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Inhibition of the retrograde axonal transport of dopamine-β-hydroxylase antibodies by the calcium ionophore A23187
62
Brain Research, ~45 ! ! 9,~5 ) 1SO- i ~)i Elsevier
BRE 11057
Inhibition of the Retrograde Axonal Transport of Dopamine-fl-Hydroxylase Antibodies by the Calcium Ionophore A23187 G. J. LEES Department of Psychiatry, School of Medicine, University of Auckland, Auckland (New Zealand)
(Accepted January 3rd, 1985) Key words: antibodies to dopamine-fl-hydroxylase - - retrograde axonal transport - - calcium ionophore
High levels of calcium, as well as calcium ionophores, have been reported to inhibit the anterograde transport of proteins, The effect of the calcium ionophore, A23187, on the retrograde axonal transport of proteins was therefore investigated. The uptake of antibodies to dopamine-fl-hydroxylase (anti-DflH) by sympathetic nerve terminals in the iris and their subsequent accumulation in the superior cervical ganglion was inhibited by up to 65% by A23187 (6 nmol. i.o.). At this dose. catecholamine fluorescence in the iris was reduced, indicating a high rate of exocytosis, but tyrosine hydroxylase levels and the capacity of the treated irides to take up noradrenaline were unaffected. Higher amounts of A23187 ( 28 nmol. i.o.) did not cause a greater degree of inhibition of retrograde transporl However, this dose was toxic to the neurons as shown by a 68% decrease in the ability of the nerve terminals in the iris to take up [3H]noradrenaline. This loss of function occurred gradually over a 12-h period. On the other hand. tyrosine hydroxylase levels were unaffected by 28 nmol A23187. The toxicity of A23187 may be a consequence of a build up in intracellular calcium but such toxicity did not lead to any apparent loss of nerve terminals within a 3-day period.
INTRODUCTION The rapid anterograde axonal transport of proteins has been shown to have a requirement for calcium 1,13. However, excess intracellular calcium is inhibitory, whether induced by high extracellular concentrations of calcium 1A3, or following stimulation of calcium uptake by ionophores 2,s. Dopamine-fl-hydroxylase (DflH), a marker for noradrenergic vesicles, is among the proteins whose anterograde transport is inhibited by the calcium ionophore. A23t872. Extracellular calcium is essential for the A23187-induced inhibition, and is accompanied by microtubular disruption 2. This disruption is probably due to a depolymerization of microtubules, which can be induced by relatively low levels of calcium ( 1 - 5 0 0 ~M) in the presence of calmodulin Is. The mechanism for the retrograde transport of proteins is less clearly defined, although somatopedal m o v e m e n t of various-sized particles has been observed (see ref. 9). Although less evidence is avail-
able. microtubules also appear to be involved in retrograde transport, as compounds inducing disruption of microtubules (such as colchicinel inhibit both anterograde and retrograde transport4, 9.19. It was therefore of interest to determme whether A23187 could also affect the retrograde transport of proteins. The transport of antibodies to dopamine-fl-hydroxylase (anti-D/3H) was investigated since these are known to be specifically taken up by sympathetic nerve terminals and subsequently undergo retrograde axonal transport 4.10-12.22 MATERIALS AND METHODS The retrograde axonal transport of anti-DflH was studied as previously described in detaiP 0.11. Antibodies to DflH, raised in rabbits, were purified on a DflH-Sepharose column. Proteins were iodinated with iodine-125 using the chloramine-T method of Greenwood et al. 5. [125I]anti-DflH (!015 X 106 cpm) was injected in a m a x i m u m volume of 20 ~1 into one
Correspondence: G. J. Lees, Department of Psychiatry, School of Medicine, University of Auckland, Private Bag, Auckland, New Zealand.
