Neuronal differentiation of Ca2+ channel by nerve growth factor

BrainResearch, 341(1985)381-384 Elsevier

381

BRE 20997

Neuronal differentiation of Ca a+ channel by nerve growth factor MASAMI TAKAHASHI, HIROKO TSUKUI and HIROSHI HATANAKA

Departmentof Neuroscience, Mitsubishi-Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194 (Japan) (Accepted April 2nd, 1985)

Key words: adrenal chromaffin cell - -

C a 2+

channel - - neuronal differentiation - - nerve growth factor - - dihydropyridine

The inhibitory effect of nicardipine, a potent Ca 2+ channel blocker in muscular cells, on the Ca2+ channel of clonal rat pheochromocytoma cells (PC12h) and cultured rat adrenal medullary cells was studied during the neuronal differentiation mediated by nerve growth factor (NGF). Nicardipine at nM-order concentrations suppressed the high-K+-evoked, CaZ+-dependent release of preloaded [3H]norepinephrine from PC12h cells and adrenal medullary cells, whereas it scarcely inhibited the release from the cultered rat brainstem cells. The inhibitory actions of nicardipine on both PC12h and newborn rat adrenal medullary cells were significantly decreased after these cells were cultured in the presence of NGF. These results suggest that the changes in Ca 2+ channel are accompanied by the neuronal differentiation mediated by NGF.

Nerve growth factor ( N G F ) is a protein essential for the d e v e l o p m e n t and m a i n t e n a n c e of the functions of p e r i p h e r a l sympathetic and sensory neurons3,10,1L The PC12 cell line was established as an N G F - r e s p o n s i v e cell from rat p h e o c h r o m o c y t o m a of adrenal medullae origin 2. W h e n e x p o s e d to N G F , PC12 cells take on a n u m b e r of differentiated phenotypic properties of sympathetic neurons 3,1s, including neurite outgrowth, enzyme induction and increases of transmitter r e c e p t o r numbers 2~7,1t. F u r t h e r m o r e , the n u m b e r of voltage-sensitive Na + channels increased by 15 to 20-fold p e r PC12 cell and the electrical excitability was r e m a r k a b l y i m p r o v e d by the N G F - t r e a t m e n t 14. In the present communication, we compare the differential p r o p e r t i e s of voltage-sensitive Ca 2+ channels, being one of the key factors in the control of the intracellular Ca 2+ level 4. W e especially focused on the differential effects of dihydropyridine derivatives, p o t e n t organic Ca 2+ channel blockers on muscular cells 20. That is, they inhibited the function of high-K+-sensitive Ca 2+ channels of P C 1 2 c e l l s 16.19, whereas they showed scarcely any suppression in neuronal p r e p a r a t i o n s such as rat brain synaptosomes and slices 12, suggesting that the high-K+-sensitive Ca 2+ channels of PC12 cells are

distinct from those of neuronal cells. Now, we found that during the neuronal differentiation m e d i a t e d by N G F , the p r o p e r t y of Ca 2+ channels of PC12 cells changed to a neuronal type less sensitive to dihydropyridine. These events also a p p e a r e d to occur in cultured newborn rat adrenal medullary cells. PC12h cells (a subclone of PC12 cells) were cultured as described previously 5. Newborn (7- to 10day-old) and adult rat adrenal medulla and fetal (16 days) brainstem were dissected from Wistar ST rats and were cultured as described in the legend to Fig. 1. We estimated Ca 2+ channel functions by measuring the high-K+-stimulated release of p r e l o a d e d [3H]-norepinephrine ([3H]NE). This release was abolished in the absence of external Ca 2+ and suppressed by the universal Ca 2t channel blocker, Co 2+, meaning that the releases are m e d i a t e d Ca 2+ influxes through Ca 2+ channels 4. As we r e p o r t e d previously 16, nicardipine, one of the dihydropyridine derivatives, suppressed the release of [3H]NE from PC12h cells in a d o s e - d e p e n d e n t m a n n e r and the I C ~ value was less than 10-8 M and nearly full suppression was achieved by 10 -6 M of nicardipine (Fig. 1), open circles). Nicardipine also suppressed the release of [3H]NE from adrenal medullary cells of adult rat as

Correspondence: M. Takahashi, Laboratory of Neurochemistry, Department of Neuroscience. Mitsubishi-Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194, Japan. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

