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Axoplasmic transport of enzymes involved in the synthesis of noradrenaline: relationship between the rate of transport and subcellular distribution
Brain Research, 62 (1973) 471475 © Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
471
AXOPLASMIC TRANSPORT OF ENZYMES INVOLVED IN THE SYNTHESIS OF N O R A D R E N A L I N E : RELATIONSHIP BETWEEN THE RATE OF TRANSPORT AND SUBCELLULAR DISTRIBUTION
H. THOENEN, U. OTTEN AND F. OESCH Department of Pharmacology, Biocenter of the University, CH-4056 Basel (Switzerland)
Until fairly recently the main activity in neurobiological research has been focused on the formation, transmission and modulation of electrical impulses and the underlying ionic events. The biochemical studies of macromolecular constituents of neurons were generally confined to static-descriptive approaches. However, in the last decade it has been recognized that although regeneration capabilities of the highly specialized neuronal cells are generally poor, their protein synthesis is remarkably rapid. Since the main site of protein synthesis is the perikaryon - - it is still a matter of controversy whether extramitochondrial synthesis also takes place in nerve terminals - - the proximo-distal transport of proteins is of great general importance for the maintenance of the functional integrity of the nerve terminals. Investigations in the last few years have shown that the peripheral sympathetic nervous system provides a useful model for the study of transsynaptic regulation of the synthesis of specific proteins by neuronal activity. A prolonged increase in the activity of the preganglionic cholinergic nerves leads to an increased synthesis of tyrosine hydroxylase (EC 1.14.16.2) and dopamine fl-hydroxylase (EC 1.14.17.1) in the postganglionic adrenergic neuron6, s-12. This specific induction of key enzymes of noradrenaline synthesis can be considered as a long-term adaptation to increased transmitter utilization 14. However, the main site of transmitter synthesis is the adrenergic nerve terminal rather than the cell body a. Therefore, the transport of these specific enzymes to the periphery is a prerequisite for rendering their induction to be of functional relevance. Thus, in view of the great importance of the proximo-distal transport of these enzymes and in view of the possible relationship between the rate of transport and the subcellular localization, we have determined the rate of transport of the enzymes involved in the synthesis of noradrenaline in the rat sciatic nerve and correlated their rate of transport with their subcellular distribution. A major part of postganglionic adrenergic fibers originating from the lower lumbar ganglia run to the periphery in the sciatic nerve. For the determination of the rate of transport we adopted a method originally described by Dahlstr6m and H~iggendal3. They determined the rate of noradrenaline accumulation above a ligature of the rat sciatic nerve. The rate of noradrenaline accumulation was taken as a measure for the rate of transport of
472
H.
l DBH
TH
DDC
/
"6 200 /
THOENEN et al.
O Non particulate I
Particulate
[~]
100"
6
12
6
12
Hours after Ligation
Fig. 1. Enzymesinvolvedin noradrenalinebiosynthesis:rate of proximo-distaltransport and subcellular distribution in the rat sciatic nerve. For the determination of the rate of transport four 1 cm segments proximal to a ligature were homogenized in 0.5 ml 0.005 M Tris-HC1 (pH 7.4) containing 0.1 ~ Triton X-100. For the determination of the subcellular distribution ten 2 cm segments were homogenized in 1 ml 0.25 M sucrose or 0.16 M KCI, centrifuged at 800 × g for 20 rain and the resuiting supernatant centrifuged at 100,000 × g for 60 min. The final supernatant is referred to as the 'non-particulate' fraction, the resuspended sediment as the 'particulate' fraction. Sciatic nerve was taken from the thigh region for both determinations. The values given are means -t- S.E.M. of at least 10 pools. Values of the curves representing accumulation of enzymes above a ligature as a function of time are expressed as percent of non-operated controls. The activities in controls were 1.28 -4- 0.11 nmoles phenylethanolamine/h/cm of sciatic nerve for dopamine fl-hydroxylase (DBH), 19.1 zE 1.0 pmoles DOPA/h/cm of sciatic nerve for tyrosine hydroxylase (TH) and 7.4 ± 0.60 nmoles dopamine/h/cm of sciatic nerve for DOPA decarboxylase (DDC). The slopes of the regression lines were calculated by the method of least squares. Each slope is significantly (P < 0.005) different from the other two.
