EFFECTS OF NERVE GROWTH FACTOR ON DIHYDROTETRABENAZINE BINDING TO PC12 CELLS

P

e

r

g

a

m

o

n

PII: S0197-0186(96)000769

Neurochem.Int.Vol. 30,Nos 4/5, pp. 411415, 1997 Copyright01997 ElsevierScienceLtd Printedin Great Britain.All rightsreserved 01974186/97$17.00+0.00

EFFECTS OF NERVE GROWTH FACTOR ON DIHYDROTETRABENAZINE BINDING TO PC12 CELLS CHRIS R. ADAMSON, TYLER E. EMLEY, LISA J. HERBIG and JOSEPH A. NEAR* Medical Sciences Program, Myers Hall, Indiana University School of Medicine, Bloomington, IN 47405, U.S.A. (Received 19 April 1996; accepted 4 June 1996) Abstract—Tetrabenazine and dihydrotetrabenazine (TBZOH) are potent inhibitors of substrate transport by the predominant forms of the vesicular monoamine transporter (VMAT) present in bovine brain synaptic vesicles and bovine adrenal medullary chromaffin vesicles. Radiolabeled TBZOH binds to these preparations with apparent dissociation constants in the low nanomolar range. However, tetrabenazine is a much less potent inhibitor of transport by rVMAT1, a form of the transporter cloned from a rat pheochromocytoma (PC12) cDNA library and expressed in CHO cells. Reported attempts to observe binding of [3H]TBZOH to rVMATl have not been successful. We examined binding of [3H]TBZOH to a crude membrane fraction from PC12 cells. Computerized nonlinear least squares curve fitting revealed two classes of binding sites (K~l = 1.5nM, R, =0.2 pmol/mg protein, K,j2= 340 nM, R*= 15.2pmol/mg protein). While the identity of the higher affinity sites is not certain, their high affinity for TBZOH suggests that they may be associated with rVMAT2. The lower affinity sites are likely to be associated with rVMATl on the basis of their affinity for TBZOH and sensitivity to inhibition of TBZOH binding by transporter substrates and inhibitors. NGF-treated PC12 cells also exhibited two classes of sites (K~l = 1.9nM, R,= 0,18 pmol/mg protein; Kd2= 370 nM, R>= 23.7 pmol/mg protein). While there were no significant differences between control and NGF-treated cells in binding capacity of the higher affinity sites, nor in apparent dissociation constants for either class of sites, there was a highly significant increase in number of lower affinity binding sites in the NGF-treated cells (p= 0.001). These results provide direct evidence that the differential sensitivity of rat brain and adrenal catecholamine stores to depletion by tetrabenazine and its derivatives is due to a much lower affinity of rVMATl for these compounds, and that NGF treatment may increase levels of rVMATl expression in PC12 cells. 0 1997 Elsevier Science Ltd. All rights reserved

The catecholamine-secreting rat adrenal pheo(PC12) cell is a useful model system chromocytoma

for neurobiologic and neurochemical studies. During embryologicw development the adrenal medulla is derived from the neural crest cells, as is the sympathetic nervous system. Greene and Tischler (1976) demonstrated that treatment of PC12 cells with nerve growth factor (NGF) leads to the formation of processes which resemble neurites found in sympathetic neurons, and to changes in catecholamine levels as well as enzymatic activities associated with catecholamine synthesis. PC12 cells are capable of storing and releasing catecholamines (Greene and Tischler, 1982), and storage is sensitive to inhibition by reserpine (Bagwell et al., 1989). Although catecholamine storage in PC12 cells has not been characterized to the same extent, it is thought to occur by *To whom all correspondence should be addressed.

the same general transport mechanism as storage in adrenal chromaffin cells. Catecholamines are concentrated in adrenal chromaffin vesicles, as well as in the synaptic vesicles of catecholamine- and seroton-containing neurons, by reserpine-sensitive vesicular monoamine transporters (VMATS) located in the vesicle membrane (Johnson, 1988). Tetrabenazine and dihydrotetrabenazine (TBZOH) block catecholamine transport in vitro and cause depletion of stored catecholamines in vivo by binding to these transporters (Scherman and Henry, 1980; Pletcher et al., 1962). Previous studies employing secretory vesicles isolated from bovine striatum and adrenal medulla have shown that TBZOH binds to storage vesicles from these two sources with apparent dissociation constants in the low nanomolar range (Scherman et al., 1982; Near, 1986). However, Peter et al. (1994) were unable to detect appreciable TBZOH binding to a transporter (rVMATl) cloned from a 411

