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NAD metabolism and induction of tyrosine aminotransferase in the rat
Int. J. Biochem. Vol. 17, No. 2, pp. 161-165, 1985 Printed in Great Britain. All rights reserved
Copyright
0
0020--711X/85 $3.00 + 0.00 198s Pergamon Press Ltd
NAD METABOLISM AND INDUCTION OF TYROSINE AMINOTRANSFERASE IN THE RAT H. Robert Koch-Institut,
KROGER,
R. GRATZ and H. GRAHN
Nordufer 20, D-1000 Berlin 65. West Germany [Tel. (030) 4503-2301
Abstract-l. The relationship between the NAD-metabolism and the induction of the tyrosine aminotransferase was studied. 2. The content of NAD+ + NADH differs markedly from organ to organ. The highest values can be found in the liver. 3. In intact animals tryptophan leads to an increase of NAD in liver and kidney, but not in brain and spleen. Nicotinamide, on the other hand, induces NAD synthesis in all the organs tested. 4. In adrenalectomized animals, however, there is practically no rise of the NAD content after application of tryptophan contrary to the effect of nicotinamide. 5. The enzyme tyrosine aminotransferase can be induced in intact animals by nicotinamide and tryptophan. This effect is much less pronounced in adrenalectomiced animals. 6. In adrenalectomized animals the induction of the tryrosine aminotransferase by tryptophan is markedly elevated by caffeine and theophylline. Under these conditions there is a significant increase of the NAD content as well. 7. The tryptophan promoted induction of the tyrosine aminotransferase is influenced by inhibitors of the ADPR-transferase, 8. The data presented give further evidence that the NAD adenoribosylation metabolism is involved in the induction of the tyrosine aminotransferase.
RESULTS
INTRODUCTION
The enzyme, tyrosine aminotransferase can be induced by different agents, i.e. by its substrate, tyrosine, cortisone and by tryptophan (Kenney and Flora, 1961; Kriiger et al., 1968). From the inducibility by tryptophan we supposed that the NAD-ADPribosylation metabolism might be involved in the induction of the tyrosine aminotransferase (Kriiger and GrStz, 1978; Kriiger and Grtitz, 1980). The experiments described in this paper have been performed for the purpose of a further elucidation of this problem.
Normal
MATERIALS ANDMETHODS
For the experiments, male Wistar rats (150 g) of the Zentrale Tierversuchsanlage of the BGA (Berlin) were used. The adrenalectomized animals were operated 5 days before starting the experiments. They were given 0.15 M NaCl for drinking. All substances were injected intraperitoneally and the rats were sacrificed 4 hr after administration. The substances were obtained from the following sources: Merck (Darmstadt): caffeine, L-tyrosine, nicotinamide, oL-tryptophan; Sigma (Miinchen): 5azacytidine, benzamide. 3-aminobenzamide, hydrocortisone, I-methylnicotinamide; Serva (Heidelberg): bromdeoxyuridine, guanosine, adenosine, cytidine, thymidine, theophylline; Schwarz-Mann (Orangebourg, N.Y.): I.methionine. Determination of NAD+ + NADH. This was performed according to Nisselbaum and Green (1969). Determination of TAT activity. The method described by Kriiger and Greuer (1965) was used. -.I____ Abbreviations:
NAD-nicotinamide adenine-dinucleotide; TAT-tyrosine aminotransferase; ADPR transferaseADP-ribosyl transferase.
animals
NAD content. Table 1 shows the NAD+ + NADH
content of different organs. There were marked differences between them. The highest values could be seen in the liver, the lowest were in the spleen and the brain, while that for the kidney was higher. Furthermore we measured the increase of the NAD content after administration of nicotinamide and tryptophan. Tryptophan provoked NAD synthesis only in the liver and the kidneys, but not in the brain and the spleen. Nicotinamide on the other hand induced NAD synthesis in all organs examined. The NAD content was higher with nicotinamide than with tryptophan. When studying the influence of different substances on the NAD synthesis we found that Lmethionine reduces the NAD synthesis initiated by nicotinamide in the liver and the kidneys. l-methyl nicotinamide reduced the NAD synthesis from trpytophan in the liver. TA Tinduction. The tyrosine aminotransferase can be induced by tryptophan and by nicotinamide. There was only a minor influence of the different substances on the induction caused by nicotinamide. However, an increase of the tryptophan induction could be noted in the presence of cytidine. guanosine, caffeine and theophyliine. Adrenalectomized NAD contents.
animals
