European Journal of Pharmacology, 42 (1977) 371--377 © Elsevier/North-Holland Biomedical Press
371
THE INHIBITORY EFFECT OF CADMIUM ON NEUROMUSCULAR TRANSMISSION IN THE RAT PHILIP J. FORSHAW Toxicology Unit, Medical Research Council Laboratories, Woodmansterne Road, Carshalton, Surrey SM5 4EF, England
Received 20 September 1976, revised MS received 14 December 1976, accepted 21 December 1976 P.J. FORSHAW, The inhibitory effect o f cadmium on neuromuscular transmission in the rat, European J. Pharmacol. 42 (1977) 371--377. Cadmium (0.125--1 mM) was found to inhibit the isometric response of the isolated rat hemidiaphragm during indirect stimulation, but not during direct stimulation. This effect of cadmium (1 mM) was completely reversed by ethyleneglycol bis-(aminoethyl)-N,N,N',N'-tetra-acetic acid (2 raM) or by L-cysteine (2 raM) but only partially by increased calcium. Cadmium (10 uM) significantly reduced the quantal release of transmitter in the isolated phrenic diaphragm and a concentration of 0.1 mM frequently caused a complete failure of the endplate response after 30 rain. The effect of cadmium on neuromuscular transmission could not be readily reversed by washing with cadmium-free solution. Miniature endplate potential frequency and amplitude were not significantly affected by cadmium (0.1 or 0.5 raM). The results suggest that the effect of cadmium on the isolated phrenic nerve-diaphragm is due largely to inhibition of calcium function at presynaptic nerve terminals. Phrenic nerve---diaphragm preparation Neuromuscular transmission
Endplate potential Acetylcholine
1. I n t r o d u c t i o n The effects of chronic exposure to cadmium (Cd 2÷) in m a n a n d a n i m a l s a n d its e p i d e m i ological c o r r e l a t i o n w i t h s o m e diseases h a v e b e e n well s t u d i e d ( B r e m n e r , 1974). H o w e v e r , t h e a c u t e e f f e c t s o f large doses o f Cd 2÷ in l a b o r a t o r y animals are n o t so well k n o w n . T h e p r o s t r a t i o n a n d r e s p i r a t o r y failure w h i c h we in this l a b o r a t o r y h a v e o b s e r v e d t o f o l l o w w i t h i n a f e w m i n u t e s o f t h e administ r a t i o n o f large d o s e s o f Cd 2÷ ( 5 - - 1 0 m g i.v.) to t h e r a t suggested t h a t it w o u l d be w o r t h investigating t h e a c t i o n o f Cd 2+ o n t h e gross t w i t c h r e s p o n s e s o f t h e isolated r a t p h r e n i c n e r v e - - d i a p h r a g m p r e p a r a t i o n . In a d d i t i o n , since o t h e r d i v a l e n t c a t i o n s including m a n g a nese (Balnave a n d Gage, 1 9 7 3 ) ; lead (Manalis a n d C o o p e r , 1 9 7 3 ) ; c o b a l t (Weakly, 1 9 7 3 ) ; nickel a n d zinc ( B e n o i t a n d M a m b r i n i , 1 9 7 0 )
Calcium
Cadmium
have all b e e n s h o w n to r e d u c e e v o k e d transm i t t e r release at t h e n e u r o m u s c u l a r j u n c t i o n , it was d e c i d e d t o e x a m i n e t h e e f f e c t s o f Cd 2÷ on t h e intracellularly r e c o r d e d e n d p l a t e potentials ( e . p . p . ' s ) a n d m i n i a t u r e e n d p l a t e potentials ( m . e . p . p . ' s ) o f t h e r a t d i a p h r a g m .
2. Materials and methods T h e left isolated h e m i d i a p h r a g m was set u p as d e s c r i b e d b y Krnjevid a n d Miledi ( 1 9 5 8 a , b). Male rats o f w e i g h t 2 5 0 - - 3 0 0 g w e r e used. T h e b a t h v o l u m e was 10 ml. 2 b a t h i n g s o l u t i o n s w e r e used. F o r experim e n t s investigating t h e e f f e c t o f Cd 2÷ o n diap h r a g m gross t w i t c h r e s p o n s e s a t r i s ( h y d r o x y m e t h y l ) m e t h y l a m i n e - b u f f e r e d s o l u t i o n was used in o r d e r t o p r e v e n t p r e c i p i t a t i o n w h e n
372 concentrations of Cd 2÷ greater than 0.5 mM were used. The composition of this solution was as follows (mM) NaC1 127; KC1 5.9; CaC12 2.0; MgC12 1.0; tris(hydroxymethyl)methylamine 10.0; dextrose 11.0. The solution was adjusted to pH 7.4 with HC1 and gassed in the fluid reservoir with 100% 02. Liley solution (Liley, 1956) was used for the intracellular experiments because it was usually difficult to detect m.e.p.p.'s in trisbuffered solution. This solution contained 1 mM Ca 2÷ and 12.5 mM Mg2÷ in order to reduce endplate potentials below threshold level. Cd 2÷ concentrations greater than 0.5 mM could not be used in this solution because otherwise precipitation of Cd 2÷ salts occurred. The solution had a pH of 7.4 when gassed with 95% 02 and 5% COs. No osmotic compensation was made for the addition of Cd 2÷ or extra Ca 2÷ in either solution in view of demonstrations that variations in osmotic pressure in the range of the present experiments do not significantly affect the quantal c o n t e n t of e.p.p.'s (Furshpan, 1956; Gage and Hubbard, 1966}. Solutions were kept at a temperature of 25°C rather than the usual 37°C since it has been shown that this minimises the occurrence of presynaptic failure due to h y p o x i a which can occur when oxygen is n o t bubbled directly onto the surface of the diaphragm (Krnjevi6 and Miledi, 1959). The solutions were allowed to flow over the preparation at a rate of 3--4 ml/min and were removed by continuous suction. The preparation was stimulated indirectly using supramaximal pulses of 0.1 msec duration and a frequency of 8/min. The isometric contractions were recorded using a transducer (Statham UC3) connected to a pen recorder (Devices M4). Direct stimulation with pulses of 1 msec duration, 8/min frequency was applied using two platinum electrodes in contact with each end of the diaphragm. E.p.p.'s and m.e.p.p.'s were recorded using conventional microelectrode techniques, displayed on an oscilloscope (Tektronix 502) and photographed with a moving film camera
P.J. FORSHAW (Nihon Kohden). Since the amplitude of e.p.p.'s recorded from preparations bathed in high Mg2÷ and/or low Ca 2÷ solution varies according to the quantal hypothesis (Del Castillo and Katz, 1954), at least 30 recordings were made of the e.p.p, as well as a 30 sec period of m.e.p.p, recording, after allowing 30 min for equilibration with each new solution, in order to obtain the mean e.p.p, and m.e.p.p. amplitudes. Wherever possible the quantal content (m) was calculated from the ratio of mean e.p.p, to mean m.e.p.p, amplitude but in cases where failure of e.p.p, response occurred the m e t h o d of failure was used (Del Castillo and Katz, 1954). No correction was made for the non linear summation of e.p.p.'s since results were discarded when m was greater than 10 (Martin, 1955). All amplitude measurements of m.e.p.p.'s were corrected to a resting membrane potential of --75 mV (Katz and Thesleff, 1957). In each experiment concerned with the effect of Cd 2÷ on evoked transmitter release an initial series of e.p.p, and m.e.p.p, recordings in control solution was obtained and a further series of records after 30 min in either control or Cd 2÷ solution. The values for m were calculated and the ratios of the 30 rain to initial value found for control and Cd 2÷ solutions. The means of these ratios for all the experiments were compared and the significance of differences between them calculated using the Student's t-test. By this means the possible significance of the effect of Cd 2+ would not be affected by deterioration of the preparations due to other factors since these would affect both control and Cd 2÷ solution 30 min values of m. M.e.p.p. frequencies were calculated directly from the photographic records; only junctions where the average frequency was less than 3/sec were used in order to avoid the use of junctions which had been damaged by impalement. Only one Cd 2÷ experiment was done on each preparation in order to avoid retention effects after fluid replacement.
