little c o n s e q u e n c e f o r the d u r a t i o n o f AP u n d e r the p r e s e n t conditions (very low [Ca2+]0 ).

A p r e l i m i n a r y r e p o r t o f s o m e o f these results has b e e n p u b l i s h e d (Miller, 1976).

M A T E R I A L S A N D M E T H O D S Pre#aration and Mounting Procedure

A total of 20 preparations were used which comprised either a single trabeculum or a

bundle of trabeculae from the ventricle of the frog liana esculenta, with diameters

between 200 and 700/~m and length of 2-4 ram. The muscle was mounted in a chamber similar to that of Chapman and Tunstall (1971) where one end of the muscle was tied to a fixed hook and the other to the beam of a force transducer (Endevco, 8107/20). The muscle was regularly stimulated (Devices Digitimer) generally at 4 min -~, with square pulses (field stimulation, duration 2 ms and twice-threshold intensity). The superfusing solutions could be rapidly exchanged by means of a threeway tap (chamber dead space clearance <0.1 s).

Superfusing Solutions

The muscle was continuously superfused with the appropriate bathing solutions whose compositions are given in Table I. The preparation was dissected and mounted for initial superfusion in 2 mM Ca Ringer solution. A lower calcium level (0.2 raM) was used as standard to reduce contractile strength and facilitate the prolonged maintenance of the microelectrode in individual cells. The 2 mM Ca Ringer solution was occasionally reapplied for several minutes, particularly after long exposures to EGTA, to provide a check that no irreversible changes had occurred.

The free concentration of the divalent cations in the EGTA-containing solutions was calculated with a knowledge of the contamination level for calcium (c. 2" 10 .5 M) and of the appropriate binding constants for the various divalent cations to EGTA (see Miller and Moisescu, 1976; Sill~n and Martell, 1964). Importance is attached to the precise definition of the experimental pH in all solutions so that [X2+]0 may be calculated (see Table 1). Although Tris is a relatively poor buffer at pH 7.0, the rapid superfusion of a

MILLER AND M6RCnEN Divalent Cations and the Cardia~ Action Potential 49 s m a l l p r e p a r a t i o n e n s u r e s s u f f i c i e n t b u f f e r c a p a c i t y . E x p e r i m e n t s w e r e m a d e a t 2 0 . 5 -+ 0 . 5 " C .

Recording and Evaluation o f Data

S t a n d a r d m i c r o e l e c t r o d e t e c h n i q u e w a s u s e d . I n t r a c e l l u l a r p o t e n t i a l s w e r e m e a s u r e d v i a 3 M K C l - f i l l e d g l a s s m i c r o e l e c t r o d e s ( r e s i s t a n c e 1 0 - 1 8 M f l , t i p p o t e n t i a l s 10 m V o r l e s s w i t h c a p a c i t y c o m p e n s a t i o n ) . P o t e n t i a l s w e r e r e c o r d e d w i t h e i t h e r a n o s c i l l o s c o p e c a m e r a o r a U V c h a r t r e c o r d e r ( g a l v a n o m e t e r r e s p o n s e f i a t to 1 k H z , m a x i m u m d e f l e c t i o n 12 c m ) . E a c h s e r i e s o f a c t i o n p o t e n t i a l s s h o w n o r e v a l u a t e d w a s o b t a i n e d d u r i n g a s i n g l e p e n e t r a t i o n . A s f a r a s p o s s i b l e , a w h o l e s e r i e s o f s o l u t i o n c h a n g e s w a s c o m p l e t e d w i t h c o n t i n u o u s r e c o r d i n g f r o m o n e c e l l a n d t h e q u a n t i t a t i v e d a t a w e r e o b t a i n e d u n d e r t h e s e T A B L E I C O M P O S I T I O N O F T H E P E R F U S I O N S O L U T I O N S

Solution CaClz Mg, CIt SrCI, EGTA [CaS+]e [S&÷]o [Mga+]o

mM (added) M (calculated) N o r m a l R i n g e r ' s 2 o r 0.2 . . . . C a - f r e e . . . . 2 . 1 0 -s* - - 0.2 m M E G T A - - - 0.2 2.2" 10 -s - - 0.2 m M E G T A + Mg - 0.2 - 0.2 2.25" 10 -8 - 1.98.10 -4 - 1 . 0 - 0.2 2.33" 1 0 - 8 - 0.99.10 -s - - 5.0 - 0.2 2.74" 10 -8 -- 4.97" 10 -s 2.0 m M E G T A - - - 2.0 2.02" 10 -D - - 2.0 m M E G T A + Sr -- -- 0.4 2.0 2.51" 10 -Q 1.58- 10 -~ - 5.0 m M E G T A -- - - 5.0 8.0" 10 -1° - -

All solutions c o n t a i n e d (raM): NaC! 117, KC1 3, Tris 2.5, glucose 5, in glass-distilled water. T h e d i v a l e n t cations w e r e varied as above.

T h e free m a g n e s i u m level in t h e E G T A free solutions was e s t i m a t e d to be ~10 -s M.

Acetylcholine, a t r o p i n e sulfate, BaCI~, a n d MnCi2 were a d d e d as r e q u i r e d f r o m stock solutions, t h e latter two s u b s t a n c e s only to C a - f r e e R i n g e r . All solutions were a d j u s t e d to a final p H o f 7.00 with HCI o r N a O H . D600 (racemate) was t h e k i n d gift o f Knoll A G . , L u d w i g s h a f e n , F e d e r a l Republic o f G e r m a n y .

* V a l u e o b t a i n e d by f l a m e p h o t o m e t r y a n d titration against E G T A (see Miller a n d Moisescu, 1976).

c o n d i t i o n s . A c t i o n p o t e n t i a l d u r a t i o n w a s m e a s u r e d a s t h e t i m e t o 9 0 % r e p o l a r i z a t i o n e x c e p t i n a f e w c a s e s w h e r e a n a f t e r p o t e n t i a l d e v e l o p e d i n t h e p r e s e n c e o f E G T A ( s e e F i g . 3), s o t h a t f a s t r e p o l a r i z a t i o n r e p r e s e n t e d o n l y a b o u t 8 0 % o f t h e t o t a l . D u r a t i o n w a s t h e n m e a s u r e d to 8 0 % o r 7 0 % r e p o l a r i z a t i o n , a l t h o u g h t h e d i f f e r e n c e s i n t r o d u c e d w e r e i n s i g n i f i c a n t as t h e f a s t p h a s e o f r e p o l a r i z a t i o n r e m a i n e d s t e e p u n d e r all c o n d i t i o n s t e s t e d . T h e r e s t i n g p o t e n t i a l w a s t a k e n as t h a t i m m e d i a t e l y b e f o r e t h e a c t i o n p o t e n t i a l u p s t r o k e . A l l t h e e f f e c t s s t u d i e d w e r e f u l l y r e v e r s i b l e . I n c a s e s w h e r e A P d u r a t i o n e x c e e d e d 15 s it w a s p o i n t l e s s to m a i n t a i n t h e s t i m u l u s f r e q u e n c y a t 4 m i n -1, s o t h e r a t e w a s r e d u c e d a s n e c e s s a r y . I n s e v e r a l f i g u r e s t h e a l t e r a t i o n s i n d u r a t i o n o f A P a r e p l o t t e d o n s e m i l o g a r i t h m i c a x e s a g a i n s t t i m e . T h i s s e r v e s to e x a g g e r a t e a n d c l a r i f y t h e s m a l l c h a n g e s o c c u r r i n g a t t h e b e g i n n i n g o r e n d o f t h e t i m e c o u r s e s i l l u s t r a t e d .