Under these conditions we found only a minor NAD synthesis in the liver and the kidney after the administration of tryptophan (Table 2). It is very interesting, however, that there has been an increase of NAD+ + NADH in the liver and the kidney in the presence of theophylline. l-methylnicotinamide and caffeine.
161
162 Table
H. KROGER era/. 1. Induction
of tyrosine aminotransferase and changes in the NAD content of the organs from intact rats under the mfluence different substances. The organs were analyzed 4hr after the intraperitoneal injection of the substances. TAT
-N
Control
(0.15 M NaCI; 10 ml/kg)
Nicotinamide
(500 mg/kg)
oL-Tryptophan L-Methionine l-Methyl
(3OOmgikg) (500mgIkg)
nicotinamide
(500 mg/kg)
Nicotinamide (500 mg/kg) + L-methionine (5OOmg/kg) or_-Tryptophan (300 mg/kg) + oL-methionine (5OOmglkg) oL-Tryptophan (3OOmg/kg) + l-methyl nicotinamide (500 mg/kg) Nicotinamide (5OOmgIkg) + l-methyl nicotinamide (500 mg/kg) oL-Tryptophan (300 mg/kg) + nicotinamide (500 mg/kg) Theophylline (50mgIkg)
4 -4 4 4 4 4 3 4 4 4
Theophylline (50 mg/kg) + nicotinamide (5OOmg/kg) Thymidine (300 mg/kg)
4
Thymidine (300 “@kg) + nicotinamide (500 mg/kg) Thymidine (300 mgikg) + oL-tryptophan (300 mg/kg) Cytidine (300 mg/kg)
4
Cytidine (300 mg/kg) + nicotinamide (500 mgikg) Cytidine (3OOmgIkg) + “L-tryptophan (300 “@kg) Adenosine (300 mg/kg)
4
Adenosine (300 + nicotinamide Adenosine (300 + owyptophan Guanosine (300
4
mg/kg) (500 mg/kg) mglkg) (3OOmgIkg) mg/kg)
4
3 4
4 4
4 4
Guanosine (300 mg/kg) + nicotinamide (500 mg/kg) Guanosine (300 mg/kg) + oL-tryptophan (300 mg/kg) Bromdeoxyuridine (300 mg/kg)
4
Bromdeoxyuridine (300 mg/kg) + nicotinamide (5OOmgIkg) Bromdeoxyuridine (300 mg/kg) + oL-tryptophan (300 mg/kg) Caffeine (50mg/kg)
4
Caffeine (50 @kg) + nicotinamide (500 mg/kg) Caffeine (50 @kg) + DL-tryptophan (3OOmg/kg)
NAD’ + NADH (pmoligwet tissue)
(p.mol/hr/mg protein)
4
4 4
4 4 4 4
i)t
0.79 f 0.06 4.64 * 0.47 3.70 *OS8 1.50 * 0.30 1.36 + 0.33 4.58 +0.16 4.05 + 0.41 3.30 + 0.56 3.89 ? 0.32 4.67 * 0.41 1.65 +0.32 5.20 f 0.31 0.99 f 0.23 5.35 + 0.03 6.57 +0.16 1.00 f 0.15 4.15 + 0.23 6.04 + 0.52 1.47 f 0.36 2.94 + 0.26 4.24 t 0.64 1.34 +0.54 3.40 f 0.26 5.89 z+0.39 0.98 f0.13 4.03 * 0.55 3.76 ? 0.96 3.32 + 0.35 4.21 + 0.48 5.81 + 0.20
N
Liver
6
0.8075 + 0.0662 2.5738 + 0.3485 1.3909 * 0.1035 0.8737 * 0.0801 0.7744 + 0.0546 1.7156 + 0.2110 1.3456 + 0.2024 1.0355 + 0.2297 2.3995 Ik 0.1868 1.9770 ? 0.2699 0.8793 f 0.0736 1.9561 +_0.5251 0.7424 ? 0.1259 1.9382 + 0.1271 1.4518 + 0.2792 0.9789 ? 0.0473 1.6290 + 1.1798 1.1777 + 0.2770 0.7574 f 0.0570 1.5175 2 0.3332 1.1442 + 0.1856 0.7214 * 0.0573 1.8421 + 0.3340 1.2394 + 0.1042 0.7894 + 0.1075 1.9714 + 0.2853 1.2094 + 0.1677 1.0363 Sz0.1376 2.1510 It 0.3589 1.4048 + 0.1821
3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
N
8 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 4 4 4 4 4
Bral”
N
Spleen
N
Kidnev
0.3293 + 0.0528 0.4150 + 0.0604 0.2951 ? 0.0141 0.2607 * 0.0171 0.2764 ?I 0.0359 0.2891 + 0.0408 0.2117 + 0.0250 0.2559 & 0.0148 0.2941 + 0.0139 0.3756 + 0.0337 0.2190 f 0.0137 0.2832 + 0.0466 0.3144 + 0.0392 0.4679 ? 0.0964 0.3461 -c 0.0378 0.3384 ? 0.0253 0.3817 ? 0.0246 0.3394 + 0.0274 0.2820 * 0.0137 0.2522 + 0.0249 0.3140 + 0.0131 0.1754 & 0.0044 0.2454 f 0.0217 0.3285 ? 0.0152 0.1987 2 0.0147 0.2844 * 0.0222 0.2843 f 0.0193 0.3139 ?z 0.0399 0.3682 + 0.0444 0.3281 + 0.0412
7
0.2908 * 0.0283 0.5084 ? 0.0522 0.3050 + 0.0080 0.3062 + 0.0281 0.3555 + 0.0604 0.4862 f 0.1493 0.2775 * 0.0143 0.2798 + 0.0099 0.4931 + 0.0362 0.4358 * 0.0236 0.3210 + 0.0191 0.4834 + 0.0426 0.3230 -c 0.0217 0.5562 + 0.1056 0.3862 + 0.0174 0.2949 * 0.0777 0.3201 + 0.0386 0.2640 t 0.0210 0.2395 * 0.0296 0.3969 f 0.0234 0.2696 t 0.0235 0.2678 f 0.0179 0.3957 + 0.0410 0.2710 * 0.0784 0.2891 + 0.0606 0.4680 + 0.0662 0.2928 + 0.0289 0.2662 ? 0.0027 0.4292 f 0.0188 0.3333 * 0.0310
3
0.6623 + 0.1369 1.6891 + 0.2705 0.8997 -+ 0.0828 0.5765 + 0.0653 0.5931 + 0.0624 1.1912 + 0.2108 0.8614 c 0.0498 0.7512 i- 0.1520 1.5490 t- 0.0871 1.3727 + 0.2259 0.6250 * 0.0520 1.589X It 0.1772 0.6796 + 0.1467 1.5795 +- 0.2768 1.1017 t 0.0603 0.6532 +_0.0475 1.3315 + 0.2297 I .1574 + 0.0748 0.5444 f 0.0692 1.2948 + 0.4778 0.7963 + 0.0835 0.6383 I,! 0.0729 1.4551 to.1487 0.8571 + 0.0618 0.6492 +_0.0935 1.5194 + 0.3691 0.8863 + 0.0493 0.6291 + 0.0627 I. 1847 f 0.0734 0.8543 ?I 0.0945
3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
4 4 4 4 4 4 4 4 4 4 4 3 4 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
NAD metabolism and induction of
TAT
163
Table 2. Induction of tyrosine aminotransferase and changes in the NAD content of the organs from adrenalectomized rats under the influence of different substances. The animals were analyzed 4hr after the intraperitoneal injection of the substances. TAT