n o r a d r e n a l i n e storage vesicles. I n the present study we ligated the right sciatic nerve o f 100-120 g male S p r a g u e - D a w l e y rats at the level o f the hip j o i n t . The animals were killed after a p p r o p r i a t e intervals ranging f r o m 2 to 12 h. The activity o f tyrosine h y d r o x y l a s e s, D O P A d e c a r b o x y l a s e ( E C 4.1.1.28) 5 a n d d o p a m i n e # - h y d r o x y l a s e 7 was d e t e r m i n e d in 1 c m segments p r o x i m a l to the ligation a c c o r d i n g to previously described procedures. The c o r r e s p o n d i n g pieces o f the n o n - l i g a t e d sciatic nerves served as 0 time controls. The use o f this m e t h o d to d e t e r m i n e the rate o f enzyme t r a n s p o r t seems to be justified only u n d e r the c o n d i t i o n t h a t the ligation o f the postganglionic adrenergic n e u r o n changes neither the rate o f synthesis a n d d e g r a d a t i o n n o r the rate o f a x o p l a s m i c t r a n s p o r t o f the specific p r o t e i n s u n d e r investigation. The o b s e r v a t i o n t h a t all the enzymes involved in the synthesis o f n o r e p i n e p h r i n e accum u l a t e d in a linear fashion d u r i n g the 12 h p e r i o d used to d e t e r m i n e the rate o f transport, a n d t h a t no time lag between ligation a n d the beginning o f enzyme a c c u m u l a t i o n c o u l d be observed (Fig. 1), m a k e s it very i m p r o b a b l e t h a t a change in the rate o f t r a n s p o r t t o o k place d u r i n g the e x p e r i m e n t a l period. Since the basic level a n d the extent o f t r a n s s y n a p t i c inducibility o f these enzymes in the l u m b a r ganglia d i d n o t change within 24 48 h after ligation, it seems also very unlikely t h a t changes in the
AXOPLASMIC TRANSPORT OF SPECIFIC ENZYMES
473
rate of synthesis or degradation in the perikaryon took place during this period. Thus, measuring the rate of accumulation of enzymes above a ligature of the sciatic nerve during the first 12 h after ligation seems to be a reliable procedure to determine the rate of proximo-distal transport. This rate represents a net rate of transport, allowing no conclusions as to whether the cell constituent under investigation is transported at a uniform rate and to what relative extent orthograde and (possibly) retrograde transport determine the measured rate of accumulation. For the 3 enzymes involved in the synthesis of noradrenaline a clear correlation between their subcellular distribution in the rat sciatic nerve and their rate of proximodistal transport became apparent (Fig. 1). The activity of dopamine fl-hydroxylase is predominantly localized in the particulate fraction and, in fact, this enzyme was transported at the fastest rate (1.94 ram/h) whereas DOPA decarboxylase, exclusively located in the post-microsomal supernatant, was transported at the slowest rate (0.63 mm/h). Tyrosine hydroxylase, predominantly located in the non-particulate fraction of the sciatic nerve, was transported much slower (0.75 mm/h) than dopamine fl-hydroxylase but still significantly (P < 0.005) faster than DOPA decarboxylase. Interestingly, the apparent difference in the transsynaptic inducibility of tyrosine hydroxylase and dopamine fl-hydroxylase as determined in the cell body of the adrenergic neuron was inversely proportional to their rate of proximo-distal transport. The increase in tyrosine hydroxylase activity in the rat lower lumbar ganglia 48 h after treatment with 5 mg/kg of reserpine was 3 times larger than that of dopamine fl-hydroxylase while the rate of transport of the latter in the sciatic nerve was 2.6 times higher than that of the former. Thus, the difference between the relative increase in the activity of these two enzymes in sympathetic ganglia does not necessarily imply that their increased rate of synthesis is different and under separate control, since this difference can be explained satisfactorily by the difference in the rate of transport of the two enzymes from the perikaryon to the periphery. The subcellular distribution of dopamine fl-hydroxylasein ganglia did not differ significantly (0.3 > P > 0.2) from that in the sciatic nerve. However, in the nerve terminals a greater proportion of dopamine fl-hydroxylase was localized in the particulate fractions. It is noteworthy that the subcellular distribution of