15. 7. 1977

Specialia

c a n d i d a t e t h a n s o m a t o s t a t i n as a s a t i e t y factor, since P P is d e t e c t e d in t h e s y s t e m i c circulation15,17, ~s (whereas s u c h m a y n o t be t h e case for s o m a t o s t a t i n ) a n d since t h e p l a s m a c o n c e n t r a t i o n of P P i n d e e d increases in r e s p o n s e to food intake15-~9. The p r e s e n t w o r k reveals t h a t e x o g e n o u s P P , w h e n a d m i n i s t e r e d to t h e h y p e r p h a g i c o b / o b mice, reduces

917

food i n t a k e a n d causes a d o s e - r e l a t e d decrease in b o d y w e i g h t gain. T h e s e results are c o m p a t i b l e w i t h t h e h y p o t h e s i s t h a t P P p a r t i c i p a t e s in t h e r e g u l a t i o n of food intake. The d e m o n s t r a t i o n t h a t h y p e r p h a g i a in obese mice is indeed associated t o d e c r e a s e d levels of circulating p a n c r e a t i c p o l y p e p t i d e awaits t h e a v a i l a b i l i t y of a m e t h o d of m e a s u r e m e n t of P P in m u r i n e p l a s m a .

Adenosine 3',5'-monophosphate in snail (Helix pomatia) nervous system: Analysis of dopamine receptors N. N. O s b o r n e 1

Max-Planck-lnstitut /i2r Experimentelle Medizin, Forschungsstelle Neurochemie, D-3400 Gdttingen (Federal Republic o/ Germany, BRD), 24 September 7976 Summary. The effect of d o p a m i n e on snail (Helix p o m a t i a ) n e r v o u s tissue a d e n o s i n e 3', 5 ' - m o n o p h o s p h a t e c o n t e n t was e x a m i n e d . The results s h o w s u p p o r t for t h e idea t h a t t h e d o p a m i n e r e c e p t o r s in t h e snail n e r v o u s s y s t e m involve a d e n y l a t e cyclase. I t is s u g g e s t e d t h a t t h e s e are t h e d o p a m i n e e x c i t a t o r y r e c e p t o r s r a t h e r t h a n t h e i n h i b i t o r y ones. Considerable e v i d e n c e has a c c u m u l a t e d i m p l i c a t i n g d o p a m i n e as a n e u r o t r a n s m i t t e r in t h e snail brain. I t is localized in a small p r o p o r t i o n of n e u r o n p e r i k a r y a , t h e i r a x o n s a n d p r e s u m e d p r e s y n a p t i c endings2-4, a n d all t h e d a t a f r o m e x p e r i m e n t s on t h e release of d o p a m i n e f r o m a single n e u r o n c o n t a i n i n g t h e a m i n e 5, t h e occurrence of d o p a m i n e r e c e p t o r s on c e r t a i n g a s t r o p o d n e u r o n s 5-7, t h e p r e s e n c e of d o p a m i n e w i t h i n s y n a p t i c - t y p e vesicles 8 a n d t h e d e m o n s t r a t i o n of a h i g h a f f i n i t y u p t a k e process for d o p a m i n e 9 tend to substantiate the hypothesis t h a t the a m i n e is i n v o l v e d in some specific a s p e c t of f u n c t i o n in t h e snail CNS. W i t h r e g a r d to t h e n a t u r e of t h e d o p a m i n e rec e p t o r in t h e g a s t r o p o d s , a n u m b e r of electrophysiological s t u d i e s h a v e b e e n carried o u t s u g g e s t i n g t h a t a t least 2, a n d p r o b a b l y more, t y p e s of d o p a m i n e r e c e p t o r e x i s t in t h e i r n e r v o u s tissues ~~ Moreover, a n a d e n y l a t e cyclase, w h i c h is a c t i v a t e d b y low c o n c e n t r a t i o n s of d o p a m i n e ~, h a s r e c e n t l y b e e n d e m o n s t r a t e d to occur in t h e g a s t r o p o d n e r v o u s s y s t e m , a n d this, w i t h o t h e r d a t a lz,13, suggests t h a t d o p a m i n e r e c e p t o r s of g a s t r o p o d n e r v o u s tissues m a y be i d e n t i c a l w i t h t h e d o p a m i n e - b i n d i n g m o i e t y of d o p a m i n e - s e n s i t i v e a d e n y l a t e cyclase, a n d t h a t t h e effects of d o p a m i n e on t h e c e n t r a l n e r v o u s s y s t e m m a y be due t o d o p a m i n e - i n d u c e d increases in a d e n o s i n e 3', 5 ' - m o n o -

