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New aspects of the localization of catecholamines in adrenergic neurons
Frontiers in Catecholcunine Research
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cervical ganglia it remains to be established whether cyclic AMP acts as second messenger under physiological conditions and which are the specific steps linking the changes in the functional state of the neuronal membrane with the regulation of the transcription of specific messenger-RNA of proteins involved in the synthesis of norepinephrine.
NEW ASPECTS OF THE LOCALIZATION OF CATECHOLAMINES IN ADRENERGIC NEURONS J.-P. Tranzer Department of Experimental Medicine, F. Hoffmann-La Roche & Co. Ltd., Basle, Switzerland Electron microscopic studies have revealed that in adrenergic nerve terminals norepinephrine (NE) is localized in two types of vesicles, the large dense core (l.d.c.) vesicles and the small dense core (s.d.c.) vesicles whereas in the axon of the sympathetic nerve trunk only l.d.c, vesicles seem to exist. Biochemical subcellular fractionation studies have led to similar conclusions. Thus, in sympathetically innervated organs two types of NE storing particles, the heavy and the light were found, whereas in the nerve trunk only heavy particles could be detected. Moreover, it is now considered likely that the heavy particles are the equivalent of the 1.d.c. vesicles and the light ones that of the s.d.c, vesicles. For current reviews see refs. (1) and (2). Recent re-investigations of the fine structure of adrenergic axons indicate that there may exist additional particulate amine storing compartments. I.
Adrenergic nerve terminals
When tissues are prepared for electron microscopy with an improved technique for the localization of biogenic amines (3), (4) it appears that in addition to 1.d.c. and s.d.c, vesicles there exist in the adrenergic nerve terminals of various tissues of rats, a peculiar tubular structure which contains an electron dense, reserpine sensitive material (5). In addition, the content of this tubular reticulum (t.r.) shows a positive chromaffin reaction in the absence of 0s0, treatment, similar to the content of the 1.d.c. and of the s.d.c, vesicles. Both of these unrelated findings strongly suggest that in adrenergic nerve terminals, this electron dense material stored in the t.r. represents catecholamines, since these techniques are commonly considered to be specific for the demonstration of biogenic amines at a fine structural level (6). No such electron dense t.r. could be observed in cholinergic axons or in any other cells. A'very similar t.r. was found in animals pretreated with 5-hydroxydopamine (7), an amine which optimally traces amine storing sites. In addition it appears that strong variations
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Frontiers in Catecholamine Research
e x i s t from one organ to another. Thus a t.r. f i l l e d with e n d o g e n o u s a m i n e s i s very a p p a r e n t in a d r e n e r g i c nerve t e r m i n a l s of the rat i r i s and m e s e n t e r i c a r t e r y but i s q u i t e rare in t h o s e of the rat v a s d e f e r e n s . At the p r e s e n t time the functional meaning of the tubular reticulum s t o r i n g a m i n e ( t . r . s . a . ) i s not c l e a r . However, it s e e m s r e a s o n a b l e to think that the t . r . s . a . , which s e e m s to be a rather l a b i l e s t r u c t u r e would l i b e r a t e i t s amine c o n t e n t during h o m o g e n i z a t i o n of the t i s s u e and thereby may account for at l e a s t a part of the "pool of free a m i n e s " found in a l l s u b c e l l u l a r f r a c t i o n a t i o n studies. 