15. 7. 1977

Specialia

923

d u c e d in cold are p r o b a b l y due to t h e differences of a c c l i m a t i s a t i o n t i m e used. This t y p e of a d a p t i v e c h a n g e s e e m s t o be t e m p o r a r y , b e c o m i n g c o m p e n s a t e d l a t e r b y o t h e r factors. E a r l i e r ~-4 we h a d a s s u m e d t h a t t h e e n h a n c e d a d r e n e r g i c a c t i v i t y in t h e r a t could be a r e a s o n for t h e s u b s e n s i t i v i t y in t h e heart. T h e p r e s e n t results i n d i c a t e t h a t t h i s ind u c t i v e effect is n o t only l i m i t e d to t h e h e a r t b u t is also p r e s e n t in t h e v a s c u l a r s y s t e m a n d p e r h a p s in all a d r e n ergically i n n e r v a t e d effectors.

80 "4 days 70 8C

E E 6C

o

4c 02

~_ 3C 2c

g lC 07654 3 -log mol.conc.NA Fig. 2. Log concentration-response curves for the perfusion pressure response to noradrenaline in isolated femoral arteries from control rats (O) and from rats exposured to cold environment for 4 days (O). Initial pressure is 30.7 mm Hg for the cold exposed group and 29.7 mm Hg for the control group. Further explanation as in figure l.

1 2 3 4 5 6 7

A c o m p a r i s o n b e t w e e n t h e figure 1 C a n d E shows t h a t t h e st~bsensitivity t o N A i n d u c e d in r a t s b y t h e cold e n v i r o n m e n t w a s e x a c t l y as g r e a t as r e s p o n s e ro P H E , t h u s i n d i c a t i n g lowered a l p h a - a d r e n e r g i c r e s p o n s e in femoral arteries. This result is similar t o t h a t f o u n d in isolated a t r i a f r o m c o l d - e x p o s e d rats, w h e r e t h e t e m p o r a r y a l p h a a d r e n e r g i c s u b s e n s i t i v i t y was d e v e l o p e d in t h e cold w i t h i n 4 d a y s ~. This i n d u c t i o n t i m e is also in a g r e e m e n t w i t h t h e results c o n c e r n i n g s u p e r s e n s i t i v i t y . The nonspecific p o s t j u n c t i o n a l s u p e r s e n s i t i v i t y to N A was s h o w n to develop in t h e dog h e a r t in 1-3 d a y s of t r e a t m e n t w i t h reserpine lS, 1.. I n r a b b i t aortic strip, significant supers e n s i t i v i t y was f o u n d 24 h a f t e r a single dose of reserpine a n d was m a x i m a l a f t e r 3 d a i l y doses XK C o n t r a d i c t i o n a m o n g t h e p r e v i o u s resultse-~ on v a s c u l a r s e n s i t i v i t y in-

T h e a c t i o n of o u a b a i n in p r o m o t i n g

8 9 10 11 12 13 14 15 16

This study was supported by the Academy of Finland. M. Harri, L. Melender and R. Tirri, Experientia 30, 1041 (1974). A. Siltovuori, R. Tirri and M. Harri, Aeta physiol, seand., 99, 457 (1977). R. Tirri, A. Siltovuori and M. Harri, Experientia 32, 1283 (1976). N.A. Kuzmicheva, I. M. Rodionov, O. V. Volkova and M. Z. Chunaeva, Experientia 29, 304 (1973). J. LeBlane, Proc. Soc. exp. Biol. Med. 105, 109 (1960). J. LeBlanc, J. Vallieres and C. Vachon, Am. J. Physiol. 222, 1043 (1972). N. Honda, W. V. Judy and L. D. Carlson, J. appl. Physiol. 17, 754 (1962). J. Himms-Hagen had I. M. Mazurkiewicz-Kwileeli, Can. J. Physiol. Pharmac. d8, 657 (1970). M.N.E. Harri and R. Tirri, Acta physiol, scand. 90, 509 (1974). J . J . MePhillips and M. S. Dar, J. Pharmae. exp. Ther. 156, 507 (1967). J. ~. MePhillips, J. Pharmae. exp. Ther. 766, 249 (1969). L.Z. Bito, K. Hyslop and J. Hyndman, J. Pharmac. exp. Ther. 157, 159 (1969). W. W. Fleming, J. J. MePhillips and D. P. Westfall, Ergebn. Physiol. 68, 55 (1973). G. Kunos, I. Vermes-Kunos and M. Nickerson, Nature 250, 779 (1974). P.M. Hudgins and W. W. Fleming, J. Pharmae. exp. Ther. 75,3, 70 (1966).