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
63 anterior eye chamber of adult guinea pigs (200300 g) under ether anaesthesia. The contralateral side served as a control for uptake from the circulation into the superior cervical ganglion (SCG). Drugs were injected intraocularly along with the iodinated protein in a single injection unless otherwise specified. The animals were subsequently killed-at various times and the SCGs and irides removed, washed with 1 ml of 0.14 M saline and counted by y-spectrometry. Tyrosine hydroxylase was estimated by a micromodification of the method of Waymire et al. 21. Each iris was homogenized in 20 ¢tl of water and 5 ¢tl aliquots were incubated for 60 min at 37 °C in a reaction mixture (total volume 12.5 ktl) containing 2.75/~mol sodium acetate buffer (pH 6.1), 1.1 nmol L-[1-14C]tyrosine (spec. act. 53.4 mCi/mmol), 10.9 nmol ferrous sulphate, 2 nmol pyridoxal phosphate, and 0.8BI of a pig kidney D O P A decarboxylase preparation 21. The incubation was carried out in a tube sealed with a rubber stopper from which was suspended a well containing 15 ,ul Protosol (New England Nuclear). The reaction was stopped by 50 ,ul 10% trichloroacetic acid and the CO 2 released collected overnight in the Protosol. The well was removed and counted by liquid scintillation spectrometry. Noradrenaline uptake was estimated by incubating each iris for 20 min at 37 °C in 200 ktl of a glucose-salt medium (Thoa et al. 20) containing 5 x 10-7 M [3H]noradrenaline (spec. act. 4.0 Ci/mmol). Each iris was then washed in two changes of glucose-saline medium (0.5 ml), dissolved in 0.2 ml Protosol, and counted by liquid scintillation spectrometry. Irides were stained for catecholamines by incubating the irides in 2% glyoxylic acid in 0.1 M sodium phosphate buffer (adjusted to pH 7) for at least 15 min. The irides were then stretched on chrome-alum slides, blotted dry and heated for 4 min at 100 °C. After mounting in paraffin oil, the fluorescence was visualized under a Leitz Ortholux 2 microscope fitted with a 200 W high pressure mercury vapour lamp and with BG 12 excitation and K 510 emission filters. In order to visualize DflH, the irides were washed with 1 ml PBS for 20 min, then incubated at 37 °C for 30 min in 0.2 ml crude anti-DflH (diluted 1:40 with 0.3% Triton X-100 in PBS). The irides were further washed with 2 changes of 1 ml 2% Triton X-100 in PBS for 40 min, followed by incubation for 30 min at 37 °C with 200 #1 fluorescein isothiocyanate-labelled
sheep anti-rabbit IgG (Wellcome Labs., diluted 1:80 in 0.3% Triton in PBS). The irides were finally washed with 2 changes 1 ml 2% Triton X-100 in PBS for 60 min, stretched on chrome-alum slides, dried for 10 min and then mounted in buffered glycerol, pH 8.6 (50:50 mixture 0.5 M sodium carbonate and glycerol). The DflH immunofluorescence was visualized under a fluorescence microscope using the conditions outlined above. RESULTS Due to the insolubility of A23187, dimethylsulphoxide (DMSO) was used to dissolve the ionophore. As shown in Fig. 1, D M S O (5 ~d) had no effect on the accumulation of [125I]anti-DflH in the SCG, 24 h following injection into the anterior eye chamber of the guinea pig. The intraocular injection of A23187 along with anti-DflH resulted in a decrease in the amount of [125I]anti-DflH which accumulated in the SCG 24 h later. No inhibition was observed with 0.6 nmol A23187, but a 65% inhibition occurred with 6 nmol A23187 (Fig. 1). Higher amounts of A23187 (28 nmol) did not increase the de-
'~-CONTROL~"'~DMSO~ '(~
--A23187
~"
120 36nmole :s
o~
~ 6nmoie ~L] 28nmole
Fig. 1. Accumulation of [t25I]anti-DflHin the SCG 24 h after injection into the ipsilateral anterior eye chamber, along with 5 kd of A23187 dissolved in DMSO. Results from groups of 4-19 animals and from three batches of [1251]anti-DflH are combined and expressed as a percentage of the [1251]-accumulation in control animals + S.E.M. (408 cpm, 777 cpm and 1512 cpm for the individual batches). Neutralization of the anti-DflH was achieved by incubation of 100 ,ul of the anti-DflH with excess DflH at 37 °C for 1 h, followed by overnight storage at 4 °C. Illl, non-injected side; IN, 5 ul DMSO plus anti-DflH; [], A23187 plus anti-DflH; [], A23187 plus neutralized anti-DflH. * P < 0.001 compared with DMSO-treated control animals.
64 transport was insensitive to the actions of A23187.
A
A decrease in the amount of retrograde transport of anti-DflH could have been due to a toxic effect of A23187 on the nerve terminals. Thus catecholamine function in the iris was assessed by estimating noradrenaline exocytosis, noradrenaline reuptake, and tyrosine hydroxylase levels. A high rate of exocytosis of noradrenaline was readily apparent as the normal catecholamine fluorescence of the nerve terminals was markedly reduced 18 h following intraocular injection of A23187 (6 nmot), and almost completely lost with higher doses (Fig. 21. Minimal loss of fluorescence occurred in control animals injected with vehicle (DMSO) alone. On a scale of 0 to -r~--r-r. catecholamine fluorescence following DMSO was + *-,-. with 6 nmol A23187. ~--r. and with 28 nmol A23187. - . In = 7-8 animals in each category). The apparent increased exocytosis induced by A23t87 would result in a higher concentration of DflH at the plasma membrane t5. Thus it was predicted that the amount of anti-DflH bound to the iris would also be increased. However. while A23187 caused a marked dose-dependent increase (5-16-fold) in the binding of [125I]anti-DflH to the iris. such binding was not 120
80
\
40
C
Fig. 2. Glyoxylic acid-induced catecholamine fluorescence m the iris following injection of 5 Izl DMSO ( A h A23187 (6 nmol in 5~1DMSO) (B), or A23187 (28 nmol in 5/~1DMSO) (C) into the anterior eye chamber, 18 h prior to sacrifice. Irides were stretched and incubated with glyoxylic acid as described in Methods.
gree of inhibition (60%). If. however, the [taSI]antiDflH was neutralized by prior incubation with pure DflH, then no transport occurred in the presence of A23187 (Fig. 1). Thus while all transport of [t25I]anti-DflH was specific, approximately 40% of such
TIME
2 4 6 8 AFTER INJECTION (DAYS)
Fig. 3. Time course for the accumulation of [125i]anti-DflH in the SCG following injection of anti-DflH and 5 gl of A23187 dissolved in DMSO into the ipsilaieral eye chamber 1, 3 and 7 days previously. Results from groups of 3 - 9 animals are expressed as a percentage ± S.E.M. of the [tzsI]-accumulation in animals treated with 5 #1 DMSO i,o., 24 h prior to sacrifice (680 cpm). Accumulation in the ipsilateral ( 0 ) and contralateral (©) ganglion following inrraoeular injection of 5 /~1 DMSO plus anti-DflH: accumulation in the ganglia fotlowmg rejection of anti-D/SH plus A23187 into the ipsilateral eye ( A ) and A23187 alone into the contralateral eye ( A ) o f the same animals.
