- Email: [email protected]
The effect of triiodthyronine on neuromuscular junctions of isolated rat diaphragm
EUROPEAN JOURNAL OF PHARMACOLOGY 2 (1967) 119-122. NORTH-HOLLAND PUBL. COMP., AMSTERDAM
THE EFFECT OF T R I I O D T H Y R O N I N E O N NEUROMUSCULAR J U N C T I O N S OF ISOLATED RAT DIAPHRAGM A. T E I T E L , M i c h a e l a C O N S T A N T I N I D I a n d I r i n a B E R C E A Laboratory of Pharmacology. Faculty of Medicine of B u c a r e s t and Institute of Endocrinology. B u c a r e s t . Roumania Accepted 1 August 1967
A . T E I T E L , M. CONSTANTINIDI and I. BERCEA, The efject of triiodthyronine on neuromuscular j u n c tions of isolated rat diaphragm, European J. P h a r m a e o l . 2 (1967) 119-122. The effects of triiodthyronine (T3) were investigated on the isolated phrenic n e r v e diaphragm p r e p aration of the rat which had been c u r a r i z e d by repeated washes with c a l c i u m - f r e e Tyrode solution (TyOCa). It was found that T 3, in 10 × /2M to 31.6 #M concentrations, r e s t o r e d the synaptic t r a n s m i s sion blocked by the c a l c i u m - f r e e bathing solution. This effect, which began a f t e r 1 to 2 rain, i n c r e a s e d p r o g r e s s i v e l y and could r e s t o r e as mueh as 90~ of the initial amplitude of contraction for s e v e r a l hours. The authors suggest that these effects result from a shift of calcium ions from i n t r a c e l l u l a r to synaptic m e m b r a n e sites; a shift aecounted for by the caleium chelating p r o p e r t i e s of the hormone. The question of a possible ealeium dependent m e c h a n i s m in thyrotoxicosis-induced hyperexeitability is raised. T riiodthyronine Calcium ions Rat phrenic n e r v e diaphragm p r e p a r a t i o n
1. I N T R O D U C T I O N Chelation, as a fundamental physico-chemic a l p r o p e r t y of s p e c i a l o r g a n i c m o l e c u l a r s t r u c t u r e s , i s of p a r t i c u l a r i n t e r e s t b o t h f o r t h e p h y s iologist and pharmacologist (Schubert, 1966; F o y e , 1961). Haemoglobin and chlorophyll are very import a n t n a t u r a l l y o c c u r r i n g c h e l a t e s of i r o n a n d of magnesium, and some antibacterial drugs exert their bacteriostatic or bactericidal actions by chelating certain metallic cations which are essential for the bacterial enzymes, or by promoti n g t h e i n f l u x of s u c h c a t i o n s a s a r e t o x i c f o r t h e i r c e l l s ( R u b i n , 1963; W i l b r a n d t , 1965). The thyroid hormones as amino-acid derivat i v e s h a v e t h e c a p a c i t y of c h e l a t i n g b i v a l e n t m e t al ions, and recently some authors (Pitt-Rivers a n d T a t a , 1959; L i t w a c k a n d K r i t c h e w s k y , 1964) have stressed this property. In p r e v i o u s e x p e r i m e n t s ( T e i t e l , B e r c e a a n d C o n s t a n t i n i d i , 1967), i t w a s p o s s i b l e to d e m o n s t r a t e on i s o l a t e d s k e l e t a l m u s c l e s of t h e f r o g a r a p i d s t i m u l a t i n g a c t i o n of t r i i o d t h y r o n i n e , d e p e n d e n t o n t h e r e m o v a l b y t h e h o r m o n e of c a l c i u m i o n s b o u n d to t h e m e m b r a n e s of t h e c o n t r a c t i l e f i b e r s . In o r d e r t o i n v e s t i g a t e t h e a c t u a l
N e u r o m u s c u l a r block Chelating by triiodthyronine Decurarization
m e c h a n i s m of t h i s e f f e c t m o r e t h o r o u g h l y , we used another experimental model.
2. M E T H O D E x p e r i m e n t s w e r e c a r r i e d out on t h e 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 of t h e r a t ( a s d e s c r i b e d b y B u r n , F i n n e y a n d G o o d w i n , 1952), w i t h t h e a d d i t i o n of two s i l v e r c h l o r i d e e l e c t r o d e s in t h e b a t h f l u i d , p l a c e d on e i t h e r s i d e of the muscle. These electrodes were used for dir e c t s t i m u l a t i o n of t h e m u s c l e , a n d m a d e i t p o s s i b l e to c o m p a r e t h e e f f e c t s of b o t h i n d i r e c t a n d d i r e c t e l e c t r i c a l s t i m u l a t i o n . A 4 v o l t 50 c y c l e s per sec alternating electric current was used; s h o c k s w e r e of 0.1 s e c d u r a t i o n a n d w e r e g i v e n a t a r a t e of 3 p e r m i n . T h e m u s c l e s w e r e m a d e calcium-free by repeated washes with Tyrode s o l u t i o n in w h i c h t h e c a l c i u m c h l o r i d e w a s r e p l a c e d b y e q u i v a l e n t a m o u n t s of s o d i u m c h l o r i d e (TyOCa); this was used both for muscles isolated initially in normal Tyrode solution, and for musc l e s i s o l a t e d f r o m t h e b e g i n n i n g of t h e e x p e r i m e n t in T y O C a . T h e r e m o v a l of t h e c a l c i u m i o n s was greatly hastened by adding carefully adjusted
120
TEITEL ET AL.
a m o u n t s of N a 2 - E D T A s o l u t i o n to t h e b a t h i n g fluid. C o n v e n i e n t a m o u n t s of t r i i o d t h y r o n i n e (T3) w e r e a d d e d to t h e b a t h a f t e r a s t e a d y s t a t e h a d been obtained. The bathing solution was kept at r o o m t e m p e r a t u r e ( b e t w e e n 22 ° a n d 2 4 ° C ) , a n d w a s o x y g e n a t e d b y a s t e a d y a i r flow.