2. S y m p a t h e t i c nerve fibres P a r a l l e l fine s t r u c t u r a l i n v e s t i g a t i o n s of the a x o n s of the p o s t g a n g l i o n i c nerve fibres of the v a s d e f e r e n s and the m e s e n t e r y of rats r e v e a l s that t h e s e a x o n s c o n t a i n a n o t i c e a b l e number of v e s i c l e s which, from their s i z e and s h a p e , a r e very s i m i l a r to t h o s e found in axon t e r m i n a l s of the s a m e organ. T h e s e v e s i c l e s are, however, at l e a s t one hundred t i m e s l e s s frequent in the former than in the latter, whether one e q u a t e s their number per s u r f a c e a r e a of a x o p l a s m or per axon profile. The proportion of the two main t y p e s of v e s i c l e s in the non terminal axon is c o m p a r a b l e to that in the terminals. Thus, in the a x o n s of the O n e r v e f i b r e s of both organs apoproximately 10% 1.d.c. v e s i c l e s (1000 A in diameter) and 90% s m a l l v e s i c l e s (500 A in diameter) were found, whereby about h a l f of the s m a l l v e s i c l e s c o n t a i n a d e n s e core and the other h a l f a p p e a r empty. By c o m p a r i s o n the axon t e r m i n a l s c o n t a i n about 5% 1.d.c. v e s i c l e s ( 1 0 0 0 A in d i a m e t e r ) , 88% s . d . c , v e s i c l e s (500 A in diameter) and 7% small empty v e s i c l e s (500)~ in diameter). Furthermore, it could be shown that the e l e c t r o n d e n s e c o r e s of the v a r i o u s v e s i c l e s of the nerve fibres are resel'pine s e n s i t i v e s i m i l a r to the d e n s e c o r e s of the v e s i c l e s of the axon t e r m i n a l s , which s t r o n g l y s u g g e s t s that t h i s r e s e r p i n e s e n s i t i v e e l e c t r o n d e n s e material r e p r e s e n t s c a t e c h o l a m i n e s , most p r o b a b l y NE. T h a t only a p p r o x i m a t e l y half of the small v e s i c l e s of the nerve fibres c o n t a i n a d e n s e core i s not s u r p r i s i n g if one t a k e s the commonly a c c e p t e d view that most of the NE is s y n t h e s i z e d in the n e r v e t e r m i n a l s rather than in the nerve trunk. On the other hand, it h a s p r e v i o u s l y been shown that the s . d . c , v e s i c l e s and the small empty v e s i c l e s of the axon t e r m i n a l s q u i t e probably r e p r e s e n t the s a m e type of v e s i c l e , differing only in their d e g r e e of filling with a m i n e s (7), (8). Thus, the major finding of t h i s i n v e s t i g a t i o n c l a i m s that in the nerve fibres, a s in the t e r m i n a l s , there e x i s t a c o m p a r a t i v e l y high proportion of small v e s i c l e s a b l e to s t o r e NE. T h i s may r e p r e s e n t a s much as 80 - 90% of the total number of v e s i c l e s .
Frontiers in Catecholamine Research
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Although t h e s e r e s u l t s confirm and extend e a r l i e r findings (9), (10) they are in o b v i o u s c o n t r a d i c t i o n with most of the recent fine s t r u c t u r a l i n v e s t i g a t i o n s (11), (12), (13) and b i o c h e m i c a l s u b c e l l u l a r f r a c t i o n a t i o n s t u d i e s of s y m p a t h e t i c n e r v e s (13), (14), (15), (16). For a recent review s e e ref. (2). As stated above u n t i l now only heavy NE s t o r i n g p a r t i c l e s have been found in the s y m p a t h e t i c nerve. However, all t h e s e s u b c e l l u l a r fractionation s t u d i e s were done on b o v i n e s p l e n i c nerve. We are p r e s e n t l y r e - i n v e s t i g a t i n g this nerve, by electron microscopy to find out whether there is an other e x p l a n a t i o n for t h e s e apparently contradictory r e s u l t s . Whether or not small v e s i c l e s a b l e to store NE e x i s t in the non-terminal axon may be of some importance not only for further work attempting to i s o l a t e , purify and a n a l y s e them but a l s o for a better comprehension of the various f u n c t i o n a l a s p e c t s of the adrenergic neuron. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. ll. 12. 13. 14. 15. 16.
