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Increased choline kinase activity in the rat superior cervical ganglion after axonal injury
420
Increased choline kinase activity in the axonal injury
Brain Research, 220 (1981) 42~426 Elsevier/North-Holland Biomedical Press
rat
superior cervical ganglion after
GAD M. GILAD and VARDA H. GILAD Department of lsotope Research, The Weizmann Institute of Science, Rehovot (Israel)
(Accepted May 7th, 1981) Key words: choline kinase - - superior cervical ganglion - - axotomy
The activity of choline kinase (CK) was examined in the rat superior cervical ganglion (SCG) during development and following postganglionic axotomy. The highest specific enzyme activity (nmol phosphorylcholine/mg protein/h) 52-1-8, is observed 5 d before birth, then it rapidly decreases by about 50~, reaching at the day of birth levels observed in the ganglion throughout life. During development the total enzyme activity per ganglion is increased steadily until it reaches a 5-fold increase which parallels the increase in protein content. Following axotomy the enzyme activity per ganglion is rapidly increased by about 2-fold between 1 and 5 d postoperative and then gradually decreases reaching control levels at 30 d. The transient increase in enzyme activity parallels the increase in protein content of the axotomized ganglia, The peak increase in enzyme activity coincides with the peak chromatolytic response of the axotomized ganglion. We conclude that choline kinase activity is transiently increased within neurons after axonal injury, and that this event represents an effort of the nerve cell body to enhance its phosphatidylcholine biosynthesis essential for new membrane synthesis during the regeneration of the cut axon. Choline kinase (CK) catalyzes the first step in the CDP-choline pathway leading to the synthesis o f phosphatidylcholine (lecithin) 1,2° a major c o m p o n e n t o f phospholipids which are important constituents o f biological membranes 17. Increased C K activity has been demonstrated in proliferating cells during active membrane synthesis and concomitant lecithin biosynthesis s,2z. Furthermore, polyamines have been demonstrated to activate the enzyme C K 9,21. These observations suggest that C K t h r o u g h lecithin synthesis is involved in m e m b r a n e biosynthesis, and in turn implicate polyamines in the regulation o f these processes. During development or after axonal injury there appears to be an alteration in metabolic activity o f the neuron to produce materials needed for axonal growth or regeneration, respectively a,a,aa,15,16, at the expense o f those required for functioning in the cell's neurotransmission. 1°,18. In addition, axonal growth involves changes in the a m o u n t and rate o f transported materials along the axon 6,14. Lecithin labeled with [SH]-choline, appears to move rapidly, at a rate comparable to that o f the fastest reported velocities for proteins 1a,22. Furthermore, lecithin has been recently reported to be transported rapidly in a proximodistal direction within injured sciatic rat nerve 7. In the present study, therefore, we have examined the effect of axonal injury o f 0006-8993/81/0000-00(O/$02.50© Elsevier/North-Holland Biomedical Press
421 rat postganglionic sympathetic nerves on the activity of CK in the superior cervical ganglion (SCG) which contains the parent nerve cell bodies. Changes in CK activity were also examined during ontogenetic growth of SCG neurons. In addition, since polyamines have been implicated in the reaction of neurons to injury, it we have examined the effect of the naturally occurring polyamines on CK activity within the SCG of intact and axotomized animals. Animals. Experiments were performed on male and female Sprague-Dawley rats (Experimental Animal Center, Weizmann Institute of Science, Rehovot, Israel) ranging in age from 16 d of gestation, dated from the time of vaginal plug formation, to 120 d postnatally. Surgery. Axotomy was performed as described before 12 by cutting the two major postganglionic nerves of the SCG unilaterally. The unoperated contralateral SCG served as control after ascertaining that the parameters under study remained unchanged as compared to unoperated or sham-operated animals. Biochemical assays. SCG was removed TM at various times during normal development and after operations, and homogenized in 10 vol of 5 mM Tris buffer (pH 7.2) containing 0.1 ~ Triton X-100 (v/v). CK activity was assayed according to Burt and
A
~
50
c 5.0 _o
~
40
4,0
3.0
"6 g ~
a~ ~ _e
2O
g_ ->'O_d
c
I0
.0 cE
2OO
B
150
/ 30 ~rth
40
6 0 t20 Do~
Fig. 1. Upper panel: activity of choline kinase (CK) per mg protein (open circle) and per ganglion (solid line) in the superior cervical ganglion (SCG) during development. Lower panel: Protein content of the ganglion during development. Each point represents the mean (:k S.E.M.) values of 6 animals. Note the interruption of the abscissa.