CADMIUM AND NEUROMUSCULAR TRANSMISSION
373
3.2. Reversal o f Cd 2÷ block by EGTA, L-cysteine and Ca 2+
3. Results 3.1. Effect o f Cd ~÷ on diaphragm contractions
Indirectly or directly stimulated hemidiaphragms bathed in the tris-buffered solution without Cd 2÷ maintained responses for at least 4 h without noticeable decline. In solutions containing Cd 2÷ (0.125--2 mM) with the normal concentration of Ca 2÷ the twitch responses to indirect stimulation gradually declined and became blocked, that is too small to be recorded, after an interval of 10---52 min. The relationship between the time interval to block and log Cd 2÷ concentration is shown in fig. 1. Block still developed if the preparation was in contact with a Cd 2÷ (1 mM) solution for only 5 min (fig. 2a).
Ethyleneglycol bis(aminoethyl ether)N,N,N',N'-tetra-acetic acid (EGTA) 2 mM and L-cysteine (2 mM) reversed the block of the diaphragm twitch responses to indirect stimulation caused by Cd 2+ (1 mM). Reversal generally took place within 20 min of adding the reversing agent (figs. 2b and 2c).
50 45 40 35 30 25 F I.--
20 15 1(] I
1
02
el~
I
0~6 Q~ 1
mM Ioglo [Cd]
Fig. 1. D o s e - - r e s p o n s e relationship b e t w e e n the log10 Cd 2+ c o n c e n t r a t i o n ( m M ) on the abscissa and on the ordinate the time taken to produce b l o c k (rains) o f the responses o f the isolated rat diaphragm to indirect stimulation. Vertical lines at each p o i n t represent the standard error.
Fig. 2. E f f e c t o f Cd 2+ on the responses o f the isolated diaphragm o f the rat to indirect s t i m u l a t i o n showing; the d e v e l o p m e n t o f block after changing from Cd 2+ (1 raM) s o l u t i o n to control s o l u t i o n (a), the reversal o f Cd 2+ (1 raM) block by E G T A (b), and by L-cysteine (c). T h e partial reversal o f Cd 2+ (0.25 raM) block by Ca 2+ (6 raM) and the failure o f a higher c o n c e n t r a t i o n o f Ca 2+ (10 raM) to c o m p l e t e l y reverse the block is s h o w n in d. Time axis in 1 rain intervals.
374 Cd2+-induced b l o c k was f o u n d t o be partially reversible b y Ca :+ ; t h e d e g r e e o f r e c o v e r y d e p e n d e d o n t h e c o n c e n t r a t i o n o f Cd 2÷ to which the preparation had been exposed. Thus in 3 o u t o f 4 e x p e r i m e n t s 6 m M Ca 2÷ gave 60--70% recovery from block induced by 0 . 2 5 m M Cd 2+ a n d 10 m M Ca 2+ did n o t increase this r e c o v e r y n o t i c e a b l y (fig. 2d). C o m p l e t e or n e a r l y c o m p l e t e r e c o v e r y f r o m 0.1 m M Cd 2+ b l o c k was o b t a i n e d b y raising Ca 2+ to 6 m M b u t a f t e r e x p o s u r e t o 0.5 m M Cd 2+ o n l y a b o u t 40% r e c o v e r y o f t h e t w i t c h r e s p o n s e to i n d i r e c t s t i m u l a t i o n c o u l d be o b t a i n e d b y p r o g r e s s i v e l y increasing t h e Ca 2+ c o n c e n t r a t i o n t o 20 raM. A f t e r 1 m M Cd 2+ t h e r e was o n l y a v e r y small degree o f r e c o v e r y ( < 1 0 % ) in m o s t e x p e r i m e n t s b y progressively increasing t h e Ca 2+ c o n c e n t r a t i o n t o as high as 40 m M . A gradual contracture of the diaphragm usually f o l l o w e d w h e n Ca 2÷ c o n c e n t r a t i o n was i n c r e a s e d a b o v e 6 m M . 3.3. Cd 2÷ and e.p.p, quantal c o n t e n t ( m ) Cd 2+ in t h e highest c o n c e n t r a t i o n used in t h e i n t r a c e l l u l a r e x p e r i m e n t s (0.5 m M ) did n o t cause a significant c h a n g e in t h e m e m b r a n e p o t e n t i a l d u r i n g a 30 m i n p e r i o d o f exposure. T h e r e was n o significant c h a n g e in t h e m e a n value o f m a f t e r 30 m i n in t h e c o n t r o l s o l u t i o n b u t in t h e case o f t h e Cd 2+ (10 p M ) s o l u t i o n t h e r e was a significant r e d u c t i o n o f m a f t e r 30 m i n (table 1). A t higher c o n c e n t r a t i o n s (0.1 m M a n d 0.5 m M ) o f Cd 2+ t h e r e was f r e q u e n t l y a partial or c o m p l e t e failure o f t h e e.p.p, r e s p o n s e t o a series o f stimuli w h i c h c o u l d n o t b e reversed w i t h i n 30 rain b y r e v e r t i n g t o t h e c o n t r o l b a t h i n g s o l u t i o n , T h e decline in t h e e.p.p. r e s p o n s e a f t e r 0.1 m M Cd 2+ c o u l d b e reversed w i t h i n 10 m i n t o a level w h i c h e x c e e d e d t h e threshold for the initiation of a muscle action p o t e n t i a l b y raising t h e Ca 2+ c o n c e n t r a t i o n t o 7 m M . T h e q u a n t a l c o n t e n t ( m ) in t h e s e cases must have exceeded the value obtained before b e g i n n i n g e x p o s u r e t o Cd 2÷ a l t h o u g h it was
P.J. FORSHAW TABLE 1 Effect of Cd 2+ (10 pM) on end plate potential quantal content (m) in the rat diaphragm. Preparation
m (initial value)
m (30 rain value)
m 30 min m initial
Control solution 1 2,80 2 6.80 3 2.79 4 4.25 5 4.80 6 4.43 7 4.85 8 3.43
2.50 8.10 3.30 3.40 5.20 3.90 4.33 2.85