5 0 T H E J O U R N A L O F G E N E R A L P H Y S I O L O G Y " V O L U M E 7 1 " 1 9 7 8

R E S U L T S

(Normal) Ringer Solution (0.2 mM Ca)

T h e initial levels (+- SD) for the resting potential, o v e r s h o o t , a n d A P d u r a t i o n after at least 30 min in n o r m a l Ringer solution were: - 7 4 . 8 +- 6.2 mV; +12.4 - 8.6 mV; a n d 0.80 + 0.19 s, respectively (47 cells). This r e p r e s e n t e d a stable state. T h e s e values are in reasonable a g r e e m e n t with those o f o t h e r a u t h o r s , e.g., N i e d e r g e r k e a n d O r k a n d (1966), a l t h o u g h the resting potential is slightly lower, possibly reflecting an effect o f p r o l o n g e d e x p o s u r e to the relatively low calcium c o n c e n t r a t i o n .

Calcium-Free Solutions

Mines (1913) a n d Daly a n d Clarke (1921) r e p o r t e d that the AP persists in calcium-free solutions even t h o u g h contraction fails. This f i n d i n g has f r e q u e n t l y been c o n f i r m e d a n d Fig. 1 a shows c o m p a r a b l e findings in the p r e s e n t study.

0

mV

0.1mN

2s

raM'1

'O'Ca

0.2 Ca

a.p. 2"OJ

~

~

(~)

F

i-s

I

~'~'~'m

I

I

I

I

I

I

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FmuRE 1. Action potentials in Ca-free Ringer's solution. (a) Upper trace, action potentials recorded in one cell immediately before, and 8 minutes after, the solution change (AP prolonged). Lower trace, tension output immediately before, and 15, 30 (---), and 120 s after solution change. (b) Action potential duration (ordinate) for successive action potentials plotted against time (abscissa) after switching from 0.2 mM Ca to Ca free Ringer solution. After 11 min 45 s 0.2 mM Ca was restored. The solid curve is an exponential with a half-time of 6 s (stimulus rate 4 min-1).

MILLER AND MORCHEN Divalent Cations and the Cardiac Action Potential 51 T h e action potentials s h o w n were r e c o r d e d in the same cell i m m e d i a t e l y b e f o r e a n d 8 min a f t e r c h a n g i n g f r o m 0.2 mM Ca to Ca-"free" solution which p r o l o n g e d the d u r a t i o n .

D u r i n g the first minutes o f p e r f u s i o n with zero Ca Ringer's solution the AP u n d e r g o e s several changes which may be s u m m a r i z e d as follows. T h e early phase o f the o v e r s h o o t falls rapidly by several millivohs (Fig. 1 a), the plateau in general is also r e d u c e d b u t assumes a m o r e "horizontal" f o r m as the onset o f the fast repolarization is progressively delayed. AP d u r a t i o n increases by between 20% a n d 200% c o m p a r e d to that in n o r m a l R i n g e r solution. This c h a n g e in d u r a t i o n continues o v e r several minutes a n d follows a n o n e x p o n e n t i a l time course (Fig. lb). In some cells the first few action potentials in Ca-free (and E G T A ) Ringer solution were slightly s h o r t e r than the value immediately b e f o r e addition o f the substance (Rougier et al., 1969). T h e resting potential is little a f f e c t e d , a slight depolarization ( - 5 mV) being n o t e d in some cells.

T h e r e t u r n to 0.2 mM Ca Ringer solution very rapidly r e d u c e s the d u r a t i o n o f the AP (t i for Fig. lb is 6 s) with a single e x p o n e n t i a l time course. T h e n o r m a l action potential f o r m is, h o w e v e r , not achieved f o r several minutes (see later). Twitch tension falls to a very low level in these calcium-free solutions ([Ca2+]0 = 2.10 -s M) with an a p p r o x i m a t e l y e x p o n e n t i a l time c o u r s e (Fig. l a ) . In the case o f Fig. 1 b ( d i f f e r e n t p r e p a r a t i o n ) tension was n o l o n g e r detected with the first stimulus (15 s) a f t e r the solution c h a n g e so that the time course could not be accurately d e t e r m i n e d (i.e. t t <4 s). In thicker p r e p a r a t i o n s tension fell m o r e slowly (e.g., Fig. 2). T h e half-time for the loss o f tension thus varied considerably f r o m o n e p r e p a r a t i o n to a n o t h e r but was always slower than the fastest phase (t t - 3 s) o b t a i n e d with the t h i n n e r atrial trabeculae ( C h a p m a n , 1971a; Miller, 1975) o r with the s u p e r f u s e d half-ventricle m e t h o d ( C h a p m a n a n d Miller, 1974). In n o r m a l Ringer solution, twitch tension recovers m o r e slowly (see Fig. 2b, d), d e p e n d i n g u p o n the d u r a t i o n and e x t e n t o f the [Ca] r e d u c t i o n , as described by C h a p m a n a n d N i e d e r g e r k e (1970).

Effects o f E G T A

T h e free calcium c o n c e n t r a t i o n can only be r e d u c e d below a b o u t 10 -5 M by the use o f calcium buffers. T o be able to give the extracellular free calcium level with any certainty it is essential that the b u f f e r capacity be a d e q u a t e for the conditions n e a r the surface o f the muscle cells. Miller a n d Moisescu (1976) have shown that in c o n t i n u o u s l y s u p e r f u s e d thin atrial trabeculae, w h e r e extracellular e x c h a n g e is r a p i d , a total [ E G T A ] o f - 0 . 5 - 1 mM is necessary to hold [Ca2+]0 constant d u r i n g the 0 Na + c o n t r a c t u r e (at 4.10 -~ M Ca02+). In the p r e s e n t e x p e r i m e n t s , w h e r e a g r e a t e r p r o p o r t i o n o f the E G T A remains in the free f o r m , 0.2 mM E G T A probably r e p r e s e n t s a m i n i m u m level for satisfactory b u f f e r i n g .

Fig. 2 shows the effect o f 0.2 mM E G T A Ringer solution ([Ca2+]o = 2.2.10 -8 M, see T a b l e I) on t h r e e p a r a m e t e r s . T h e records in Fig, 2a show the f o r m o f the action potentials f r o m a cell d u r i n g , a n d in Fig. 2b, after, s u p e r f u s i o n with 0.2 mM E G T A R i n g e r solution. It should be n o t e d how slowly the p r o l o n g a t i o n o f the AP occurs (Fig. 2a) a n d how quickly the effect is r e v e r s e d by calcium

52 0 - mV -80 - 0 . I r n N [ T H E J O U R N A L O F G E N E R A L P H Y S I O L O G Y ' V O L U M E 71" 1978 5 e I' a,

/ ' x

" ' " "

F.

fO0 ~ ~----i § a.p,

cOntraction 0 G:x~OCCC~ 50 e'" •

!

FIGURE 2. Membrane potentials in 0.2 mM EGTA Ringer's solution (see Table

I). (a) T h e action potentials immediately before, a n d 2, 4, a n d 6 min after changing to Ringer + 0.2 mM E G T A (prolonged AP); a n d (b) from the same cell immediately before a n d 15, 30, 45, a n d 150 s after r e t u r n i n g to normal Ringer solution. T h e lower trace in (a) a n d (b) shows the tension o u t p u t from the muscle associated with the action potentials. (c, d) Data from one cell from another preparation (O) for action potential overshoot (upper curve, c) and resting potential (lower curve, c) a n d AP duration (d, •, right-hand ordinate) for the first 6 min (abscissa, d, also applies to c) of perfusion with Ringer + 0.2 mM EGTA. Peak twitch tension recorded simultaneously (left-hand ordinate, d, ©) fell to zero.