422 Brody5. A 20-#1 aliquot of tissue homogenate was incubated for 1 h at 37 °C with 20 #1 of a mixture containing, in final concentrations: 0.1 M 2-amino-2-methyl-l,3propanediol buffer (pH 9.0), 10 mM MgCI2, 10 mM ATP, 10 mM choline chloride, 0.2 #Ci choline chloride (methyl-14C) (New England Nuclear, specific activity 50 mCi/mmol), 1.0 mM oubain, 20 mM phospho(enol) pyruvate, and 81 units/ml pyruvate kinase (Sigma). The reaction was stopped by addition of 50/A of chilled water. The nonphosphorylated choline was removed by three extractions each with 200 #1 of allyl cyanide containing 30 mg/ml sodium tetraphenylboron. A 50-/zl aliquot of the remaining aqueous phase was removed and radioactivity measured by liquid scintillation spectrometry. The concentration of proteins was determined according to the method of Lowry et al. 19 using bovine serum albumin as a standard. Histology. Ganglia were removed and placed in formalin solution made out of phosphate-buffered saline (pH 7.4) containing 10 7oformaldehyde. After 7 d the ganglia, following dehydration, were embedded in paraffin. Sections of 10/~m thickness were cut and stained with cresyl violet. Choline kinase activity in the superior cervical ganglion during development. The specific activity of CK (expressed per mg protein) was highest at 16 d of gestation (--5 d) (Fig. 1, upper panel). Immediately thereafter, the activity fell sharply, by about 50 ~, reaching at the day of birth levels observed in the ganglion throughout life. The total enzyme activity (expressed per ganglion) however, was low at 16 d of gestation, but steadily increased approximately 5-fold (Fig. 1, upper panel) reaching adult levels between 15 and 20 d postnatally. The pattern of increase in enzyme activity paralleled the increase in total protein content of the ganglion (Fig. 1, lower panel). Choline kinase activity in the superior cervical ganglion following axotomy. CK activity (expressed per ganglion) remained unchanged for up to 24 h after postganglionic nerve injury. The activity then rapidly increased, reaching a peak of nearly 200 of control levels, by 5 d postoperative (Fig. 2). A gradual decrease followed, and by 30 d postoperative enzyme activity returned back to control levels. The changes in CK activity were in parallel with changes in total protein content of the SCG after axotomy for up to 20 d postoperative. But later the enzyme activity returned back to control levels, while protein content remained elevated for up to 35 d after axonal injury (Fig. 3). Effect of axotomy on cellular morphology of the superior cervical ganglion. The biochemical changes observed are reflected in morphological changes elicited in the injured ganglia. The morphological alterations are most conspicuous 5 d after axotomy (Fig., 4), the time when both CK activity and protein content have also reached their high peak. At this time the wet weight of the injured ganglion nearly doubles (control ~ 2.4 4. 0.2 mg; experimental = 4.2 4- 0.2 mg; n ~ 10) as well. This is due to an increase in the size of the injured ganglion (Fig. 4), which is in turn the result of first, an increase in the number of non-neuronal cells in the injured ganglia (Fig. 4, middle panel). And, second, an increase in the size of nerve cell bodies, a change concomitant of the prominent chromatolytic response observed (Fig. 4, lower panel).
423
500
\
400 A
§ .>_
W
2OO
150
~//////~/~///////.~ ~-.~//.~///////////////////////////////////////.~
J JJJJ/JJJ CK
1(3(2
0
Ibh 2~1 I 5:d 2d
Ibd
2bd
3bd
1
Time postoperative
Fig. 2. Changes in choline kinase (CK) activity in the superior cervical ganglion following unilateral axotomy. The results are expressed as per cent of unoperated contralateral side (±S.E.M.) (shaded area), and are calculated per ganglion basis. Each point represents the mean (=ES.E.M.) value of 6 animals. ** = P < 0.001. The smooth line represents ornithine decarboxylase (ODC) activity as reported elsewhere 11, and displayed here for the sake of completion. Note the interruption of the ordinates.