0.89 1.19 1.18 0.80 1.08 0.88 0.89 0.83 mean + S.E.M. = 0.97 ~ 0.06
Cd 2+ solution 9 3.30 10 1.42 11 8.23 12 9.25 13 1.41 14 7.13 15 5.55 16 2.00 17 5.95 18 5.53
1.05 0.69 3.75 7.08 1.06 2.87 1.87 1.09 4.30 3.45
0.32 0.49 0.46 0.77 0.75 0.40 0.34 0.55 0.72 0.62 mean ± S.E.M. = 0,54 + 0.05 1
i Difference from control significant at p < 0.01. n o t possible t o a c t u a l l y m e a s u r e m a f t e r increasing t h e Ca 2÷ c o n c e n t r a t i o n to 7 m M b e c a u s e t w i t c h i n g o f m u s c l e fibres r e s u l t e d in d i s l o d g e m e n t o f t h e m i c r o - e l e c t r o d e . I t was difficult to select a Ca 2÷ c o n c e n t r a t i o n w h i c h reversed t h e e f f e c t o f a given c o n c e n t r a t i o n o f Cd 2÷ o n m at an individual n e u r o m u s c u l a r j u n c t i o n w i t h o u t initiating m e c h a n i c a l activi t y in s o m e fibres. R e d u c t i o n o f m d u e t o a p r e - s y n a p t i c cond u c t i o n b l o c k b y Cd 2÷ is u n l i k e l y b e c a u s e in all e x p e r i m e n t s w h e r e failures o f t h e e.p.p. r e s p o n s e o c c u r r e d t h e r e was g o o d a g r e e m e n t between the observed and expected number o f failures (Krnjevi6 a n d Miledi, 1 9 5 9 ; Weakly, 1973).
CADMIUM AND NEUROMUSCULAR TRANSMISSION TABLE 2 Effect of Cd 2+ (0.1 and 0.5 raM) on miniature end plate potential (m.e.p.p.) f r e q u e n c y and amplitude. N u m b e r in parentheses represents the no. of observations contributing to the comparisons. N.S. Difference from initial value n o t significant. m.e.p.p, freq u e n c y (no./ sec) ± S.E.M.
m.e.p.p, amplitude (mV ± S.E.M.
Initial value in control solution Value after 30 rain in Cd 2+ 0.1 mM
1.18 ± 0.46 (5) 0.81 ± 0.18 N.S.
0.53 ± 0.05 (5) 0.50 ± 0.04 N.S.
Initial value in controlsolution Value after 3 0 r a i n i n C d 2+ 0 . 5 r a M
1.62 ± 0.40 (4) 0.83 ± 0.07 N.S.
0.33 ± 0.02 (4) 0.29 ± 0.02 N.S.
3.4. C d 2+ a n d m . e . p . p . 's
A 30 min exposure to 0.1 or 0.5 mM Cd :+ did not significantly affect either the mean frequency or the mean amplitude of m.e.p.p.'s (table 2). Higher concentrations were n o t tested in order to avoid precipitation of Cd 2+ salts.
4. Discussion
These experiments show that at the mammalian neuromuscular junction, Cd ~+ has a depressant action which is not readily reversed by washing b u t is reversed by EGTA or by L-cysteine and partially reversed b y an increased Ca 2+ concentration. Toda (1973b) has shown that Cd 2+ can reduce the influx of Ca 2+ ions across K+-depo larised membranes in smooth muscle and it is conceivable that Cd 2+ m a y also reduce the influx of Ca 2+ ions across the presynaptic nerve terminal membrane during depolarization. The reversal of the inhibitory action of Cd 2+ on the diaphragm twitch responses by EGTA and L-cysteine is in agreement with the results o f mammalian heart conduction
375
experiments by Toda (1973b), and is und o u b t e d l y due to complexing of Cd :÷ by these compounds, presumably by effectively competing with receptors for Cd 2÷. The present findings show that the inhibitory effect of Cd 2+, at concentrations of up to 0.1 mM, on the twitch response of the phrenic--diaphragm to indirect stimulation can be completely reversed by increasing the Ca 2÷ concentration in the bathing fluid. The effect of higher concentrations of Cd 2+ could only be partially reversed by increasing Ca2+; this result seems to agree with the work of Toda (1973a) who showed that Ca 2+ cannot fully reverse the inhibitory effect of Cd :+ on the electrical activity of the rabbit sinoatrial node pacemaker fibres. In the present experiments Cd 2+ (10/~M) significantly depressed the evoked quantal release of transmitter at the neuromuscular junction. Concentrations of Cd :+ which completely inhibited the e.p.p, response were without significant effect on the post-synaptic membrane as shown by the lack of change of m.e.p.p, amplitude. Thus Cd 2+ behaves in a similar way to some other cations including L a 3+, C o 2+, Mn 2+, Pb 2+, Mg 2+, Ni 2+ and Zn :+ (Heuser and Miledi, 1971; Weakly, 1973; Meiri and Rahaminoff, 1972; Balnave and Gage, 1973; Manalis and Cooper, 1973; Del Castillo and Engbaek, 1954; Benoit and Mambrini, 1970). Since raising the Ca 2+ concentration of the bathing fluid from 1 to 7 mM was effective in reversing the decline in the e.p.p, response due to 0.1 mM Cd 2+ it is suggested that Cd inhibits neuromuscular transmission by binding to Ca 2+ receptor sites on the presynaptic terminal nerve membrane. Cd 2+ m a y competitively inhibit the formation of a Ca-receptor complex or the entry of such a complex inta the nerve terminal during depolarization. Toda (1973b) has suggested that Cd 2+ interferes with inward Ca 2+ movements across membranes by binding to sulphydryl groups in membranes. Triggle (1972) has reviewed the important role of Ca 2+ in membrane and neurotransmitter events.