p h o s p h a t e (cyclic AMP) in t h e p o s t - s y n a p t i c cells. S t r o n g e v i d e n c e exists to s u p p o r t t h e o p i n i o n t h a t cyclic A M P m e d i a t e s t h e effect of d o p a m i n e in t h e m a m m a l i a n superior cervical ganglion, r e t i n a a n d c a u d a t e nucleus 14 a n d also in t h e t h o r a c i c ganglia of t h e c o c k r o a c h 16. I n view of t h e i m p r e s s i v e d a t a s u p p o r t i n g a t r a n s m i t t e r role for d o p a m i n e in t h e g a s t r o p o d c e n t r a l n e r v o u s s y s t e m , I e x a m i n e d m o r e closely t h e effects of d o p a m i n e o n cyclic A M P c o n t e n t w i t h t h e aim of c h a r a c t e r i z i n g t h e receptors. Material and methods. A c t i v e snails c a u g h t d a i l y in t h e s u r r o u n d i n g w o o d s of G 6 t t i n g e n were used. A d e n y l a t e cyclase a c t i v i t y was m e a s u r e d in h o m o g e n a t e s of tissue a c c o r d i n g to t h e m e t h o d of K e b a b i a n e t al. 16 w i t h some modifications. The s t a n d a r d a s s a y s y s t e m (final v o l u m e 500 ~xl) c o n t a i n e d (in mmoles/litre) : tris ( h y d r o x y m e t h y l ) a m i n o m e t h a n e - m a l e a t e 90; A T P , 0.5; Mg SO4, 2.0; a s c o r b a t e , 0.5 ; t h e o p h y l l i n e , 5 ; E G T A , 0.2 ; plus t e s t subs t a n c e s as indicated. The r e a c t i o n was i n i t i a t e d b y t h e add i t i o n of A T P . I n c u b a t i o n was for p e r i o d s of 15 m i n in a

1 2

9

Dopamine

3 4

i=n

_E

5

o

o

6 7 3

8 9

.=

10-6

~0~

~0'

i

]0

10.3 M

Concentration of agonist

Increase in cyclic AMP in homogenates of snail central ganglia as a function of the concentration of dopamine and apomorphine. The data represent the mean ! SEM for 30 replicate samples in the case of dopamine, and 20 replicate samples in the instance of apomorphine. In the absence of added agonist, the cyclic AMP content was 2.78 -40.33 pmoles/mg wet weight of tissue' (4- SEM for 30 experiments).

11 12 13 14 15 16

Acknowledgments. I thank Dr Kuschinsky and Prof. V. Neuhoff for reading through the manuscript and Miss E. Priggemeier for her technical help. E. Dahl, B. Falck, C, von Meklenburg, H. Myhrberg and E. Rosengren, Z. Zellforsch. 71,489 (1966). N. Osborne and G. A. Cottrell, Z. Zellforsch. 112, 15 (1971). N.N. Osborne, E. Priggemeier and V. Neuhoff, Brain Res. 90, 261 (1975). M.S. Berry and G. A. Cottrell, Nature New Biology 242, 250 (1973). P. Ascher, J. Physiol. 22.5, 173 (1972). R . J . Walker, G. N. Woodruff, B. Glaizner, C. B. Sedden and G. A. Kerkut, Comp. Biochem. Physiol. 24, 455 (1968). V.W. Pentreath and G. A. Cottrell, Experientia 30, 293 (1974). N.N. Osborne, L. Hiripi and V. Neuhoff, Bioehem. Pharmac. 24, 2141 (1975). P. Ascher and J. S. Kehoe, in: Handbook of Psychopharmacology, vol. 4, p. 265. Ed. L. L. Iversen, S. Iversen and S. Snyder, 1975. H. Cedar and J. H. Schwartz, J. gen. Physiol. 60, 570 (1972). H. Cedar, E. R. Kandel and J. H. Schwartz gen. Physiol. 60, 558 (1972). S.N. Treistan and I. B. Levitan, Nature 261, 62 (1976). D.H. York, Brain Res. 37, 91 (1972). J . A . Nathanson and P. Greengard, Science 180, 308 (1973). J. W. Kebabian, G. L. Petzold and P. Greengard, Proe. nat. Acad. Sci. USA 69, 2145 (1972).

