changes (e.g., in resting potential and overshoot) are, however, only more slowly ..... plateau may result from the electrotonic influence of a "decoupled" cell or.
On the Effects of Divalent Cations and Ethylene Glycol-bis-(/3-Aminoethyl Ether) N,N,N',N'-Tetraacetate on Action Potential Duration in Frog Heart 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 (EGTA) prolongs the action potential (AP). The 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 (Mg2+, 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. The cell membrane does not become excessively "leaky" in EGTA solutions. INTRODUCTION 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 cardiac action potential (AP) is influenced by the b a t h i n g calcium concentration. Generally, a reduction in calcium p r o l o n g s the d u r a t i o n o f the AP, a l t h o u g h this effect is, for 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 have r e p o r t e d that calcium r e d u c t i o n by the use o f chelating agents, e.g., e t h y l e n e d i a m i n e tetraacetic acid, a n d ethylene glycol-bis-(fl-aminoethyl ether)N,N,N',N'-tetraacetate ( E D T A , E G T A ) will evoke p r o l o n g e d action potentials ( T r i t t h a r t et al., 1973), s o m e t i m e s o f several seconds' 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). This effect is o f i m m e d i a t e interest because b o t h intracellular a n d extracellular calcium levels have b e e n f o u n d to affect potassium c o n d u c t a n c e in a n u m b e r o f cell types (Meech, 1974; Meech a n d S t a n d e n , 1975; I s e n b e r g , 1975; Kass and Tsien, 1975, 1976; Lew a n d THE JOURNAL
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Ferreira, 1976), quite a p a r t f r o m the d e p e n d e n c e o f the "slow inward c u r r e n t " o f cardiac muscle o n the 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 reduction on h e a r t muscle is also interesting 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 larger molecules into the cell ( W i n e g r a d , 1971). Until now t h e r e has b e e n no detailed observation o f tlae time course o f the effects o f E G T A on the h e a r t or 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 on the responses. T h e p r e s e n t e x p e r i m e n t s on f r o g ventricle fibers were 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 on the h e a r t u n d e r precisely d e f i n e d conditions (e.g., extracellular e x c h a n g e , p H , extracellular divalent cation c o n c e n t r a t i o n , IX2+]0). Special attention was paid to obtaining continuous 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 sequence o f solution changes. T h e results show that resting a n d action potentials are well m a i n t a i n e d in divalent c a t i o n - p o o r media, a l t h o u g h the d u r a t i o n o f the AP is dramatically increased. T h e observations indicate that the extracellular divalent cation concentration IX2+]0 affects the time o f onset o f repolarization in f r o g ventricle. C h a n g e s in [Ca~+]t are concluded to be o f little c o n s e q u e n c e for 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 some o f these results has b e e n published (Miller, 1976). MATERIALS
AND
METHODS
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