MILLER AND ~¢[6RCHEN Divalent Cations and the Cardiac Action Potential 53

(Fig. 2 b). T h e resting potential is r e d u c e d h e r e by a b o u t 10 mV a n d the fall in o v e r s h o o t a n d the changes in the plateau can be distinguished. In almost all the cells studied, the fast r e p o l a r i z a t i o n phase was simply shifted a l o n g the time axis without a substantial c h a n g e in its steepness. T h e potential at which fast repolarization o c c u r r e d in this cell was m o r e positive in E G T A . T h e f i n d i n g is, h o w e v e r , not consistent as in m a n y cases almost n o c h a n g e o c c u r r e d (see, e.g., Fig. 3a). Evidently, calcium ions quickly restore the d u r a t i o n to n o r m a l , b u t the resting potential (Fig. 2c) a n d the f o r m a n d h e i g h t (overshoot) o f the n o r m a l plateau d e v e l o p s o m e w h a t later.

In this cell, a m a r k e d a f t e r p o t e n t i a l d e v e l o p e d d u r i n g the r e c o v e r y to n o r m a l AP d u r a t i o n . T h i s m a y reflect a phase o f potassium a c c u m u l a t i o n n e a r the cell m e m b r a n e (see C l e e m a n a n d M o r a d , 1976; Discussion), t h o u g h it was not o b s e r v e d in all p r e p a r a t i o n s .

T h e m i d d l e p a r t o f the f i g u r e (Fig. 2c) shows the time c o u r s e o f the changes in o v e r s h o o t a n d resting potential which o c c u r r e d d u r i n g a n d a f t e r a 6-min e x p o s u r e to E G T A . It can be seen that both p a r a m e t e r s c h a n g e slowly in r e s p o n s e to the a l t e r e d [Ca2+]0: the effects are fully r e v e r s e d a b o u t 10 rain a f t e r r e s t o r a t i o n o f the calcium ions.

T h e continually r e c o r d e d values o f AP d u r a t i o n are p r e s e n t e d in the lowest part (d) o f Fig. 2. A l t h o u g h the values achieved are well in excess o f those in the Ca-free solutions (Fig. 1 b), the time course o f AP p r o l o n g a t i o n is similar. In the case w h e r e n o r m a l R i n g e r solution was r e s t o r e d a f t e r 6 min (filled circles, same cell as in Fig. 2c), d u r a t i o n r e t u r n e d to n o r m a l with an e x p o n e n t i a l time course (Fig. 2d; the points fall o n a solid c u r v e which r e p r e s e n t s an e x p o n e n t i a l with t t = 22 s). This value is very close to the half-time f o r the fall in twitch tension (Fig. 2d, o p e n circles, t t = 21 s) associated with the c h a n g e to E G T A R i n g e r solution. Twitch tension falls to zero, the points a p p r o x i m a t i n g a straight line in Fig. 2 d (semilogarithmic plot) indicating an e x p o n e n t i a l rate o f fall.

( T e n s i o n n o r m a l l y falls with t h r e e e x p o n e n t i a l phases w h e n [Ca2+]0 is r e d u c e d in f r o g ventricle [ C h a p m a n a n d Miller, 1974] b u t w h e r e [Ca2+]0 is t h e r e b y r e d u c e d to below the m i n i m u m necessary f o r steady-state tension p r o d u c t i o n , the a p p e a r a n c e o f the two slowest phases is s u p p r e s s e d . T h i s kind o f b e h a v i o r indicates that internal Ca stores act cooperatively with a Ca influx d u r i n g the AP [see C h a p m a n , 1971b; Miller, 1974].)

L a t e r in the same e x p e r i m e n t the action potentials were o b s e r v e d in a n o t h e r cell f o r 25 min in 0.2 m M E G T A (Fig. 2d, s q u a r e symbols) a f t e r which a steady level for AP d u r a t i o n was achieved. T h e subsequent fall in d u r a t i o n (t~ = 23 s) in n o r m a l R i n g e r solution paralleled that seen a f t e r the s h o r t e x p o s u r e to

The solid line is an exponential, tlt~ 21 s. Normal Ringer (0.2 mM Ca) was restored

after 6 min (arrows, c). Final values for the curves in (c) were obtained 9 min later. The fall in AP duration (d, 0) fitted by an exponential curve (tll~ 22 s). Twitch tension also recovers (©). Recordings made subsequently from another cell in the same preparation (tl) were extended to 25 min in EGTA when a steady level for duration was reached. The fall in duration upon restoring normal Ringer solution is fitted by an exponential curve (tl/, 23 s). (Stimulus rate, 4 min-1).

54 T H E J O U R N A L O F G E N E R A L P H Y S I O L O G Y • V O L U M E 7 1 - 1 9 7 8 EGTA. The slow decline of both resting potential and overshoot was completed in this cell after 25 min at values of - 2 mV (peak of the AP) and - 6 0 mV (resting potential). However, such a marked depolarization was not observed in every preparation, or even in all the cells tested in any one preparation during successive exposures to EGTA. Repeated cycles of EGTA and normal Ringer solution produced very similar responses each time with continuous impalement of one cell.

Although restoring Ca0 may be expected to alter [Ca] near the cells with the same simple time course as is seen with Ca0 removal, the recovery of tension after exposure to EGTA is a complex phenomenon. Drastic changes in action potential duration after removing EGTA are superposed on the interaction of three "stores" for calcium normally detected in the kinetics of tension response to [Ca2+]0 change (Chapman and Miller, 1974). T h e time course of twitch recovery gives no clear information on the rate of [Ca] change near the cells as the former is subject to too many variables to permit detailed analysis. It may be noted that tension returns within 15-30 s (four beats per minute, see Fig. 2d) at which time the still abnormally long action potentials evoke twitches of corresponding duration which peak within 1 s and then show a slow relaxation; the onset of fast repolarization then initiates a phase of relaxation at the normal rate (see for example, Fig. 2b). A complete return to normal twitch strength only occurs after - 1 0 min stimulation in Ringer's solution.

Higher EGTA Concentrations (2 and 5 mM: Table I)

Fig. 3a shows an example of a cell where a marked afterpotential developed in the presence of EGTA (2 mM) although a near-normal resting potential was maintained throughout perfusion with EGTA Ringer solution. This phase o f slow repolarization was often observed and prevented the resting potential from reaching a maximum when interrupted by the subsequent action potential. In this case reduction of the stimulus frequency allowed the slow repolarization to reach a steady level within about 30 s. Reducing the stimulus frequency also had the effect of prolonging the subsequent action potentials still further, as is observed in heart at more normal [Ca2+]0 (e.g., Bassingthwaighte et al., 1976). The higher concentrations of EGTA produced qualitatively similar effects, although a large variation occurred from cell to cell even in the same prepara- tion. The time course for the development of the prolonged AP in EGTA Ringer solution in two cells from the same preparation was frequently quite different although the first few AP's were nearly identical in both. This was one of several indications that some deficiences in the electrical synchronization or coordination between individual cells may develop in EGTA solutions. A possibly related phenomenon was the appearance o f "humps" during the otherwise smooth prolonged plateau of the action potential.

This p h e n o m e n o n was noted by Hoffman and Suckling (1956). Examples from the present experiments are given in Fig. 3b and c, which shows the development in the first six beats (Fig. 3b) and after 5 rain (Fig. 3c) of the response of a cell to 2 mM EGTA Ringer solution. This odd feature in the plateau may result from the electrotonic influence of a "decoupled" cell or group of cells which repolarized earlier than that under observation.