NAD+ + NADH (Fmol/g wet tissue)
N (~moUhr/mg protein)
Control(O.lSMNaCI; Nicotinamide DL-Tryptophan L-Methionine I-Methyl
10mVkg
(5OOmg/kg) (3OOmgIkg) (5OOmg/kg)
nicotinamide
(500 mgikg)
8 4 4 4 4
Nicotinamide (5OOmgIkg) + L-methionine (500 mg/kg) rx-Tryptophan &_Nlm&g) + r-methionine (500 mg/kg) m-Tryptophan (3OOmg/kg) + I-methyl nicotinamide (500mg/kg) Nicotinamide (5OOmgnig) + l-methyl nicotinamide (500 mg/kg) oL-Tryptophan (3OOmg/kg) + nicotinamide (5OOmg/kg) Theophylline (50mg/kg)
4
Theoohvlline
4
150melke)
4 4 4 3 4
2 + m.-iryptophan (300 mg/kg) Prednisolone (10 mg/kg + 5 mg/kg))
4
Prednisolone (10 mg/kg + 5 mg/kg) + nicotinamide (500 mg/kg) Prednisolone (10 mg/kg + 5 mg/kg) + oL-tryptophan (300mgIkg) Thymidine (3OOmgIkg)
4
Thymidine (300 mg/kg) + nicotinamide (5COmgIkg) Thymidine (300 mg/kg) + rx-tryptophan (3OOmgikg) Cytidine (300 mg/kg)
4
Cytidine (300 mgikg) + nicotinamide (500 mg/kg) Cytidine (3OOmgIkg) + IX -tryptophan (300 mglkg) Adenosine (300 mg/kg)
4
Adenosine (300 mg/kg) + nicotinamide (500mp/kg) Adenosine (300&kg) + rx-tryptophan (3OOmgIkg) Guanosine (3OOmg/kg)
2
Guanosine (300 mg/kg) + nicotinamide (5OOmgIkg) Guanosine (3OOmg/kg) + lx-tryptophan (300mgikg) Bromdeoxyuridine (300mg/kg)
4
Bromdeoxyuridine (300 mg/kg) + nicotinamide (5OOmgIkg) Bromdeoxyuridine (300 mg/kg) + nL-tryptophan (3OOmg/kg) Caffeine (50mgikg)
4
Caffeine (50 mg/kg) + nicotinamide (5OOmg/kg) Caffeine (50 mg/kg) + or-tryptophan(3OOmgIkg)
2
4 4
4 4
4 4
3 4
4 4
3 4
4
0.65 _+O.lO 1.80 + 0.20 1.24 kO.51 0.68 f 0.04 0.60 50.11 2.57 f0.93 2.14 + 0.33 2.58 rt 0.50 1.76 z!z0.38 1.39 +0.14 0.93 +0.15 0.96 + Ef 2 1.05 7.64 * 1.90 6.75 + 0.05 10.65 + 1.24 0.40 + 0.05 1.61 ? 0.36 1.25 * 0.33 0.53 * 0.07 0.71 f0.18 1.25 ro.ll 0.77 * 0.28 0.67 +0.15 0.95 _+0.28 0.58 +0.10 0.96 f 0.25 1.07 * 0.38 0.96 f 0.28 2.19 f 0.67 1.03 kO.10 0.83 f 0.08 1.59 _+O.lO 3.15 f 0.20
N
Liver
N
9
0.7056 _+0.0897 2.5134 f 0.6376 0.8645 -f: 0.1303 0.7853 + 0.1005 0.7388 + 0.0786 2.4341 It 0.2113 0.8594 + 0.0491 1.1507 & 0.0756 1.6041 jr 0.1926 2.3427 + 0.9428 0.8308 * 0.0590 2.8339 + y:t;;;
9
3 3 4 4 4 4 4 3 3 3 4 3
4 4 4 4 4 4 4 4 3 4 4 3
3 4 4 4 4 4 4 3 4 4 2 3 4 4 4 4 4 3 4 3 4
lf:o.1109 0.9147 to.1182 2.3196 + 0.6425 1.1837 + 0.1695 0.6618 lr 0.0671 2.0759 * 0.2412 1.4339 ?I 0.1621 0.6886 C 0.0461 2.1242 + 0.3698 1.0224 ? 0.2335 0.7414 + 0.0886 1.9244 * 0.0931 0.8270 * 0.0920 0.7488 f 0.08SO 2.4009 + 0.5775 0.9681 + 0.2717 0.6183 * 0.0805 2.0878 + 0.2362 1.0731 + 0.1667 0.9037 + 0.1385 1.9540 _+0.2461 1.3244 + 0.1386
4 4 4 4 4 4 3 3 4 4 2 3 4 3 3 4 4 3 4 3 4
Brain
0.2550 _+0.0476 0.3526 + 0.0423 0.2809 * 0.0204 0.4430 + 0.0215 0.3237 c 0.0139 0.4092 + 0.0263 0.2821 * 0.0157 0.3032 + 0.0324 0.3316 _+0.0243 0.3604 + 0.0716 0.2795 * 0.0047 0.3793 f 0.0431 0.3303 c 0.0145 0.3230 + 0.0269 0.3737 * 0.0050 0.3353 + 0.0445 0.2644 C 0.0286 0.3565 &.0.0229 0.3393 & 0.0427 0.2071 f 0.0085 0.4571 C 0.0240 0.3874 If- 0.031 1 0.2691 & 0.0291 0.3536 f 0.0350 0.2222 f 0.0327 0.2376 + 0.0234 0.2126 f 0.0312 0.2153 * 0.0947 0.2466 * 0.0162 0.3074 t 0.0334 0.2582 +- 0.0094 0.3263 * 0.0130 0.3957 _t 0.1026 0.3243 + 0.1241
N
Spleen
N
Kidney
9
0.2521 f 0.0391 0.3743 f 0.0427 0.2286 * 0.0285 0.3707 + 0.1720 0.3024 + 0.0307 0.3895 5 0.0291 0.2615 f 0.0374 0.2179 * 0.0266 0.4157 + 0.0668 0.4233 + 0.0598 0.3042 + 0.0155 0.4878
9
0.6429 f0.1948 1.1284 ~0.1090 0.5883 * 0.0459 0.5468 t 0.1093 0.6508 + 0.0514 1.1018 +- 0.0989 0.7082 It 0.0876 0.7074 & 0.0122 I .2257 _t 0.2079 1.3820 & 0.1357 0.7057 * 0.0383 I. 1570 t ;:;;j:
4 4 4 4 4 4 4 4 3 4 4 3 4 4 4 4 4 4 4 3 4 4 2 3 4 4 4 4 4 3 4 3 4
* 11:%1 + 0.0632 0.1944 + 0.0380 0.3066 +0.1152 0.2318 t 0.0640 0.2016 + 0.0131 0.3466 + 0.0576 0.3246 + 0.0444 0.3443 i: 0.0229 0.4215 + 0.0663 0.31 13 + 0.0181 0.207! + 0.0176 0.2643 + 0.0640 0.3023 + 0.0198 0.3125 ?I 0.0281 0.4264 ? 0.0446 0.3064 * 0.0345 0.2643 + 0.0331 0.4738 * O.OSOR 0.2809 5 0.0126 0.2798 ? 0.0163 0.4100 _+0.0244 0.2857 + 0.0339
4 3 3 J 4 4 4 4 3 1 J 3
1 4 1 1 4 3 4 3 1 J 7 3 4 4 4 4 -I 3 3 3 4
IO.0451 0.5404 c 0.2353 0.9787 i 0.2259 0.5489 f 0.1086 0.582 I c 0.0789 1.0046 tO.lhlY 0.7604 + 0.09OY 0.3777 IO.0544 1.0414 t 0.1257 0.9392 + 0 1204 0.5837 + 0.0333 1.091 I + 0.2403 0.5564 _t 0.027Y 0.4227 -t- 0.0522 0.8014 rt 0.2203 0.5879 +o 1296 0.6120 i 0.0677 1.0591 i 0.0949 I 3710 + 0.7868 0.7301 + 0.0981 1.3362 * 0 0x43 0.Y4.58 rtO.lY71
H. KROC~ER et al.
164
TAT induction. The tyrosine aminotransferase can be induced only to a small extent by tryptophan and nicotinamide in adrenalectomized rats. The enzyme is stimulated by substances which lead to an increase of NAD such as theophylline, caffeine and l-methyl nicotinamide.
