Properties of the nerve growth factor receptor of a clonal line of rat pheochromocytoma (PC12) cells

Printed in Sweden Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-2827/79/070071-08$02.00/0

Experimental

PROPERTIES

OF THE

OF A CLONAL

NERVE

LINE

GROWTH

OF RAT (PC12)

KARL

121 (1979) 71-78

Cell Research

HERRUP’

FACTOR

RECEPTOR

PHEOCHROMOCYTOMA

CELLS and HANS THOENENZ

Departmentof Pharmacology, Biocenter of the University, Basle, Switzerland

SUMMARY The interaction of nerve growth factor (NGF) with its receptor on cells of the PC 12 cell line was studied. All experiments were done at O.S“C to minimize degradation and processes requiring membrane mobility. Under these conditions, a single class of high affinity binding sites with a dissociation constant of 2.9~ lobs M was observed. The number of receptors per cell was 58000. The binding was linear with the number of cells in the assay and was not displaced by proteins other than native nerve growth factor. Trypsin treatment of the cells destroyed the specific binding. The removal of divalent cations had no effect on the binding. Culturing the cells for 2 weeks in NGF prior to assay did not change the receptor number or receptor afftnity and there was a similar lack of effect of the density of the culture from which the cells were taken for assay. The present findings are compared with previous studies on the dorsal root ganglia and sympathetic ganglia neurons, and the implication for the use of PC 12 as a model for the study of NGF action are discussed.

Nerve growth factor (NGF) is a protein which has profound effects on peripheral sympathetic and developing sensory neurons [cf 1, 2, 31. Both in vivo and in vitro NGF causes responsive neurons to increase in size and to extend neurites having the characteristics of axons. Moreover, NGF treatment of sympathetic neurons causes a selective induction of tyrosine hydroxylase and dopamine fl-hydroxylase [4, 51, enzymes which are characteristic for adrenergic neurons and adrenal medullary cells. The importance of NGF for the developing nervous system is further illustrated by the observation that injection of NGF antiserum into newborn rodents causes an extensive destruction of the peripheral sympathetic nervous system [cf 1, 2, 31. Since the protein chemistry of NGF is well known [cf 2, 31 it would seem to be an excellent

probe with which to study selective aspects of the development of the peripheral sympathetic and sensory nervous systems. However, although the pleiotypic actions of NGF have been described in great detail [cf 1, 2, 31 the underlying molecular mechanism(s) remains largely unknown. PC 12 is a clonally derived cell line from a rat adrenal medullary pheochromocytoma [6] which can synthesize, store and release both catecholamines [7] and acetylcholine [8, 91 and which responds to nanomolar concentrations of NGF by ceasing cell division and extending neurite-like processes ’ Present address: Department of Human Genetics, Yale University School of Medicine, 333 Cedar Str., New Haven, CT 04510, USA. * Present address: Department of Neurochemistry, Max-Planck-Institute for Psychiatry, 80033 Martinsried, Germany. Exp Cell

Res 121 (1979)

72

Herrup

and Thoenen

how its properties compare with those reported for the sensory and sympathetic neurons [ 12-141. Our findings demonstrate that high affinity binding sites for NGF do exist on PC 12 cells, but their properties are distinctly different from those reported for the cell body receptors on NGF-responsive neurons. MATERIALS 10

20

30

LO

I. Abscissa: cells/ml (X lo+); ordinate: specific pg bound. Specific binding of ‘Z51-NGF to PC 12 cells as a function of cell concentration in the assay. Varying numbers of PC 12 cells were incubated for 90 min in the presence of 50 rig/ml ‘*I-NGF. Incubation was terminated by centrifugation as described in the Methods. The values are the means of triplicate determinations. Each point represents the difference of total minus non-specific binding (that seen in the presence of 5 &ml non-radioactive NGF). The line represents the equation determined by the least squares tit method. Fig.

