Journal of Neurochemistry, 1977. Vol. 28, pp. 843-849. Pergamon Press. Printed in Great Britain.
THE DE NOVO SYNTHESIS OF TYROSINE HYDROXYLASE IN RAT SUPERIOR CERVICAL GANGLIA I N VITRO: THE EFFECT OF NERVE GROWTH FACTOR P. MACDONNELL,~ N. TOLSON,M. W. Yu and G. GUROFF Section on Intermediary Metabolism, Laboratory of Biomedical Sciences, National Institute of Child Health and Human Development, National Institutes of Health, Bet%sda, MD 20014, U.S.A. (Received 15 July 1976. Accepted 27 October 1976)
Abstract-An immunoprecipitation technique has been employed to measure the rate of synthesis of tyrosine hydroxylase in organ cultures of rat superior cervical ganglia and the effect of nerve growth factor on that rate. Ganglia which have been maintained in culture for 16 h without nerve growth factor synthesizetyrosine hydroxylase; the hydroxylase comprises approx 0.2%of the newly synthesized soluble protein. While the total amount of tyrosine hydroxylase synthesized de novo increases in the presence of physiological levels of nerve growth factor, the differential rate of tyrosine hydroxylase synthesis is essentially unchanged. At higher levels of nerve growth factor (3-10 pg/ml) there is a small increase in the differential rate of tyrosine hydroxylase synthesis. The major action of nerve growth factor appears to be on the survival of the tissue, but a small effect on the induction of tyrosine hydroxylase is evident at high levels of nerve growth factor. NERVEgrowth factor (NGF), a protein isolated from the submaxillary glands of adult male mice, causes hypertrophy and hyperplasia of the superior cervical ganglia (SCG) when injected into neonatal rats and & mice (LEVI-MONTALCINI, 1966; LEVI-MONTALCINI ANGELETTI, 1968). Along with these marked growth responses there is a concomitant and selective rise in the levels of tyrosine hydroxylase (TH) (L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2) and dopamine 8-hydroxylase (DBH) (3,4-dihydroxyphenylethylamine,ascorbate: oxygen oxidoreductase (8-hydroxylating); EC 1.14.17.1) (THOENEN et al., 1971). This increase appears to be specific since neither monoamine oxidase (MAO) (monoamine:oxygen oxidoreductase (deaminating):EC 1.4.3.4), DOPA decarboxylase (DDC) (3,4-dihydroxy-~-phenylalaninecarboxy-lyase; EC 4.1.1.28) (THOENEN et al., 1971), nor dihydropteridine et al., 1977), show a specific reductase (NIKODUEVIC increase. Such enzyme induction may reflect an action of NGF at the level of transcription or translation. In order to understand the action of NGF on the regulation of the norepinephrine pathway enzymes more clearly, we have studied the action of N G F on the levels of T H in the SCG in uitro under a number of defined conditions. This data appears in an accom-
' Supported during a part of this work by postdoctoral fellowship, NIH 1 F22 CA 04011-01. Abbreviations used: NGF, nerve growth factor; SCG, superior cervical ganglia; TH, tyrosine hydroxylase; DBH, dopamine B-hydroxylase; MAO, monoamine oxidase; DDC, DOPA decarboxylase; DOC, deoxycholate; PBS, phosphate-bufferedsaline. 843
panying paper (Yu et al., 1977). Under the conditions of those experiments, the major action of NGF appeared to be in maintaining viable neurons. However, the specific activity of TH did rise somewhat above the levels found in uiuo, i.e. induction of T H in uitro. To prove that NGF has a specific effect on T H synthesis in any system it would be desirable to measure the de nouo synthesis of the enzyme directly. In the present work we have used an immunological approach to measure the synthesis of TH in uitro, and we present here the results of experiments on the effect of NGF on this synthesis.
MATERIALS AND METHODS Methods. Rats of the Sprague-Dawley strain (ZivicMiller, Allison Park, PA) were killed by a blow to the head. The SCG and the nodose ganglia were removed and decapsulated by blunt dissection with the aid of a binocular microscope. Samples of the liver and kidney were cut into 1-2mg cubes; the adrenal medulla was freed of the overlying cortex. All tissues were cultured in 0.35 ml of BGJ, media, Fitton-Jackson modification, without phenol red, and supplemented with 0.1% bovine albumin fraction V and an antibiotic-antimycotic mixture which included penicillin, streptomycin, and fungizone at 100 units/ml, 100pg/ml, and 0.25 pg/ml, respectively. Tissues were maintained at 37°C in tissue culture clusters in a humidified atmosphere of 95% oxygen-5% carbon dioxide. At appropriate times tissues were removed from culture, rinsed in 0.25 M-sucrose, homogenized in ground glass homogenizers (Kontes Glass) with 5 mM-Tris, pH 7.4, containing 0.1% Triton X-100,and centrifuged at 20,ooOg for 20 min. In some experiments, homogenates were centri-
844
P.MACDONNELL, N. TOLSON,M. W. Yu and G. GUROFF
fuged at 150,OOOg for 60min in an SW 50.1 Spinco rotor. with PBS, each time being vigorously vortexed and microThe supernatant fluids were used for TH assay, immuno- fuged for 4min. precipitation of de nouo synthesized TH, and estimation SDS-polyacrylamide gel electrophoresis was performed of ['Hlleucine incorporation into total soluble protein. on 7.5% gels by the procedure of WEBERet al. (1972). The The activity of TH was determined by measuring the antigen-antibody pellet was solubilized by boiling for formation of 3H,0 from ~-[3,5-~H]tyrosineas described 15min in 100pI of a solution containing 1% SDS, 1% by NAGATSU et al. (1965) and modified by OESCHet al. 2-mercaptoethanol, 0.092% Bromphenol blue tracking dye, (1973).A unit is the amount of enzyme which will produce and 50% glycerol. Electrophoresis was carried out for 4 h 1 nmol 'H20 per h at 30°C. Protein was measured by at 8mA per gel. Gels were sliced into 1.2mm sections; the procedure of LOWRYet al. (1951) using bovine serum the slices were solubilized by heating at 55°C overnight albumin as standard. The efficiency of tritium counting in 1 ml portions of hydrogen peroxide containing 1% in Aquasol was 25-30% in all experiments. ammonium hydroxide, and counted. TH synthesis was The incorporation of ['Hlleucine into the total soluble quantitated by determining the counts under the peak. The protein of the ganglia was estimated as follows. To aliquots molecular weight of newly synthesized TH was determined of the 20,OOOg supernatant fluid were added 200pg of on SDS gels by comparison with a series of marker proteins. bovine serum albumin (BSA) and 1 ml of 10% TCA. After Materials. ~-[4,5-'H(N)]Leucine (specific activity of 60 15min at 0°C samples were filtered in a Yeda Filteriing Apparatus over GF/C glass fiber paper (Whatman). The Ci/mmol) and ~-[3,5-~H]tyrosine(specific activity of 60.3 filters were washed 5 times with 5 ml of 5% TCA and dried. Ci/mmol) were obtained from the New England Nuclear The precipitates were solubilized by heating the filters at Corp. Albumin, y-globulin, and cytochrome c were pur55°C for 2 h in l m l of 1% SDS, and were counted in chased from Schwarz Mann. Ovalbumin and ovalbumin antiserum were from Miles, lactic acid dehydrogenase was Aquasol. NGF and NGF antibody were prepared as described from Sigma Chemical Co., and carbonic anhydrase was in the previous paper (Yu et al., 1977). Monospecific anti- from Calbiochem. Culture media and the antibiotieantibody to TH was kindly supplied by Dr. T. LLOYD(Hershey Medical Center, Hershey, PA). Briefly, this antiserum was produced in sheep by repeated injections of a purified fragment of TH obtained by chymotryptic digestion of bovine adrenal medulla chromaffin granules (LLOYD& KAUFMAN, 1973). The y-globulin fraction of the serum was obtained by precipitation at 40% of saturation with ammonium sulfate, and was diluted to the original serum volume with 20 mM-KPO, buffer, pH 6.8, containing 