65 specific. Binding of non-immune [125I]IgG to the iris was also increased by 28 nmol A23187 (3.7-fold). Even if, following injection of A23187, part of the increased binding of anti-D/3H to the iris was due to absorption to DflH trapped on the neuronal surface, at no stage up to 7 days later did this result in a corresponding increase in the amount transported to the SCG (Fig. 3). Immunofluorescence staining for D/3H in the nerve terminals in the eye was not affected by A23187 (28 nmol) (Fig. 4), indicating that nerve terminals remained intact in spite of the loss of noradrenaline. Three days after the injection of 28 nmol A23187, the nerve terminals in the irides still stained normally for D/3H. The absence of nerve degeneration following A23187 was confirmed by estimating the iris content of tyrosine hydroxylase. While the injection of vehicle ( D M S O ) resulted in a 38% reduction of tyrosine hydroxylase activity, low or high doses of A23187 dissolved in the vehicle did not result in a further loss (Table I). The functional activity of sympathetic nerve terminals, as measured by [3H]noradrenaline uptake, was also reduced by D M S O treatment (33%). The addition of 6 nmol A23187 did not reduce this activity further, but with higher doses, there was a major loss of activity (62% less than vehicle control) (Table II). This loss of function occurred gradually, with a maximum effect by 12 h.
A
-05 mm B
Fig. 4. Immunofluorescence staining for D/3H in the irides following injection of 5 #1 DMSO (A) or A23187 (28 nmol in 5 ul DMSO) (B) into the anterior eye chamber 24 h prior to sacrifice. No staining occurred if exogenous anti-D/3H or Triton X-100 was omitted from the staining reagents.
DISCUSSION While anterograde axonal transport requires calciuml, 6,13, its mechanism of action has not been deTABLE I Effect of A23187 on levels of tyrosine hydroxylase in guinea pig irides
5 JA DMSO, or A23187 dissolved in DMSO was injected into the anterior eye chamber of guinea pigs. 18 h later the irides were dissected out and assayed for tyrosine hydroxylase as described in Methods. Results are expressed as means + S.E.M. for the numbers of determinations in parentheses. Treatment in vivo
Tyrosine hydroxylase activi~ (pmol.h 1.iris-O
DMSO A23187, 6 nmol A23187, 28 nmol
20.4 + 2.7 (13) 12.6 -+ 3.2 (8) 11.8 + 2.2 (10) 11.3 + 1.3 (9)
fined. A role in the initiation of transport in the cell perikayron at the level of the Golgi apparatus or beyond 6, as well as in the transport process itself 1,13, has been suggested. Excess calcium, however, inhibits anterograde transport, and such inhibition becomes irreversible at high concentrations~. The inhibition caused by the calcium ionophores A23187 and X-537A is calcium-dependent and results in an increased axonal calcium content 2,~. As shown in these results, the retrograde transport of anti-DflH was also inhibited by A23187. Inhibition occurred at doses of A23187 which caused no change in other catecholamine parameters (noradrenaline uptake and tyrosine hydroxylase levels) and hence was not due to a general toxic effect of A23187. It thus seems likely that anterograde and retrograde transport are similarly affected by high intracellular
66 TABLE II Effect ofA23187 on noradrenaline uptake into,guinea pig irides
5/~1 DMSO, or A23187 dissolved in DMSO was injected into the anterior eye chamber of guinea pigs. At various times subsequently the irides were dissected out and the uptake of noradrenaline (NA) measured as described in Methods. Results are expressed as means + S.E.M. for the number of determinations in parentheses. Treatment in vivo
Time of sacrifice
NA uptake (pmol. h -1, iris-l)
Control Control Control DMSO A23187, 6 nmol A23187, 30 nmol A23187, 30 nmol A23187, 30 nmol A23187, 30 nmol A23187, 30 nmol
-
1.9 + 0.3 (4) ~ 3.6 + 0.5 (3)b 30.9 + 2.6 (14) 20.8 + 4.0 (5)** 21.0 + 2.2 (4) 18.5 + t.8 (4) 14.2 + 2.9 (7) 11.0 + 1.6 (4) 7,9 + 0.7 (4)* 9.2 + 1.0 (7)*
18 h 18 h 1h 2h 6h 12 h 18 h
Incubated at 0 °C. b Incubated with 10-4 M desipramine. * P < 0.025, compared with vehicle injected, 2-tailed Student's t-test. ** P < 0.05, compared with non-injected animals, 2-tailed Student's t-test.