3. RESULTS In n o r m a l T y r o d e s o l u t i o n , t h e m u s c u l a r c o n t r a c t i o n s e l i c i t e d b y e l e c t r i c a l s t i m u l a t i o n of t h e phrenic nerve had an amplitude which was about 9 / 1 0 of t h a t p r o d u c e d b y d i r e c t s t i m u l a t i o n of t h e m u s c l e . If T 3 w a s a d d e d to s u c h a p r e p a r a t i o n , it h a d no e f f e c t e i t h e r on t h e d i r e c t l y o r on t h e i n directly stimulated muscle. Repeated washes with TyOCa impaired the a m p l i t u d e of c o n t r a c t i o n of t h e d i r e c t l y s t i m u lated muscle only slightly while the indirectly stimulated contractions were progressively depressed, and finally completely abolished. The few e x p e r i m e n t s i n w h i c h t h e r e w a s a t e n d e n c y to s p o n t a n e o u s r e c o v e r y of c o n t r a c t i l e e f f e c t s were discarded. This recovery always occurred in t h e f i r s t h a l f h o u r a f t e r t h e l a s t w a s h . In e x p e r i m e n t s w h e r e r e p e a t e d w a s h e s a b o l i s h e d t h e r e s p o n s e of t h e m u s c l e to i n d i r e c t stimulation or where the response at the most w a s o n l y 1-5% of t h a t of t h e d i r e c t l y s t i m u l a t e d m u s c l e , T 3 a d d e d to t h e b a t h , in a f i n a l c o n c e n t r a t i o n of 10 ~M, r a p i d l y e n h a n c e d t h e a m p l i t u d e of t h e r e s p o n s e s (fig. 1). T h i s e f f e c t b e g a n w i t h i n 1 to 2 m i n of t h e a d d i t i o n of t h e h o r m o n e a n d , i n t h e e x p e r i m e n t s h o w n i n fig. 1, a f t e r 100 m i n t h e a m p l i t u d e r e a c h e d 45% of t h a t p r o d u c e d b y t h e initial direct stimulation and was still rising. T h e a d d e d T 3 h a d n o e f f e c t on t h e a m p l i t u d e of c o n t r a c t i o n of t h e d i r e c t l y e x c i t e d m u s c l e . In s u c h e x p e r i m e n t s a s t h e o n e s h o w n i n fig. 1, a f t e r s e v e r a l h o u r s of c o n t i n u a l r h y t h m i c a l s t i m u l a t i o n t h e e f f e c t of d i r e c t m u s c u l a r s t i m u l a t i o n was slightly but steadily decreasing, while the indirectly elicited contractions were still improving. W h e n t h e c o n t r a c t i o n s of a n i n d i r e c t l y s t i m u lated muscle were abolished by repeated washes w i t h T y O C a , t h e p r o g r e s s i v e a d d i t i o n of c a l c i u m to t h e b a t h i n g f l u i d g r a d u a l l y r e s t o r e d t h e e f f e c t s of n e r v e e x c i t a t i o n . T h i s r e s t o r a t i o n i s d i r e c t l y r e l a t e d to t h e c a l c i u m c o n c e n t r a t i o n . T a b l e 1 s h o w s r e s u l t s of t h e a b o v e - m e n t i o n e d e f f e c t s on t h e c o n t r a c t i o n a m p l i t u d e s of a d i r e c t ly and indirectly stimulated muscle. After addit i o n of 1 to 3.16 g M of T 3 t h e g a i n of a m p l i t u d e
6
8
I0
IIIIIrllfltlfllfllrtllliElllitllffftll~ff~
Fig, 1. 5.I. 1967. P h r e n i c n e r v e diaphragm p r e p a r a tion. E l e c t r i c a l stimulation (direct and indirect) = 4 volt ~ , 50 Hz 3 X/min. T i m e m a r k s = 15 min. The muscle was washed 6 t i m e s and isolated in TyOCa solution. At 5 (= 14:00/, 5 hr after beginning or r e c o r d ing, change from indirect to direct stimulation. At 6 ( 14:30), r e t u r n to indirect stimulation. At 7 (= 16:00), 1 /~M T 3 (with no effect). At 8 ( 16:30), again direct stimulation. At 9 (= 17:00), back to indirect s t i m u l a tion. At 10 (- 17:30), 10 ~M and at 11 (- 18:45), 31.6 /IM T3. The amplitude of contraction elicited by indirect stimulation rose from 0 to a steady value amounting (after 6¼ hr) to 68.4~ of the contractions to direct stimulation. p r o d u c e d b y 0.2 m M CaC12 on d i r e c t l y s t i m u l a t e d c o n t r a c t i o n s r e a c h e d v a l u e s of 39 to 48% of t h e d i r e c t l y s t i m u l a t e d o n e s . T h e a d d i t i o n of a s l i t t l e a s 1 /zM of T 3 s h o r t l y b e f o r e t h e a d d i t i o n of c a l c i u m e n h a n c e d t h e r e c o v e r y of t h e a m p l i t u d e of c o n t r a c t i o n s a n d s h i f t e d t h e t h r e s h o l d of t h e a c t i v e c a l c i u m c o n c e n t r a t i o n to t h e l e f t . T h e b l o c k of n e u r o - r n u s c u l a r transmission achieved by repeated washes with TyOCa was g e n e r a l l y l o n g l a s t i n g . E v e n 8 to 14 h r of r h y t h m i c a l s t i m u l a t i o n of t h e n e r v e , i n t h e c a l c i u m free solution, did not restore synaptic transmiss i o n . In m a n y s u c h p r e p a r a t i o n s t h e a d d i t i o n of T 3 to the bathing solution, up to a final concent r a t i o n of 31.6 /aM, r a p i d l y r e s t o r e d t h e s y n a p t i c Table 1 Effect of i n c r e a s i n g Ca concentration on contraction amplitudes of a directly and indirectly stimulated phrenic n e r v e diaphragm preparation. Kind of m u s c l e stimulation
CaC12 concentration in the b a t h ing Tyrode solution 0
0.1
0.2
0.4
mM
20.2
20.4
22.6
25.8
mm
Indirectly
0
0
3.1
5.3
mm
Percentage
0
0
13.7
18.9
%
Directly
TRIIODTHYRONINE AND NEUROMUSCULAR JUNCTIONS t r a n s m i s s i o n and s t a r t e d the i n d i r e c t l y s t i m u la te d c o n t r a c t i o n s again. T h e s e c o n t r a c t i o n s w e r e of s t e a d i l y i n c r e a s i n g a m p li tu d e and r o s e to 9/10, or even m o r e , of those e l i c i t e d by d i r e c t s t i m u l a t i n g of the m u s c l e (fig. 2).