t h e r e l e a s e of c a t e c h o l a m i n e s

c. J. Duncan Department o/ Zoology, University o/Liverpool, P. O. Box 147, Liverpool L69 3 B X (England), 20 December 1976 Summary. I t is s u g g e s t e d t h a t o u a b a i n p r o m o t e s c a t e c h o l a m i n e release b y c a u s i n g a rise in i n t r a c e l l u l a r N a + which, in t u r n , causes a n e l e v a t e d s t e a d y - s t a t e level of i n t r a c e l l u l a r Ca 2+. I t is s u g g e s t e d t h a t t h e N a + - K + - A T P a s e is n o t d i r e c t l y i n v o l v e d in e x o c y t o s i s a t e i t h e r adrenergic or cholinergic synapses. K a t s u r a g i a n d Suzuki 1 in a r e c e n t p a p e r in E x p e r i e n t i a h a v e s h o w n clearly t h a t o u a b a i n (the i n h i b i t o r of t h e N a + - K + - A T P a s e ) a t a c o n c e n t r a t i o n of 10-SM is effective in releasing e x t r a n e u r o n a l c a t e c h o l a m i n e in t h e guineapig vas deferens. This s t u d y c o m p l e m e n t s t h e i r earlier findings 2 in w h i c h t h e y s u g g e s t e d t h a t o u a b a i n was also able t o facilitate c a t e c h o l a m i n e release f r o m t h e n e u r o n a l site. O u a b a i n has b e e n k n o w n t o p r o m o t e c a t e c h o l a m i n e release since t h e earlier s t u d i e s of B a n k s 3 on s p o n t a n e o u s release f r o m b o v i n e a d r e n a l gland. K a t s u r a g i a n d Suzuki h a v e s u g g e s t e d t h a t t h e N a + - K + - A T P a s e is essential for s t o r a g e a t b o t h e x t r a n e u r o n a l 1 a n d n e u r o n a l sites 2, c a t e c h o l a m i n e release t a k i n g place w h e n t h e A T P a s e is i n h i b i t e d b y o u a b a i n . This h y p o t h e s i s is similar to t h a t p r o p o s e d b y Garcia a n d K i r p e k a r 4 , 5 who h a v e s h o w n t h a t o u a b a i n causes a d o s e - d e p e n d e n t release of n o r a d r e n aline f r o m c a t spleen slices a n d s u g g e s t 4 t h a t t h e N a + - K +A T P a s e serves to m a i n t a i n t h e i n t e g r i t y of t h e a x o n a l

m e m b r a n e ; procedures t h a t depress enzyme activity (e.g. i n t r a c e l l u l a r a c c u m u l a t i o n of Ca 2+, Garcia e t al. s) w o u l d cause t r a n s m i t t e r release b y p r o d u c i n g a t e m p o r a r y d i s t u r b a n c e in t h e m e m b r a n e . C o m p a r a b l e s u g g e s t i o n s c o n c e r n i n g t h e i n v o l v e m e n t of t h e N a + - K + - A T P a s e in e x o c y t o s i s h a v e b e e n a d v a n c e d for t h e release of t r a n s m i t t e r s f r o m s y n a p t o s o m e s 6 a n d of a c e t y l c h o l i n e in t h e m y e n t e r i c p l e x u s of t h e l o n g i t u d i n a l

1 2 3 4 5 6

T. Katsuragi and T. Suzuki, Experientia 32, 727 (1976). H. Ozawa and T. Katsuragi, Eur. J. Pharmae. 25, 147 (1974). P. Banks, J. Physiol. (Lond.) 193, 631 (1967). A.G. Gareia and S. M. Kirpekar, Br. J. Pharmae. 47, 729 (1973). A.G. Garcia and S. M. Kirpekar, J. Physiol. (Lond.) 235, 693 (1973). J.C. Gilbert, M. G. Wyllie and D. V. Davison, Nature (Lond.) 258, 237 (1975).