concentrations of calcium induced by A23t87. The mechanism for such inhibition m a y welt be via a calcium-dependent depolymerization of microtubulesl3,17,18, which are generally believed to be involved in axonal t r a n s p o r t ~9. In addition to effects on axonal transport, A23187 and o t h e r calcium i o n o p h o r e s also cause an increased rate of exocytosis 20. The loss of catecholamine fluorescence in the iris following injection of A23187 is consistent with a high rate of exocytosis~ Since drugs thought to increase exocytosis result in a higher amount of anti-DflH t r a n s p o r t e d to the ganglion10-12, A23187 might have also been expected to increase r e t r o g r a d e t r a n s p o r t by this mechanism. The results
REFERENCES 1 Chan, S. Y., Ochs, S. and Worth, R. M., The requirement for calcium ions and the effect of other ions on axoplasmic transport in mammalian nerve, J. Physiol. (London), 301 (1980) 477-504. 2 Esquerro, E., Garcia, A. G. and Sanchez-Garcia, P., The effects of the calcium ionophore, A23187, on the axoplas-
presented here indicate that any increase m the uptake of anti-DflH does not e v o k e a subsequent rise in the amount t r a n s p o r t e d to the ganglion. A further possibility was that the action of A23187 on uptake of anti-DflH would lessen the o b s e r v e d degree of inhibition of r e t r o g r a d e transport. H o w e v e r , 28 nmol A23187 also caused a similar percentage reduction in the r e t r o g r a d e transport of nerve growth factor from the iris (unpublished observations). Results of o t h e r investigations support the conclusion that axonal transport is at least as sensitive as exocytosis to changes in calcium fluxes induced by A23187. Thus inhibition of a n t e r o g r a d e transport of DflH in the cat hypogastric nerve occurs at lower concentrations of A23187 (10-33/~M) 2 than those required for the stimulation of the release of n o r a d r e n aline and DflH from nerve endings in rat atria (30-300 p M ) 2°. In contrast to the selective effects at low doses, higher amounts of A23187 (28 n m o l ) a p p e a r e d to more generally affect the functioning of the neurons, as shown by losses in the capacity of the iris to take up noradrenaline. Such losses m a y be due to excess calcium, which has been suggested to be toxic to cells, producing cellular death3,7, t6. In these experiments, however, the reduction in the capacity of the terminals to take up n o r a d r e n a l i n e does not a p p e a r to be due to a loss of nerve terminals, as shown by the absence of an affect on tyrosine hydroxylase levels and by a normal p a t t e r n of DflH immunofluorescence (for up to 3 days p o s t - t r e a t m e n t following A23187). ACKNOWLEDGEMENTS This research was s u p p o r t e d by a grant from the Medical Distribution C o m m i t t e e of New Z e a l a n d . The gift of antibodies to dopamine-fl-hydroxylase from Dr, R. A. Rush is gratefully a c k n o w l e d g e d
mic transport of dopamine fl-hydroxylase. Br. J Pharmacol., 70 (1980) 375-381. 3 Farber, L. F., The role of calcium in cell death. Life Sci. 29 (1981) 1289-1295. 4 Fillenz, M., Gagnon, C., Stoeckel. K. and Thoenen, H.. Selective uptake and retrograde axonal transport of dopamine-fl-hydroxylase antibodies in peripheral adrenergic neurons, Brain Research, 114 (1976"1293-303.
67
5 Greenwood, F. C., Hunter, W. M. and Glover, J. S., Preparation of 131I-labelled human growth hormone of high specific radioactivity, Biochem. J., 89 (1963) 114-123. 6 Hammerschlag, R. and Lavoie, P.-A., Initiation of fast axonal transport: involvement of calcium during transfer of proteins from Golgi apparatus to the transport system, Neuroscience, 4 (1979) 1195-1201. 7 Jancs6, G., Karcsfi, S., Kirfily, E., Szebeni, A., T6th, L., Bficsy, E., Jo6, F. and Pfirducz, ,~.., Neurotoxin induced nerve cell degeneration: possible involvement of calcium, Brain Res'earch, 295 (1984) 211-216. 8 Kanjc, M., Edstrom, A. and Hanson, M., Inhibition of rapid axonal transport in vitro by the ionophores X-537A and A23187, Brain Research, 21)4 (1981) 43-50. 9 Kristensson, K., Retrograde transport of macromolecules in axons, Annu. Rev. Pharmacol., 18 (1978) 97-110. 10 Lees, G., Chubb, I., Freeman, C., Geffen, L. and Rush, R., Effect of nerve activity on transport of nerve growth factor and dopamine fl-hydroxylase antibodies in sympathetic neurons, Brain Research, 214 (1981) 186-189. 11 Lees, G. J., Geffen, L. B. and Rush, R. A., Phentolamine increases neuronal binding and retrograde transport of dopamine fl-hydroxylase antibodies, Neurosci. Lett., 22 (1981) 115-118. 12 Lees. G. J. and Horsburgh, R. J., Retrograde transport of antibodies to dopamine fl-hydroxylase in sympathetic neurons: effects of drugs modifying noradrenergic transmission, Brain Research, 301 (1984) 281-286, 13 Ochs, S., Worth, R. M. and Chan, S.-Y., Calcium requirement for axoplasmic transport in mammalian nerve,