[6]. Although tyrosine hydroxylase activity is not induced [lo], as would be expected if the cells were to respond to NGF in the same way as adrenergic neurons [4, 51 or adrenal medullary cells [5], the enzymes choline acetyltransferase and omithine decarboxylase [&ll] are induced by NGF. One important advantage of PC 12 cells is that unlike most NGF-responsive neurons, they do not die in culture in the absence of NGF. These cells therefore represent a model system in which certain specific properties of neurons derived from the neural crest may be studied. In embryonic dorsal root ganglia and in sympathetic neurons, the first step in the interaction of NGF with the cells is the binding of the protein to a high affinity cell surface receptor [12-14). We therefore asked whether PC 12 cells also possess a cell surface receptor for NGF and, if so, Exp Cell Res 121 (1979)

AND METHODS

NGF was isolated in the 2.5 S form by a modification [15] of the method of Bocchini & Angeletti [16] from the salivarv glands of male mice. Enidermal growth factor (EGF) was isolated from the same source by the method of Savage & Cohen r171. Lactoperoxidase was obtained from Boehringer,- trypsin from Worthington, bovine serum albumin from Sigma, insulin and glucagon from Calbiochem and pronase from Serva. Fetal calf serum and horse serum were obtained from Gibco. Sodium iodide (Na*%I-carrier free) was from E.I.R., Wtirenlingen. The buffer used for all binding experiments was a modified Krebs Ringer solution buffered with 0.025 M HEPES (KRH) according to the formula of Greene [7] omitting ascorbate. In most instances the KRH was supplemented with 1 mg/ml of bovine serum albumin (KRHIA) to prevent non-specific adsorption of NGF to the vessel surfaces. Iodination of NGF was performed both by the use of Chloramine T [18] and iactoperoxidase [is], as described in [ 131. Since the latter procedure gave a more stable product, most of the experiments reported here used lactoperoxidase iodinated NGF. No preparation was kept longer than 2 weeks. The PC 12 cells were grown from an original stock generously provided by-m Lloyd Greene-The cells were grown as described by Edgar t Tboenen [lo]. All binding experiments were performed using cells from dishes which had become relatively dense (3-8~ 106 cells per 10 cm dish). The cells were harvested by removing their medium and replacing it with 10 ml KRH/A. Rapid ejection of this solution from a Pasteur pipette was sufficient to detach the cells from the dish. After the cells were in susnension thev were washed by centrifugation at 800 g ior 5 mm and resuspended in 10 ml fresh KRHIA. Following this they were recentrifuged and suspended in the appropriate volume of KRHIA. PC 12 cells tend to grow in large clumps which are not disrupted by simple trituration and which might restrict access of NGF, to the cells in the center. These clumps were reduced to small aggregates of fewer than 5 cells by rapidly drawing them in and out of a disposible syringe fitted with three plastic a-pipette tips in series. Following this treatment the viability of the cells, as judged by Trypan blue exclusion, remained greater than 95 %. The binding studies were performed according to the protocol of Herrup & Shooter [13]. To determine total binding, cells were incubated with the appropriate con-

NGF receptors

in pheochromocytoma

cells

73

300 t 240

.

t

Fig. 2. Abscissa: (a) &ml NGF; (b) pg bound/lP cells; ordinate: (a) pg bound/loS cells; (b) pg NGF bound/l05 cells/rig/ml NGF free. Specitic binding of Y-NGF as a function of NGF concentration at O.S’C. (a) Total (0) and non-specific binding (A) were determined as described in the Methods. The incubation time was 60 min with 25x10s cells/ml. Specific binding (0) was calculated by sub-

tractina the value of the equation of the least squares tit of tie non-specific poinis from the value observed for the total binding points. The line is the theoretical curve for a simple Michaelis-Menton reaction using the values for Ko and B,., from the Scatchard plot. (b) Scatchard plot of the data in fig. 2 a. The line shown is the equation of the least squares tit of the points.

