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Biphasic effect of norepinephrine in the regulation of hepatic tyrosine transaminase activity
ARCHIVES
OF
Biphasic
BIOCHEMISTRY
Effect
AND
BIOPHYSICS
of Norepinephrine Tyrosine
Laboratory
614-619 (1970)
138,
in the Regulation
Transaminase
IRA B. BLACK
AND
Science, National
Institute
of Clinical
Received
January
JULIUS of Mental
26, 1970; accepted
of Hepatic
Activity AXELROD Health, April
Bethesda, Maryland
20014
7, 1970
Norepinephrine injection is associated with a biphasic response of heptatic tyroL-tyrosine: 2-oxoglutarate aminosine transaminase (tyrosine aminotransferase, transferase EC 2.6.1.5) activity in the adrenalectomized and hypophysectomized rat. After an initial increase, enzyme activity falls below control levels. The increase in tyrosine transaminase activity is blocked by cycloheximide but not actinomycin D, ergotamine tartrate, or propranalol. Depression of tyrosine transaminase activity by norepinephrine is maximal at night when enzyme activity normally rises to peak levels, resulting in a suppression of the circadian enzyme rhythm. The decreased activity at night occurs without prior induction, indicating the independence of these two effects of norepinephrine. Evidence suggesting that norepinephrine depresses tyrosine transaminase activity by competing with apoenzyme for the pyridoxal-5’phosphate cofactor is presented. These observations indicate that a regulator molecule may have multiple effects on an enzyme system varying not only with dosage but with time of administration.
Norepinephrine suppresses the activity of rat liver tyrosine transaminase in vitro and in vivo (l-5). This neuromediator, by forming a complex with the pyridoxal-5’phosphate cofactor, appears to decrease the rate of apoenzyme synthesis (4, 5). In addition, treatment with norepinephrine suppressed the daily enzyme rhythm by abolishing the evening peak in tyrosine transaminase activity while daytime, basal activity was not altered (5). These observations suggested that the effects of norepinephrine on the hepatic tyrosine transaminase system vary with time of day. This communication reports that norepinephrine has a biphasic, time-related effect on hepatic tyrosine transaminase activity. MATERIALS
AND
used and were fasted for the duration of each experiment. The efficacy of adrenalectomy was established by the inability of randomly chosen rats to survive for 1 week on tap water, and by inspection of the retroperitoneal space at the time of death. Purina Chow and 1% NaCl solution were provided ad Zibitum except during experimentation. Hypophysectomized rats were maintained on 1% saline, oranges, and Purina Chow and used 1 week after surgery. At the termination of each experiment rats were killed by a blow to the head, and livers were immediately removed and frozen. After homogenization in isotonic KC1 solution, livers were assayed for tyrosine transaminase activity by a modification of the method of Diamondstone (8). (-) Arterenol bitartrate (norepinephrine) was obtained from Calbiochem. Cycloheximide and actinomycin D were generous gifts of Harry B. Wood, Chief, Drug Development Branch, NIH. Pyridoxine hydrochloride (Nutritional Biochemicals Corp.) was dissolved in water and brought to neutral pH before administration. Propranalol was purchased as Inderal from Ayerst Lab. Phenoxybenzamine was generously donated by Harry Green, Head, Neurobiochemistry Section at Smith, Kline and French Labs.