Geffen, L. B. and Livett, B. G., Physiological Reviews, 51, 98-157 (1971). Smith, A. D., Pharmacological Reviews, 24, 435-457 (1972}. Tranzer, J. P. and Snipes, R., Proc. European Beg. Conf. Elect. Microscop., 4th, Rome, Vol. 2, 519-520 (1968L Tranzer, J. P. and Richards, J. G., In preparation Tranzer, J. P., Nature New Biology, 237, 57-,r'~ (1972). Bloom, F. E., intern. Rev. Neurobiol., 13, 27-66 (1970). Tranzer, J. P. and Thoenen, H., Experientia (Basel), 23, 743-745 (1967). Tranzer, J. P. and Thoenen, H., Experientia (Basel}, 23, 123--124 (1967). Elfvin, L. G., Ultrastruet. Res., 1, 428-454 (1958). HSkfelt, T.. Acta physiol. Scand., 76, 427--440 (1969). Fillenz, M., Proe. Roy. Soe. Ser. B Biol. Sci., 174, 459-468 (1970). Geffen, L. B. and Ostberg, A., d. Physiol. (London), 204, 583-592 (1969). Roth, R. H., Sterne, L., Bloom F. E. and Giarman, N. J., J. Pharmaeol. Exp. Ther., 162, 203-212 (1968}. Burger, A., Phillipu, A. and Sch~amann, H. J., Naunyn-Schmiedebergs Arch. Pharmakol. Exp. Pathol., 262, 208--220 (1969). De Potter, W. P., Smith, A. D. and De Schaepdriver, A. F., Tissue and Cell., 2, 520--546 (1970). Lagercrantz, H., Acta Physil. Scand., 82, suppl. 366 (1971L THE EXTRANEURONAL REMOVAL, ACCUMULATION AND O-METHYLATION O F H-ISOPROTERENOL IN THE PERFUSED RAT AND GUINEA-PIG H E A R T U. T r e n d e l e n b u r g and H. BGnisch Department of Pharmacology and Toxicology, University of Wlirzburg, Germany During perfusion of the heart with 200 n g / m l of 3H-dl-isoproterenol the rate
clxi
cervical ganglia it remains to be established whether cyclic AMP acts as second messenger under physiological conditions and which are the specific steps linking the changes in the functional state of the neuronal membrane with the regulation of the transcription of specific messenger-RNA of proteins involved in the synthesis of norepinephrine.
NEW ASPECTS OF THE LOCALIZATION OF CATECHOLAMINES IN ADRENERGIC NEURONS J.-P. Tranzer Department of Experimental Medicine, F. Hoffmann-La Roche & Co. Ltd., Basle, Switzerland Electron microscopic studies have revealed that in adrenergic nerve terminals norepinephrine (NE) is localized in two types of vesicles, the large dense core (l.d.c.) vesicles and the small dense core (s.d.c.) vesicles whereas in the axon of the sympathetic nerve trunk only l.d.c, vesicles seem to exist. Biochemical subcellular fractionation studies have led to similar conclusions. Thus, in sympathetically innervated organs two types of NE storing particles, the heavy and the light were found, whereas in the nerve trunk only heavy particles could be detected. Moreover, it is now considered likely that the heavy particles are the equivalent of the 1.d.c. vesicles and the light ones that of the s.d.c, vesicles. For current reviews see refs. (1) and (2). Recent re-investigations of the fine structure of adrenergic axons indicate that there may exist additional particulate amine storing compartments. I.