that benzamide increased the activity provoked tyrosine and by tryptophan + methionine. DISCUSSION
Warburg (1935) demonstrated for the first time the function of NAD as coenzyme in oxydation-reduction-processes. During the last years it could be shown that the overwhelming amount of NAD serves as substrate for the enzyme ADPR-transferase (Shall, personal communication). The ADP-ribosylation is involved in processes of differentiation and regulation (Hilz and Stone, 1976; Hayaishi and Ueda, 1977; Purnell et al., 1980). From earlier publications we supposed that the NAD-ADP ribosylation metabolism takes part in the induction of the tyrosine aminotransferase (Kroger and Gratz, 1978, 1980). In this paper it was shown that substances which inhibit the ADPR transferase influence the induction of the TAT by tryosine and by tryptophan
TAT induction by tryptophan under the influence of ADPR transferase antagonists. From Table 3 it can be
seen that the tryptophan induction of TAT was stimulated by these substances after 4 hr. Measurements of the TAT activity 8 hr after administration of antagonists showed less activity in comparision to DLtryptophan alone. When looking for the data after 12 hr it seemed that there was some delay in the decrease of the activity in the presence of the antagonists. lnjluence of benzamide on the induction of TAT by tyrosine, hydrocortisone and tryptophan + methionine. The TAT induction by hydrocortisone is
not influenced by benzamide
Table 3. Influence
by
(Table 4). Contrary to
of antagonists of the ADPR transferase on the induction of TAT by oL-tryptophan and nicotinamide. were used after a fasting period of 18 hr. All substances were administered by the i.p. route
The normal animals
TAT (pmol/hrlmgprotein) after application Substance NaCl(O.15
M) (10mVkg)
oL-Tryptophan Benzamide
(5OOmg/kg)
(25 mg/kg)
3.Aminobenzamide
(25 mg/kg)
12hr _________0.84 * 0.09
4hr
8hr
1.41 * 0.17
1.3 * 0.35
(4) 2.75 ? 2.00
(5) 7.07 t 0.34
1.18?0.15
(4) 1.72 + 0.68
(5) .46 f 0.20
(5) 1.32 -c 0.41
(5) 1.40f0.42
(5) .s4 + 0.30
(4) 1.95 +0.55
Theophylline
(50mg/kg)
(4) 2.73 ? 0.14
(5) 3.33 ? 0.23
(5) .68 + 0.55
Nicotinamide
(500 mg/kg)
(4) 2.74 + 1.34
(4) 4.00 + 0.16
(5) -72 ? 0.34
(5) 4.81 k 1.46
(5) 6.51 + 2.53
(4) 2.47? 0.62
(4) 6.97 + 0.78
(5) 3.48k1.15
(6) 2.87 + 0.26
4.23 t 1.50
(4) 5.09? 0.62
(5) 5.96 + 0.77
(4) 4.16 & 1.46
(5) 6.37 IO.62
(5) 5.23 t 0.24
(6) 2.55 * 0.45 (5) 3.64 + 0.50 (5) 5.11 + 0.79 (5)
m-Tryptophan (500 mg/kg) + benzamide (25 mg/kg) m.-Tryptophan (300mg/kg) + 3-aminobenzamide (25 mg/kg) m-Tryptophan (500 mg/kg) + theophylline (50mg/kg) oL-Tryptophan (500 mg/kg) + thymidine (300 mg/kg) oL-Tryptophan (500 mg/kg) + nicotinamide (500 mg/kg)
(4)
(4)
Table 4. Influence of benzamide on the induction of tyrosine aminotransferase by L-tyrosine, methionine in normal male rats. The substances were administered to the animals intraperitoneally killed 4 hr after application
hydrocortisone and L-tryptophan + Lafter fasting for 18 hr. The animals were
TAT (~mollhrlmgprotein) NaCl(0.15M; lOml/kg) Benzamide (25 mg/kg) L-Tyrosine (300 mg/kg) L-Tyrosine (300 mg/kg) + benzamide (25 mg/kg) Hydrocortisone (10 mg/kg) Hydrocortisone (10 mgikg) + benzamide (25 mg/kg) L-Tryptophan (300 mg/kg) + L-methionine (300 mg/kg) L-Tryptophan (3OOmg/kg) + L-methionine (3OOmg/kg) + benzamide (25 mg/kg)
1.81 2.27 2.44 3.87
* f + +
0.21 0.39 0.62 0.76
4.78 f 0.26 4.56 f 0.60 3.35i
1.1’)
6.37 + 0.61
NAD metabolism
(Tables l-4). These substances lead to an increase of NAD+ + NADH in adrenalectomized animals also after injection of tryptophan (Table 2). The effects seen may be due to an interference with the NAD ADP-ribosylation metabolism. Earlier, we have found sound indications that galactosamine influences the liver also via the NAD ADP-ribosylation metabolism (Kroger and Gratz, 1983). For some time, it has been known that xanthins are potent inhibitors of the ADPR transferase in vitro (Williams and Johnstone, 1983). Kitamura et al. (1979) discovered that the activity of the ADPR transferase is markedly reduced in nuclei of embryos which were treated with theophylline. It has been shown that the amount of poly ADPR molecules are reduced in the cell under the influence of this substance. Purnell et al. (3980) have demonstrated that the polymer formation can be inhibited by thymidine and by 3-aminobenzamide. It is known that thymidine and nicotinamide are potent inhibitors of ADPR transferase in vitro (Romer et af., 1968). This is also the case in vivo (see Williams and Johnstone, 1983). Taking the data in the literature and our own results together, we would conclude that the effect of the ADPR antagonists on the TATinduction and the NAD synthesis is caused by their influence on the ADPR metabolism. REFERENCES
Bernofsky C. (1980) Physiologic aspects of pyridine nucleotide regulation in mammals. Molec. Cell. Biochem. 33,135-143. Dietrich L. S. (1971) Regulation of nicotinamide metabolism. Am. J. din. Nutr. 24,800-804. Grunicke H., Keller H. J., Liersch M. and Benaguid A. (1974) New aspects of the mechanism and regulation of pyridine nucleotide metabolism. Adv. Enz. Reg. 12, 397-418. Hayaishi 0. and Ueda K. (1977) Poly(ADP-ribose) and
and induction
of TAT
165
ADP-ribosylation of proteins. A. Rev. biochem. 46, 95-l 16. Hilz H. and Stone P. R. (1976) Poly(ADP-ribose) and ADP-ribosylation of proteins. Rev. Physiol. Biochem. Pharmac. 76, 1-59. Jacobson E. L., Lange R. A. and Jacobson M. K. (1979) Pyridine nucleotide synthesis in 3T3 cells. .l Cell. Physiol. 99,417-426. Kenney F. T. and Flora R. M. (1961) Induction of tyrosineu-ketoglutarate transaminase in rat liver. I. Hormonal nature. J. biol. Chem. 236, 2699-2702. Kitamura A., Tanigawa Y., Ohamoto S., Miyake Y. and Shimoyama M. (1979) Theophylline reduces poly(ADPR) synthetase from chick embryo liver nuclei. Biochim. biophys. Acta 677, 6M8. Kroger H. and Greuer B. (1965) EinfluR von Antagonisten des Nucleinsaure-Stoffwechsels und von Rontgenstrahlen auf die Induktion von Enzymen. Biochem. 2. 341, 190-198. Kroger H. and Gratz R. (1978) Induktion der TyrosinAminotransferase (EC. 2.1.5.1) in der Rattenleber durch Nikotinsaureamid. J. clin. Chem. clin. Eiochem. 16. 525-529. Kroger H. and Grltz R. (1980) On the induction of tyrosine aminotransferase (TAT) by tryptophan and methionine. Int. J. Biochem. 12,575-582. Kroger H., Lowe1 M. and Kessel H. (1968) Induktion der Tyrosin-Transaminase in Gegenwart von t.-Tryptophan. Hoppe-Seyler’s Z. physiol. Chem. 349, 1221-1224. Nisselbaum J. S. and Green S. (1969) A simple ultramicro method for determination of pyridine nucleotides in tissues. Analyt. Biochem. 27, 212-217. Purnell M. R., Stone P. R. and Whish W. J. D. (1980) ADPBiochem. Sot. Trans. ribosylation of nuclear protein. f&215-227. Romer V., Lambrecht J., Kittler M.. and Hilz H. (1968) Identity of nuclear NAD nucleosidase with a poly ADPribose forming enzyme in Ehrlich ascites tumor cells. Hoppe-Syler’s Z. physiol. Chem. 349, 109-l 12. Warburg 0. and Christian W. (1975) Co-Fermentproblem. Biochem. Z. 275,464. Williams G. T. and Johnstone A. P. (1983) ADP-ribosyl transferase, rearangement of DNA and cell differentiation. Biosci. Rep. 3. 815-830.