4. DISCUSSION In the e x p e r i m e n t s d e s c r i b e d h e r e a r a t h e r p e c u l i a r kind of e l e c t r i c a l s t i m u l a t i o n was used viz. a low voltage a l t e r n a t i n g c u r r e n t of 50 c y c l e s p e r s e c . This unusual s t i m u l a t i o n was found to be p a r t i c u l a r l y suitable for long e x p e r i m e n t s b e c a u s e of its lack of p o l a r i z i n g e f f e c t s on the m e t a l l i c e l e c t r o d e s . It is obvious that in t h e s e e x p e r i m e n t s the d e p r e s s i o n , or block, of t r a n s m i s s i o n at the n e u r o m u s c u l a r junction is to be a s c r i b e d in the f i r s t p l a c e if not e x c l u s i v e l y to the lack of c a l c i u m in the e x t r a c e l l u l a r fluid (Kuffler, 1944). Thus addition of c a l c i u m to this fluid, up to its p h y s i o l o g i c a l l e v e l , r a p i d l y r e s t o r e d synaptic t r a n s m i s s i o n . The lack of c a l c i u m ions in the bathing fluid c a u s e s the p r o g r e s s i v e e x t r a c e l l u t a r diffusion of m o s t of the l a b i l e c a l c i u m f r o m the functional m e m b r a n e s i t e s . This r e s u l t s in the synaptic c o n c e n t r a t i o n of c a l c i u m ions dropping to below the c r i t i c a l l e v e l n e c e s s a r y for t r a n s m i s s i o n to occur. Lack of c a l c i u m in the bathing solution may a l s o i n t e r f e r e s e c o n d a r i l y with the lo c a l conditions of c a l c i u m ion mobility. T h i s i n t e r f e r e n c e could follow the discontinuation of r a n d o m l y o c c u r r i n g ex ch an g es between the c a l c i u m ions l o cated in the m u s c l e m e m b r a n e s and those which a r e f r e e in the e x t r a c e l l u l a r fluid. This d i s c o n tinuation could, in its turn, induce a r e l a t i v e , and p o s s i b l y p r o g r e s s i v e s t a b i l i z a t i o n of the m e r e -
T3(A5) ......................................
J~,,,,J.....,J ......................................................................
~. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fig. 2. 16.XII. 1966. Phrenic nerve diaphragm preparation. Same experimental conditions as for fig. 1. Time marks = 1 rain. The muscle was washed 6 times and isolated in TyOCa solution. The figure represents the contractile responses of the muscle 6 hr after beginning of the indirect stimulation. At mark T3(A5) , a 10 gM concentration of T 3 was added to the bathing solution. The curve presented covers a period of 2 hr before, and 13 hr after, the addition of T 3.
121
b r a n e - b o u n d c a l c i u m ions which could p a r t i a l l y account f o r the p r o g r e s s i v e block of n e u r o m u s cular transmission. The exact n a t u r e of the action of e x t r a c e l l u l a r c a l c i u m ions at this l e v e l is st i l l unknown ( F r a n k , 1961). It may be m en t i o n ed h e r e that, if the e x t r a c e l l u l a r c a l c i u m is only r e d u c e d synaptic t r a n s m i s s i o n is g r e a t l y enhanced and s o m e t i m e s spontaneous b u r s t s of synaptic f i r i n g o c c u r . This is the s p e c i f i c n e u r o m u s c u l a r h y p e r e x c i t a b i l i t y c h a r a c t e r i s t i c s of c a l c i u m - l a c k tetany. Conversely, calcium chloride concentrations g r e a t e r than the p h y si o l o g i cal ones i m p a i r 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 , and may lead to a c o m p l e t e synaptic block (Copp~e, 1946). In sh ar p c o n t r a s t to t h ese a l t e r a t i o n s in t r a n s m i s s i o n o c c u r r i n g at the synaptic l e v e l , the lack of c a l c i u m ions in the e x t r a c e l l u l a r fluid does not a l t e r , in a significant way, the c h a r a c t e r i s t i c s of the c o n t r a c t i o n s in r e s p o n s e to dir e c t s t i m u l a t i o n of the m u s c l e . The s a m e o p t i m al e l e c t r i c a l c u r r e n t , which b e c a m e i n e f f e c t i v e if applied to the p h r e n i c n e r v e , p r o d u c e d a n o r m a l m u s c u l a r c o n t r a c t i o n if d i r e c t l y applied. This e m p h a s i z e s the fact that the p r e s e n c e of c a l c i u m ions in the bathing solution, and consequently at the s y n a p t i c l e v e l , is not e s s e n t i a l for the c o n t r a c t i o n i t s e l f , but only for the 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 . C o n v e r s e l y , the p r e s e n c e of c a l cium ions inside the m u s c l e f i b e r s is s t r i c t l y ind i s p e n s a b l e both for the c a r r y i n g out of the m e c h a n i c a l events of c o n t r a c t i o n , and for those of r el ax at i o n . The i n t r a c e l l u l a r c a l c i u m ions, although i d e n t i cal so long as they a r e f r e e ions, fulfil d i f f e r ent and s o m e t i m e s opposing p h y