2OO
I
Time Postopemii~ Fig. 3. Changes in protein content of the superior cervical ganglion following unilateral axotomy. The results are expressed as per cent of unoperatexl contralateral side (±S.E.M.) (shaded area). Each point represents the mean (~= S.E.M.) value of 6 animals. * P < 0.01 ; ** P < 0.001. Note the interruption o f the abscissa.
424
Fig. 4. Photomicrographs of sections through the superior cervical ganglion stained with cresyl violet. A (left column): control ganglion ; B (right colunm): ganglion after postganglionic axotomy. Original magnifications: upper panel x 63; middle panel x 250; lower panel x 400. Note: (a) the extensive proliferation of non-neuronal cells at the root of the postganglionic nerve, and (b) the classic chromatolytic response of axonally injured neurons, with enlarged cell body, clear cytoplasm, and enlarged and eccentric nucleus.
Effect of polyamines on CK activity in the superior cervical ganglion. Changes in ornithine decarboxylase (ODC) activity ~1 are displayed in Fig. 2 for the sake of completion, to show that C K activity was increased while O D C activity was still elevated in the injured ganglia. This fact has suggested the involvement of polyamines in the control of C K activity. The three naturally occuring polyamines; putrescine, spermidine and spermine, both at low (0.5 mM) and high (4 mM) concentrations did not effect C K activity in homogenates of intact or injured ganglia (Table I). Discussion. During developmental growth total C K activity in the SCG is increased steadily in parallel with protein content, a measure of general growth of the
425 TABLE I The effect of polyamines on choline kinase activity in the superior cervical ganglion 4 days after axotomy
Tissue homogenateswere pre-incubatedfor 15 min with the various concentrations of polyaminesand then assayedfor CK activity.Resultsare the mean valuesof duplicate determinations in tissues pooled from 10 animals. Polyamine (mM)
None Putrescine Putrescine Spermidine Spermidine Spermine Spermine
CK activity (nmol product/mg protein~h)
(0.5) (4.0) (0.5) (4.0) (0.5) (4.0)
Contralateral side
4d Postoperative
35.7 35.2 40.6 38.2 39.1 32.8 32.3
41.7 32.5 37.6 39.2 45.2 44.3 43.2
ganglion. Interestingly, while the specific activity of the enzyme is constant throughout adult life, it is however, 2-fold higher at about 5 d before birth, a period when there is still growth of terminal axons2,12. Following postganglionic axotomy there is a large increase in total CK activity within the SCG starting after the first postoperative day, reaching a high peak at 5 d and then gradually falling back to control levels by 30 d postoperative. This increase in enzyme activity parallels almost exactly the increase in protein content of the ganglion for up to 20 d after the injury. It follows that during this time the specific activity of CK (per mg protein) does not change in the injured ganglion. This may indicate that the increase in CK activity is an integral part of an enhanced but selective accumulation of proteins needed for neuronal repair after axonal injury10,14,is. After 20 d postoperative CK activity returns to control levels faster than general protein content. The increase in protein content is a reflection of major cellular alterations within the injured ganglion, in which both neurons and non-neuronal cells participate. This raises the question of the origin of the increased CK activity. The resolution of this question still awaits the proper methodology. Recent studies, however, have demonstrated rapid axonal transport of phospholipids18, 22 and furthermore that their transport is rapidly enhanced within injured axons 7. Therefore, it is reasonable to assume that an increased demand for phospholipids may lead to increased CK activity intraneuronally. Furthermore, the signal for this increase in CK activity may be the depletion of lecithin (phosphahydylcholine) from the nerve cell body due to the enhanced axonal transport of phospholipids after the injury. Polyamines were found to activate directly CK in various tissuesP. However, in the SCG polyamines are not involved in CK activation. Since polyamine biosynthesis may be enhanced in the SCG within the first few hours after axotomy 11 they may still be involved in the increased accumulation of CK and not in its activation. It may be concluded that increased CK activity is an integral part of the reaction of the SCG to postganglionic nerve injury. Furthermore, the changes in CK activity
426 p r o b a b l y occur within axonaUy injured nerve cell bodies as a part of a selective r e a r r a n g e m e n t of protein biosynthesis aimed at increasing synthesis of proteins required for regeneration of structural c o m p o n e n t s of the d a m a g e d axon. This research was supported in part by a g r a n t from the M u s c u l a r D y s t r o p h y Association (V.H.G.).