dopamine fl-hydroxylase in the sciatic nerve proximal to a ligation approached that in the nerve terminals (Fig. 2), indicating that ligation of an axon mimics the physiological conditions present in nerve endings, although the underlying mechanism is unknown. Tyrosine hydroxylase and DOPA decarboxylase were found exclusively in the non-particulate fractions of the ganglia. In the nerve endings of effector organs a small but consistent portion of tyrosine hydroxylase was found in particulate fractions, whereas DOPA decarboxylase was exclusively in the high-speed supernatant. Changes in the isotonic medium (non-ionic or ionic) or in the homogenization procedure (6 or 20 strokes) failed to change the subcellular distribution of the enzymes studied in the various tissues, indicating that the subcellular distribution observed under these experimental conditions can be considered to be representative for the physiological subcellular distribution. During the progress of these experiments, a study of Brimijoin1 on the rate of
474
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~
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~
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"Microsomal "
,,oo,oooxo,
Fig. 2. Subcellular distribution of dopamine fl-hydroxylase in cell bodies, axons and nerve endings of the peripheral adrenergic nervous system of the rat. One pair of ganglia (superior cervical or stellate) was homogenized in 0.5 ml, ten 2 cm segments of sciatic nerve in 1 ml, hearts or salivary glands in 20 vol. (w/v) of 0.25 M sucrose. The homogenate was centrifuged at 800 × g for 20 rain and the resulting supernatant at 10,000 × g for 20 rain to yield a 'mitochondrial' pellet. The postmitochondrial supernatant was centrifuged at 100,000 × g for 60 min to yield a 'microsomal' pellet. The post-microsomal supernatant is referred to as the 'non-particulate' fraction. Values represent means ~: S.E.M. of at least 6 preparations. Bars, which are placed below the corresponding region of a schematically drawn adrenergic neuron, represent total dopamine fl-hydroxylase activity, the empty part indicating the 'non-particulate', the dotted part the 'mitochondrial' and the hatched part the 'microsomal' portion. Total dopamine /~-hydroxylase activities amounted to 75 ~k 8.4 nmoles phenylethanolamine/h!mg protein in superior cervical ganglia, to 86.8 ~: 5.5 nmoles/h/mg protein in stellate ganglia, to 1.08 ± 0.03 nmoles/h/cm of non-ligated sciatic nerve and to 3.18 ± 0.22 nmoles/h/cm 24 h after ligation, to 31.7 :k 0.9 nmoles/h/submaxillary gland and to 31.0 4- 1.1 nmoles/h/heart.
t r a n s p o r t o f d o p a m i n e fl-hydroxylase in the rat sciatic nerve a p p e a r e d which r e p o r t e d a c o n s i d e r a b l y faster rate. The l a t t e r results were o b t a i n e d by use o f the same m e t h o d as described a b o v e but with older animals. Since age differences could possibly influence the rate o f t r a n s p o r t , we r e p e a t e d o u r experiments with 250-300 g rats, but the rate did n o t differ significantly ( P > 0.1) f r o m that in 100-120 g rats. In contrast, we could n o t observe a linear a c c u m u l a t i o n o f tyrosine h y d r o x y l a s e in these o l d e r animals. Coyle a n d W o o t e n 2 recently r e p o r t e d for the latter enzyme a rate o f t r a n s p o r t o f 1.6 m m / h which is c o n s i d e r a b l y faster t h a n t h a t d e t e r m i n e d in our experiments. However, the values o b t a i n e d in o u r experiments are close to those calculated f r o m the rate o f p r o x i m o - d i s t a l progress o f tyrosine h y d r o x y l a s e activity in the r a t sciatic nerve after t r a n s s y n a p t i c i n d u c t i o n o f this enzyme in the l u m b a r ganglia by a d m i n i s t r a t i o n o f reserpine 13. The rate o f t r a n s p o r t , as calculated by the l a t t e r procedure, is m a i n l y d e t e r m i n e d by the faster m o v i n g m o i e t y o f the enzyme, whereas the rate o f a c c u m u l a tion a b o v e a ligature represents the s u m m a t i o n o f the fast a n d slow m o v i n g moieties. Accordingly, the rate o f t r a n s p o r t o f t y r o s i n e hydroxylase, as d e t e r m i n e d by m e a s u r i n g the a c c u m u l a t i o n a b o v e a ligature, was slightly lower t h a n t h a t d e t e r m i n e d in unligated sciatic nerves after induction.
