756

Specialia

degree of contraction. This blocking effect of propranolol on t h e phenylephrine-induced contraction seemed to be non-specific, because the same concentration of propranolol also inhibited the contraction induced by serotonin. W h e n d i b e n a m i n e in t h e concentration of 2.5• 10 -7 g/ml was added to the b a t h 30 min before phenyl-

Rata

15og~

Mr

1

Adr

I:'ropranolol

l~og ~

A~r

I Dibenamine

Fig. 2. Change in response to 5 • 10 s g/mI of adrenaline (Adr) in the presence of 2.5 • 10-v g/ml of propranoloI (Rat A), and that in the presence of 2.5 • 10 ~ g/ml of dibenamine (Rat B).

EXPERIENTIA26/7

ephrine, b o t h phases of relaxation (if present) and contraction were completely abolished. The foregoing results suggest t h a t stimulation of greceptors produces an initial relaxation followed b y a secondary contraction in t h e rat j e j u n u m , because this typical response can be seen b y the addition of adrenaline in the presence of propranolol. P h e n y l e p h r i n e also produced similar response. The fact t h a t this t y p e of reaction to adrenaline, or to phenylephrine is almost c o m p l e t e l y abolished b y the p r e t r e a t m e n t of the j e j u n u m w i t h dibenamine, indicates t h a t t h e response m e n t i o n e d a b o v e is produced b y stimulation of g-receptors. I t is a p p a r e n t t h a t stimulation of fl-receptors produces relaxation of t h e j e j u n u m , because in our e x p e r i m e n t isoproterenol produced relaxation, which was not inhibited b y the pret r e a t m e n t w i t h d i b e n a m i n e b u t was completely abolished b y propranolol. So, t h e relaxation of t h e rat j e j u n u m could be p r o d u c e d b y stimulation of either g- or flreceptors. B u t t h e contraction could be p r o d u c e d b y stimulation of g-receptors.

Zusammen/assung. Es wird gezeigt, dass die Stimulation der g-Rezeptoren a m isolierten R a t t e n j e j u n u m E r schlaffung und K o n t r a k t i o n verursacht, wghrend die S t i m u l a t i o n der fl-Rezeptoren n u t Erschlaffung bewirkt. A. MINAMIDATE

Department o/ Pharmacology, Fukushima Medical College, Fukushima (Japan), 70 January 7970.

Autoradiographic Localization of 5-Hydroxytryptamine in Monkey Pineal Gland The pineal gland of m a n and other m a m m a l s contains a high c o n c e n t r a t i o n of 5 - h y d r o x y t r y p t a m i n e (5-HT) and the highest levels recorded h a v e been in the r a t and m o n k e y 1,~. I t appears 3 t h a t t h e principal intrapineal p a t h w a y for the m e t a b o l i s m of 5 - H T depends on t h e a c t i v i t y of the e n z y m e m o n o a m i n e oxidase (MAO) which is present in large a m o u n t s in pineal gland tissue ~. The present report deals w i t h a morphological s t u d y of t h e m o n k e y pineal gland e m p l o y i n g an autoradiographic technique. The i n v e s t i g a t i o n is concerned w i t h first, d e m o n s t r a t i n g in v i v o u p t a k e of tritium-labelled 5h y d r o x y t r y p t o p h a n (3H-5-tITP) and its localization within the gland, secondly, t h e conversion of a t t - 5 - H T P to aH-5-HT and thirdly, detecting alterations in intrapineal levels of isotope following interference w i t h MAO activity. Because t h e level of 5 - H T in m o n k e y pineal gland fluct u a t e s over a 24 h period, being at its highest concentration during the daily light period 1, an a p p r o p r i a t e t i m e in the m o r n i n g was chosen for t h e a d m i n i s t r a t i o n of t h e isotope. Three h e a l t h y adult male cynomolgus monkeys, Macaca irus, housed at the C o m m o n w e a l t h S e r u m Laboratories, Melbourne, weighing 4000-5000 g each, were used. 2 animals were given 5 mCi/kg aH-5-HTPS, specific activ i t y 3.3 Ci/mM, at 11.00 h. P r e v i o u s l y 1 of the animals received at 10.00 h 20 m g / k g t r a n y l c y p r o m i n e sulphate, a rapidly acting MAO inhibitor 6. B o t h substances were administered b y i.p. injection. Tissues from the third animal, which was not injected, were used as controls to exclude possible chemical artefacts in t h e nuclear emulsion of t h e autoradiographs. 2 h after t h e a d m i n i s t r a t i o n