376 La 3÷, Co 2÷, Mn 2÷, Pb 2÷ a n d Zn 2÷ h a v e b e e n s h o w n t o increase t h e s p o n t a n e o u s release o f t r a n s m i t t e r at t h e resting a m p h i b i a n n e u r o m u s c u l a r j u n c t i o n ( H e u s e r a n d Miledi, 1 9 7 1 ; W e a k l y , 1 9 7 3 ; Meiri a n d R a h a m i n o f f , 1 9 7 2 ; Balnave and Gage, 1 9 7 3 ; Manalis a n d C o o p e r , 1 9 7 3 ; B e n o i t a n d M a m b r i n i , 1970). In t h e p r e s e n t e x p e r i m e n t s Cd 2÷ did n o t increase t h e f r e q u e n c y o f m . e . p . p . ' s at c o n c e n t r a t i o n s o f 0.1 and 0.5 m M . H o w e v e r , these c o n c e n t r a t i o n s are 2 - - 2 0 t i m e s l o w e r t h a n t h o s e o f t h e previously mentioned cations which have b e e n s h o w n t o increase m . e . p . p , f r e q u e n c y . T h e e f f e c t o f Cd 2÷ c o n c e n t r a t i o n s higher t h a n 0.5 m M o n m . e . p . p , f r e q u e n c y w e r e n o t investigated b e c a u s e p r e c i p i t a t i o n o f Cd 2÷ salts o c c u r r e d . Cd 2÷ differs f r o m t h e a f o r e m e n t i o n e d c a t i o n s in t w o respects. Firstly, it a p p e a r s to be m o r e p o t e n t in i n h i b i t i n g t h e e v o k e d q u a n t a l release o f t r a n s m i t t e r ; a c o n c e n t r a t i o n o f 10 p M was s u f f i c i e n t to cause a significant i n h i b i t i o n c o m p a r e d w i t h 50 pM f o r Co 2÷ ( W e a k l y , 1 9 7 3 ) , 70 pM f o r Mn 2÷ (Meiri a n d R a h a m i n o f f , 1 9 7 2 ) , 0.5 m M f o r Ni 2÷ a n d 0.1 m M f o r Zn 2÷ ( B e n o i t a n d M a m b r i n i , 1970). S e c o n d l y , in t h e p r e s e n t e x p e r i m e n t s t h e e f f e c t s o f Cd 2÷ o n t h e d i a p h r a g m t w i t c h r e s p o n s e to i n d i r e c t s t i m u l a t i o n a n d on t h e e.p.p, r e s p o n s e c o u l d n o t be readily reversed b y r e p e a t e d washing w i t h Cd2+-free s o l u t i o n unlike t h e o t h e r c a t i o n s m e n t i o n e d above. It s e e m s likely t h a t this is b e c a u s e Cd 2÷ has a greater a f f i n i t y f o r m e m b r a n e c o n s t i t u e n t s and c o n t r a c t i l e p r o t e i n s c o n t a i n i n g s u l p h y d r y l g r o u p s t h a n t h e o t h e r c a t i o n s (Passow et al., 1 9 6 1 ; Begenisich a n d L y n c h , 1 9 7 4 ) . I t is o f i n t e r e s t t h a t it has b e e n f o u n d t h a t t h e d e p r e s s a n t e f f e c t o f Cd 2÷ o n t h e r a t h e a r t is n o t reversible b y washing a f t e r 20 m i n e x p o sure t o Cd 2÷ ( H a w l e y a n d K o p p , 1 9 7 5 ) . T h e p r e s e n t e x p e r i m e n t s also s h o w t h a t n e u r o m u s c u l a r b l o c k c o n t i n u e s to d e v e l o p s u b s e q u e n t to t h e r e m o v a l o f Cd 2÷ s o l u t i o n suggesting t h a t Cd 2÷ b i n d s r a p i d l y to m u s c l e p r o t e i n s a n d this f o r m s a reservoir o f Cd 2÷ w i t h i n t h e p r e p a r a t i o n ; Cd 2* f r o m this reservoir m i g h t t h e n gradually e q u i l i b r a t e w i t h
P.J. FORSHAW b i n d i n g sites at t h e n e u r o m u s c u l a r j u n c t i o n . This m i g h t also explain w h y t h e i n h i b i t o r y e f f e c t o f Cd 2÷ at c o n c e n t r a t i o n s g r e a t e r t h a n about 0.1mM c o u l d n o t be c o m p l e t e l y reversed b y raising t h e Ca 2÷ c o n c e n t r a t i o n . In c o n c l u s i o n , it seems possible t h a t t h e e f f e c t s o f Cd 2÷ on d i f f e r e n t p h y s i o l o g i c a l s y s t e m s m a y be the result o f a c o m m o n m o d e o f a c t i o n on cell m e m b r a n e s in w h i c h Cd 2÷ inhibits Ca 2÷ influx. F u r t h e r w o r k is n o w being done on the quantitative relationship b e t w e e n Cd 2÷ and Ca 2÷ in o r d e r t o d e t e r m i n e the affinity and dissociation constants for Cd 2÷ in t h e r a t d i a p h r a g m .
References Balnave, R.J. and P.W. Gage, 1973, The inhibitory effect of manganese on transmitter release at the neuromuscular junction of the toad, Brit. J. Pharmacol. 47,339. Begenisich, E. and C. Lynch, 1974, Effects of internal divalent cations on voltage-clamped squid axons, J. Gen. Physiol. 63,675. Benoit, P.R. and J. Mambrini, 1970, Modification of transmitter release by ions which prolong the presynaptic action potential, J. Physiol., London 210, 681. Bremner, I., 1974, Heavy metal toxicities, Quart. Rev. Biophys. 7, 75. Del Castillo, J. and L. Engbaek, 1954, The nature of the neuromuscular block produced by magnesium, J. Physiol., London 124,370. Del Castillo, J. and B. Katz, 1954, Quantal components of the end-plate potential, J. Physiol., London 124,560. Furshpan, E.J., 1956, The effects of osmotic pressure changes on the spontaneous activity at motor nerve endings, J. Physiol., London 134,689. Gage, P.W. and J.I. Hubbard, 1966, An investigation of the post-tetanic potentiation of end-plate potentials at a mammalian neuromuscular junction, J. Physiol., London 184,353. Hawley, P.L. and S.J. Kopp, 1975, Extension of PR interval in isolated rat heart by cadmium, Proc. Soc. Exptl. Biol. Med. 150,669. Heuser, J. and R. Miledi, 1971, Effect of lanthanum ions on function and structure of frog neuromuscular junctions, Proc. R. Soc. B. 179,247. Katz, B. and S. Thesleff, 1957, On the factors which determine the amplitude of the "miniature endplate potential", J. Physiol., London 137,267. Krnjevid, K. and R. Miledi, 1958a, Motor units in the
CADMIUM AND NEUROMUSCULAR TRANSMISSION rat diaphragm, J. Physiol., London 140,427. Krnjevid, K. and R. Miledi, 1958b, Failure of neuromuscular propagation in rats, J. Physiol., London 140,440. Krnjevi~, K. and R. Miledi, 1959, Presynaptic failure of neuromuscular propagation in rats, J. Physiol., London 149, 1. Lfley, A.W., 1956, An investigation of spontaneous activity at the neuromuscular junction of the rat, J. Physiol., London 132,650. Manalis, R.S. and G.P. Cooper, 1973, Presynaptic and postsynaptic effects of lead at the frog neuromuscular junction, Nature, London 243,354. Martin, A.R., 1955, A further study of the statistical composition of the end-plate potential, J. Physiol., London 1 3 0 , 1 1 4 . Meiri, U. and R. Rahaminoff, 1972, Neuromuscular transmission: inhibition by manganese ions, Science 176,308.