AXOPLASMIC TRANSPORT OF SPECIFIC ENZYMES
475
In conclusion, it has been d e m o n s t r a t e d t h a t the t r a n s s y n a p t i c i n d u c t i o n o f t y r o s i n e h y d r o x y l a s e a n d d o p a m i n e fl-hydroxylase is o f functional significance as an a d a p t a t i o n to increased t r a n s m i t t e r utilization, since these enzymes are t r a n s p o r t e d f r o m the site o f their e n h a n c e d synthesis, the p e r i k a r y o n , to the m a i n site o f their action, the nerve endings. A clear c o r r e l a t i o n between the rate o f t r a n s p o r t o f the enzymes involved in n o r a d r e n a l i n e synthesis a n d their subcellular localization b e c a m e a p p a r e n t . D o p a m i n e fl-hydroxylase, p r e d o m i n a n t l y localized in p a r t i c u l a t e fractions o f sciatic nerve, is t r a n s p o r t e d at the highest rate, tyrosine hydroxylase, p r e d o m i n a n t l y localized in n o n - p a r t i c u l a t e fractions, at a lower rate a n d D O P A decarboxylase, exclusively localized in n o n - p a r t i c u l a t e fractions, at the lowest rate. The excellent technical assistance o f Mrs. H i l a r y W o o d , Miss Vreni F o r s t e r a n d Mr. Ueli J/iggi is gratefully a c k n o w l e d g e d . This study was s u p p o r t e d by the Swiss N a t i o n a l F o u n d a t i o n for Scientific R e s e a r c h ( G r a n t No. 3.653.71).
1 BRIMIJOIN,S., Transport and turnover of dopamine fl-hydroxylase (EC 1.14.2.1) in sympathetic nerves of the rat, J. Neurochem., 19 (1972) 2183-2193. 2 COVLE,J. T., AND WOOTEtq,G. F., Rapid axonal transport of tyrosine hydroxylase and dopamine fl-hydroxylase, Brain Research, 44 (1972) 701-704. 3 DAHLSTROM,A., AND H.~GGENDAL,J., Studies on the transport and life-span of amine storage granules in a peripheral adrenergic neuron system, Acta physiol, scand., 76 (1966) 278-288. 4 GEFFEN,L. B., AND RUSH, R. A., Transport of noradrenaline in sympathetic nerves and the effect of nerve impulses on its contribution to transmitter stores, J. Neurochem., 15 (1968) 925-930. 5 H~KANSON, R., AND OWMAN,CH., Effect of denervation and enzyme inhibition on DOPA decarboxylase and monoamine oxidase activities of rat pineal gland, J. Neurochem., 12 (1965) 417-429. 6 MOLINOFF,P. B., BRIMIJOIN,S., WEINSHILBOUM,R., ANDAXELROD,J., Neurally mediated increase in dopamine fl-hydroxylase activity, Proc. nat. Acad. Sci. (Wash.), 66 (1970) 453-458. 7 MOLINOFF,P. B., WEINSHILBOUM,R., ANDAXELROD,J., A sensitive enzymatic assay for dopamine fl-hydroxylase, J. Pharmacol. exp. Ther., 178 (1971)425--431. 8 MUELLER,R. A., THOENEN, H., AND AXELROD,J., Increase in tyrosine hydroxylase activity after reserpine administration, J. Pharmacol. exp. Ther., 169 (1969) 74-79. 9 MUELLER, R.A., THOENEN, H., AND AXELROD, J., Inhibition of trans-synaptically increased tyrosine hydroxylase activity by cycloheximide and actinomycin D, Molec. PharmacoL, 5 (1969) 463-469. 10 THOENEN,H., Induction of tyrosine hydroxylase in peripheral and central adrenergic neurones by cold exposure of rats, Nature (Lond.), 228 (1970) 861-862. 11 THOENEN,H., KETTLER, R., BURKARD,W., AND SANER, A., Neurally mediated control of enzymes involved in the synthesis of norepinephrine; are they regulated as an operational unit? Naunyn-Schmiederberg's Arch. exp. Path. Pharmak., 270 (1971) 146-160. 