Journal of Nrurochemisfry, 1977. Vol. 28, pp. 835-842. Pergamon Press. Printed in Great Britain.
NERVE GROWTH FACTOR AND THE ACTIVITY OF TYROSINE HYDROXYLASE IN ORGAN CULTURES OF RAT SUPERIOR CERVICAL GANGLIA M. W.
YU,
B.
NIKODUEVIC,'
J. LAKSHMANAN, V. Row,*P.MACDONNELL3 and G. GUROFF
Section on Intermediary Metabolism, Laboratory of Biomedical Sciences, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20014, U.S.A. (Received 15 July 1976. Accepted 27 October 1976) Abstract-Superior cervical ganglia from young rats were cultured in the absence of serum. The effect of nerve growth factor on the level of tyrosine hydroxylase was studied. In the absence of nerve growth factor the specific activity of tyrosine hydroxylase fell by more than 50% within 48 h. In the presence of nerve growth factor the total and specific activities were maintained and even increased in the same period. Both the 2.5 S and the 7 S forms of nerve growth factor were effective. Oxidized nerve growth factor had no effect except when present in very high concentration. Purified antibody to nerve growth factor was inhibitory. Insulin had only a slight effect in this system, but dibutyryl CAMP elevated tyrosine hydroxylase activity substantially. Propranolol inhibited the action of nerve growth factor but its action appeared to be nonspecific and unrelated to its action on the 8-adrenergic receptor. Changes in the activity of dihydropteridine reductase paralleled those seen in tyrosine hydroxylase.
TYROSINE hydroxylase (TH; L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2), which catalyzes the hydroxylation of tyrosine to DOPA, is a rate-limiting step in the biosynthesis of norepinephrine (LEVITT et al., 1965). The activity of this enzyme can be selectively increased by nerve growth factor (NGF) in rat superior cervical et al., 1971). This inganglia (SCG) in uiuo (THOENEN crease is the most specific effect of NGF known and one of the very few in which an influence on a characterized enzyme has been shown. It may indicate that at least one of the effects of NGF, directly or indirectly, is on the level of transcription. A comparable effect of NGF on TH in uitro has not yet been shown. The demonstration of such an effect in a chemically defined system in uitro would provide a convenient model for the study of the action of NGF. In this study we describe the effects of NGF on TH activity in SCG organ cultures in a completely defined medium. No serum was added in the culture system. This is important since serum has been shown to mimic the effects of NGF on SCG anabolism Present address: Department of Pharmacology, Medical School, University of Skopje, Yugoslavia. Present address: Department of Neurology, Johns Hopkins University, Baltimore, MD 21205, U.S.A. Supported during a part of this work by postdoctoral fellowship NIH 1 F22 CA04011-01. Abbreviations used: TH, tyrosine hydroxylase; NGF, nerve growth factor; SCG, superior cervical ganglia; DHPR, dihydropteridine reductase; 1-oxi-, 2-0xi-, and 3-oxi-NGF, 2.5 S NGF with 1, 2, or 3 of the tryptophans of each peptide chain oxidized, respectively.
(BURNHAM et al., 1974) and on neurite outgrowth in dorsal root ganglia and sympathetic ganglia of chick embryos (MIZEL& BAMEWRG,1976). Further, some experiments suggest that serum also contains inhibitors of N G F activity (BANKSet al., 1973). We have studied the specificity of the NGF effect and the influence of various agents on the level of TH activity. The results are discussed with respect to the action of NGF, the possible involvement of cyclic nucleotides and /3-adrenergic agents in that action, and the appropriateness of organ culture for the study of the action of NGF. MATERIALS AND METHODS
Organ cultures. The conditions were essentially as described by NIKODUEVIC et al. (1975) with minor modification. Rats, 5-7 days of age (Sprague-Dawley), were obtained from Zivic-Miller (Allison Park, PA). The animals were stunned by a blow to the head and the superior cervical ganglia were removed and decapsulated. Three or four ganglia were incubated in 0.25 ml BGJ, medium (Fitton-Jackson modification) in an atmosphere of 95% O2 and 5% COz. The incubations were carried out at 37°C in a humidified incubator for 40-48 h in Costar tissue culture cluster dishes (17.8 x 16mm). NGF was added in a vol of 5-lop1 after appropriate dilution with BGJ, medium. For experiments with cyclic nucleotides, the SCG were pre-incubated with theophylline (2 mM) for 30 min before the addition of the nucleotides or of NGF. Adrenergic blockers were also added to the SCG in BGJ, medium before the addition of NGF or vehicle. After incubation the ganglia were rinsed briefly in 0.25 M-sucrose and blotted on filter paper. They were homogenized in 5 mM-Tris-HC1, pH 7.4, containing 0.1% Triton X-100,using a volume of 0.1 ml per ganglion. The homo-
835
M.W. Yu et
836
a1
genate was centrifuged at 18,000 g for 15 min. The supernar tant portion was used for the assay of tyrosine hydroxylase and dihydropteridine reductase, and for the estimation of protein content. Zero time controls are ganglia homogenized immediately after removal from the rats and culture control refers to ganglia cultured for a comparable period of time in the absence of NGF. Tyrosine hydroxylase assay. The enzyme was assayed according to the method of NAGATSU et a/. (1964) as modified by OESCHet a/. (1973). Fifty pl of ganglia homogenate were used for assay. The results are expressed as total activity (nmol 'H20 formed/h/ganglion) or as specific acTIME IN CULTURE (hours) tivity (nmol 3 H 2 0 formed/h/mg protein). Reductase assay. Dihydropteridine reductase (DHPR) FIG.1. Time course of changes in the specific activity of was assayed using a 50 pl portion of the supernatant frac- tyrosine hydroxylase in rat superior cervical ganglia in tion. The spectrophotometric method of NSELSEN ef u/. organ culture without nerve growth factor (0,culture control), in the presence of 2.5 S nerve growth factor,.( NGF) (1969) was employed. at a concentration of 1 pg/ml, and in the presence of pure N G F preparation. Nerve growth factor (2.5 S form) was prepared from the submaxillary glands of adult male mice antibody to nerve growth factor (A, Ab) at a concentration (Swiss-Webster) by the method of BOCCHINI & ANGELETTI of 460 pg protein/ml. Each point represents the average of two determinations. (1969). Unless otherwise stated, 2.5 S NGF was. used in all experiments. The NGF was stored frozen at a concentration of 1 mg/ml. Early preparations were assayed using stable for about 15 h and then began to decrease (zero the dorsal root ganglia outgrowth assay (FENTON,1970). time level: 16.2 nmol j H 2 0 formed/h/mg protein). More recent preparations have been quantitated on the Addition of purified NGF antibody (Ab) did not basis of their absorption at 280nm where 1 mg/ml accelerate the decrease in the specific activity of TH. = l.6AzBOin 0.05 M-acetate, pH 5.0. N G F antibody. Anti-NGF serum was produced in sheep Culture in the presence of N G F (1 pg/ml) resulted by injection of 2.5 S NGF. One mg of NGF in complete in some increase in the specific activity of TH within Freund's adjuvent was injected subcutaneously. Four 15 h with the maximum occurring after approx 24 h. weeks later 500pg of the same preparation in complete The difference in the specific activity of TH between Freund's adjuvent was injected as booster. Serum was col- the control culture ganglia and those with NGF, howlected 2 weeks after the boost. The NGF antibody was ever, was greatest at 4 W 8 h. When N G F and excess purified by affinity chromatography as described by NGF antibody were added to the culture simulet a/. (1976). STOCKEL taneously, the increase in the TH level was abolished Oxidized N G F . NGF was oxidized with N-bromosuc(data not shown) and the time course of TH activity cinimide as described by ANGELETTI(1970). The number of tryptophan residues oxidized was determined by the for- was the same as that of the culture control. The total activity of TH after 24 and 48 h in culture mula of SPANDE& WITKOP(1967). Protein determination. Protein was assayed by the control was about 90% and 30% of the zero time method of LOWRY et u/. (1951), using bovine serum albu- control, respectively (zero time level: 0.343 nmol min as a standard. 