The S-100 protein causes an increase of intracellular calcium and death of PC12 cells

0306-4522/93$6.00+ 0.00 Pergamon PressLtd 0 1993IBRO

Neuroscience Vol. 53, No. 4, pp. 919-925, 1993 Printed in Great Britain

THE S-100 PROTEIN CAUSES AN INCREASE OF INTRACELLULAR CALCIUM AND DEATH OF PC12 CELLS G.

FANo’,*t M. A. MARIGGIO’,* P. ANGELELLA,*I. NICOLETTI,$A. ANTONICA,* S. FULLE* and P. CALISSAN@

*Istituto di Biologia Cellulare, Universita di Perugia, via Elcc di sotto, 06100 Pert&a, Italy $Istituo di Clinica Medica I, Universitl di Perugia, Policlinico Monteluce, 06100 Perugia, Italy §Istituto di Neurobiologia C.N.R., via K. Marx 15, 00156 Roma, Italy

Abstract-The S-100 protein-PC12 cell interaction has been studied as a model system of the possible physiological role played by S-100 protein in the nervous system. The data reported demonstrate that S-100 exerts a cytotoxic action which eventually leads to PC12 cell death, regardless of the cell cycle phase. The effect is specific for the S-100 isoforms, which are made up of two identical subunits and is abolished by a monoclonal antibody directed against the same isoforms. Other isoforms and/or calcium-binding proteins, such as troponin or calmodulin, do not induce the same effects. The action of S-100 on cell viability is not detectable in other ccl1 lines of different embryological origin, such as 3T3, L1210, GH3. S-100 causes a rapid and considerable increase (two- to three-fold) of intracellular Ca2+ concentration in PC12 cells accompanied by cytostatic and cytotoxic action. It is postulated that this action also occurs in uiuo, as part of the physiological action of this protein.

S-100 is a dimeric protein (a, B) which was first purified from bovine brain by Moore.” It has a molecular weight of 21,000, is characterized by its peculiar interaction with Ca*+, and has two highaffinity and several low-affinity binding sites.5 S-100 is present in three isoforms: tlc~,(S-lOOa,,>; /I/?, (S-1OOb); crZ?,(S-lOOa), and in tissues of different origin. ” In the nervous system, S-100 is much more abundant than in other organs or tissuesz8 where it is present as a mixture of S-100a (20%) and S-1OOb (SO%), S-100 is present in, and secretred from, both normal and transformed glial cell~.~i*~~~~~ Furthermore, it is detectable in cerebrospinal fluid and in blood, and is found in abnormally high levels in

tTo whom correspondence should be addressed. Abbreuiutions: C6, glioma cell line; DMSO, dimethylsulfoxide; DTNB, dithio-bis-nitrobenzoic acid; DTT, 1,4-dithio-o,L-threitol; EGTA, ethylenediaminetetraacetic acid; Fura 2/AM, I-[2-(5-carboxyoxazol2-y])6 - aminobenzofuran - 5 - oxy ]- 2 - (2’ - amino - 5’ -methylphenoxy)-ethane-N,N,N’,N’-tetracetic acid, pentaacetoxymethyl ester; GH3, clone line from rat pituitary tumor; GICAN, cell line from human neuroblastoma; HEPES, N-[2-hydroxyetyl)piperazine-N’(2-ethanesulfonic acid); L1210, cell line from murine leukemia; mAb, monoclonal antibody; MIT, 3(4,5-dimethylthiaziol-21-yl)-2,5-diphenyltetrazolium bromide; NGF, nerve growth factor; PBS, phosphate-buffered saline; PC12, cell line from rat pheochromocytoma; S-lOOa, calcium binding protein isoform; S-lOOb, calcium binding protein isoform; S-IOOab, mixture of%1OOa and S-l&%; S-lOOc1,S-IOOab cross-linked; SDS. sodium dodecvl sulfate: 3T3-NIH. clone of murine fibroblasts; Tris, tris(hydroxy: methyl)aminomethane.