377 Passow, H., A. Rothstein and T.W. Clarkson, 1961, The general pharmacology of the heavy metals, Pharmacol. Rev. 13,185. Toda, N., 1973a, Inhibition by cadmium ions of the electrical activity of sino-atrial nodal pacemaker fibers and their response to norepinephrine, J. Pharmacol. Exptl. Therap. 184,357. Toda, N., 1973b, Influence of cadmium ions on the transmembrane potential and contractility of isolated rabbit left atria, J. Pharmacol. Exptl. Therap. 186, 60. Triggle, D.J., 1972, Effect of calcium on excitable membranes and neurotransmitter action, Progr. Surf. Mere. Sci. 5 , 2 6 7 . Weakly, J.N., 1973, The action of cobalt ions on neuromuscular transmission in the frog, J. Physiol., London 2 3 4 , 5 9 7 .
371
THE INHIBITORY EFFECT OF CADMIUM ON NEUROMUSCULAR TRANSMISSION IN THE RAT PHILIP J. FORSHAW Toxicology Unit, Medical Research Council Laboratories, Woodmansterne Road, Carshalton, Surrey SM5 4EF, England
Received 20 September 1976, revised MS received 14 December 1976, accepted 21 December 1976 P.J. FORSHAW, The inhibitory effect o f cadmium on neuromuscular transmission in the rat, European J. Pharmacol. 42 (1977) 371--377. Cadmium (0.125--1 mM) was found to inhibit the isometric response of the isolated rat hemidiaphragm during indirect stimulation, but not during direct stimulation. This effect of cadmium (1 mM) was completely reversed by ethyleneglycol bis-(aminoethyl)-N,N,N',N'-tetra-acetic acid (2 raM) or by L-cysteine (2 raM) but only partially by increased calcium. Cadmium (10 uM) significantly reduced the quantal release of transmitter in the isolated phrenic diaphragm and a concentration of 0.1 mM frequently caused a complete failure of the endplate response after 30 rain. The effect of cadmium on neuromuscular transmission could not be readily reversed by washing with cadmium-free solution. Miniature endplate potential frequency and amplitude were not significantly affected by cadmium (0.1 or 0.5 raM). The results suggest that the effect of cadmium on the isolated phrenic nerve-diaphragm is due largely to inhibition of calcium function at presynaptic nerve terminals. Phrenic nerve---diaphragm preparation Neuromuscular transmission
Endplate potential Acetylcholine
1. I n t r o d u c t i o n The effects of chronic exposure to cadmium (Cd 2÷) in m a n a n d a n i m a l s a n d its e p i d e m i ological c o r r e l a t i o n w i t h s o m e diseases h a v e b e e n well s t u d i e d ( B r e m n e r , 1974). H o w e v e r , t h e a c u t e e f f e c t s o f large doses o f Cd 2÷ in l a b o r a t o r y animals are n o t so well k n o w n . T h e p r o s t r a t i o n a n d r e s p i r a t o r y failure w h i c h we in this l a b o r a t o r y h a v e o b s e r v e d t o f o l l o w w i t h i n a f e w m i n u t e s o f t h e administ r a t i o n o f large d o s e s o f Cd 2÷ ( 5 - - 1 0 m g i.v.) to t h e r a t suggested t h a t it w o u l d be w o r t h investigating t h e a c t i o n o f Cd 2+ o n t h e gross t w i t c h r e s p o n s e s o f t h e isolated r a t p h r e n i c n e r v e - - d i a p h r a g m p r e p a r a t i o n . In a d d i t i o n , since o t h e r d i v a l e n t c a t i o n s including m a n g a nese (Balnave a n d Gage, 1 9 7 3 ) ; lead (Manalis a n d C o o p e r , 1 9 7 3 ) ; c o b a l t (Weakly, 1 9 7 3 ) ; nickel a n d zinc ( B e n o i t a n d M a m b r i n i , 1 9 7 0 )
Calcium
Cadmium
have all b e e n s h o w n to r e d u c e e v o k e d transm i t t e r release at t h e n e u r o m u s c u l a r j u n c t i o n , it was d e c i d e d t o e x a m i n e t h e e f f e c t s o f Cd 2÷ on t h e intracellularly r e c o r d e d e n d p l a t e potentials ( e . p . p . ' s ) a n d m i n i a t u r e e n d p l a t e potentials ( m . e . p . p . ' s ) o f t h e r a t d i a p h r a g m .