3 H 2 0 formed/h/ganglion). In the presence of NGF, Materials. Nerve growth factor in the 7 S form was pur- the total enzyme activity was 190% and 180% of zero chased from Burroughs Wellcome and Co. BGJ, medium time control after 24 and 48 h. The content of soluble was obtained from Grand Island Biological Co. and protein per ganglion, as measured in the 18,OOOg ~-[3S-~H]tyrosine(sp. act. = 60.3 Ci/mmol) from New supernatant, in the culture control was 100% and 65% England Nuclear, N6,02'-dibutyryl adenosine 3',5'-cycIic monophosphate (sodium salt), NL,02'-dibutyrylguanosine of the zero-time value at 24 and 48h, respectively 3',5'-cyclic monophosphate (sodium salt), insulin, DL-pro- (zero time level: 21.2 pg protein/ganglion). In the prespranolol-HCI, L-isoproterenol D-bitartrate, theophylline, ence of NGF the comparable figures were 130% and and concanavalin A were from Sigma Chemical Co. 145%. By way of comparison the specific activity of TH Phenoxybenzamine-HCI was from Smith, Kline & French Labs., Sotalol-HCI from Regis Co. and phytohemagglu- in the ganglia increased very little in uiuo during this tinin and hydrocortisone acetate from Calbiochem. Soy- 48 h period, i.e. from the 5th to the 7th postnatal bean agglutinin and wheat germ agglutinins were pur- day. The specific activity of the enzyme after 24 and chased from Miles-Yeda, Ltd. L- and D-Propranolol were 48 h in vivo was not significantly different from the gifts from Ayerst Labs. Dexamethasone sodium phosphate zero time control. was from Merck, Sharp & Dohme. RESULTS
Time course of changes in tyrosine hydroxylase activity in organ culture of superior cervical ganglia As shown in Fig. 1 the specific activity of TH in ganglia cultured in the absence of NGF (CC) was
In view of the observation that T H levels in cultured ganglia remained stable for 15 h, it seemed possible that some endogenous NGF was present in the ganglia when they were removed. Attempts were made to preincubate the SCG without N G F for 15 h in order to remove any endogenous N G F (LARRABEE, 1972). The response to N G F with preincubated gang-
NGF and tyrosine hydroxylase activity in uitro
837
7
NGF
48
12 24 36 TIME IN CULTURE (hours)
NGF Inglmll FIG. 2. Time course of changes in the specific activity of FIG. 3. Effect of the concentration of 2.5 S NGF (0)or tyrosine hydroxylase without nerve growth factor (M, CC) and in the presence of 2.5 S NGF (1 pg/ml) without of 7 S NGF (A) on the specific activity of tyrosine hydroxy(u or with )(+-+) 15 h preincubation period. The lase in 48 h organ culture. Abscissa indicates ng/ml of arrows indicate NGF (1 pg/ml) addition after preincuba- either 2.5s or the 7 s . Each point represents the tion period. Each point represents the average of two mean S.E.(in brackets) of at least three determinations. determinations.
lia was never as great as the response obtained without preincubation. However, the T H activity from such ganglia did rise above the zero time control after 2 4 4 8 h of culture (Fig. 2). The experiment indicates that ganglia are still responsive to N G F even after overnight incubation in its absence. Dependence on NGF concentration The activity of T H showed a roughly linear response to 2.5 S N G F at concentrations between 10 ng (4 x 1 0 - l ' ~ ) and 250ng/ml (1 x ~ O - * M )(Fig. 3). Below lOng/ml N G F did not significantly increase the TH activity. At concentrations above 250ng/ml there was a gradual increase in TH activity. Maximal response occurred at 1 pg/ml of NGF. Higher concentrations of N G F did not appear to depress the TH level. This is different from the response in certain of the bioassay methods where higher concentrations of N G F have been shown to inhibit neurite outOF TABLE1. EFFECTS
Addition None NGF I-Oxi-NGF
2-Oxi-NGF
growth (FENTON, 1970; GREENE,1974; MIZEL& BAMBURG, 1976). Both the 2.5 S and the 7 S forms of N G F are active (Fig. 3). 7 S NGF at 25 ng/ml (2 x lo-'' M) produced a response equivalent to that of 2.5s N G F at 25ng/ml (1 x 1 0 - 9 ~ )The . dose-response curve of 7 s was similar in shape to that of 2 . 5 s NGF, although the maximal response to 7 s was reached at a lower concentration, approx 0.5 pg/ml. Specijicity of the action of N G F on tyrosine hydroxylase There are three tryptophan residues in each peptide chain of 2.5 S N G F (FRAZIER et al., 1973~).Preparations of NGF in which one (1-oxi-NGF) or two (2-oxi-NGF) tryptophan residues had been oxidized by N-bromosuccinimide were assayed in the organ culture system. The results are shown in Table 1. Neither 1-oxi-NGF nor 2-oxi-NGF increased the T H
OXIDIZED 2.5 S NGF ON THE SPECIFIC ACTIVITY OF TYROSINE HYDROXYLASE IN CULTURED GANGLIA
Concentration (ng/ml)
TH activity* (nmol/h/mg protein)
% of control
-
8.89 0.28 11.6 f 0.32t 20.7 f 0.69t 1.os.t 21.2 8.36 f 0.18 9.55 f 0.16 0.87t 18.5 21.2 f 0.81t 9.35 k 0.17 8.91 k 0.21 10.8 f 0.511 12.5 f 0.291
100 130 233 239 94 107 208 238 105 100 122 141
50 500 1000 50 500 lo00 loo00 50 500 lo00 loo00
* *
* Tyrosine hydroxylase activity was measured 40 h after the addition of 2.5 S NGF or its oxidized derivatives. Each value represents the mean f S.E. from at least three determinations. t P < 0.001 compared with minus NGF control. 1P < 0.01 compared with minus NGF control.
M. W. YU et al.
838
TAHLE2. EFFECTS OF
INSULIN ON THE SPECIFIC ACTIVITY OF TYROSINE HYDROXYLASE IN CULTURED GANGLIA
Addition
Concentration (pgiml)
TH activity (nmol/h/mg protein) Minus NGF Plus NGF*
~~
None Insulin
0.1 1.0
10
8.80 0.14 9.95 L- 0.14 10.5 k 0.33 10.1 & 0.40
18.5 f 1.23 20.7 f 1.49 17.2 k 0.29 17.5 0.29
* 1 pg/ml 2.5 S NGF final concentration. Each value represents the mean k S.E. from four determinations. activity when used at concentrations lower than 0.5pg/ml. 1-Oxi-NGF produced almost full T H activity when used at a concentration of 1 pg/ml. 2-0x1NGF at 1 pg/ml increased T H activity slightly while at 10 pg/ml, it increased the enzyme activity to about the same extent as native N G F at 0.05 pg/ml. Therefore, the oxidation of the tryptophan residues in N G F markedly lowers its ability to maintain or increase the levels of T H in cultured SCG. The data are similar to those obtained in studies of the culture of embryonic dorsal root ganglia, where 1-oxi-NGF retained almost full activity, 2-oxi-NGF was partially active, and 3-oxi-NGF had no biological activity (ANGELETTI, 1970; GREENE,1974). This is different from the effect observed by MERRELL et al. (1975) in the chick optic tectal system in which the tryptophan residues were not essential for the activity of NGF. Dexamethasone, cortisol, and glucagon at various concentrations were tried, but had no effect on the T H activity. Insulin, which has some structural similarity to N G F (FRAZIER et al., 1972), had only minimal effect in increasing TH activity at concentrations at which N G F was fully effective (Table 2). Insulin has been reported to markedly increase several anabolic processes in cultured chick dorsal root gnaglia (BURNHAMet al., 1974). A group of plant lectins that have binding, agglutinating, and mitogenic properties have been widely
TIME OF ADDITION OF NGF ANTIBODY (hours)