patients with Down’s syndrome or Alzheimer’s disease.26,32It has been shown that the addition of beta S-100 subunit to the growth medium stimulates differentiation in neurons and neuroblastoma cell lines.22,35More recently, Selinfreund et al. reported that beta S-100 subunit induces proliferation of glioma cell lines (C6) and primary astrocytes, while it does not cause the same effect on neuroblastoma cell lines.30 In uitro, S- 100 protein has been shown to modulate the assembly and disassembly of microtubules,‘J’ the activation of adenylate cyclase of skeletal muscle membranes’2~‘3 and the increase of cation fluxes in both liposomes4ss and natural membranes.14 On the basis of these findings, it has been proposed that the beta S-100 subunit plays an important role in CNS development by synchronously stimulating the differentiation of neurons and the proliferation of astrocytes.‘O Mitotic arrest is a crucial prerequisite for the subsequent expression of a neuronal phenotype in all cells committed to differentiate into neurons. This is also true for the clonal cell line PC12 which, in the presence of nerve growth factor (NGF),‘,” undergoes mitotic arrest and differentiates into sympathetic-like cells. This complex change of cell’ phenotype is accompanied by an early and late expression of several mRNAs.23 Among these are two mRNAs coding for two proteins of 95 and 101 amino acids which share several sequence homologies with the S-100 protein and other Ca*+ binding proteins.24 Expression of these genes could be causally related to two major physiological effects induced by NGF in PC12 cells: mitotic arrest and neurite outgrowth.

919

We therefore tested the action of exogenously supplied S-l 00 on these ceils and found that S-100 exerts a cell-specific cytotoxic action. EXPERIMENTAL PROCEDURES

Materials

MTT (3-[4,5-dimethylthiaziol-2yl)-?,S diphenyl tetrazolium bromide), caimodulin (16,723 mol. wt from bovine brain) and Fura Z/AM {l-[2-(S~arboxyox~ol-2-yl)-6aminobenzofuran-S-oxy]-2-(2’-amino-S’-methyl-phenoxy)ethane-N,N,N’N’-tetraacetic acid. penta-acetoxymethyl ester]} were obtained from Calbiochem (Canada); troponin C from bovine muscle. S- lOOaand S- 1OObfrom bovine brain from SIGMA (St Louis, U.S.A.); monoclonal antibody anti-S-100 (mouse) was from PEL-FREEZ (Wisconsin, U.S.A.) and 45CaZ+(IO--4OmCi~mg Ca’+f from Amersham (U.K.). Purification

NGF was prepared by the Bocchini and Angeletti method.’ S-lOOab, a mixture of the two isoforms a and b, was isolated from bovine brain with the procedure described by Moore.27 Its state of purity was checked either under denaturing 140mM l,4-dithio-D,L-threitol (DTT), 0.05% sodium dodecylsulfate (SDS), 12.5% acrylamide] or nondenaturing (10% acrylamide) gel electrophoresis.6 Subunit cross-linking of S-100 (S-1OOcl)was performed according to the procedure described by Calissano et al.,’ which is based on disulfide bond formation of S-1OOabcatalysed by dithiobis-nitro~nzo~c acid (DTNB). With this procedure the disulfide bridge between the two S-100 subunits stabilizes the quaternary structure of S-100 without altering other physicochemical properties of the dimer. The tritiumlabeled form of S-1OOab(specific activity 4.8 Ci;mmol) was prepared as previously described’ under nondenaturing conditions. The method is based on the reductive methylation of lysine residues and does not alter the physicochemchanges) ical prop&ties (Ca r+ binding and confo~ati~nal nor the effect on Ca*+ fluxes in PC12 cells. Cell cultures PC12 (clone Al), a clonal cell line derived from a rat pheochromocytoma, were cultured as described by Greene and Tischler” in RPM1 1640 supplemented with 10% horse serum and 5% fetal bovine serum. All the experiments, if not otherwise indicated, were carried out on PC12 cells previously synchronized by serum starvation and then resuspended in medium containing 10% horse serum and 5% fetal bovine serum, at time 0. GH3 cells, a clone line originating from a rat pituitary tumor, were grown in Ham’s F-10 medium supplemented with 15% horse serum and 2.5% fetal calf serum. Ll210 cells, derived from a murine leukemia, were cultured in RPM1 1640 supplemented with 10% fetal bovine serum. 3T3-NIH, a clone of murine fibroblasts from mouse embryos. were cultured in Dulbecco’s modification of Eagle’s medium supplemented with 10% fetal bovine serum. GICAN cells, stabilized cell lines deriving from a human neuroblastoma, were cultured in PRMI 1640 supplemented with 10% fetal bovine serum. All experiments on GH3, L1210, 3T3-NIH and GICAN cells were carried out on confluent cultures. Growth cunv