Adrenergic nerve terminals
When tissues are prepared for electron microscopy with an improved technique for the localization of biogenic amines (3), (4) it appears that in addition to 1.d.c. and s.d.c, vesicles there exist in the adrenergic nerve terminals of various tissues of rats, a peculiar tubular structure which contains an electron dense, reserpine sensitive material (5). In addition, the content of this tubular reticulum (t.r.) shows a positive chromaffin reaction in the absence of 0s0, treatment, similar to the content of the 1.d.c. and of the s.d.c, vesicles. Both of these unrelated findings strongly suggest that in adrenergic nerve terminals, this electron dense material stored in the t.r. represents catecholamines, since these techniques are commonly considered to be specific for the demonstration of biogenic amines at a fine structural level (6). No such electron dense t.r. could be observed in cholinergic axons or in any other cells. A'very similar t.r. was found in animals pretreated with 5-hydroxydopamine (7), an amine which optimally traces amine storing sites. In addition it appears that strong variations
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Frontiers in Catecholamine Research
e x i s t from one organ to another. Thus a t.r. f i l l e d with e n d o g e n o u s a m i n e s i s very a p p a r e n t in a d r e n e r g i c nerve t e r m i n a l s of the rat i r i s and m e s e n t e r i c a r t e r y but i s q u i t e rare in t h o s e of the rat v a s d e f e r e n s . At the p r e s e n t time the functional meaning of the tubular reticulum s t o r i n g a m i n e ( t . r . s . a . ) i s not c l e a r . However, it s e e m s r e a s o n a b l e to think that the t . r . s . a . , which s e e m s to be a rather l a b i l e s t r u c t u r e would l i b e r a t e i t s amine c o n t e n t during h o m o g e n i z a t i o n of the t i s s u e and thereby may account for at l e a s t a part of the "pool of free a m i n e s " found in a l l s u b c e l l u l a r f r a c t i o n a t i o n studies. 2. S y m p a t h e t i c nerve fibres P a r a l l e l fine s t r u c t u r a l i n v e s t i g a t i o n s of the a x o n s of the p o s t g a n g l i o n i c nerve fibres of the v a s d e f e r e n s and the m e s e n t e r y of rats r e v e a l s that t h e s e a x o n s c o n t a i n a n o t i c e a b l e number of v e s i c l e s which, from their s i z e and s h a p e , a r e very s i m i l a r to t h o s e found in axon t e r m i n a l s of the s a m e organ. T h e s e v e s i c l e s are, however, at l e a s t one hundred t i m e s l e s s frequent in the former than in the latter, whether one e q u a t e s their number per s u r f a c e a r e a of a x o p l a s m or per axon profile. The proportion of the two main t y p e s of v e s i c l e s in the non terminal axon is c o m p a r a b l e to that in the terminals. Thus, in the a x o n s of the O n e r v e f i b r e s of both organs apoproximately 10% 1.d.c. v e s i c l e s (1000 A in diameter) and 90% s m a l l v e s i c l e s (500 A in diameter) were found, whereby about h a l f of the s m a l l v e s i c l e s c o n t a i n a d e n s e core and the other h a l f a p p e a r empty. By c o m p a r i s o n the axon t e r m i n a l s c o n t a i n about 5% 1.d.c. v e s i c l e s ( 1 0 0 0 A in d i a m e t e r ) , 88% s . d . c , v e s i c l e s (500 A in diameter) and 7% small empty v e s i c l e s (500)~ in diameter). Furthermore, it could be shown that the e l e c t r o n d e n s e c o r e s of the v a r i o u s v e s i c l e s of the nerve fibres are resel'pine s e n s i t i v e s i m i l a r to the d e n s e c o r e s of the v e s i c l e s of the axon t e r m i n a l s , which s t r o n g l y s u g g e s t s that t h i s r e s e r p i n e s e n s i t i v e e l e c t r o n d e n s e material r e p r e s e n t s c a t e c h o l a m i n e s , most p r o b a b l y NE. T h a t only a p p r o x i m a t e l y half of the small v e s i c l e s of the nerve fibres c o n t a i n a d e n s e core i s not s u r p r i s i n g if one t a k e s the commonly a c c e p t e d view that most of the NE is s y n t h e s i z e d in the n e r v e t e r m i n a l s rather than in the nerve trunk. On the other hand, it h a s p r e v i o u s l y been shown that the s . d . c , v e s i c l e s and the small empty v e s i c l e s of the axon t e r m i n a l s q u i t e probably r e p r e s e n t the s a m e type of v e s i c l e , differing only in their d e g r e e of filling with a m i n e s (7), (8). Thus, the major finding of t h i s i n v e s t i g a t i o n c l a i m s that in the nerve fibres, a s in the t e r m i n a l s , there e x i s t a c o m p a r a t i v e l y high proportion of small v e s i c l e s a b l e to s t o r e NE. T h i s may r e p r e s e n t a s much as 80 - 90% of the total number of v e s i c l e s .