s i o l o g i c a l f u n ctions, depending on the d i f f e r e n t and i n t r i c a t e o r g a n i c m o l e c u l e s to which they a r e bound at their various strategic sites. These different bindings a r e not i n d i s s o c i a b l e and p e r m a n e n t , but a r e m o b i l e and dynamic. The r e d i s t r i b u t i o n of the c a l c i u m ions is d e t e r m i n e d by t h ei r l o cal a f f i n i t i e s and c o n c e n t r a t i o n g r a d i e n t s , and r e p r e s e n t s the r e s u l t of i n t r a c e l l u l a r c a l c i u m h o m e o s t a s i s p r o v i d i n g one of the m o s t i m p o r t a n t functional p a r a m e t e r s of m u s c u l a r contraction. A f t e r a few w a s h e s with TyOCa it is p o s s i b l e that, for a c e r t a i n length of t i m e , this r e d i s t r i bution could c o m p e n s a t e for the initial l o s s e s of l a b i l e c a l c i u m ions f r o m t h e i r synaptic s i t e s and s t i l l p r o v i d e s u f f i c i e n t ions for 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 to o c c u r . H o w e v e r , when the w a s h e s a r e r e p e a t e d al l the r e d i s t r i b u t e d c a l c i u m ions a r e g r a d u a l l y r e m o v e d and the n e u r o m u s c u l a r block develops.
122
TEITEL ET AL.
In t h e s e s p e c i a l c o n d i t i o n s , t h e r e d i s t r i b u t i o n of t h e m o r e s t r o n g l y l i n k e d a n d t h e r e f o r e l e s s labile calcium ions could be brought about only a s a n e f f e c t of c e r t a i n p h a r m a c o d y n a m i c a l l y a c t i v e d r u g s , a d d e d to t h e b a t h i n g f l u i d , d r u g s w h i c h , b y t h e i r s p e c i f i c p r o p e r t i e s , a r e a b l e to r e m o v e a n d to m o b i l i z e t h e c a l c i u m f r o m t h e various intracellular structures where it is firmly bound. The authors believe that triiodthyronine, added in the TyOCa bathing solution, bec a u s e of i t s c a l c i u m c h e l a t i n g a b i l i t y , r e m o v e s the calcium from the less differentiated muscle f i b e r m e m b r a n e s a n d t r a n s f e r s i t to t h e s y n a p t i c ones. T h e c o r r e l a t i o n s w h i c h t h e y w e r e a b l e to find on t h e i s o l a t e d 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 , b e t w e e n t h e r e l a t i v e l y r a p i d e f f e c t of t r i i o d t h y r o n i n e a n d t h e c o n t r o l of n e u r o m u s c u l a r transmission by a calcium dependent mechan i s m , r a i s e s t h e p r o b l e m of a s c e r t a i n i n g t h e i r character more accurately. They may have some t h e r a p e u t i c i m p l i c a t i o n s in t h y r o t o x i c o s i s , w h e r e t h e n e r v o u s h y p e r e x c i t a b i l i t y m i g h t d e p e n d on a l t e r a t i o n s of t h e d i s t r i b u t i o n , o r t h e l a b i l i t y , of s y n a p t i c c a l c i u m c a u s e d b y t h e e x c e s s of c i r c u lating thyroid hormones. T h e c a l c i u m d e p e n d e n t s y n a p t i c e f f e c t s of t r i i o d t h y r o n i n e m a y a l s o h e l p to e l u c i d a t e t h e f u n c tional correlations between the thyroid and parathyroid glands and explain the presence, in the former, of t h y r o c a l c i t o n i n e a n d i t s s p e c i f i c functions.
REFERENCES Burn, J.H., D.J. Finney and L.G. Goodwin, 1952, Biological standardization (Oxford University P r e s s , London) p. 347. Copp~e, G., 1946, Le rSle des ions calcium dans la t r a n s m i s s i o n n e u r o m u s c u l a i r e , Arch. Int. Physiol. 54, 323. Foye, W.O., 1961, Role of metal-binding in the biological activities of drugs, J. P h a r m a c e u t . Sci. 50, 93. Frank, G.B., 1961, in: Biophysics of Physiological and Pharmacological actions, ed. A.M. Shanes ( A m e r i can Association for the Advancement of Science, Washington) p. 293. Kuffler, S.W., 1944, The effect of calcium on the neur o m u s c u l a r junction, J. Neurophysiol. 7, 17. Litwack and D.Kritchewsky, 1964, Actions of h o r m o n e s on m o l e c u l a r p r o c e s s e s (J.Wiley and Sons, New York) pp. 58, 132. P i t t - R i v e r s , R. and J. R. Tata, 1959, The thyroid h o r mones I ( P e r g a m o n P r e s s , Oxford). Rubin, M., 1963, The biological implications of alkaline e a r t h chelation, in: The t r a n s f e r of calcium and s t r o n t i u m a c r o s s biological m e m b r a n e s , ed. R. H . W a s s e r m a n (Academic P r e s s , New York) p. 25. Schubert, J., 1966, Chelation in medicine, Sci. Amer. 214, 4O. Teitel, A., I. B e r c e a and M. Constantinidi, 1967, Calcium-dependent stimulating action of triiodthyronine on the isolated muscle of the frog, J. P h a r m . P h a r macol. 19, 262. Wilbrandt, W., 1965, P h a r m a k o l o g i s c h e Aspekte des M e m b r a n t r a n s p o r t e s , P h a r m a c e u t . Acta Helv. 40, 641.