Cell growth was tested by a calorimetric assay based on the use of MTT, an insoluble tetrazolic salt whose ring is hydrolysed by the dehydrogenases present in the mitochondria of living cell~.~ The assay was performed by plating 10,000 cells/well in a fmal volume of 200 &‘weif of 96-well plates (Falcon). At timed intervals, the incu~t~on was stopped by adding 20 pl of an MTT solution [S mg/ml in

phosphate-buffered saline (PBS)] to each well li~ld then incubating at 37’C for 3 h. After incubation. the supernatant was removed and 200 ~1 dim~thylsulfoxide tl?MSO) was added to each well. The plate was shaken for i nun to dissolve the crystals formed by the action of cciluiar dehydrogenase and incubated for 30 min at 37 C. Finally, the plate was read at 540 nm on a Titertek Multiscan Microelisa Reader (Flow Laboratories. tlrvinc 1Jtah.

U.S.A.I. The percentage of cells in the various phases of the cell cycle was determined by incubating controls and S-lOOtreated cells with a fluorochromic solution containing 0.05 mg/ml propidium iodide, 0.1% sodium citrate and 0.1% Triton X-100, for 24 h at 4 C. The suspension was read in a cytofluorimeter (FACSCAN Becton Dickinson. Mountain View, CA) connected to a Hewlett Packard computer (HP 9000 model 310).15Cell viability was assayed using a solution of PBS containing Song/ml propidium iodide. After 1 h of incubation, the suspension was read in the cytofluorimeter.rO

The concentration of intracellular Ca?+ was measured with Fura 2/AM (a calcium fluorescent ester chelator and indicator).rO PC12 cells were washed and resuspended in a standard medium containing: 125 mM NaCl, 5 mM KCI. 1 mM MgSO,, 1mM KHrPO,, S.SmM glucose, 1 mM CaCl,. 20 mM HEPES (final pH 7.4). Fura Z/AM (3 PM) and sulphinpyrazone (25OnM) were added to the cell suspension which was subsequently incubated at 37-C for 30min. The cells were then washed, resuspended in the standard medium containing 250 pM sulphinpyr~one and transferred to thermostatically controlled cuvettes equipped with a magnetic stirrer. Fluorescence measurements were made on a Perkin-Elmer LS-SB (A excitation; 340 nm, i emission: 486nm. Inner Cal+ concentration [Ca’+& was calculated according to the general formula:

where K,, is the dissociation constant for Ca’+ binding (224 nM) for Fura-2; F is the fluorescence of the intracellular indicator; F,,, isthe fluorescence after lysis of the cells with 0.05% Triton X-100 in the oresence of 6mM EGTA and 40 m,M Tris (final pH 8.5); Fix is the fluorescence of the lysed cells after addition of 4 mM CaCl,. In prelimina~ experiments, we tested the possibiljty that the fluorescence could vary according to the method employed (simple or dual wavelength recording). In both cases the performed tests showed the same qualitative and quantitative time-course (data not shown).