Nature (London), 270 (1977) 748-750. 14 Pressman, B. C. and Fahim, M., Pharmacology and toxicology of the monovalent carboxylic ionophores, Annu. Rev. Pharmacol., 22 (1982) 465-490. 15 Rush, R. A. and Geffen, L. B., Dopamine fl-hydroxylase in health and disease, Crit. Rev. Clin. Lab. Sci., 12 (1980) 241-277. 16 Schanne, F. A. X., Kane, A. B., Young, E. E. and Farber, J. L., Calcium dependence of toxic cell death: a final common pathway, Science, 206 (1979) 700-702. 17 Schlaepfer, W., Structural alterations of peripheral nerve induced by the calcium ionophore A23187, Brain Research, 136 (1977) 1-9. 18 Schliwa, M., Euteneuer, U., Bulinski, J. C. and Izant, J. G., Calcium lability of cytoplasmic microtubules and its modulation by microtubule-associated protein, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 1037-1041. 19 Schwartz, J. H., Axonal transport: components, mechanisms, and specificity, Annu. Rev. Neurosci., 2 (1979) 467-504. 20 Thoa, N. B., Costa, J. L., Moss, J. and Kopin, I. J., Mechanism of release of norepinephrine from peripheral adrenergic neurons by the calcium ionophores X537A and A23187, LifeSci., 14 (1974) 1705-1719. 2l Waymire, J. C., Bjur, R. and Weiner, N., Assay of tyrosine hydroxylase by coupled decarboxylation of dopa formed from 1-14C-L-tyrosine, Anal. Biochem., 43 (1971) 588-600. 22 Ziegler, M. G., Thomas, J. A. and Jacobowitz, D. M., Retrograde axonal transport of antibody to dopamine-fl-hydroxylase, Brain Research, 104 (1976) 390-395.
Brain Research, ~45 ! ! 9,~5 ) 1SO- i ~)i Elsevier
BRE 11057
Inhibition of the Retrograde Axonal Transport of Dopamine-fl-Hydroxylase Antibodies by the Calcium Ionophore A23187 G. J. LEES Department of Psychiatry, School of Medicine, University of Auckland, Auckland (New Zealand)
(Accepted January 3rd, 1985) Key words: antibodies to dopamine-fl-hydroxylase - - retrograde axonal transport - - calcium ionophore
High levels of calcium, as well as calcium ionophores, have been reported to inhibit the anterograde transport of proteins, The effect of the calcium ionophore, A23187, on the retrograde axonal transport of proteins was therefore investigated. The uptake of antibodies to dopamine-fl-hydroxylase (anti-DflH) by sympathetic nerve terminals in the iris and their subsequent accumulation in the superior cervical ganglion was inhibited by up to 65% by A23187 (6 nmol. i.o.). At this dose. catecholamine fluorescence in the iris was reduced, indicating a high rate of exocytosis, but tyrosine hydroxylase levels and the capacity of the treated irides to take up noradrenaline were unaffected. Higher amounts of A23187 ( 28 nmol. i.o.) did not cause a greater degree of inhibition of retrograde transporl However, this dose was toxic to the neurons as shown by a 68% decrease in the ability of the nerve terminals in the iris to take up [3H]noradrenaline. This loss of function occurred gradually over a 12-h period. On the other hand. tyrosine hydroxylase levels were unaffected by 28 nmol A23187. The toxicity of A23187 may be a consequence of a build up in intracellular calcium but such toxicity did not lead to any apparent loss of nerve terminals within a 3-day period.
INTRODUCTION The rapid anterograde axonal transport of proteins has been shown to have a requirement for calcium 1,13. However, excess intracellular calcium is inhibitory, whether induced by high extracellular concentrations of calcium 1A3, or following stimulation of calcium uptake by ionophores 2,s. Dopamine-fl-hydroxylase (DflH), a marker for noradrenergic vesicles, is among the proteins whose anterograde transport is inhibited by the calcium ionophore. A23t872. Extracellular calcium is essential for the A23187-induced inhibition, and is accompanied by microtubular disruption 2. This disruption is probably due to a depolymerization of microtubules, which can be induced by relatively low levels of calcium ( 1 - 5 0 0 ~M) in the presence of calmodulin Is. The mechanism for the retrograde transport of proteins is less clearly defined, although somatopedal m o v e m e n t of various-sized particles has been observed (see ref. 9). Although less evidence is avail-
able. microtubules also appear to be involved in retrograde transport, as compounds inducing disruption of microtubules (such as colchicinel inhibit both anterograde and retrograde transport4, 9.19. It was therefore of interest to determme whether A23187 could also affect the retrograde transport of proteins. The transport of antibodies to dopamine-fl-hydroxylase (anti-D/3H) was investigated since these are known to be specifically taken up by sympathetic nerve terminals and subsequently undergo retrograde axonal transport 4.10-12.22 MATERIALS AND METHODS The retrograde axonal transport of anti-DflH was studied as previously described in detaiP 0.11. Antibodies to DflH, raised in rabbits, were purified on a DflH-Sepharose column. Proteins were iodinated with iodine-125 using the chloramine-T method of Greenwood et al. 5. [125I]anti-DflH (!015 X 106 cpm) was injected in a m a x i m u m volume of 20 ~1 into one
Correspondence: G. J. Lees, Department of Psychiatry, School of Medicine, University of Auckland, Private Bag, Auckland, New Zealand.