centration of NGF in a final volume of 0.5 ml KRH/A. To determine low affinity ‘non-specific’ binding, duplicate tubes were incubated with the addition of 5 &ml non-radioactive NGF. The incubation time vi&d from 60 to 120 min. Bound NGF was separated from free NGF by sedimenting the cells through a twostep sucrose gradient. The gradients contained 50 ~1 0.3 M sucrose in KRH/A, 100 ~1 0.15 M sucrose in KRH/A (containing phenol red to help visualize the interfaces) and 150 ~1 of sample in a 400 gl microcentrifuge tube. The tubes were spun either in a microcentrifuge (M.S.E. microhematocrit centrifuge) for 10 min or in special adapters in a Sorvall HB-4 swinging bucket rotor for 10 min at 16000 R. Following centrifugation the tubes were frozen in crushed d;y ice and the bottom 4 mm (ca 5 ~1) was cut off with a razor blade and counted in a Padk&d gamma-counter at an efficiency of 65%. As the binding values determined with the two centrifuges did not differ significantly, we used the Sorvall centrifuge for the majority of the experiments, for reasons of convenience.-Preliminary experiments with the microfuge showed that more than 75 % of the counts were pelletted after 1 min of centrifugation and 100% after 3 min. However, even these times are overestimates as a maioritv of the cells are still in equilibrium with the g&en concentration of NGF in the incubation medium (upper half of the gradient) during the initial part of the sedimentation period. That dissociation during and after sedimentation does not significantly affect the results using the given experimental conditions can be deduced from the fact that the radioactivity in the upper part of the sucrose gradient accounted for
the apical part of the microtube cut off in the experimental scheme contains a volume that is many times larger than the volume of the actual pellet.. Thus, ‘9-NGF that might have dissociated from the cells after sedimentation would be contained in this volume (about 5 ul). Without cells, 0.1% to 0.3 % of the counts in the s&&e were found in the “pellet” when centrifugation was done in the microcentrifuge and only 0.01% to 0.03 % when the swinging bucket rotor was used. Standard errors were consistently less than 6%. For normal binding curves the plot of non-specific binding vs NGF concentration was linear. Specific binding was calculated in these experiments by computing the best equation for a straight line through the non-specific binding points (using the least-squares methods) and subtracting the value of the equation at any one concentration from the value obtained for total binding. In all other experiments specific binding was determined by subtraction of the observed nonspecific from the observed total binding.

RESULTS At room temperature and at 37°C preliminary experiments (not shown) revealed that the interaction of 1251-NGF with PC 12 cells is more complex than simple associationdissociation kinetics. Since our objective was to examine only the properties of the Exp Cell

Res 121 (1979)

74

Herrup and Thoenen Table 1. Displacement of lz51-NGF from PC 12 cells by various substance Compound Fetal calf serum 10% 3% 1% Glucagon (10 Z.&ml) Insulin (50 j&ml) Enidermal erowth factor (20 Z&ml j Cytochrome c (50 pglml) NGF (5 dml)

IlllllllllllC 1.0

2.0

30

4.0

5.0

6.0-

Fig. 3. Abscissa: pg/ml native NGF; (inset) total pg/ ml NGF; ordinate: pg l*JI-NGF bound/W cells; (inset) specific pg bound/W cells. Displacement of ‘WNGF by increasing concentrations of native NGF. PC 12 cells at a concentration of 15X 105 cells/ml were incubated for 60 min with 50 ng/ ml ‘“I-NGF plus increasing concentrations of native (non-radioactive) NGF. The values given represent total binding. (Inset) Specific binding of NGF as a function of NGF concentrations using the value of the 6 pg/ml point as non-specific binding and correcting for the altered specific activity.

NGF-receptor interaction, the experiments reported here were done in ice (O.YC) to eliminate steps requiring membrane mobility such as internalization and to minimize enzymatic processes such as degradation. Under these experimental conditions specific binding (as defined in Methods) increased linearly with increasing cell concentration (fig. 1). Although it was technically impossible to increase the cell concentration enough to bind all of the NGF in solution, the values obtained when analyzed by a double reciprocal plot suggest that all of the labeled NGF was capable of binding to the cells. The specific binding of NGF as a function of NGF concentration (fig. 2~) is consistent with a single class of binding sites. When the data are replotted in a Scatchard plot (fig. 2b), the intercept on the abscissa is Exp Cell

Res 121 (1979)

% displacement <5 C.5 <5 <5 <5 <5 <5 73

PC 12 cells at a concentration of 14X 105 cells/ml were incubated with 50 rig/ml ‘T-NGF and the compounds listed above for 90 min at 0.5”C. Percent displacement bound in the abwas calculated as the pg ‘TNGF sence of any addition minus pg bound in the presence of the additive divided by the first number times 100.

approx. 250 pg per 105 cells. This corresponds to 58000 binding sites per cell. The dissociation constant, KD is approx. 75 ngl ml or 2.9~ 10mgM. The binding of lz51-NGF as a function of increasing concentration of non-radioactive NGF is shown in fig. 3. The data also can be expressed as specific binding (inset) using the 6 pg/rnl point as the amount of non-specific binding and correcting for the dilution of the specific activity. Though these data are less accurate for the higher non-radioactive NGF concentrations, the close correspondence of the curve to that predicted by the Scatchard plot in fig. 2 b demonstrates that the receptors on these cells do not distinguish between iodinated and non-iodinated NGF and further supports the observation that there is a single class of saturable binding sites on these cells. The specific binding was found to be specific for NGF. Table 1 shows that proteins other than NGF but with similar physical chemical properties (e.g. cytochrome c) [20] do not compete with NGF for its bind-