The method adopted for me~uring ion fluxes was essentially that previously described.z5 In brief, 0.5 x IO’ cells, plated in 24well plates in a final volume of 600 pl of medium, were cultured in RPM1 medium (containing 0.4 x lo-’ M Ca2+ and supplemented with 10% horse serum and 5% fetal bovine serum) in presence of 0.6nmoliml “‘Ca2+. After 1 h at 37C. 2uM S-IOOab was added to the medium. After one, two and three days, the cells were detached, filtered on a Millipo~ apparatus (0.45 pm filters) and washed three times with PBS solution containing 5 mM EGTA. The solubilized filters were read in a /I-counter. Binding assay

To measure the binding of [‘I-&+100 to PC12 cells, 5.0 x lo6 cells/ml were incubated in medium plus sera containing different con~trations (lo-300 nM) of tritiated S-1OOab. After 10 min of incubation at 37°C. the cells

921

The S-100 protein in PC12 cells 801

50

40

Fig. I. Effect of different concentrations of S-IOOab (0.05~2.OpM) on growth of PC12 cells synchronized by serum starvation. The arrow indicates the time of addition of 10% horse serum and 5% fetal bovine serum and 2 _uM SlOOab. Each point is the mean of 12 determinations of three reproducible experiments; s.d. c 10%. There is a statistical difference (P i 0.01) between treated (0.5-2.0 PM S-1OOab) and untreated samples from the second day of incubation.

were rapidly filtered (Whatman GF/C) through a Bio-Rad apparatus. The filters were washed twice with cold PBS buffer, placed in scintillation liquid and the radioactivity bound to the membranes was read in a scintillator. Parallel experiments were performed in the presence of a lOO-fold excess of unlabeled S-100 to calculate the extent of nonspecific binding which was subtracted from the total binding. RESULTS

The growth trend of PC12 cells, previously synchronized by serum starvation and then incubated in 10% horse serum and 5% fetal bovine serum with different concentrations of S-100ab is shown in Fig. 1. As can be seen, in the presence of l-2 PM of S-100, the number of viable cells decreased progressively throughout the incubation period. The action that

oorunl 0

+

s-100

I

0

1

2

day83

4

6

-r4-

00”lro’

-+

S-100

+

s-1Oo.b

dep1M.d *jaw 1--

Fig. 3. The antiproliferative action of S-IOOab is reversible. (0) PC12 cells pretreated with 1 PM S-1OOab and then shifted to S-lOO-free medium on the fourth day; (+) cells cultured continuously in 1 FM S-1OOab;(A) controls incubated in the absence of S-100. The arrows indicate the medium shiftings, Each point represents the mean f s.d. of 12 determinations, There is a significant difference (P < 0.01) between treated and control cultures from the fourth day and between the two experimental groups from the 1lth day.

S-1OOab exerted on synchronized cells became evident 48 h after it was added to the medium. An anajogous effect also occurred when PC12 cells were kept, for several days, in mitotic arrest with antimitotic agents such as cytosine arabinoside, hydroxyurea or antimicrotubular agents. I9 When the S-1OOab protein, which is a combination of the a and b isoforms, was substituted by b (Fiji) or a, (aa), the action was equally detectable. On the contrary (Fig. 2), when the cells were incubated with S-100a (a/?) or with ab isoform stabilized in its quaternary structure by disulfide bonds,$ no effect was detected. When S-1OOab was removed from the medium after a four-day incubation period, PC12 cells regained their normal proliferative capacity (Fig. 3). Even when S-100 was added during cell growth, the inhibitory effect was still detectable and was followed by an increased cell death (Fig. 4).

