Excitation of Skeletal Muscle by Fluoride

H.Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science,Volume 27, pp. 277-284 0 1986 Elsevier Science PublishersB.V., Amsterdam - Printed in The Netherlands

EXCITATION

OF

SKELETAL MUSCLE

277

BY FLUORIDE

TOSHIMI HATTORI AND HIROSHI MAEHASHI Department of Dental Pharmacology, Matsumoto Dental College, Shiojiri 39907. Japan

ABSTRACT The effects of NaF on the mechanical and electrical responses of sciatic nerve-sartorius muscle preparations from the bullfrog were investigated i n an attempt to elucidate the mechanism of excitation of skeletal muscle induced by fluoride. NaF at concentrations above 0.1 mM augmented the twitch and tetanus evoked by indirect stimulation of the partially fatigued muscle, and above 5.0 m M it induced fibrillation. Sodium oxalate at 2.5 sodium citrate at 1.7 m M , and calcium-deficient Ringer's solution neither induced fibrillation nor augmented the twitch. NaF increased the

mM.

amp1 itude of the miniature endplate potential (m.e.p.p.) even under pretreatment with 16 uM neostigmine. but the extent of the increment was only one half as much as that by NaF alone. NaF, 0.1 m M adrenaline, 1.0 m M theophylline, and 4.0 m M dibutyryl cyclic A M P , raised the m.e.p.p. frequency and increased the quanta1 content of the endplate potential.

These

findings suggest that fluoride may induce excitation of skeletal muscle in a manner unrelated to decalcification and that such excitation may be due to the activation of adenylate cyclase in the nerve ending, the inhibition of cholinesterase, and to increased sensitivity of the ZndPlate to acetylcholine.

INTRODUCTION Fluoride affects the l i v i n g cell i n various ways, for example, decalcification, inhibition o f enzymes, and activation of adenylate cyclase, etc. It also excites skeletal muscles. Fibrillation is observed in acute fluoride poisoning (1). and fluoride decurarizes muscles blocked by 4tubocurarine (2). Many investigators have ascribed acute fluoride poisoning to decalcification (3). However, there are some reports indicating that it is due rather to other properties of fluoride, e.g., its inhibitory effect on cholinesterase (4).

Most studies on fluoride toxicity have been

performed in vivo. The experimental data are complex, and the mechanism of

278 t h e poisoning i s s t i l l unclear.

An i n v i t r o s t u d y h a s b e e n m a d e o n t h e

e f f e c t s o f s o d i u m f l u o r i d e on m e c h a n i c a l a n d e l e c t r i c a l r e s p o n s e s o f t h e skeletal

muscle o f t h e frog,

t o d e t e r m i n e w h e t h e r o r n o t f l u o r i d e causes

muscle e x c i t a t i o n t h r o u g h a mechanism independent o f d e c a l c i f i c a t i o n . MATERIALS AND METHODS Sciatic nerve-sartorius

m u s c l e p r e p a r a t i o n s f r o m t h e b u l l f r o g Rana c a t e s The

b e i a n a ( w e i g h i n g b e t w e e n 1 0 0 and 2 0 0 g) w e r e u s e d a s t h e m a t e r i a l . p r e p a r a t i o n s h a v e been d e s c r i b e d p r e v i o u s l y (5). The p r e p a r a t i o n was (volume:

v e r t i c a l l y f i x e d o n t h e b o t t o m o f a Magnus t u b e

10 m l ) c o n t a i n i n g a e r a t e d R i n g e r ' s s o l u t i o n .

T w i t c h and t e t a n u s

were evoked b y e l e c t r i c a l s t i m u l a t i o n o f t h e n e r v e o r t h e muscle. n e r v e was s t i m u l a t e d w i t h a s u c t i o n e l e c t r o d e ( v o l t a g e :

supramaximum;

d u r a t i o n , 0.1 m s e c ; f r e q u e n c y , 0.1 a n d 50 Hz f o r t w i t c h a n d t e t a n u s , pectively).

res-

D i r e c t s t i m u l a t i o n o f t h e m u s c l e was done t r a n s v e r s e l y b y

f i e l d s t i m u l a t i o n w i t h p l a t i n u m p l a t e s ( 2 0 0 V, 0.1-1.0 l o w i n g t r e a t m e n t w i t h d - t u b o c u r a r i n e c h l o r i d e (6.0-30

m s e c , 0.1 H z ) f o l -

ugiml).

Tensions i n

t h e muscle were measured i s o m e t r i c a l l y w i t h a f o r c e - d i s p l a c e m e n t (SB-IT.