of a H - 5 - H T P the m o n k e y s were e x s a n g u i n a t e d under deep i.v. b a r b i t u r a t e anaesthesia and t h e pineal glands removed. After fixation in 10% (v/v) phosphate-buffered formol-saline, p H 7.0, the glands were d e h y d r a t e d in ethanol, e m b e d d e d in paraffin wax, cut serially at 4 b~ thickness and m o u n t e d on acid-cleaned glass microscope slides. Sections were dewaxed, dipped in liquid nuclear emulsion (Ilford K5) diluted 1 : 4 w i t h glass-distilled w a t e r and exposed for 10 weeks at 4 ~ The slides were developed for 4 m i n in N e u t o l - S (Agfa) diluted w i t h w a t e r (1 : 7), fixed in ' A m f i x ' solution (May and Baker) for 3 rain and t h e n washed for 20 min in filtered t a p water. The sections were stained w i t h nuclear fast red and t a r t r a z i n O (Chroma) and m o u n t e d in polystyrene. Precise localization of r a d i o a c t i v i t y was d e m o n s t r a t e d o v e r the pineal gland (Figure A). V e r y few silver grains were present over pinealocytes in the autoradiographs prepared from the m o n k e y given a H - 5 - H T P alone (Figure B), b u t in those prepared from the animal given MAO inhibitor, in addition to aH-5-HTP, dense collections of grains had developed o v e r these cells (Figure C).

1 W. B. QUAY,Proc. Soc. exp. Biol. and Med. 121,946 (1966). 2 R. J. WURT~aAN, J. AXELROD and D. E. KELLY, The Pineal (Academic Press, New York 1968), p. 63. a W. B. QUAY, Proc. Sot. exp. Biol. and Ned. 115, 710 (1964). R. J. WURTMAN,J. AXELRODand L. S. PHILLIPS, Scienee 7d2, 1071 (1963). 5 Obtained from the Radiochemical Centre, Amersham, Bucks. 6 Donated by Smith Kline and French Laboratories.

15. 7. 1970

757

Specialia

(A) Autoradiogram of section of pineai gland and adjacent brain tissue from cynomolgus monkey injected 25 mCi 3H-5-HTP 60 rain after administration of MAO inhibitor. It shows dense silver grain deposition precisely localized over gland tissue indicating presence of high concentration of isotope. Nuclear fast red and tartrazin (• 30). (B) Autoradiogram of section of pineal gland from cynomolgus monkey injected 25 mCi SH-5-HTP with no MAO inhibitor showing sparse silver grain deposition. Compare with (C) and (D). Nuclear fast red and tartrazin (• 350). (C) and (D) Autoradiograms of section of pineal gland from the same monkey as shown in (A) showing silver grain distribution concentrated mainly over cytoplasm and cytoplasmic processes. Nuclear fast red and tartrazin ( • 300).