2. Materials and methods T h e left isolated h e m i d i a p h r a g m was set u p as d e s c r i b e d b y Krnjevid a n d Miledi ( 1 9 5 8 a , b). Male rats o f w e i g h t 2 5 0 - - 3 0 0 g w e r e used. T h e b a t h v o l u m e was 10 ml. 2 b a t h i n g s o l u t i o n s w e r e used. F o r experim e n t s investigating t h e e f f e c t o f Cd 2÷ o n diap h r a g m gross t w i t c h r e s p o n s e s a t r i s ( h y d r o x y m e t h y l ) m e t h y l a m i n e - b u f f e r e d s o l u t i o n was used in o r d e r t o p r e v e n t p r e c i p i t a t i o n w h e n
372 concentrations of Cd 2÷ greater than 0.5 mM were used. The composition of this solution was as follows (mM) NaC1 127; KC1 5.9; CaC12 2.0; MgC12 1.0; tris(hydroxymethyl)methylamine 10.0; dextrose 11.0. The solution was adjusted to pH 7.4 with HC1 and gassed in the fluid reservoir with 100% 02. Liley solution (Liley, 1956) was used for the intracellular experiments because it was usually difficult to detect m.e.p.p.'s in trisbuffered solution. This solution contained 1 mM Ca 2÷ and 12.5 mM Mg2÷ in order to reduce endplate potentials below threshold level. Cd 2÷ concentrations greater than 0.5 mM could not be used in this solution because otherwise precipitation of Cd 2÷ salts occurred. The solution had a pH of 7.4 when gassed with 95% 02 and 5% COs. No osmotic compensation was made for the addition of Cd 2÷ or extra Ca 2÷ in either solution in view of demonstrations that variations in osmotic pressure in the range of the present experiments do not significantly affect the quantal c o n t e n t of e.p.p.'s (Furshpan, 1956; Gage and Hubbard, 1966}. Solutions were kept at a temperature of 25°C rather than the usual 37°C since it has been shown that this minimises the occurrence of presynaptic failure due to h y p o x i a which can occur when oxygen is n o t bubbled directly onto the surface of the diaphragm (Krnjevi6 and Miledi, 1959). The solutions were allowed to flow over the preparation at a rate of 3--4 ml/min and were removed by continuous suction. The preparation was stimulated indirectly using supramaximal pulses of 0.1 msec duration and a frequency of 8/min. The isometric contractions were recorded using a transducer (Statham UC3) connected to a pen recorder (Devices M4). Direct stimulation with pulses of 1 msec duration, 8/min frequency was applied using two platinum electrodes in contact with each end of the diaphragm. E.p.p.'s and m.e.p.p.'s were recorded using conventional microelectrode techniques, displayed on an oscilloscope (Tektronix 502) and photographed with a moving film camera
P.J. FORSHAW (Nihon Kohden). Since the amplitude of e.p.p.'s recorded from preparations bathed in high Mg2÷ and/or low Ca 2÷ solution varies according to the quantal hypothesis (Del Castillo and Katz, 1954), at least 30 recordings were made of the e.p.p, as well as a 30 sec period of m.e.p.p, recording, after allowing 30 min for equilibration with each new solution, in order to obtain the mean e.p.p, and m.e.p.p. amplitudes. Wherever possible the quantal content (m) was calculated from the ratio of mean e.p.p, to mean m.e.p.p, amplitude but in cases where failure of e.p.p, response occurred the m e t h o d of failure was used (Del Castillo and Katz, 1954). No correction was made for the non linear summation of e.p.p.'s since results were discarded when m was greater than 10 (Martin, 1955). All amplitude measurements of m.e.p.p.'s were corrected to a resting membrane potential of --75 mV (Katz and Thesleff, 1957). In each experiment concerned with the effect of Cd 2÷ on evoked transmitter release an initial series of e.p.p, and m.e.p.p, recordings in control solution was obtained and a further series of records after 30 min in either control or Cd 2÷ solution. The values for m were calculated and the ratios of the 30 rain to initial value found for control and Cd 2÷ solutions. The means of these ratios for all the experiments were compared and the significance of differences between them calculated using the Student's t-test. By this means the possible significance of the effect of Cd 2+ would not be affected by deterioration of the preparations due to other factors since these would affect both control and Cd 2÷ solution 30 min values of m. M.e.p.p. frequencies were calculated directly from the photographic records; only junctions where the average frequency was less than 3/sec were used in order to avoid the use of junctions which had been damaged by impalement. Only one Cd 2÷ experiment was done on each preparation in order to avoid retention effects after fluid replacement.
CADMIUM AND NEUROMUSCULAR TRANSMISSION
373
3.2. Reversal o f Cd 2÷ block by EGTA, L-cysteine and Ca 2+
3. Results 3.1. Effect o f Cd ~÷ on diaphragm contractions
Indirectly or directly stimulated hemidiaphragms bathed in the tris-buffered solution without Cd 2÷ maintained responses for at least 4 h without noticeable decline. In solutions containing Cd 2÷ (0.125--2 mM) with the normal concentration of Ca 2÷ the twitch responses to indirect stimulation gradually declined and became blocked, that is too small to be recorded, after an interval of 10---52 min. The relationship between the time interval to block and log Cd 2÷ concentration is shown in fig. 1. Block still developed if the preparation was in contact with a Cd 2÷ (1 mM) solution for only 5 min (fig. 2a).
Ethyleneglycol bis(aminoethyl ether)N,N,N',N'-tetra-acetic acid (EGTA) 2 mM and L-cysteine (2 mM) reversed the block of the diaphragm twitch responses to indirect stimulation caused by Cd 2+ (1 mM). Reversal generally took place within 20 min of adding the reversing agent (figs. 2b and 2c).
50 45 40 35 30 25 F I.--
20 15 1(] I
1
02
el~
I
0~6 Q~ 1
mM Ioglo [Cd]
Fig. 1. D o s e - - r e s p o n s e relationship b e t w e e n the log10 Cd 2+ c o n c e n t r a t i o n ( m M ) on the abscissa and on the ordinate the time taken to produce b l o c k (rains) o f the responses o f the isolated rat diaphragm to indirect stimulation. Vertical lines at each p o i n t represent the standard error.
Fig. 2. E f f e c t o f Cd 2+ on the responses o f the isolated diaphragm o f the rat to indirect s t i m u l a t i o n showing; the d e v e l o p m e n t o f block after changing from Cd 2+ (1 raM) s o l u t i o n to control s o l u t i o n (a), the reversal o f Cd 2+ (1 raM) block by E G T A (b), and by L-cysteine (c). T h e partial reversal o f Cd 2+ (0.25 raM) block by Ca 2+ (6 raM) and the failure o f a higher c o n c e n t r a t i o n o f Ca 2+ (10 raM) to c o m p l e t e l y reverse the block is s h o w n in d. Time axis in 1 rain intervals.