918

Specialia

s h a k i n g w a t e r - b a t h a t 25 ~ T h e r e a c t i o n was t e l m i n a t e d b y p l a c i n g a s s a y t u b e s in a boiling w a t e r - b a t h for 3 min. T h e a m o u n t of cyclic A M P p r e s e n t in e a c h t u b e w a s m e a s u r e d o n 50 ~1 a l i q u o t s b y t h e m e t h o d of G i l m a n ~7. Results. I n t h i s s t u d y 350 ~xM d o p a m i n e c a u s e d m a x i m a l s t i m u l a t i o n of a d e n y l a t e cyclase in h o m o g e n a t e s of snail c e n t r a l ganglia. I n several e x p e r i m e n t s , t h e m a x i m a l increase was a b o u t 3fold. T h i s agrees well w i t h t h e v a l u e r e p o r t e d for d o p a m i n e - i n d u c e d cyclic A M P f o r m a t i o n in A p l y s i a ganglia:L T h e figure i l l u s t r a t e s t h e effects of v a r i ous c o n c e n t r a t i o n s of d o p a m i n e a n d also a p o m o r p h i n e , a d o p a m i n e a g o n i s t is o n snail a d e n y l a t e cyclase. T h e conc e n t r a t i o n s c a u s i n g a h a l f - m a x i m a l s t i m u l a t i o n (ECs0) were a p p r o x i m a t e l y 50 ~M for d o p a m i n e a n d 180 ~xM for apomorphine. In the vertebrates, half-maximal stimul a t i o n s of a d e n y l a t e cyclase for d o p a m i n e a n d a p o m o r p h i n e in slices of c a u d a t u s n u c l e u s are in tile region of 60 a M a n d 150 a M r e s p e c t i v e l y 19. T h e o b s e r v a t i o n t h a t a p o m o r p h i n e a c t i v a t e s snail a d e n y l a t e cyclase, b u t to a lesser e x t e n t t h a n d o p a m i n e , is of p a r t i c u l a r i n t e r e s t , since S t r u y k e r B o n d i e r e t al. ~0 h a v e r e c e n t l y s h o w n t h a t apom o r p h i n e c a n m i m i c t h e e x c i t a t o r y effects of d o p a m i n e in

Table 1. Effect of various substances on the dopamine stilnulated adenylate cyclase in snail central ganglia Substance

Dopamine Dopamine Dopamine Dopamine Dopamine Dopamine Dopamine Dopanfine Dopamine Dopamine Dopamine Dopamine

Cyclic AMP content (pmolesflng wet weight tissue) (120 ~/.:u)alone i (120 Exm)+tubocurarine 100 ~m) (120 a m) + tubocurarine (10 ~ x m ) (120 ~m)+strychnine (100 a m) (120 ~xm)+chlorpromazine (100 tzm) (120 am)+propranolol (100 ~xm) (120 ~m)+ergometrine (100 ~nl) (120 am) +ergometrine (10 ~ t l n ) (120 ~m)+haloperidol (100 t x m ) (120 ~xm)+haloperidol (5 [xm) (120 ~xm)+ fluphenazine (100 ~xm) (120 ~zm)+fluphenazine (5 a m)

10.6:[-0.31 2.94-0.29 7.3~0.29 7.84-0.33 8.8~0.29 10.44-0.42" 8.32:0.28 10.84-0.33 * 2.82:0.34 7.74-0.36 2.6~-0.34 3.84-0.26

* Not significantly different from dopanfine alone. The basal level of cyclic AMP (without dopamine added to the incubation medium) was 2.9 J_ 0.31 pmoles/mg wet weight tissue. Each value is the mean ~- SEM for at least 10 experiments.

Table 2. Effect of dopanfine and other substances on the stimulation of cyclic AMP production in bomogenates of snail central ganglia Substance

ECs0 values for snail ganglia

Dopamine 50 a m Apomorphine 180 a m 5-Hydroxytryptamine 40 [xm N-Methyl dopamine 85 ~xm

ECs0 values for striatum and caudate nucleus 2 ~in*, 160 a m** 150 a m** 1.5 a m*

Basal level of cyclic AMP in snail central ganglia was 2.7• pmoles/mg wet weight tissue ( • SEM for 6 experiments). * Values for striatum homogenates of rat, from Miller et al. ~5. ** Values for caudate nucleus slices of rat, from Forn et al. is. ECs0 values refer to the concentrations required to give half maximal stimulation. :t_ Isoprenaline, p-tyramine, octopamine, isoterenol, noradrenaline and histamine were all without effect at 120 [xm.