FIG.5. Time course of NGF antibody effect on the specific activity of tyrosine hydroxylase in the presence of 2.5s NGF (1 pg/ml). NGF antibody was added to the medium at various time intervals at a final concentration of 460 pg protein per ml. Each column represents the mean & S.E. (in brackets) of three determinations. used as probes for membrane-related phenomena (SHARON& LIS, 1972). Concanavalin A has been shown to interact with insulin receptors of fat cells and liver membranes and mimic insulin action in those cells (CUATRECASAS & TELL,1973). Concanavalin A alone has also been shown to stimulate the incorporation of radioactive uridine and radioactive leucine into chick embryo dorsal root ganglia to the same extent as N G F although there did not seem to be any interaction when the two were mixed together (BURNHAM et al., 1974). None of the lectins had any significant effect on ganglionic T H activity either alone or in the presence of N G F (Fig. 4). Purified antibody to N G F prevents the action of N G F on TH. As shown in Fig. 5, when antibody was added at zero time to medium containing 1 pg/ml of N G F the activity of T H after 48 h in culture was the same as in the culture control. When antibody was added between 1 and 18 h after the addition of NGF, the final T H activity was roughly proportional to the time interval. Addition of antibody after 18 h no longer influenced the action of N G F on the enzyme activity. The efSect of dibutyryl cyclic A M P (dbcAMP) on tyrosine hydroxylase
"T
NIKODUEVIC et al. (1975) have recently reported the elevation of the cAMP level of SCG in vitro immediately after N G F addition. In the present experiments, an attempt was made to determine if cAMP can mimic the effect of N G F on TH. Since dbcAMP can pass through the plasma membrane more easily and is not as susceptible to hydrolytic degradation, this derivative of cAMP was used. Theophylline ( 2 mM), tN PHVTOan inhibitor of phosphodiesterase, was also included lpglml Oluglml AGGL HEMAGGL VALlN AGGL in the medium to protect dbcAMP from enzymatic 5r10'M 7110'M 1xlO.M 2110.M degradation. The results are shown in Fig. 6. The FIG.4. Effect of plant lectins either alone or in the presence of 2.5 S NGF on the specific activity of tyrosine hy- presence of theophylline increased the specific activity droxylase. The ganglia were preincubated with the lectins of T H about 10% either in culture control or in gangfor 30 min before the addition of 2.5 S NGF. Each point lia cultured in the presence of 0.1 pg/ml NGF. The maximal effect on TH activity was obtained at 1 represents the average of two determinations. + NGF0.1pQhl
fl
N G F and tyrosine hydroxylase activity in uitro
+
0 lpglml
Olpglml
10-'M
1O'M
THEOPH
THEOPH
THEOPH
t THEOPH
THEOPH
+
839
Olpglml
1O'M
10'M
+
+
+
THEOPH
THEOPH
THEOPH
NGF 0 luglml
FIG. 6. Etfects of dibutyryl cyclic AMP (dbcAMP) either alone or in the presence of 2.5 S NGF on the specific activity of tyrosine hydroxylase. Theophylline (2 KIM) was added and pre-incubated with the ganglia for 30 min prior to the addition of dbcAMP and/or NGF. Each column represents the mean f S.E. (in brackets) of three determinations.
mM-dbcAMP which increased the TH level to about the same extent as NGF at 0.1 pg/ml. However, the combination of NGF, at either 0.1 pg/ml or 1 pg/ml, and dbcAMP did not further increase the enzyme acXivity. To determine if the increase produced by dbcAMP is specific, dibutyryl cyclic GMP (dbcGMP) was also used. As shown in Fig. 7, dbcGMP, even at a concentration of 10 mM, did not increase the TH activity either alone or in combination with NGF. The influence of P-adrenergic blockers on the action of NGF on tyrosine hydroxylase Recently it has been shown that the NGF effect on adenylate cyclase activity of adrenal granule membranes is blocked by L-propranolol, a blocker of the fl-adrenergic receptor (NIKODUEVIC et al., 1976). It, therefore, seemed appropriate to study the effects of the isomers of propranolol and other related comTABLE3: INFLUENCE OF
NGF
FIG.7. Effects of dibutyryl cyclic G M P (dbcGMP) either alone or in the presence of 2.5 S NGF on the specific activity of tyrosine hydroxylase. Theophylline (2 mM) was added in all the medium. Each column represents the mean f S.E. of three determinations.
pounds in the present system. The results are summarized in Table 3. Although the presence of L-propranolol lowered the TH activity, D-propranolol appeared to have a similar inhibitory effect. L-Propranolol is a P-blocker (POTTER, 1967; BARRETT, 1969; BARRETT & CARTER,1970); D-propranolol is virtually devoid of 8-receptor blocking activity, but is reported to have non-specific cell membrane activity. Therefore, the inhibitory effect of propranolol appears to be due to non-specific cell membrane activity. Sotalol, which has been classified as a weak 8-antagonist with essentially no nonspecific cell membrane, i.e. local anesthetic, activity (LISHet al., 1965; BARRETT8z CARTER, 1970), had little influence on the NGF effect even at 1mM. None of the blockers had any effect when presented alone. Phenoxybenzamine, an a-blocker, at a concentration of M,did not block the NGF effect (data not shown). Isoproterenol, a 8-agonist, when used at concentrations between M and M,had no effect either alone or with NGF (data not shown).
P-ADRENERGIC
Addition
(M)
None L-Propranolol
-
10-4
D-Propranolol Sotalol
10-5 10-4 10-5 10-4 10-3
1O'M
THEOPH
0 lgglrnl
BLOCKERS ON THE EFFECT OF 2.5s NGF ON THE SPECIFIC ACTIVITY OF TYROSINE HYDROXYLASE IN CULTURED GANGLIA
Concentration
10.M
THEOPH
TH activity (nmol/h/mg protein) Minus N G F Plus NGF* 9.44 f 0.36 9.53 k 0.30 10.8 k 0.23 7.14 2 0.18 10.6 k 0.60 10.9 k 0.71 8.32 k 0.43 8.09 k 0.27 8.15 & 0.04 7.65 k 0.08
20.1 f 0.51 19.8 _+ 0.63 18.1 0.25 12.7 0.50 18.7 & 0.64 16.9 k 1.40 12.3 i 0.65 18.7 0.62 17.9 +. 0.09 16.7 k 0.55
Each value represents mean k S.E. from at least three determinations. * 1pg/ml 2.5 S N G F final concentration.
M.W. Yu et al.
840
TABLE 4. EFFECTS OF 2.5 S NGF, OXIDIZED NGF, AND NGF ANTIBODY
ON THE SPECIFIC ACTIVITY OF TYROSINE HYDROXY-
LAST AND DIHYDROPTFR1I)INl RFDUCTASE IN CULTURED GANGLIA
Concentration
TH activity
bg/ml)
(nmol/h/mg protein)
Addition
~
None (zero time control) None (culture control) 2.5 S NGF 1-oxi-NGF 2-oxl-NGF NGF Ab NGF + NGF Ab
-
1 .o 5.O 5.0 460 1 .o 460
DHPR activity (nmol NADH oxidized/ 5 min/mg protein) ~~
13.8 If: 1.21 8.81 0.55 19.1 k 0.70* 17.3 f 0.98* 9.71 k 0.56 9.07 k 0.48 9.55 k 0.38
524 k 10.6 458 f 53.4 1496 k 1.55* 1389 5 65.0* 480 k 22.9 613 k 1.81 710 f 11.2
Each value represents the mean f S.E. of at least three determinations. * P < 0001 compared to culture control Dihydropteridine reductase activity
Dihydropteridine reductase (DHPR) is the enzyme which recycles the reduced pteridine cofactor for tyrosine hydroxylation. The activity of this enzyme is higher in ganglia cultured in the presence of NGF than in the culture control (Table 4). In addition, its activity is increased by 1-oxi-NGF, but not by 2-oxiNGF, and the presence of NGF antibody blocks the action of NGF. Generally, the changes in DHPR are comparable to those in TH.
DISCUSSION Superior cervical ganglia in culture have been used before to investigate some aspects of the regulation of TH by NGF (MACKAY, 1974). However, in these experiments, as in most in uitro studies on NGF, serum was added to the culture media. The present experiments were done in the absence of serum which seems quite important since serum contains some NGF (HENDRY, 1972), has been shown to mimic some of the effects of NGF (BURNHAM et al., 1974; MIZEL & BAMBURG, 1976), and also seems to contain some factors which inhibit the actions of NGF (BANKSet al., 1973). STICKCOLD & SHOOTER (1974) have also presented data in preliminary form on NGF and TH levels in SCG cultured in the apparent absence of serum but no full paper on the subject has appeared. The methodology presented in this study represents a possible approach to the quantitative assay of NGF. The applicable range for measuring 2.5 S N G F is between 10 and 250 ng/ml. The method seems simple, accurate, and, as described below, specific. We have encountered some variability in the dose-response curve from experiment to experiment but no more, certainly, than in the other bioassay methods for NGF. More detailed studies need to be done before it is known whether it can be widely applied. However, this assay would seem to offer a more strictly quantitative measure than the widely used bioassay method for N G F (LEVI-MONTALCINI et al., 1954; COHEN,1959; FENTON, 1970; GREENE,1974; MIZEL & BAMBURG,1976).