0.3 M-glycine. Prior to immunoprecipitation the serum was centrifuged at 16,000 g for 4 min (Brinkman Microfuge) to remove insoluble protein. The antibody was previously shown (LLOYD& KAUFMAN, 1973) to cross-react with rat and bovine adrenal medulla TH and, in the present studies, with rat SCG TH (see Results section). The antigen-antibody equivalence point was determined as described by FEIGELSON & GREENGARD (1962). In a volume of 0.3 ml containing 0.5% Triton X-100, a constant volume of antiserum (50 or 100 pl) was incubated with increasing volumes of the supernatant fraction of SCG or adrenal medulla. After 1 h at 30°C and 16 h at 4°C samples were microfuged for 4min at 16,0009; TH activity was measured in aliquots of the supernatant fluid. One unit of enzyme was inactivated by approximately loop1 of reconstituted antiserum. TYROSINE HYDROXYLASE ADDED (units) Immunoprecipitation of newly synthesized radioactive TH was performed in a volume of 1.0ml in the presence FIG.1. Immunotitration of tyrosine hydroxylase of the rat of 1% Triton X-100, 1% freshly prepared sodium deoxy- superior cervical ganglia and adrenal medulla. In a reaccholate (DOC), and 500 pl of antiserum. Sufficient carrier tion volume of 0.3 ml, containing 0.5% Triton X-100, aliTH from the 150,OOOg supernatant fluid of rat adrenal quots of the 20,000 g supernatant fluid of detergent-treated glands was added to adjust the final concentration of TH tissues (see Materials and Methods) were added to 5 0 ~ 1 in the reaction mixtures to 2.5 units. Samples were incu- or 100 pl of antiserum. After 1 h at 30°C and 16 h at 4°C bated at 30°C for 1 h and then for 16 h at 4°C. The antisamples were centrifuged at 16,000 g for 4 min and aliquots gen-antibody precipitates were then collected using a of the supernatant fluid were assayed for T H activity. The modification of the procedures of RHOADS et al. (1973) and data has been corrected to represent a constant antibody concentration (100 pl). Each value represents a single titraof SIPPELet al. (1975). The sample was layered onto a tion. 0.5 ml sucrose cushion (1 M-sucrose, 150 mM-NaC1, 1% TriSymbols: 0 SCG from 7-day-old rats, 24 h in culture ton X-100, 10 mM-NaP04, pH 7.0) and centrifuged at without 2.5 S NGF. 0 SCG from 7-day-old rats, 24 h in 16,000 g for 15 min in an HB-4 Sorvall rotor. The top of culture with 2.5s NGF. A SCG from 8-day-old rats. A the sucrose cushion was gently rinsed twice with phosphate SCG from adult rats. 0 Adrenal medulla from 5-day-old buffered saline (PBS) (10 mM-NaPO,, pH 7.0, containing rats. Adrenal medulla from adult rats. 140 mM-NaC1): the pellet itself was then washed 2-3 times
Nerve growth factor and tyrosine hydroxylase synthesis in uitro mycotic mixture were purchased from Grand Island Biological Co.; tissue culture clusters were from Castor Plastics, Cambridge, MA, or from Microbiological Associates, Bethesda, MD. RESULTS Data presented in the previous paper (Yu et al., 1977) showed that rat superior cervical ganglia, in culture for 48 h, exhibit a loss of TH activity when maintained in NGF-free media. In media supplemented with NGF some increase in the total and the specific activity of TH is, in fact, observed. In order to determine if this increase in the activity of TH in ganglia cultured with NGF is due to more TH or to an increase in the activity of existing TH, immunoinhibition studies were performed. Titration of TH from day 7 and from adult rat ganglia, as well as from ganglia cultured in the presence or the absence of NGF (1 pg/ml) for 16 h, were performed by the addition of a constant volume of antiserum to increasing volumes of ganglia supernatant fluid. The data in Fig. 1 illustrates that the equivalence points from all the ganglia are the same, indicating that TH is immunologically identical in young, adult, and cultured ganglia. Similar observations have been made by BLACKet al. (1974). The fact that the equivalence points of the various plots are identical also shows that the increased activity of TH in the presence of NGF (Yu et al., 1977) represents an increase in the number of enzyme molecules (FEIGELSON & GREENGARD, 1962). In addition, the fact that the equivalence points of TH from the adrenal medulla of young and of adult rats is identical to that of TH from ganglia shows that TH is antigenically identical in the two tissues. Most of the tissue TH is found in the 20,000g supernatant fluid of the detergent-treated tissues. At least SS% of ganglionic TH and 68% of medullary TH is present in the solubilized portion of the homogenate. The actual numbers, in nmol/h per mg tissue, were as follows: superior cervical ganglia, 0.303 f 0.029 soluble, 0.042 & 0.009 particulate; adrenal medulla: 0.204 0.049 soluble, 0.098 f 0.007 particulate. No activity was found in either the soluble or the particulate fractions of liver, kidney, or nodose ganglia. Ganglia cultured for 16 h in the presence of NGF (1 pg/ml) and 50 pCi C3H]leucine are capable of synthesizing TH de nouo as illustrated in Fig. 2(a). After immunoprecipitation and solubilization, only one major peak of radioactivity is present on SDS polyacrylamide gels; the area under the peak in Fig. 2(a) corresponds to approx 0.243% of the newly synthesized protein (Table 1). Essentially all of the newly synthesized TH is removed by immunoprecipitation. No additional radioactive peak corresponding to that in Fig. 2(a) is detected if a second immunoprecipitate is collected after the addition of carrier TH and fresh antiserum to the supernatant fluid (Fig. 2(b)).
845
All immunological experiments were performed in a range of protein and of TH activity (0.4-4.0 units) within which the radioactivity in the TH peak was proportional to the amount of SCG protein added (Fig. 3). The minimum amount of enzyme necessary for complete precipitation by antibody is about 0.4 units. Approximately 0.2 and 0.6 units of enzyme are present in a single 5-8 day rat SCG and adrenal medulla, respectively. All immunological reactions contained sufficient carrier TH to bring the total TH to between 0.4 and 4.0 units of enzyme when 500p1 of antibody was present. Substantial radioactivity is also found spread over other regions of the gel, precluding the use of an assay based upon a direct measurement of the radioactivity in the antigen-antibody precipitate. Nonspecific trapping of proteins in immunologically precipitated samples can be a potentially serious problem if the samples are not purified by gel electrophoresis prior to quantitation, especially when a low titer of antibody requires the use of large amounts of serum. One way to overcome the problem of contamination is to purify the immune complex, as in the present work, by SDS-polyacrylamide gel electrophoresis. In the present work, the extent of protein contamination in immunologically isolated samples varied considerably depending upon the tissue, but was relatively uniform in the same tissue. For example, in immunoprecipitated samples from the SCG only 3040% of the pro-
+
l o r
0
10 FRACTION NUMBER
FIG.2. Sodium didecyl sulfatepolyacrylamide gel electrophoretic profile of immunoprecipitated TH synthesized in
superior cervical ganglia in culture. Ganglia from 7-dayold rats were maintained in culture for 16 h in the presence of 1 jig/ml of 2.5 S NGF and 50 pCi of [3H]leucine. (a) The soluble protein (20,243 x c.p.m./mg protein) from 12 ganglia (containing 2.5 units of TH and 382pg of protein) was immunoprecipitated with 5 0 0 ~ 1of immune serum in a volume of 1.0 ml; (b) after the immunoprecipitate was collected the supernatant fluid was reimmunoprecipitated with 500j11 of immune serum and 1.3 units of rat adrenal TH as carrier (final volume of 2.0 ml). Direction of migration is from left to right.