MILLER AND M6RCHEN Divalent Cations and the Cardiac Action Potential 55

This p o i n t c o u l d be resolved by stimultaneous r e c o r d i n g f r o m several cells in o n e p r e p a r a t i o n but, because o f the rarity o f this p h e n o m e n o n a n d the technical difficulties in m a k i n g multiple r e c o r d i n g s with small p r e p a r a t i o n s , this e x p e r i m e n t was n o t a t t e m p t e d . Fig. 3d shows a similar h u m p , but in the d e p o l a r i z i n g direction which a p p e a r s to p r o l o n g the plateau. This f e a t u r e was m a i n t a i n e d f o r the first beat u p o n restoration o f Ca0 ~-+ w h e n a d o u b l e contrac- tion was e v o k e d , r e i n f o r c i n g the idea that two p o p u l a t i o n s o f cells were

m V - 8 0 - i I | S I I I @ 2 EGTA _ _ ~ " 0.2 Ca , a . . . - 6 0 S s O 10 48 5iS s - 8 | L J Ss

FXGVRE 3. Action potentials in 2 mM EGTA (see Table 1). (a) Recorded immedi- ately before, and 1, 2, 4, and 5 min after the solution change (4 min-t). (b) Upper

trace, action potential before and 15, 30, 45, 60, and 75 s after changing to 2 mM

EGTA. Lower trace, twitch tension associated with the first four action potentials. (c) The same cell as in (b) (mV calibration as in b, note changed time base) after 5 min in EGTA. The inflection which developed at the end of the plateau in (b) is here a clear "jump" in the plateau (stimulus frequency reduced to 2 min-l). (d) Depolarizing jump in the plateau which appears to delay the repolarization. AP recorded after 5 min in EGTA. (e) AP of indefinite duration which was terminated after 52 s by removing EGTA and adding calcium.

0 -

m V :

(temporarily) established by the E G T A solution, in o n e o f which activity was delayed or s p o n t a n e o u s .

Examples o f the d e v e l o p m e n t o f A P d u r a t i o n with d i f f e r i n g time courses in 2 mM E G T A are given in Figs. 4b a n d 6 a which illustrate two d i f f e r e n t r u n s with o n e p r e p a r a t i o n . As n o t e d earlier, this d i f f e r e n c e f r o m r u n to r u n was n o t seen if the i m p a l e m e n t o f o n e cell was m a i n t a i n e d t h r o u g h o u t , again i m p l y i n g that action potential d u r a t i o n s m a y differ f r o m o n e individual cell to a n o t h e r in E G T A solutions.

5 6 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 71 • 1 9 7 8

T h e half-time f o r the increase o f action potential d u r a t i o n t h e r e f o r e varied considerably. In addition, in the h i g h e r E G T A c o n c e n t r a t i o n s AP d u r a t i o n could s u d d e n l y rise to values in excess o f 1 min. In these cases the n o r m a l slow d e v e l o p m e n t o f AP d u r a t i o n c o n t i n u e d until a value o f a b o u t 10 s had b e e n reached; t h e r e a f t e r an action potential lasting 1 min o r m o r e was r e c o r d e d , followed by successive action potentials o f similar length. T h u s no average value could be given f o r the d e v e l o p m e n t o f AP d u r a t i o n . In a few p r e p a r a t i o n s , the AP lasted f o r m o r e than 3 min a n d could be b r o u g h t to repolarize as desired by the a d d i t i o n o f calcium (Fig. 3e) a n d by Mg ~+ o r acetylcholine (see later sections).

T h e s e findings d e m o n s t r a t e the necessity f o r a c o n t i n u o u s observation o f the potential f r o m a single cell since a t t e m p t s to impale a new cell d u r i n g such a plateau yield no reliable indication o f a successful p e n e t r a t i o n a n d might give the false impression that the cells had p e r m a n e n t l y d e p o l a r i z e d to n e a r zero potential.

T h e o c c u r r e n c e o f s p o n t a n e o u s t h o u g h r e g u l a r action potentials was o f t e n n o t e d in E G T A solutions. S p o n t a n e o u s activity r e n d e r e d the observations invalid f o r quantitative t r e a t m e n t , but qualitative observations could be m a d e .

Action of Other Divalent Cations

W h e n a m o r e or less stable action potential had b e e n achieved in E G T A R i n g e r solution a n u m b e r o f agents were a p p l i e d in an a t t e m p t to influence the AP d u r a t i o n . It was o f interest to see w h e t h e r a n o t h e r divalent cation could c o m p e n s a t e f o r the lack o f calcium ions.

Magnesium

Magnesium was tested first as it is k n o w n to have a stabilizing effect similar to that o f calcium in b o t h n e r v e (e.g., F r a n k e n h a e u s e r a n d H o d g k i n , 1957) a n d muscle (D6rrscheidt-K/ifer, 1976). Magnesium also has the a d v a n t a g e that it binds r a t h e r weakly to E G T A so that complications o f the final free m a g n e s i u m and calcium c o n c e n t r a t i o n s are largely avoided (see T a b l e I).

Fig. 4a shows that progressive increases in the c o n c e n t r a t i o n o f m a g n e s i u m in the p r e s e n c e o f 0.2 mM E G T A r e d u c e d the AP d u r a t i o n stepwise to a level very n e a r that subsequently m a i n t a i n e d in n o r m a l R i n g e r solution. T h e rate o f c h a n g e o f AP d u r a t i o n was a p p r o x i m a t e l y e x p o n e n t i a l in each case with half- times close to the half-time f o r the action o f calcium. In the p r e p a r a t i o n s tested in this way, 5 m M Mg 2+ was necessary to make the d u r a t i o n r e t u r n to n o r m a l . H o w e v e r , switching directly f r o m 0.2 mM Ca 2+ to 0.2 m M E G T A + 1 mM Mg 2+ p r o d u c e d no significant c h a n g e in AP d u r a t i o n , so that m a g n e s i u m action is to some e x t e n t d e p e n d e n t u p o n the i m m e d i a t e history o f the p r e p a r a t i o n . This behavior may reflect some changes resulting f r o m p r o l o n g e d e x p o s u r e to E G T A . T h a t Mg can c o m p e n s a t e f o r Ca at a ratio o f 5:1 was also f o u n d by H o f f m a n a n d Suckling (1956) for d o g heart.

Strontium

S t r o n t i u m ions are known to be able to replace Ca in a n u m b e r o f physiological processes, such as the slow inward c u r r e n t in the h e a r t (Vereecke a n d Carmeliet,

MILLER AND MORCHEN Divalent Cations and the Cardiac Action Potential 57 1971). I s o l a t e d c o n t r a c t i l e p r o t e i n s c a n b e f u l l y a c t i v a t e d b y S r ~+ i o n s ( M o i s e s c u a n d T h i e l e c z e k , 1975). E G T A b i n d s s t r o n t i u m s o m e w h a t less s t r o n g l y t h a n c a l c i u m so t h a t , b y s u i t a b l e c h o i c e o f t h e r e s p e c t i v e c o n c e n t r a t i o n s , a low [Ca2+]0 c a n b e m a i n t a i n e d 0.2 mM EGTA 0-1 0.2 I 1 I s 0.2 mM Ca 2÷ 0 MO 2÷ mM a,p. 4 duration $ 3 1 - 0 -- I I i I | I 0 2 4 6 | 10 rain (~ 2.si i 21 1.S 1 0.6 _ .eeeeeet,

•

l **.-....

