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Changes in potential difference and short-circuit current produced by electrical stimulation in a nerve-skin preparation of the toad
186
PRELIMINARY NOTES
I J. D. JUDAH, K. AHMED AND A. E. M. MCLEAN, Biochim. Biophys. Acta, 65 (1962) 472. 2 K. NAGAI, F. IZUMI AND H. YOSHIDA, J. Biochem., 59 (19661 991. 3 Z. NAKAO, Y. TASHIMA, K. NAGANO AND M. NAKAO, Biochem. Biophys. Res. Commun., 19 (19651 755. 4 M. FuJrrA, T. NAKAO, Y. TASHIMA, N. MIZUNO, I~. NAGANO AND ,'V[. NAKAO, tO be published. 5 S. GRISOLIA, J. CARAVACA AND B. JOYCE, Biochim. Biophys. Acta, 29 (1958) 432. 6 ],. E. I-IOKIN, P. S. SASTRY, P. t{. GALSWORTHY AND A. YODA, Proc. Nall. Acad. Sci. U.S., 54 (19651 I77, 7 K. NAGANO, T. IX~ANAZAWA, N. MIZUNO, Y. TASHIMA, T. NAKAO AND M. NAKAO, tJiochem. Biophys. Res. Commun., 19 (19651 759-
Received J a n u a r y I7th, 1966 Biochim. Biophys. dcta, 12o (1900) I 8 3 - i 8 6
BBA 41 O43
Changes in potential difference and short-circuit current produced by electrical stimulation in a nerve-skin preparation of the toad I n the present work the effect of electrical s t i m u l a t i o n a n d L-noradrenalin on t h e p o t e n t i a l difference a n d short-circuit current across isolated t o a d skin was studied. I n t h e foreleg region of the t o a d (Bufo spinulosus) it was possible to isolate a piece of skin i n n e r v a t e d b y a b r a n c h of t h e brachial nerve. The piece of skin was m o u n t e d as a flat sheet between two c o m p a r t m e n t s of a lucite chamber, b o t h containing t o a d - R i n g e r solution. The nerve was placed on a pair of s t i m u l a t i n g electrodes which were connected to the isolation unit of a Grass s t i m u l a t o r (model $4C). The characteristics of t h e stimulus were pulses of I msec d u r a t i o n at a frequency of i o sec -1 a n d an a m p l i t u d e of i o V. The p o t e n t i a l difference across t h e skin was measured b y means of calomel electrodes placed in c o n t a c t with the internal a n d t h e e x t e r n a l sides of t h e skin t h r o u g h a g a r - R i n g e r solution bridges. The calomel electrodes were connected to a T e k t r o n i x oscilloscope T y p e 502 or a Grass D.C. channel Model 5 P I K . The shortcircuit current was m e a s u r e d following t h e m e t h o d s of USSING AND ZERAHN 1. The area of the exposed skin was 1.5 c m 2. Stabilization of the p o t e n t i a l difference a n d short-circuit c u r r e n t was o b t a i n e d after approx. 4 ° rain, a n d in this s t a t e s t i m u l a t i o n of t h e nerve was performed, or t h e chemical substances being s t u d i e d were a d d e d to t h e internal c o m p a r t m e n t of the lucite chamber. I n IO e x p e r i m e n t s it was o b s e r v e d t h a t t h e s t i m u l a t i o n of t h e t o a d b r a c h i a l nerve produces a l t e r a t i o n s of p o t e n t i a l difference consisting of depolarizations a n d hyperpolarizations2; these were r e p e a t e d under the effect of subseq u e n t s t i m u l a t i o n s after allowing the p r e p a r a t i o n to rest for 3 ° min. S i m u l t a n e o u s l y t h e short-circuit current was m e a s u r e d a n d it was observed t h a t it u n d e r w e n t variations t h a t were similar to the profile of the results given b y t h e p o t e n t i a l difference. A similar effect was observed when a dose of i / z g of L-noradrenalin (0.4/zg/ml) was placed in the i n t e r n a l c o m p a r t m e n t of the lucite c h a m b e r (Fig. I). The skin from one of the forelegs of the t o a d was placed in a lucite c h a m b e r a n d t h e p r e p a r a t i o n was s t i m u l a t e d under o x y g e n a t i o n , while t h e skin from t h e other foreleg (of the same toad) was placed in a n o t h e r c h a m b e r a n d s t i m u l a t e d w i t h o u t oxygenation. Biochim. Biophys. Acta, I2o (I966) 186-188
PRELIMINARY NOTES
I~ 7
Fig. 2 shows the response, as a percentage (potential difference) of the preparation under oxygenation, to be a hyperpolarization of about 8o % above the initial level. The response decreases relatively rapid with respect to the non-oxygenated preparation, which reaches almost the same level of response and remains in hyperpolarization for a more prolonged period of time. f o , O-o-o-o-Q,~_o.o~o-°-o-o-o-o
mV
P
z w
o . . . . .