P. MACDONNELL, N. TOLSON, M. W.Y U and G. GURCIFF
846
TABLE1. SYNTHESIS OF TYROSINE
HYDROXYLASE IN RAT TISSUES IN CULTURE
Radioactivity in: Soluble protein TH (c.p.m./mg protein) x lo-' 25,752 F 5,906 (20) 12,852 8475 5347 14,066
Superior cervical ganglia Nodose ganglia Liver Kidney
7789 Adrenal medulla ~
~
10,226 9222
69.8 F 13.8 (14) 0 0 0 0
0 124.8 & 4.7 (6) 109.2 5.2 (4)
% Radioactivity in TH 0.27 0 0 0 0 0 1.22 1.18
~~~
Tissues from 5-7-day-old rats were cultured for 16-18 h in BGJ, media containing 1pg/ml of 2.5 S NGF (SCG and nodose ganglia only) and 50 pCi C3H]leucine (all tissues). The concentration of leucine in BGJ, medium is 0.38 mM; thus, the specific activity of C3H]leucine is 0.44 Ci/mmol. The mean weight of the explants was 0.58, 0.40, 1.3, 1.7, and 1.8 mg for SCG, nodose ganglia, liver, kidney, and adrenal medulla, respectively. Tissues were homogenized in 5 mM-Tris, pH 7.4, containing 0.1% Triton X-100, and centrifuged at either 20,000g or 150,000g (no difference was observed in the radioactivity of the 20,OOOg or 150,OOOg supernatant fluids). Carrier TH was added to aliquots of each supernatant fluid to a final concentration of 1.3-2.5 units, and TH was immunologically precipitated with a 24-fold excess of antiserum (500 PI) and purified by SDS-polyacrylamide gel electrophoresis. The SCG values are . number of observations in brackets), each experiment expressed as means of the individual experiments ~ s . D (the containing a pool of 15-50 ganglia. Radioactivity in the nodose ganglia, liver, and kidney represents individual experiments each containing a pool of 7-30 explants. The adrenal medulla results represent the radioactivity from 2 experiments each containing a pool of 1623 medullae; various aliquots of each supernatant fluid were immunoprecipitated with 500 pl of antiserum. The S.D. of TH in the adrenal medulla samples illustrates the reproducibility of the immunoprecipitation technique in 2 samples each of which employed a 4-6-fold difference in radioactivity in the immunological reaction mixture. tein in the antigen-antibody precipitate was T H (calculated by determining the area under the peak of SDS gels) while in the adrenal medulla samples 60-70% of the immunoprecipitated protein was identified as TH. Attempts to remove these contaminating proteins were only partially effective. Immunoprecipitation reactions conducted in the presence of 1% Triton X-100 104 DOC were far superior to those
+
pg PROTEIN
FIG. 3. Quantitation of immunologically precipitated de now synthesized TH in SCG cultures. Ganglia were cultured as described in Fig. 2. Aliquots of SCG soluble protein (31,569 x 1 O - j c.p.m./mg protein) were immunoprecipitated with 5OOpI of antiserum in a volume of 1.0ml. Antigen-antibody precipitates were collected by centrifugation, thoroughly, washed with PBS, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioactivity in TH from each gel was quantitated by subtracting the background radioactivity from the peak area.
done in the presence of either 0.5% Triton X-100, 1% Triton X-100, or 1% Triton X-100 0.5 NaCl (data not shown). Contamination was further diminished by collecting the antigen-antibody precipitate over a 1 M-sucrose cushion as described by RHOADSet al. (1973) and SIPPELet al. (1975). Because of this nonspecific trapping of proteins in the immune complex all immunoprecipitates were routinely purified by SDS-polyacrylamide gel electrophoresis prior to quantitation of TH. The immunoprecipitation reaction is specific to T H in that the addition of 4 pg of ovalbumin and sufficient ovalbumin antiserum did not precipitate any significant amounts of radioactivity corresponding to the position of TH. After the ovalbumin-antiovalbumin complex is removed by centrifugation ['HlTH can still be precipitated from solution by addition of antiserum to TH. The synthesis of T H in culture occurred only in those tissues that contain T H in viuo (Table 1). No radioaetive peak corresponding to T H (Fig. 4(a)) is detected on SDS gels after liver, kidney, and nodose ganglia were cultured for 16 h with 50 pCi of C3H]leucine and then immunoprecipitated with nonradioactive carrier T H and antiserum (Fig. 4(b,c,d)). The only tissue, other than the SCG, which synthesized significant amounts of TH in culture is the adrenal medulla (Fig. 4(e)). Although the incorporation of [3H]leucine into the soluble protein of the adrenal medulla was considerably less than into ganglia protein, the differential rate of TH synthesis was approx 4 times greater (Table 1). The apparent molecular weight of newly synthesized T H from both the SCG and adrenal medulla
+
Nerve growth factor and tyrosine hydroxylase synthesis in vitro
K
2
847
H Chain, y Globulin Ovalbumin
4l
Carbonic Anhydrase
L Chain, yGlobulin
P x
z
u
'Orc 5
0
0.2
0.4
0.6
0.8
1.0
MOBILITY
FRACTION NUMBER
FIG.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic profiles of immunoprecipitated TH from rat tissues in culture. Superior cervical ganglia, nodose ganglia (both in the presence of 1 pg/ml of 2.5 S NGF), liver, kidney, and adrenal medulla from 5-day-old rats were cultured as described in Fig. 2, and in the Materials and Methods section. De nouo synthesized TH was immunoprecipitated with 500 pl antiserum in a volume of 1.0 ml; 1.3 units of nonradioactive carrier TH from rat adrenal medulla was added to the liver, kidney, and nodose ganglia immune reactions. (a) 357pg protein (31,569 x c.p.m./mg protein) containing 3.2 units of TH from 15 superior cervical ganglia; (b) 158pg of liver protein c.p.m./mg protein); (c) 227 pg of kidney pro(5344 x tein (7786 x c.p.m./mg protein); (d) 91 pg of nodose ganglia protein (12,852 x c.p.m./mg protein); and (e) 134pg of adrenal medulla protein (9215 x c.p.m./mg protein) containing 3.7 units of TH. Direction of migration is from left to right. was approx 6M5,OOO (Fig. 5). This data was obtained by the procedure of WEBERet al. (1972) for SDS gel electrophoresis after dissociation of the immune complex by boiling in 1% SDS and 1% 2-mercaptoethanol. Protein synthesis in ganglia cultured under these conditions is increased by the presence of N G F in the medium (Table 2). The maximum incorporation of C3H]leucine into soluble protein was at lpg/ml N G F ; at this N G F concentration approximately twice as much incorporation was observed as in those ganglia which did not receive NGF. At concentrations greater than 1 pg/ml of N G F soluble protein synthesis was depressed and, in fact, at lOpg/ml of N G F the extent of protein synthesis was essentially identical to that seen in ganglia cultured either without N G F or with N G F antibody (Table 2).
FIG. 5. Determination of the molecular weight of newly synthesized TH from SCG and adrenal medulla in organ culture. Ganglia and adrenal medulla from 5-day-old rats were cultured as in Fig. 2. Tyrosine hydroxylase from the SCG and adrenal medulla was immunoprecipitated with 500 pl antiserum in a volume of 1.0 ml. Immune precipitates and standard proteins were dissociated by boiling for 15min in 1% SDS + 1% 2-mercaptoethanol (see Materials and Methods section) prior to SDS-polyacrylamide gel electrophoresis. After the gels were stained and destained the mobilities of the standard proteins were determined. Radioactive gels were then sliced, care being taken to match protein bands to individual slices; gel slices were solubilized for counting as described in the Materials and Methods section. The molecular weight of TH from the SCG and adrenal medulla was calculated to be approx 60,oo(r65,000. The absolute synthesis of T H increased in proportion to the synthesis of soluble protein (compare columns 2 and 3, Table 2) up to a concentration of 3pg/ml of NGF. However, the ratio of TH synthesized to total protein synthesized (column 4, Table 2) did not vary appreciably in ganglia cultured without NGF, with N G F at concentrations of 0.05-1 pg/ml, or with N G F antibody. Somewhat higher levels of TH synthesis were observed at 3 pg/ml and substantially higher rates of T H synthesis were repeatedly observed at 10 pg/ml of NGF.