NGF receptors 10

in pheochromocytoma

cells

75

r

Fig. 4. Abscissa: pg/ml trypsin in the digestion step; ordinate: pg bound/lW cells. Sensitivity of ‘2JI-NGF binding to trypsin treatment of the cells. 43 x 105 PC 12 cells per ml were incubated with various concentrations of trypsin for 30 min at 37°C in KRH without albumin. The cells were then washed 3 times in ice-cold KRH/A, resuspended in KRH/A with 50 &ml V-NGF, incubated for 60 min at O.s”C and the amount of bound ‘251-NGF determined.

ing site on the PC 12 cells. In addition other hormones such as insulin, whose primary amino acid sequence has some similarities with NGF [cf 31, and EGF, which is a growth factor also isolated from the male mouse submaxillary gland [ 171, do not compete for the binding site. These incubations were carried out for 90 min with the cells being exposed to 1251-NGF and the additive simultaneously. For native NGF, even after pre-equilibration for 60 min with lz51-NGF alone specific binding was reduced over 90% 5 min after the addition of non-radioactive NGF. The binding was also specific for NGF-responsive cells. A cell line derived from PC 12 which has lost its NGF responsiveness showed no specific binding of NGF (Lucas et al., unpublished observations) . The binding of NGF to PC 12 cells is sensitive to trypsin treatment of the cells. Fig. 4 shows the results of an experiment in

20 60 200 Fig. 5. Abscissa: rig/ml NGF; ordinate: specific pg bound/ lo5 cells. Effects of divalent cations on the specific binding of 129-NGF. 13x 105 PC 12 cells were incubated at the concentrations shown either in normal KRH/A (Fa) (control) or in the same buffer made up without calcium or magnesium and supplemented with 5 mM EDTA (Cl) (EDTA). Specific binding was determined (as described in the Methods) after 60 min at 0.W.

which PC 12 cells were incubated in the presence of various concentrations of trypsin at 37°C for 30 min. The cells were then washed three times and resuspended at 0.5”C in 50 rig/ml 1251-NGF. The viability of the cells (as judged by trypan blue exclusion) was unaffected by trypsin treatment. Concentrations of trypsin as low as 0.1 pg/ ml significantly decreased the binding under these conditions. Divalent cations are not necessary for the specific binding of NGF to PC 12 cells. Cells were harvested by trituration in KRHl A buffer without calcium or magnesium. Normal amounts of these two cations were added back to control incubations while EDTA at a final concentration of 5 mM was added to experimental incubations. The results shown in fig. 5 demonstrate that there is no noticeable effect of the presence or absence of divalent cations on the amount or affinity of the specific binding of NGF. Various culture conditions were ex-

76

Herrup

and Thoenen

v” 40 80

120

160

200

Fia. 6. Abscissa: nalml NGF: ordinate: svecific DP Ibound/l05 cells. -. Effects of NGF oretreatment on the uronerties of the PC 12 NGF receptbr. PC 12 cells were-p&ted in plastic Petri dishes to which they do not adhere and were subsequently grown for 2 weeks without addition (A-A), or in the presence of 0.5 rig/ml NGF (0.. .O), 5 &ml NGF (O---O) or 50 rig/ml NGF (A- * -A). The cells were then harvested and assayed in parallel for specific binding as a function of increasing NGF concentration.

amined for their effects on the properties of the PC 12 cell NGF receptors. Several cell types are known to change their number of receptors in response to pretreatment with the hormone to which they are responsive (e.g. insulin [21,22] and human growth hormone [23]). Therefore PC 12 cells were grown in the presence of concentrations of NGF from zero to 50 rig/ml (0.5, 5.0 and 50) for 2 weeks. When grown on collagen under these conditions, the cells would have produced extensive fiber networks at the two highest NGF concentrations. To avoid the problems which would arise from trying to compare receptor number per cell among cells with and without fibers, fiber outgrowth was suppressed by plating the cells in plastic Petri dishes to which they do not adhere. Clumps with large numbers of cells formed during this culture procedure. They were successfully broken up as described in Methods. Subsequently, the four binding curves were done in parallel. No differE.yp Cdl