The

N i h o n Kohden).

transducer

The c o n c e n t r a t i o n o f c a l c i u m i n t h e R i n g e r ' s s o l u -

t i o n was d e t e r m i n e d b y a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y . E l e c t r i c a l r e s p o n s e s o f t h e m u s c l e f i b e r were i n t r a c e l l u l a r l y recorded w i t h a n o s c i l l o s c o p e (VC-10. f i l l e d w i t h 3M KC1. extracellularly. p o t e n t i a l (e.p.p.).

N i h o n Kohden)

a glass microelectrode

The compound a c t i o n p o t e n t i a l o f t h e n e r v e was d e r i v e d

For

d e t e r m i n a t i o n o f t h e quanta1 content o f t h e endplate

t h e m u s c l e was t r e a t e d w i t h a p e r f u s a t e c o n t a i n i n g 0.5

m M CaC12 a n d 6.0 m M MgC12 t h r o u g h o u t t h e e x p e r i m e n t a n d t h e e.p.p. m i n i a t u r e e n d p l a t e p o t e n t i a l (m.e.p.p.)

w e r e r e c o r d e d (6).

and

Quanta1 c o n t e n t

w a s o b t a i n e d b y b o t h t h e d i r e c t m e t h o d ( m e a n a m p l i t u d e o f e.p.p./mean a m p l i t u d e o f m.e.p.p.)

and t h e f a i l u r e s method,

i m p u l s e s / n u m b e r o f f a i l u r e s o f e.p.p.)

(6).

log,

(number o f nerve

The v a l u e s o b t a i n e d b y t h e

d i r e c t m e t h o d w e r e c o r r e c t e d f o r n o n l i n e a r s u m m a t i o n (7). The c o m p o s i t i o n o f n o r m a l R i n g e r ' s s o l u t i o n ( i n m M ) was a s f o l l o w s : NaC1, 5.6.

1 1 0 ; KC1,

1.9;

CaC12,

1.1;

T h e pH w a s a d j u s t e d t o 7.3.

s o d i u m f l u o r i d e (NaF),

NaH2P04,

0.4;

NaHC03.

2.4:

and g l u c o s e ,

The f o l l o w i n g c h e m i c a l s w e r e used:

sodium o x a l a t e , sodium c i t r a t e . and t h e o p h y l l i n e

(Na k a r a i ); n e o s t i g m i n e m e t h y l s u 1f a t e ( S h io n o g i); d - t u b o c u r a r in e c h 1 o r id e (d-Tc. (Sigma).

Yoshitomi);

adrenaline (Daiichi);

and d i b u t y r y l c y c l i c AMP (dbcAMP)

A l l e x p e r i m e n t s w e r e c a r r i e d o u t a t r o o m t e m p e r a t u r e (20-25°C).

219 RESULTS NaF (0.1-10

mM) dose-dependently

a u g m e n t e d t h e t w i t c h and t e t a n u s e v o k e d

b y i n d i r e c t s t i m u l a t i o n o f t h e p a r t i a l l y f a t i g u e d m u s c l e whose t w l t c h t e n s i o n had d e c r e a s e d t o 60-70% o f t h e m a x i m a l v a l u e .

The e f f e c t o f NaF on

t h e t w i t c h i s i l l u s t r a t e d i n F i g u r e 1.

even a t a concentra-

t i o n a s h i g h a s 1 0 mM,

However,

NaF.

augmented n e i t h e r t h e maximal t w i t c h n o r t h e t w i t c h

induced by d i r e c t stimulation.

NaF a b o v e 5.0 m M i n d u c e d f i b r i l l a t i o n o f

t h e u n s t i m u l a t e d muscle. T h e Ca c o n c e n t r a t i o n i n t h e R i n g e r ' s s o l u t i o n w a s l o w e r e d f r o m 1.1 t o

0.94 mM b y 5 mM NaF.

To c o m p a r e NaF w i t h v a r i o u s c h e m i c a l a g e n t s f o r t h e i r

Ca-reducing e f f e c t ,

we i n v e s t i g a t e d t h e e f f e c t s o f t h e d e c a l c i f y i n g d r u g s ,

s o d i u m o x a l a t e (2.5

m M ) and s o d i u m c i t r a t e (1.7

t o 5.0 m M NaF i n t e r m s o f C a - b i n d i n g c a p a c i t y , c o n t a i n i n g 0.94

m M Ca.

mM),

which are equivalent

and C a - d e f i c i e n t R i n g e r

None o f t h e s e i n d u c e d f i b r i l l a t i o n o r a u g m e n t e d t h e

twitch.