12 THOENEtq, H., MUELLER,R. A., AND AXELROD,J., Trans-synaptic induction of adrenal tyrosine hydroxylase, J. Pharmacol. exp. Ther., 169 (1969) 249-254. 13 THOENEN,H., MUELLER,R. A., AND AXELROD,J., Phase difference in the induction of tyrosine hydroxylase in cell body and nerve terminals of synaptic neurones, Proc. nat. Acad. Sci. (Wash.), 65 (1970) 58-62. 14 TrtOENEN, H., AND OESC., F., New enzyme synthesis as a long-term adaptation to increased transmitter utilization. In A. J. MANDELL(Ed.), New Concepts in Neurotransmitter Regulation, Plenum Press, New York, 1973, pp. 33-52.
471
AXOPLASMIC TRANSPORT OF ENZYMES INVOLVED IN THE SYNTHESIS OF N O R A D R E N A L I N E : RELATIONSHIP BETWEEN THE RATE OF TRANSPORT AND SUBCELLULAR DISTRIBUTION
H. THOENEN, U. OTTEN AND F. OESCH Department of Pharmacology, Biocenter of the University, CH-4056 Basel (Switzerland)
Until fairly recently the main activity in neurobiological research has been focused on the formation, transmission and modulation of electrical impulses and the underlying ionic events. The biochemical studies of macromolecular constituents of neurons were generally confined to static-descriptive approaches. However, in the last decade it has been recognized that although regeneration capabilities of the highly specialized neuronal cells are generally poor, their protein synthesis is remarkably rapid. Since the main site of protein synthesis is the perikaryon - - it is still a matter of controversy whether extramitochondrial synthesis also takes place in nerve terminals - - the proximo-distal transport of proteins is of great general importance for the maintenance of the functional integrity of the nerve terminals. Investigations in the last few years have shown that the peripheral sympathetic nervous system provides a useful model for the study of transsynaptic regulation of the synthesis of specific proteins by neuronal activity. A prolonged increase in the activity of the preganglionic cholinergic nerves leads to an increased synthesis of tyrosine hydroxylase (EC 1.14.16.2) and dopamine fl-hydroxylase (EC 1.14.17.1) in the postganglionic adrenergic neuron6, s-12. This specific induction of key enzymes of noradrenaline synthesis can be considered as a long-term adaptation to increased transmitter utilization 14. However, the main site of transmitter synthesis is the adrenergic nerve terminal rather than the cell body a. Therefore, the transport of these specific enzymes to the periphery is a prerequisite for rendering their induction to be of functional relevance. Thus, in view of the great importance of the proximo-distal transport of these enzymes and in view of the possible relationship between the rate of transport and the subcellular localization, we have determined the rate of transport of the enzymes involved in the synthesis of noradrenaline in the rat sciatic nerve and correlated their rate of transport with their subcellular distribution. A major part of postganglionic adrenergic fibers originating from the lower lumbar ganglia run to the periphery in the sciatic nerve. For the determination of the rate of transport we adopted a method originally described by Dahlstr6m and H~iggendal3. They determined the rate of noradrenaline accumulation above a ligature of the rat sciatic nerve. The rate of noradrenaline accumulation was taken as a measure for the rate of transport of
472
H.
l DBH
TH
DDC
/
"6 200 /
THOENEN et al.