DISCUSSION The culture of rat superior cervical ganglia in oitro offers many advantages to the conventional in vivo studies for investigating tyrosine hydroxylase regulation. In a chemically defined medium one can manipulate the environment of the SCG and measure the resultant synthesis of TH in response to exogenously added agents. Similarly, interpretation of the data is simplified inasmuch as the normal regulatory events occurring in oioo are absent in culture. The disadvantages of such a system are obvious in that damage to the adrenergic neurones may result from transection of the preganglionic cholinergic fibers. Likewise, lack of suitable serum and growth promoting factors, and differences in the normal metabolite concentrations may play an important role in the normal
P. MACDONNELL, N. TOLSON, M. W. Yu and G. GUROFF
848
TABLE2. THEEFFECT OF NGF NGF concentration (Pg/ml)
ON THE
de nouo
16,862 2 1950 17,668 f 2701
0.33
22,176 28,754 29,955 & 6403 23,762 19,171 16,802 5 2942 14,351 13,328
10.00 0 + NGF antibody
TH
I N SUPERIOR CERVICAL GANGLIA IN CULTURE
Radioactivity in: Soluble protein TH (c.p.m./mg protein x 10- 7
0 0.05
1.oo 3.00
SYNTHESIS OF
25.2 k 4.1 36.5 31.6 36.0 53.3 44.2 k 3.0 50.0 58.7 50.5 k 7.9 28.1 19.7
”/, Radioactivity in enzyme
*
0.15 0.03 0.18 0.22 0.16 0.19 0.16 0.05 0.21 0.30 0.30 k 0.02 0.20 0.15
*
Ganglia, in groups of 3, were maintained in culture for 20 h in the presence of 50 pCi [3H]leucine and the indicated concentrations of 2.5 S NGF or 115 pg of NGF antibody. Ganglia were processed as described in Materials and Methods and 2.3 units of carrier TH and 500 p1 of antiserum were added to the supernatant fluid from 1 ganglion; immunoprecipitated TH was purified by SDS gel electrophoresis. The values represent the means S.D. of 3. experiments; other values are from individual experiments.
regulation of T H synthesis during maturation of the SCG. These factors cannot be accurately duplicated in uitro. With the above in mind, in the previous paper (Yu et al., 1977) we attempted to define the action of N G F on the levels of T H in SCG. The data presented indicated that N G F had some action on TH induction independent of its overall action in allowing the survival of adrenergic neurons. To determine the actual extent of TH induction it is necessary to measure the true rate of the synthesis of T H in relation to total synthesized protein. We have followed this approach by immunologically isolating the T H synthesized de novo. The present experiments show that T H is being synthesized in SCGs and adrenal medulla in organ culture. The evidence that the radioactive protein being measured is, in fact, T H is as follows: (1) The TH antiserum used in the present study is monospecific, having been raised to a pure chymotryptic fragment of bovine adrenal medulla TH. (2) The antibody completely inhibits TH enzyme activity from rat SCG’s and adrenal medulla while comparable amounts of control serum have no effect on enzyme activity. (3) The radioactive peak is only present in immunoprecipitates derived from soluble proteins of the SCG and adrenal medulla. N o radioactive peaks corresponding to T H are observed when the soluble proteins of liver, kidney, or nodose ganglia are immunoprecipitated in the presence of carrier TH. (4) The formation of an ovalbumin-antiovalbumin precipitate does not precipitate the major radioactive peak which can be subsequently precipitated by T H antibody. ( 5 ) After the immunoprecipitate from ganglia is collected no additional radioactive peak is seen on SDS gels after nonradioactive carrier T H and fresh TH antibody were added to the supernatant fluid.
(6) In experiments not presented here, it has been shown that immunoprecipitation of radioactive TH from ganglia is abolished when purified carrier bovine adrenal medulla T H is present in excess in the immunological reaction. Native TH has been difficult to purify and purification from ganglia is not feasible. Thus, n o satisfactory marker is available for the SDS gels. It seems clear, however, on the basis of the evidence cited above that the synthesis of T H is being measured here. A report by CHUANG et al. (1975) has appeared in which the de nouo synthesis of TH in adrenal in uiuo has been evaluated by techniques similar to those used here. The amount of T H synthesized by adrenal in that study appears to be somewhat higher than that reported in the present data. The newly synthesized T H had two subunits with molecular weights of 33,000 and 38,000. The reason for the difference in these parameters is not clear, but it could be due to the minor differences in our preparative techniques, or to the fact that the previous work was done in adults in uiuo while our experiments were done with tissues from young rats in uitro. This question is currently being explored. The data in this paper is consistent with that from the previous work (Yu ef at., 1977). In the previous paper it was shown that the total activity of T H of ganglia cultured in the presence of N G F is greater than that in its absence. Here we show that the total T H synthesis is greater also, as is the total incorporation of radioactive amino acid into protein. Further, it was shown that there is a small increase in the specific activity of the enzyme at maximal levels of NGF. Here we show that at very high levels of N G F there is a small increase in the differential rate of synthesis of TH. Whether this small increase at high N G F levels is a reflection of the in vivo action of N G F is still an open question. The interpretation of the present data is consistent
Nerve growth factor and tyrosine hydroxylase synthesis in vitro
849
REFERENCES with that advanced previously. The action of N G F in this system is primarily on survival of the TH-conBLACKI. B., JOH T. H. & REIS D. J. (1974) Bruin Res. taining neurons (LEVI-MONTALCINI, 1966 ; LEVI-MON75, 133-144. TALCINI & ANGELETTI, 1968). There seems, in addition, CHUANG D., ZSILLA G. & COSTAE. (1975) Molec. Pharmac. to be a small selective action on T H synthesis, es11, 784794. pecially at high levels of NGF. This conclusion was FEIGELSON P. & GREENGARD 0. (1962) J . biol. Chem. 237, also reached by STICKGOLD & SHOOTER (1974) on 37163717. R. (1966) Harvey Lect. 60, 217-259. the basis of TH activity measurements reported, so LEVI-MONTALCINI R. & ANGELETTI P. U. (1968) Physiol. far, only in preliminary form. Although the present LEVI-MONTALCINI Rev. 48, 536569. system would seem to be of substantial value for studies on the mechanism of N G F action on the sur- LLOYDT. & KAUFMANS. (1973) Molec. Pharmac. 9, 438444. vival of sympathetic neurons, the action of NGF on LOWRY0.H., ROSEBROUGHN. J., FARRA. L. & RANDALL TH in uitro does not seem to be comparable to its R. J. (1951) J . biol. Chem. 193, 265-275. action on TH in uiuo. NAGATSU T., LEVITT M. & UDENFRIEND S. (1964) Analyt. That the conclusions from this and the previous Biochem. 9, 122-126. paper (Yu et al., 1977) are valid is borne out by recent NIKODIJEVIC B., Yu M. W. & GUROFF G. (1977) J . Neuroexperiments using a different protocol (MACDONNELL chern. 28, 851-852. H. (1973) J . Neurochem. et al., in preparation). When NGF is given in uiuo OESCHF., OTTENU. & THOENEN 20, 1691-1706. and TH synthesis measured subsequently in uitro the G. S. & SCHIMKE R. T . (1973) differential rate of TH synthesis is much greater than RHOADSR. E., MCKNIGHT J . biol. Chem. 248, 2031-2039. when NGF is given in uitro as described in the present A. E., FEIGELSON P. & ROY A. K. (1975) Biochemiswork. Thus, the action of NGF on TH synthesis in SIPPEL try 14, 825-829. uiuo is in some way different than it is in uitro. HopeSTICKWLD R. & SHOOTER E. M. (1974) Fedn Proc. Fedn fully, this explant system will provide an approach Am. SOCS exp. Biol. 33, 1495. to an understanding of the mechanism by which NGF THOENENH., ANGELETTI P. U., LEVI-MONTALCINI R. & influences the specific synthesis of TH. KETTLER R. (1971) Proc. Natn. Acad. Sci., U.S.A. 68,
Acknowledgement-The continued interest and advice of Dr. T . LLOYDis appreciated.
1598-1602. WERER K.. PRINGLE J. R . & OSBORN M. (1972) in Methods in Enzymology (HIRSC. H. W. & TIMASHEFF S. N.., eds.) 26, 3-27. Yu M. W., NIKODIJEVIC B., LAKSHMANAN J., ROWE V., MACDONNELL P. & GUROFFG. (1977) J . Neurochem. 28, 843-849.