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
63 anterior eye chamber of adult guinea pigs (200300 g) under ether anaesthesia. The contralateral side served as a control for uptake from the circulation into the superior cervical ganglion (SCG). Drugs were injected intraocularly along with the iodinated protein in a single injection unless otherwise specified. The animals were subsequently killed-at various times and the SCGs and irides removed, washed with 1 ml of 0.14 M saline and counted by y-spectrometry. Tyrosine hydroxylase was estimated by a micromodification of the method of Waymire et al. 21. Each iris was homogenized in 20 ¢tl of water and 5 ¢tl aliquots were incubated for 60 min at 37 °C in a reaction mixture (total volume 12.5 ktl) containing 2.75/~mol sodium acetate buffer (pH 6.1), 1.1 nmol L-[1-14C]tyrosine (spec. act. 53.4 mCi/mmol), 10.9 nmol ferrous sulphate, 2 nmol pyridoxal phosphate, and 0.8BI of a pig kidney D O P A decarboxylase preparation 21. The incubation was carried out in a tube sealed with a rubber stopper from which was suspended a well containing 15 ,ul Protosol (New England Nuclear). The reaction was stopped by 50 ,ul 10% trichloroacetic acid and the CO 2 released collected overnight in the Protosol. The well was removed and counted by liquid scintillation spectrometry. Noradrenaline uptake was estimated by incubating each iris for 20 min at 37 °C in 200 ktl of a glucose-salt medium (Thoa et al. 20) containing 5 x 10-7 M [3H]noradrenaline (spec. act. 4.0 Ci/mmol). Each iris was then washed in two changes of glucose-saline medium (0.5 ml), dissolved in 0.2 ml Protosol, and counted by liquid scintillation spectrometry. Irides were stained for catecholamines by incubating the irides in 2% glyoxylic acid in 0.1 M sodium phosphate buffer (adjusted to pH 7) for at least 15 min. The irides were then stretched on chrome-alum slides, blotted dry and heated for 4 min at 100 °C. After mounting in paraffin oil, the fluorescence was visualized under a Leitz Ortholux 2 microscope fitted with a 200 W high pressure mercury vapour lamp and with BG 12 excitation and K 510 emission filters. In order to visualize DflH, the irides were washed with 1 ml PBS for 20 min, then incubated at 37 °C for 30 min in 0.2 ml crude anti-DflH (diluted 1:40 with 0.3% Triton X-100 in PBS). The irides were further washed with 2 changes of 1 ml 2% Triton X-100 in PBS for 40 min, followed by incubation for 30 min at 37 °C with 200 #1 fluorescein isothiocyanate-labelled
sheep anti-rabbit IgG (Wellcome Labs., diluted 1:80 in 0.3% Triton in PBS). The irides were finally washed with 2 changes 1 ml 2% Triton X-100 in PBS for 60 min, stretched on chrome-alum slides, dried for 10 min and then mounted in buffered glycerol, pH 8.6 (50:50 mixture 0.5 M sodium carbonate and glycerol). The DflH immunofluorescence was visualized under a fluorescence microscope using the conditions outlined above. RESULTS Due to the insolubility of A23187, dimethylsulphoxide (DMSO) was used to dissolve the ionophore. As shown in Fig. 1, D M S O (5 ~d) had no effect on the accumulation of [125I]anti-DflH in the SCG, 24 h following injection into the anterior eye chamber of the guinea pig. The intraocular injection of A23187 along with anti-DflH resulted in a decrease in the amount of [125I]anti-DflH which accumulated in the SCG 24 h later. No inhibition was observed with 0.6 nmol A23187, but a 65% inhibition occurred with 6 nmol A23187 (Fig. 1). Higher amounts of A23187 (28 nmol) did not increase the de-
'~-CONTROL~"'~DMSO~ '(~
--A23187
~"
120 36nmole :s
o~
~ 6nmoie ~L] 28nmole
Fig. 1. Accumulation of [t25I]anti-DflHin the SCG 24 h after injection into the ipsilateral anterior eye chamber, along with 5 kd of A23187 dissolved in DMSO. Results from groups of 4-19 animals and from three batches of [1251]anti-DflH are combined and expressed as a percentage of the [1251]-accumulation in control animals + S.E.M. (408 cpm, 777 cpm and 1512 cpm for the individual batches). Neutralization of the anti-DflH was achieved by incubation of 100 ,ul of the anti-DflH with excess DflH at 37 °C for 1 h, followed by overnight storage at 4 °C. Illl, non-injected side; IN, 5 ul DMSO plus anti-DflH; [], A23187 plus anti-DflH; [], A23187 plus neutralized anti-DflH. * P < 0.001 compared with DMSO-treated control animals.
64 transport was insensitive to the actions of A23187.