4

Fig. 2. Effect of different S-100 isoforms on proliferation of PC12 cells. After serum starvation, PC12 cells were transferred in medium containing 10% horse serum and 5% fetal bovine serum plus different types of S-100 protein. The curves are plotted as polynomial regressions and each point is the mean of 12 determinations carried out in triplicate (sd. c 10%). There is a statistically significant difference (P < 0.01 between test and control cultures from the second day only when the isoform contained at least two identical monomers (S-100%, S-lOOb, S-1OOab).

Fig. 4. inhibition induced by different concentrations of S-1OOab (1 and 5pM) during the growth of PC12 cells. Synchronized cells were cultured in medium containing 10% horse seand 5% fetal bovine serum. S-1OOabwas added after 48 h. The difference between treated and untreated samples becomes significant (P c 0.01) 48 h after S-1OOab had been added. Each point represents the mean f s.d. of 12 determinations of two reoroducible exmriments. rmm~

Table

1. Cell growth

in the presence

of monoclonal antibody or calcium binding proteins.

Day

Control

Calmodulin

Troponin C

s-100

mAb

SILO + mAh

0

IO + 0.6

10 f_ 0.6

IO 3_ 0.6

10 * 0.6

IO & 0.6

I

13+ 1.3

2 3 4

18, I.5 23 & 3.6 3 I & 4.0

14 * 1.5 16k2.I 20 + 4.2 28 & 3.1

131 1.5 IX _t 2.7 ‘8 & 2.5 33 F 2.0

II * If?+ IX + l4jI

I:! ): 0.8 I!1 i: 1.3 32 f 3.7 38 & 3.8

IO _+0.6 I2iO.7 21 + 3.5 262 3.5 32 * 4.4

I.0 3.1 2.9 3.3

Cell growth, expressed as number of ~~ls~lOOO,after incubation with 2 PM troponin C, 2 I’M catmodulin (CaM). 5 pg/mi monoclonal antibody (mAb) anti S-100 (mAb), 2pM S-1OOab (SIOO) and 5 ,rrg/ml monoclonal antibody pius 2 FM S-IOOab (SIOO + mAb). Each number represents the means + s.d. of I2 determinations of two separate experimental sets. A statistical difference between test and control cultures exists only if S- 100 or monoclonal antibody alone is present in the medium starting from the third day of incubation

We found that S-100 also exerted its cytotoxic action, when PC12 cells were contemporarily incubated with NGF (not shown). Neither calmodulin nor troponin C, two other Ca’+-binding proteins, were able to mimic the action of S-100 (Table 1). This finding indicates that the cytotoxic action exerted by S-100 is not a general property of Ca2+ binding polypeptides. An anti-S-100 monoclonal antibody (see Experimental Procedures) completely blocked the action of S-100 (Table 1). When the monoclonal antibody (mAb) was added to the PC12 cells in the absence of exogenously supplied S-100, the number of cells increased slightly but reproducibly.

*) = :

80 -

78.2 15%

6

80-

; 2

40-

t

46

This finding suggests that PC12 cells produce a polypeptide, possibly structurally related to S-100, whose function in PC12 cell division was neutralized by the antibody employed in these experiments. The modulation of PC12 ceil proliferation by S-100 could be the result of either a forced entry into a G, phase or a drastic action on their viability. The two possibilities were tested by carrying out a cytofluorimetric analysis on PC12 cells preincubated with or without S-lOOab, at different stages of the cell cycle. The percentage of cells in the various phases (Co/G,, S, C&/M) was similar (Fig. 5). but the number of dead cells at 48 and 72 h (24 and 39% vs 8 and 15%) was markedly higher in the presence of S-100. The effect was even more pronounced when incubation was extended beyond 72 h (data not shown). It seems that S-100 exerts its action at any phase of the cell cycle. Since Ca’+ involvement is known to be critical in mechanisms leading to cell death,Y.3hthe variation in its intracellular concentration in PC12 cells exposed to S-100 was determined. S-100 caused a two- to three-fold increase of intracellular Ca’+ within few minutes after its addition to PC12 cells, as shown in Fig. 6. 1 PM troponin C, another Cal+-binding protein, and the cross-linked form of S-100 (SlOOcl) failed to elicit the same response. The total amount of divalent cation reached its maximum in untreated