O Non particulate I
Particulate
[~]
100"
6
12
6
12
Hours after Ligation
Fig. 1. Enzymesinvolvedin noradrenalinebiosynthesis:rate of proximo-distaltransport and subcellular distribution in the rat sciatic nerve. For the determination of the rate of transport four 1 cm segments proximal to a ligature were homogenized in 0.5 ml 0.005 M Tris-HC1 (pH 7.4) containing 0.1 ~ Triton X-100. For the determination of the subcellular distribution ten 2 cm segments were homogenized in 1 ml 0.25 M sucrose or 0.16 M KCI, centrifuged at 800 × g for 20 rain and the resuiting supernatant centrifuged at 100,000 × g for 60 min. The final supernatant is referred to as the 'non-particulate' fraction, the resuspended sediment as the 'particulate' fraction. Sciatic nerve was taken from the thigh region for both determinations. The values given are means -t- S.E.M. of at least 10 pools. Values of the curves representing accumulation of enzymes above a ligature as a function of time are expressed as percent of non-operated controls. The activities in controls were 1.28 -4- 0.11 nmoles phenylethanolamine/h/cm of sciatic nerve for dopamine fl-hydroxylase (DBH), 19.1 zE 1.0 pmoles DOPA/h/cm of sciatic nerve for tyrosine hydroxylase (TH) and 7.4 ± 0.60 nmoles dopamine/h/cm of sciatic nerve for DOPA decarboxylase (DDC). The slopes of the regression lines were calculated by the method of least squares. Each slope is significantly (P < 0.005) different from the other two.
n o r a d r e n a l i n e storage vesicles. I n the present study we ligated the right sciatic nerve o f 100-120 g male S p r a g u e - D a w l e y rats at the level o f the hip j o i n t . The animals were killed after a p p r o p r i a t e intervals ranging f r o m 2 to 12 h. The activity o f tyrosine h y d r o x y l a s e s, D O P A d e c a r b o x y l a s e ( E C 4.1.1.28) 5 a n d d o p a m i n e # - h y d r o x y l a s e 7 was d e t e r m i n e d in 1 c m segments p r o x i m a l to the ligation a c c o r d i n g to previously described procedures. The c o r r e s p o n d i n g pieces o f the n o n - l i g a t e d sciatic nerves served as 0 time controls. The use o f this m e t h o d to d e t e r m i n e the rate o f enzyme t r a n s p o r t seems to be justified only u n d e r the c o n d i t i o n t h a t the ligation o f the postganglionic adrenergic n e u r o n changes neither the rate o f synthesis a n d d e g r a d a t i o n n o r the rate o f a x o p l a s m i c t r a n s p o r t o f the specific p r o t e i n s u n d e r investigation. The o b s e r v a t i o n t h a t all the enzymes involved in the synthesis o f n o r e p i n e p h r i n e accum u l a t e d in a linear fashion d u r i n g the 12 h p e r i o d used to d e t e r m i n e the rate o f transport, a n d t h a t no time lag between ligation a n d the beginning o f enzyme a c c u m u l a t i o n c o u l d be observed (Fig. 1), m a k e s it very i m p r o b a b l e t h a t a change in the rate o f t r a n s p o r t t o o k place d u r i n g the e x p e r i m e n t a l period. Since the basic level a n d the extent o f t r a n s s y n a p t i c inducibility o f these enzymes in the l u m b a r ganglia d i d n o t change within 24 48 h after ligation, it seems also very unlikely t h a t changes in the
AXOPLASMIC TRANSPORT OF SPECIFIC ENZYMES
473
rate of synthesis or degradation in the perikaryon took place during this period. Thus, measuring the rate of accumulation of enzymes above a ligature of the sciatic nerve during the first 12 h after ligation seems to be a reliable procedure to determine the rate of proximo-distal transport. This rate represents a net rate of transport, allowing no conclusions as to whether the cell constituent under investigation is transported at a uniform rate and to what relative extent orthograde and (possibly) retrograde transport determine the measured rate of accumulation. For the 3 enzymes involved in the synthesis of noradrenaline a clear correlation between their subcellular distribution in the rat sciatic nerve and their rate of proximodistal transport became apparent (Fig. 1). The activity of dopamine fl-hydroxylase is predominantly localized in the particulate fraction and, in fact, this enzyme