A
A decrease in the amount of retrograde transport of anti-DflH could have been due to a toxic effect of A23187 on the nerve terminals. Thus catecholamine function in the iris was assessed by estimating noradrenaline exocytosis, noradrenaline reuptake, and tyrosine hydroxylase levels. A high rate of exocytosis of noradrenaline was readily apparent as the normal catecholamine fluorescence of the nerve terminals was markedly reduced 18 h following intraocular injection of A23187 (6 nmot), and almost completely lost with higher doses (Fig. 21. Minimal loss of fluorescence occurred in control animals injected with vehicle (DMSO) alone. On a scale of 0 to -r~--r-r. catecholamine fluorescence following DMSO was + *-,-. with 6 nmol A23187. ~--r. and with 28 nmol A23187. - . In = 7-8 animals in each category). The apparent increased exocytosis induced by A23t87 would result in a higher concentration of DflH at the plasma membrane t5. Thus it was predicted that the amount of anti-DflH bound to the iris would also be increased. However. while A23187 caused a marked dose-dependent increase (5-16-fold) in the binding of [125I]anti-DflH to the iris. such binding was not 120
80
\
40
C
Fig. 2. Glyoxylic acid-induced catecholamine fluorescence m the iris following injection of 5 Izl DMSO ( A h A23187 (6 nmol in 5~1DMSO) (B), or A23187 (28 nmol in 5/~1DMSO) (C) into the anterior eye chamber, 18 h prior to sacrifice. Irides were stretched and incubated with glyoxylic acid as described in Methods.
gree of inhibition (60%). If. however, the [taSI]antiDflH was neutralized by prior incubation with pure DflH, then no transport occurred in the presence of A23187 (Fig. 1). Thus while all transport of [t25I]anti-DflH was specific, approximately 40% of such
TIME
2 4 6 8 AFTER INJECTION (DAYS)
Fig. 3. Time course for the accumulation of [125i]anti-DflH in the SCG following injection of anti-DflH and 5 gl of A23187 dissolved in DMSO into the ipsilaieral eye chamber 1, 3 and 7 days previously. Results from groups of 3 - 9 animals are expressed as a percentage ± S.E.M. of the [tzsI]-accumulation in animals treated with 5 #1 DMSO i,o., 24 h prior to sacrifice (680 cpm). Accumulation in the ipsilateral ( 0 ) and contralateral (©) ganglion following inrraoeular injection of 5 /~1 DMSO plus anti-DflH: accumulation in the ganglia fotlowmg rejection of anti-D/SH plus A23187 into the ipsilateral eye ( A ) and A23187 alone into the contralateral eye ( A ) o f the same animals.
65 specific. Binding of non-immune [125I]IgG to the iris was also increased by 28 nmol A23187 (3.7-fold). Even if, following injection of A23187, part of the increased binding of anti-D/3H to the iris was due to absorption to DflH trapped on the neuronal surface, at no stage up to 7 days later did this result in a corresponding increase in the amount transported to the SCG (Fig. 3). Immunofluorescence staining for D/3H in the nerve terminals in the eye was not affected by A23187 (28 nmol) (Fig. 4), indicating that nerve terminals remained intact in spite of the loss of noradrenaline. Three days after the injection of 28 nmol A23187, the nerve terminals in the irides still stained normally for D/3H. The absence of nerve degeneration following A23187 was confirmed by estimating the iris content of tyrosine hydroxylase. While the injection of vehicle ( D M S O ) resulted in a 38% reduction of tyrosine hydroxylase activity, low or high doses of A23187 dissolved in the vehicle did not result in a further loss (Table I). The functional activity of sympathetic nerve terminals, as measured by [3H]noradrenaline uptake, was also reduced by D M S O treatment (33%). The addition of 6 nmol A23187 did not reduce this activity further, but with higher doses, there was a major loss of activity (62% less than vehicle control) (Table II). This loss of function occurred gradually, with a maximum effect by 12 h.
A
-05 mm B
Fig. 4. Immunofluorescence staining for D/3H in the irides following injection of 5 #1 DMSO (A) or A23187 (28 nmol in 5 ul DMSO) (B) into the anterior eye chamber 24 h prior to sacrifice. No staining occurred if exogenous anti-D/3H or Triton X-100 was omitted from the staining reagents.