THE DE NOVO SYNTHESIS OF TYROSINE HYDROXYLASE IN RAT SUPERIOR CERVICAL GANGLIA I N VITRO: THE EFFECT OF NERVE GROWTH FACTOR P. MACDONNELL,~ N. TOLSON,M. W. Yu and G. GUROFF Section on Intermediary Metabolism, Laboratory of Biomedical Sciences, National Institute of Child Health and Human Development, National Institutes of Health, Bet%sda, MD 20014, U.S.A. (Received 15 July 1976. Accepted 27 October 1976)
Abstract-An immunoprecipitation technique has been employed to measure the rate of synthesis of tyrosine hydroxylase in organ cultures of rat superior cervical ganglia and the effect of nerve growth factor on that rate. Ganglia which have been maintained in culture for 16 h without nerve growth factor synthesizetyrosine hydroxylase; the hydroxylase comprises approx 0.2%of the newly synthesized soluble protein. While the total amount of tyrosine hydroxylase synthesized de novo increases in the presence of physiological levels of nerve growth factor, the differential rate of tyrosine hydroxylase synthesis is essentially unchanged. At higher levels of nerve growth factor (3-10 pg/ml) there is a small increase in the differential rate of tyrosine hydroxylase synthesis. The major action of nerve growth factor appears to be on the survival of the tissue, but a small effect on the induction of tyrosine hydroxylase is evident at high levels of nerve growth factor. NERVEgrowth factor (NGF), a protein isolated from the submaxillary glands of adult male mice, causes hypertrophy and hyperplasia of the superior cervical ganglia (SCG) when injected into neonatal rats and & mice (LEVI-MONTALCINI, 1966; LEVI-MONTALCINI ANGELETTI, 1968). Along with these marked growth responses there is a concomitant and selective rise in the levels of tyrosine hydroxylase (TH) (L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2) and dopamine 8-hydroxylase (DBH) (3,4-dihydroxyphenylethylamine,ascorbate: oxygen oxidoreductase (8-hydroxylating); EC 1.14.17.1) (THOENEN et al., 1971). This increase appears to be specific since neither monoamine oxidase (MAO) (monoamine:oxygen oxidoreductase (deaminating):EC 1.4.3.4), DOPA decarboxylase (DDC) (3,4-dihydroxy-~-phenylalaninecarboxy-lyase; EC 4.1.1.28) (THOENEN et al., 1971), nor dihydropteridine et al., 1977), show a specific reductase (NIKODUEVIC increase. Such enzyme induction may reflect an action of NGF at the level of transcription or translation. In order to understand the action of NGF on the regulation of the norepinephrine pathway enzymes more clearly, we have studied the action of N G F on the levels of T H in the SCG in uitro under a number of defined conditions. This data appears in an accom-
' Supported during a part of this work by postdoctoral fellowship, NIH 1 F22 CA 04011-01. Abbreviations used: NGF, nerve growth factor; SCG, superior cervical ganglia; TH, tyrosine hydroxylase; DBH, dopamine B-hydroxylase; MAO, monoamine oxidase; DDC, DOPA decarboxylase; DOC, deoxycholate; PBS, phosphate-bufferedsaline. 843
panying paper (Yu et al., 1977). Under the conditions of those experiments, the major action of NGF appeared to be in maintaining viable neurons. However, the specific activity of TH did rise somewhat above the levels found in uiuo, i.e. induction of T H in uitro. To prove that NGF has a specific effect on T H synthesis in any system it would be desirable to measure the de nouo synthesis of the enzyme directly. In the present work we have used an immunological approach to measure the synthesis of TH in uitro, and we present here the results of experiments on the effect of NGF on this synthesis.
MATERIALS AND METHODS Methods. Rats of the Sprague-Dawley strain (ZivicMiller, Allison Park, PA) were killed by a blow to the head. The SCG and the nodose ganglia were removed and decapsulated by blunt dissection with the aid of a binocular microscope. Samples of the liver and kidney were cut into 1-2mg cubes; the adrenal medulla was freed of the overlying cortex. All tissues were cultured in 0.35 ml of BGJ, media, Fitton-Jackson modification, without phenol red, and supplemented with 0.1% bovine albumin fraction V and an antibiotic-antimycotic mixture which included penicillin, streptomycin, and fungizone at 100 units/ml, 100pg/ml, and 0.25 pg/ml, respectively. Tissues were maintained at 37°C in tissue culture clusters in a humidified atmosphere of 95% oxygen-5% carbon dioxide. At appropriate times tissues were removed from culture, rinsed in 0.25 M-sucrose, homogenized in ground glass homogenizers (Kontes Glass) with 5 mM-Tris, pH 7.4, containing 0.1% Triton X-100,and centrifuged at 20,ooOg for 20 min. In some experiments, homogenates were centri-
844
P.MACDONNELL, N. TOLSON,M. W. Yu and G. GUROFF
fuged at 150,OOOg for 60min in an SW 50.1 Spinco rotor. with PBS, each time being vigorously vortexed and microThe supernatant fluids were used for TH assay, immuno- fuged for 4min. precipitation of de nouo synthesized TH, and estimation SDS-polyacrylamide gel electrophoresis was performed of ['Hlleucine incorporation into total soluble protein. on 7.5% gels by the procedure of WEBERet al. (1972). The The activity of TH was determined by measuring the antigen-antibody pellet was solubilized by boiling for formation of 3H,0 from ~-[3,5-~H]tyrosineas described 15min in 100pI of a solution containing 1% SDS, 1% by NAGATSU et al. (1965) and modified by OESCHet al. 2-mercaptoethanol, 0.092% Bromphenol blue tracking dye, (1973).A unit is the amount of enzyme which will produce and 50% glycerol. Electrophoresis was carried out for 4 h 1 nmol 'H20 per h at 30°C. Protein was measured by at 8mA per gel. Gels were sliced into 1.2mm sections; the procedure of LOWRYet al. (1951) using bovine serum the slices were solubilized by heating at 55°C overnight albumin as standard. The efficiency of tritium counting in 1 ml portions of hydrogen peroxide containing 1% in Aquasol was 25-30% in all experiments. ammonium hydroxide, and counted. TH synthesis was The incorporation of ['Hlleucine into the total soluble quantitated by determining the counts under the peak. The protein of the ganglia was estimated as follows. To aliquots molecular weight of newly synthesized TH was determined of the 20,OOOg supernatant fluid were added 200pg of on SDS gels by comparison with a series of marker proteins. bovine serum albumin (BSA) and 1 ml of 10% TCA. After Materials. ~-[4,5-'H(N)]Leucine (specific activity of 60 15min at 0°C samples were filtered in a Yeda Filteriing Apparatus over GF/C glass fiber paper (Whatman). The Ci/mmol) and ~-[3,5-~H]tyrosine(specific activity of 60.3 filters were washed 5 times with 5 ml of 5% TCA and dried. Ci/mmol) were obtained from the New England Nuclear The precipitates were solubilized by heating the filters at Corp. Albumin, y-globulin, and cytochrome c were pur55°C for 2 h in l m l of 1% SDS, and were counted in chased from Schwarz Mann. Ovalbumin and ovalbumin antiserum were from Miles, lactic acid dehydrogenase was Aquasol. NGF and NGF antibody were prepared as described from Sigma Chemical Co., and carbonic anhydrase was in the previous paper (Yu et al., 1977). Monospecific anti- from Calbiochem. Culture media and the antibiotieantibody to TH was kindly supplied by Dr. T. LLOYD(Hershey Medical Center, Hershey, PA). Briefly, this antiserum was produced in sheep by repeated injections of a purified fragment of TH obtained by chymotryptic digestion of bovine adrenal medulla chromaffin granules (LLOYD& KAUFMAN, 1973). The y-globulin fraction of the serum was obtained by precipitation at 40% of saturation with ammonium sulfate, and was diluted to the original serum volume with 20 mM-KPO, buffer, pH 6.8, containing 0.3 M-glycine. Prior to immunoprecipitation the