374 Cd2+-induced b l o c k was f o u n d t o be partially reversible b y Ca :+ ; t h e d e g r e e o f r e c o v e r y d e p e n d e d o n t h e c o n c e n t r a t i o n o f Cd 2÷ to which the preparation had been exposed. Thus in 3 o u t o f 4 e x p e r i m e n t s 6 m M Ca 2÷ gave 60--70% recovery from block induced by 0 . 2 5 m M Cd 2+ a n d 10 m M Ca 2+ did n o t increase this r e c o v e r y n o t i c e a b l y (fig. 2d). C o m p l e t e or n e a r l y c o m p l e t e r e c o v e r y f r o m 0.1 m M Cd 2+ b l o c k was o b t a i n e d b y raising Ca 2+ to 6 m M b u t a f t e r e x p o s u r e t o 0.5 m M Cd 2+ o n l y a b o u t 40% r e c o v e r y o f t h e t w i t c h r e s p o n s e to i n d i r e c t s t i m u l a t i o n c o u l d be o b t a i n e d b y p r o g r e s s i v e l y increasing t h e Ca 2+ c o n c e n t r a t i o n t o 20 raM. A f t e r 1 m M Cd 2+ t h e r e was o n l y a v e r y small degree o f r e c o v e r y ( < 1 0 % ) in m o s t e x p e r i m e n t s b y progressively increasing t h e Ca 2+ c o n c e n t r a t i o n t o as high as 40 m M . A gradual contracture of the diaphragm usually f o l l o w e d w h e n Ca 2÷ c o n c e n t r a t i o n was i n c r e a s e d a b o v e 6 m M . 3.3. Cd 2÷ and e.p.p, quantal c o n t e n t ( m ) Cd 2+ in t h e highest c o n c e n t r a t i o n used in t h e i n t r a c e l l u l a r e x p e r i m e n t s (0.5 m M ) did n o t cause a significant c h a n g e in t h e m e m b r a n e p o t e n t i a l d u r i n g a 30 m i n p e r i o d o f exposure. T h e r e was n o significant c h a n g e in t h e m e a n value o f m a f t e r 30 m i n in t h e c o n t r o l s o l u t i o n b u t in t h e case o f t h e Cd 2+ (10 p M ) s o l u t i o n t h e r e was a significant r e d u c t i o n o f m a f t e r 30 m i n (table 1). A t higher c o n c e n t r a t i o n s (0.1 m M a n d 0.5 m M ) o f Cd 2+ t h e r e was f r e q u e n t l y a partial or c o m p l e t e failure o f t h e e.p.p, r e s p o n s e t o a series o f stimuli w h i c h c o u l d n o t b e reversed w i t h i n 30 rain b y r e v e r t i n g t o t h e c o n t r o l b a t h i n g s o l u t i o n , T h e decline in t h e e.p.p. r e s p o n s e a f t e r 0.1 m M Cd 2+ c o u l d b e reversed w i t h i n 10 m i n t o a level w h i c h e x c e e d e d t h e threshold for the initiation of a muscle action p o t e n t i a l b y raising t h e Ca 2+ c o n c e n t r a t i o n t o 7 m M . T h e q u a n t a l c o n t e n t ( m ) in t h e s e cases must have exceeded the value obtained before b e g i n n i n g e x p o s u r e t o Cd 2÷ a l t h o u g h it was
P.J. FORSHAW TABLE 1 Effect of Cd 2+ (10 pM) on end plate potential quantal content (m) in the rat diaphragm. Preparation
m (initial value)
m (30 rain value)
m 30 min m initial
Control solution 1 2,80 2 6.80 3 2.79 4 4.25 5 4.80 6 4.43 7 4.85 8 3.43
2.50 8.10 3.30 3.40 5.20 3.90 4.33 2.85
0.89 1.19 1.18 0.80 1.08 0.88 0.89 0.83 mean + S.E.M. = 0.97 ~ 0.06
Cd 2+ solution 9 3.30 10 1.42 11 8.23 12 9.25 13 1.41 14 7.13 15 5.55 16 2.00 17 5.95 18 5.53
1.05 0.69 3.75 7.08 1.06 2.87 1.87 1.09 4.30 3.45
0.32 0.49 0.46 0.77 0.75 0.40 0.34 0.55 0.72 0.62 mean ± S.E.M. = 0,54 + 0.05 1
i Difference from control significant at p < 0.01. n o t possible t o a c t u a l l y m e a s u r e m a f t e r increasing t h e Ca 2÷ c o n c e n t r a t i o n to 7 m M b e c a u s e t w i t c h i n g o f m u s c l e fibres r e s u l t e d in d i s l o d g e m e n t o f t h e m i c r o - e l e c t r o d e . I t was difficult to select a Ca 2÷ c o n c e n t r a t i o n w h i c h reversed t h e e f f e c t o f a given c o n c e n t r a t i o n o f Cd 2÷ o n m at an individual n e u r o m u s c u l a r j u n c t i o n w i t h o u t initiating m e c h a n i c a l activi t y in s o m e fibres. R e d u c t i o n o f m d u e t o a p r e - s y n a p t i c cond u c t i o n b l o c k b y Cd 2÷ is u n l i k e l y b e c a u s e in all e x p e r i m e n t s w h e r e failures o f t h e e.p.p. r e s p o n s e o c c u r r e d t h e r e was g o o d a g r e e m e n t between the observed and expected number o f failures (Krnjevi6 a n d Miledi, 1 9 5 9 ; Weakly, 1973).
CADMIUM AND NEUROMUSCULAR TRANSMISSION TABLE 2 Effect of Cd 2+ (0.1 and 0.5 raM) on miniature end plate potential (m.e.p.p.) f r e q u e n c y and amplitude. N u m b e r in parentheses represents the no. of observations contributing to the comparisons. N.S. Difference from initial value n o t significant. m.e.p.p, freq u e n c y (no./ sec) ± S.E.M.
m.e.p.p, amplitude (mV ± S.E.M.
Initial value in control solution Value after 30 rain in Cd 2+ 0.1 mM
1.18 ± 0.46 (5) 0.81 ± 0.18 N.S.
0.53 ± 0.05 (5) 0.50 ± 0.04 N.S.
Initial value in controlsolution Value after 3 0 r a i n i n C d 2+ 0 . 5 r a M
1.62 ± 0.40 (4) 0.83 ± 0.07 N.S.
0.33 ± 0.02 (4) 0.29 ± 0.02 N.S.
3.4. C d 2+ a n d m . e . p . p . 's
A 30 min exposure to 0.1 or 0.5 mM Cd :+ did not significantly affect either the mean frequency or the mean amplitude of m.e.p.p.'s (table 2). Higher concentrations were n o t tested in order to avoid precipitation of Cd 2+ salts.
4. Discussion
These experiments show that at the mammalian neuromuscular junction, Cd ~+ has a depressant action which is not readily reversed by washing b u t is reversed by EGTA or by L-cysteine and partially reversed b y an increased Ca 2+ concentration. Toda (1973b) has shown that Cd 2+ can reduce the influx of Ca 2+ ions across K+-depo larised membranes in smooth muscle and it is conceivable that Cd 2+ m a y also reduce the influx of Ca 2+ ions across the presynaptic nerve terminal membrane during depolarization. The reversal of the inhibitory action of Cd 2+ on the diaphragm twitch responses by EGTA and L-cysteine is in agreement with the results o f mammalian heart conduction
375
experiments by Toda (1973b), and is und o u b t e d l y due to complexing of Cd :÷ by these compounds, presumably by effectively competing with receptors for Cd 2÷. The present findings show that the inhibitory effect of Cd 2+, at concentrations of up to 0.1 mM, on the twitch response of the phrenic--diaphragm to indirect stimulation can be completely reversed by increasing the Ca 2÷ concentration in the bathing fluid. The effect of higher concentrations of Cd 2+ could only be partially reversed by increasing Ca2+; this result seems to agree with the work of Toda (1973a) who showed that Ca 2+ cannot fully reverse the inhibitory effect of Cd :+ on the electrical activity of the rabbit sinoatrial node pacemaker fibres. In the present experiments Cd 2+ (10/~M) significantly depressed the evoked quantal release of transmitter at the neuromuscular junction. Concentrations of Cd :+ which completely inhibited the e.p.p, response were without significant effect on the post-synaptic membrane as shown by the lack of change of m.e.p.p, amplitude. Thus Cd 2+ behaves in a similar way to some other cations including L a 3+, C o 2+, Mn 2+, Pb 2+, Mg 2+, Ni 2+ and Zn :+ (Heuser and Miledi, 1971; Weakly, 1973; Meiri and Rahaminoff, 1972; Balnave and Gage, 1973; Manalis and Cooper, 1973; Del Castillo and Engbaek, 1954; Benoit and Mambrini, 1970). Since raising the Ca 2+ concentration of the bathing fluid from 1 to 7 mM was effective in reversing the decline in the e.p.p, response due to 0.1 mM Cd 2+ it is suggested that Cd inhibits neuromuscular transmission by binding to Ca 2+ receptor sites on the presynaptic terminal nerve membrane. Cd 2+ m a y competitively inhibit the formation of a Ca-receptor complex or the entry of such a complex inta the nerve terminal during depolarization. Toda (1973b) has suggested that Cd 2+ interferes with inward Ca 2+ movements across membranes by binding to sulphydryl groups in membranes. Triggle (1972) has reviewed the important role of Ca 2+ in membrane and neurotransmitter events.