THE EFFECT OF T R I I O D T H Y R O N I N E O N NEUROMUSCULAR J U N C T I O N S OF ISOLATED RAT DIAPHRAGM A. T E I T E L , M i c h a e l a C O N S T A N T I N I D I a n d I r i n a B E R C E A Laboratory of Pharmacology. Faculty of Medicine of B u c a r e s t and Institute of Endocrinology. B u c a r e s t . Roumania Accepted 1 August 1967
A . T E I T E L , M. CONSTANTINIDI and I. BERCEA, The efject of triiodthyronine on neuromuscular j u n c tions of isolated rat diaphragm, European J. P h a r m a e o l . 2 (1967) 119-122. The effects of triiodthyronine (T3) were investigated on the isolated phrenic n e r v e diaphragm p r e p aration of the rat which had been c u r a r i z e d by repeated washes with c a l c i u m - f r e e Tyrode solution (TyOCa). It was found that T 3, in 10 × /2M to 31.6 #M concentrations, r e s t o r e d the synaptic t r a n s m i s sion blocked by the c a l c i u m - f r e e bathing solution. This effect, which began a f t e r 1 to 2 rain, i n c r e a s e d p r o g r e s s i v e l y and could r e s t o r e as mueh as 90~ of the initial amplitude of contraction for s e v e r a l hours. The authors suggest that these effects result from a shift of calcium ions from i n t r a c e l l u l a r to synaptic m e m b r a n e sites; a shift aecounted for by the caleium chelating p r o p e r t i e s of the hormone. The question of a possible ealeium dependent m e c h a n i s m in thyrotoxicosis-induced hyperexeitability is raised. T riiodthyronine Calcium ions Rat phrenic n e r v e diaphragm p r e p a r a t i o n
1. I N T R O D U C T I O N Chelation, as a fundamental physico-chemic a l p r o p e r t y of s p e c i a l o r g a n i c m o l e c u l a r s t r u c t u r e s , i s of p a r t i c u l a r i n t e r e s t b o t h f o r t h e p h y s iologist and pharmacologist (Schubert, 1966; F o y e , 1961). Haemoglobin and chlorophyll are very import a n t n a t u r a l l y o c c u r r i n g c h e l a t e s of i r o n a n d of magnesium, and some antibacterial drugs exert their bacteriostatic or bactericidal actions by chelating certain metallic cations which are essential for the bacterial enzymes, or by promoti n g t h e i n f l u x of s u c h c a t i o n s a s a r e t o x i c f o r t h e i r c e l l s ( R u b i n , 1963; W i l b r a n d t , 1965). The thyroid hormones as amino-acid derivat i v e s h a v e t h e c a p a c i t y of c h e l a t i n g b i v a l e n t m e t al ions, and recently some authors (Pitt-Rivers a n d T a t a , 1959; L i t w a c k a n d K r i t c h e w s k y , 1964) have stressed this property. In p r e v i o u s e x p e r i m e n t s ( T e i t e l , B e r c e a a n d C o n s t a n t i n i d i , 1967), i t w a s p o s s i b l e to d e m o n s t r a t e on i s o l a t e d s k e l e t a l m u s c l e s of t h e f r o g a r a p i d s t i m u l a t i n g a c t i o n of t r i i o d t h y r o n i n e , d e p e n d e n t o n t h e r e m o v a l b y t h e h o r m o n e of c a l c i u m i o n s b o u n d to t h e m e m b r a n e s of t h e c o n t r a c t i l e f i b e r s . In o r d e r t o i n v e s t i g a t e t h e a c t u a l
N e u r o m u s c u l a r block Chelating by triiodthyronine Decurarization
m e c h a n i s m of t h i s e f f e c t m o r e t h o r o u g h l y , we used another experimental model.
2. M E T H O D E x p e r i m e n t s w e r e c a r r i e d out on t h e 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 of t h e r a t ( a s d e s c r i b e d b y B u r n , F i n n e y a n d G o o d w i n , 1952), w i t h t h e a d d i t i o n of two s i l v e r c h l o r i d e e l e c t r o d e s in t h e b a t h f l u i d , p l a c e d on e i t h e r s i d e of the muscle. These electrodes were used for dir e c t s t i m u l a t i o n of t h e m u s c l e , a n d m a d e i t p o s s i b l e to c o m p a r e t h e e f f e c t s of b o t h i n d i r e c t a n d d i r e c t e l e c t r i c a l s t i m u l a t i o n . A 4 v o l t 50 c y c l e s per sec alternating electric current was used; s h o c k s w e r e of 0.1 s e c d u r a t i o n a n d w e r e g i v e n a t a r a t e of 3 p e r m i n . T h e m u s c l e s w e r e m a d e calcium-free by repeated washes with Tyrode s o l u t i o n in w h i c h t h e c a l c i u m c h l o r i d e w a s r e p l a c e d b y e q u i v a l e n t a m o u n t s of s o d i u m c h l o r i d e (TyOCa); this was used both for muscles isolated initially in normal Tyrode solution, and for musc l e s i s o l a t e d f r o m t h e b e g i n n i n g of t h e e x p e r i m e n t in T y O C a . T h e r e m o v a l of t h e c a l c i u m i o n s was greatly hastened by adding carefully adjusted
120
TEITEL ET AL.
a m o u n t s of N a 2 - E D T A s o l u t i o n to t h e b a t h i n g fluid. C o n v e n i e n t a m o u n t s of t r i i o d t h y r o n i n e (T3) w e r e a d d e d to t h e b a t h a f t e r a s t e a d y s t a t e h a d been obtained. The bathing solution was kept at r o o m t e m p e r a t u r e ( b e t w e e n 22 ° a n d 2 4 ° C ) , a n d w a s o x y g e n a t e d b y a s t e a d y a i r flow.