METHODS
Female, adrenalectomized, Sprague-Dawley, 160-180-g rats were housed in clear plastic cages and exposed to a lighting schedule, with lights on from 5 A.M. to 7 P.M. Since glucocorticoids (6) and various amino acids (7) induce tyrosine transaminase, only adrenalectomized rats were 614
BIPHASIC
EFFECTS
TABLE
RESULTS
Treatment with norepinephrine resulted in a biphasic response of tyrosine transaminase activity (Fig. 1). Enzyme activity increased for the first hour after injection but subsequently fell below control levels at 3 hr. By 4 hr, activity was returning to baseline levels. To preclude the possibility that one, or both, of the effects of norepinephrine was mediated by growth hormone secretion (9, lo), the experiment was also performed in hypophysectomized rats. The results were similar (Fig. 1). To elucidate the nature of this unusual time course, the elevation and depression of enzyme activity were characterized separately. Norepinephrine over the range of 1.0 to 4.0 mg/kg caused a 50-70 % increase in tyrosine transaminase activity in 1 hr (Table I). Treatment with higher doses resulted in a significant mortality rate. This
FIG. 1. Tyrosine transaminase activity after norepinephrine. Rats were treated with norepinephrine, 1.0 mg/kg subcutaneously at time zero (10 A.M.), and groups of six to eight were sacrificed at the indicated times. Results are expressed as mean f SE (vertical bars). Solid line represents adrenalectomized animals, and dashed line represents hypophysectomized rats. Control groups differ from l- and 3-hr groups at p < .Ol.
615
OF NOREPINEPHRINE I
ELEVATION OF TYHOSINE TRANS~MIN~SE ACTIVITY 1 HR AFTER NOREPINEPHRINE ADMINISTRATION” Dose (w/kg) 0 0.5 1.0 2.0 4.0
Tyrosine transaminase activity (pm&s product/g/hr)
67.6 84.5 88.3 89.7 102.6
f dz zt -L &
2.5 7.7 2.6b 3.5b 2.9*
a Groups of eight rats were treated with the indicated doses of norepinephrine subcutaneously in a volume of 1 ml and were killed 1 hr later. Results are expressed as mean f SE. b Differs from control at p < ,001.
increase in enzyme activity could be due to the activation of preexistent tyrosine transaminase molecules or to the accumulation of increased enzyme protein. To distinguish between these alternatives, rats were treated with norepinephrine and/or the protein synthesis inhibitor cycloheximide (11). Abolition of the increase in tyrosine transaminase activity by inhibition of protein synthesis suggests that norepinephrine induces enzyme (Table II). To determine whether on-going RNA synthesis is necessary for tyrosine transaminase induction by norepinephrine, rats were treated with actinomycin D (12). Inhibition of RNA synthesis did not prevent the rise in tyrosine transaminase activity after norepinephrine (Table II), suggesting that the neuromediator increases tyrosine transaminase activity through a process independent of RNA synthesis. Tyrosine transaminase has been reported to respond to cyclic AMP in fetal liver explants (13) and the rat fetus in utero (14). Since cyclic AMP is associated with the b-receptor (15), the possible role of /3receptor mediation was examined. Treatment with the P-receptor blocking agent, propranalol, had no effect on norepinephrine induction (Table III). Ergotamine tartrate, which specifically blocks hepatic P-receptor activity (16), was also without effect on induction. In addition, a-receptor blockade with phenoxybenzamine failed to alter the enzyme rise after norepinephrine. The decline of enzyme activity at 3 hr was related to the initial level of enzyme
616
BLACK
AND
activity: the higher the control level, the greater the decrease. It was of interest, therefore, to compare the effect of norepinephrine during the day when enzyme activity is low, with its action at night when enzyme activity is high. Maximal depression of enzyme activity occurred during the evening when tyrosine transaminase was at peak levels (Table IV) with resultant suppression of the enzyme rhythm. To further investigate this difference in norepinephrine effect with time of day, a time course was performed during the evening. Since enzyme activity rises to a sharp peak at night, norepinephrine was administered at various times before normal peak activity. All rats were sacrificed at 11 P.M. In the evening, the suppressive effects of the amine were apparent, with greatest depression of enzyme activity 4 hr after injection (Fig. 2). In contrast to daytime injection (Fig. l), no induction was observed 1 hr after the nightTABLE
II
EFFECT OF INHIBITION OF PROTEIN OR RNA SYNTHESIS ON ELEVATION OF TYROSINE TRANSAMINASE ACTIVITY BY NOREPINEPHRINE" Tyrosine transaminase Group
Experiment 1 Control Norepinephrine Cycloheximide Norepinephrine + Cycloheximide Experiment 2 Control Norepinephrine Actinomycin D Norepinephrine + Actinomycin D
activity &nmles product/g/hr)
77.8 137.9 69.8 74.1
f f f *
7.7 12.5* 6.6 7.9
85.4 130.6 92.9 116.4
f f f f
3.6 9.5 3.9 9.5c
a In Experiment 1 groups of eight rats were treated with norepinephrine, 1.0 mg/kg subcutaneously at 10 A.M. and/or cycloheximide, 50 mg/kg intraperitoneally. In Experiment 2 rats were treated in a similar fashion with norepinephrine and/or actinomycin D, 1.5 mg/kg intraperitoneally 15 min earlier. Controls were treated at appropriate times with saline. Results are expressed as mean f SE. * Differs from control and group treated with both drugs at p < .Ol. c Does not differ significantly from group treated with norepinephrine alone.