FIGURE 4. (a) AP d u r a t i o n was r e d u c e d f r o m an initial steady level (achieved after 10 min in 0.2 mM EGTA) by successive increases in [Mg]0 to 0.2, 1, and 5 mM (total) in the presence o f E G T A . Associated changes in [Ca2+]0 are negligible ([Ca2+]0 = 2.2-2.7 × 10 -s M; see Table I). After 8 min, E G T A a n d Mg were r e m o v e d simultaneously and Ca was raised to 0.2 mM (arrow), p r o d u c i n g only a slight f u r t h e r r e d u c t i o n in AP d u r a t i o n . (b) Development o f AP d u r a t i o n in 2 mM E G T A . A f t e r 2 min 45 s strontium was a d d e d (arrow, 0.4 mM SrC12; 1.6 × 10 -s M Sr2+; see Table I) r e d u c i n g AP d u r a t i o n with a time course similar to that o f Mg 2+ (4 min-l).

in t h e p r e s e n c e o f S r z+. I n 2 m M E G T A , s t r o n t i u m (0.4 m M SrCI2 = 0.016 m M S r 2+, see T a b l e I) r e s t o r e s t h e a c t i o n p o t e n t i a l d u r a t i o n a l m o s t to n o r m a l (Fig. 4b) a n d p r o d u c e s a r e c o v e r y o f t w i t c h t e n s i o n . H i g h e r l e v e l s o f S r 2+ p r o l o n g t h e n o r m a l A P ( e . g . , V e r e e c k e a n d C a r m e l i e t , 1971) p r o b a b l y as a r e s u l t o f

5 8 T H E J O U R N A L O F G E N E R A L P H Y S I O L O G Y ' V O L U M E 71 - 1 9 7 8

altered inactivation kinetics f o r the slow c h a n n e l ( K o h l h a r d t et al., 1973), t h e r e f o r e a c o m p l e t e r e s t o r a t i o n o f the AP d u r a t i o n to n o r m a l is not to be e x p e c t e d .

Manganese

M a n g a n e s e ions a r e well k n o w n as inhibitors o f calcium c u r r e n t in several tissues ( N a r a h a s h i , 1974) b u t h a v e also b e e n r e p o r t e d to c a r r y a c u r r e n t in the calcium c h a n n e l to m a m m a l i a n h e a r t (Ochi, 1975). U n f o r t u n a t e l y , m a n g a n e s e binds m o r e s t r o n g l y to E G T A t h a n Ca so that in g e n e r a l a d d i t i o n o f Mn 2+ to E G T A solutions results in an increase in [Ca2+]o as Mn 2+ is p r e f e r e n t i a l l y b o u n d . T h u s Mn 2+ can readily be tested only with [Ca2+]0 a b o v e - 1 0 -5 M if Mn 2÷ c o n c e n t r a t i o n s a b o v e m i c r o m o l a r levels are o f interest. 1 m M MnCl~ h a d no detectable effect o n the action potential in n o r m a l R i n g e r solution a l t h o u g h c o n t r a c t i o n was blocked. H o w e v e r , action potentials in C a - f r e e R i n g e r solution ( d u r a t i o n 2.1 s) w e r e r a p i d l y r e s t o r e d to n o r m a l d u r a t i o n (0.8 s) by 0.4 m M Mn, whose action thus r e s e m b l e s that o f Ca, Mg a n d Sr.

T h e r e l a t e d divalent cations nickel a n d cobalt w e r e n o t tested since they also b i n d strongly to E G T A .

Barium

B a r i u m was tested o n o n e p r e p a r a t i o n . C o n c e n t r a t i o n s o f 0.4 a n d 0.04 m M p r o d u c e d a d e p o l a r i z a t i o n o f 10-15 m V a n d a p r o l o n g a t i o n o f the actin potential in C a - f r e e solution by several seconds. T h e l o w e r b a r i u m c o n c e n t r a - tion, h o w e v e r , p r o d u c e d a s h o r t e n i n g o f the first AP (Fig. 5). T h e s h o r t e n i n g

mV

- | 0

I I

2 5

FIGURE 5. Action potentials r e c o r d e d in a C a - f r e e R i n g e r solution i m m e d i a t e l y

b e f o r e (0), 30 s (1), a n d 135 s (2) a f t e r a d d i t i o n o f 0.04 m M BaCl2 (4 m i n - l ) .

action m a y reflect an action o f Ba 2+ like that o f the o t h e r divalent cations tested. T h e p r o l o n g a t i o n is p r e s u m e d to be r e l a t e d to the well-known action o f Ba 2+ to r e d u c e the p o t a s s i u m c o n d u c t a n c e (cf. R e u t e r , 1973), an action which evidently finally m a s k s any similarity b e t w e e n Ba ~+ a n d the o t h e r divalent cations.

D600

D600 is widely r e p o r t e d to block the slow inward c u r r e n t in h e a r t muscle in a m o r e o r less specific m a n n e r ( K o h l h a r d t et al., 1972; Einw/ichter et al., 1972).

MILLER AND MORCHEN Divalent Cations and the Cardiac Action Potential 59

T h e effects o f D600 (1, 5, a n d 10 mg/liter = 2, 10, a n d 20 p.M) were tested o n the c o n t r a c t i o n a n d action potential. T h e s e levels o f D600 r e d u c e the twitch to u n d e r 5% o f n o r m a l in 2 m M Ca o r 0.2 mM Ca R i n g e r solution within 5 min. T h e action potential is s h o r t e n e d f r o m the n o r m a l value (0.8 +-- 0.19 s) to ~ 0 . 3 s (range 0.25-0.55 s, six cells in 0.2 mM Ca). T h i s effect is r e v e r s e d only a f t e r ~2 h o f washout time a l t h o u g h the c o n t r a c t i o n a p p e a r s again within a few minutes o f r e m o v i n g D600. This p o o r reversibility m e a n t that D600 could reliably be tested only once o n each p r e p a r a t i o n a n d in t h r e e muscles the d r u g was first a d d e d to E G T A solutions w h e n a steady level o f AP d u r a t i o n h a d already b e e n r e a c h e d . Fig. 6 a shows the time c o u r s e o f the r e s p o n s e to r e s t o r a t i o n o f calcium ions (0.2 mM) a f t e r e x p o s u r e to 2 mM E G T A with a n d without D600 (10 mg/liter). D600 h a d no significant effect on the p r o l o n g e d action potentials in E G T A or o n the time course o f the recovery in C a - c o n t a i n i n g solutions.

Stimulus Frequency

E x p e r i m e n t s were m a d e w h e r e the stimulus f r e q u e n c y was c h o s e n in the r a n g e 1-30 rain -1 f o r successive observations o f the effect o f calcium restoration. No significant d i f f e r e n c e was d e t e c t e d in e i t h e r the time r e q u i r e d f o r d u r a t i o n to r e a c h n o r m a l , o r in the time c o u r s e o f this r e c o v e r y o v e r the whole r a n g e o f frequencies. T h u s in the e x a m p l e shown in Fig. 6b, 1 m i n u t e a f t e r the solution c h a n g e , the 1st (at 1 min -1) or 20th (at 20 min -1) action potential had the same d u r a t i o n . T h e n u m b e r o r f r e q u e n c y o f the action potentials a n d thus the associated Ca influx are not factors which influence the time c o u r s e o f the m e m b r a n e ' s r e s p o n s e to [Ca2+]0 increase over this c o n c e n t r a t i o n r a n g e . T h i s is consistent with the effects o f D600 described above.