3. RESULTS In n o r m a l T y r o d e s o l u t i o n , t h e m u s c u l a r c o n t r a c t i o n s e l i c i t e d b y e l e c t r i c a l s t i m u l a t i o n of t h e phrenic nerve had an amplitude which was about 9 / 1 0 of t h a t p r o d u c e d b y d i r e c t s t i m u l a t i o n of t h e m u s c l e . If T 3 w a s a d d e d to s u c h a p r e p a r a t i o n , it h a d no e f f e c t e i t h e r on t h e d i r e c t l y o r on t h e i n directly stimulated muscle. Repeated washes with TyOCa impaired the a m p l i t u d e of c o n t r a c t i o n of t h e d i r e c t l y s t i m u lated muscle only slightly while the indirectly stimulated contractions were progressively depressed, and finally completely abolished. The few e x p e r i m e n t s i n w h i c h t h e r e w a s a t e n d e n c y to s p o n t a n e o u s r e c o v e r y of c o n t r a c t i l e e f f e c t s were discarded. This recovery always occurred in t h e f i r s t h a l f h o u r a f t e r t h e l a s t w a s h . In e x p e r i m e n t s w h e r e r e p e a t e d w a s h e s a b o l i s h e d t h e r e s p o n s e of t h e m u s c l e to i n d i r e c t stimulation or where the response at the most w a s o n l y 1-5% of t h a t of t h e d i r e c t l y s t i m u l a t e d m u s c l e , T 3 a d d e d to t h e b a t h , in a f i n a l c o n c e n t r a t i o n of 10 ~M, r a p i d l y e n h a n c e d t h e a m p l i t u d e of t h e r e s p o n s e s (fig. 1). T h i s e f f e c t b e g a n w i t h i n 1 to 2 m i n of t h e a d d i t i o n of t h e h o r m o n e a n d , i n t h e e x p e r i m e n t s h o w n i n fig. 1, a f t e r 100 m i n t h e a m p l i t u d e r e a c h e d 45% of t h a t p r o d u c e d b y t h e initial direct stimulation and was still rising. T h e a d d e d T 3 h a d n o e f f e c t on t h e a m p l i t u d e of c o n t r a c t i o n of t h e d i r e c t l y e x c i t e d m u s c l e . In s u c h e x p e r i m e n t s a s t h e o n e s h o w n i n fig. 1, a f t e r s e v e r a l h o u r s of c o n t i n u a l r h y t h m i c a l s t i m u l a t i o n t h e e f f e c t of d i r e c t m u s c u l a r s t i m u l a t i o n was slightly but steadily decreasing, while the indirectly elicited contractions were still improving. W h e n t h e c o n t r a c t i o n s of a n i n d i r e c t l y s t i m u lated muscle were abolished by repeated washes w i t h T y O C a , t h e p r o g r e s s i v e a d d i t i o n of c a l c i u m to t h e b a t h i n g f l u i d g r a d u a l l y r e s t o r e d t h e e f f e c t s of n e r v e e x c i t a t i o n . T h i s r e s t o r a t i o n i s d i r e c t l y r e l a t e d to t h e c a l c i u m c o n c e n t r a t i o n . T a b l e 1 s h o w s r e s u l t s of t h e a b o v e - m e n t i o n e d e f f e c t s on t h e c o n t r a c t i o n a m p l i t u d e s of a d i r e c t ly and indirectly stimulated muscle. After addit i o n of 1 to 3.16 g M of T 3 t h e g a i n of a m p l i t u d e
6
8
I0
IIIIIrllfltlfllfllrtllliElllitllffftll~ff~
Fig, 1. 5.I. 1967. P h r e n i c n e r v e diaphragm p r e p a r a tion. E l e c t r i c a l stimulation (direct and indirect) = 4 volt ~ , 50 Hz 3 X/min. T i m e m a r k s = 15 min. The muscle was washed 6 t i m e s and isolated in TyOCa solution. At 5 (= 14:00/, 5 hr after beginning or r e c o r d ing, change from indirect to direct stimulation. At 6 ( 14:30), r e t u r n to indirect stimulation. At 7 (= 16:00), 1 /~M T 3 (with no effect). At 8 ( 16:30), again direct stimulation. At 9 (= 17:00), back to indirect s t i m u l a tion. At 10 (- 17:30), 10 ~M and at 11 (- 18:45), 31.6 /IM T3. The amplitude of contraction elicited by indirect stimulation rose from 0 to a steady value amounting (after 6¼ hr) to 68.4~ of the contractions to direct stimulation. p r o d u c e d b y 0.2 m M CaC12 on d i r e c t l y s t i m u l a t e d c o n t r a c t i o n s r e a c h e d v a l u e s of 39 to 48% of t h e d i r e c t l y s t i m u l a t e d o n e s . T h e a d d i t i o n of a s l i t t l e a s 1 /zM of T 3 s h o r t l y b e f o r e t h e a d d i t i o n of c a l c i u m e n h a n c e d t h e r e c o v e r y of t h e a m p l i t u d e of c o n t r a c t i o n s a n d s h i f t e d t h e t h r e s h o l d of t h e a c t i v e c a l c i u m c o n c e n t r a t i o n to t h e l e f t . T h e b l o c k of n e u r o - r n u s c u l a r transmission achieved by repeated washes with TyOCa was g e n e r a l l y l o n g l a s t i n g . E v e n 8 to 14 h r of r h y t h m i c a l s t i m u l a t i o n of t h e n e r v e , i n t h e c a l c i u m free solution, did not restore synaptic transmiss i o n . In m a n y s u c h p r e p a r a t i o n s t h e a d d i t i o n of T 3 to the bathing solution, up to a final concent r a t i o n of 31.6 /aM, r a p i d l y r e s t o r e d t h e s y n a p t i c Table 1 Effect of i n c r e a s i n g Ca concentration on contraction amplitudes of a directly and indirectly stimulated phrenic n e r v e diaphragm preparation. Kind of m u s c l e stimulation
CaC12 concentration in the b a t h ing Tyrode solution 0
0.1
0.2
0.4
mM
20.2
20.4
22.6
25.8
mm
Indirectly
0
0
3.1
5.3
mm
Percentage
0
0
13.7
18.9
%
Directly
TRIIODTHYRONINE AND NEUROMUSCULAR JUNCTIONS t r a n s m i s s i o n and s t a r t e d the i n d i r e c t l y s t i m u la te d c o n t r a c t i o n s again. T h e s e c o n t r a c t i o n s w e r e of s t e a d i l y i n c r e a s i n g a m p li tu d e and r o s e to 9/10, or even m o r e , of those e l i c i t e d by d i r e c t s t i m u l a t i n g of the m u s c l e (fig. 2).