382

•

6

ff'j

g o --It

[-,

I0 8

~

I0

g-, I0

(Nieardipine) (M) Fig. 1. Inhibitory effects of nicardipine on the high-K+-stimulated [3H]NE release from NGF-untreated PC12h cells (O), NGF-treated PC12h cells (O), adult rat adrenal medullary cells (f-l), NGF-treated newborn rat adrenal medullary cells (11) and rat brainstem cells ( A ) in culture. PC12h cells were cultured in Dulbecco's modified Eagle's medium and adrenal medullary and brainstem cells were in DF medium. DF medium consisted of a 1:1 mixture of Dulbecco's modified Eagle's and Ham's F12 media supplemented with 15 mM HEPES buffer, 30 nM selenium, 50 IU/ml penicillin and 200 ug/ml streptomycin. Each culture medium contained 5% (v/v) precolostrum newborn calf serum (Mitsubishi Chemical Industry, Tokyo) and 5% (v/v) heat-inactivated horse serum (Gibco Laboratories). NGF-untreated PC12h cells were plated on 35mm polylysine-coated culture dishes (Falcon) at a density ol 1.3 x 105 cells/cm2 and cultured for two days before [3H]NE release assay. For NGF treatment, PC12h cells were plated on collagen-coated 35-mm culture dishes at a density of 3.8 x 105 cells/cm 2 and cultured for 25 days in the presence of 50 ng/ml ot NGF. NGF (2.5S form) was prepared from male mouse salivary gland by the method of Suda et al. ~5. In order to kill the dividing cells, 1 mM of cytosine arabinonucleoside was added two days after plating. The medium was changed every other day and cytosine arabinonucleoside was supplemented every other medium change. Adrenal medulla dissected from adult rat was digested with 0.4 mg/ml collagenase (Wako Pure Chemical Industries, Tokyo) and 33 ~g/ml deoxyribonuclease 1 (Sigma Chemical Co.) in Hank's basal salt solution for 90 min at 37 °C. After dissociation into single cells by gentle pipetting, the ceils were purified by Percoll density gradient, washed two times with culture medium and plated on 35-mm collagen-coated culture dishes at a density of 0.9 x 105 ceUs/cm2. The release was assayed 6 days after plating. Newborn rat adrenal medullary cells were obtained from 7- to 10-day-old rats~L After dissection, medulla was digested with 1 mg/ml collagenase and 20 ug/ml deoxyribonuclease I in Hank's basal salt solution for 60 min at 37 °C and the dissociated cells were plated on collagencoated multi-16-mm well plates (Nunc) at a density of 105 cells/cm 2 and cultured for 17 days in the presence or absence of 5(1 ng/ml NGF. Rat brainstem was dissected from 1f-day fetal

rats and digested with 0.25% trypsin (Difco Laboratories) anti 20 ~g/ml deoxyribonuclease I in CMF-Hank's basal salt sotu. tion for 15 min at 37 °C. Trypsinization was terminated by adding horse serum and culture medium and the tissue was dissociated into single cells by gentle pipetting. After filtration through two-layered lens-paper, the cells were plated on potylysine-coated 35-mm culture dishes at a density of 3.5 × Hr' cells/cm 2. [3H]NErelease was assayed as described before 17 in the assay medium containing 130 mM NaCI, 5.4 mM KCI, 1.8 mM CaCI2, [1.8 mM MgSO 4, 5.5 mM glucose, 1.2 mM ascorbic acid and 50 mM HEPES buffer, pH 7.3 at 37 °C. BOvine serum albumin (1 mg/ml) was supplemented in the experiment of newborn adrenal medullary cells. 46 mM KCI was supplemented in the high-K + solution. Nicardipine and Co 2* were treated 5 min before high-K ~ treatment. The amount ol [3H]NE released for 1 min was expressed as percent of remaining radioactivity in the cells and the high-K ~-stimulated releasc was calculated by subtracting the basal release in 5.4 mM KCI from total release in 50.4 mM KCI. All values in this figure were expressed as relative to the release in the absence of nicardipine. The high-K+-stimulated releases in the absence of nicardipine were 2.32 + 0.04% for NGF-untrcated PC12h cells, 0.69 + 0.03% for NGF-treated PC12h cells, 4.92 .+_ 0.14% for adult adrenal medullary cells, 3.17 ~ 0.02% lbr NGF-treated newborn adrenal medullary cells and 24,2 ± 3.3% for brainstem cells.

e f f e c t i v e l y as t h a t f r o m P C 1 2 h cells, a n d full i n h i b i t i o n was o b s e r v e d at 10-6 M (Fig. 1, o p e n s q u a r e s ) . On the other hand, nicardipine scarcely inhibited the h i g h - K + - s t i m u l a t e d r e l e a s e of [ 3 H ] N E f r o m b r a i n s t e m cells in c u l t u r e a n d o n l y 7 . 9 % of i n h i b i t i o n was o b s e r v e d b y 10 -6 M o f n i c a r d i p i n e (Fig. 1, c l o s e d triangles). These results further confirm that the pharm a c o l o g i c a l p r o p e r t i e s of t h e h i g h - K + - s e n s i t i v e C a 2+ c h a n n e l of P C 1 2 h cells r e s e m b l e n o t t h o s e o f n e u r o n s b u t t h o s e of a d r e n a l m e d u l l a r y cells. The pharmacological properties of high-K+-sensi tive C a 2÷ c h a n n e l s o f P C 1 2 h cells w e r e f o u n d to dramatically change during the NGF-mediated neuronal d i f f e r e n t i a t i o n . N i c a r d i p i n e at .3 x 10-8 M , w h i c h inh i b i t e d t h e h i g h - K + - s t i m u l a t e d [ 3 H ] N E r e l e a s e by 80% from NGF-untreated

