1614
Specialia
d e p e n d i n g on t h e a m o u n t of food i n t a k e . T h i s influences in a crucial w a y t h e still u n d i f f e r e n t i a t e d h y p o t h a l a m i c c e n t e r s a n d stabilizes t h e t h r e s h o l d to p e r i p h e r a l feedb a c k s t i m u l i a t a d i f f e r e n t level. Accordingly, a t a prep u b e r t a l age ( b e t w e e n 10 a n d 16 y e a r s of age) a d i e t r e l a t i v e l y p o o r in calories b u t o t h e r w i s e b a l a n c e d in all its c o m p o n e n t s m i g h t b e one d e t e r m i n a n t f a c t o r in pro-
EXPERIENTIA 33/12
g r a m m i n g t h e species-specific life s p a n of h u m a n s a n d p r o l o n g i t t o its m a x i m a l e x p e c t a n c y . T h i s k i n d of i n t e r v e n t i o n will n o t p r e v e n t ageing, b u t i t m i g h t well p o s t p o n e t h e o n s e t of some of its m o s t t y p i c a l d e g e n e r a t i v e p h e n o m e n a (artheriosclerosis, h y p e r t e n s i o n , increased genesis of t u m o r s , a.o.) w h i c h m o s t p r o b a b l y r e l a t e to an imbalanced hormonal status.
Potentials in m a m m a l i a n skeletal m u s c l e f r o m c o l l a g e n a s e - t r e a t e d t i s s u e W. T. S t a u b e r , C. T. J a n u a r y a n d B. A. S c h o t t e l i u s
Dept. o[ Physiology and Biophysics, College o/Medicine, University o/ Iowa, Iowa City (Iowa 52242, USA), 7? May 1977 Summary. T r a n s m e m b r a n e p o t e n t i a l s were r e c o r d e d f r o m s k e l e t a l muscle fibres dissected w i t h t h e aid of collagenase perfusion. Collagenase t r e a t m e n t h a d little or no effect o n t h e a c t i o n or r e s t i n g p o t e n t i a l s . B e c a u s e dissection of single s k e l e t a l muscle fibres or s m a l l b u n d l e s of fibres o f t e n is difficult d u e to t o u g h c o n n e c t i v e tissue, we h a v e looked for a t e c h n i q u e t h a t w o u l d r e d u c e t h i s c o m p o n e n t w i t h o u t cellular i n j u r y . Collagenases h a v e been shown to digest primarily intercellular proteins and h a v e allowed t h e i s o l a t i o n of i n t a c t s m o o t h muscle i a n d c a r d i a c cells 2. W e h a v e used collagenases t o p r e p a r e s k e l e t a l muscle for h o m o g e n i z a t i o n s, t h u s we decided to explore t h e possibilities of e n z y m e - a s s i s t e d dissection of m a m m a l i a n s k e l e t a l muscle fibres a n d b u n d l e s . P r e l i m i n a r y e x p e r i m e n t s d e m o n s t r a t e d t h a t simple s u b m e r s i o n of t h e t i s s u e in a n e n z y m e - c o n t a i n i n g s o l u t i o n was ina d e q u a t e for d i s r u p t i o n . Therefore, a h i n d - l i m b p e r f u s i o n technique similar to the liver perfusion protocol employed in t h e isolation of l i v e r cells* was a d o p t e d . T h e i n t e g r i t y of p r e p a r a t i o n s s u b j e c t e d t o t h i s t e c h n i q u e was assessed by examining the transmembrane action potential.
Electrical potentials from rat semi-tendinosus muscle
Resting potential Overshoot Action potential
Normal *
Enzyme-treated *
p
68.9 :t_ 1.0 my (15) +20.4 -t- 2.4 my (15) 89.4 :~= 2.5 mv (15)
--66.6 4- 1.0 mv (23) +17.6 4- 1.2 mv (16) 84.1 :j= 1.9 mv (16)
0.09 0.33 0.10
* Indicates that 3 animals were used and numbers in parentheses indicate the number of penetrations recorded. Electrical potentials are the means i SE of all the penetrations recorded.
L
Control
Enzymedissected
Tracings of action potential recorded from control and enzyme dissected muscle. The dashed line in each represents the is0potential line. Vertical bar is 20 inV. Horizontal bar is 2 msec.