Copyright
0
0020--711X/85 $3.00 + 0.00 198s Pergamon Press Ltd
NAD METABOLISM AND INDUCTION OF TYROSINE AMINOTRANSFERASE IN THE RAT H. Robert Koch-Institut,
KROGER,
R. GRATZ and H. GRAHN
Nordufer 20, D-1000 Berlin 65. West Germany [Tel. (030) 4503-2301
Abstract-l. The relationship between the NAD-metabolism and the induction of the tyrosine aminotransferase was studied. 2. The content of NAD+ + NADH differs markedly from organ to organ. The highest values can be found in the liver. 3. In intact animals tryptophan leads to an increase of NAD in liver and kidney, but not in brain and spleen. Nicotinamide, on the other hand, induces NAD synthesis in all the organs tested. 4. In adrenalectomized animals, however, there is practically no rise of the NAD content after application of tryptophan contrary to the effect of nicotinamide. 5. The enzyme tyrosine aminotransferase can be induced in intact animals by nicotinamide and tryptophan. This effect is much less pronounced in adrenalectomiced animals. 6. In adrenalectomized animals the induction of the tryrosine aminotransferase by tryptophan is markedly elevated by caffeine and theophylline. Under these conditions there is a significant increase of the NAD content as well. 7. The tryptophan promoted induction of the tyrosine aminotransferase is influenced by inhibitors of the ADPR-transferase, 8. The data presented give further evidence that the NAD adenoribosylation metabolism is involved in the induction of the tyrosine aminotransferase.
RESULTS
INTRODUCTION
The enzyme, tyrosine aminotransferase can be induced by different agents, i.e. by its substrate, tyrosine, cortisone and by tryptophan (Kenney and Flora, 1961; Kriiger et al., 1968). From the inducibility by tryptophan we supposed that the NAD-ADPribosylation metabolism might be involved in the induction of the tyrosine aminotransferase (Kriiger and GrStz, 1978; Kriiger and Grtitz, 1980). The experiments described in this paper have been performed for the purpose of a further elucidation of this problem.
Normal
MATERIALS ANDMETHODS
For the experiments, male Wistar rats (150 g) of the Zentrale Tierversuchsanlage of the BGA (Berlin) were used. The adrenalectomized animals were operated 5 days before starting the experiments. They were given 0.15 M NaCl for drinking. All substances were injected intraperitoneally and the rats were sacrificed 4 hr after administration. The substances were obtained from the following sources: Merck (Darmstadt): caffeine, L-tyrosine, nicotinamide, oL-tryptophan; Sigma (Miinchen): 5azacytidine, benzamide. 3-aminobenzamide, hydrocortisone, I-methylnicotinamide; Serva (Heidelberg): bromdeoxyuridine, guanosine, adenosine, cytidine, thymidine, theophylline; Schwarz-Mann (Orangebourg, N.Y.): I.methionine. Determination of NAD+ + NADH. This was performed according to Nisselbaum and Green (1969). Determination of TAT activity. The method described by Kriiger and Greuer (1965) was used. -.I____ Abbreviations:
NAD-nicotinamide adenine-dinucleotide; TAT-tyrosine aminotransferase; ADPR transferaseADP-ribosyl transferase.
animals
NAD content. Table 1 shows the NAD+ + NADH
content of different organs. There were marked differences between them. The highest values could be seen in the liver, the lowest were in the spleen and the brain, while that for the kidney was higher. Furthermore we measured the increase of the NAD content after administration of nicotinamide and tryptophan. Tryptophan provoked NAD synthesis only in the liver and the kidneys, but not in the brain and the spleen. Nicotinamide on the other hand induced NAD synthesis in all organs examined. The NAD content was higher with nicotinamide than with tryptophan. When studying the influence of different substances on the NAD synthesis we found that Lmethionine reduces the NAD synthesis initiated by nicotinamide in the liver and the kidneys. l-methyl nicotinamide reduced the NAD synthesis from trpytophan in the liver. TA Tinduction. The tyrosine aminotransferase can be induced by tryptophan and by nicotinamide. There was only a minor influence of the different substances on the induction caused by nicotinamide. However, an increase of the tryptophan induction could be noted in the presence of cytidine. guanosine, caffeine and theophyliine. Adrenalectomized NAD contents.
animals
Under these conditions we found only a minor NAD synthesis in the liver and the kidney after the administration of tryptophan (Table 2). It is very interesting, however, that there has been an increase of NAD+ + NADH in the liver and the kidney in the presence of theophylline. l-methylnicotinamide and caffeine.