serum was centrifuged at 16,000 g for 4 min (Brinkman Microfuge) to remove insoluble protein. The antibody was previously shown (LLOYD& KAUFMAN, 1973) to cross-react with rat and bovine adrenal medulla TH and, in the present studies, with rat SCG TH (see Results section). The antigen-antibody equivalence point was determined as described by FEIGELSON & GREENGARD (1962). In a volume of 0.3 ml containing 0.5% Triton X-100, a constant volume of antiserum (50 or 100 pl) was incubated with increasing volumes of the supernatant fraction of SCG or adrenal medulla. After 1 h at 30°C and 16 h at 4°C samples were microfuged for 4min at 16,0009; TH activity was measured in aliquots of the supernatant fluid. One unit of enzyme was inactivated by approximately loop1 of reconstituted antiserum. TYROSINE HYDROXYLASE ADDED (units) Immunoprecipitation of newly synthesized radioactive TH was performed in a volume of 1.0ml in the presence FIG.1. Immunotitration of tyrosine hydroxylase of the rat of 1% Triton X-100, 1% freshly prepared sodium deoxy- superior cervical ganglia and adrenal medulla. In a reaccholate (DOC), and 500 pl of antiserum. Sufficient carrier tion volume of 0.3 ml, containing 0.5% Triton X-100, aliTH from the 150,OOOg supernatant fluid of rat adrenal quots of the 20,000 g supernatant fluid of detergent-treated glands was added to adjust the final concentration of TH tissues (see Materials and Methods) were added to 5 0 ~ 1 in the reaction mixtures to 2.5 units. Samples were incu- or 100 pl of antiserum. After 1 h at 30°C and 16 h at 4°C bated at 30°C for 1 h and then for 16 h at 4°C. The antisamples were centrifuged at 16,000 g for 4 min and aliquots gen-antibody precipitates were then collected using a of the supernatant fluid were assayed for T H activity. The modification of the procedures of RHOADS et al. (1973) and data has been corrected to represent a constant antibody concentration (100 pl). Each value represents a single titraof SIPPELet al. (1975). The sample was layered onto a tion. 0.5 ml sucrose cushion (1 M-sucrose, 150 mM-NaC1, 1% TriSymbols: 0 SCG from 7-day-old rats, 24 h in culture ton X-100, 10 mM-NaP04, pH 7.0) and centrifuged at without 2.5 S NGF. 0 SCG from 7-day-old rats, 24 h in 16,000 g for 15 min in an HB-4 Sorvall rotor. The top of culture with 2.5s NGF. A SCG from 8-day-old rats. A the sucrose cushion was gently rinsed twice with phosphate SCG from adult rats. 0 Adrenal medulla from 5-day-old buffered saline (PBS) (10 mM-NaPO,, pH 7.0, containing rats. Adrenal medulla from adult rats. 140 mM-NaC1): the pellet itself was then washed 2-3 times
Nerve growth factor and tyrosine hydroxylase synthesis in uitro mycotic mixture were purchased from Grand Island Biological Co.; tissue culture clusters were from Castor Plastics, Cambridge, MA, or from Microbiological Associates, Bethesda, MD. RESULTS Data presented in the previous paper (Yu et al., 1977) showed that rat superior cervical ganglia, in culture for 48 h, exhibit a loss of TH activity when maintained in NGF-free media. In media supplemented with NGF some increase in the total and the specific activity of TH is, in fact, observed. In order to determine if this increase in the activity of TH in ganglia cultured with NGF is due to more TH or to an increase in the activity of existing TH, immunoinhibition studies were performed. Titration of TH from day 7 and from adult rat ganglia, as well as from ganglia cultured in the presence or the absence of NGF (1 pg/ml) for 16 h, were performed by the addition of a constant volume of antiserum to increasing volumes of ganglia supernatant fluid. The data in Fig. 1 illustrates that the equivalence points from all the ganglia are the same, indicating that TH is immunologically identical in young, adult, and cultured ganglia. Similar observations have been made by BLACKet al. (1974). The fact that the equivalence points of the various plots are identical also shows that the increased activity of TH in the presence of NGF (Yu et al., 1977) represents an increase in the number of enzyme molecules (FEIGELSON & GREENGARD, 1962). In addition, the fact that the equivalence points of TH from the adrenal medulla of young and of adult rats is identical to that of TH from ganglia shows that TH is antigenically identical in the two tissues. Most of the tissue TH is found in the 20,000g supernatant fluid of the detergent-treated tissues. At least SS% of ganglionic TH and 68% of medullary TH is present in the solubilized portion of the homogenate. The actual numbers, in nmol/h per mg tissue, were as follows: superior cervical ganglia, 0.303 f 0.029 soluble, 0.042 & 0.009 particulate; adrenal medulla: 0.204 0.049 soluble, 0.098 f 0.007 particulate. No activity was found in either the soluble or the particulate fractions of liver, kidney, or nodose ganglia. Ganglia cultured for 16 h in the presence of NGF (1 pg/ml) and 50 pCi C3H]leucine are capable of synthesizing TH de nouo as illustrated in Fig. 2(a). After immunoprecipitation and solubilization, only one major peak of radioactivity is present on SDS polyacrylamide gels; the area under the peak in Fig. 2(a) corresponds to approx 0.243% of the newly synthesized protein (Table 1). Essentially all of the newly synthesized TH is removed by immunoprecipitation. No additional radioactive peak corresponding to that in Fig. 2(a) is detected if a second immunoprecipitate is collected after the addition of carrier TH and fresh antiserum to the supernatant fluid (Fig. 2(b)).
845
All immunological experiments were performed in a range of protein and of TH activity (0.4-4.0 units) within which the radioactivity in the TH peak was proportional to the amount of SCG protein added (Fig. 3). The minimum amount of enzyme necessary for complete precipitation by antibody is about 0.4 units. Approximately 0.2 and 0.6 units of enzyme are present in a single 5-8 day rat SCG and adrenal medulla, respectively. All immunological reactions contained sufficient carrier TH to bring the total TH to between 0.4 and 4.0 units of enzyme when 500p1 of antibody was present. Substantial radioactivity is also found spread over other regions of the gel, precluding the use of an assay based upon a direct measurement of the radioactivity in the antigen-antibody precipitate. Nonspecific trapping of proteins in immunologically precipitated samples can be a potentially serious problem if the samples are not purified by gel electrophoresis prior to quantitation, especially when a low titer of antibody requires the use of large amounts of serum. One way to overcome the problem of contamination is to purify the immune complex, as in the present work, by SDS-polyacrylamide gel electrophoresis. In the present work, the extent of protein contamination in immunologically isolated samples varied considerably depending upon the tissue, but was relatively uniform in the same tissue. For example, in immunoprecipitated samples from the SCG only 3040% of the pro-
+
l o r
0
10 FRACTION NUMBER
FIG.2. Sodium didecyl sulfatepolyacrylamide gel electrophoretic profile of immunoprecipitated TH synthesized in
superior cervical ganglia in culture. Ganglia from 7-dayold rats were maintained in culture for 16 h in the presence of 1 jig/ml of 2.5 S NGF and 50 pCi of [3H]leucine. (a) The soluble protein (20,243 x c.p.m./mg protein) from 12 ganglia (containing 2.5 units of TH and 382pg of protein) was immunoprecipitated with 5 0 0 ~ 1of immune serum in a volume of 1.0 ml; (b) after the immunoprecipitate was collected the supernatant fluid was reimmunoprecipitated with 500j11 of immune serum and 1.3 units of rat adrenal TH as carrier (final volume of 2.0 ml). Direction of migration is from left to right.