180,

F i g u r e 1. L o g d o s e - r e s p o n s e c u r v e o f NaF o n t h e t w i t c h t e n s i o n o f t h e z a r t o r i u s muscle evoked b y s c i a t i c n e r v e s t i m u l a t i o n o f a p r e p a r a t i o n f r o m the bullfrog. NaF a u g m e n t e d t h e t w i t c h d o s e - d e p e n d e n t l y . Each p o i n t r e p r e s e n t s t h e mean o f r e l a t i v e v a l u e s (8-9 e x p e r i m e n t s ) o f t h e t w i t c h t e n s i o n s o b s e r v e d 3 m i n u t e s a f t e r a p p l i c a t i o n o f NaF c o m p a r e d w i t h t h a t before the application. The v e r t i c a l b a r s i n d i c a t e t h e s t a n d a r d d e v i a t i o n . +$, ?yric: S i g n i f i c a n t l y d i f f e r e n t f r o m t h e v a l u e b e f o r e t h e a p p l i c a t i o n a t p<0.05 and p
280

200 r

-2

180

-

u

$e

-

ti d

f

140120-

looL0:5 1:O 210 5:O NgF concentration (mM) Figure 2. Rise of m.e.p.p. frequency produced by NaF. Each polnt represents the mean o f flve relatlve values compared with those before the application. )ep~0.05,i'"p
The effects of NaF on the nerve and muscle were studied to ascertain that they were not the sites of NaF action. As expected, NaF (5.0 and 10 mM) had no of the (r.p.), muscle

effect on their electrical responses, i.e., the alpha-wave amplitude compound action potential of the nerve and the resting potential membrane resistance, and wave form of the action potential of fiber remained unchanged by NaF treatment.

The influence of NaF on the electrical phenomena in the endplate was also NaF at 0.5-5.0 m M raised the m.e.p.p. frequency (i.e. the rate of spontaneous release of the transmitter) and increased the amplitude investigated.

of not only the m.e.p.p. but also the e.p.p. The increase of the m.e.p.p. frequency produced by NaF is shown in Figure 2. Since the elevation of m.e.p.p. frequency and e.p.p. amplitude seemed to be related to the improvement of transmitter release from the nerve ending, the effect of NaF on the quantal content of e.p.p. (i.e., the amount of transmitter released in response to a single nerve impulse) was studied. NaF increased the quantal contents calculated by both methods, as illustrated in Figure 3. The quantal content obtained by the direct method was corrected by Martin's method (7); namely, the value was multiplied by (r.p.-15)/(r.p.-15-e.p.p.) after r.p. was substituted by 89.4 or 92.0, obtained from measurement during the control or test period. In addition, the effects of adrenaline. an activator of adenylate cyclase. theophylline, an inhibitor of phosphodiesterase, and dbcAMP, a derivative o f cyclic AMP, on the transmitter release were studied to see how NaF affected the nerve ending. The quantal content and m.e.p.p.

frequency were increased by 0.1 mM adrenaline, 1.0 mM

theophylline, and 4.0 mM dbcAMP (Table I).

TABLE I

E F F E C T S O F ADRENALINE, THEOPHYLLINE. A N D dbcAMP

ON T H E

QUANTAL CONTENT O F e.p.p.

A N D O N THE

m.e. p. p. F R E Q U E N C Y . Treatment

Mean A m p l i t u d e o f e.p.p. ( m V )

23.7 30.0

1.50

0.31 0.36

4.53 5.15

29.3 51.0

0.13 0.87''

0.26 0.23

3.06 4.35

49.3 61.3

A d r e n a l i n e , 0.1 mM

0.85

Control

1.37

Control DbcAMP, "p
4.0 mM

Frequency o f m.e.p.p. (/min)

2.52 3.31

0.70

1.0 mM

Corrected Quanta1 Content

0.30 0.30

Control

Theophylline,

Mean A m p l i t u d e o f m.e.p.p. (mV)

282

-t140r c

C

d

130 -

5 120. -

10010:5 1:O 2:O 5:O NaF concentration (mM) Figure 3. Increment o f quanta1 content of e.p.p. produced by NaF. Each point represents the same as in Figure 2. 0 : direct method, A : failures m e t h od. 3c p < 0.0 5, 3*"p<0.0 1.