161
162 Table
H. KROGER era/. 1. Induction
of tyrosine aminotransferase and changes in the NAD content of the organs from intact rats under the mfluence different substances. The organs were analyzed 4hr after the intraperitoneal injection of the substances. TAT
-N
Control
(0.15 M NaCI; 10 ml/kg)
Nicotinamide
(500 mg/kg)
oL-Tryptophan L-Methionine l-Methyl
(3OOmgikg) (500mgIkg)
nicotinamide
(500 mg/kg)
Nicotinamide (500 mg/kg) + L-methionine (5OOmg/kg) or_-Tryptophan (300 mg/kg) + oL-methionine (5OOmglkg) oL-Tryptophan (3OOmg/kg) + l-methyl nicotinamide (500 mg/kg) Nicotinamide (5OOmgIkg) + l-methyl nicotinamide (500 mg/kg) oL-Tryptophan (300 mg/kg) + nicotinamide (500 mg/kg) Theophylline (50mgIkg)
4 -4 4 4 4 4 3 4 4 4
Theophylline (50 mg/kg) + nicotinamide (5OOmg/kg) Thymidine (300 mg/kg)
4
Thymidine (300 “@kg) + nicotinamide (500 mg/kg) Thymidine (300 mgikg) + oL-tryptophan (300 mg/kg) Cytidine (300 mg/kg)
4
Cytidine (300 mg/kg) + nicotinamide (500 mgikg) Cytidine (3OOmgIkg) + “L-tryptophan (300 “@kg) Adenosine (300 mg/kg)
4
Adenosine (300 + nicotinamide Adenosine (300 + owyptophan Guanosine (300
4
mg/kg) (500 mg/kg) mglkg) (3OOmgIkg) mg/kg)
4
3 4
4 4
4 4
Guanosine (300 mg/kg) + nicotinamide (500 mg/kg) Guanosine (300 mg/kg) + oL-tryptophan (300 mg/kg) Bromdeoxyuridine (300 mg/kg)
4
Bromdeoxyuridine (300 mg/kg) + nicotinamide (5OOmgIkg) Bromdeoxyuridine (300 mg/kg) + oL-tryptophan (300 mg/kg) Caffeine (50mg/kg)
4
Caffeine (50 @kg) + nicotinamide (500 mg/kg) Caffeine (50 @kg) + DL-tryptophan (3OOmg/kg)
NAD’ + NADH (pmoligwet tissue)
(p.mol/hr/mg protein)
4
4 4
4 4 4 4
i)t
0.79 f 0.06 4.64 * 0.47 3.70 *OS8 1.50 * 0.30 1.36 + 0.33 4.58 +0.16 4.05 + 0.41 3.30 + 0.56 3.89 ? 0.32 4.67 * 0.41 1.65 +0.32 5.20 f 0.31 0.99 f 0.23 5.35 + 0.03 6.57 +0.16 1.00 f 0.15 4.15 + 0.23 6.04 + 0.52 1.47 f 0.36 2.94 + 0.26 4.24 t 0.64 1.34 +0.54 3.40 f 0.26 5.89 z+0.39 0.98 f0.13 4.03 * 0.55 3.76 ? 0.96 3.32 + 0.35 4.21 + 0.48 5.81 + 0.20
N
Liver
6
0.8075 + 0.0662 2.5738 + 0.3485 1.3909 * 0.1035 0.8737 * 0.0801 0.7744 + 0.0546 1.7156 + 0.2110 1.3456 + 0.2024 1.0355 + 0.2297 2.3995 Ik 0.1868 1.9770 ? 0.2699 0.8793 f 0.0736 1.9561 +_0.5251 0.7424 ? 0.1259 1.9382 + 0.1271 1.4518 + 0.2792 0.9789 ? 0.0473 1.6290 + 1.1798 1.1777 + 0.2770 0.7574 f 0.0570 1.5175 2 0.3332 1.1442 + 0.1856 0.7214 * 0.0573 1.8421 + 0.3340 1.2394 + 0.1042 0.7894 + 0.1075 1.9714 + 0.2853 1.2094 + 0.1677 1.0363 Sz0.1376 2.1510 It 0.3589 1.4048 + 0.1821
3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
N
8 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 4 4 4 4 4
Bral”
N
Spleen
N
Kidnev
0.3293 + 0.0528 0.4150 + 0.0604 0.2951 ? 0.0141 0.2607 * 0.0171 0.2764 ?I 0.0359 0.2891 + 0.0408 0.2117 + 0.0250 0.2559 & 0.0148 0.2941 + 0.0139 0.3756 + 0.0337 0.2190 f 0.0137 0.2832 + 0.0466 0.3144 + 0.0392 0.4679 ? 0.0964 0.3461 -c 0.0378 0.3384 ? 0.0253 0.3817 ? 0.0246 0.3394 + 0.0274 0.2820 * 0.0137 0.2522 + 0.0249 0.3140 + 0.0131 0.1754 & 0.0044 0.2454 f 0.0217 0.3285 ? 0.0152 0.1987 2 0.0147 0.2844 * 0.0222 0.2843 f 0.0193 0.3139 ?z 0.0399 0.3682 + 0.0444 0.3281 + 0.0412
7
0.2908 * 0.0283 0.5084 ? 0.0522 0.3050 + 0.0080 0.3062 + 0.0281 0.3555 + 0.0604 0.4862 f 0.1493 0.2775 * 0.0143 0.2798 + 0.0099 0.4931 + 0.0362 0.4358 * 0.0236 0.3210 + 0.0191 0.4834 + 0.0426 0.3230 -c 0.0217 0.5562 + 0.1056 0.3862 + 0.0174 0.2949 * 0.0777 0.3201 + 0.0386 0.2640 t 0.0210 0.2395 * 0.0296 0.3969 f 0.0234 0.2696 t 0.0235 0.2678 f 0.0179 0.3957 + 0.0410 0.2710 * 0.0784 0.2891 + 0.0606 0.4680 + 0.0662 0.2928 + 0.0289 0.2662 ? 0.0027 0.4292 f 0.0188 0.3333 * 0.0310
3
0.6623 + 0.1369 1.6891 + 0.2705 0.8997 -+ 0.0828 0.5765 + 0.0653 0.5931 + 0.0624 1.1912 + 0.2108 0.8614 c 0.0498 0.7512 i- 0.1520 1.5490 t- 0.0871 1.3727 + 0.2259 0.6250 * 0.0520 1.589X It 0.1772 0.6796 + 0.1467 1.5795 +- 0.2768 1.1017 t 0.0603 0.6532 +_0.0475 1.3315 + 0.2297 I .1574 + 0.0748 0.5444 f 0.0692 1.2948 + 0.4778 0.7963 + 0.0835 0.6383 I,! 0.0729 1.4551 to.1487 0.8571 + 0.0618 0.6492 +_0.0935 1.5194 + 0.3691 0.8863 + 0.0493 0.6291 + 0.0627 I. 1847 f 0.0734 0.8543 ?I 0.0945
3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
4 4 4 4 4 4 4 4 4 4 4 3 4 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
NAD metabolism and induction of
TAT
163
Table 2. Induction of tyrosine aminotransferase and changes in the NAD content of the organs from adrenalectomized rats under the influence of different substances. The animals were analyzed 4hr after the intraperitoneal injection of the substances. TAT