412

C. R. Adamson etal

PC 12 cDNA library and expressed in in chinese hamster ovary fibrobktst (CHO) cells, while binding to

Binding of [3H]TBZOH was assayed by incubating PC 12 membranes with 50 mM K Hepes, 100mM KC-I, 10mM NaCl, s nlM MgC12,I mM EC,TA, pH 7.6, in a total volume of 300 vi, for 2 h at room ternperaturc. For nonspecific binding determinations, 300 P~M unlabeled tetraberrazine was included. The mixture was filtered over polyethylcncimine treated GF/B glass fiber filters (Vincent and Near, 1989) and the filters were placed in scintillation vials containing Bic>Safe 11 at least 12 h prior to liquid scintillation counting. Analysis of saturation binding and inhibition experiments was performed as described by McPherson ( 1985) using a commercially available version of LIGAND (Munson and Rodbard, 1980). Protein concentrations were determined by the method of Markwell e( cr/. ( 1978) using bovine serum albumin as standard.

the homologous rat brain transporter (rVMAT2) was observed. Tetr~ibenazine, a structural analog of TBZOH, is able to block both substrate transport (Liu etu/., 1992) and [3H]reserpine binding (Schtddiner et a/.. 1993) associated with rVMAT 1, but higher concentrations are required than for inhibition of the corresponding activities associated with rVMAT2 or any other vesicular monoamine transporter studied to date. Because the apparent absence of TBZOH binding to the PC.I2-derived transporter might be due to a much lower affinity for the Iigand, we examined binding of’IJH]TBZOH to crude membrane fractions RESULTS from control and NGF-treated PC 12cells using much higher concentrations of the ligand than reported in TBZOH binding to PC 12 cells previous studies. The raw data for total, nonspecific, and specific (tetrabenazine-sensitive) binding of [’H]TBZOH to PC 12cells at various Iigand concentrations are shown EXPERIMENTAL PROCEDURES in Fig. 1. Nonspecific binding was linearly dependent M(irl,rfu[.$ on Iigand concentration throughout the range tested. At the highest ligand concentration employed PC12 cells were obtained from American Type Culture Collection (Rockville, MD, U.S.A.). Horse serum and fetal (500 nM), nonspecific binding accounted for approxiboline serum were purchased from Atlanta Biological (Normately 70”%of the total binding. The same data were cross, GA, U.S.A.). NGF, rcserpine, Dulbecco’s modilied used to construct the modified Scatchard plot shown Eagle’s media (DME) and the hydrochloride salts of serotonin, dopamine and norepinephrine were from Sigma Chemical Co. (St Louis, MO. U.S.A.). Tetmbenazine was obtained from Fluka Chemical Corp. (Ronkonkoma, NY, U.S.A.). Bio-Safe 1I from Research Products International Corp. (Mount Prospect. IL. U.S.A.) and glass fiber filters (GF/B) from Whatman (Fairfield, NJ, U.S.A.). Cell cullurc und menlbrune preparutiw PC I2 CCIISwere grown under 7(Y0(’02 in uncoated plastic culture flasks containing 10°/0horse serum, 50/. fetal bovine serum, 50 ~g,ml streptomycin, 50 U/ml penicillin and, where indicated, 10ngrnl NGF. Media were changed every 4 days. and NGF treated cells were harvested for analysis after 1216days of exposure to NGF. At this time more than 90°Aof’ the NGF-treated cells exhibited processes. Crude membranes were prepared hy the method of Schuldiner et al. (1993). Cells were scraped from the flask, ccntrif’uged at 500 g for 10min. the pellet was suspended in ice cold 0.01 M K HEPES, pH 7.4, 0.15 M NaCl, 5 mM MgC12,and homogenized with a Branson 450 Sonitier with microprobe (Branson Ultrasonics Corp.. Danbury. CT. U.S.A.). The homogenate was centrifuged for 5 min at 500 g and the resulting supernatant centrifuged for 45 min at 150,000g. The pellet was resuspended in IOmM K HEPES, pH 7.4, homogenized in a tight fitting teflon/glass homogenizer, and stored for Icss than 1 week at —20”C bef’ore analysis. Previous work has shown that storage for up to 2 months under these conditions had no effect on binding activity. TBZOII hkli)l,y ami dura unul~si.$ [’H]’fBzoFf (I5 Ci/mmol) was prepared and its purity assayed periodically as described previously (Near, 1986).