P C 1 2 h cells, s u p p r e s s e d

t h e r e l e a s e o n l y by 3 7 % f r o m N G F - t r e a t e d cells (Fig. 1, c o s e d circles). F u r t h e r m o r e ,

this p a r t i a l s u p p r e s -

sion level was n o t e n h a n c e d w i t h f u r t h e r i n c r e a s e in t h e n i c a r d i p i n e c o n c e n t r a t i o n a n d a s i g n i f i c a n t release was still o b s e r v e d in t h e p r e s e n c e o f 10-6 M nic a r d i p i n e . A s s h o w n in Fig. 2, a u n i v e r s a l C a z+ c h a n nel b l o c k e r , C o ~+, at 5 m M a b o l i s h e d t h e s e r e l e a s e s from both NGF-treated

and NGF-untreated

cells,

w h e r e a s n i c a r d i p i n e at 10 (' M p a r t i a l l y s u p p r e s s e d the release from NGF-treated

cells. T h e s e r e s u l t s

s u g g e s t t h a t a n e w t y p e of C a 2 + c h a n n e l , r e s e m b l i n g

383 NGF- treated

NGF-untreated •

IOO -

8

£: uJ 50Z

i "I-

._~ "~ ._~

0-

-I-

+Co~+ + Nicar

+Co2+ +Nicat

(5raM) (19M)

(5raM) (IJJM)

Fig. 2. Inhibition of high-K+-stimulated [3H]NE release from NGF-treated (for 23 days) and untreated PC12h ceils by Co 2+ (5 mM) and nicardipine (10 6 M). The [3H]NE releases were assayed as described in the legend to Fig. 1 and were expressed as relative to the release in the absence of inhibitors. High-K +stimulated [3H]NE releases in the absence of inhibitors were 1.90 + 0.07% and 0,84 + 0.03% for NGF-untreated and NGFtreated ceils, respectively.

I00 .0 o

z j

"5

'~o 5O 1O

.c ¢-

I0 Day

of

20 NGF

:5 Treatment

Fig. 3. The decrease in the inhibitory effect of nicardipine on the high-K* stimulated [3H]NE release from PC12h cells (O) and newborn rat adrenal medullary cells (11) during the cultivation in the presence of NGF (50 ng/ml). High-K+-stimulated [3H]NE release in the presence or absence of 10_6 M nicardipine was measured as described in the legend to Fig. 1 at the indicated day after NGF treatment and the percent inhibition by l0 6 M nicardipine was calculated and plotted against the NGF-treated days. Each value represents the means of 2-4 experiments.

that of neuronal cells, appeared in the course of neuronal differentiation mediated by NGF. Adrenal medullary cells of newborn rat have been reported to undergo a neuronal differentiation when cultured in the presence of N G F 21. The NGF-mediated change of the pharmacological properties of high-K+-sensi tive Ca 2÷ channels also occurred in these cells. Nicardipine at 10-6 M suppressed high-K+-stimulated [3H]NE release by 89 + 4% in NGF-untreated cells, whereas it suppressed only by 42.5 _+ 0.5% in NGFtreated cells (Fig. 1, closed squares). In Fig. 3, the extents of suppression by 10-6 M of nicardipine on the high-K+-stimulated [3H]NE release from PC12h cells and newborn rat adrenal medullary cells were plotted against the number of days of NGF treatment. The inhibitory action of nicardipine gradually decreased in both cells during the time of NGF treatment up to about one month. Sympathetic neurons and adrenal medullary cells share a common neural crest origin and the differentiation of these cells is known to be controlled by various factors including N G P .13. The present study suggests that pharmacological types of Ca 2+ channel are also regulated by NGF. There have been several other reports on the developmental changes in ion channel properties. Uninnervated and denervated mammalian skeletal muscles have only tetrodotoxin-insensitive Na + channels and after innervation Na + channels are changed to the tetrodotoxin-sensitive type s. The slow Ca2+/Na + channels of embryonic chick hearts reduced their sensitivity to D600 during development 1. On the other hand, the Ca 2+ channel of ascidian egg is changed in its ion selectivity and inactivation properties during the development after fertilization 6. It is the problem to be solved in future what kinds of changes in the structure and/or the microenvironment (for example, membrane lipid composition) of ion channel molecules are responsible for these phenomena. Previously, we reported that the high-K+-stimulated [3H]GABA release from cultured 6-day embryonic chick neural retina cells was partially suppressed by nicardipine 16, suggesting that the developmental changes in Ca 2+ channel property might occur in these cells. It is very interesting to know what kinds of factors regulate this development since the neurogenesis of the central nervous system is believed to be controlled by factor(s) other than NGF.