The effect of neostigmine, an inhibitor of cholinesterase (ChE), was investigated to find out whether the increase in the m.e.p.p.

amplitude

induced by NaF was related to C h E inhibition. Figure 4 shows that 5.0 m M NaF increased the m.e.p.p. amplitude about twice as much as neostigmine (16 uM). a concentration 8 times higher than that which inhibited ChE up to

80%. NaF increased the amplitude even after neostigmine application. but the increase was only one half as much as that by NaF alone (Fig. 4).

180r

T

"i

I d 120

100

NaF

Figure 4. Effects of NaF and neostigmine on m.e.p.p. amplitude. NaF increased the m.e.p.p. amplitude more markedly than neostigmine. The extent of the increment o f m.e.p.p. amplitude by N a f under pretreatment with neostigmine was only half as much as that by NaF alone. Each column represents the same as in Figure 2.

283

DISCUSSION NaF a t c o n c e n t r a t i o n s above 0.1 stimulation,

mM augmented t h e t w i t c h evoked by n e r v e

a n d a b o v e 5.0 mM i n d u c e d f i b r i l l a t i o n .

l a t e o r sodium c i t r a t e ,

However,

s o d i u m oxa-

b o t h a t a c o n c e n t r a t i o n e q u i v a l e n t t o 5.0 m M NaF,

or C a - d e f i c i e n t R i n g e r caused n e i t h e r f i b r i l l a t i o n n o r augmentation o f These r e s u l t s i n d i c a t e t h a t NaF a t c o n c e n t r a t i o n s l o w e r t h a n 5.0

twitch.

mM induces e x c i t a t i o n t h r o u g h a m e c h a n i s m u n r e l a t e d t o d e c a l c i f i c a t i o n . Moreover,

s i n c e NaF h a d no e f f e c t o n t h e t w i t c h e v o k e d b y d i r e c t s t i m u l a -

t i o n n o r on t h e e l e c t r i c a l responses o f t h e muscle, i t i s u n l i k e l y t o a c t on t h e muscle f i b e r d i r e c t l y .

S i n c e d e c r e a s i n g t h e e x t e r n a l Ca c o n c e n t r a -

t i o n raises the e x c i t a b i l i t y o f the nerve

(8). t h e e f f e c t o f NaF o n t h e

compound a c t i o n p o t e n t i a l o f t h e n e r v e was i n v e s t i g a t e d . wave f o r m r e m a i n e d unchanged.

Therefore,

I t s a m p l i t u d e and

t h e s i t e o f a c t i o n o f NaF seems

t o be n e i t h e r t h e n e r v e n o r m u s c l e b u t t h e n e u r o m u s c u l a r j u n c t i o n .

P.s t h e m.e.p.p.

frequency i s e n t i r e l y c o n t r o l l e d by t h e conditions o f t h e

p r e s y n a p t i c membrane (9),

i n c r e a s e i n f r e q u e n c y b y NaF s h o w e d t h a t NaF

a c t e d on t h e n e r v e e n d i n g and i n c r e a s e d t h e s p o n t a n e o u s r a t e o f t r a n s m i t t e r release.

I n addition,

i t a l s o s u g g e s t e d t h a t t h e i n c r e a s e i n t h e e.p.p.

a m p l i t u d e may b e d u e t o i m p r o v e m e n t o f t h e e v o k e d r e l e a s e o f t h e t r a n s mitter.

Thus,

t h e q u a n t a l c o n t e n t o f t h e e.p.p.

was measured.

5.0 m M i n c r e a s e d t h e q u a n t a l c o n t e n t , i n d i c a t i n g t h a t

NaF a t

0.5-

NaF i n c r e a s e d t h e

amount o f t r a n s m i t t e r r e l e a s e d b y a n e r v e impulse. S i n c e NaF a c t i v a t e s a d e n y l a t e c y c l a s e , various t i s s u e s (lo),

a l l o f w h l c h show a s i m i l a r a c t i o n , increased the

t h u s r a i s i n g t h e cAMP l e v e l i n

t h e e f f e c t s o f adrenaline,

theophylline,

were studied.

and dbcAMP,

Each o f t h e t e s t e d d r u g s

q u a n t a l c o n t e n t a n d r a i s e d t h e m.e.p.p.

frequency.

i m p l i e s t h a t cAMP may be i n v o l v e d i n t h e t r a n s m i t t e r r e l e a s e .