Materials and methods. S p r a g u e - D a w l e y r a t s 200-300 g were a n a e s t h e t i z e d w i t h e t h e r a n d a s m a l l c a n n u l a was i n t r o d u c e d i n t o t h e r i g h t f e m o r a l a r t e r y . A f t e r t h e cann u l a was t i e d in place, r o o m t e m p e r a t u r e (22 ~ -t- 2 ~ calcium-free H a n k ' s s o l u t i o n was p e r f u s e d t h r o u g h t h e l i m b b y m e a n s of a p e r i s t a l t i c p u m p u n t i l t h e veins were cleared of blood. T h e n , t h e f e m o r a l v e i n was s e v e r e d a n d t h e p e r f u s a t e allowed to escape for a 5-rain-period. S u b s e q u e n t l y , collagenase ( T y p e ' I , S i g m a Co.) in comp l e t e H a n k ' s s o l u t i o n ( c o n t a i n i n g calcium) t h a t h a d b e e n filtered t h r o u g h G e l m a n 0.45 ~ m filtres was perfused, a t r o o m t e m p e r a t u r e , for 5 mill. T h e r e a f t e r , n o r m a l H a n k ' s s o l u t i o n w i t h o u t e n z y m e was p e r f u s e d for 10 rain to rem o v e t h e collagenase a n d d i g e s t i o n p r o d u c t s . Dissection a n d r e m o v a l of s m a l l b u n d l e s of fibres f r o m t h e semit e n d i n o s u s m u s c l e was p e r f o r m e d in H a n k ' s solution. C o n t r o l p r e p a r a t i o n s were dissected f r o m n o n p e r f u s e d , e t h e r a n a e s t h e t i z e d a n i m a l s a n d p l a c e d d i r e c t l y i n t o complete, r o o m t e m p e r a t u r e , H a n k ' s solution. F o r electrical recording, c o n t r o l a n d t e s t muscle b u n d l e p r e p a r a t i o n s were t r a n s f e r r e d to a h o r i z o n t a l muscle c h a m b e r c o n t a i n i n g o x y g e n a t e d H a n k ' s solution. T r a n s m e m b r a n e p o t e n t i a l s were m e a s u r e d w i t h 3 1V[ KC1 filled glass m i c r o e l e c t r o d e s h a v i n g 5 t o 20 M/2 r e s i s t a n c e a n d c o n n e c t e d t h r o u g h calomel h a l f cells to a T r a n s i d y n e MPA-6 preamplifier with stray capacitance neutralization. S t i m u l a t i o n was a c c o m p l i s h e d w i t h s u p r a t h r e s h o l d s q u a r e w a v e pulses of 0.2 to 0.4 msec d u r a t i o n applied t h r o u g h p l a t i n u m electrodes. A f l o a t i n g m i c r o e l e c t r o d e a r r a n g e m e n t was utilized to m i n i m i z e m e c h a n i c a l a r t i facts. Signals were d i s p l a y e d o n a T e k t r o n i c T y p e 503 oscilloscope e q u i p p e d for P o l a r o i d p h o t o g r a p h y . Results. W h e n p e r f u s e d w i t h e n z y m e - c o n t a i n i n g solution, h i n d - l i m b muscles g r a d u a l l y swelled t o n e a r l y twice t h e i r i n i t i a l d i a m e t e r . U n d e r d i r e c t microscopic view, i t w a s a p p a r e n t t h a t m u s c l e fibre b u n d l e s were well dispersed a n d t h a t t h e y r e s p o n d e d t o electrical s t i m u l a t i o n w i t h vigorous contractions. Subsequently, minimal mechanical dissection was n e c e s s a r y to o b t a i n s m a l l b u n d l e s of muscle fibres t h a t were less r e a d i l y o b t a i n e d f r o m u n 1 T . J . Peters, M. Miiller and C. de Duve, J. exp. Med. 136, 1117 (1972). 2 D . J . Cavanaugh, W. O. Berndt and T. E. Smith, Nature 200, 261 (1963). 3 W.T. Stauber and B. A. Sehottelius, Cytobios 14, 87 (1975). 4 T. Berg, D. Boman and P. O. Seglen, Exp. Cell Res. 72, 571 (1972).