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muscle of guinea pig i l e u m 7 a n d ill cortical slices f r o m r a t b r a i n s, b u t it s e e m s t o be i m p o r t a n t t o e m p h a s i z e t h a t t h e r e m i g h t be a n a l t e r n a t i v e e x p l a n a t i o n for t h i s effect of o u a b a i n on c a t e c h o l a m i n e release. Release of c a t e c h o l a m i n e s h a s m a n y f e a t u r e s in c o m m o n w i t h t h e release of a c e t y l c h o l i n e f r o m p r e s y n a p t i c terminalsg, 1~ a n d it is clear t h a t Ca 2+ acts as t h e link in s t i m u l u s - s e c r e t i o n c o u p l i n g in b o t h s y s t e m s n , 1~. I t has also b e e n s u g g e s t e d t h a t t h e f r e q u e n c y of t h e s p o n t a n e o u s r a t e of release of q u a n t a of a c e t y l c h o l i n e (recorded as t h e miniature endplate potentials, MEPPs) at the amphibian n e u r o m u s c u l a r j u n c t i o n is largely d e t e r m i n e d b y [Ca~+~i a t t h e p r e s y n a p t i c terminals~3-~7. F a c t o r s t h a t serve t o e l e v a t e [Ca2+]j p r o d u c e an increase in M E P P f r e q u e n c y . Cardiac glycosides also cause a rise in t h e r a t e of s p o n t a n e o u s release a t t h e frog n e u r o m u s c u l a r j u n c t i o n 18-~~ a n d similar effects are f o u n d w h e n Li+ is allowed t o a c c u m u l a t e i n t r a c e l l u l a r l y a t t h e p r e s y n a p t i c t e r m i n a l s 21-~5. Such e x p e r i m e n t s s u g g e s t t h a t a rise in t h e i n t r a c e l l u l a r conc e n t r a t i o n of e i t h e r N a + (by t h e a c t i o n of o u a b a i n in supp r e s s i n g Na+-efflux) or Li+ (which is n o t r e a d i l y r e m o v e d b y t h e c a t i o n p u m p ) p r o m o t e s s p o n t a n e o u s release a 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 a n d t h e r e is n o w e v i d e n c e t h a t a rise in [Li+]l or [Na+li causes a rise in [Ca2+]l. Such effects h a v e b e e n r e p o r t e d in s y n a p t o s o m e s 26, C h i r o n o m u s saliv a r y g l a n d cells ~7 a n d in t h e isolated islets of L a n g e r h a n s , a p p a r e n t l y b y release of Ca 2+ f r o m i n t r a c e l l u l a r stores zS. I t is n o t e w o r t h y t h a t b o t h N a + a n d Li+ h a v e b e e n s h o w n t o p r o m o t e Ca 2+ release f r o m isolated h e a r t m i t o c h o n dria~% I t t h e r e f o r e s e e m s p r o b a b l e t h a t t h e a c t i o n of o u a b a i n in p r o m o t i n g c a t e c h o l a m i n e release is also a c h i e v e d i n d i r e c t l y b y a rise in [Ca~+li a n d t h a t one n e e d n o t n e c e s s a r i l y p o s t u l a t e t h a t t h e N a + - K + - A T P a s e is d i r e c t l y c o n c e r n e d w i t h exocytosis.

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

EXPERIENTIA 33/7 W.D.M. Paton, E. S. Vizi and M. Aboo Zar, J. Physiol. (Lond.) 215, 819 (1971). E.S. Vizi, J. Physiol. (Lond.) 226, 95 (1972). C.J. Duncan, The Molecular Properties and Evolution of Excitable Cells. Pergamon Press, Oxford 1967. M.J. Berridge, in: Advances in Cyclic Nueleotide Research, vol. 6, p. 1. Ed. P. Greengard and G. A. Robison. Raven Press, New York 1975. W.W. Douglas, Biochem. Soc. Symmp.39, 1 (1974). 13. Katz, The Release of Neural Transmitter Substances. Liverpool University Press, Liverpool 1969. P . F . Baker, Prog. ]3iophys. inol. Biol. 2d, 177 (1972). E. Alnaes and R. Rahamililoff, J. Physiol. (Lond.) 248, 285 (1975). H . E . Stathaiii and C. J. Duncan, Life Sci. 17, 1401 (1975). H . E . Stathaul and C. J. Duncan, Eur. J. Pharmae. 39, 143 (1976). H . E . Statham and C..1. Duncan, J. Physiol. (Lond.)268, in press (1977). R.I. Birks and M. W. Cohen, Proc. R. Soc. B. 170, 381 (1968). R.I. ]3irks and M. VV. Cohen, Proc. R. Soc. ]3. 770, 401 (1968). P . F . Baker and A. C. Crawford, J. Physiol. (Lond.) 2d7, 209 (1975). K. Onodera and K. Yamakawa, Jap. J. Physiol. 16, 541 (1966). P.N. Benoit, M. L. Audibert-Benoit and M. Peyrot, Archo. ital. Biol. 3, 323 (1973). J..1. Carmody and P. W. Gage, Brain Res. 50, 476 (1973). D.D. Branisteanu and R. L. Voile, J. Pharmae. exp. Ther. 194, 362 (1975). A.C. Crawford, J. Physiol. (Lond.) 246, 109 (1975). M. P. ]31austein and C. J. Oborn, J. Physiol. (Lond.) 247, 657 (1975). ]3. Rose and W. R. Loewenstein, J. Memb. Biol. 5, 20 (1971). D. A. Lowe, B. P. Richardson, P. Taylor and P. Donatsch, Nature (Lond.) 260, 337 (1976). M. Crommpton,M. Capano and E. Carafoli, Enr. J. ]3iochem. 69, 453 (1976).