This

This idea i s

s u p p o r t e d b y m a n y r e p o r t s d e s c r i b i n g t h e r o l e o f cAMP i n n e u r o m u s c u l a r t r a n s m i s s i o n (11). dbcAMP,

F o r example, W i l s o n (12) i n v e s t i g a t e d t h e e f f e c t s o f

theophylline,

and a m i n o p h y l l i n e ( a n i n h i b i t o r o f p h o s p h o d i e s t e r a s e ) and r e p o r t e d t h a t a l l o f t h e t e s t e d

o n t h e t r a i n s o f r e p e t i t i v e e.p.p.s

d r u g s i n c r e a s e d t h e q u a n t a l c o n t e n t o f t h e f i r s t e.p.p.. and m o b i l i z a t i o n r a t e ,

releasable store,

s u g g e s t i n g t h a t cAMP was i n v o l v e d i n t h e s y n t h e s i s ,

m o b i l i z a t i o n , and s t o r a g e o f Ach.

We b e l i e v e , t h e r e f o r e ,

t h a t NaF i m p r o v e s

Ach r e l e a s e t h r o u g h e l e v a t i o n o f cAMP c o n c e n t r a t i o n s i n t h e n e r v e e n d i n g . B e c a u s e NaF r e d u c e s t h e a c t i v i t y o f ChE, m.e.p.p.

a m p l i t u d e was i n v e s t i g a t e d .

t h e i n f l u e n c e o f NaF o n t h e

T h e i n c r e a s e i n t h e m.e.p.p.

amp1 i-

t u d e b y NaF u n d e r p r e t r e a t m e n t w i t h n e o s t i g m i n e was o n l y o n e h a l f a s much as t h a t b y NaF a l o n e ;

therefore,

t h e p o t e n t i a t i o n o f t h e m.e.p.p.

may be p a r t i a l l y due t o t h e a n t i - C h E

a c t i o n o f NaF.

amplitude

284 NaF i n c r e a s e d t h e m.e.p.p. stigmine. t o Ach,

a m p l i t u d e e v e n u n d e r p r e t r e a t m e n t w i t h neo-

T h i s i n d i c a t e s t h a t NaF r a i s e d t h e s e n s i t i v i t y o f t h e e n d p l a t e

f o r t h e a m p l i t u d e o f t h e m.e.p.p.

t h e p o s t s y n a p t i c membrane (9). t h e i n c r e a s e i n t h e m.e.p.p.

i s c o n t r o l l e d by p r o p e r t i e s o f

Koketsu and Gerard ( 2 ) have d e s c r i b e d t h a t

a m p l i t u d e induced by NaF i s due t o t h e r i s e o f

Ach s e n s i t i v i t y r a t h e r t h a n t o ChE i n h i b i t i o n . F r o m t h e r e s u l t s d e s c r i b e d above,

t h e mechanism o f e x c i t a t i o n o f t h e

s k e l e t a l muscle by NaF i s t h o u g h t t o be as f o l l o w s : a t e cyclase,

spontaneous o r evoked r e l e a s e o f t h e t r a n s m i t t e r ; raised,

ChE i s i n h i b i t e d ,

increased;

NaF a c t i v a t e s adenyl-

t h u s r a i s i n g t h e cAMP l e v e l i n t h e n e r v e ending: a n d t h e Ach

t h e m.e.p.p.

cAMP improves frequency i s

s e n s i t i v i t y o f the endplate i s

d e p o l a r i z a t i o n o f t h e e n d p l a t e r e g i o n by Ach i s made so i n t e n -

s i v e t h a t t h e a c t i o n p o t e n t i a l i s generated;

f i n a l l y f i b r i l l a t i o n occurs o r

t h e t w i t c h i s a u g m e n t e d as a r e s u l t o f r e c r u i t m e n t o f t h e m u s c l e f i b e r s . It

IS

concluded t h a t f l u o r i d e may induce e x c i t a t i o n o f s k e l e t a l muscles i n

a fashion unrelated t o d e c a l c i f i c a t i o n ;

t h a t i s , e x c i t a t i o n may be due t o

t h e a c t i v a t i o n o f a d e n y l a t e c y c l a s e i n t h e nerve ending, ChE,

the i n h i b i t i o n o f

and t o t h e i n c r e a s e d s e n s i t i v i t y o f t h e e n d p l a t e t o Ach.

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2. Koketsu K, Gerard RW (1956) Am J P h y s i o l 186:278-282 3. Haynes RC. M u r a d F ( 1 9 8 0 ) I n : Goodman c o l o g i c a l B a s i s o f Therapeutics, 1550

4.

6 t h ed.

LS.

G i l m a n A ( e d s ) The PharmaM a c M i l l a n . New York, pp 1525-

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