1 Ansell, G. B. and Spanner, S., The origin and metabolism of brain choline. In P. G. Waser (Ed.), Cholinergic mechanisms, Raven Press, New York, 1975, pp. 117-129. 2 Black, I. B. and Mytilineou, C., Transsynaptic regulation of the development of end-organ innervation by sympathetic neurons, Brain Research, 101 (1976) 503-521. 3 Bodian, D., Nucleic acid in nerve regeneration, Soc. Exp. Symp., 1 (1947) 163-178. 4 Brattgard, S. O., Hyden, H. and Sjostrand, J., The chemical changes in regenerating neurons, J. Neurochem, 1 (1957) 316-325. 5 Burt, A. M. and Brody, S. A., The measurement of choline kinase activity in rat brain: the problem of alternate pathways of ATP metabolism, Anal. Biochem., 65 (1975) 215-224. 6 Dahlstr6m, A., Axoplasmic transport (with particular respect to adrenergic neurons), Phil. Trans. B, 261 (1971) 325-358. 7 Dziegielewska, K. M. Evans, C. A. N. and Saunders, N. R., Rapid effect of nerve injury upon axonal transport of phospholipids, J. PhysloL (Lond.), 304 (1980) 83-98. 8 Farrell, P. M., Lundgren, D. W. and Adams, A. J., Choline kinase and choline phosphotransferase in developing fetal rat lung, Biochem. biophys. Res. Commun., 57 (1974) 696-701 9 Fukuyama, H. and Yamashita, S., Activation of rat liver choline kinase by polyamines, FEBS Lett., 71 (1976) 33-36. 10 Gilad, G. M. and Reis, D. J., Reversible reduction of tyrosine hydroxylase enzyme protein during the retrograde reaction in mesolimbic dopaminergic neurons, Brain Research, 149 (1978) t41-153. 11 Gilad, G. M. and Kopin, I. J., Ornithine decarboxylase (ODC) activity in the rat superior cervical ganglion SCG : Rapid changes following axonal injury, Int. Soc. Neurochem., 7th. meeting. (1979) 353. 12 Gilad, G. M., Gagnon, C. and Kopin, I. J., Protein carboxymethylase activity in the rat superior cervical ganglion during development and after axonal injury, Brain Research, 183 (1980) 393-402. 13 Grafstein, B., Miller, J. A., Ledeen, R. W., Haley, J. and Specht, S. C., Axonal transport of phospholipid in goldfish optic system, Exp. neuroL, 46 (1975) 261-281. 14 Grafstein, B. and McQuarrie, I. G., Role of the nerve cell body in axonal regeneration. In C. W. Cotman (Ed.), Neuronal Plasticity, Raven Press, New York, 1978, pp. 155-195. 15 Griffith, A. and LaVelle, A., Developmental protein changes in normal and chromatolytic facial nerve nuclear regions, Exp. Neurol., 33 (1971) 360-371. 16 Harkonen, M. H. A. and Kauffman, F. C., Metabolic alterations in the axotomized superior cervical ganglion of the rat. I. Energy metabolism, Brain Research 65 (1973) 127-139. 17 Jackson, R. L. and Gotto, A. M., Phospholipids in biology and medicine, N. Engl. J. Med., 290 (1974) 24-29. 18 Kopin, I. J. and Silberstein, S. D., Axons of sympathetic neurons: transport of enzymes in vivo and properties of axonal sprouts in vitro, Pharmacol. Rev., 24 (1972) 245-254. 19 Lowry, O. H., Rosebrough, N. J. Farr, A. L. and Randall, R. J., Protein measurement with the folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 20 McCaman, R. E., and Cook, R., Intermediary metabolism of phospholipids in brain tissue, J. biol. Chem. 241 (1966) 339-3394. 21 Oka, T. and Perry, J. W., Glucocorticoid stimulation of choline kinase activity during the development of mouse mammary gland, Develop. Biol., 68 (1979) 311-318. 22 Toews, A. D., Goodrum. J. F. and Morell, P., Axonal transport of phospholipids in rat visual system, J. Neurochem., 32 (1979) 1165-1173. 23 Weinhold, P. A., Skinner, R. S. and .Sanders, R. D., Activity and some properties of choline kinase, cholinephosphate cytidyltranaferase and choline phosphotransferase during liver development in the rat, Biochem. biophys. Acta, 326 (1973) 43-51.