/ ;4

/=

/

/

nz : m F Z2 m

P’ / -

/

..A

/

~

0 0

200

400 ITBZOH],.M

Fig. 1. Concentration dependence of’[3H]TBZOH binding to PC12 cell membranes. Various concentrations of [’H]TBZOH were incubated with PC12 mcmbrancs (0.23 mg protein) and bound radioactivity was dctcrmincd as described in Experimental Procedures. Nonspecific binding (circles) was determined by inclusion of 300 WM tetrabenazine in the assay. Specific binding (A) was determined by subtraction of nonspecific binding from total binding (W). Each point is the mean of three determinations, and standard deviations were less than 3% of the indicated value. Curves for total binding (- -), specific binding ( ) and nonspecific ~~~~N -) were calculated using binding constants binding ( obtained from nonlinear least squares curve fitting to a twosite model.

Effects of NGF on TBZOH binding to PC12 cells

in Fig. 2. Visual inspection of the plot suggests two classes of binding sites, and computerized nonlinear curve fitting confirmed that the best fit of the data is to a two site model. The capacity of the lower affinity class of sites (K~2= 340 nM, R2 = 15.2pmol/mg protein) was more than 50-fold higher than that of the higher affinity sites (K~l = 1.5nM, RI =0.2 pmol/mg protein). In order to gain some information about the identity of TBZOH binding sites, the inhibitory potencies of several substrates and inhibitors were assessed (Fig. 3) by experiments using a TBZOH concentration of 50 nM. Using the dissociation constants and site densities cited above, we calculate that approximately 90Y0 of the specific binding in the inhibition assays was to the lower affinity class of sites at the concentration of [3H]TBZOH employed (50 nM). Attempts to fit the inhibition data to a two-site model were unsuccessful. Reserpine and tetrabenazine were effective inhibitors of TBZOH binding when present at concentrations in the micromolar range, whereas the substrates serotonin, dopamine and norepinephrine inhibited binding in the millimolar range. Considerations of pH, in the case of norepinephrine and dopamine, imposed an upper limit on the concentrations that could be used in these studies. None of the inhibitors at the highest concentrations tested here gave additive inhibition when combined with 300 PM tetrabenazine and compared with assays containing tetrabenazine alone (data not shown), indicating that all of the inhibitors were blocking only tetrabenazinesensitive sites.

Kq = 1.5 nM K2 = 340 nM RI = 0.2 pmol/mg R2 = 15.2 pmol/mg 0.02 H ~ a z

413

I

1

(),()~ -9

-8

-7

-6

-5

-4

-3

-2

-1

LOG(INHIBITOR CONCENTRATION)

Fig. 3. Inhibitor sensitivities of [3H]TBZOH binding. PC12 membranes (0.21 mg protein) were incubated with 50 nM [3H]TBZOH, and various concentrations of the indicated inhibitors and bound radioactivity were determined. Each point is the mean of at least two determinations, and was calculated as the ratio of specific binding in the presence of inhibitor over specific binding in its absence. Curves represent the best fit to a one-site model, assuming complete and competitive inhibition using binding constants obtained from saturation binding experiments with [3H]TBZOH, and assuming all binding was to the lower affinity class of sites. In the absence of any inhibitor, 7023+ 168cpm were bound, whereas in the presence of 300 LM tetrabenazine 2300f 37 cpm were nonspecifically bound. The inhibitors used were tetrabenazine (0), reserpine (A), serotonin (o), dopamine (n) and norepinephrine (u). Effects of NGF on TBZOH

binding

NGF treatment had no significant effect on the apparent dissociation constant or binding capacity of the higher affinity sites (Kdl = 1.9nM, R,= 0.18 pmol/mg protein), nor did it change the apparent dissociation constants for the lower affinity class of sites (K~2= 370 nM) (Fig. 4). However, NGF treatment significantly increased the number of low affinity binding sites when compared with control @= O.001). In two additional experiments qualitatively similar results were obtained, with increases in binding capacity for the lower affinity sites of 1.3and 1.5-fold and no significant effects of NGF on the other binding constants.