15. 12. 1977
1615
Specialia
t r e a t e d p r e p a r a t i o n s . H o w e v e r , b e c a u s e s o m e of t h e s u p porting structure had been removed by enzyme treatment, these bundles were easily damaged. R e s u l t s of t h e m i c r o e l e c t r o d e s t u d i e s a r e p r e s e n t e d i n tile table and the figure. Although a slight reduction was noted in the resting potential with the enzymetreated m u s c l e s , t h e a c t i o n p o t e n t i a l o v e r s h o o t of t h e i s o e l e c t r i c line a n d t h e a c t i o n p o t e n t i a l a m p l i t u d e w e r e n o t s i g n i f i c a n t l y (p < 0.05) d i f f e r e n t f r o m t h e u n t r e a t e d p r e p arations. Discussion. P r o t e o l y t i c e n z y m e s s u c h a s t r y p s i n h a v e been shown to have deleterious effects on skeletal muscle transmembrane potentialsK Collagenases are more spe-. cific in c l e a v i n g p r i m a r i l y i n t e r c e l l u l a r p r o t e i n s a n d h a v e b e e n u s e d t o r e c o v e r v i a b l e cells f r o m a v a r i e t y of t i s s u e s ~,2,6. I n t h e e x p e r i m e n t s r e p o r t e d h e r e , w e a p p l i e d
collagenase to rat skeletal muscle using a hind-limp perfusion technique to facilitate enzyme distribution and subsequent fibre bundle dispersion. The results indicate t t l a t c o l l a g e n a s e p r e t r e a t m e n t h a s o n l y a s l i g h t , or n e g l i gible, e f f e c t o n t h e c e l l u l a r r e s t i n g p o t e n t i a l a n d a c t i o n potential magnitudes. Action potential durations were observed in some preparations to become prolonged. However, this occurred only when the action potential a n d r e s t i n g p o t e n t i a l a m p l i t u d e s b e g a n t o d e c a y . W e feel this technique offers a simple adjunct to mammalian skeletal muscle dissection, and thus may be particularly u s e f u l i n s t u d i e s of e l e c t r o p h y s i c a l a n d t h e r m o m e c h a n i c a l phenomena. 5 6
D. Holtzlnan and D. Agin, Nature 205, 911 (1965). M. Robell, J. biol. Chem. 239, 375 (1964).
T h e effect of d e h y d r a t i o n on the n e u r o h y p o p h y s e a l b l o o d f l o w in rats w i t h hereditary diabetes insipidus 1 J. K a p i t o l a 2
H . D l o u h A , J. K ~ e ~ e k a a n d J. Z i c h a
Institute o/ Physiology, Czechoslovak Academy o/Sciences, Kr6 BudJ~ovickd 1083, CS-142 20 Praha 4 (Czechoslovakia), 25 May 1977 Summary. N e u r o h y p o p h y s e a l b l o o d f l o w i n c r e a s e s i n w a t e r - d e p r i v e d r a t s . T h i s i n c r e a s e is i n d e p e n d e n t r e l e a s e , s i n c e i t o c c u r s e v e n in r a t s w i t h h e r e d i t a r y d e f e c t o f h y p o t h a l a m i c v a s o p r e s s i n s y n t h e s i s . U s i n g l ~ 5 I - a n t i p y r i n , L i c h a r d u s e t a l J f o u n d a n i n c r e a s e of neurohypophyseal blood flow in rats after water deprivation, and they concluded that it accompanied the release o f v a s o p r e s s i n (VP). E a r l i e r it w a s r e p o r t e d t h a t a n y s t i m u l a t i o n of n e u r o h y p o p h y s e a l h o r m o n e s r e l e a s e c a u s e s a vasodilatation in posterior pituitaryK Furthermore, water deprivation causes the release, from the neurohypop h y s i s , of b o t h o x y t o c i n a n d V P 6. A n a t t e m p t is p r e s e n t e d to ascertain whether neurohypophyseal blood flow increases after water deprivation even in rats which are u n a b l e t o s y n t h e t i z e V P , i.e. in h o m o z y g o u s B r a t t l e b o r o r a t s w i t h h e r e d i t a r y d i a b e t e s i n s i p i d u s ~.
1 2 3 4 5 6 7
of v a s o p r e s s i n
Acknowledgment. The technical assistance of D. Vitimovsk& is acknowledged. Laboratory for Endocrinology and Metabolism, Faculty of General Medicine, Charles University, Prague. Reprint requests: Dr J. Kfe6ek, Institute of Physiology, Czechoslovak Academy of Sciences, 14220, Prague 4. B. Lichardus, I. Albrecht, J. Ponce, L. Linhart, Endocr. exp. 11, 99 (1977). T. Sooriyamoorthy and A. Livingstone, J. Endocr. 5d, 407 (1972). C . W . Jones and B. T. Picketing, J. Physiol., Lond. 203, 449 (1969). H. Valtin and H. A. Sehroeder, Am. J. Physiol. 206, 425 (1964).