Frontiers in Catecholamine Research
clxiii
Although t h e s e r e s u l t s confirm and extend e a r l i e r findings (9), (10) they are in o b v i o u s c o n t r a d i c t i o n with most of the recent fine s t r u c t u r a l i n v e s t i g a t i o n s (11), (12), (13) and b i o c h e m i c a l s u b c e l l u l a r f r a c t i o n a t i o n s t u d i e s of s y m p a t h e t i c n e r v e s (13), (14), (15), (16). For a recent review s e e ref. (2). As stated above u n t i l now only heavy NE s t o r i n g p a r t i c l e s have been found in the s y m p a t h e t i c nerve. However, all t h e s e s u b c e l l u l a r fractionation s t u d i e s were done on b o v i n e s p l e n i c nerve. We are p r e s e n t l y r e - i n v e s t i g a t i n g this nerve, by electron microscopy to find out whether there is an other e x p l a n a t i o n for t h e s e apparently contradictory r e s u l t s . Whether or not small v e s i c l e s a b l e to store NE e x i s t in the non-terminal axon may be of some importance not only for further work attempting to i s o l a t e , purify and a n a l y s e them but a l s o for a better comprehension of the various f u n c t i o n a l a s p e c t s of the adrenergic neuron. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. ll. 12. 13. 14. 15. 16.
Geffen, L. B. and Livett, B. G., Physiological Reviews, 51, 98-157 (1971). Smith, A. D., Pharmacological Reviews, 24, 435-457 (1972}. Tranzer, J. P. and Snipes, R., Proc. European Beg. Conf. Elect. Microscop., 4th, Rome, Vol. 2, 519-520 (1968L Tranzer, J. P. and Richards, J. G., In preparation Tranzer, J. P., Nature New Biology, 237, 57-,r'~ (1972). Bloom, F. E., intern. Rev. Neurobiol., 13, 27-66 (1970). Tranzer, J. P. and Thoenen, H., Experientia (Basel), 23, 743-745 (1967). Tranzer, J. P. and Thoenen, H., Experientia (Basel}, 23, 123--124 (1967). Elfvin, L. G., Ultrastruet. Res., 1, 428-454 (1958). HSkfelt, T.. Acta physiol. Scand., 76, 427--440 (1969). Fillenz, M., Proe. Roy. Soe. Ser. B Biol. Sci., 174, 459-468 (1970). Geffen, L. B. and Ostberg, A., d. Physiol. (London), 204, 583-592 (1969). Roth, R. H., Sterne, L., Bloom F. E. and Giarman, N. J., J. Pharmaeol. Exp. Ther., 162, 203-212 (1968}. Burger, A., Phillipu, A. and Sch~amann, H. J., Naunyn-Schmiedebergs Arch. Pharmakol. Exp. Pathol., 262, 208--220 (1969). De Potter, W. P., Smith, A. D. and De Schaepdriver, A. F., Tissue and Cell., 2, 520--546 (1970). Lagercrantz, H., Acta Physil. Scand., 82, suppl. 366 (1971L THE EXTRANEURONAL REMOVAL, ACCUMULATION AND O-METHYLATION O F H-ISOPROTERENOL IN THE PERFUSED RAT AND GUINEA-PIG H E A R T U. T r e n d e l e n b u r g and H. BGnisch Department of Pharmacology and Toxicology, University of Wlirzburg, Germany During perfusion of the heart with 200 n g / m l of 3H-dl-isoproterenol the rate