$ 0.01

DISCUSSION

0.00 [

1.0

2,0

3.0

ITBZOH] BOUND, nM

Fig. 2. Scatchard plot of [3H]TBZOH binding to PC12 cell membranes. Data for specific binding from Fig. 1 were used. (—) Best fit to the two-site model with the indicated bindingconstants; (- --) individual binding sites as calculated from individual binding parameters.

Results presented here demonstrate that, in a crude membrane fraction from PC12 cells, the most abundant binding site for TBZOH exhibits an apparent dissociation constant of 340nM for this compound. Although this value is higher by several orders of magnitude than those observed previously for TBZOH binding in a variety of other tissues, tetrabenazine is a much less potent inhibitor of serotonin

414

C. R

KI = 1.9 nM

K2 = 370 nM

Rq = 0.18 pmol/mg R2= 23.7 pmol/mg 002 Lu

u K u. z z 3 g 0.01

0.00 0.0

1.0

2.0

3.0

4.0

ITBZOH] BOUND, nM

Fig. 4. Scatchard plot of [3H]TBZOH binding to membranes from NGF treated PC’12cells. Data were obtained and anaIysed as in Fig. 2. The solid curve represents the best fits 10 a two-site model, and dashed lines represent individual binding sites as calculated from the indicated binding parameters,

and reserpine binding in CHO cells expressing a vesicular monoamine transporter cloned from PC12 cells (rVMATl ) than in cells expressing a similar transporter cloned from rat brain (rVMAT2) (Liu et al., 1992; Schuldiner et a/., 1993; Peter ef al., 1994). [n PCl 2 cells, rVMATl appears almost exclusively in large dense core vesicles, with a small amount of immunoreactivity in clear vesicles (Liu eta/.. 1994). The pharmacological sensitivities of lower afhnity binding reported herein are also consistent with assignment of this class of sites to rVMAT 1. Tetrabenazine inhibited binding to PC12 membranes at micromolar concentrations and higher, a range previously observed to inhibit reserpine binding and substrate transport in CUHOcells expressing rVMATl, but much higher than required for inhibition of the same activities of other VMATS (Schuldiner et al., 1993; Liu eta/., 1995). All substrates exhibited millimolar potencies as inhibitors of [qH]TBZOH binding, while reserpine was effective in the low micromolar range. These values are in general agreement with those reported for other vesicular monoamine transporters (Near, 1986; Liu et al., 1995). The identity of the higher affinity class of [’H]TBZOH binding sites is more problematic, A pharmacological characterization was not attempted because these sites represent such a small fraction of the total binding capacity for the Iigand in PC12 membranes. On the basis of their high affinity for TBZOH, they are most likely associated with rVMAT2. Using anti-peptide antibodies against rVMAT 1 and rVMAT2, two groups observed transport