hours (b) e

,oo-

0 m

dead cells Go/G1

i

S

(%) UQ2lM

48

hours

Fig. 5. Cytofluorimetric analysis of cell cycle phase distribution (G,/G,, S, G,/M) of PC12 cells treated without (a) and with 2 PM S-1OOab (b). Cell death and the number of cells in different phases is calculated as described in the methods section. All experiments were carried out on at least five randomly selected samples of i(t4 c&s; s.d. ~3%.

Fig. 6. Dose-response effect of S-100 on [Caz+li in PC12 cells preloaded with Fura 2/AM. Notice that the addition of 1 FM troponin C or 1 PM S-1Ofkl does not change the intracellular level of Ca2+. The arrows indicate the time of addition and final concentration of S-100. Data are derived from one reproducible experiment carried out in triplicate (s.d. < 10%).

923

The S-100 protein in PC12 cells Table 2. Levels of intracellular Ca*+

Day

Control

S-IOOab

0

26 + 3.0 55 f 1.3 52* 1.7 40f2.1

26 f 3.0 64k5.1 83 + 9.0 101 + 5.9

1 2 3

35

a

3~

n 26.

Wa*+ levels in living PC12 cells cultured in medium containing 0.4 x IO-’ M Ca*+ and 0.6nmol/ml 45Ca2+ in the

absence (Control) and presence of 2pM S-1OOabfor three days. Each value is the mean f s.d. of 15 determinations in three separate experiments. The difference is statistically significant (*P < 0.01) from the first day. PC12 cells one day after they had been re-exposed to serum. This was followed by a decline on the second and third days (Table 2), probably due to the progression of the cells in the cycle, characterized by fluctuations in intracellular Ca*+ levels.‘* In contrast, the Ca*+ concentration of cells exposed to S-100 progressively increased with incubation time, while the number of viable cells progressively decreased below that of the controls. In order to test whether the S-100 effect on PC12 cells is confined to these cells, analogous experiments were performed with cell lines derived from different embryonic tissues (3T3, L1210, GICAN, GH3). As can be seen in Table 3, none of these cell lines, with the exception of neuroblastoma (GICAN), responded to s-100. It should also be noted that in all these cell lines S-100 increased intracellular calcium with the exception of GH3. Thus, an intracellular rise of this divalent cation appears to be a necessary, but not sufficient condition, for the cytostatic and/or cytotoxic action of S-100. Other intracellular events, specific to each cell line, must be activated or repressed for the onset of cell death. The action of S-100 on PC12 cells was characterized by a binding which became saturated at concentrations of 200-300nM, as shown in Fig. 7. Scatchard plot analysis of these preliminary data Table 3. Effect of S-1OOabon proliferation and [Ca2+li level Cell lines 3T3 L1210 GH3 GICAN PC12

Proliferation (T/C) 1.10*0.07 0.93 + 0.06 0.99 f 0.05 0.80 + 0.05* 0.45 f 0.03;

[Ca2+li increase (%) 224+21* 274 f 35* 102 k 18 234 f 34+ 330 * 302

The treated/control ratio (T/C) is measured after three days (3T3, L1210) or five days (GH3, GICAN, PC12) of incubation of each cell line with 1 PM S-1OOab (mean + s.d., n = 12). The [Ca*+], is determined by Fura 2/AM method starting 2 mm after the addition of 1 PM S-1OOabto cell suspensions (when the fluorimetric value was stabilized). The data are reported as percentage of increment with respect to the basal value normalized to 100% (mean + s.d., n = 3).