AXELROD TABLE
III
ROLES OF CC- AND &RECEPTORS IN NOREPINEPHRINE INDUCTION OF TYROSINE TRANSAMINaSE" Tyrosine transaminase Group
Experiment 1 Control Norepinephrine Phenoxybenzamine Propranalol Norepinephrine + Phenoxybenzamine Norepinephrine + Propranalol Experiment 2 Control Norepinephrine Ergotamine Norepinephrine + Ergotamine
activity (pm&s/ product/g/hr)
61.8 99.3 64.2 68.5 123.5
f It f zt f
3.9 4.23 8.3 6.3 ll.l*
105.6 f
8.7*
92.9 144.9 87.2 128.7
12.1 10.4c 12.3 9.e
i f f f
a Groups of eight rats were treated with norepinephrine as in Fig. 2 and/or in Experiment 1 with either phenoxybenzamine or propranalol, 10 mg/kg intraperitoneally 1 hr earlier. In Experiment 2 groups were treated with norepinephrine as above and/or with ergotamine tartrate, 1.0 mg/kg intraperitoneally 30 min earlier. Rats were killed 1 hr after norepinephrine injections. Results are expressed as mean f SE. b Differs from control at p < ,001. e Differs from control at p < .Ol.
time administration of norepinephrine. This indicates that depression of enzyme activity by norepinephrine is independent of prior induction. Previous work has demonstrated that norepinephrine inhibits tyrosine transaminase activity in vitro by competing with apoenzyme for the pyridoxald’-phosphate cofactor (4). If norepinephrine suppresses enzyme activity in vivo through combination with cofactor, the neuromediator should block the previously described induction (17) of enzyme by pyridoxine. This was found to be the case (Table V). Complex formation between norepii,ephrine and pyridoxal-5’-phosphate increases in a hyperbolic fashion with time, reaching a pleateau in 1 hr (4), the time after which tyrosine transaminase activity begins declining after injection of norepinephrine. To establish the period of time, if any, during
BIPHASIC TABLE SUPPRESSION
EFFECTS
TYROSINE
TRANSAMINASE RHYTHM NOREPINEPHRINE~ Tyrosine
which tyrosine transaminase is refractory to the stimulatory effects of norepinephrine, the amine was administered at hourly (Fig. 3) or two-hourly (Fig. 4) intervals.
IV
OF THE DAILY
BY
transaminase activity product/g/hr
qmles/
TABLE
DWi-eastafter
Day
2 P.M.
“i%
.
. l1
product/ dhr
Control
15.9 f
10.4
Norepinephrine
j!j.4 f
4.7
30.5
norepinephrine rmoles/ product/ dhr
184.1 f17.4b 81.6 f11.3”
V
NOREPINEPHRINE BLOCKADE OF TYROSINE TRANSAMINASE INDUCTION BY PYRIDOXINE DURING THE DAY”
De,cfteea;se norepinephrine /.mmles/
617
OF NOREPINEPHRINE
Tyrosine transminase activity (fimoles product/g/hr)
Group
Control Pyridoxine Norepinephrine Pyridoxine + Norepinephrine
102.5
(1Groups of six rats were treated with either saline or norepinephrine, 1.0 mg/kg subcutaneously 4 hr before death. Rats were killed .- at 2 P.M. and 11 P.M. Results are expressed as mean f SE. b Differs from daytime control and from those treated with norepinephrine at night at p < 901. c Does not significantly differ from those treated with norepinephrine during the day.