2!
a
/
./
no 2( o
O..o.O.O.o/ tNorodrenalin (1.tJg)
No!odrenalin (l~g)
b
2'o 3b ~b
sb
6'o ~o
io
9'0
0
10
20
30
40
50
60
70
80
90
TIME(rain) TIME (mln) Fig. I. Effect of L - n o r a d r e n a l i n (o. 4/2g/ml) in t h e i n t e r n a l c o m p a r t m e n t of t h e lucite c h a m b e r o n t h e p o t e n t i a l difference a n d s h o r t - c i r c u i t current, m e a s u r e d s i m u l t a n e o u s l y .
160. LU1~" 01.~C. 0:1'3( 12( 11( 10(
S
fo
f5
2'0 25 3b ~5 4"0 TIME(rain) Fig. 2. Effect of electrical s t i m u l a t i o n of t h e b r a c h i a l n e r v e b r a n c h on t h e p o t e n t i a l difference e x p r e s s e d as per c e n t response. O - - O , w i t h Os; O - - D , w i t h o u t 02. T w o p r e p a r a t i o n s from t h e s a m e t o a d ( o x y g e n a t e d a n d n o n - o x y g e n a t e d ) were e x a m i n e d s i m u l t a n e o u s l y .
A similar experiment was carried out in which, instead of using electrical stimulation, L-noradrenalin was added (0.4 Fg/ml) to the internal compartment of each chamber. It was observed (Fig. 3) that in the preparation under oxygenation a hyperpolarization of about 40 % above the initial level was produced which then started to diminish, while in the non-oxygenated preparation the hyperpolarization reached about 80 % above the initial level, and then remained in this state. Biochim. Biophys. Acta, 12o (1966) 186-188
i8~
PRELIMINARY NOTES
The present experiments show that the electrical stimulation of one of the branches of the brachial nerve has an effect on the potential difference and on shortcircuit current of the isolated skin of the toad foreleg. L-noradrenalin has a similar effect (at o.4/,g/ml) when placed in the internal c o m p a r t m e n t of the lucite chamber a.
170.
/t /.
160 /
150
,~.oo.o.o o,o.o.o.o.o.o.o.oo.°
140
i
~o" /
130
cz120
/
•
110
/ /
100 .~e-e-e-e-eo-~H~-~,. 0~ / t ~ -,¢ fl Norcxtren~lin°.o /~ 90 (t~g) \o*' 5
10
15
/
o-o-o-c-o
"0, ¢~o-c-~o.o.°
o,°
/
/ c,
20
25 30 TIME ( rain )
35
40
z,5
50
55
60
Fig. 3. Effect of L-noradrenalin (0.4 #g/ml) in the internal c o m p a r t m e n t of the lucite c h a m b e r s on the potential difference, expressed as per cent response. O - - © , with O2; I - - Q , w i t h o u t O 2. Two preparations from the same toad (oxygenated and non-oxygenated) were e x a m i n e d simultaneously.
If the electrical stinmlation of the brachial nerve branch exerts its effect by means of a chemical mediator liberated at the nerve endings, it could be concluded that the mediator is sensitive to oxidation (Fig. 2). The results obtained with L-noradrenalin (Fig. 3) lead us to think t h a t this substance could be tile mediator. This assumption is supported by the fact that tissue from toads treated with reserpine for a period of 24 h prior to operating manifested no effect at all on electrical stimulation. Certainly further experiments arc required to ascertain whether L-noradrenalin is the substance t h a t is liberated by the stimulation. The authors wish to acknowledge the excellent technical assistance of Mr. A. C~xrol~.
Departamento de Fisiologla, Escuda de Medici~a y Departamento de Bioflsica, Escuela de Farmacia, Universidad de Concepci6n, Concepci6~, (Chile)
C. (;oxzXLuz S. J. SXNCHEZ O.
I H. H. USSlN(; AND I~. ZERAHN, Acta Physiol. Scan&, 23 (I95t) 1Io. 2 E. SCHOFFENIELS AND M. L. SALEE, Comp. Biochem. Physiol., 14 (1965) 587 . 3 J- 1V[ARCELIN PLAISANCE AND J. CONCHA B., J. Physiol. Paris, 55 (1963) 425.
Received J a n u a r y 24th, 1966 Biochirn. Biophys. dcta, 12o (1966) 186-188
J . CONCHA B.