Sucrose activation of mitosis in lemon fruit explants (Citrus limon L.) I-I. A. K o r d a n 1

Department o/ Plant Biology, University o] Birmingham B15 2 T T (England), 22 December 7976 Summary. L e m o n f r u i t e x p l a n t s m a n i f e s t m i t o t i c a c t i v i t y w h e n i n c u b a t e d on a single c o m p o n e n t n u t r i e n t m e d i u m c o n s i s t i n g solely of a n a q u e o u s sucrose solution. E x c i s e d juice vesicle stalks f r o m m a t u r e l e m o n fruits were o b s e r v e d to b e c a p a b l e of m a n i f e s t i n g m i t o t i c act i v i t y w h e n i n c u b a t e d on a c a l c i u m - p o t a s s i u m - s u c r o s e agar n u t r i e n t m e d i u m S. A l t h o u g h t h i s n u t r i e n t m e d i u m was far s i m p l e r in c o m p o s i t i o n t h a n a n y used p r e v i o u s l y for b r i n g i n g a b o u t m i t o t i c a c t i v i t y in l e m o n f r u i t exp l a n t s , t h e p o s s i b i l i t y could n o t be ruled o u t t t l a t Oxoid A g a r No. 3 m a y h a v e s u p p l i e d t h e tissue e x p l a n t s w i t h o t h e r m i n e r a l n u t r i e n t s k n o w n t o be essential for p l a n t g r o w t h s . T h e m a n i f e s t a t i o n of m i t o t i c a c t i v i t y in l e m o n f r u i t e x p l a n t s i n c u b a t e d o n sucrose alone on a n all-glass p h y s i c a l s u b s t r a t u m is r e p o r t e d here. Materials and methods. E n t i r e juice vesicles (sac plus stalk) were r e m o v e d a s e p t i c a l l y f r o m f i r m m a t u r e yellow l e m o n fruits w i t h green b u t t o n s (Citrus l i m o n L.) as d e s c r i b e d p r e v i o u s l y 3 a n d f l o a t e d on t h e surface of sterile glassdistilled w a t e r in ' P y r e x ' P e t r i dishes i m m e d i a t e l y u p o n r e m o v a l f r o m t h e fruit. T h e stalks were s e v e r e d f r o m t h e sacs, t h e sacs were d i s c a r d e d , a n d t h e stalks were t r a n s ferred t o ' P y r e x ' P e t r i dishes lined w i t h W h a t m a n G F / A glass fibre p a p e r s a t u r a t e d w i t h a) sterile glass-distilled

w a t e r , a n d b) sterile 4 % sucrose solution. The P e t r i dishes of b o t h t r e a t m e n t s were sealed w i t h ' P a r a f i l m ' a n d p l a c e d in c o n t i n u o u s d a r k n e s s a t 26-27 ~ A f t e r 6 d a y s of i n c u b a t i o n , all stalks of b o t h t r e a t m e n t s were fixed in Lillie's A A F solution 4, d e h y d r a t e d w i t h i s o p r 0 p a n o l a n d e m b e d d e d in p a r a f f i n w a x as d e s c r i b e d p r e v i o u s l y 5, a n d s e c t i o n e d a t 10 a m thickness. The sections were m o u n t e d on glass m i c r o s c o p e slides w i t h o u t a n y adhesive, d e w a x e d in xylene, b r o u g h t d o w n to w a t e r t h r o u g h a g r a d e d i s o p r o p a n o l series, a n d s t a i n e d w i t h 1% a q u e o u s S a f r a n i n - O for 30 min. The S a f r a n i n - s t a i n e d sections were rinsed t h o r o u g h l y w i t h glass-distilled water, s t a i n e d w i t h 1% alcoholic F a s t - G r e e n F C F for 5 sec, a n d t h e F a s t - G r e e n s t a i n i n g a c t i o n s t o p p e d b y q u i c k l y imm e r s i n g a n d g e n t l y a g i t a t i n g t h e slide ill a b s o l u t e isopropanol. T h e sections were t h o r o u g h l y d e h y d r a t e d in 2 m o r e c h a n g e s of a b s o l u t e i s o p r o p a n o l a n d m o u n t e d in Euparal. Results and discussion. T h e r e was no e v i d e n c e of m i t o t i c a c t i v i t y in a n y of t h e e x p l a n t s i n c u b a t e d on glass-distilled w a t e r ( t r e a t m e n t a) w h e r e a s m i t o t i c figures r e p r e -