Body weight, adenohypophyseal and neurohypophyseaI weight, flow fraction of cardiac output and S~Rb/g-uptake in control (C) and water deprived (WD) Wistar, heterozygous (non-DI) and homozygous (DI) Brattleboro male rats Wistar C
WD
Non-DI C
WD
DI C
WD
204.4 4- 5.56 (9)
187.5" 4- 2.50 (8)
247.1 4- 7.47 (7)
216.3~ • 6.25 (8)
190.7 4- 7.35 (7)
160.6 9 4- 5.21 (8)
Adenohypophysis Weight (mg/100 g b.wt)
-
-
Flow fraction of cardiac output (% • 10 3)
_
_
S6Rb-uptake (%/g)
-
-
1.98 4- 0.061 2.49 4- 0.257 0.62 4- 0.056
1.93 4- 0.071 2.31 4- 0.206 0.60 4- 0.046
2.05 4- 0.061 2.74 4- 0.229 0.67 4- 0.048
2.29 ~ 4- 0.108 3.04~ 4- 0.175 0.67 4- 0.046
0.52 4- 0.026 4.09 q- 0.268 3.94 i 0.243
0.55 4- 0.027 5.04" _t_ 0.352 4.58" 4- 0.167
0.43 b 4- 0.027 3.90 4- 0.350 4.55 4- 0.354
0.48 4- 0.022 4.71 4-4-0.212 4.90 4- 0.210
0.68 b~ -4- 0.034 4.92 bc 4- 0.256 3.67 4- 0.229
0.81 ~bc 4- 0.016 7.53 ~bc 4- 0.657 4.68~ 4- 0;390
Body weight (g)
Neurohypophysis Weight (mg/100 g b.wt) Flow fraction of cardiac output (% • 10 8) S6Rb-uptake (%/g)
Data are given as mean 4- SEM ( ) indicates number of animals. significantly different from control animals (p
Specialia
d e p e n d i n g on t h e a m o u n t of food i n t a k e . T h i s influences in a crucial w a y t h e still u n d i f f e r e n t i a t e d h y p o t h a l a m i c c e n t e r s a n d stabilizes t h e t h r e s h o l d to p e r i p h e r a l feedb a c k s t i m u l i a t a d i f f e r e n t level. Accordingly, a t a prep u b e r t a l age ( b e t w e e n 10 a n d 16 y e a r s of age) a d i e t r e l a t i v e l y p o o r in calories b u t o t h e r w i s e b a l a n c e d in all its c o m p o n e n t s m i g h t b e one d e t e r m i n a n t f a c t o r in pro-
EXPERIENTIA 33/12
g r a m m i n g t h e species-specific life s p a n of h u m a n s a n d p r o l o n g i t t o its m a x i m a l e x p e c t a n c y . T h i s k i n d of i n t e r v e n t i o n will n o t p r e v e n t ageing, b u t i t m i g h t well p o s t p o n e t h e o n s e t of some of its m o s t t y p i c a l d e g e n e r a t i v e p h e n o m e n a (artheriosclerosis, h y p e r t e n s i o n , increased genesis of t u m o r s , a.o.) w h i c h m o s t p r o b a b l y r e l a t e to an imbalanced hormonal status.