PRELIMINARY NOTES
I J. D. JUDAH, K. AHMED AND A. E. M. MCLEAN, Biochim. Biophys. Acta, 65 (1962) 472. 2 K. NAGAI, F. IZUMI AND H. YOSHIDA, J. Biochem., 59 (19661 991. 3 Z. NAKAO, Y. TASHIMA, K. NAGANO AND M. NAKAO, Biochem. Biophys. Res. Commun., 19 (19651 755. 4 M. FuJrrA, T. NAKAO, Y. TASHIMA, N. MIZUNO, I~. NAGANO AND ,'V[. NAKAO, tO be published. 5 S. GRISOLIA, J. CARAVACA AND B. JOYCE, Biochim. Biophys. Acta, 29 (1958) 432. 6 ],. E. I-IOKIN, P. S. SASTRY, P. t{. GALSWORTHY AND A. YODA, Proc. Nall. Acad. Sci. U.S., 54 (19651 I77, 7 K. NAGANO, T. IX~ANAZAWA, N. MIZUNO, Y. TASHIMA, T. NAKAO AND M. NAKAO, tJiochem. Biophys. Res. Commun., 19 (19651 759-
Received J a n u a r y I7th, 1966 Biochim. Biophys. dcta, 12o (1900) I 8 3 - i 8 6
BBA 41 O43
Changes in potential difference and short-circuit current produced by electrical stimulation in a nerve-skin preparation of the toad I n the present work the effect of electrical s t i m u l a t i o n a n d L-noradrenalin on t h e p o t e n t i a l difference a n d short-circuit current across isolated t o a d skin was studied. I n t h e foreleg region of the t o a d (Bufo spinulosus) it was possible to isolate a piece of skin i n n e r v a t e d b y a b r a n c h of t h e brachial nerve. The piece of skin was m o u n t e d as a flat sheet between two c o m p a r t m e n t s of a lucite chamber, b o t h containing t o a d - R i n g e r solution. The nerve was placed on a pair of s t i m u l a t i n g electrodes which were connected to the isolation unit of a Grass s t i m u l a t o r (model $4C). The characteristics of t h e stimulus were pulses of I msec d u r a t i o n at a frequency of i o sec -1 a n d an a m p l i t u d e of i o V. The p o t e n t i a l difference across t h e skin was measured b y means of calomel electrodes placed in c o n t a c t with the internal a n d t h e e x t e r n a l sides of t h e skin t h r o u g h a g a r - R i n g e r solution bridges. The calomel electrodes were connected to a T e k t r o n i x oscilloscope T y p e 502 or a Grass D.C. channel Model 5 P I K . The shortcircuit current was m e a s u r e d following t h e m e t h o d s of USSING AND ZERAHN 1. The area of the exposed skin was 1.5 c m 2. Stabilization of the p o t e n t i a l difference a n d short-circuit c u r r e n t was o b t a i n e d after approx. 4 ° rain, a n d in this s t a t e s t i m u l a t i o n of t h e nerve was performed, or t h e chemical substances being s t u d i e d were a d d e d to t h e internal c o m p a r t m e n t of the lucite chamber. I n IO e x p e r i m e n t s it was o b s e r v e d t h a t t h e s t i m u l a t i o n of t h e t o a d b r a c h i a l nerve produces a l t e r a t i o n s of p o t e n t i a l difference consisting of depolarizations a n d hyperpolarizations2; these were r e p e a t e d under the effect of subseq u e n t s t i m u l a t i o n s after allowing the p r e p a r a t i o n to rest for 3 ° min. S i m u l t a n e o u s l y t h e short-circuit current was m e a s u r e d a n d it was observed t h a t it u n d e r w e n t variations t h a t were similar to the profile of the results given b y t h e p o t e n t i a l difference. A similar effect was observed when a dose of i / z g of L-noradrenalin (0.4/zg/ml) was placed in the i n t e r n a l c o m p a r t m e n t of the lucite c h a m b e r (Fig. I). The skin from one of the forelegs of the t o a d was placed in a lucite c h a m b e r a n d t h e p r e p a r a t i o n was s t i m u l a t e d under o x y g e n a t i o n , while t h e skin from t h e other foreleg (of the same toad) was placed in a n o t h e r c h a m b e r a n d s t i m u l a t e d w i t h o u t oxygenation. Biochim. Biophys. Acta, I2o (I966) 186-188
PRELIMINARY NOTES
I~ 7
Fig. 2 shows the response, as a percentage (potential difference) of the preparation under oxygenation, to be a hyperpolarization of about 8o % above the initial level. The response decreases relatively rapid with respect to the non-oxygenated preparation, which reaches almost the same level of response and remains in hyperpolarization for a more prolonged period of time. f o , O-o-o-o-Q,~_o.o~o-°-o-o-o-o
mV
P
z w
o . . . . .