G r a i n c o u n t s p e r p i n e a l o e y t e were 5 t i m e s g r e a t e r i n t h i s a n i m a l t h a n in t h e f o r m e r a n d g r a i n d i s t r i b u t i o n was conc e n t r a t e d o v e r t h e c y t o p l a s m a n d c y t o p l a s m i c processes of t h e c e l l T h e r e w a s m i n i m a l l a b e l l i n g o v e r t h e n u c l e u s (Figure D). I n t h e a u t o r a d i o g r a p h s p r e p a r e d f r o m t h e u n i n j e c t e d a n i m a l n o e v i d e n c e of c h e m i c a l fogging of t h e emulsion was found. Silver g r a i n d e p o s i t i o n o v e r p i n e a l o c y t e s shows t h a t t h e s e s t r u c t u r e s c o n t a i n isotope. I n t h e p r e s e n t s t u d y t h e s p a r s e n e s s of g r a i n s in t h e g l a n d of t h e m o n k e y n o t g i v e n M A O i n h i b i t o r i n d i c a t e s a low tissue c o n c e n t r a t i o n of isot o p e a n d implies t h a t isotope is b e i n g r e m o v e d . H o w e v e r , r e m o v a l of a n y isotope f r o m t h e s e cells b y M A O d e p e n d s on t h e c o n v e r s i o n of a H - 5 - H T P to 3H-5-HT b y t h e enz y m e a r o m a t i c L - a m i n o acid d e c a r b o x y l a s e 7 b e c a u s e aH5 - H T P is n o t a s u b s t r a t e for M A O s. Since t h e p i n e a l g l a n d c o n t a i n s t h e e n z y m e s n e c e s s a r y to s y n t h e s i z e a n d d e s t r o y 5 - H T 3,7, t h e low c o n c e n t r a t i o n of isotope in tl~is a n i m a l is v e r y likely d u e t o t h e u n i n h i b i t e d b r e a k d o w n of s y n t h e sized 3 H - 5 - H T b y MAO. T h i s h y p o t h e s i s is s u p p o r t e d b y t h e d e m o n s t r a t i o n of a h i g h c o n c e n t r a t i o n of isotope i n t h e a n i m a l in w h i c h M A O a c t i v i t y was i n h i b i t e d . T h e s e f i n d i n g s are similar to t h o s e m a d e earlier 9 o n r a t b r a i n

n e u r o n e s in v i v o a n d in t i s s u e c u l t u r e a n d are c o n s i s t e n t w i t h t h e k n o w n r a p i d t u r n o v e r of 5 - H T 1~

Zusammen/assung. Die V e r t e i l u n g v o n a u s 5 - H y d r o x y T r y p t o p h a n e n t s t a n d e n e m S e r o t o n i n in d e r Zirbeldrfise yon Alien wird beschrieben. C. J. Louls, G. C. KENNY and R. McD. ANDERSON

Depa~lments o/ Pathology and Anatomy, University o/Melbourne, Parkville (Vicloria, Auslralia), 23 January 7970.

7 S. I2s SNYDER, J. AXELROD, J. E. FISCHER and R. J. WURTMAN,

J. Pharmac. exp. Ther. 1d7, 371 (1965). a S. UDENFRIEND, H. WEISSBACH and D. F. BOGDANSKI, J. biol. Chem. 224, 803 (1957). 9 C. J. Louis, J. Histochem., 2, 29 (1970). 10 M. BULATand Z. SUPEK, J. Neurochem. 75, 383 (1968). 1i t3. FALCK, CH. OWMANand E. ROSENGREN, Aeta. physiol, stand. 67, 300 (1966). ;3 Supported by National Health and Medical Research Council of Australia grant No. 150 (C.J.L.).

Comparison of fl-Adrenergic Blocking Activity of DCI, H56/28, ICI50172, LB46, Methoxamine, MJ 1999 and Propranolol in the Blood Perfused Canine Papillary Muscle Preparation Since fl-adrenergic b l o c k i n g a c t i o n of D C I 1 was found, m a n y a c t i v e c o m p o u n d s h a v e b e e n successively s y n t h e sized in t h e p a s t decade. Q u a l i t a t i v e differences, h o w e v e r , were f o u n d a m o n g d i f f e r e n t / 3 - a d r e n e r g i c receptors~. Prev i o u s l y we c o m p a r e d t h e r e l a t i v e p o t e n c y of DCI, m e t h o x amine, propranolol, MJ1999, H56/28, LB46 and ICI 50172 t o b l o c k t h e p o s i t i v e c h r o n o t r o p i c effect of iso-

p r o t e r e n o I w h i c h was g i v e n i n t r a - a r t e r i a l l y i n t o t h e sinus n o d e a r t e r y 3. I n t h i s s t u d y we c o m p a r e d t h e p o t e n c i e s of t h e s e c o m p o u n d s t o b l o c k t h e p o s i t i v e i n o t r o p i c effect of norepinephrine using the blood perfused papillary muscle p r e p a r a t i o n of dogs. T h e h e a r t was r e m o v e d f r o m a dog, a n e s t h e t i z e d w i t h e t h e r a n d p l u n g e d i n t o t h e cold T y r o d e ' s solution. T h e