The action of NGF in this system is quite specific. Oxidized NGF, insulin, glucagon, glucocorticoids, and lectins have been studied. They are all virtually without activity. This is generally true in NGF-responsive systems. On the other hand, it is interesting that insulin, which stimulates the anabolic activities of some NGF-sensitive ganglia (BURNHAM et a!., 1974) has no substantial effect on TH levels in this system. Dibuturyl cAMP has some ability to mimic the effect of NGF on TH activity in the ganglia. This has been observed before (MACKAY & IVERSEN,1972; GOODMAN et al., 1974), but the presence of serum and, thus, perhaps NGF, in these previous cultures allowed some latitude in the interpretation of that data. Here there is no question that dbcAMP, and dbcAMP alone, is causing an increased TH activity after 48 h. There is, however, a question as to whether a rise in cAMP in the cells is obligatory in the action of NGF on TH. That is, is cAMP the ‘second messenger’ for N G F action? In favor of such a relationship are the following points. First, NIKODIJEVIC et al. (1975) found that NGF produced an increase in cAMP level in SCG in uitro. An increase could be seen within 5 min of N G F addition, and with as little as 40 ng/ml, and the effect is quite specific. Second, our present data indicate that dbcAMP, but not dbcGMP, can mimic the NGF effect on TH activity to some substantial extent in a similar SCG organ culture system. This would seem to exclude certain non-specific effects such as induction by sodium butyrate. Third, NGF has now been shown to have an effect on cAMP levels in a totally different system, namely, isolated membranes of adrenal medulla vesicles (NIKODIJEVIC et a!., 1976). Finally, a very similar response in cAMP levels has been recently produced by the addition of a related factor, the epidermal growth factor (EGF), to cultures of lens epithelial cells ( A m et al., 1976). On the other hand, the following points suggest that the relationship is not obligatory. First the levels of N G F necessary to produce a rise in intracellular cAMP are quite high (NIKODIJEVIC et al., 1975) compared to those required to produce an increase in the TH level. Second, the cAMP response is rather
N G F and tyrosine hydroxylase activity in uitro
small and quite transitory. Third, the present data show that dbcAMP had no additional influence when submaximal amounts of N G F was used, and dbcAMP itself was not able to elicit the maximal response. Finally, the antibody studies show that the presence of N G F is necessary long after cAMP levels are back to normal to obtain its effect on TH. I t should be added that other investigators have failed to detect changes in cAMP levels in cultured dorsal root ganglia upon addition of N G F (FRAZIERet al., 1973b; HER rt al., 1973). Thus, although NGF produces a rise in cAMP levels, and dbcAMP, but not dbcGMP, mimics the N G F effect, and although others have shown that treatment of SCG with dbcAMP under other conditions increases T H (MACKAY & IVERSEN, 1972; GOODMAN et al., 1974) and that treatment of sensory ganglia with dbcAMP produces neurite outgrowth (ROISENet al., 1972; HAASet al., 1972), the role of cAMP as the ‘second messenger’ for N G F remains questionable. Our present data show that the effect of N G F on ganglionic TH in organ culture can be blocked by /l-adrenergic blocker but not by a-adrenergic blocker. However, the lack of steric specificity in the propranolol effect, the high concentration of Sotalol required, and the lack of effect by isoproterenol suggest that the inhibition by /?-blockers is non-specific, perhaps a result of membrane effects. Still, it is intriguing that 1-adrenergic response results in activation of adenylate cyclase and accumulation of cAMP (ROBINSON et al., 1971), and some studies indicate that the induction of T H in SCG by stress is dependent on increases of cAMP (GUIDOTTIet al., 1975). The increase of TH activity in our organ culture of rat SCG is a specific and quantifiable property of NGF. It is not clear, however, whether N G F is selectively inducing TH, as it does in uiuo, or whether it is merely allowing the survival of the neurons in the ganglia, which are, after all, the TH-containing cells. The results with dihydropteridine reductase would argue the latter as the major effect. DHPR is not selectively induced in uiuo and does not follow changes in the TH level (NIKODIJEVIC et al., 1977), but in the present work it seems to reflect the changes seen in TH. Thus, it must be concluded that selective induction of T H is not a major effect of N G F in ganglia cultured under these conditions. Other results with SCG in culture have previously been interpreted to mean that it is unsuitable as a model, e.g. for transsynaptic induction, for events in uiuo (GOODMANet al., 1974). It is impossible, however, to avoid the conclusion that N G F has some, albeit minor, effect on TH synthesis in this system. Since the specific activity of TH rises substantially above the zero-time control level in uitro, it is hard to see how such data can be interpreted as due only to neuronal survival. Certainly the specific activity changes are quantitatively less than those seen with N G F in uiuo (THOENEN et al., 1971;
841
NIKODIJEVIC et al., 1977), but they seem quite real. That N G F has some selective action on the T H level in uitro has abo been the conclusion of STICKGOLD & SHOOTER(1974). The accompanying paper (MACDONNELL et al., 1977) presents further information on this question. Acknowledgement-The authors appreciate the advice and interest of Dr. D. C. KLEIN.
REFERENCES AHNH., HOROWITZ S. G., HOLLENBERG M. D. & MAKMAN M. H. (1976) Fedn Proc. Fedn Am. SOCSexp. B i d . 35, 1731. ANGELETTI R. A. (1970) Biochirn. hiophys. Acta 214, 479482. BANKSB., BANTHORPE D., CHARLWOOD K. A,, PEARCEF. L., VERNONC. A. & EDWARDSD. C. (1973) Nature, Lond. 246, 503-504. BARRETT A. M. (1969) J . Pharm. Pharrnac. 21, 241-247. BARRETT A. M. & CARTERJ. (1970) Br. J . Pharrnac. Chemother. 40, 373-381. BOCCHINI V. & AN~ELETTI P. U. (1969) Proc. Natn. Acad. Sci., U.S.A. 64. 787-794. BURNHAM P. A,, SILVAJ. A. & VARONS. (1974) J . Neurochern. 23, 689-695. COHENS . (1959) J. b i d . Chern. 234, 1129-1137. CUTRACASAS P. & TELLG. P. E. (1973) Proc. Natn. Acad. Sci., U.S.A. 70, 485489. FENTONE. L. (1970) Exptl Cell Res. 59, 383-392. FRAZIER W. A,, ANGELETTI R. A. & BRADSHAW R. A. (1972) Science 176, 482487. FRAZIER W. A., HOGUE-ANGELETTI R. A., SHERMAN R. & BRADSHAW R. A. (19730) Biochemistry 12, 3281-3293. FRAZIER W. A., OHLENDORF C. E., BOYDL. R., ALOEL., JOHNSON E. M., FARRENDELL J. A. & BRADSHAW R. A. (1973b) Proc. Natn. Acad. Sci., U.S.A. 70, 2448-2452. GOODMAN R., OESCHF. & THOENENH. (1974) J . Neurochern. 23, 369-378. GREENE L. A. (1974) Neurobiology 4, 286292. GUIDOTTI A., HANBAUER I. & COSTAE. (1975) in Advances G. I., GREENin Cyclic Nucleotide Research (DRUMMOND GARD P. & ROBINSON G. A., eds.), Vol. 5, pp. 619-639. Raven Press, New York. HAASD. C., HIERD. B., ARNASONB. G. W. & YOUNG M. (1972) Proc. SOC.exp. Biol. Med. 140, 4 5 4 7 . HENDRYI. A. (1972) Biochem. J . 128, 1265-1272. HIERD. B., ARNASON B. G. W. & YOUNGM. (1973) Science 182, 79-81. LARRABEE M. G. (1972) in Nerve Growth Factor and its E. & KNIGHTJ., eds.), p. 71. Athone Antiserum (ZAIMIS Press of the Univ. of London. LEVI-MONTALCINI R., MEYERH. & HAMBURGER U. (1954) Cancer R e x 14, 49-57. . LEVITTM., SPECTOR S., SJOERDSMA A. & UDENFRIEND S. (1965) J . Pharrnac. exp. Ther. 148, 1-8. LISHP. M., WEIKELJ. H. & DUNGAN K. W. (1965) J . Pharmac. exp. Ther. 149, 161-173. LOWRYD. H., ROSEBROUGH N. J., FARRA. L. & RANDALL R. T . (1951) J . biol. Chem. 193, 265-275. MACDONNELL P., TOLSONN., Yu M. W. & GUROFF G. (1977) J . Neurochern. 28, 843-852. MACKAYA. V. P. & IVERSEN L. L. (1972) Brain Res. 48, 424-426.