376 La 3÷, Co 2÷, Mn 2÷, Pb 2÷ a n d Zn 2÷ h a v e b e e n s h o w n t o increase t h e s p o n t a n e o u s release o f t r a n s m i t t e r at t h e resting a m p h i b i a n n e u r o m u s c u l a r j u n c t i o n ( H e u s e r a n d Miledi, 1 9 7 1 ; W e a k l y , 1 9 7 3 ; Meiri a n d R a h a m i n o f f , 1 9 7 2 ; Balnave and Gage, 1 9 7 3 ; Manalis a n d C o o p e r , 1 9 7 3 ; B e n o i t a n d M a m b r i n i , 1970). In t h e p r e s e n t e x p e r i m e n t s Cd 2÷ did n o t increase t h e f r e q u e n c y o f m . e . p . p . ' s at c o n c e n t r a t i o n s o f 0.1 and 0.5 m M . H o w e v e r , these c o n c e n t r a t i o n s are 2 - - 2 0 t i m e s l o w e r t h a n t h o s e o f t h e previously mentioned cations which have b e e n s h o w n t o increase m . e . p . p , f r e q u e n c y . T h e e f f e c t o f Cd 2÷ c o n c e n t r a t i o n s higher t h a n 0.5 m M o n m . e . p . p , f r e q u e n c y w e r e n o t investigated b e c a u s e p r e c i p i t a t i o n o f Cd 2÷ salts o c c u r r e d . Cd 2÷ differs f r o m t h e a f o r e m e n t i o n e d c a t i o n s in t w o respects. Firstly, it a p p e a r s to be m o r e p o t e n t in i n h i b i t i n g t h e e v o k e d q u a n t a l release o f t r a n s m i t t e r ; a c o n c e n t r a t i o n o f 10 p M was s u f f i c i e n t to cause a significant i n h i b i t i o n c o m p a r e d w i t h 50 pM f o r Co 2÷ ( W e a k l y , 1 9 7 3 ) , 70 pM f o r Mn 2÷ (Meiri a n d R a h a m i n o f f , 1 9 7 2 ) , 0.5 m M f o r Ni 2÷ a n d 0.1 m M f o r Zn 2÷ ( B e n o i t a n d M a m b r i n i , 1970). S e c o n d l y , in t h e p r e s e n t e x p e r i m e n t s t h e e f f e c t s o f Cd 2÷ o n t h e d i a p h r a g m t w i t c h r e s p o n s e to i n d i r e c t s t i m u l a t i o n a n d on t h e e.p.p, r e s p o n s e c o u l d n o t be readily reversed b y r e p e a t e d washing w i t h Cd2+-free s o l u t i o n unlike t h e o t h e r c a t i o n s m e n t i o n e d above. It s e e m s likely t h a t this is b e c a u s e Cd 2÷ has a greater a f f i n i t y f o r m e m b r a n e c o n s t i t u e n t s and c o n t r a c t i l e p r o t e i n s c o n t a i n i n g s u l p h y d r y l g r o u p s t h a n t h e o t h e r c a t i o n s (Passow et al., 1 9 6 1 ; Begenisich a n d L y n c h , 1 9 7 4 ) . I t is o f i n t e r e s t t h a t it has b e e n f o u n d t h a t t h e d e p r e s s a n t e f f e c t o f Cd 2÷ o n t h e r a t h e a r t is n o t reversible b y washing a f t e r 20 m i n e x p o sure t o Cd 2÷ ( H a w l e y a n d K o p p , 1 9 7 5 ) . T h e p r e s e n t e x p e r i m e n t s also s h o w t h a t n e u r o m u s c u l a r b l o c k c o n t i n u e s to d e v e l o p s u b s e q u e n t to t h e r e m o v a l o f Cd 2÷ s o l u t i o n suggesting t h a t Cd 2÷ b i n d s r a p i d l y to m u s c l e p r o t e i n s a n d this f o r m s a reservoir o f Cd 2÷ w i t h i n t h e p r e p a r a t i o n ; Cd 2* f r o m this reservoir m i g h t t h e n gradually e q u i l i b r a t e w i t h
P.J. FORSHAW b i n d i n g sites at t h e n e u r o m u s c u l a r j u n c t i o n . This m i g h t also explain w h y t h e i n h i b i t o r y e f f e c t o f Cd 2÷ at c o n c e n t r a t i o n s g r e a t e r t h a n about 0.1mM c o u l d n o t be c o m p l e t e l y reversed b y raising t h e Ca 2÷ c o n c e n t r a t i o n . In c o n c l u s i o n , it seems possible t h a t t h e e f f e c t s o f Cd 2÷ on d i f f e r e n t p h y s i o l o g i c a l s y s t e m s m a y be the result o f a c o m m o n m o d e o f a c t i o n on cell m e m b r a n e s in w h i c h Cd 2÷ inhibits Ca 2÷ influx. F u r t h e r w o r k is n o w being done on the quantitative relationship b e t w e e n Cd 2÷ and Ca 2÷ in o r d e r t o d e t e r m i n e the affinity and dissociation constants for Cd 2÷ in t h e r a t d i a p h r a g m .
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377 Passow, H., A. Rothstein and T.W. Clarkson, 1961, The general pharmacology of the heavy metals, Pharmacol. Rev. 13,185. Toda, N., 1973a, Inhibition by cadmium ions of the electrical activity of sino-atrial nodal pacemaker fibers and their response to norepinephrine, J. Pharmacol. Exptl. Therap. 184,357. Toda, N., 1973b, Influence of cadmium ions on the transmembrane potential and contractility of isolated rabbit left atria, J. Pharmacol. Exptl. Therap. 186, 60. Triggle, D.J., 1972, Effect of calcium on excitable membranes and neurotransmitter action, Progr. Surf. Mere. Sci. 5 , 2 6 7 . Weakly, J.N., 1973, The action of cobalt ions on neuromuscular transmission in the frog, J. Physiol., London 2 3 4 , 5 9 7 .