68.1 154.2 54.2 70.3
f f f f
8.0 10.gb 5.5 6.7
n Groups of eight rats were injected with pyridoxine hydrochloride 109 mg/lOO g in a volume of 1 ml, intraperitoneally at 8 A.M. and 10 A.M. Norepinephrine, 1.0 mg/kg, was injected subcutaneously at 8 A.M. Controls were treated with saline at appropriate times. Rats were killed at noon. b Differs from all other groups at p < .OOl. MULTIPLE
120 r
NE INJECTIONS T
160
I
I
I
I
2
3
4
5
TIME
I
6
(hours)
FIG. 2. Effect of norepinephrine at night. Groups of six to eight adrenalectomized rats were injected with norepinephrine, 1.0 mg/kg subcutaneously at varying times before the normal enzyme peak (11 P.M.), e.g., the 4-hr group was treated at 7 P.M. All rats were killed at 11 P.M. Results are expressed as mean f SE.
‘1
2
0
TIME
3
(hours)
FIG. 3. Effect of norepinephrine injections every hour. Adrenalectomized rats were treated with norepinephrine, 1 mg/kg subcutaneously at 0, 1 and 2 hr (arrows). Groups of six to eight were sacrificed as indicated, e.g., those killed at 3 hr received three injections. Results are expressed as mean f SE.
618
BLACK
I
1
I
I
0
I
2
3
TIME
AND
(hours)
4. Effect of norepinephrine injections 2 hr apart. Adrenalectomized rats were treated with norepinephrine, 1 mg/kg subcutaneously at zero time. The dotted line represents those which received a second injection at 2 hr (see arrows). Results are expressed as mean f SE. FIG.
One hour after an initial injection of norepinephrine, enzyme is unresponsive to an additional exposure to the amine: not only is there no further increase, but enzyme activity actually falls from the induced level. However, 2 hr after injection, another injection of norepinephrine results in enzyme elevation, identifying a refractory period as restricted to the second hour after injection. DISCUSSION
Norepinephrine alters hepatic tyrosine transaminase activity in a biphasic fashion in the adrenalectomized rat. After an initial induction, enzyme activity is depressed below control values. The elevation of enzyme activity requires on-going protein synthesis but appears to be independent of RNA synthesis. 3’) 5’Cyclic AMP has been reported to induce tyrosine transaminase in fetal and neonatal rats. However, in the present experiments performed on adult, adrenalectomized rats neither (Y- nor /3blockade abolished induction by norepinephrine. Tyrosine transaminase activity displays a
AXELROD
circadian rhythm with peak levels at night and lowest values during the day. The magnitude of the depressive effect of norepinephrine varies with level of basal (control) enzyme activity. I\linimal depression occurred during the day when enzyme activity was lowest, while the greatest decline of enzyme activity resulted at night when this enzyme normally rises to peak levels. Pyridoxine induces tyrosine transaminase by increasing the rate of enzyme synthesis (5, IS), and norepinephrine blocks this induction (Table V). These data suggest that suppression of enzyme activity by norepinephrine occurs as a result of formation of a complex with the pyridoxald’-phosphate cofactor with consequent inhibition of tyrosine transaminase synthesis. The marked decrease in enzyme activity after norepinephrine at night, in the absence of an initial induction, demonstrates that these two phases of norepinephrine action are separate and independent. Variation of the effects of the neuromediator with time of administration suggests temporal alterations in the state of the tyrosine transaminase system. These observations suggest that in the interaction of a regulator with an enzyme system one must consider time of administration in addition to dose. During the biphasic time course of norepinephrine action there is a refractory period when a repeated administration fails to induce the enzyme. During the second hour after administration of the catecholamine enzyme activity falls and cannot be elevated by a second injection of norepinephrine. By 2 hr, however, norepinephrine again causes an inductive response. The magnitude of the inductive and suppressive effects of norepinephrine on this enzyme system may vary with experimental model dependent, for example, on local concentrations of pyridoxaW-phosphate. In addition, results may vary as a function of time after norepinephrine exposure. The data presented indicate that a regulator molecule may have both inhibitory and stimulatory effects on an enzyme system in vivo, either directly or through a number of mediators.