P. MACDONNELL, N. TOLSON, M. W.Y U and G. GURCIFF
846
TABLE1. SYNTHESIS OF TYROSINE
HYDROXYLASE IN RAT TISSUES IN CULTURE
Radioactivity in: Soluble protein TH (c.p.m./mg protein) x lo-' 25,752 F 5,906 (20) 12,852 8475 5347 14,066
Superior cervical ganglia Nodose ganglia Liver Kidney
7789 Adrenal medulla ~
~
10,226 9222
69.8 F 13.8 (14) 0 0 0 0
0 124.8 & 4.7 (6) 109.2 5.2 (4)
% Radioactivity in TH 0.27 0 0 0 0 0 1.22 1.18
~~~
Tissues from 5-7-day-old rats were cultured for 16-18 h in BGJ, media containing 1pg/ml of 2.5 S NGF (SCG and nodose ganglia only) and 50 pCi C3H]leucine (all tissues). The concentration of leucine in BGJ, medium is 0.38 mM; thus, the specific activity of C3H]leucine is 0.44 Ci/mmol. The mean weight of the explants was 0.58, 0.40, 1.3, 1.7, and 1.8 mg for SCG, nodose ganglia, liver, kidney, and adrenal medulla, respectively. Tissues were homogenized in 5 mM-Tris, pH 7.4, containing 0.1% Triton X-100, and centrifuged at either 20,000g or 150,000g (no difference was observed in the radioactivity of the 20,OOOg or 150,OOOg supernatant fluids). Carrier TH was added to aliquots of each supernatant fluid to a final concentration of 1.3-2.5 units, and TH was immunologically precipitated with a 24-fold excess of antiserum (500 PI) and purified by SDS-polyacrylamide gel electrophoresis. The SCG values are . number of observations in brackets), each experiment expressed as means of the individual experiments ~ s . D (the containing a pool of 15-50 ganglia. Radioactivity in the nodose ganglia, liver, and kidney represents individual experiments each containing a pool of 7-30 explants. The adrenal medulla results represent the radioactivity from 2 experiments each containing a pool of 1623 medullae; various aliquots of each supernatant fluid were immunoprecipitated with 500 pl of antiserum. The S.D. of TH in the adrenal medulla samples illustrates the reproducibility of the immunoprecipitation technique in 2 samples each of which employed a 4-6-fold difference in radioactivity in the immunological reaction mixture. tein in the antigen-antibody precipitate was T H (calculated by determining the area under the peak of SDS gels) while in the adrenal medulla samples 60-70% of the immunoprecipitated protein was identified as TH. Attempts to remove these contaminating proteins were only partially effective. Immunoprecipitation reactions conducted in the presence of 1% Triton X-100 104 DOC were far superior to those
+
pg PROTEIN
FIG. 3. Quantitation of immunologically precipitated de now synthesized TH in SCG cultures. Ganglia were cultured as described in Fig. 2. Aliquots of SCG soluble protein (31,569 x 1 O - j c.p.m./mg protein) were immunoprecipitated with 5OOpI of antiserum in a volume of 1.0ml. Antigen-antibody precipitates were collected by centrifugation, thoroughly, washed with PBS, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioactivity in TH from each gel was quantitated by subtracting the background radioactivity from the peak area.
done in the presence of either 0.5% Triton X-100, 1% Triton X-100, or 1% Triton X-100 0.5 NaCl (data not shown). Contamination was further diminished by collecting the antigen-antibody precipitate over a 1 M-sucrose cushion as described by RHOADSet al. (1973) and SIPPELet al. (1975). Because of this nonspecific trapping of proteins in the immune complex all immunoprecipitates were routinely purified by SDS-polyacrylamide gel electrophoresis prior to quantitation of TH. The immunoprecipitation reaction is specific to T H in that the addition of 4 pg of ovalbumin and sufficient ovalbumin antiserum did not precipitate any significant amounts of radioactivity corresponding to the position of TH. After the ovalbumin-antiovalbumin complex is removed by centrifugation ['HlTH can still be precipitated from solution by addition of antiserum to TH. The synthesis of T H in culture occurred only in those tissues that contain T H in viuo (Table 1). No radioaetive peak corresponding to T H (Fig. 4(a)) is detected on SDS gels after liver, kidney, and nodose ganglia were cultured for 16 h with 50 pCi of C3H]leucine and then immunoprecipitated with nonradioactive carrier T H and antiserum (Fig. 4(b,c,d)). The only tissue, other than the SCG, which synthesized significant amounts of TH in culture is the adrenal medulla (Fig. 4(e)). Although the incorporation of [3H]leucine into the soluble protein of the adrenal medulla was considerably less than into ganglia protein, the differential rate of TH synthesis was approx 4 times greater (Table 1). The apparent molecular weight of newly synthesized T H from both the SCG and adrenal medulla
+
Nerve growth factor and tyrosine hydroxylase synthesis in vitro
K
2
847
H Chain, y Globulin Ovalbumin
4l
Carbonic Anhydrase
L Chain, yGlobulin
P x
z
u
'Orc 5
0
0.2
0.4
0.6
0.8
1.0
MOBILITY
FRACTION NUMBER
FIG.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic profiles of immunoprecipitated TH from rat tissues in culture. Superior cervical ganglia, nodose ganglia (both in the presence of 1 pg/ml of 2.5 S NGF), liver, kidney, and adrenal medulla from 5-day-old rats were cultured as described in Fig. 2, and in the Materials and Methods section. De nouo synthesized TH was immunoprecipitated with 500 pl antiserum in a volume of 1.0 ml; 1.3 units of nonradioactive carrier TH from rat adrenal medulla was added to the liver, kidney, and nodose ganglia immune reactions. (a) 357pg protein (31,569 x c.p.m./mg protein) containing 3.2 units of TH from 15 superior cervical ganglia; (b) 158pg of liver protein c.p.m./mg protein); (c) 227 pg of kidney pro(5344 x tein (7786 x c.p.m./mg protein); (d) 91 pg of nodose ganglia protein (12,852 x c.p.m./mg protein); and (e) 134pg of adrenal medulla protein (9215 x c.p.m./mg protein) containing 3.7 units of TH. Direction of migration is from left to right. was approx 6M5,OOO (Fig. 5). This data was obtained by the procedure of WEBERet al. (1972) for SDS gel electrophoresis after dissociation of the immune complex by boiling in 1% SDS and 1% 2-mercaptoethanol. Protein synthesis in ganglia cultured under these conditions is increased by the presence of N G F in the medium (Table 2). The maximum incorporation of C3H]leucine into soluble protein was at lpg/ml N G F ; at this N G F concentration approximately twice as much incorporation was observed as in those ganglia which did not receive NGF. At concentrations greater than 1 pg/ml of N G F soluble protein synthesis was depressed and, in fact, at lOpg/ml of N G F the extent of protein synthesis was essentially identical to that seen in ganglia cultured either without N G F or with N G F antibody (Table 2).
FIG. 5. Determination of the molecular weight of newly synthesized TH from SCG and adrenal medulla in organ culture. Ganglia and adrenal medulla from 5-day-old rats were cultured as in Fig. 2. Tyrosine hydroxylase from the SCG and adrenal medulla was immunoprecipitated with 500 pl antiserum in a volume of 1.0 ml. Immune precipitates and standard proteins were dissociated by boiling for 15min in 1% SDS + 1% 2-mercaptoethanol (see Materials and Methods section) prior to SDS-polyacrylamide gel electrophoresis. After the gels were stained and destained the mobilities of the standard proteins were determined. Radioactive gels were then sliced, care being taken to match protein bands to individual slices; gel slices were solubilized for counting as described in the Materials and Methods section. The molecular weight of TH from the SCG and adrenal medulla was calculated to be approx 60,oo(r65,000. The absolute synthesis of T H increased in proportion to the synthesis of soluble protein (compare columns 2 and 3, Table 2) up to a concentration of 3pg/ml of NGF. However, the ratio of TH synthesized to total protein synthesized (column 4, Table 2) did not vary appreciably in ganglia cultured without NGF, with N G F at concentrations of 0.05-1 pg/ml, or with N G F antibody. Somewhat higher levels of TH synthesis were observed at 3 pg/ml and substantially higher rates of T H synthesis were repeatedly observed at 10 pg/ml of NGF.