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MACKAY A. V. P. (1974) Br. J. Pharmac. 51, 509-520. MERRELL R., PULLIAM M. W., RANWNS L., BOYDL., BRADSHAW R. A. & GLASER L. (1975) Proc. Natn. Acad. Sci., U.S.A. 72, 427M274. MIZELS. B. & BAMBURG J. B. (1976) D e n B i d . 49, 11-19. NAGATSU T., LEVITTM. & UDENFRIEND S. (1964) Analyt. Biochem. 9, 122-126. NIELSENK. H., SIMONSEN V. & LIND K. E. (1969) Eur. J . Biochem. 9, 497-502. NIKODIJEVIC B., NIKODIJEVIC O., Yu M. W., POLLARD H. & GUROFFG. (1975) Proc. Natn. Acad. Sci., U.S.A. 7 2 , 4769477 1.
NIKODIJEVIC O., NIKODIJEVIC B., ZINDER0.. Yu M. W., GUROFFG. & POLLARDH. B. (1976) Proc. Natn. Acad. Sci., U.S.A. 13, 711-714. NIKODIJFVIC B.. YII M. W. & GIIROFF,G. (1977) J . Nrtrrochem. 28, 851-852. OESCHF., OTTEN[J. & THOENEN H. (1973) J. Neurochem. 20, 1691-1706.
POTTERL. T. (1967) J. Pharmac. exp. Ther. 155, 91-100. ROBINSON G. A., BUTCHERR. W. & SUTHERLAND E. W. (1971) in Cyclic A M P . Academic Press, New York. ROISENF. J., MURPHYR. A,, PICHICHERO M. E. & BRADEN W. G. (1972) Science 175, 13-74. SHARON N. & LIS H. (1972) Science 177, 949-959. STICKGOLD R. & SHOOTERE. M. (1974) Fedn Proc. Fedn Am. SOCSexp. Biol. 33, 1495. STOCKELK., GAGNON C., GUROFF G. & THOENEN H. (1976) J. Neurochem. 26, 1207-1211. SPANDET. F. & WITKOPB. (1967) in Methods in EnzymoS. P. & KAPLANN. O., eds.), Vol. VII, logy (COLOWICK pp. 5-13. Academic Press, New York. THOENEN H., ANGELETTIP. U., LEVI-MONTALCINI R. & KETTLERR. (1971) Proc. Natn. Acad. Sci., U.S.A. 6 8 , 1598-1602.
NERVE GROWTH FACTOR AND THE ACTIVITY OF TYROSINE HYDROXYLASE IN ORGAN CULTURES OF RAT SUPERIOR CERVICAL GANGLIA M. W.
YU,
B.
NIKODUEVIC,'
J. LAKSHMANAN, V. Row,*P.MACDONNELL3 and G. GUROFF
Section on Intermediary Metabolism, Laboratory of Biomedical Sciences, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20014, U.S.A. (Received 15 July 1976. Accepted 27 October 1976) Abstract-Superior cervical ganglia from young rats were cultured in the absence of serum. The effect of nerve growth factor on the level of tyrosine hydroxylase was studied. In the absence of nerve growth factor the specific activity of tyrosine hydroxylase fell by more than 50% within 48 h. In the presence of nerve growth factor the total and specific activities were maintained and even increased in the same period. Both the 2.5 S and the 7 S forms of nerve growth factor were effective. Oxidized nerve growth factor had no effect except when present in very high concentration. Purified antibody to nerve growth factor was inhibitory. Insulin had only a slight effect in this system, but dibutyryl CAMP elevated tyrosine hydroxylase activity substantially. Propranolol inhibited the action of nerve growth factor but its action appeared to be nonspecific and unrelated to its action on the 8-adrenergic receptor. Changes in the activity of dihydropteridine reductase paralleled those seen in tyrosine hydroxylase.
TYROSINE hydroxylase (TH; L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2), which catalyzes the hydroxylation of tyrosine to DOPA, is a rate-limiting step in the biosynthesis of norepinephrine (LEVITT et al., 1965). The activity of this enzyme can be selectively increased by nerve growth factor (NGF) in rat superior cervical et al., 1971). This inganglia (SCG) in uiuo (THOENEN crease is the most specific effect of NGF known and one of the very few in which an influence on a characterized enzyme has been shown. It may indicate that at least one of the effects of NGF, directly or indirectly, is on the level of transcription. A comparable effect of NGF on TH in uitro has not yet been shown. The demonstration of such an effect in a chemically defined system in uitro would provide a convenient model for the study of the action of NGF. In this study we describe the effects of NGF on TH activity in SCG organ cultures in a completely defined medium. No serum was added in the culture system. This is important since serum has been shown to mimic the effects of NGF on SCG anabolism Present address: Department of Pharmacology, Medical School, University of Skopje, Yugoslavia. Present address: Department of Neurology, Johns Hopkins University, Baltimore, MD 21205, U.S.A. Supported during a part of this work by postdoctoral fellowship NIH 1 F22 CA04011-01. Abbreviations used: TH, tyrosine hydroxylase; NGF, nerve growth factor; SCG, superior cervical ganglia; DHPR, dihydropteridine reductase; 1-oxi-, 2-0xi-, and 3-oxi-NGF, 2.5 S NGF with 1, 2, or 3 of the tryptophans of each peptide chain oxidized, respectively.