Fig. 7. [3H]S-100ab binding on non-synchronized PC12 cells (5 x 106/ml) after 20min of incubation with different concentrations (lo-300 nM) of [‘HIS-100 (specific activity = 4.8 Ci/mmol). Nonspecific binding is assayed in the presence of 20 PM unlabeled S-1OOab(n = 8). B and F are the concentrations (nM) of bound and free [3H]S-100, respectively. The Kd values, derived from Scatchard analysis, are Kd, = 9.45 x 10-s M and Kd2 = 4.95 x lo-’ M. indicated the existence of two distinct binding sites, one having a & of 78 nM, the other endowed with a

much lower affinity. It is interesting to note that the concentration of S-100 which causes one-half the maximal Ca’+ influx is similar to that half saturating the high-affinity sites. DISCUSSION

The experiments reported clearly show that some forms of S-100 protein cause the death of PC12 cells. The effect is detectable in both synchronized and in growing PC12 cells. The action is restricted to dimers with homologous subunits, is conformationdependent, and is mediated by a specific interaction with PC12 cell plasma membrane constituents. While it is not possible, at the present time, to assess whether the component is a binding site or a functionally active receptor, Ca*+ ions could be involved in this effect, since the cytoplasmic concentration of this intracellular messenger rises immediately when PC12 cells are incubated with 0.05-2.0 ~1M S- 100 and increases steadily thereafter (Table 2). Two mechanisms could be proposed to explain the marked rise of intracellular Ca*+: an action exerted on some component(s) (e.g. G-proteins) of the Ca*+ channels8 and a direct effect of S-100 on cell permeability. It has been demonstrated that S-100 activates adenylate cyclase probably by an interaction with a G-protein from plasma membranes of the cerebral cortex.i6 Thus, S-100 could affect the permeability of G-protein-modulated Ca2+ channels and favor Ca2+ influx with consequent perturbation of the cell cycle, eventually leading to cell death. On the other hand, the finding that S-100 favors the entry of Ca*+ and monovalent cations into liposomes of various compositions,5 suggests that this Ca2+ binding protein may also act as a Ca*+ ionophore. In both instances, the end result would be an increased Ca*+ influx.

A third possibility, i.e. that S-100 acts in a detergent-like fashion, can be ruled out on the basis of the data concerning the specificity of cell interaction, the subunit composition and the conformational state of the protein. It is also important to note that during short periods of incubating PC12 cell with 2 PM S-100ab (O-240 min), the lactate dehydrogenasc levels, measured in the medium, were not significantly different with respect to the controls (not shown). However, it is possible, that S-100 alters the intracellular concentration and/or membrane permeability of other cations, such as Na+ and K +, as previously shown in artificial lipid membranes.’ Whatever the precise mechanism(s), it is tempting to postulate that the action exerted on PC1 2 cells mimics some function(s) played by S-100 in vivo. This effect could be exerted by a pool of extracellularly released S-100 as has been shown to occur in tissue cultures.‘3 This pool of S-100 may play a physiological role during development. such as in the process of programmed-cell death when large populations of neurons are doomed to die. When terminal differentiation has been achieved, an S-loo-triggered Ca” increase may play a crucial role in all processes

whereby this divalent cation operates as an rntracellular messenger. These functions may include the already documented neurite outgrowth promoting activityj5 3Hand proliferative effect on astroglia,“’ as well as on other cell lines,” via an altered permeability or intracellular distribution of C’:r’’ within the cellular compartments.

CONCLUSION

Thus, the abundant and widespread distribution of S-100 within the nervous system suggests that its release in the extracellular compartment is an event that may have varying relevance depending upon the cell type, developmental stage and functional state of the target cell upon which this polypeptide may happen to interact. wish to thank Dr B. W. Moore for his valuable advice and Dr P. Cornaglia (Istituto G. Gaslini, Genova) for kindly supplying GICAN cell culture. This work was supported by research grants from M.U.R.S.T. to G. Fano’ and from Progetto Finalizzato Biotecnologie e Biostrumentazione and Chimica Fine to P. Calissano. Acknowledgements-We

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