Acetylcholine

Acetylcholine h y p e r p o l a r i z e s the resting m e m b r a n e a n d shortens the action potential in m a n y cardiac tissues. T h e s e effects are t h o u g h t to be primarily d u e to an increased potassium permeability (e.g., T e n Eick et al., 1976) a l t h o u g h some effects on the slow inward c u r r e n t have b e e n r e p o r t e d for f r o g atrial tissue (Giles a n d Noble, 1976). Acetylcholine was tested in E G T A a n d Ca-free Ringer solution at 10 -~, 5.10 -7, a n d 10 -7 M a n d p r o d u c e d in each case a rapid r e d u c t i o n o f the d u r a t i o n o f AP. T h e time course is illustrated in Fig. 7a. A m i n i m u m is r e a c h e d within 1 min o f a d d i n g acetylcholine a n d is followed by a slow increase in AP d u r a t i o n to a slightly g r e a t e r level. This biphasic time course has also been r e p o r t e d for g u i n e a pig atria ( U e n o , 1973). T h e f o r m o f the action potentials u n d e r acetylcholine is shown in Fig. 7b w h e r e it can be seen that its addition brings a b o u t the repolarization phase within a few seconds. Successive action potentials are progressively s h o r t e n e d . T h e dose effectiveness, a l t h o u g h not extensively studied, was similar in both Ca-free solutions a n d E G T A R i n g e r solution, implying that extracellular calcium ions are not essential to the action o f acetylcholine. T h e s e effects c o u l d be blocked by a b r i e f (3 min) e x p o s u r e to a t r o p i n e (10 -7 M) b e f o r e application o f the acetylcholine solution (also containing atropine). In combining observations m a d e with acetylcholine with the results o b t a i n e d with D600, o n e may safely conclude that the s h o r t e n i n g o f AP d u r a t i o n by acetylcholine reflects an increase in potassium permeability which can be achieved even in the virtual absence o f

60 a.p, duration s ( ~ 01 a . p . d u r a t i o n s

®

THE J O U R N A L OF GENERAL PHYSIOLOGY • VOLUME 7 1 • 1 9 7 8

_ _ I D 600 [GTAJ Ca EGTA [ Ca ' - - m o o ""°°°eee°e°'" "°°o : • o o • • ° o o ° ° o ° o ° O o ~ • o o I I I . . ] J . . I I 0 2 0 2 6 8 rain 5 O 4 3 xo 2 1 0 Solution C han ge X 2 0 / m i n O 1 2 / m i n • l / m i n o x x O x X ° x ~ - - x ' R ~ O x x ~ x C K ) o . . . • X I I I I 15 30 45 60 s e c o n d s

FIGURE 6. (a) Effect o f D600 on AP d u r a t i o n . AP d u r a t i o n (ordinate) is plotted against time (abscissa). On the left a steady level has been achieved after 10 min in E G T A (2 mM). C h a n g i n g to 0.2 mM Ca Ringer solution p r o d u c e d the n o r m a l r a p i d fall towards n o r m a l values (see Fig. 2). Later, in a second cell from the same p r e p a r a t i o n , the sequence was r e p e a t e d (right) but first D600 (10 mg/liter) was a d d e d in the presence o f E G T A (2 mM). Even after 5 min in D600, only minimal changes in AP d u r a t i o n had occurred. Restoring calcium (0.2 mM) p r o d u c e d the typical r a p i d fall in AP d u r a t i o n in the continuous presence o f D600. (b) Effect of stimulus frequency on recovery o f AP d u r a t i o n . A p r e p a r a t i o n was e x p o s e d to 0.2 mM E G T A until a steady AP d u r a t i o n was established. Stimulus frequency was then set to the desired rate for a few minutes a n d 0.2 Ca Ringer restored. T h e fall o f AP d u r a t i o n after the solution change is plotted. All t h r e e records were obtained in the same cell after successive e x p o s u r e s to E G T A . (Note that at high frequencies stimuli often occurred d u r i n g an A P plateau in the E G T A solution or immediately after its removal when AP d u r a t i o n exceeded 1/stimulus frequency.) c a l c i u m o r o t h e r e x t r a c e l l u l a r d i v a l e n t c a t i o n s . T h e p r o t r a c t e d p h a s e o f i n c r e a s e in A P d u r a t i o n a f t e r t h e a d d i t i o n o f E G T A fails to o c c u r w h e n a c e t y l c h o l i n e is a p p l i e d . T h e r e s u l t s w i t h a c e t y l c h o l i n e s e r v e to d e m o n s t r a t e t h a t a n i n c r e a s e d p o t a s s i u m p e r m e a b i l i t y will p r o d u c e s h o r t e n i n g o f A P D in E G T A - t r e a t e d cells.

MILLER AND M 6 R C H E N Divalent Cations and the Cardiac Action Potential | ~ - I 4 ~ L p . ~ a t ~ t - o O.i - o I 0 o o o ~-.~ ~ ° 0 0 0 0 0 0 o o A C h o ® 2m41 EGTA J I [ I I I | 4 I 8 10 12 ~n 61 ACh m V - 6 0 | D S s

FIGURE 7. (a) Development of AP duration monotonically in 2 mM EGTA. After 6 rain acetylcholine (5.10 -7 M) was added (arrow) (same preparation as Fig. 4b). (b) For the same solution change, the action potentials immediately before acetylcholine addition, (0) and the first, second, and third in the presence of acetylcholine. The drug was added during the action potential labeled no. 1, along the plateau as indicated by the arrow. Repolarization was thereby accelerated, following within 3 s (4 rain-l).

D I S C U S S I O N

It has previously b e e n r e p o r t e d f o r m a m m a l i a n h e a r t ( H o f f m a n a n d Suckling, 1956; Surawicz et al., 1961) a n d f r o g h e a r t ( J u n c k e r et al., 1972; Chesnais et al., 1975) t h a t r e d u c e d levels o f calcium in the b a t h i n g m e d i u m p r o m o t e p r o l o n g e d action potentials. H o w e v e r , a l t h o u g h the e f f e c t o f e x t r e m e [Ca2+]0 r e d u c t i o n by use o f Ca c h e l a t o r s has b e e n r e p o r t e d ( C h a n g a n d S c h m i d t , 1960; T r i t t h a r t et al., 1973), it has n o t b e e n extensively investigated. T h e f o r e g o i n g results indicate that AP p r o l o n g a t i o n is r e l a t e d to the [Ca2+]0: in 0.2 m M E G T A ( ~ 10 -8

M Ca 2+) d u r a t i o n s r e a c h 2-10 s; in 2 a n d 5 m M E G T A ( < ~ 1 0 -9 M Ca 2+, see T a b l e I) this value o f t e n e x c e e d s 1 min. T h e o n s e t o f r e p o l a r i z a t i o n is, h o w e v e r , a c c e l e r a t e d by several divalent cations (and acetylcholine) e v e n with [Ca2+]0

6 2 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 7 1 • 1 9 7 8

below 10 -8 M. Blocking the slow inward c u r r e n t with D600 has no significant effect on restoration o f action potential d u r a t i o n when calcium is r e a d m i t t e d after e x p o s u r e to E G T A . T h e s e effects o f calcium r e d u c t i o n d e v e l o p only as long as no o t h e r divalent cations are p r e s e n t in significant a m o u n t s . (Fig. 4).

Divalent Cation Chelators on Frog Heart

B e f o r e an i n t e r p r e t a t i o n o f the results in terms o f the u n d e r l y i n g ionic m o v e m e n t s is m a d e , some points arising f r o m the use o f E G T A n e e d to be discussed.

It is w o r t h noting that despite e x p o s u r e to [Ca2+]0 o f less t h a n 10 -9 M for periods o f t e n e x c e e d i n g 0.5 h, the cell m e m b r a n e does not a p p e a r to b e c o m e excessively leaky: even the longest-lasting action potentials were t e r m i n a t e d by a phase o f rapid repolarization to n e a r - n o r m a l resting potentials, a l t h o u g h some cells exhibited m a r k e d afterpotentials (see Figs. 2, 3a, a n d 7). It seems that, at least for frog h e a r t , e x p e r i m e n t s e m p l o y i n g E G T A (e.g., Miller and Moisescu, 1976) can be m a d e without risking severe d i s r u p t i o n o f the mem- b r a n e . T h e p r o b l e m o f skinning o f cardiac cells by E G T A / E D T A t r e a t m e n t is dealt with e l s e w h e r e ? T h e various binding constants o f E G T A are all highly p H sensitive in the physiological r a n g e (Portzehl et al., 1964). Tris has been used as the p H b u f f e r in the p r e s e n t e x p e r i m e n t s . A l t h o u g h it is a relatively p o o r b u f f e r at the e x p e r i m e n t a l p H (7.00), the effective b u f f e r capacity is very large as a result o f the r a p i d s u p e r f u s i o n o f the p r e p a r a t i o n ( - 1 0 - 2 0 ml/min). T h e p r o b l e m s o f the a d e q u a c y o f the C a - b u f f e r capacity o f E G T A solutions were discussed in c o n n e c t i o n with Fig. 2. It should be n o t e d that toxic effects o f E G T A are difficult to distinguish f r o m those o f [Ca 2+] r e d u c t i o n associated with h i g h e r [EGTA]. H o w e v e r , at h i g h e r f r e e [Ca], variation o f the total b u f f e r c o n c e n t r a t i o n does not have m a r k e d effects (Miller and Moisescu, 1976).