(BURNHAM et al., 1974) and on neurite outgrowth in dorsal root ganglia and sympathetic ganglia of chick embryos (MIZEL& BAMEWRG,1976). Further, some experiments suggest that serum also contains inhibitors of N G F activity (BANKSet al., 1973). We have studied the specificity of the NGF effect and the influence of various agents on the level of TH activity. The results are discussed with respect to the action of NGF, the possible involvement of cyclic nucleotides and /3-adrenergic agents in that action, and the appropriateness of organ culture for the study of the action of NGF. MATERIALS AND METHODS
Organ cultures. The conditions were essentially as described by NIKODUEVIC et al. (1975) with minor modification. Rats, 5-7 days of age (Sprague-Dawley), were obtained from Zivic-Miller (Allison Park, PA). The animals were stunned by a blow to the head and the superior cervical ganglia were removed and decapsulated. Three or four ganglia were incubated in 0.25 ml BGJ, medium (Fitton-Jackson modification) in an atmosphere of 95% O2 and 5% COz. The incubations were carried out at 37°C in a humidified incubator for 40-48 h in Costar tissue culture cluster dishes (17.8 x 16mm). NGF was added in a vol of 5-lop1 after appropriate dilution with BGJ, medium. For experiments with cyclic nucleotides, the SCG were pre-incubated with theophylline (2 mM) for 30 min before the addition of the nucleotides or of NGF. Adrenergic blockers were also added to the SCG in BGJ, medium before the addition of NGF or vehicle. After incubation the ganglia were rinsed briefly in 0.25 M-sucrose and blotted on filter paper. They were homogenized in 5 mM-Tris-HC1, pH 7.4, containing 0.1% Triton X-100,using a volume of 0.1 ml per ganglion. The homo-
835
M.W. Yu et
836
a1
genate was centrifuged at 18,000 g for 15 min. The supernar tant portion was used for the assay of tyrosine hydroxylase and dihydropteridine reductase, and for the estimation of protein content. Zero time controls are ganglia homogenized immediately after removal from the rats and culture control refers to ganglia cultured for a comparable period of time in the absence of NGF. Tyrosine hydroxylase assay. The enzyme was assayed according to the method of NAGATSU et a/. (1964) as modified by OESCHet a/. (1973). Fifty pl of ganglia homogenate were used for assay. The results are expressed as total activity (nmol 'H20 formed/h/ganglion) or as specific acTIME IN CULTURE (hours) tivity (nmol 3 H 2 0 formed/h/mg protein). Reductase assay. Dihydropteridine reductase (DHPR) FIG.1. Time course of changes in the specific activity of was assayed using a 50 pl portion of the supernatant frac- tyrosine hydroxylase in rat superior cervical ganglia in tion. The spectrophotometric method of NSELSEN ef u/. organ culture without nerve growth factor (0,culture control), in the presence of 2.5 S nerve growth factor,.( NGF) (1969) was employed. at a concentration of 1 pg/ml, and in the presence of pure N G F preparation. Nerve growth factor (2.5 S form) was prepared from the submaxillary glands of adult male mice antibody to nerve growth factor (A, Ab) at a concentration (Swiss-Webster) by the method of BOCCHINI & ANGELETTI of 460 pg protein/ml. Each point represents the average of two determinations. (1969). Unless otherwise stated, 2.5 S NGF was. used in all experiments. The NGF was stored frozen at a concentration of 1 mg/ml. Early preparations were assayed using stable for about 15 h and then began to decrease (zero the dorsal root ganglia outgrowth assay (FENTON,1970). time level: 16.2 nmol j H 2 0 formed/h/mg protein). More recent preparations have been quantitated on the Addition of purified NGF antibody (Ab) did not basis of their absorption at 280nm where 1 mg/ml accelerate the decrease in the specific activity of TH. = l.6AzBOin 0.05 M-acetate, pH 5.0. N G F antibody. Anti-NGF serum was produced in sheep Culture in the presence of N G F (1 pg/ml) resulted by injection of 2.5 S NGF. One mg of NGF in complete in some increase in the specific activity of TH within Freund's adjuvent was injected subcutaneously. Four 15 h with the maximum occurring after approx 24 h. weeks later 500pg of the same preparation in complete The difference in the specific activity of TH between Freund's adjuvent was injected as booster. Serum was col- the control culture ganglia and those with NGF, howlected 2 weeks after the boost. The NGF antibody was ever, was greatest at 4 W 8 h. When N G F and excess purified by affinity chromatography as described by NGF antibody were added to the culture simulet a/. (1976). STOCKEL taneously, the increase in the TH level was abolished Oxidized N G F . NGF was oxidized with N-bromosuc(data not shown) and the time course of TH activity cinimide as described by ANGELETTI(1970). The number of tryptophan residues oxidized was determined by the for- was the same as that of the culture control. The total activity of TH after 24 and 48 h in culture mula of SPANDE& WITKOP(1967). Protein determination. Protein was assayed by the control was about 90% and 30% of the zero time method of LOWRY et u/. (1951), using bovine serum albu- control, respectively (zero time level: 0.343 nmol min as a standard. 3 H 2 0 formed/h/ganglion). In the presence of NGF, Materials. Nerve growth factor in the 7 S form was pur- the total enzyme activity was 190% and 180% of zero chased from Burroughs Wellcome and Co. BGJ, medium time control after 24 and 48 h. The content of soluble was obtained from Grand Island Biological Co. and protein per ganglion, as measured in the 18,OOOg ~-[3S-~H]tyrosine(sp. act. = 60.3 Ci/mmol) from New supernatant, in the culture control was 100% and 65% England Nuclear, N6,02'-dibutyryl adenosine 3',5'-cycIic monophosphate (sodium salt), NL,02'-dibutyrylguanosine of the zero-time value at 24 and 48h, respectively 3',5'-cyclic monophosphate (sodium salt), insulin, DL-pro- (zero time level: 21.2 pg protein/ganglion). In the prespranolol-HCI, L-isoproterenol D-bitartrate, theophylline, ence of NGF the comparable figures were 130% and and concanavalin A were from Sigma Chemical Co. 145%. By way of comparison the specific activity of TH Phenoxybenzamine-HCI was from Smith, Kline & French Labs., Sotalol-HCI from Regis Co. and phytohemagglu- in the ganglia increased very little in uiuo during this tinin and hydrocortisone acetate from Calbiochem. Soy- 48 h period, i.e. from the 5th to the 7th postnatal bean agglutinin and wheat germ agglutinins were pur- day. The specific activity of the enzyme after 24 and chased from Miles-Yeda, Ltd. L- and D-Propranolol were 48 h in vivo was not significantly different from the gifts from Ayerst Labs. Dexamethasone sodium phosphate zero time control. was from Merck, Sharp & Dohme. RESULTS
Time course of changes in tyrosine hydroxylase activity in organ culture of superior cervical ganglia As shown in Fig. 1 the specific activity of TH in ganglia cultured in the absence of NGF (CC) was
In view of the observation that T H levels in cultured ganglia remained stable for 15 h, it seemed possible that some endogenous NGF was present in the ganglia when they were removed. Attempts were made to preincubate the SCG without N G F for 15 h in order to remove any endogenous N G F (LARRABEE, 1972). The response to N G F with preincubated gang-
NGF and tyrosine hydroxylase activity in uitro
837
7
NGF
48
12 24 36 TIME IN CULTURE (hours)
NGF Inglmll FIG. 2. Time course of changes in the specific activity of FIG. 3. Effect of the concentration of 2.5 S NGF (0)or tyrosine hydroxylase without nerve growth factor (M, CC) and in the presence of 2.5 S NGF (1 pg/ml) without of 7 S NGF (A) on the specific activity of tyrosine hydroxy(u or with )(+-+) 15 h preincubation period. The lase in 48 h organ culture. Abscissa indicates ng/ml of arrows indicate NGF (1 pg/ml) addition after preincuba- either 2.5s or the 7 s . Each point represents the tion period. Each point represents the average of two mean S.E.(in brackets) of at least three determinations. determinations.
lia was never as great as the response obtained without preincubation. However, the T H activity from such ganglia did rise above the zero time control after 2 4 4 8 h of culture (Fig. 2). The experiment indicates that ganglia are still responsive to N G F even after overnight incubation in its absence. Dependence on NGF concentration The activity of T H showed a roughly linear response to 2.5 S N G F at concentrations between 10 ng (4 x 1 0 - l ' ~ ) and 250ng/ml (1 x ~ O - * M )(Fig. 3). Below lOng/ml N G F did not significantly increase the TH activity. At concentrations above 250ng/ml there was a gradual increase in TH activity. Maximal response occurred at 1 pg/ml of NGF. Higher concentrations of N G F did not appear to depress the TH level. This is different from the response in certain of the bioassay methods where higher concentrations of N G F have been shown to inhibit neurite outOF TABLE1. EFFECTS
Addition None NGF I-Oxi-NGF
2-Oxi-NGF
growth (FENTON, 1970; GREENE,1974; MIZEL& BAMBURG, 1976). Both the 2.5 S and the 7 S forms of N G F are active (Fig. 3). 7 S NGF at 25 ng/ml (2 x lo-'' M) produced a response equivalent to that of 2.5s N G F at 25ng/ml (1 x 1 0 - 9 ~ )The . dose-response curve of 7 s was similar in shape to that of 2 . 5 s NGF, although the maximal response to 7 s was reached at a lower concentration, approx 0.5 pg/ml. Specijicity of the action of N G F on tyrosine hydroxylase There are three tryptophan residues in each peptide chain of 2.5 S N G F (FRAZIER et al., 1973~).Preparations of NGF in which one (1-oxi-NGF) or two (2-oxi-NGF) tryptophan residues had been oxidized by N-bromosuccinimide were assayed in the organ culture system. The results are shown in Table 1. Neither 1-oxi-NGF nor 2-oxi-NGF increased the T H
OXIDIZED 2.5 S NGF ON THE SPECIFIC ACTIVITY OF TYROSINE HYDROXYLASE IN CULTURED GANGLIA
Concentration (ng/ml)
TH activity* (nmol/h/mg protein)
% of control
-
8.89 0.28 11.6 f 0.32t 20.7 f 0.69t 1.os.t 21.2 8.36 f 0.18 9.55 f 0.16 0.87t 18.5 21.2 f 0.81t 9.35 k 0.17 8.91 k 0.21 10.8 f 0.511 12.5 f 0.291
100 130 233 239 94 107 208 238 105 100 122 141
50 500 1000 50 500 lo00 loo00 50 500 lo00 loo00
* *
* Tyrosine hydroxylase activity was measured 40 h after the addition of 2.5 S NGF or its oxidized derivatives. Each value represents the mean f S.E. from at least three determinations. t P < 0.001 compared with minus NGF control. 1P < 0.01 compared with minus NGF control.