2!
a
/
./
no 2( o
O..o.O.O.o/ tNorodrenalin (1.tJg)
No!odrenalin (l~g)
b
2'o 3b ~b
sb
6'o ~o
io
9'0
0
10
20
30
40
50
60
70
80
90
TIME(rain) TIME (mln) Fig. I. Effect of L - n o r a d r e n a l i n (o. 4/2g/ml) in t h e i n t e r n a l c o m p a r t m e n t of t h e lucite c h a m b e r o n t h e p o t e n t i a l difference a n d s h o r t - c i r c u i t current, m e a s u r e d s i m u l t a n e o u s l y .
160. LU1~" 01.~C. 0:1'3( 12( 11( 10(
S
fo
f5
2'0 25 3b ~5 4"0 TIME(rain) Fig. 2. Effect of electrical s t i m u l a t i o n of t h e b r a c h i a l n e r v e b r a n c h on t h e p o t e n t i a l difference e x p r e s s e d as per c e n t response. O - - O , w i t h Os; O - - D , w i t h o u t 02. T w o p r e p a r a t i o n s from t h e s a m e t o a d ( o x y g e n a t e d a n d n o n - o x y g e n a t e d ) were e x a m i n e d s i m u l t a n e o u s l y .
A similar experiment was carried out in which, instead of using electrical stimulation, L-noradrenalin was added (0.4 Fg/ml) to the internal compartment of each chamber. It was observed (Fig. 3) that in the preparation under oxygenation a hyperpolarization of about 40 % above the initial level was produced which then started to diminish, while in the non-oxygenated preparation the hyperpolarization reached about 80 % above the initial level, and then remained in this state. Biochim. Biophys. Acta, 12o (1966) 186-188
i8~
PRELIMINARY NOTES
The present experiments show that the electrical stimulation of one of the branches of the brachial nerve has an effect on the potential difference and on shortcircuit current of the isolated skin of the toad foreleg. L-noradrenalin has a similar effect (at o.4/,g/ml) when placed in the internal c o m p a r t m e n t of the lucite chamber a.
170.
/t /.
160 /
150
,~.oo.o.o o,o.o.o.o.o.o.o.oo.°
140
i
~o" /
130
cz120
/
•
110
/ /
100 .~e-e-e-e-eo-~H~-~,. 0~ / t ~ -,¢ fl Norcxtren~lin°.o /~ 90 (t~g) \o*' 5
10
15
/
o-o-o-c-o
"0, ¢~o-c-~o.o.°
o,°
/
/ c,
20
25 30 TIME ( rain )
35
40
z,5
50
55
60
Fig. 3. Effect of L-noradrenalin (0.4 #g/ml) in the internal c o m p a r t m e n t of the lucite c h a m b e r s on the potential difference, expressed as per cent response. O - - © , with O2; I - - Q , w i t h o u t O 2. Two preparations from the same toad (oxygenated and non-oxygenated) were e x a m i n e d simultaneously.
If the electrical stinmlation of the brachial nerve branch exerts its effect by means of a chemical mediator liberated at the nerve endings, it could be concluded that the mediator is sensitive to oxidation (Fig. 2). The results obtained with L-noradrenalin (Fig. 3) lead us to think t h a t this substance could be tile mediator. This assumption is supported by the fact that tissue from toads treated with reserpine for a period of 24 h prior to operating manifested no effect at all on electrical stimulation. Certainly further experiments arc required to ascertain whether L-noradrenalin is the substance t h a t is liberated by the stimulation. The authors wish to acknowledge the excellent technical assistance of Mr. A. C~xrol~.
Departamento de Fisiologla, Escuda de Medici~a y Departamento de Bioflsica, Escuela de Farmacia, Universidad de Concepci6n, Concepci6~, (Chile)
C. (;oxzXLuz S. J. SXNCHEZ O.
I H. H. USSlN(; AND I~. ZERAHN, Acta Physiol. Scan&, 23 (I95t) 1Io. 2 E. SCHOFFENIELS AND M. L. SALEE, Comp. Biochem. Physiol., 14 (1965) 587 . 3 J- 1V[ARCELIN PLAISANCE AND J. CONCHA B., J. Physiol. Paris, 55 (1963) 425.
Received J a n u a r y 24th, 1966 Biochirn. Biophys. dcta, 12o (1966) 186-188
J . CONCHA B.