EXPERIENTIA 33/7

snail n e u r o n s despite p r e v i o u s p r o o f t h a t i t does n o t imit a t e t h e d o p a m i n e i n h i b i t o r y effect in snail n e u r o n s 21 A t least 2 t y p e s of d o p a m i n e r e c e p t o r h a v e b e e n r e p o r t e d to o c c u r in t h e g a s t r o p o d moIluscs 6,10. E r g o m e t r i n e select i v e l y a n t a g o n i z e s t h e i n h i b i t o r y effects of d o p a m i n e a t m o d e r a t e doses of a b o u t 10 ~M 7, t h o u g h a t h i g h e r conc e n t r a t i o n s it h a s d i f f e r e n t i a l effects o n t h e e x c i t a t o r y d o p a m i n e response1~ a n d r e c e n t studies h a v e also s h o w n e r g o m e t r i n e to a n t a g o n i z e t h e i n h i b i t o r y s e r o t o n i n effects in g a s t r o p o d neuronsS. E x c i t a t o r y effects of d o p a m i n e in t h e g a s t r o p o d n e r v o u s s y s t e m seem in c o n t r a s t to b e selectively blocked b y t u b o c u r a r i n e a n d s t r y c h n i n e 2~,23. C h l o r p r o m a z i n e a n d haloperidol, selective blockers of d o p a m i n e r e c e p t o r s in t h e v e r t e b r a t e s 2., h a v e n o effect upon the inhibitory dopamine responses but depress the e x c i t a t o r y responses in t h e g a s t r o p o d s e,7. As s h o w n in t a b l e 1, t u b o c u r a r i n e (100 ~xM) was f o u n d to a n t a g o n i z e t h e a c t i v a t i n g r e s p o n s e of d o p a m i n e (120 a M) t o snail a d e n y l a t e cyclase a l m o s t c o m p l e t e l y , w h i l s t e r g o m e t r i n e , chlorpromazine and strychnine at the same concentration (100 ~M) o n l y p a r t l y d e p r e s s e d t h e increase in t h e a d e n y l a t e cyclase a c t i v i t y c a u s e d b y 120/xM d o p a m i n e . P r o p r a n o l o l (100 aM) was w i t h o u t effect. E r g o m e t r i n e a t a c o n c e n t r a t i o n of 10 ~M, w h i c h is k n o w n to depress select i v e l y t h e d o p a m i n e i n h i b i t o r y responses7, h a d n o effect on snail a d e n y l a t e cyclase. H o w e v e r , h a l o p e r i d o l a n d f l u p h e n a z i n e , a n a n t i p s y c h o t i c a g e n t of t h e p h e n o t h i a z i n e class, b l o c k e d t h e increase in cyclic A M P r e s p o n s e c a u s e d b y d o p a m i n e a t 100 a M. H a l o p e r i d o l a t 5 a M was n o t w i t h o u t effect, while f l u o p h e n a z i n e still h a d a p o t e n t influence, e v e n a t 5 ttM. T h e a b i l i t y of o t h e r s u b s t a n c e s to a c t i v a t e snail a d e n y l a t e cyclase was also a n a l y z e d (table 2). Dose r e s p o n s e c u r v e s for N - m e t h y l d o p a m i n e a n d 5 - h y d r o x y t r y p t a m i n e were o b t a i n e d w i t h ECs0 v a l u e s of 85 a M a n d 40 a M r e s p e c t i v e ly w i t h m a x i m a l s t i m u l a t i o n of t h e s a m e o r d e r as t h a t for d o p a m i n e . T h e 5 - h y d r o x y t r y p t a m i n e (120 a M) a d e n y l a t e cyclase a c t i v a t i n g r e s p o n s e was n o t i n h i b i t e d b y t u b o c u r a r i n e (100 txM), h a l o p e r i d o l (10 ~M) or f l u p h e n a z i n e (10 ~xM). I s o p r e n a l i n e , p - t y r a m i n e , o c t o p a m i n e , isoterenol, n o r a d r e n a l i n e a n d h i s t a m i n e were all i n a c t i v e on snail a d e n y l a t e cyclase a t 120 a M. Discussion. T h e r e s u l t s r e p o r t e d h e r e i n d i c a t e t h a t t h e r e are d i s t i n c t d o p a m i n e r e c e p t o r s in t h e snail b r a i n , t h e s t i m u l a t i o n of w h i c h r e s u l t s in i n c r e a s e d a m o u n t s of cyclic A M P . T h i s s u p p o r t s t h e a l r e a d y i m p r e s s i v e evid e n c e t h a t d o p a m i n e is a t r a n s m i t t e r in t h e g a s t r o p o d central nervous system. From the present data it would a p p e a r t h a t a d e n y l a t e cyclase is i n v o l v e d in t h e d o p a m i n e e x c i t a t o r y receptors, since d r u g s k n o w n to i n f l u e n c e t h e d o p a m i n e e x c i t a t o r y responses, viz. t u b o c u r a r i n e , s t r y c h n i n e a n d h a l o p e r i d o l ~,v,2~,2a, a n t a g o n i z e t h e d o p a m i n e i n d u c e d i n c r e a s e of cyclic AMP. I n c o n t r a s t , e r g o m e t r i n e ,