Potentials in m a m m a l i a n skeletal m u s c l e f r o m c o l l a g e n a s e - t r e a t e d t i s s u e W. T. S t a u b e r , C. T. J a n u a r y a n d B. A. S c h o t t e l i u s
Dept. o[ Physiology and Biophysics, College o/Medicine, University o/ Iowa, Iowa City (Iowa 52242, USA), 7? May 1977 Summary. T r a n s m e m b r a n e p o t e n t i a l s were r e c o r d e d f r o m s k e l e t a l muscle fibres dissected w i t h t h e aid of collagenase perfusion. Collagenase t r e a t m e n t h a d little or no effect o n t h e a c t i o n or r e s t i n g p o t e n t i a l s . B e c a u s e dissection of single s k e l e t a l muscle fibres or s m a l l b u n d l e s of fibres o f t e n is difficult d u e to t o u g h c o n n e c t i v e tissue, we h a v e looked for a t e c h n i q u e t h a t w o u l d r e d u c e t h i s c o m p o n e n t w i t h o u t cellular i n j u r y . Collagenases h a v e been shown to digest primarily intercellular proteins and h a v e allowed t h e i s o l a t i o n of i n t a c t s m o o t h muscle i a n d c a r d i a c cells 2. W e h a v e used collagenases t o p r e p a r e s k e l e t a l muscle for h o m o g e n i z a t i o n s, t h u s we decided to explore t h e possibilities of e n z y m e - a s s i s t e d dissection of m a m m a l i a n s k e l e t a l muscle fibres a n d b u n d l e s . P r e l i m i n a r y e x p e r i m e n t s d e m o n s t r a t e d t h a t simple s u b m e r s i o n of t h e t i s s u e in a n e n z y m e - c o n t a i n i n g s o l u t i o n was ina d e q u a t e for d i s r u p t i o n . Therefore, a h i n d - l i m b p e r f u s i o n technique similar to the liver perfusion protocol employed in t h e isolation of l i v e r cells* was a d o p t e d . T h e i n t e g r i t y of p r e p a r a t i o n s s u b j e c t e d t o t h i s t e c h n i q u e was assessed by examining the transmembrane action potential.
Electrical potentials from rat semi-tendinosus muscle
Resting potential Overshoot Action potential
Normal *
Enzyme-treated *
p
68.9 :t_ 1.0 my (15) +20.4 -t- 2.4 my (15) 89.4 :~= 2.5 mv (15)
--66.6 4- 1.0 mv (23) +17.6 4- 1.2 mv (16) 84.1 :j= 1.9 mv (16)
0.09 0.33 0.10
* Indicates that 3 animals were used and numbers in parentheses indicate the number of penetrations recorded. Electrical potentials are the means i SE of all the penetrations recorded.
L
Control
Enzymedissected
Tracings of action potential recorded from control and enzyme dissected muscle. The dashed line in each represents the is0potential line. Vertical bar is 20 inV. Horizontal bar is 2 msec.
Materials and methods. S p r a g u e - D a w l e y r a t s 200-300 g were a n a e s t h e t i z e d w i t h e t h e r a n d a s m a l l c a n n u l a was i n t r o d u c e d i n t o t h e r i g h t f e m o r a l a r t e r y . A f t e r t h e cann u l a was t i e d in place, r o o m t e m p e r a t u r e (22 ~ -t- 2 ~ calcium-free H a n k ' s s o l u t i o n was p e r f u s e d t h r o u g h t h e l i m b b y m e a n s of a p e r i s t a l t i c p u m p u n t i l t h e veins were cleared of blood. T h e n , t h e f e m o r a l v e i n was s e v e r e d a n d t h e p e r f u s a t e allowed to escape for a 5-rain-period. S u b s e q u e n t l y , collagenase ( T y p e ' I , S i g m a Co.) in comp l e t e H a n k ' s s o l u t i o n ( c o n t a i n i n g calcium) t h a t h a d b e e n filtered t h r o u g h G e l m a n 0.45 ~ m filtres was perfused, a t r o o m t e m p e r a t u r e , for 5 mill. T h e r e a f t e r , n o r m a l H a n k ' s s o l u t i o n w i t h o u t e n z y m e was p e r f u s e d for 10 rain to rem o v e t h e collagenase a n d d i g e s t i o n p r o d u c t s . Dissection a n d r e m o v a l of s m a l l b u n d l e s of fibres f r o m t h e semit e n d i n o s u s m u s c l e was p e r f o r m e d in H a n k ' s solution. C o n t r o l p r e p a r a t i o n s were dissected f r o m n o n p e r f u s e d , e t h e r a n a e s t h e t i z e d a n i m a l s a n d p l a c e d d i r e c t