DISCUSSION While anterograde axonal transport requires calciuml, 6,13, its mechanism of action has not been deTABLE I Effect of A23187 on levels of tyrosine hydroxylase in guinea pig irides
5 JA DMSO, or A23187 dissolved in DMSO was injected into the anterior eye chamber of guinea pigs. 18 h later the irides were dissected out and assayed for tyrosine hydroxylase as described in Methods. Results are expressed as means + S.E.M. for the numbers of determinations in parentheses. Treatment in vivo
Tyrosine hydroxylase activi~ (pmol.h 1.iris-O
DMSO A23187, 6 nmol A23187, 28 nmol
20.4 + 2.7 (13) 12.6 -+ 3.2 (8) 11.8 + 2.2 (10) 11.3 + 1.3 (9)
fined. A role in the initiation of transport in the cell perikayron at the level of the Golgi apparatus or beyond 6, as well as in the transport process itself 1,13, has been suggested. Excess calcium, however, inhibits anterograde transport, and such inhibition becomes irreversible at high concentrations~. The inhibition caused by the calcium ionophores A23187 and X-537A is calcium-dependent and results in an increased axonal calcium content 2,~. As shown in these results, the retrograde transport of anti-DflH was also inhibited by A23187. Inhibition occurred at doses of A23187 which caused no change in other catecholamine parameters (noradrenaline uptake and tyrosine hydroxylase levels) and hence was not due to a general toxic effect of A23187. It thus seems likely that anterograde and retrograde transport are similarly affected by high intracellular
66 TABLE II Effect ofA23187 on noradrenaline uptake into,guinea pig irides
5/~1 DMSO, or A23187 dissolved in DMSO was injected into the anterior eye chamber of guinea pigs. At various times subsequently the irides were dissected out and the uptake of noradrenaline (NA) measured as described in Methods. Results are expressed as means + S.E.M. for the number of determinations in parentheses. Treatment in vivo
Time of sacrifice
NA uptake (pmol. h -1, iris-l)
Control Control Control DMSO A23187, 6 nmol A23187, 30 nmol A23187, 30 nmol A23187, 30 nmol A23187, 30 nmol A23187, 30 nmol
-
1.9 + 0.3 (4) ~ 3.6 + 0.5 (3)b 30.9 + 2.6 (14) 20.8 + 4.0 (5)** 21.0 + 2.2 (4) 18.5 + t.8 (4) 14.2 + 2.9 (7) 11.0 + 1.6 (4) 7,9 + 0.7 (4)* 9.2 + 1.0 (7)*
18 h 18 h 1h 2h 6h 12 h 18 h
Incubated at 0 °C. b Incubated with 10-4 M desipramine. * P < 0.025, compared with vehicle injected, 2-tailed Student's t-test. ** P < 0.05, compared with non-injected animals, 2-tailed Student's t-test.
concentrations of calcium induced by A23t87. The mechanism for such inhibition m a y welt be via a calcium-dependent depolymerization of microtubulesl3,17,18, which are generally believed to be involved in axonal t r a n s p o r t ~9. In addition to effects on axonal transport, A23187 and o t h e r calcium i o n o p h o r e s also cause an increased rate of exocytosis 20. The loss of catecholamine fluorescence in the iris following injection of A23187 is consistent with a high rate of exocytosis~ Since drugs thought to increase exocytosis result in a higher amount of anti-DflH t r a n s p o r t e d to the ganglion10-12, A23187 might have also been expected to increase r e t r o g r a d e t r a n s p o r t by this mechanism. The results
REFERENCES 1 Chan, S. Y., Ochs, S. and Worth, R. M., The requirement for calcium ions and the effect of other ions on axoplasmic transport in mammalian nerve, J. Physiol. (London), 301 (1980) 477-504. 2 Esquerro, E., Garcia, A. G. and Sanchez-Garcia, P., The effects of the calcium ionophore, A23187, on the axoplas-
presented here indicate that any increase m the uptake of anti-DflH does not e v o k e a subsequent rise in the amount t r a n s p o r t e d to the ganglion. A further possibility was that the action of A23187 on uptake of anti-DflH would lessen the o b s e r v e d degree of inhibition of r e t r o g r a d e transport. H o w e v e r , 28 nmol A23187 also caused a similar percentage reduction in the r e t r o g r a d e transport of nerve growth factor from the iris (unpublished observations). Results of o t h e r investigations support the conclusion that axonal transport is at least as sensitive as exocytosis to changes in calcium fluxes induced by A23187. Thus inhibition of a n t e r o g r a d e transport of DflH in the cat hypogastric nerve occurs at lower concentrations of A23187 (10-33/~M) 2 than those required for the stimulation of the release of n o r a d r e n aline and DflH from nerve endings in rat atria (30-300 p M ) 2°. In contrast to the selective effects at low doses, higher amounts of A23187 (28 n m o l ) a p p e a r e d to more generally affect the functioning of the neurons, as shown by losses in the capacity of the iris to take up noradrenaline. Such losses m a y be due to excess calcium, which has been suggested to be toxic to cells, producing cellular death3,7, t6. In these experiments, however, the reduction in the capacity of the terminals to take up n o r a d r e n a l i n e does not a p p e a r to be due to a loss of nerve terminals, as shown by the absence of an affect on tyrosine hydroxylase levels and by a normal p a t t e r n of DflH immunofluorescence (for up to 3 days p o s t - t r e a t m e n t following A23187). ACKNOWLEDGEMENTS This research was s u p p o r t e d by a grant from the Medical Distribution C o m m i t t e e of New Z e a l a n d . The gift of antibodies to dopamine-fl-hydroxylase from Dr, R. A. Rush is gratefully a c k n o w l e d g e d
mic transport of dopamine fl-hydroxylase. Br. J Pharmacol., 70 (1980) 375-381. 3 Farber, L. F., The role of calcium in cell death. Life Sci. 29 (1981) 1289-1295. 4 Fillenz, M., Gagnon, C., Stoeckel. K. and Thoenen, H.. Selective uptake and retrograde axonal transport of dopamine-fl-hydroxylase antibodies in peripheral adrenergic neurons, Brain Research, 114 (1976"1293-303.
67
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