Increased choline kinase activity in the axonal injury
Brain Research, 220 (1981) 42~426 Elsevier/North-Holland Biomedical Press
rat
superior cervical ganglion after
GAD M. GILAD and VARDA H. GILAD Department of lsotope Research, The Weizmann Institute of Science, Rehovot (Israel)
(Accepted May 7th, 1981) Key words: choline kinase - - superior cervical ganglion - - axotomy
The activity of choline kinase (CK) was examined in the rat superior cervical ganglion (SCG) during development and following postganglionic axotomy. The highest specific enzyme activity (nmol phosphorylcholine/mg protein/h) 52-1-8, is observed 5 d before birth, then it rapidly decreases by about 50~, reaching at the day of birth levels observed in the ganglion throughout life. During development the total enzyme activity per ganglion is increased steadily until it reaches a 5-fold increase which parallels the increase in protein content. Following axotomy the enzyme activity per ganglion is rapidly increased by about 2-fold between 1 and 5 d postoperative and then gradually decreases reaching control levels at 30 d. The transient increase in enzyme activity parallels the increase in protein content of the axotomized ganglia, The peak increase in enzyme activity coincides with the peak chromatolytic response of the axotomized ganglion. We conclude that choline kinase activity is transiently increased within neurons after axonal injury, and that this event represents an effort of the nerve cell body to enhance its phosphatidylcholine biosynthesis essential for new membrane synthesis during the regeneration of the cut axon. Choline kinase (CK) catalyzes the first step in the CDP-choline pathway leading to the synthesis o f phosphatidylcholine (lecithin) 1,2° a major c o m p o n e n t o f phospholipids which are important constituents o f biological membranes 17. Increased C K activity has been demonstrated in proliferating cells during active membrane synthesis and concomitant lecithin biosynthesis s,2z. Furthermore, polyamines have been demonstrated to activate the enzyme C K 9,21. These observations suggest that C K t h r o u g h lecithin synthesis is involved in m e m b r a n e biosynthesis, and in turn implicate polyamines in the regulation o f these processes. During development or after axonal injury there appears to be an alteration in metabolic activity o f the neuron to produce materials needed for axonal growth or regeneration, respectively a,a,aa,15,16, at the expense o f those required for functioning in the cell's neurotransmission. 1°,18. In addition, axonal growth involves changes in the a m o u n t and rate o f transported materials along the axon 6,14. Lecithin labeled with [SH]-choline, appears to move rapidly, at a rate comparable to that o f the fastest reported velocities for proteins 1a,22. Furthermore, lecithin has been recently reported to be transported rapidly in a proximodistal direction within injured sciatic rat nerve 7. In the present study, therefore, we have examined the effect of axonal injury o f 0006-8993/81/0000-00(O/$02.50© Elsevier/North-Holland Biomedical Press
421 rat postganglionic sympathetic nerves on the activity of CK in the superior cervical ganglion (SCG) which contains the parent nerve cell bodies. Changes in CK activity were also examined during ontogenetic growth of SCG neurons. In addition, since polyamines have been implicated in the reaction of neurons to injury, it we have examined the effect of the naturally occurring polyamines on CK activity within the SCG of intact and axotomized animals. Animals. Experiments were performed on male and female Sprague-Dawley rats (Experimental Animal Center, Weizmann Institute of Science, Rehovot, Israel) ranging in age from 16 d of gestation, dated from the time of vaginal plug formation, to 120 d postnatally. Surgery. Axotomy was performed as described before 12 by cutting the two major postganglionic nerves of the SCG unilaterally. The unoperated contralateral SCG served as control after ascertaining that the parameters under study remained unchanged as compared to unoperated or sham-operated animals. Biochemical assays. SCG was removed TM at various times during normal development and after operations, and homogenized in 10 vol of 5 mM Tris buffer (pH 7.2) containing 0.1 ~ Triton X-100 (v/v). CK activity was assayed according to Burt and
A
~
50
c 5.0 _o
~
40
4,0
3.0
"6 g ~
a~ ~ _e
2O
g_ ->'O_d
c
I0
.0 cE
2OO
B
150
/ 30 ~rth
40
6 0 t20 Do~
Fig. 1. Upper panel: activity of choline kinase (CK) per mg protein (open circle) and per ganglion (solid line) in the superior cervical ganglion (SCG) during development. Lower panel: Protein content of the ganglion during development. Each point represents the mean (:k S.E.M.) values of 6 animals. Note the interruption of the abscissa.