rVMAT 1-like immunoreactivity in all chromaflin cells of’the rat adrenal medulla, but some cells also exhibit rVMAT2-like immunoreactivity (Peter ef cd., 1995; 1994). However. sympathetic ganglion Weihe etcd.. cells contain only rVM AT2. Further exploration of the pharmacological sensitivities of TBZOH binding to PC 12 cells and to transformed cells expressing rVMATs, as well as immunochemical studies designed to detect the presence of’rVMAT2 in PC12 cells, will be required before the higher affinity class of [’H]TBZOH binding sites can be attributed to rVMAT2 with any degree of certainty. Treatment ofPC12 cells with NGF led to a 1.5-fold increase in lower affinity TBZOH binding capacity, while having no effect on higher affinity binding, Greene and Tischler ( 1976) observed a decrease in enzymatic activities associated with catecholamine biosynthesis, and a decrease in catecholamine level per milligram of protein, in NGF-treated PC12 cells. Although the effects of NGF treatment on TBZOH binding has not been examined previously to our knowledge, Desnos etal. (1992)Desnos etal. (1995) observed decreased catecholamine content but increased TBZOH binding capacity in bovine chromafhn cells cultured in the presence of depolarizing concentrations of potassium. NGF has numerous effects on PC 12cells via multiple mechanisms (Maness etal., 1993). NGF’ induces changes in phosphorylation of serine/threonine residues in several proteins (Ohmichi etal., 1992), and it rapidly activates cellular immediate early genes such as (:/b.!’, C:jzitz, ,jun-B, and q}’- 1 (Ito et al., 1990). Protein kinase A may be modulated by NGF via activation of’ protein kinase N (Volonte e{al., 1995), and activation of protein kinase C is also a likely consequence of NGF treatment (Maness et al., 1993). VMATS contain consensus sequences for phosphorylation by protein kinases A and C (Surratt e( a/., 1993; Liu et al., 1992).Nakanishi eta/. (1995a) provided evidence of a down-regulation of catecholamine storage in response to dibutyryl cyclic AMP, presumably owing to phorphorylation of VMAT1. Treatment of PC12 cells with a phosphoprotein phosphatase inhibitor enhanced cyclic AMP-mediated down-regulation, whereas treatment with a protein kinase inhibitor increased vesicular amine transport (Nakanishi cf al., 1995b). Although phosphorylation state may modulate the transport activity of rVMATl, eflects of phosphorylation on TBZOH binding have not been described. Whether the NGF-induced increase in TBZOH binding sites described here was due to increased levels of rVMATl, or to exposure of previously inaccessible binding sites as a consequence of phosphorylation

Effects of NGF on TBZOH binding to PC12 cells

of the transporter, cannot be determined from the available evidence. Immunochemical determination of the levels of rVMATl in control and NGF-treated PC12 cells, coupled with studies of the effects of phosphorylation on TBZOH binding, could resolve this question. Acknowledgernenrs-This study was generously supported by the National Institutes of Health, grant NS20784, to J. A, N.

REFERENCES Bagwell, E. E., Webb, J. G., Wane, T. and Gaffney, T. E. (1989) Stereoselective uptake of atenelol into storage granules isolated from PC12 cells. 1 Pharmacol. Exp. Thera, 249, 47&482. Desnos, C., Laran, M. P., Langley, K., Aunis, D. and Henry, J. P. (1995) Long term stimulation changes the vesicular monoamine transporter content ofchromaffin granules. J. biol. Chenr. 270, 16030-16038. Desnos, C,, Laran, M. P. and Scherman, D. (1992) Regulation of the chromaffin granule catecholamine transporter in cultured bovine adrenal medullary cells: stimulusbiosynthesis coupling. J. Neurochern. 59,2105-2112. Greene, L. A. and Tischler, A. S. (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. natn. Acad. Sci. USA 73, 2424-2428. Greene, L. A. and Tischler, A. S. (1982) PC12 pheochromocytoma cultures in neurobiological research. In Advances in Cellular Neurobiology, eds S. Federotf and L. Hertz, pp. 373414. Academic Press, New York.. Ito, E., Sweterlitsch, L. A., Tran, P. B., Rauscher, F. J. and Narayanan, R. (1990) Inhibition of PC12 cell differentiation by the immediate early gene fra-1. Oncogene 5, 1755–1760. Johnson, R. G. (1988) Accumulation of biological amines into chromaffin granules: a model for hormone and neurotransmitter transport. Physiol. Rev. 68, 232–306. Liu, Y. J., Peter, D., Merickel, A., Krantz, D., Finn, J. P. and Edwards, R. H. (1995) A molecular analysis of vesicular amine transport. Behav. Brain Res. 73, 51–58. Liu, Y., Peter, D., Roghani, A., Schuldiner, S., Prive, G. G., Eisenberg, D., Brecha, N. and Edwards, R. H. (1992) A cDNA that suppresses MPP+ toxicity encodes a vesicular amine transporter. Cell 70, 539–551. Liu, Y., Schweitzer, E. S., Nirenberg, M. J., Pickel, V. M., Evans, C. J. and Edwards, R. H. (1994) Preferential localization ofa vesicular monoamine transporter to dense core vesicles in PC12 cells. J. cell. Biol. 127, 1419–1433. Maness, L. M., Kastin, A. J., Weber, J. T., Banks, W. A., Beckman, B. S. and Zadina, J. E. (1993) Theneurotrophins and their receptors: structure, function and neuropathology. Neurosci., Biobehav. Rev. 18, 143–159. Markwell, M. A., Haas, S. M., Bieber, L. L. and Tolbert, N. E. (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 87, 2OG21O.