l y i n t o complete, r o o m t e m p e r a t u r e , H a n k ' s solution. F o r electrical recording, c o n t r o l a n d t e s t muscle b u n d l e p r e p a r a t i o n s were t r a n s f e r r e d to a h o r i z o n t a l muscle c h a m b e r c o n t a i n i n g o x y g e n a t e d H a n k ' s solution. T r a n s m e m b r a n e p o t e n t i a l s were m e a s u r e d w i t h 3 1V[ KC1 filled glass m i c r o e l e c t r o d e s h a v i n g 5 t o 20 M/2 r e s i s t a n c e a n d c o n n e c t e d t h r o u g h calomel h a l f cells to a T r a n s i d y n e MPA-6 preamplifier with stray capacitance neutralization. S t i m u l a t i o n was a c c o m p l i s h e d w i t h s u p r a t h r e s h o l d s q u a r e w a v e pulses of 0.2 to 0.4 msec d u r a t i o n applied t h r o u g h p l a t i n u m electrodes. A f l o a t i n g m i c r o e l e c t r o d e a r r a n g e m e n t was utilized to m i n i m i z e m e c h a n i c a l a r t i facts. Signals were d i s p l a y e d o n a T e k t r o n i c T y p e 503 oscilloscope e q u i p p e d for P o l a r o i d p h o t o g r a p h y . Results. W h e n p e r f u s e d w i t h e n z y m e - c o n t a i n i n g solution, h i n d - l i m b muscles g r a d u a l l y swelled t o n e a r l y twice t h e i r i n i t i a l d i a m e t e r . U n d e r d i r e c t microscopic view, i t w a s a p p a r e n t t h a t m u s c l e fibre b u n d l e s were well dispersed a n d t h a t t h e y r e s p o n d e d t o electrical s t i m u l a t i o n w i t h vigorous contractions. Subsequently, minimal mechanical dissection was n e c e s s a r y to o b t a i n s m a l l b u n d l e s of muscle fibres t h a t were less r e a d i l y o b t a i n e d f r o m u n 1 T . J . Peters, M. Miiller and C. de Duve, J. exp. Med. 136, 1117 (1972). 2 D . J . Cavanaugh, W. O. Berndt and T. E. Smith, Nature 200, 261 (1963). 3 W.T. Stauber and B. A. Sehottelius, Cytobios 14, 87 (1975). 4 T. Berg, D. Boman and P. O. Seglen, Exp. Cell Res. 72, 571 (1972).
15. 12. 1977
1615
Specialia
t r e a t e d p r e p a r a t i o n s . H o w e v e r , b e c a u s e s o m e of t h e s u p porting structure had been removed by enzyme treatment, these bundles were easily damaged. R e s u l t s of t h e m i c r o e l e c t r o d e s t u d i e s a r e p r e s e n t e d i n tile table and the figure. Although a slight reduction was noted in the resting potential with the enzymetreated m u s c l e s , t h e a c t i o n p o t e n t i a l o v e r s h o o t of t h e i s o e l e c t r i c line a n d t h e a c t i o n p o t e n t i a l a m p l i t u d e w e r e n o t s i g n i f i c a n t l y (p < 0.05) d i f f e r e n t f r o m t h e u n t r e a t e d p r e p arations. Discussion. P r o t e o l y t i c e n z y m e s s u c h a s t r y p s i n h a v e been shown to have deleterious effects on skeletal muscle transmembrane potentialsK Collagenases are more spe-. cific in c l e a v i n g p r i m a r i l y i n t e r c e l l u l a r p r o t e i n s a n d h a v e b e e n u s e d t o r e c o v e r v i a b l e cells f r o m a v a r i e t y of t i s s u e s ~,2,6. I n t h e e x p e r i m e n t s r e p o r t e d h e r e , w e a p p l i e d
collagenase to rat skeletal muscle using a hind-limp perfusion technique to facilitate enzyme distribution and subsequent fibre bundle dispersion. The results indicate t t l a t c o l l a g e n a s e p r e t r e a t m e n t h a s o n l y a s l i g h t , or n e g l i gible, e f f e c t o n t h e c e l l u l a r r e s t i n g p o t e n t i a l a n d a c t i o n potential magnitudes. Action potential durations were observed in some preparations to become prolonged. However, this occurred only when the action potential a n d r e s t i n g p o t e n t i a l a m p l i t u d e s b e g a n t o d e c a y . W e feel this technique offers a simple adjunct to mammalian skeletal muscle dissection, and thus may be particularly u s e f u l i n s t u d i e s of e l e c t r o p h y s i c a l a n d t h e r m o m e c h a n i c a l phenomena. 5 6