17 A. Gilman, Proc. nat. Acad. Sci. USA 67, 305 (1970). 18 N.E. Anden, A. Rubenson, K. Fuxe and T. H6kfeld, J. Pharm. Pharmac. 19, 627 1967}. 19 J. Forn, B. K. Krueger and P. Greengard, Science 186, 118 (1974). 20 H . A . J . Struyker Bondier, W. Gielen and J. M. van Rossum, in: Frontiers in Catecholamine Research, p. 673. Ed. E. Usdin and S. Snyder. Pergamon Press, New York 1973. 21 G.N. Woodruff and R. J. Walker, Int. J. Neuropharmac. 8, 279 (1969). 22 M.R. Klee and D. S. Faber, Experientia 27, 8 (1971). 23 D.S. Faber and M. R. Klee, Brain Res. 65, 109 (1974). 24 D.H. York, Brain Res. 37, 91 (1972). 25 R. Miller, A. Horn, L. Iversen and R. Pinder, Nature 250, 238 (1974).

15.7. 1977

Specialia

k n o w n t o block d o p a m m e i n h i b i t o r y effectsT, l~ c a u s e d n o a l t e r a t i o n in t h e s n a i l cyclic A M P . T h e s n a i l d o p a m i n e r e c e p t o r s also s e e m t o be s i m i l a r to t h o s e of t h e v e r t e b r a t e s ~9, since f l u p h e n a z i n e s t r o n g l y c o u n t e r a c t s t h e inc r e a s e in cyclic A M P c a u s e d b y d o p a m i n e , a n d a p o m o r p h i n e also m i m i c s t h e effect of d o p a m i n e . M o r e o v e r , t h e s e r e c e p t o r s are u n l i k e t h e fl-adrenergic r e c e p t o r s of t h e v e r t e b r a t e s 1~ in t h a t t h e y are n o t i n f l u e n c e d b y n o r a d r e n a l i n e or p r o p r a n o l o l . A l t h o u g h 5 - h y d r o x y t r y p t a m i n e is m o r e p o t e n t t h a n d o p a m i n e in s t i m u l a t i n g a d e n y l a t e c y c l a s e in t h e s n a i l n e r v o u s s y s t e m , w h i c h s u p p o r t s t h e r e s u l t s of C e d a r a n d S c h w a r t z n , it a p p e a r s a s if t h e r e c e p t o r s for

919

t h e 2 a m i n e s are different, since t u b o c u r a r i n e , f l u p h e n a zine a n d h a l o p e r i d o l h a d n o i n f l u e n c e u p o n t h e 5 - h y d r o x y t r y p t a m i n e effect a t c o n c e n t r a t i o n s w h i c h d r a s t i c a l l y inh i b i t e d d o p a m i n e s t i m u l a t i o n of a d e n v l a t e c y c l a s e . To c o n c l u d e , t h e p r e s e n t r e s u l t s p r o v i d e s u p p o r t for t h e idea t h a t a dopamine-sensitive adenylate cyclase m a y be the e x c i t a t o r y r e c e p t o r for d o p a m i n e in t h e snail n e r v o u s s y s t e m . F u r t h e r s t u d i e s on t h e b i o c h e m i s t r y a n d p h a r m a c o l o g y of t h i s d o p a m i n e - s e n s i t i v e a d e n y l a t e c y c l a s e a n d its r e l a t i o n s h i p w i t h p h y s i o ! o g i c a l p r o c e s s e s in n e u r o n s are, h o w e v e r , n e c e s s a r y n o t o n l y to c l a r i f y t h e t y p e of rec e p t o r , b u t also to locate t h e i r sites.