Rr.\ I21 11979,

ences in either the dissociation constant or the number of receptors per cell were detected (fig. 6). Similarly, the density of the culture from which cells were taken for assay did not affect the receptor affinity although at very high cell densities (greater than 10’ cells per 10 cm dish) decreases in the apparent number of receptors per cells were observed with no change in KD. The interpretation of this latter finding is difficult, however, since these cultures contained many floating cells and it cannot be excluded that the poor general conditions of the cells are responsible for the apparent decrease. DISCUSSION The characterization of the properties of the NGF receptors of PC 12 pheochromocytoma cells as compared with the results of previous studies with sympathetic and dorsal root ganglia allows a better delineation of the extent to which these tumor cells can be used as a model for the physiological targets of NGF. These studies also raise some new questions with respect to the mechanism of action of NGF in PC 12 cells. Several reports have demonstrated that the density of the cultures in which the PC 12 cells are growing affects their responsiveness to NGF. Edgar & Thoenen [IO] for example have shown that dense cultures (3.6~ 104 cells/ cm2) exhibit no induction of choline acetyltransferase, while less dense cultures (0.36~104 cells/cm2) showed marked enzyme induction. This change in NGF sensitivity has to occur at a point subsequent to the initial NGF binding to the receptor, since our studies have shown that culture densities (below 13x 104 cells/cm2) do not affect the number or affinity of the receptors. Moreover, Greene has shown [26]

NGF receptors

that, if PC 12 cells are pretreated with NGF in culture, when they are harvested and replated they respond to NGF more quickly (one day instead of one week) and at lower concentrations (maximal effect at 1 .O rig/ml instead of 50 nglml). Our inability to detect any change in either the number of NGF receptors or their affinity after 2 weeks of culture in NGF means that also in this case the mechanism responsible for this shift in responsiveness is found at a step (or steps) subsequent to the binding. The cell line, PC 12, was cloned from a rat pheochromocytoma [6]. The embryological origin of the adrenal medullary cells is the neural crest. Thus, the origin of these cells is the same as those of the neurons of both the sympathetic (SG), and dorsal root ganglia (DRG) as well as the adrenal chromaffii cells. The NGF responsiveness of sympathetic and dorsal root ganglion neurons is well established [cf 1, 2, 31 and cells of the adrenal medulla, while not demonstrating gross morphological changes in vivo, respond to NGF with specific enzyme induction [S]. Given the similar nature of the NGF response of PC 12 cells and these other non-tumor cells it might be expected that the mode of NGF action would be same in all of them. The first step in the action of NGF in DRG and SG neurons is the binding of NGF to a cell surface receptor. The properties of the receptors on DRG and SG neurons are virtually identical [ 12, 13, 141. If the mode of action of NGF in PC 12 cells is similar to SG and DRG neurons then the properties of its receptor should be the same as those on the neuronal cell body. Indeed, some basic properties of the PC 12 receptor are similar to those reported for other NGF responsive cells. The binding is specific for the NGF protein; no other protein tested will compete with lz51-NGF for the PC 12 binding site. The binding of

in pheochromocytoma

cells

77

NGF is saturable, and seems to result from the interaction with a single class of binding sites. In addition, the binding is sensitive to trypsin treatment of the cells. These basic properties are the same as those reported previously for the SG and DRG cell body receptor [12, 13, 141. In several important aspects, however, the PC 12 receptor differs markedly from the others. The affinity of the PC 12 receptor (2.9~10~~ M) is a full order of magnitude lower than that reported by Herrup & Shooter [13] (2.6~10~lo M) for DRG neurons or by Banerjee et al. [12] (2x lo-lo M) for SG neurons. While these experiments were performed at room temperature and the present one at O.K, investigations with PC 12 cells at 22°C demonstrated (Herrup, unpublished observations) that the large disparity in KD was also present at this temperature. This affinity of 2.9~10~~ M is comparable to that reported by Sutter et al. [24] for the type II receptor in embryonic DRG’s (1.5~ lOa M) and to the middle of the range of affinities reported by Frazier et al. [ 141. The type II receptor of Sutter et al. [24] is reported to be non-neuronal in origin. The lack of any requirement for divalent cations also distinguishes the PC 12 NGF receptor from the neuronal type. In their study of SG neurons Banejee et al. [25] report that in the presence of EDTA, specific binding of NGF is reduced to zero. The experiments reported here show that for PC 12 the presence of 5 mM EDTA has no effect on the specific binding either below (20 nglml) near (60 nglml) or above (200 nglml) the K,,. This finding suggests that there are major chemical differences in the nature of the NGF binding reaction in PC 12 cells as compared with SG cells. It should be noted, however, that Banerjee et al. [12, 251 used the microsomal fraction of homogenized rabbit SG, while the work Exp