NAD+ + NADH (Fmol/g wet tissue)
N (~moUhr/mg protein)
Control(O.lSMNaCI; Nicotinamide DL-Tryptophan L-Methionine I-Methyl
10mVkg
(5OOmg/kg) (3OOmgIkg) (5OOmg/kg)
nicotinamide
(500 mgikg)
8 4 4 4 4
Nicotinamide (5OOmgIkg) + L-methionine (500 mg/kg) rx-Tryptophan &_Nlm&g) + r-methionine (500 mg/kg) m-Tryptophan (3OOmg/kg) + I-methyl nicotinamide (500mg/kg) Nicotinamide (5OOmgnig) + l-methyl nicotinamide (500 mg/kg) oL-Tryptophan (3OOmg/kg) + nicotinamide (5OOmg/kg) Theophylline (50mg/kg)
4
Theoohvlline
4
150melke)
4 4 4 3 4
2 + m.-iryptophan (300 mg/kg) Prednisolone (10 mg/kg + 5 mg/kg))
4
Prednisolone (10 mg/kg + 5 mg/kg) + nicotinamide (500 mg/kg) Prednisolone (10 mg/kg + 5 mg/kg) + oL-tryptophan (300mgIkg) Thymidine (3OOmgIkg)
4
Thymidine (300 mg/kg) + nicotinamide (5COmgIkg) Thymidine (300 mg/kg) + rx-tryptophan (3OOmgikg) Cytidine (300 mg/kg)
4
Cytidine (300 mgikg) + nicotinamide (500 mg/kg) Cytidine (3OOmgIkg) + IX -tryptophan (300 mglkg) Adenosine (300 mg/kg)
4
Adenosine (300 mg/kg) + nicotinamide (500mp/kg) Adenosine (300&kg) + rx-tryptophan (3OOmgIkg) Guanosine (3OOmg/kg)
2
Guanosine (300 mg/kg) + nicotinamide (5OOmgIkg) Guanosine (3OOmg/kg) + lx-tryptophan (300mgikg) Bromdeoxyuridine (300mg/kg)
4
Bromdeoxyuridine (300 mg/kg) + nicotinamide (5OOmgIkg) Bromdeoxyuridine (300 mg/kg) + nL-tryptophan (3OOmg/kg) Caffeine (50mgikg)
4
Caffeine (50 mg/kg) + nicotinamide (5OOmg/kg) Caffeine (50 mg/kg) + or-tryptophan(3OOmgIkg)
2
4 4
4 4
4 4
3 4
4 4
3 4
4
0.65 _+O.lO 1.80 + 0.20 1.24 kO.51 0.68 f 0.04 0.60 50.11 2.57 f0.93 2.14 + 0.33 2.58 rt 0.50 1.76 z!z0.38 1.39 +0.14 0.93 +0.15 0.96 + Ef 2 1.05 7.64 * 1.90 6.75 + 0.05 10.65 + 1.24 0.40 + 0.05 1.61 ? 0.36 1.25 * 0.33 0.53 * 0.07 0.71 f0.18 1.25 ro.ll 0.77 * 0.28 0.67 +0.15 0.95 _+0.28 0.58 +0.10 0.96 f 0.25 1.07 * 0.38 0.96 f 0.28 2.19 f 0.67 1.03 kO.10 0.83 f 0.08 1.59 _+O.lO 3.15 f 0.20
N
Liver
N
9
0.7056 _+0.0897 2.5134 f 0.6376 0.8645 -f: 0.1303 0.7853 + 0.1005 0.7388 + 0.0786 2.4341 It 0.2113 0.8594 + 0.0491 1.1507 & 0.0756 1.6041 jr 0.1926 2.3427 + 0.9428 0.8308 * 0.0590 2.8339 + y:t;;;
9
3 3 4 4 4 4 4 3 3 3 4 3
4 4 4 4 4 4 4 4 3 4 4 3
3 4 4 4 4 4 4 3 4 4 2 3 4 4 4 4 4 3 4 3 4
lf:o.1109 0.9147 to.1182 2.3196 + 0.6425 1.1837 + 0.1695 0.6618 lr 0.0671 2.0759 * 0.2412 1.4339 ?I 0.1621 0.6886 C 0.0461 2.1242 + 0.3698 1.0224 ? 0.2335 0.7414 + 0.0886 1.9244 * 0.0931 0.8270 * 0.0920 0.7488 f 0.08SO 2.4009 + 0.5775 0.9681 + 0.2717 0.6183 * 0.0805 2.0878 + 0.2362 1.0731 + 0.1667 0.9037 + 0.1385 1.9540 _+0.2461 1.3244 + 0.1386
4 4 4 4 4 4 3 3 4 4 2 3 4 3 3 4 4 3 4 3 4
Brain
0.2550 _+0.0476 0.3526 + 0.0423 0.2809 * 0.0204 0.4430 + 0.0215 0.3237 c 0.0139 0.4092 + 0.0263 0.2821 * 0.0157 0.3032 + 0.0324 0.3316 _+0.0243 0.3604 + 0.0716 0.2795 * 0.0047 0.3793 f 0.0431 0.3303 c 0.0145 0.3230 + 0.0269 0.3737 * 0.0050 0.3353 + 0.0445 0.2644 C 0.0286 0.3565 &.0.0229 0.3393 & 0.0427 0.2071 f 0.0085 0.4571 C 0.0240 0.3874 If- 0.031 1 0.2691 & 0.0291 0.3536 f 0.0350 0.2222 f 0.0327 0.2376 + 0.0234 0.2126 f 0.0312 0.2153 * 0.0947 0.2466 * 0.0162 0.3074 t 0.0334 0.2582 +- 0.0094 0.3263 * 0.0130 0.3957 _t 0.1026 0.3243 + 0.1241
N
Spleen
N
Kidney
9
0.2521 f 0.0391 0.3743 f 0.0427 0.2286 * 0.0285 0.3707 + 0.1720 0.3024 + 0.0307 0.3895 5 0.0291 0.2615 f 0.0374 0.2179 * 0.0266 0.4157 + 0.0668 0.4233 + 0.0598 0.3042 + 0.0155 0.4878
9
0.6429 f0.1948 1.1284 ~0.1090 0.5883 * 0.0459 0.5468 t 0.1093 0.6508 + 0.0514 1.1018 +- 0.0989 0.7082 It 0.0876 0.7074 & 0.0122 I .2257 _t 0.2079 1.3820 & 0.1357 0.7057 * 0.0383 I. 1570 t ;:;;j:
4 4 4 4 4 4 4 4 3 4 4 3 4 4 4 4 4 4 4 3 4 4 2 3 4 4 4 4 4 3 4 3 4
* 11:%1 + 0.0632 0.1944 + 0.0380 0.3066 +0.1152 0.2318 t 0.0640 0.2016 + 0.0131 0.3466 + 0.0576 0.3246 + 0.0444 0.3443 i: 0.0229 0.4215 + 0.0663 0.31 13 + 0.0181 0.207! + 0.0176 0.2643 + 0.0640 0.3023 + 0.0198 0.3125 ?I 0.0281 0.4264 ? 0.0446 0.3064 * 0.0345 0.2643 + 0.0331 0.4738 * O.OSOR 0.2809 5 0.0126 0.2798 ? 0.0163 0.4100 _+0.0244 0.2857 + 0.0339
4 3 3 J 4 4 4 4 3 1 J 3
1 4 1 1 4 3 4 3 1 J 7 3 4 4 4 4 -I 3 3 3 4
IO.0451 0.5404 c 0.2353 0.9787 i 0.2259 0.5489 f 0.1086 0.582 I c 0.0789 1.0046 tO.lhlY 0.7604 + 0.09OY 0.3777 IO.0544 1.0414 t 0.1257 0.9392 + 0 1204 0.5837 + 0.0333 1.091 I + 0.2403 0.5564 _t 0.027Y 0.4227 -t- 0.0522 0.8014 rt 0.2203 0.5879 +o 1296 0.6120 i 0.0677 1.0591 i 0.0949 I 3710 + 0.7868 0.7301 + 0.0981 1.3362 * 0 0x43 0.Y4.58 rtO.lY71