422 Brody5. A 20-#1 aliquot of tissue homogenate was incubated for 1 h at 37 °C with 20 #1 of a mixture containing, in final concentrations: 0.1 M 2-amino-2-methyl-l,3propanediol buffer (pH 9.0), 10 mM MgCI2, 10 mM ATP, 10 mM choline chloride, 0.2 #Ci choline chloride (methyl-14C) (New England Nuclear, specific activity 50 mCi/mmol), 1.0 mM oubain, 20 mM phospho(enol) pyruvate, and 81 units/ml pyruvate kinase (Sigma). The reaction was stopped by addition of 50/A of chilled water. The nonphosphorylated choline was removed by three extractions each with 200 #1 of allyl cyanide containing 30 mg/ml sodium tetraphenylboron. A 50-/zl aliquot of the remaining aqueous phase was removed and radioactivity measured by liquid scintillation spectrometry. The concentration of proteins was determined according to the method of Lowry et al. 19 using bovine serum albumin as a standard. Histology. Ganglia were removed and placed in formalin solution made out of phosphate-buffered saline (pH 7.4) containing 10 7oformaldehyde. After 7 d the ganglia, following dehydration, were embedded in paraffin. Sections of 10/~m thickness were cut and stained with cresyl violet. Choline kinase activity in the superior cervical ganglion during development. The specific activity of CK (expressed per mg protein) was highest at 16 d of gestation (--5 d) (Fig. 1, upper panel). Immediately thereafter, the activity fell sharply, by about 50 ~, reaching at the day of birth levels observed in the ganglion throughout life. The total enzyme activity (expressed per ganglion) however, was low at 16 d of gestation, but steadily increased approximately 5-fold (Fig. 1, upper panel) reaching adult levels between 15 and 20 d postnatally. The pattern of increase in enzyme activity paralleled the increase in total protein content of the ganglion (Fig. 1, lower panel). Choline kinase activity in the superior cervical ganglion following axotomy. CK activity (expressed per ganglion) remained unchanged for up to 24 h after postganglionic nerve injury. The activity then rapidly increased, reaching a peak of nearly 200 of control levels, by 5 d postoperative (Fig. 2). A gradual decrease followed, and by 30 d postoperative enzyme activity returned back to control levels. The changes in CK activity were in parallel with changes in total protein content of the SCG after axotomy for up to 20 d postoperative. But later the enzyme activity returned back to control levels, while protein content remained elevated for up to 35 d after axonal injury (Fig. 3). Effect of axotomy on cellular morphology of the superior cervical ganglion. The biochemical changes observed are reflected in morphological changes elicited in the injured ganglia. The morphological alterations are most conspicuous 5 d after axotomy (Fig., 4), the time when both CK activity and protein content have also reached their high peak. At this time the wet weight of the injured ganglion nearly doubles (control ~ 2.4 4. 0.2 mg; experimental = 4.2 4- 0.2 mg; n ~ 10) as well. This is due to an increase in the size of the injured ganglion (Fig. 4), which is in turn the result of first, an increase in the number of non-neuronal cells in the injured ganglia (Fig. 4, middle panel). And, second, an increase in the size of nerve cell bodies, a change concomitant of the prominent chromatolytic response observed (Fig. 4, lower panel).
423
500
\
400 A
§ .>_
W
2OO
150
~//////~/~///////.~ ~-.~//.~///////////////////////////////////////.~
J JJJJ/JJJ CK
1(3(2
0
Ibh 2~1 I 5:d 2d
Ibd
2bd
3bd
1
Time postoperative
Fig. 2. Changes in choline kinase (CK) activity in the superior cervical ganglion following unilateral axotomy. The results are expressed as per cent of unoperated contralateral side (±S.E.M.) (shaded area), and are calculated per ganglion basis. Each point represents the mean (=ES.E.M.) value of 6 animals. ** = P < 0.001. The smooth line represents ornithine decarboxylase (ODC) activity as reported elsewhere 11, and displayed here for the sake of completion. Note the interruption of the ordinates.