Extracellular Exchange

T h e i n t e r p r e t a t i o n o f the results hinges to some e x t e n t u p o n a knowledge o f the rate o f extracellular e x c h a n g e a f t e r a c h a n g e in the p e r f u s i n g solution for this p r e p a r a t i o n o f the f r o g ventricle. T h e half-time for extracellular calcium e x c h a n g e u n d e r ideal conditions has been estimated to be a b o u t 3 s (Page and N i e d e r g e r k e , 1972; C h a p m a n a n d Miller, 1974). T h e fall o f twitch tension in Ca-free solutions (Fig. 2) gives a r o u g h estimate o f this p a r a m e t e r f o r individual p r e p a r a t i o n s (Miller, 1975). F r o m the present e x p e r i m e n t s it seems that the extracellular calcium level, a f t e r a c h a n g e to E G T A solutions, will be within a few p e r c e n t o f its final level within 20-60 s. (Although the E G T A b u f f e r will diffuse s o m e w h a t m o r e slowly t h a n Ca 2+ ions, the b u f f e r i n g action will accelerate the rate o f fall o f [Ca~+]0.) This contrasts with the d e v e l o p m e n t to the steady level o f AP d u r a t i o n which e x t e n d s o v e r m a n y minutes (Figs. 2, 4). A similar d e v e l o p m e n t occurs in Ca-free Ringer solution so that the cause c a n n o t lie in e i t h e r the m u c h lower [Ca2+]0 o f the E G T A solutions, o r in the p r e s e n c e o f E G T A itself. It seems possible that intracellular calcium is leached out o f the cells in C a - p o o r m e d i a and thus the a m o u n t o f calcium b o u n d in the m e m b r a n e

Miller, D. J. Submitted for publication.

MILLER AND MORCHEN Divalent Cations and the Cardiac Action Potential 63

may fall slowly even t h o u g h the e x t e r n a l c h a n g e is r a p i d . Such a process has b e e n suggested by N i e d e r g e r k e a n d O r k a n d (1966) who also o b s e r v e d that in nominally calcium-free solutions, o v e r s h o o t and resting potential fell only very slowly. In contrast, restoration o f Ca 2+ (Figs. 1, 2) o r the introduction o f o t h e r divalent cations (Fig. 4) restores AP d u r a t i o n with a r a p i d , e x p o n e n t i a l time course very close to that f o r the extracellular e x c h a n g e in the individual p r e p a r a t i o n s (see Fig. 2).

( T h e fall in twitch tension represents an "average" v a l u e - a l l cells contribute to the tension o u t p u t . T h i s is not so f o r the action potential d u r a t i o n o f an individual cell unless s t r o n g electrotonic c o u p l i n g e n s u r e s n e a r - s y n c h r o n o u s repolarization o f the whole p r e p a r a t i o n . While this is likely it is by no m e a n s definite, especially in Ca-poor media [see text in connection with Fig. 3].)

In this respect t h e rate o f action o f successive [Mg~+]0 increases (Fig. 4 a ) o r that o f Sr 2+ (Fig. 4b) is i m p o r t a n t since it shows that levels o f the cations which are suboptimal in t h e i r e f f e c t o n AP d u r a t i o n act as rapidly as 0.2 mM calcium; i.e., the rate o f action o f calcium is not accelerated by a saturation effect.

T h e rate o f r e s t o r a t i o n o f n o r m a l AP d u r a t i o n , like that f o r the fall o f twitch tension in C a - f r e e m e d i a (Miller, 1975), is i n d e p e n d e n t o f stimulus f r e q u e n c y o v e r a wide r a n g e (Fig. 6b). T h e similarity o f this time c o u r s e to that f o r tension fall indicates that both are strongly, even if only indirectly, d e p e n d e n t u p o n extracellutar c a l c i u m - t h e f o r m e r directly f r o m [Ca~+]0, the latter f r o m the r e q u i r e m e n t f o r a Ca-influx f o r contraction (see above) a n d h e n c e a d e p e n d e n c e o n [Ca2+]0.

Origin of the Plateau Prolongation: External or Internal Site of Action for Divalent Cations?

T h e e x t r e m e d u r a t i o n o f the action potential in m e d i a p o o r in divalent cations could be i n t e r p r e t e d in several ways. T h r e e o f t h e m are briefly c o n s i d e r e d .

T h e first possibility is that a Ca-influx a n d / o r intracellular release with a s u b s e q u e n t b i n d i n g to the i n n e r side o f the s a r c o l e m m a leads to an increase in the potassium c o n d u c t a n c e , GK. T h i s would be in k e e p i n g with the observations o f Meech (1974) a n d Meech a n d S t a n d e n (1975) o n snail n e u r o n s a n d o f I s e n b e r g (1975) o n cardiac Purkinje fibers, that intracellularly a p p l i e d Ca 2+ (or a C a - E G T A b u f f e r ) can increase GK. T h e failure o f the calcium influx in E G T A solutions would t h e r e f o r e limit o r delay the increase in GK necessary f o r repolarization.

T h e results d e m o n s t r a t e that calcium can be mimicked by several divalent cations in r e d u c i n g AP d u r a t i o n ; thus Ca a n d Mg acted alike ( c o m p a r e Figs. 2 a n d 4a). This is not in k e e p i n g with an intracellular c o n t r o l o f GK since it is unlikely that Mg ions can c a r r y a c u r r e n t t h r o u g h the cell m e m b r a n e (Reuter, 1973; but see K o h l h a r d t et al., 1973) o r p e n e t r a t e it to any significant extent. Any s h o r t - t e r m e n t r y o f Mg 2+ (and the ion acts within seconds, Fig. 4) is likely to be negligible in terms o f an intracellular [Mg z+] change. ([Mg2+]l ~1 mM; Polimeni a n d Page, 1973). In any case, m a g n e s i u m had the opposite effect to calcium on GK in snail n e u r o n s (Meech, 1974). T h e effect o f Mg 2+ is t h e r e f o r e c o n c l u d e d to be on the o u t e r side o f the m e m b r a n e .

T h e second i n t e r p r e t a t i o n is that AP d u r a t i o n is p r o l o n g e d as a result o f

6 4 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 71 - 1978

slowed inactivation kinetics for the slow i n w a r d c u r r e n t in C a - p o o r m e d i a . I n the p r e s e n t e x p e r i m e n t s this c u r r e n t could not be c a r r i e d by Ca 2+ in E G T A solutions, b e c a u s e the d r i v i n g force f o r calcium ions is t h e n very small or e v e n o u t w a r d l y d i r e c t e d d u r i n g the p l a t e a u o f the action potential. R o u g i e r et al. (1969) a n d Chesnais et al. (1975) have s u g g e s t e d , on the basis o f v o l t a g e - c l a m p e x p e r i m e n t s with f r o g atrial t r a b e c u l a e , that s o d i u m ions carry the slow inward c u r r e n t in C a - f r e e solutions b u t that the inactivation is slowed.