Cell

Res

121 (1979~

78

Herrup

and Thoenen

presented here was performed on whole living cells. Our failure to observe a calcium dependency might stem from these experimental differences. The effects of temperature on the binding of NGF to PC 12 cells were not persued in this study since our aim was to characterize the properties of the binding alone. The fact that at room temperature and 37°C the binding was more complex than simple association-dissociation suggests that there are further significant differences between the mode of NGF action on PC 12 and neuronal cells since such properties have not been reported in any other NGF binding study. The dissimilarity of the PC 12 and neuronal receptors with respect to affinity for NGF, requirement for divalent cations, and response to temperature suggest that beginning with the first step the systems reach similar responses (e.g. neurite outgrowth) by different means. This conclusion has important implications, limiting the use of PC 12 as a model for the action of NGF on SG and DRG neurons cell bodies, although it may be that normal adrenal medullary cells (which gave rise to the PC 12 line) have receptors which differ from the nerve cell body and are more similar to those on PC 12. One other possibility is that, analogous to the action of NGF following retrograde transport [cf 271 NGF must be internalized to be active in PC 12 cells and thus, receptor binding is simply a prerequisite for internalization and not per se an integral part of the mechanism of action.

1. Levi-Montalcini, R & Angeletti, P U, Physiol rev 48 (1968) 534. 2. Mobley, W C, Server, A C, Ishi, D N, Riopelle, R J & Shooter, E M, New eng j med 297 (1977) IO%, 1149, 1211. 3. Bradshaw, R A, Ann rev biochem 47 (1978) 191. 4. Thoenen, H, Angeletti, P U, Levi-Montalcini, R & Kettler, R, Proc natl acad sci US 68 (1971) 1598. 5. Otten, U, Schwab, M, Gagnon, C & Thoenen, H, Brain res 133 (1977) 291. 6. Green, L A & Tischler, A S, Proc natl acad sci US 73 (1976) 2424. 7. Green, L A & Rein, G, Brain res 129 (1977) 247. 8. - Nature 268 (1977) 349. 9. Schubert, D, Heineman, S & Kidokoro, Y, Proc natl acad sci US 74 (1977) 2579. 10. Edgar, D H & Thoenen; H, Brain res 154 (1978) 186. 11. Hatanaka, H, Otten, U & Thoenen, H, FEBS lett 92 (1978) 313. 12. Banejee, S P, Snyder, S H, Cuatrecasas, P & Green. L A. Proc natl acad sci US 70 (1973) 2519. 13. Herrup, K & Shooter, E M, Proc natl acad’sci US 70 (1973) 3884. 14. Frazier, W A, Boyd, L F & Bradshaw, R A, J biol them 249 (1974) 5513. 15. Suda, K, Bard& Y-A & Thoenen, H, Proc natl acad sci US 75 (1978) 4042. 16. Bocchini, V & kngeletti, P U, Proc natl acad sci US 64 (1969) 787. 17. Savaee. R & Cohen. S. J biol them 247 (1972), 7609,7612. ’ 18. Greenwood, F C, Hunter, W M & Glover, J S, Biochemistry 89 (1963) 114. 19. Marchalonis, J J, Biochem j 113 (1969) 299. 20. Stockel, K, Paravicini, U & Thoenen, H, Brain res 76 (1974) 413. 21. Kahn, C R, Neville, D M & Roth, U, J biol them 245 (1973) 244. 22. Gavin, J R, Roth, J, Neville, D M, DeMeyts, P & Buell, D N, Proc natl acad sci US 71 (1974) 84. 23. Leshiak, M A & Roth, J, J biol them 251 (1976) 3720. 24. Sutter, A, Riopelle, R J, Harris-Warrick, R M & Shooter, E M. Neurosci abst 3 (1977) 461. 25. Banetjee, S P, Cuatrecasas, P’& Snyder, S H, J biol them 250 (1975) 1427. 26. Green, L A, Brain res 133 (1977) 350. 27. Thoenen, H & Schwab, M, 7th Int congr pharmacol, Paris (1978). In press.

This work was supported by a US National Science Foundation National Needs Fellowship (SMI7712340).

Received October 5, 1978 Revised version received December 27, 1978 Accepted January 2, 1979

Exp Cell Res 121 (1979)

REFERENCES