Investigation of the microstructure of kidney stones (oxalate type) by high voltage electron microscopy and electron diffraction K. E 1 - S a y e d 1,2 a n d V. E. C o s s l e t t

Department of Physics, Electron Microscopy Section, Old Cavendish Laboratory, Free School Lane, GB-Cambridge CB2 3RQ (England), 26 November 1976 Summary. T h e o r g a n i c m a t r i x of t h i s t y p e of u r i n a r y c a l c u l u s c o n t a i n s 3 c o m p o n e n t s , w h i c h differ in f o r m a n d in a m o r p h o u s , c r y s t a l l i n e c o n t e n t . D e p o s i t i o n of c r y s t a l l i n e m a t e r i a l in t h e e a r l y s t a g e s of m i n e r a l i s a t i o n s e e m s to be e p i t a x i a l l y r e l a t e d to t h e o r i e n t a t i o n of t h e o r g a m c m a t r i x . Optical microscopy and X-radiography show that urinary calculi are c o m p o s e d of a series of l a y e r s of c r y s t a l l i n e c o m p o n e n t s a n d a lesser a m o u n t of o r g a n i c m a t r i x 3-5. The crystalline layers contain oxalates and p h o s p h a t e s of c a l c i u m , v a r y i n g in calculi of differing p r o v e n a n c e . B o y c e a n d G a r v e y a f o u n d t h a t t h e m a t r i x is m a d e u p of fibrils a n d a m o r p h o u s i n t e r f i b r i l l a r m a t e r i a l . X - r a y diffraction a n d microprobe analysis h a v e been succ e s s f u l in i n v e s t i g a t i n g t h e n a t u r e a n d o r i e n t a t i o n of t h e c r y s t a l l i n e r e g i o n s ~. 5.7 b u t e l e c t r o n m i c r o s c o p y h a s b e e n little u s e d , b e c a u s e of t h e d i f f i c u l t y of c u t t i n g s u f f i c i e n t l y t h i n s e c t i o n s . W a t s o n s a p p l i e d a replica t e c h n i q u e a n d o b t a i n e d m i c r o g r a p h s of h e x a g o n a l c r y s t a l s f r o m s o m e specimens, provisionally identified as cystine. Catalina and Cifuentes Delatte 9 examined the calcium oxalate s e d i m e n t s d e p o s i t e d f r o m urine, a n d o b t a i n e d s o m e electron micrographs and diffraction patterns. These showed a t y p e of s p h e r u l i t i c c r y s t a l g r o w t h , b u t t h e d i f f r a c t i o n p a t t e r n s were d i f f u s e d a n d p r o b a b l y d o m i n a t e d b y o r g a n i c matrix material deposited with the crystalline compon e n t s s. M o h a m m e d et al.10, tl s u c c e e d e d in o b t a i n i n g t h i n s e c t i o n s of calculi a n d s t u d i e d t h e m b y e l e c t r o n m i c r o s c o p y a t 80 kV a n d b y e l e c t r o n d i f f r a c t i o n . T h e y s h o w e d t h a t t h e o r g a n i c m a t r i x is o f t e n a r r a n g e d in l a y e r s a l t e r n a t i n g w i t h c r y s t a l l i n e c o m p o n e n t s , b u t s o m e t i m e s s e e m s to be pres e n t in screw d i s l o c a t e d c r y s t a l s . T h e y were u n a b l e to d i s t i n g u i s h s t r u c t u r e in t h e o r g a n i c m a t r i x , or t o f i n d e v i d e n c e for t h e n a t u r e of i t s role in c r y s t a l g r o w t h . T h e a v a i l a b i l i t y of e l e c t r o n m i c r o s c o p e s o p e r a t i n g a t v e r y h i g h v o l t a g e n o w m a k e s it p o s s i b l e to s t u d y r e l a t i v e l y t h i c k s e c t i o n s , a n d a d v a n c e s in t h e t e c h n i q u e s of m i c r o t o m y f a c i l i t a t e t h e c u t t i n g of s u i t a b l e s e c t i o n s f r o m friable m a t e r i a l s s u c h as calculi. T h e p r e s e n t s t u d y u t i l i z e d t h e s e d e v e l o p m e n t s to obtain detailed information on the struct u r e of t h e o r g a n i c m a t r i x a n d its m o r p h o l o g i c a l r e l a t i o n s with the crystalline regions. Materials and methods. S e c t i o n s w e r e p r e p a r e d f r o m 3 s t o n e s collected b y s u r g i c a l o p e r a t i o n f r o m 3 d i f f e r e n t p a t i e n t s , 1 w o m a n a n d 2 m e n . S o m e 200 s e c t i o n s , c u t