DISCUSSION The culture of rat superior cervical ganglia in oitro offers many advantages to the conventional in vivo studies for investigating tyrosine hydroxylase regulation. In a chemically defined medium one can manipulate the environment of the SCG and measure the resultant synthesis of TH in response to exogenously added agents. Similarly, interpretation of the data is simplified inasmuch as the normal regulatory events occurring in oioo are absent in culture. The disadvantages of such a system are obvious in that damage to the adrenergic neurones may result from transection of the preganglionic cholinergic fibers. Likewise, lack of suitable serum and growth promoting factors, and differences in the normal metabolite concentrations may play an important role in the normal
P. MACDONNELL, N. TOLSON, M. W. Yu and G. GUROFF
848
TABLE2. THEEFFECT OF NGF NGF concentration (Pg/ml)
ON THE
de nouo
16,862 2 1950 17,668 f 2701
0.33
22,176 28,754 29,955 & 6403 23,762 19,171 16,802 5 2942 14,351 13,328
10.00 0 + NGF antibody
TH
I N SUPERIOR CERVICAL GANGLIA IN CULTURE
Radioactivity in: Soluble protein TH (c.p.m./mg protein x 10- 7
0 0.05
1.oo 3.00
SYNTHESIS OF
25.2 k 4.1 36.5 31.6 36.0 53.3 44.2 k 3.0 50.0 58.7 50.5 k 7.9 28.1 19.7
”/, Radioactivity in enzyme
*
0.15 0.03 0.18 0.22 0.16 0.19 0.16 0.05 0.21 0.30 0.30 k 0.02 0.20 0.15
*
Ganglia, in groups of 3, were maintained in culture for 20 h in the presence of 50 pCi [3H]leucine and the indicated concentrations of 2.5 S NGF or 115 pg of NGF antibody. Ganglia were processed as described in Materials and Methods and 2.3 units of carrier TH and 500 p1 of antiserum were added to the supernatant fluid from 1 ganglion; immunoprecipitated TH was purified by SDS gel electrophoresis. The values represent the means S.D. of 3. experiments; other values are from individual experiments.
regulation of T H synthesis during maturation of the SCG. These factors cannot be accurately duplicated in uitro. With the above in mind, in the previous paper (Yu et al., 1977) we attempted to define the action of N G F on the levels of T H in SCG. The data presented indicated that N G F had some action on TH induction independent of its overall action in allowing the survival of adrenergic neurons. To determine the actual extent of TH induction it is necessary to measure the true rate of the synthesis of T H in relation to total synthesized protein. We have followed this approach by immunologically isolating the T H synthesized de novo. The present experiments show that T H is being synthesized in SCGs and adrenal medulla in organ culture. The evidence that the radioactive protein being measured is, in fact, T H is as follows: (1) The TH antiserum used in the present study is monospecific, having been raised to a pure chymotryptic fragment of bovine adrenal medulla TH. (2) The antibody completely inhibits TH enzyme activity from rat SCG’s and adrenal medulla while comparable amounts of control serum have no effect on enzyme activity. (3) The radioactive peak is only present in immunoprecipitates derived from soluble proteins of the SCG and adrenal medulla. N o radioactive peaks corresponding to T H are observed when the soluble proteins of liver, kidney, or nodose ganglia are immunoprecipitated in the presence of carrier TH. (4) The formation of an ovalbumin-antiovalbumin precipitate does not precipitate the major radioactive peak which can be subsequently precipitated by T H antibody. ( 5 ) After the immunoprecipitate from ganglia is collected no additional radioactive peak is seen on SDS gels after nonradioactive carrier T H and fresh TH antibody were added to the supernatant fluid.
(6) In experiments not presented here, it has been shown that immunoprecipitation of radioactive TH from ganglia is abolished when purified carrier bovine adrenal medulla T H is present in excess in the immunological reaction. Native TH has been difficult to purify and purification from ganglia is not feasible. Thus, n o satisfactory marker is available for the SDS gels. It seems clear, however, on the basis of the evidence cited above that the synthesis of T H is being measured here. A report by CHUANG et al. (1975) has appeared in which the de nouo synthesis of TH in adrenal in uiuo has been evaluated by techniques similar to those used here. The amount of T H synthesized by adrenal in that study appears to be somewhat higher than that reported in the present data. The newly synthesized T H had two subunits with molecular weights of 33,000 and 38,000. The reason for the difference in these parameters is not clear, but it could be due to the minor differences in our preparative techniques, or to the fact that the previous work was done in adults in uiuo while our experiments were done with tissues from young rats in uitro. This question is currently being explored. The data in this paper is consistent with that from the previous work (Yu ef at., 1977). In the previous paper it was shown that the total activity of T H of ganglia cultured in the presence of N G F is greater than that in its absence. Here we show that the total T H synthesis is greater also, as is the total incorporation of radioactive amino acid into protein. Further, it was shown that there is a small increase in the specific activity of the enzyme at maximal levels of NGF. Here we show that at very high levels of N G F there is a small increase in the differential rate of synthesis of TH. Whether this small increase at high N G F levels is a reflection of the in vivo action of N G F is still an open question. The interpretation of the present data is consistent
Nerve growth factor and tyrosine hydroxylase synthesis in vitro
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REFERENCES with that advanced previously. The action of N G F in this system is primarily on survival of the TH-conBLACKI. B., JOH T. H. & REIS D. J. (1974) Bruin Res. taining neurons (LEVI-MONTALCINI, 1966 ; LEVI-MON75, 133-144. TALCINI & ANGELETTI, 1968). There seems, in addition, CHUANG D., ZSILLA G. & COSTAE. (1975) Molec. Pharmac. to be a small selective action on T H synthesis, es11, 784794. pecially at high levels of NGF. This conclusion was FEIGELSON P. & GREENGARD 0. (1962) J . biol. Chem. 237, also reached by STICKGOLD & SHOOTER (1974) on 37163717. R. (1966) Harvey Lect. 60, 217-259. the basis of TH activity measurements reported, so LEVI-MONTALCINI R. & ANGELETTI P. U. (1968) Physiol. far, only in preliminary form. Although the present LEVI-MONTALCINI Rev. 48, 536569. system would seem to be of substantial value for studies on the mechanism of N G F action on the sur- LLOYDT. & KAUFMANS. (1973) Molec. Pharmac. 9, 438444. vival of sympathetic neurons, the action of NGF on LOWRY0.H., ROSEBROUGHN. J., FARRA. L. & RANDALL TH in uitro does not seem to be comparable to its R. J. (1951) J . biol. Chem. 193, 265-275. action on TH in uiuo. NAGATSU T., LEVITT M. & UDENFRIEND S. (1964) Analyt. That the conclusions from this and the previous Biochem. 9, 122-126. paper (Yu et al., 1977) are valid is borne out by recent NIKODIJEVIC B., Yu M. W. & GUROFF G. (1977) J . Neuroexperiments using a different protocol (MACDONNELL chern. 28, 851-852. H. (1973) J . Neurochem. et al., in preparation). When NGF is given in uiuo OESCHF., OTTENU. & THOENEN 20, 1691-1706. and TH synthesis measured subsequently in uitro the G. S. & SCHIMKE R. T . (1973) differential rate of TH synthesis is much greater than RHOADSR. E., MCKNIGHT J . biol. Chem. 248, 2031-2039. when NGF is given in uitro as described in the present A. E., FEIGELSON P. & ROY A. K. (1975) Biochemiswork. Thus, the action of NGF on TH synthesis in SIPPEL try 14, 825-829. uiuo is in some way different than it is in uitro. HopeSTICKWLD R. & SHOOTER E. M. (1974) Fedn Proc. Fedn fully, this explant system will provide an approach Am. SOCS exp. Biol. 33, 1495. to an understanding of the mechanism by which NGF THOENENH., ANGELETTI P. U., LEVI-MONTALCINI R. & influences the specific synthesis of TH. KETTLER R. (1971) Proc. Natn. Acad. Sci., U.S.A. 68,
Acknowledgement-The continued interest and advice of Dr. T . LLOYDis appreciated.
1598-1602. WERER K.. PRINGLE J. R . & OSBORN M. (1972) in Methods in Enzymology (HIRSC. H. W. & TIMASHEFF S. N.., eds.) 26, 3-27. Yu M. W., NIKODIJEVIC B., LAKSHMANAN J., ROWE V., MACDONNELL P. & GUROFFG. (1977) J . Neurochem. 28, 843-849.