T h e divalent cations which mimic calcium in its effect on AP d u r a t i o n ( a p a r t f r o m m a g n e s i u m , see above) are s t r o n t i u m a n d m a n g a n e s e . B o t h these ions can p r o b a b l y cross the m e m b r a n e to s o m e e x t e n t via the slow or calcium c h a n n e l ( V e r e e c k e a n d C a r m e l i e t , 1971; Ochi, 1975; R e u t e r , 1973). T h i s was almost certainly the case f o r Sr 2+ in the p r e s e n t e x p e r i m e n t s since c o n t r a c t i o n was r e s t o r e d ( t o g e t h e r with AP d u r a t i o n ) by the cation. M a n g a n e s e did not h a v e this effect e v e n t h o u g h Mn 2+ will activate the contractile p r o t e i n s (D. G. Moisescu, p e r s o n a l c o m m u n i c a t i o n ) .

B o t h the first a n d s e c o n d i n t e r p r e t a t i o n s f o r the o b s e r v e d c h a n g e s in AP d u r a t i o n r e q u i r e t h a t the slow i n w a r d c u r r e n t play a significant role in the action potentials in E G T A - c o n t a i n i n g solutions. T h e possibility o f such a role can be e x c l u d e d o n the basis o f the findings with D600. T h i s substance is r e p o r t e d to block the slow c h a n n e l fairly specifically in b o t h m a m m a l i a n ( K o h l h a r d t et al., 1972) a n d f r o g h e a r t (Einwfichter et al., 1972), but h a d no significant effect on the p r o l o n g e d action potentials in E G T A m e d i a o r on the c h a n g e s in AP d u r a t i o n c o n s e q u e n t u p o n [Ca2+]o increase (Fig. 6). T h e c o n c e n - trations used were sufficient to block c o n t r a c t i o n in n o r m a l R i n g e r solution a n d u p to 10 times h i g h e r t h a n those u s e d in v o l t a g e - c l a m p e x p e r i m e n t s to block / c a . It m a y be c o n c l u d e d t h a t the slow i n w a r d c u r r e n t (in E G T A media) or calcium influx as p a r t o f the c u r r e n t ( u p o n r e s t o r i n g [Ca2+]o ) a r e not necessary for the m o d u l a t i o n o f AP d u r a t i o n that was o b s e r v e d . T h i s is s u p p o r t e d by the lack o f a m a r k e d effect o f stimulus f r e q u e n c y u p o n the t i m e c o u r s e o f AP d u r a t i o n r e c o v e r y a f t e r E G T A (Fig. 6b).

T h e t h i r d possibility to be c o n s i d e r e d is that the [Ca2+]o has a direct effect u p o n p o t a s s i u m c u r r e n t s . T h i s suggestion has also b e e n m a d e by Kaas a n d T s i e n (1975, 1976) on the basis o f v o l t a g e - c l a m p e x p e r i m e n t s o n cardiac Purkinje fibers. T h e s e investigators r e p o r t that the b a c k g r o u n d p o t a s s i u m c u r r e n t , iKl, is r e d u c e d w h e n [Ca2+]o is r e d u c e d . Also significant is their f i n d i n g t h a t Ca- d e p e n d e n t c h a n g e s in iKl w e r e similar with or without activation o f the slow i n w a r d c u r r e n t . T h i s f i n d i n g is in h a r m o n y with the p r e s e n t results with D600 a n d stimulus f r e q u e n c y which a r e also consistent with the i d e a that the p o t a s s i u m c u r r e n t can be i n f l u e n c e d w i t h o u t involving the slow i n w a r d c u r r e n t . T h i s t h i r d e x p l a n a t i o n o f the results s e e m s m o s t satisfactory since a n u m b e r o f conclusions d r a w n earlier p o i n t to an extracellular influence o f the divalent cations on the t i m i n g o f AP t e r m i n a t i o n .

T h e r a p i d a n d large effects o f acetylcholine in the E G T A m e d i a serve p r i m a r i l y to d e m o n s t r a t e t h a t increased GK can a c c o u n t f o r a l t e r e d AP d u r a t i o n u n d e r these conditions. T h e results also reveal that acetylcholine effectiveness is not m a r k e d l y Ca d e p e n d e n t .

MILLER AND M6RCHEN Divalent Cations and the Cardiac Action Potential 65 A m o s t i n t e r e s t i n g f e a t u r e o f these long-lasting action potentials is t h a t r e p o l a r i z a t i o n is so steep e v e n a f t e r a p l a t e a u p h a s e o f m a n y seconds' d u r a t i o n (e.g., Fig. 3). A f t e r p o t e n t i a l s were occasionally seen in the E G T A m e d i a ( a l t h o u g h they a r e m o r e f r e q u e n t l y seen d u r i n g the r e c o v e r y p e r i o d a f t e r r e m o v a l o f E G T A , e.g., Fig. 2a) a n d a r e thus not s t r o n g l y c o r r e l a t e d with a l o n g p l a t e a u . T h i s e x c l u d e d the likelihood o f a significant p o t a s s i u m a c c u m u l a - tion n e a r the s a r c o l e m m a d u r i n g these l o n g action potentials o f the kind d e s c r i b e d by C l e e m a n a n d M o r a d (1976), b u t this m i g h t be e x p e c t e d , as the o u t w a r d c u r r e n t t h a t t h e y m e a s u r e d is low at the potential level f o u n d h e r e f o r the p r o l o n g e d p l a t e a u in E G T A (ca. - 1 0 mV). Similarly, an e l e c t r o g e n i c Ca-K e x c h a n g e o f the t y p e s u g g e s t e d by these a u t h o r s is unlikely to play a role in d e t e r m i n i n g AP d u r a t i o n in E G T A solutions. As the f o r e g o i n g sections show, A P D p r o l o n g a t i o n u p o n e x p o s u r e to E G T A is not c o r r e l a t e d to the t i m e c o u r s e o f e x t r a c e l l u l a r e x c h a n g e , i.e. p l a t e a u p r o l o n g a t i o n does not c o r r e l a t e with [Ca]o d r o p .

C h a n g e s in the fast s o d i u m c o n d u c t a n c e c o n c o m i t a n t on e x t r e m e [Ca2+] o r e d u c t i o n have not b e e n c o n s i d e r e d h e r e . It is possible t h a t e x t r e m e delay in Na inactivation could account for the findings, a l t h o u g h a nonspecific r e s p o n s e to e x t r a c e l l u l a r divalent cations o f the t y p e f o u n d h e r e has not b e e n p u t f o r w a r d in the l i t e r a t u r e . T h i s possibility c a n n o t , h o w e v e r , be e x c l u d e d .

I n s u m m a r y , the results a r e consistent with the idea t h a t m e m b r a n e K c o n d u c t a n c e can be i n f l u e n c e d by sites o n the o u t e r face o f the s a r c o l e m m a which display a relatively nonspecific affinity f o r divalent cations. Since only relatively small c h a n g e s in AP d u r a t i o n a r e o b s e r v e d w h e n [Ca2+]o increases f r o m 0.2 to 2.0 m M o r m o r e (e.g., N i e d e r g e r k e a n d O r k a n d , 1966), these sites are p r o b a b l y nearly s a t u r a t e d at physiological [Ca2+] o, a l t h o u g h this point can be definitely resolved only by m e a s u r i n g the Cao-activated GK c h a n g e s o v e r a wide r a n g e o f [Ca2+] o. T h i s system o f c o n t r o l f o r GK c o m p l e m e n t s the better- d o c u m e n t e d intracellular r e g u l a t i o n o f p o t a s s i u m p e r m e a b i l i t y which, in con- trast, s e e m s to be Ca specific.

We wish to thank Prof. H. G. Glitsch, Dr. M. D6rrscheidt-K/ifer, and Dr. R. W. Tsien for their stimulating c o m m e n t s , and Mrs. J. Zwoycyk for excellent technical assistance.

This work was s u p p o r t e d in part by Sonderforschungsbereich 114 Bionach, B o c h u m .

Received for publication 14 March 1977.

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