f r o m all p a r t s of a s t o n e f r o m t h e c e n t r e t o t h e p e r i p h e r y , were e x a m i n e d in a n A E I E M 6 B e l e c t r o n m i c r o s c o p e at 80 k V a n d in t h e C a v e n d i s h h i g h v o l t a g e m i c r o s c o p e a t 60O kV. E m b e d d i f i g : A r a l d i t e w a s u s e d as e m b e d d i n g m a t e r i a l , s e v e r a l r a t i o s of resin, h a r d e n e r a n d a c c e l e r a t o r b e i n g tried in o r d e r to o b t a i n g o o d c o h e r e n t s e c t i o n s . B e s t r e s u l t s were o b t a i n e d w i t h a c o m p o s i t i o n of 5 c ~ resin, 8 c ~ h a r d e n e r a n d 0.4 c 3 a c c e l e r a t o r , i n s t e a d of t h a t u s u a l l y u s e d for biological t i s s u e s : 10, 10 a n d 0.5 c a r e s p e c t i v e l y . S e c t i o n i n g : G r e a t d i f f i c u l t y w a s e n c o u n t e r e d in c u t t i n g a n d c o l l e c t i n g t h i n s e c t i o n s f r o m calculi. Glass k n i v e s p r e p a r e d w i t h d i f f e r e n t w e d g e a n g l e s were i n v e s t i g a t e d . A 40 ~ a n g l e w a s f o u n d s a t i s f a c t o r y for c u t t i n g , a n d allowed s e c t i o n s to be collected safely. T r o u b l e w a s experienced f r o m t h e w a t e r in t h e c o l l e c t i n g t r o u g h w e t t i n g t h e s u r f a c e of t h e s t o n e so t h a t t h e l a t t e r d r a g g e d s e c t i o n s w i t h it d u r i n g t h e m o t i o n of t h e m i c r o t o m e . T o a v o i d t h i s t h e level of t h e w a t e r in t h e t r o u g h m u s t be lowered, a n d

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Address: Dept. of Physics, Ein-Shams University, Cairo (Egypt). Acknowledgments. The authors are indebted to Ass. Prof. N. Allouba of Ein-Shams University (Cairo) for supplying the kidney stones and medical information, and to the Physiology and Zoology Departments of Cambridge University for assistance in cutting the sections. The work was performed during the tenure by K. E. S. of a research bursary from the British Council. J . A . Carr. Br. J. Urol. 25.26 (1953). K. Lonsdale. Nature Lond. 217. 56 (1968 . K. Lonsdale, Science 159, 1199 11968). W . H . Boyce and F. K. Garvey, J. Urol. 76, 213 11965l. M.H. Nguyen, S. Offret and C. Laurence Micron 7, 37 I1976). J . H . L . Watson, J. Urol. 75, 940 (1956). F. Catalina and L. Cifuentes Delatte, Arehos esp. Urol. 2. 147 (1958~. A.A. Mohammed, K. E1-Sayed, A. M. Ibrahim and N. Allouha, Proe. Egypt. Acad. Sei. 23, 2 {1970). A.A. Mohammed. K. E1-Sayed and A. M. Ibrahim, Ein-Shams Bulletin {Cairo), No. 17, 10 11972).

Adenosine 3',5'-monophosphate in snail (Helix pomatia) nervous system: analysis of dopamine receptors.

15. 7. 1977 Specialia c a n d i d a t e t h a n s o m a t o s t a t i n as a s a t i e t y factor, since P P is d e t e c t e d in t h e s y s t e m...
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