D. Holtzlnan and D. Agin, Nature 205, 911 (1965). M. Robell, J. biol. Chem. 239, 375 (1964).
T h e effect of d e h y d r a t i o n on the n e u r o h y p o p h y s e a l b l o o d f l o w in rats w i t h hereditary diabetes insipidus 1 J. K a p i t o l a 2
H . D l o u h A , J. K ~ e ~ e k a a n d J. Z i c h a
Institute o/ Physiology, Czechoslovak Academy o/Sciences, Kr6 BudJ~ovickd 1083, CS-142 20 Praha 4 (Czechoslovakia), 25 May 1977 Summary. N e u r o h y p o p h y s e a l b l o o d f l o w i n c r e a s e s i n w a t e r - d e p r i v e d r a t s . T h i s i n c r e a s e is i n d e p e n d e n t r e l e a s e , s i n c e i t o c c u r s e v e n in r a t s w i t h h e r e d i t a r y d e f e c t o f h y p o t h a l a m i c v a s o p r e s s i n s y n t h e s i s . U s i n g l ~ 5 I - a n t i p y r i n , L i c h a r d u s e t a l J f o u n d a n i n c r e a s e of neurohypophyseal blood flow in rats after water deprivation, and they concluded that it accompanied the release o f v a s o p r e s s i n (VP). E a r l i e r it w a s r e p o r t e d t h a t a n y s t i m u l a t i o n of n e u r o h y p o p h y s e a l h o r m o n e s r e l e a s e c a u s e s a vasodilatation in posterior pituitaryK Furthermore, water deprivation causes the release, from the neurohypop h y s i s , of b o t h o x y t o c i n a n d V P 6. A n a t t e m p t is p r e s e n t e d to ascertain whether neurohypophyseal blood flow increases after water deprivation even in rats which are u n a b l e t o s y n t h e t i z e V P , i.e. in h o m o z y g o u s B r a t t l e b o r o r a t s w i t h h e r e d i t a r y d i a b e t e s i n s i p i d u s ~.
1 2 3 4 5 6 7
of v a s o p r e s s i n
Acknowledgment. The technical assistance of D. Vitimovsk& is acknowledged. Laboratory for Endocrinology and Metabolism, Faculty of General Medicine, Charles University, Prague. Reprint requests: Dr J. Kfe6ek, Institute of Physiology, Czechoslovak Academy of Sciences, 14220, Prague 4. B. Lichardus, I. Albrecht, J. Ponce, L. Linhart, Endocr. exp. 11, 99 (1977). T. Sooriyamoorthy and A. Livingstone, J. Endocr. 5d, 407 (1972). C . W . Jones and B. T. Picketing, J. Physiol., Lond. 203, 449 (1969). H. Valtin and H. A. Sehroeder, Am. J. Physiol. 206, 425 (1964).
Body weight, adenohypophyseal and neurohypophyseaI weight, flow fraction of cardiac output and S~Rb/g-uptake in control (C) and water deprived (WD) Wistar, heterozygous (non-DI) and homozygous (DI) Brattleboro male rats Wistar C
WD
Non-DI C
WD
DI C
WD
204.4 4- 5.56 (9)
187.5" 4- 2.50 (8)
247.1 4- 7.47 (7)
216.3~ • 6.25 (8)
190.7 4- 7.35 (7)
160.6 9 4- 5.21 (8)
Adenohypophysis Weight (mg/100 g b.wt)
-
-
Flow fraction of cardiac output (% • 10 3)
_
_
S6Rb-uptake (%/g)
-
-
1.98 4- 0.061 2.49 4- 0.257 0.62 4- 0.056
1.93 4- 0.071 2.31 4- 0.206 0.60 4- 0.046
2.05 4- 0.061 2.74 4- 0.229 0.67 4- 0.048
2.29 ~ 4- 0.108 3.04~ 4- 0.175 0.67 4- 0.046
0.52 4- 0.026 4.09 q- 0.268 3.94 i 0.243
0.55 4- 0.027 5.04" _t_ 0.352 4.58" 4- 0.167
0.43 b 4- 0.027 3.90 4- 0.350 4.55 4- 0.354
0.48 4- 0.022 4.71 4-4-0.212 4.90 4- 0.210
0.68 b~ -4- 0.034 4.92 bc 4- 0.256 3.67 4- 0.229
0.81 ~bc 4- 0.016 7.53 ~bc 4- 0.657 4.68~ 4- 0;390
Body weight (g)
Neurohypophysis Weight (mg/100 g b.wt) Flow fraction of cardiac output (% • 10 8) S6Rb-uptake (%/g)
Data are given as mean 4- SEM ( ) indicates number of animals. significantly different from control animals (p