M. W. YU et al.
838
TAHLE2. EFFECTS OF
INSULIN ON THE SPECIFIC ACTIVITY OF TYROSINE HYDROXYLASE IN CULTURED GANGLIA
Addition
Concentration (pgiml)
TH activity (nmol/h/mg protein) Minus NGF Plus NGF*
~~
None Insulin
0.1 1.0
10
8.80 0.14 9.95 L- 0.14 10.5 k 0.33 10.1 & 0.40
18.5 f 1.23 20.7 f 1.49 17.2 k 0.29 17.5 0.29
* 1 pg/ml 2.5 S NGF final concentration. Each value represents the mean k S.E. from four determinations. activity when used at concentrations lower than 0.5pg/ml. 1-Oxi-NGF produced almost full T H activity when used at a concentration of 1 pg/ml. 2-0x1NGF at 1 pg/ml increased T H activity slightly while at 10 pg/ml, it increased the enzyme activity to about the same extent as native N G F at 0.05 pg/ml. Therefore, the oxidation of the tryptophan residues in N G F markedly lowers its ability to maintain or increase the levels of T H in cultured SCG. The data are similar to those obtained in studies of the culture of embryonic dorsal root ganglia, where 1-oxi-NGF retained almost full activity, 2-oxi-NGF was partially active, and 3-oxi-NGF had no biological activity (ANGELETTI, 1970; GREENE,1974). This is different from the effect observed by MERRELL et al. (1975) in the chick optic tectal system in which the tryptophan residues were not essential for the activity of NGF. Dexamethasone, cortisol, and glucagon at various concentrations were tried, but had no effect on the T H activity. Insulin, which has some structural similarity to N G F (FRAZIER et al., 1972), had only minimal effect in increasing TH activity at concentrations at which N G F was fully effective (Table 2). Insulin has been reported to markedly increase several anabolic processes in cultured chick dorsal root gnaglia (BURNHAMet al., 1974). A group of plant lectins that have binding, agglutinating, and mitogenic properties have been widely
TIME OF ADDITION OF NGF ANTIBODY (hours)
FIG.5. Time course of NGF antibody effect on the specific activity of tyrosine hydroxylase in the presence of 2.5s NGF (1 pg/ml). NGF antibody was added to the medium at various time intervals at a final concentration of 460 pg protein per ml. Each column represents the mean & S.E. (in brackets) of three determinations. used as probes for membrane-related phenomena (SHARON& LIS, 1972). Concanavalin A has been shown to interact with insulin receptors of fat cells and liver membranes and mimic insulin action in those cells (CUATRECASAS & TELL,1973). Concanavalin A alone has also been shown to stimulate the incorporation of radioactive uridine and radioactive leucine into chick embryo dorsal root ganglia to the same extent as N G F although there did not seem to be any interaction when the two were mixed together (BURNHAM et al., 1974). None of the lectins had any significant effect on ganglionic T H activity either alone or in the presence of N G F (Fig. 4). Purified antibody to N G F prevents the action of N G F on TH. As shown in Fig. 5, when antibody was added at zero time to medium containing 1 pg/ml of N G F the activity of T H after 48 h in culture was the same as in the culture control. When antibody was added between 1 and 18 h after the addition of NGF, the final T H activity was roughly proportional to the time interval. Addition of antibody after 18 h no longer influenced the action of N G F on the enzyme activity. The efSect of dibutyryl cyclic A M P (dbcAMP) on tyrosine hydroxylase
"T
NIKODUEVIC et al. (1975) have recently reported the elevation of the cAMP level of SCG in vitro immediately after N G F addition. In the present experiments, an attempt was made to determine if cAMP can mimic the effect of N G F on TH. Since dbcAMP can pass through the plasma membrane more easily and is not as susceptible to hydrolytic degradation, this derivative of cAMP was used. Theophylline ( 2 mM), tN PHVTOan inhibitor of phosphodiesterase, was also included lpglml Oluglml AGGL HEMAGGL VALlN AGGL in the medium to protect dbcAMP from enzymatic 5r10'M 7110'M 1xlO.M 2110.M degradation. The results are shown in Fig. 6. The FIG.4. Effect of plant lectins either alone or in the presence of 2.5 S NGF on the specific activity of tyrosine hy- presence of theophylline increased the specific activity droxylase. The ganglia were preincubated with the lectins of T H about 10% either in culture control or in gangfor 30 min before the addition of 2.5 S NGF. Each point lia cultured in the presence of 0.1 pg/ml NGF. The maximal effect on TH activity was obtained at 1 represents the average of two determinations. + NGF0.1pQhl
fl
N G F and tyrosine hydroxylase activity in uitro
+
0 lpglml
Olpglml
10-'M
1O'M
THEOPH
THEOPH
THEOPH
t THEOPH
THEOPH
+
839
Olpglml
1O'M
10'M
+
+
+
THEOPH
THEOPH
THEOPH
NGF 0 luglml
FIG. 6. Etfects of dibutyryl cyclic AMP (dbcAMP) either alone or in the presence of 2.5 S NGF on the specific activity of tyrosine hydroxylase. Theophylline (2 KIM) was added and pre-incubated with the ganglia for 30 min prior to the addition of dbcAMP and/or NGF. Each column represents the mean f S.E. (in brackets) of three determinations.
mM-dbcAMP which increased the TH level to about the same extent as NGF at 0.1 pg/ml. However, the combination of NGF, at either 0.1 pg/ml or 1 pg/ml, and dbcAMP did not further increase the enzyme acXivity. To determine if the increase produced by dbcAMP is specific, dibutyryl cyclic GMP (dbcGMP) was also used. As shown in Fig. 7, dbcGMP, even at a concentration of 10 mM, did not increase the TH activity either alone or in combination with NGF. The influence of P-adrenergic blockers on the action of NGF on tyrosine hydroxylase Recently it has been shown that the NGF effect on adenylate cyclase activity of adrenal granule membranes is blocked by L-propranolol, a blocker of the fl-adrenergic receptor (NIKODUEVIC et al., 1976). It, therefore, seemed appropriate to study the effects of the isomers of propranolol and other related comTABLE3: INFLUENCE OF
NGF
FIG.7. Effects of dibutyryl cyclic G M P (dbcGMP) either alone or in the presence of 2.5 S NGF on the specific activity of tyrosine hydroxylase. Theophylline (2 mM) was added in all the medium. Each column represents the mean f S.E. of three determinations.
pounds in the present system. The results are summarized in Table 3. Although the presence of L-propranolol lowered the TH activity, D-propranolol appeared to have a similar inhibitory effect. L-Propranolol is a P-blocker (POTTER, 1967; BARRETT, 1969; BARRETT & CARTER,1970); D-propranolol is virtually devoid of 8-receptor blocking activity, but is reported to have non-specific cell membrane activity. Therefore, the inhibitory effect of propranolol appears to be due to non-specific cell membrane activity. Sotalol, which has been classified as a weak 8-antagonist with essentially no nonspecific cell membrane, i.e. local anesthetic, activity (LISHet al., 1965; BARRETT8z CARTER, 1970), had little influence on the NGF effect even at 1mM. None of the blockers had any effect when presented alone. Phenoxybenzamine, an a-blocker, at a concentration of M,did not block the NGF effect (data not shown). Isoproterenol, a 8-agonist, when used at concentrations between M and M,had no effect either alone or with NGF (data not shown).
P-ADRENERGIC
Addition
(M)
None L-Propranolol
-
10-4
D-Propranolol Sotalol
10-5 10-4 10-5 10-4 10-3
1O'M
THEOPH
0 lgglrnl
BLOCKERS ON THE EFFECT OF 2.5s NGF ON THE SPECIFIC ACTIVITY OF TYROSINE HYDROXYLASE IN CULTURED GANGLIA
Concentration
10.M
THEOPH
TH activity (nmol/h/mg protein) Minus N G F Plus NGF* 9.44 f 0.36 9.53 k 0.30 10.8 k 0.23 7.14 2 0.18 10.6 k 0.60 10.9 k 0.71 8.32 k 0.43 8.09 k 0.27 8.15 & 0.04 7.65 k 0.08
20.1 f 0.51 19.8 _+ 0.63 18.1 0.25 12.7 0.50 18.7 & 0.64 16.9 k 1.40 12.3 i 0.65 18.7 0.62 17.9 +. 0.09 16.7 k 0.55
Each value represents mean k S.E. from at least three determinations. * 1pg/ml 2.5 S N G F final concentration.