4. DISCUSSION In the e x p e r i m e n t s d e s c r i b e d h e r e a r a t h e r p e c u l i a r kind of e l e c t r i c a l s t i m u l a t i o n was used viz. a low voltage a l t e r n a t i n g c u r r e n t of 50 c y c l e s p e r s e c . This unusual s t i m u l a t i o n was found to be p a r t i c u l a r l y suitable for long e x p e r i m e n t s b e c a u s e of its lack of p o l a r i z i n g e f f e c t s on the m e t a l l i c e l e c t r o d e s . It is obvious that in t h e s e e x p e r i m e n t s the d e p r e s s i o n , or block, of t r a n s m i s s i o n at the n e u r o m u s c u l a r junction is to be a s c r i b e d in the f i r s t p l a c e if not e x c l u s i v e l y to the lack of c a l c i u m in the e x t r a c e l l u l a r fluid (Kuffler, 1944). Thus addition of c a l c i u m to this fluid, up to its p h y s i o l o g i c a l l e v e l , r a p i d l y r e s t o r e d synaptic t r a n s m i s s i o n . The lack of c a l c i u m ions in the bathing fluid c a u s e s the p r o g r e s s i v e e x t r a c e l l u t a r diffusion of m o s t of the l a b i l e c a l c i u m f r o m the functional m e m b r a n e s i t e s . This r e s u l t s in the synaptic c o n c e n t r a t i o n of c a l c i u m ions dropping to below the c r i t i c a l l e v e l n e c e s s a r y for t r a n s m i s s i o n to occur. Lack of c a l c i u m in the bathing solution may a l s o i n t e r f e r e s e c o n d a r i l y with the lo c a l conditions of c a l c i u m ion mobility. T h i s i n t e r f e r e n c e could follow the discontinuation of r a n d o m l y o c c u r r i n g ex ch an g es between the c a l c i u m ions l o cated in the m u s c l e m e m b r a n e s and those which a r e f r e e in the e x t r a c e l l u l a r fluid. This d i s c o n tinuation could, in its turn, induce a r e l a t i v e , and p o s s i b l y p r o g r e s s i v e s t a b i l i z a t i o n of the m e r e -
T3(A5) ......................................
J~,,,,J.....,J ......................................................................
~. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fig. 2. 16.XII. 1966. Phrenic nerve diaphragm preparation. Same experimental conditions as for fig. 1. Time marks = 1 rain. The muscle was washed 6 times and isolated in TyOCa solution. The figure represents the contractile responses of the muscle 6 hr after beginning of the indirect stimulation. At mark T3(A5) , a 10 gM concentration of T 3 was added to the bathing solution. The curve presented covers a period of 2 hr before, and 13 hr after, the addition of T 3.
121
b r a n e - b o u n d c a l c i u m ions which could p a r t i a l l y account f o r the p r o g r e s s i v e block of n e u r o m u s cular transmission. The exact n a t u r e of the action of e x t r a c e l l u l a r c a l c i u m ions at this l e v e l is st i l l unknown ( F r a n k , 1961). It may be m en t i o n ed h e r e that, if the e x t r a c e l l u l a r c a l c i u m is only r e d u c e d synaptic t r a n s m i s s i o n is g r e a t l y enhanced and s o m e t i m e s spontaneous b u r s t s of synaptic f i r i n g o c c u r . This is the s p e c i f i c n e u r o m u s c u l a r h y p e r e x c i t a b i l i t y c h a r a c t e r i s t i c s of c a l c i u m - l a c k tetany. Conversely, calcium chloride concentrations g r e a t e r than the p h y si o l o g i cal ones i m p a i r 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 , and may lead to a c o m p l e t e synaptic block (Copp~e, 1946). In sh ar p c o n t r a s t to t h ese a l t e r a t i o n s in t r a n s m i s s i o n o c c u r r i n g at the synaptic l e v e l , the lack of c a l c i u m ions in the e x t r a c e l l u l a r fluid does not a l t e r , in a significant way, the c h a r a c t e r i s t i c s of the c o n t r a c t i o n s in r e s p o n s e to dir e c t s t i m u l a t i o n of the m u s c l e . The s a m e o p t i m al e l e c t r i c a l c u r r e n t , which b e c a m e i n e f f e c t i v e if applied to the p h r e n i c n e r v e , p r o d u c e d a n o r m a l m u s c u l a r c o n t r a c t i o n if d i r e c t l y applied. This e m p h a s i z e s the fact that the p r e s e n c e of c a l c i u m ions in the bathing solution, and consequently at the s y n a p t i c l e v e l , is not e s s e n t i a l for the c o n t r a c t i o n i t s e l f , but only for the 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 . C o n v e r s e l y , the p r e s e n c e of c a l cium ions inside the m u s c l e f i b e r s is s t r i c t l y ind i s p e n s a b l e both for the c a r r y i n g out of the m e c h a n i c a l events of c o n t r a c t i o n , and for those of r el ax at i o n . The i n t r a c e l l u l a r c a l c i u m ions, although i d e n t i cal so long as they a r e f r e e ions, fulfil d i f f e r ent and s o m e t i m e s opposing p h y s i o l o g i c a l f u n ctions, depending on the d i f f e r e n t and i n t r i c a t e o r g a n i c m o l e c u l e s to which they a r e bound at their various strategic sites. These different bindings a r e not i n d i s s o c i a b l e and p e r m a n e n t , but a r e m o b i l e and dynamic. The r e d i s t r i b u t i o n of the c a l c i u m ions is d e t e r m i n e d by t h ei r l o cal a f f i n i t i e s and c o n c e n t r a t i o n g r a d i e n t s , and r e p r e s e n t s the r e s u l t of i n t r a c e l l u l a r c a l c i u m h o m e o s t a s i s p r o v i d i n g one of the m o s t i m p o r t a n t functional p a r a m e t e r s of m u s c u l a r contraction. A f t e r a few w a s h e s with TyOCa it is p o s s i b l e that, for a c e r t a i n length of t i m e , this r e d i s t r i bution could c o m p e n s a t e for the initial l o s s e s of l a b i l e c a l c i u m ions f r o m t h e i r synaptic s i t e s and s t i l l p r o v i d e s u f f i c i e n t ions for 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 to o c c u r . H o w e v e r , when the w a s h e s a r e r e p e a t e d al l the r e d i s t r i b u t e d c a l c i u m ions a r e g r a d u a l l y r e m o v e d and the n e u r o m u s c u l a r block develops.