was transported at the fastest rate (1.94 ram/h) whereas DOPA decarboxylase, exclusively located in the post-microsomal supernatant, was transported at the slowest rate (0.63 mm/h). Tyrosine hydroxylase, predominantly located in the non-particulate fraction of the sciatic nerve, was transported much slower (0.75 mm/h) than dopamine fl-hydroxylase but still significantly (P < 0.005) faster than DOPA decarboxylase. Interestingly, the apparent difference in the transsynaptic inducibility of tyrosine hydroxylase and dopamine fl-hydroxylase as determined in the cell body of the adrenergic neuron was inversely proportional to their rate of proximo-distal transport. The increase in tyrosine hydroxylase activity in the rat lower lumbar ganglia 48 h after treatment with 5 mg/kg of reserpine was 3 times larger than that of dopamine fl-hydroxylase while the rate of transport of the latter in the sciatic nerve was 2.6 times higher than that of the former. Thus, the difference between the relative increase in the activity of these two enzymes in sympathetic ganglia does not necessarily imply that their increased rate of synthesis is different and under separate control, since this difference can be explained satisfactorily by the difference in the rate of transport of the two enzymes from the perikaryon to the periphery. The subcellular distribution of dopamine fl-hydroxylasein ganglia did not differ significantly (0.3 > P > 0.2) from that in the sciatic nerve. However, in the nerve terminals a greater proportion of dopamine fl-hydroxylase was localized in the particulate fractions. It is noteworthy that the subcellular distribution of dopamine fl-hydroxylase in the sciatic nerve proximal to a ligation approached that in the nerve terminals (Fig. 2), indicating that ligation of an axon mimics the physiological conditions present in nerve endings, although the underlying mechanism is unknown. Tyrosine hydroxylase and DOPA decarboxylase were found exclusively in the non-particulate fractions of the ganglia. In the nerve endings of effector organs a small but consistent portion of tyrosine hydroxylase was found in particulate fractions, whereas DOPA decarboxylase was exclusively in the high-speed supernatant. Changes in the isotonic medium (non-ionic or ionic) or in the homogenization procedure (6 or 20 strokes) failed to change the subcellular distribution of the enzymes studied in the various tissues, indicating that the subcellular distribution observed under these experimental conditions can be considered to be representative for the physiological subcellular distribution. During the progress of these experiments, a study of Brimijoin1 on the rate of
474
~
H. ~HOEXEN el aL
~
"~ ~ . . . . . . . . . . . .....
~
[] Nonparticulate N(l"Mi O,Otochondri OOxg) al" []
"Microsomal "
,,oo,oooxo,
Fig. 2. Subcellular distribution of dopamine fl-hydroxylase in cell bodies, axons and nerve endings of the peripheral adrenergic nervous system of the rat. One pair of ganglia (superior cervical or stellate) was homogenized in 0.5 ml, ten 2 cm segments of sciatic nerve in 1 ml, hearts or salivary glands in 20 vol. (w/v) of 0.25 M sucrose. The homogenate was centrifuged at 800 × g for 20 rain and the resulting supernatant at 10,000 × g for 20 rain to yield a 'mitochondrial' pellet. The postmitochondrial supernatant was centrifuged at 100,000 × g for 60 min to yield a 'microsomal' pellet. The post-microsomal supernatant is referred to as the 'non-particulate' fraction. Values represent means ~: S.E.M. of at least 6 preparations. Bars, which are placed below the corresponding region of a schematically drawn adrenergic neuron, represent total dopamine fl-hydroxylase activity, the empty part indicating the 'non-particulate', the dotted part the 'mitochondrial' and the hatched part the 'microsomal' portion. Total dopamine /~-hydroxylase activities amounted to 75 ~k 8.4 nmoles phenylethanolamine/h!mg protein in superior cervical ganglia, to 86.8 ~: 5.5 nmoles/h/mg protein in stellate ganglia, to 1.08 ± 0.03 nmoles/h/cm of non-ligated sciatic nerve and to 3.18 ± 0.22 nmoles/h/cm 24 h after ligation, to 31.7 :k 0.9 nmoles/h/submaxillary gland and to 31.0 4- 1.1 nmoles/h/heart.