415

McPherson, G. A. (1985) Analysis of radiologand binding experiments: a collection of computer programs for the IBM PC, J. pharmacol, A4eth. 14, 213–228, Munson, P. J. and Rodbard, D. (1980) LIGAND: a versatile computerized approach for characterization of ligandbinding systems. Anal. Biochem. 107, 220–239. Nakanishi, N,, Onozawa, S., Matsumoto, R., Hasegawa, H. and Yamada, S. (1995) Cyclic AMP-dependent modulation of vesicular monoamine transport in pheochromocytoma cells. J, Neurochem. 64, 60G607. Nakanishi, N., Onozawa, S., Matsumoto, R., Kurihara, K,, Ueha, T., Hasegawa, H, and Minami, N. (1995) Effects of protein kinase inhibitors and protein phosphatase inhibitors on cyclic AMP-dependent down-regulation of vesicular monoamine transport in pheochromocytoma PC12 cells. FEBS Lett. 368, 411F414. Near, J, A, (1986) [3H]-Dihydrotetrabenazine binding to bovine striatal synaptic vesicles, Mol. Pharmacol. 30, 252– 257. Ohmichi, M., Pang, L., Decker, S. J. and Saltiel, A. R. (1992) Nerve growth factor stimulates the activities of the raf1 and the mitogen-activated protein kinases via the trk protooncogene. J. biol. Chern. 267, 1460414610. Peter, D., Jiminez, J., Liu, Y., Kim, J. and Edwards, R. H. (1994) The chromaffin granule and synaptic vesicle amine transporters differ in substrate recognition and sensitivity to inhibitors. J. biol. Chem. 269, 7231–7237. Peter, D., Liu, Y., Sternini, C., de Giorgio, R,, Brecha, N, and Edwards, R. H. (1995) Differential expression of two vesicular monoamine transporters. J. Neurosci. 15, 6179– 6188. Pletcher, A., Brossi, A. and Gey, K. F. (1962) Benzoquinolizine derivatives: a new class of monoamine decreasing drugs with psychotropic action. Int. Rev. Neurol. Bioi, 4,275-306. Scherman, D. and Henry, J.-P. (1980) Effect of drugs on the ATP-induced and pH-gradient-driven monoamine transport by bovine chromaffin granules. Biochem. Pharmacol. 29, 1883-1890. Scherrnan, D., Jaudon, P. and Henry, J. P. (1982) Characterization of the monoamine carrier of chromaffin granule membrane by binding of [2-3H]-dihydrotetrabenazine, Proc. natn. Acad. Sci. USA 80, 584-588. Schuldiner, S., Liu, Y. and Edwards, R. H. (1993) Reserpine binding to a vesicular amine transporter expressed in chinese hamster ovary fibroblasts. J. biol. Chem. 268, 29–34. Surratt, C. K., Persico, A. M., Yang, X. D., Edgar, S. R., Bird, G, S,, Hawkins, A. L., Griffin, C. A., Li, X., Jabs, E. W. and Uhl, G. R. (1993) A human synaptic vesicle monoamine transporter cDNA predicts posttranslational modifications, reveals chromosome 10 gene localization and identifies TaqI RFLPs. FEBS Lett. 318, 325–330. Vincent, M. S. and Near, J. A. (1991) Purification of a [3H]dihydrotetrabenazine-binding protein from bovine adrenal medulla. Mol. Pharmacol. 40, 889–894. Volonte, C, and Greene, L. A. (1995) Nerve growth factoractivated protein kinase N modulates the cAMP-dependent protein kinase. J. Neurosci. Res. 40, 108–116. Weihe, E., Schafer, M, K., Erickson, J. D. and Eiden, L. E. (1994) Localization of vesicular monoamine transporter isoforms (VMAT1 and VMAT2) to endocrine cells and neurons in rat. J. Mol. Neurosci. 5, 149–164.