122
TEITEL ET AL.
In t h e s e s p e c i a l c o n d i t i o n s , t h e r e d i s t r i b u t i o n of t h e m o r e s t r o n g l y l i n k e d a n d t h e r e f o r e l e s s labile calcium ions could be brought about only a s a n e f f e c t of c e r t a i n p h a r m a c o d y n a m i c a l l y a c t i v e d r u g s , a d d e d to t h e b a t h i n g f l u i d , d r u g s w h i c h , b y t h e i r s p e c i f i c p r o p e r t i e s , a r e a b l e to r e m o v e a n d to m o b i l i z e t h e c a l c i u m f r o m t h e various intracellular structures where it is firmly bound. The authors believe that triiodthyronine, added in the TyOCa bathing solution, bec a u s e of i t s c a l c i u m c h e l a t i n g a b i l i t y , r e m o v e s the calcium from the less differentiated muscle f i b e r m e m b r a n e s a n d t r a n s f e r s i t to t h e s y n a p t i c ones. T h e c o r r e l a t i o n s w h i c h t h e y w e r e a b l e to find on t h e i s o l a t e d 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 , b e t w e e n t h e r e l a t i v e l y r a p i d e f f e c t of t r i i o d t h y r o n i n e a n d t h e c o n t r o l of n e u r o m u s c u l a r transmission by a calcium dependent mechan i s m , r a i s e s t h e p r o b l e m of a s c e r t a i n i n g t h e i r character more accurately. They may have some t h e r a p e u t i c i m p l i c a t i o n s in t h y r o t o x i c o s i s , w h e r e t h e n e r v o u s h y p e r e x c i t a b i l i t y m i g h t d e p e n d on a l t e r a t i o n s of t h e d i s t r i b u t i o n , o r t h e l a b i l i t y , of s y n a p t i c c a l c i u m c a u s e d b y t h e e x c e s s of c i r c u lating thyroid hormones. T h e c a l c i u m d e p e n d e n t s y n a p t i c e f f e c t s of t r i i o d t h y r o n i n e m a y a l s o h e l p to e l u c i d a t e t h e f u n c tional correlations between the thyroid and parathyroid glands and explain the presence, in the former, of t h y r o c a l c i t o n i n e a n d i t s s p e c i f i c functions.
REFERENCES Burn, J.H., D.J. Finney and L.G. Goodwin, 1952, Biological standardization (Oxford University P r e s s , London) p. 347. Copp~e, G., 1946, Le rSle des ions calcium dans la t r a n s m i s s i o n n e u r o m u s c u l a i r e , Arch. Int. Physiol. 54, 323. Foye, W.O., 1961, Role of metal-binding in the biological activities of drugs, J. P h a r m a c e u t . Sci. 50, 93. Frank, G.B., 1961, in: Biophysics of Physiological and Pharmacological actions, ed. A.M. Shanes ( A m e r i can Association for the Advancement of Science, Washington) p. 293. Kuffler, S.W., 1944, The effect of calcium on the neur o m u s c u l a r junction, J. Neurophysiol. 7, 17. Litwack and D.Kritchewsky, 1964, Actions of h o r m o n e s on m o l e c u l a r p r o c e s s e s (J.Wiley and Sons, New York) pp. 58, 132. P i t t - R i v e r s , R. and J. R. Tata, 1959, The thyroid h o r mones I ( P e r g a m o n P r e s s , Oxford). Rubin, M., 1963, The biological implications of alkaline e a r t h chelation, in: The t r a n s f e r of calcium and s t r o n t i u m a c r o s s biological m e m b r a n e s , ed. R. H . W a s s e r m a n (Academic P r e s s , New York) p. 25. Schubert, J., 1966, Chelation in medicine, Sci. Amer. 214, 4O. Teitel, A., I. B e r c e a and M. Constantinidi, 1967, Calcium-dependent stimulating action of triiodthyronine on the isolated muscle of the frog, J. P h a r m . P h a r macol. 19, 262. Wilbrandt, W., 1965, P h a r m a k o l o g i s c h e Aspekte des M e m b r a n t r a n s p o r t e s , P h a r m a c e u t . Acta Helv. 40, 641.