384 1 Galper, J. B. and Catterall, W. A., Developmental changes in the sensitivity of embryonic heart cells to tetrodotoxin and D600, Develop. Biol., 65 11978) 216-227. 2 Greene. L. A. and Tischler, A. S., Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor, Proc. nat. Acad. Sci. U.S.A., 73 (1976) 2424-2428. 3 Greene, L. A. and Shooter. E. M., The nerve growth factor: biochemistry, synthesis, and mechanism of action, Ann. Rev. Neurosci., 3 (1980) 353-402. 4 Hagiwara, S. and Byerly, L., Calcium channel, Ann. Rev. Neurosci., 4 11981) 69-125, 5 Hatanaka, H., Nerve growth factor-mediated stimulation of tyrosine hydroxylase activity in a clonal rat pheochromocytoma cell line, Brain Research, 222 (1981) 225-233. 6 Hirano, T. and Takahashi, K., Comparison of properties of calcium channels between the differentiated 1-cell embryo and the egg cell of ascidians, J. Physiol. (Lond.). 347 (1984) 327-344. 7 Inoue, N. and Hatanaka, H., Nerve growth factor induces specific enkephalin binding sites in a nerve cell line, J. Biol. Chem., 257 (t982) 9238-9241. 8 Lawrence, J. C. and Catterall, W. A., Tetrodotoxin-insensitive sodium channels. Ion flux studies of neurotoxin action in a clonal rat muscle cell line, J. Biol. Chem., 256 (1981) 6213-6222. 9 LeDouarin, N. M., The ontogeny of the neural crest in avian embryo chimaeras, Nature (Lond.), 286 (1980) 663-669. 10 Levi-Montalcini, R. and Angeletti, P. U., Nerve growth factor, Physiol. Rev., 48 (1968) 534-569. 11 Mitsuka, M. and Hatanaka, H., Increase of carbamylcholine-induced 22Na+ influx into pheochromocytoma PC12h cells by nerve growth factor, Develop. Brain Res., 12 (1984) 255-260.

12 Ogura, A. and Takahashi, M.. Differential elicc~ ot a dih~ dropyridine derivative to Ca ~'~ entry pathways in ncuronai preparations, Brain Research, 3111 (1984) 323 .330. 13 Patterson, P. H., Environmental determination of autonomic neurotransmitter function~, 4nn Re~. Veurow,.. 1 11978) 1-17. 14 Rudy, B., Kirschenbaum, B, and Greene. [. A., .Nerve growth factor-induced increase in ,saxitoxm binding to rat PC12 pheochromocytoma cells. J. Neuros~,/. 2 11982) 14115-1411. 15 Suda, K., Barde, Y.-A. and l h o e n e n , H.. Nerve growth factor in mouse and rat serum: correlation between bioassy and radioimmunoassay determinations, Proc. nac Acad. Sci. U.S.A., 75 11978) 4042-4040. 16 Takahashi, M. and Ogura, A., Dihydropyridines as potent calcium channel blockers in neuronal cells, FEBS Lett., 152 (1983) 191-194. 17 Takahashi, M., Tatsumi, M., Ohizumi, Y. and Yasumoto, T., Ca-'* channel activating function of maitotoxin, the most potent marine toxin known, in clonal rat pheochromocytoma cells, J. biol. Chem., 258 (1983) 10944-11t949. 18 Thoenen, H. and Barde, Y.-A., Physiology of nerve growth factor, Physiol. Rev., 60 11980) 1284-t335. 19 Toll, L. Calcium antagonists. High-affinity binding and inhibition of calcium transport in clonal cell line. J. biol. Chem.. 257 11982) 13189-13192. 20 Triggle, D. J., Calcium antagonists: basic chemical and pharmacological aspects. In G. B. Weiss (Ed.), New Perspectives on Calcium Antagonists. American Physiological Society, Bethesda, 1981, pp. 1-18. 21 Unsicker, K., Krisch, B., Otten, U. and Thoenen, H., Nerve growth factor-induced outgrowth from isolated rat adrenal chromaffin cells: impairment by glucocortieoids, Proc. nat. Acad. Sci. U.S.A.. 75 (1976) 3498-3502