2OO
I
Time Postopemii~ Fig. 3. Changes in protein content of the superior cervical ganglion following unilateral axotomy. The results are expressed as per cent of unoperatexl contralateral side (±S.E.M.) (shaded area). Each point represents the mean (~= S.E.M.) value of 6 animals. * P < 0.01 ; ** P < 0.001. Note the interruption o f the abscissa.
424
Fig. 4. Photomicrographs of sections through the superior cervical ganglion stained with cresyl violet. A (left column): control ganglion ; B (right colunm): ganglion after postganglionic axotomy. Original magnifications: upper panel x 63; middle panel x 250; lower panel x 400. Note: (a) the extensive proliferation of non-neuronal cells at the root of the postganglionic nerve, and (b) the classic chromatolytic response of axonally injured neurons, with enlarged cell body, clear cytoplasm, and enlarged and eccentric nucleus.
Effect of polyamines on CK activity in the superior cervical ganglion. Changes in ornithine decarboxylase (ODC) activity ~1 are displayed in Fig. 2 for the sake of completion, to show that C K activity was increased while O D C activity was still elevated in the injured ganglia. This fact has suggested the involvement of polyamines in the control of C K activity. The three naturally occuring polyamines; putrescine, spermidine and spermine, both at low (0.5 mM) and high (4 mM) concentrations did not effect C K activity in homogenates of intact or injured ganglia (Table I). Discussion. During developmental growth total C K activity in the SCG is increased steadily in parallel with protein content, a measure of general growth of the
425 TABLE I The effect of polyamines on choline kinase activity in the superior cervical ganglion 4 days after axotomy
Tissue homogenateswere pre-incubatedfor 15 min with the various concentrations of polyaminesand then assayedfor CK activity.Resultsare the mean valuesof duplicate determinations in tissues pooled from 10 animals. Polyamine (mM)
None Putrescine Putrescine Spermidine Spermidine Spermine Spermine
CK activity (nmol product/mg protein~h)
(0.5) (4.0) (0.5) (4.0) (0.5) (4.0)
Contralateral side
4d Postoperative
35.7 35.2 40.6 38.2 39.1 32.8 32.3
41.7 32.5 37.6 39.2 45.2 44.3 43.2
ganglion. Interestingly, while the specific activity of the enzyme is constant throughout adult life, it is however, 2-fold higher at about 5 d before birth, a period when there is still growth of terminal axons2,12. Following postganglionic axotomy there is a large increase in total CK activity within the SCG starting after the first postoperative day, reaching a high peak at 5 d and then gradually falling back to control levels by 30 d postoperative. This increase in enzyme activity parallels almost exactly the increase in protein content of the ganglion for up to 20 d after the injury. It follows that during this time the specific activity of CK (per mg protein) does not change in the injured ganglion. This may indicate that the increase in CK activity is an integral part of an enhanced but selective accumulation of proteins needed for neuronal repair after axonal injury10,14,is. After 20 d postoperative CK activity returns to control levels faster than general protein content. The increase in protein content is a reflection of major cellular alterations within the injured ganglion, in which both neurons and non-neuronal cells participate. This raises the question of the origin of the increased CK activity. The resolution of this question still awaits the proper methodology. Recent studies, however, have demonstrated rapid axonal transport of phospholipids18, 22 and furthermore that their transport is rapidly enhanced within injured axons 7. Therefore, it is reasonable to assume that an increased demand for phospholipids may lead to increased CK activity intraneuronally. Furthermore, the signal for this increase in CK activity may be the depletion of lecithin (phosphahydylcholine) from the nerve cell body due to the enhanced axonal transport of phospholipids after the injury. Polyamines were found to activate directly CK in various tissuesP. However, in the SCG polyamines are not involved in CK activation. Since polyamine biosynthesis may be enhanced in the SCG within the first few hours after axotomy 11 they may still be involved in the increased accumulation of CK and not in its activation. It may be concluded that increased CK activity is an integral part of the reaction of the SCG to postganglionic nerve injury. Furthermore, the changes in CK activity
426 p r o b a b l y occur within axonaUy injured nerve cell bodies as a part of a selective r e a r r a n g e m e n t of protein biosynthesis aimed at increasing synthesis of proteins required for regeneration of structural c o m p o n e n t s of the d a m a g e d axon. This research was supported in part by a g r a n t from the M u s c u l a r D y s t r o p h y Association (V.H.G.).
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