M.W. Yu et al.
840
TABLE 4. EFFECTS OF 2.5 S NGF, OXIDIZED NGF, AND NGF ANTIBODY
ON THE SPECIFIC ACTIVITY OF TYROSINE HYDROXY-
LAST AND DIHYDROPTFR1I)INl RFDUCTASE IN CULTURED GANGLIA
Concentration
TH activity
bg/ml)
(nmol/h/mg protein)
Addition
~
None (zero time control) None (culture control) 2.5 S NGF 1-oxi-NGF 2-oxl-NGF NGF Ab NGF + NGF Ab
-
1 .o 5.O 5.0 460 1 .o 460
DHPR activity (nmol NADH oxidized/ 5 min/mg protein) ~~
13.8 If: 1.21 8.81 0.55 19.1 k 0.70* 17.3 f 0.98* 9.71 k 0.56 9.07 k 0.48 9.55 k 0.38
524 k 10.6 458 f 53.4 1496 k 1.55* 1389 5 65.0* 480 k 22.9 613 k 1.81 710 f 11.2
Each value represents the mean f S.E. of at least three determinations. * P < 0001 compared to culture control Dihydropteridine reductase activity
Dihydropteridine reductase (DHPR) is the enzyme which recycles the reduced pteridine cofactor for tyrosine hydroxylation. The activity of this enzyme is higher in ganglia cultured in the presence of NGF than in the culture control (Table 4). In addition, its activity is increased by 1-oxi-NGF, but not by 2-oxiNGF, and the presence of NGF antibody blocks the action of NGF. Generally, the changes in DHPR are comparable to those in TH.
DISCUSSION Superior cervical ganglia in culture have been used before to investigate some aspects of the regulation of TH by NGF (MACKAY, 1974). However, in these experiments, as in most in uitro studies on NGF, serum was added to the culture media. The present experiments were done in the absence of serum which seems quite important since serum contains some NGF (HENDRY, 1972), has been shown to mimic some of the effects of NGF (BURNHAM et al., 1974; MIZEL & BAMBURG, 1976), and also seems to contain some factors which inhibit the actions of NGF (BANKSet al., 1973). STICKCOLD & SHOOTER (1974) have also presented data in preliminary form on NGF and TH levels in SCG cultured in the apparent absence of serum but no full paper on the subject has appeared. The methodology presented in this study represents a possible approach to the quantitative assay of NGF. The applicable range for measuring 2.5 S N G F is between 10 and 250 ng/ml. The method seems simple, accurate, and, as described below, specific. We have encountered some variability in the dose-response curve from experiment to experiment but no more, certainly, than in the other bioassay methods for NGF. More detailed studies need to be done before it is known whether it can be widely applied. However, this assay would seem to offer a more strictly quantitative measure than the widely used bioassay method for N G F (LEVI-MONTALCINI et al., 1954; COHEN,1959; FENTON, 1970; GREENE,1974; MIZEL & BAMBURG,1976).
The action of NGF in this system is quite specific. Oxidized NGF, insulin, glucagon, glucocorticoids, and lectins have been studied. They are all virtually without activity. This is generally true in NGF-responsive systems. On the other hand, it is interesting that insulin, which stimulates the anabolic activities of some NGF-sensitive ganglia (BURNHAM et a!., 1974) has no substantial effect on TH levels in this system. Dibuturyl cAMP has some ability to mimic the effect of NGF on TH activity in the ganglia. This has been observed before (MACKAY & IVERSEN,1972; GOODMAN et al., 1974), but the presence of serum and, thus, perhaps NGF, in these previous cultures allowed some latitude in the interpretation of that data. Here there is no question that dbcAMP, and dbcAMP alone, is causing an increased TH activity after 48 h. There is, however, a question as to whether a rise in cAMP in the cells is obligatory in the action of NGF on TH. That is, is cAMP the ‘second messenger’ for N G F action? In favor of such a relationship are the following points. First, NIKODIJEVIC et al. (1975) found that NGF produced an increase in cAMP level in SCG in uitro. An increase could be seen within 5 min of N G F addition, and with as little as 40 ng/ml, and the effect is quite specific. Second, our present data indicate that dbcAMP, but not dbcGMP, can mimic the NGF effect on TH activity to some substantial extent in a similar SCG organ culture system. This would seem to exclude certain non-specific effects such as induction by sodium butyrate. Third, NGF has now been shown to have an effect on cAMP levels in a totally different system, namely, isolated membranes of adrenal medulla vesicles (NIKODIJEVIC et a!., 1976). Finally, a very similar response in cAMP levels has been recently produced by the addition of a related factor, the epidermal growth factor (EGF), to cultures of lens epithelial cells ( A m et al., 1976). On the other hand, the following points suggest that the relationship is not obligatory. First the levels of N G F necessary to produce a rise in intracellular cAMP are quite high (NIKODIJEVIC et al., 1975) compared to those required to produce an increase in the TH level. Second, the cAMP response is rather
N G F and tyrosine hydroxylase activity in uitro
small and quite transitory. Third, the present data show that dbcAMP had no additional influence when submaximal amounts of N G F was used, and dbcAMP itself was not able to elicit the maximal response. Finally, the antibody studies show that the presence of N G F is necessary long after cAMP levels are back to normal to obtain its effect on TH. I t should be added that other investigators have failed to detect changes in cAMP levels in cultured dorsal root ganglia upon addition of N G F (FRAZIERet al., 1973b; HER rt al., 1973). Thus, although NGF produces a rise in cAMP levels, and dbcAMP, but not dbcGMP, mimics the N G F effect, and although others have shown that treatment of SCG with dbcAMP under other conditions increases T H (MACKAY & IVERSEN, 1972; GOODMAN et al., 1974) and that treatment of sensory ganglia with dbcAMP produces neurite outgrowth (ROISENet al., 1972; HAASet al., 1972), the role of cAMP as the ‘second messenger’ for N G F remains questionable. Our present data show that the effect of N G F on ganglionic TH in organ culture can be blocked by /l-adrenergic blocker but not by a-adrenergic blocker. However, the lack of steric specificity in the propranolol effect, the high concentration of Sotalol required, and the lack of effect by isoproterenol suggest that the inhibition by /?-blockers is non-specific, perhaps a result of membrane effects. Still, it is intriguing that 1-adrenergic response results in activation of adenylate cyclase and accumulation of cAMP (ROBINSON et al., 1971), and some studies indicate that the induction of T H in SCG by stress is dependent on increases of cAMP (GUIDOTTIet al., 1975). The increase of TH activity in our organ culture of rat SCG is a specific and quantifiable property of NGF. It is not clear, however, whether N G F is selectively inducing TH, as it does in uiuo, or whether it is merely allowing the survival of the neurons in the ganglia, which are, after all, the TH-containing cells. The results with dihydropteridine reductase would argue the latter as the major effect. DHPR is not selectively induced in uiuo and does not follow changes in the TH level (NIKODIJEVIC et al., 1977), but in the present work it seems to reflect the changes seen in TH. Thus, it must be concluded that selective induction of T H is not a major effect of N G F in ganglia cultured under these conditions. Other results with SCG in culture have previously been interpreted to mean that it is unsuitable as a model, e.g. for transsynaptic induction, for events in uiuo (GOODMANet al., 1974). It is impossible, however, to avoid the conclusion that N G F has some, albeit minor, effect on TH synthesis in this system. Since the specific activity of TH rises substantially above the zero-time control level in uitro, it is hard to see how such data can be interpreted as due only to neuronal survival. Certainly the specific activity changes are quantitatively less than those seen with N G F in uiuo (THOENEN et al., 1971;
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NIKODIJEVIC et al., 1977), but they seem quite real. That N G F has some selective action on the T H level in uitro has abo been the conclusion of STICKGOLD & SHOOTER(1974). The accompanying paper (MACDONNELL et al., 1977) presents further information on this question. Acknowledgement-The authors appreciate the advice and interest of Dr. D. C. KLEIN.
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