t r a n s p o r t o f d o p a m i n e fl-hydroxylase in the rat sciatic nerve a p p e a r e d which r e p o r t e d a c o n s i d e r a b l y faster rate. The l a t t e r results were o b t a i n e d by use o f the same m e t h o d as described a b o v e but with older animals. Since age differences could possibly influence the rate o f t r a n s p o r t , we r e p e a t e d o u r experiments with 250-300 g rats, but the rate did n o t differ significantly ( P > 0.1) f r o m that in 100-120 g rats. In contrast, we could n o t observe a linear a c c u m u l a t i o n o f tyrosine h y d r o x y l a s e in these o l d e r animals. Coyle a n d W o o t e n 2 recently r e p o r t e d for the latter enzyme a rate o f t r a n s p o r t o f 1.6 m m / h which is c o n s i d e r a b l y faster t h a n t h a t d e t e r m i n e d in our experiments. However, the values o b t a i n e d in o u r experiments are close to those calculated f r o m the rate o f p r o x i m o - d i s t a l progress o f tyrosine h y d r o x y l a s e activity in the r a t sciatic nerve after t r a n s s y n a p t i c i n d u c t i o n o f this enzyme in the l u m b a r ganglia by a d m i n i s t r a t i o n o f reserpine 13. The rate o f t r a n s p o r t , as calculated by the l a t t e r procedure, is m a i n l y d e t e r m i n e d by the faster m o v i n g m o i e t y o f the enzyme, whereas the rate o f a c c u m u l a tion a b o v e a ligature represents the s u m m a t i o n o f the fast a n d slow m o v i n g moieties. Accordingly, the rate o f t r a n s p o r t o f t y r o s i n e hydroxylase, as d e t e r m i n e d by m e a s u r i n g the a c c u m u l a t i o n a b o v e a ligature, was slightly lower t h a n t h a t d e t e r m i n e d in unligated sciatic nerves after induction.
AXOPLASMIC TRANSPORT OF SPECIFIC ENZYMES
475
In conclusion, it has been d e m o n s t r a t e d t h a t the t r a n s s y n a p t i c i n d u c t i o n o f t y r o s i n e h y d r o x y l a s e a n d d o p a m i n e fl-hydroxylase is o f functional significance as an a d a p t a t i o n to increased t r a n s m i t t e r utilization, since these enzymes are t r a n s p o r t e d f r o m the site o f their e n h a n c e d synthesis, the p e r i k a r y o n , to the m a i n site o f their action, the nerve endings. A clear c o r r e l a t i o n between the rate o f t r a n s p o r t o f the enzymes involved in n o r a d r e n a l i n e synthesis a n d their subcellular localization b e c a m e a p p a r e n t . D o p a m i n e fl-hydroxylase, p r e d o m i n a n t l y localized in p a r t i c u l a t e fractions o f sciatic nerve, is t r a n s p o r t e d at the highest rate, tyrosine hydroxylase, p r e d o m i n a n t l y localized in n o n - p a r t i c u l a t e fractions, at a lower rate a n d D O P A decarboxylase, exclusively localized in n o n - p a r t i c u l a t e fractions, at the lowest rate. The excellent technical assistance o f Mrs. H i l a r y W o o d , Miss Vreni F o r s t e r a n d Mr. Ueli J/iggi is gratefully a c k n o w l e d g e d . This study was s u p p o r t e d by the Swiss N a t i o n a l F o u n d a t i o n for Scientific R e s e a r c h ( G r a n t No. 3.653.71).
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