H. KROC~ER et al.
164
TAT induction. The tyrosine aminotransferase can be induced only to a small extent by tryptophan and nicotinamide in adrenalectomized rats. The enzyme is stimulated by substances which lead to an increase of NAD such as theophylline, caffeine and l-methyl nicotinamide.
that benzamide increased the activity provoked tyrosine and by tryptophan + methionine. DISCUSSION
Warburg (1935) demonstrated for the first time the function of NAD as coenzyme in oxydation-reduction-processes. During the last years it could be shown that the overwhelming amount of NAD serves as substrate for the enzyme ADPR-transferase (Shall, personal communication). The ADP-ribosylation is involved in processes of differentiation and regulation (Hilz and Stone, 1976; Hayaishi and Ueda, 1977; Purnell et al., 1980). From earlier publications we supposed that the NAD-ADP ribosylation metabolism takes part in the induction of the tyrosine aminotransferase (Kroger and Gratz, 1978, 1980). In this paper it was shown that substances which inhibit the ADPR transferase influence the induction of the TAT by tryosine and by tryptophan
TAT induction by tryptophan under the influence of ADPR transferase antagonists. From Table 3 it can be
seen that the tryptophan induction of TAT was stimulated by these substances after 4 hr. Measurements of the TAT activity 8 hr after administration of antagonists showed less activity in comparision to DLtryptophan alone. When looking for the data after 12 hr it seemed that there was some delay in the decrease of the activity in the presence of the antagonists. lnjluence of benzamide on the induction of TAT by tyrosine, hydrocortisone and tryptophan + methionine. The TAT induction by hydrocortisone is
not influenced by benzamide
Table 3. Influence
by
(Table 4). Contrary to
of antagonists of the ADPR transferase on the induction of TAT by oL-tryptophan and nicotinamide. were used after a fasting period of 18 hr. All substances were administered by the i.p. route
The normal animals
TAT (pmol/hrlmgprotein) after application Substance NaCl(O.15
M) (10mVkg)
oL-Tryptophan Benzamide
(5OOmg/kg)
(25 mg/kg)
3.Aminobenzamide
(25 mg/kg)
12hr _________0.84 * 0.09
4hr
8hr
1.41 * 0.17
1.3 * 0.35
(4) 2.75 ? 2.00
(5) 7.07 t 0.34
1.18?0.15
(4) 1.72 + 0.68
(5) .46 f 0.20
(5) 1.32 -c 0.41
(5) 1.40f0.42
(5) .s4 + 0.30
(4) 1.95 +0.55
Theophylline
(50mg/kg)
(4) 2.73 ? 0.14
(5) 3.33 ? 0.23
(5) .68 + 0.55
Nicotinamide
(500 mg/kg)
(4) 2.74 + 1.34
(4) 4.00 + 0.16
(5) -72 ? 0.34
(5) 4.81 k 1.46
(5) 6.51 + 2.53
(4) 2.47? 0.62
(4) 6.97 + 0.78
(5) 3.48k1.15
(6) 2.87 + 0.26
4.23 t 1.50
(4) 5.09? 0.62
(5) 5.96 + 0.77
(4) 4.16 & 1.46
(5) 6.37 IO.62
(5) 5.23 t 0.24
(6) 2.55 * 0.45 (5) 3.64 + 0.50 (5) 5.11 + 0.79 (5)
m-Tryptophan (500 mg/kg) + benzamide (25 mg/kg) m.-Tryptophan (300mg/kg) + 3-aminobenzamide (25 mg/kg) m-Tryptophan (500 mg/kg) + theophylline (50mg/kg) oL-Tryptophan (500 mg/kg) + thymidine (300 mg/kg) oL-Tryptophan (500 mg/kg) + nicotinamide (500 mg/kg)
(4)
(4)
Table 4. Influence of benzamide on the induction of tyrosine aminotransferase by L-tyrosine, methionine in normal male rats. The substances were administered to the animals intraperitoneally killed 4 hr after application
hydrocortisone and L-tryptophan + Lafter fasting for 18 hr. The animals were
TAT (~mollhrlmgprotein) NaCl(0.15M; lOml/kg) Benzamide (25 mg/kg) L-Tyrosine (300 mg/kg) L-Tyrosine (300 mg/kg) + benzamide (25 mg/kg) Hydrocortisone (10 mg/kg) Hydrocortisone (10 mgikg) + benzamide (25 mg/kg) L-Tryptophan (300 mg/kg) + L-methionine (300 mg/kg) L-Tryptophan (3OOmg/kg) + L-methionine (3OOmg/kg) + benzamide (25 mg/kg)
1.81 2.27 2.44 3.87
* f + +
0.21 0.39 0.62 0.76
4.78 f 0.26 4.56 f 0.60 3.35i
1.1’)
6.37 + 0.61
NAD metabolism
(Tables l-4). These substances lead to an increase of NAD+ + NADH in adrenalectomized animals also after injection of tryptophan (Table 2). The effects seen may be due to an interference with the NAD ADP-ribosylation metabolism. Earlier, we have found sound indications that galactosamine influences the liver also via the NAD ADP-ribosylation metabolism (Kroger and Gratz, 1983). For some time, it has been known that xanthins are potent inhibitors of the ADPR transferase in vitro (Williams and Johnstone, 1983). Kitamura et al. (1979) discovered that the activity of the ADPR transferase is markedly reduced in nuclei of embryos which were treated with theophylline. It has been shown that the amount of poly ADPR molecules are reduced in the cell under the influence of this substance. Purnell et al. (3980) have demonstrated that the polymer formation can be inhibited by thymidine and by 3-aminobenzamide. It is known that thymidine and nicotinamide are potent inhibitors of ADPR transferase in vitro (Romer et af., 1968). This is also the case in vivo (see Williams and Johnstone, 1983). Taking the data in the literature and our own results together, we would conclude that the effect of the ADPR antagonists on the TATinduction and the NAD synthesis is caused by their influence on the ADPR metabolism. REFERENCES
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