BIPHASIClEFFECTS ACKNOWLEDGMENTS The authors thank Mrs. Dorothy Rutherford for their assistance.
Helen Hunt and excellent technical
REFERENCES 1. BLSCK, I:. B., AND AXELROD, J., Proc. Nat. Acad. Sci. U.S. 69, 1231 (1968). 2. AXELROD, J., AND BLACK, I. B., Nature, London 220, 161 (1968). 3. BLOCK, I. B., AND AXELROD, J., Fed. Proc. Amer. Sot. Exp. Biol. 28, 2632 (1969). 4. BLACK, I. B.. AND AXELROD, J., J. Biol. Chem. 244, 6124 (1969). 5. BLXK, I. B., J. Pharmacol. Exp.JTherap., in press. 6. LIN, E. C. C., AND KNOX, W. E., Biochim. Biophys. Acta 26, 85 (1957). 7. KNOX, W. E., Brit. J. Exp. Pathol. 32, 462 (1951).
OF NOREPINEPHRINE
619
8. DIAMONDSTONE, T. I., Anal. Biochem. 16, 395 (1966). 9. KENNEY, F. T., J. Biol. Chem. 343, 4367 (1967). 10. OTTOLENGHI, C., AND CAVAGNA, R., Endocrinology 83, 924 (1968). 11. TRAKATELLIS, A. C., MONTJAR, M., AND AXELROD, A. E., Biochemistry 4,2065 (1965). 12. REICH, E., FRANKLIN, R. M., SHATKIN, A. J., AND TATUM, E. L., Proc. Nat. Acad. Sci. U.S. 48. 1238 (1962). 13. WICKS, W.., S&&e 160, 997 (1968). 14. GREENGARD, O., Science 163, 891 (1969). 15. ROBISON, G. A., BUTCHER, R. W., AND SUTHERLAND, E. W., Ann. N.Y. Acad. Sci. 139, 703 (1967). 16. ELLIS, S., ANDERSON, H. L., JR., AND COLLINS, M., Proc. Sot. Exp. Biol. Med. 84, 383 (1953). 17. GREENGARD, O., AND GORDON, M., J. Biol. Chem. 238, 3708 (1963). 18. H OLTEN, D., WICKS, W., AND KENNEY, F. T., J. Biol. Chem. 242, 1053 (1967).
OF
Biphasic
BIOCHEMISTRY
Effect
AND
BIOPHYSICS
of Norepinephrine Tyrosine
Laboratory
614-619 (1970)
138,
in the Regulation
Transaminase
IRA B. BLACK
AND
Science, National
Institute
of Clinical
Received
January
JULIUS of Mental
26, 1970; accepted
of Hepatic
Activity AXELROD Health, April
Bethesda, Maryland
20014
7, 1970
Norepinephrine injection is associated with a biphasic response of heptatic tyroL-tyrosine: 2-oxoglutarate aminosine transaminase (tyrosine aminotransferase, transferase EC 2.6.1.5) activity in the adrenalectomized and hypophysectomized rat. After an initial increase, enzyme activity falls below control levels. The increase in tyrosine transaminase activity is blocked by cycloheximide but not actinomycin D, ergotamine tartrate, or propranalol. Depression of tyrosine transaminase activity by norepinephrine is maximal at night when enzyme activity normally rises to peak levels, resulting in a suppression of the circadian enzyme rhythm. The decreased activity at night occurs without prior induction, indicating the independence of these two effects of norepinephrine. Evidence suggesting that norepinephrine depresses tyrosine transaminase activity by competing with apoenzyme for the pyridoxal-5’phosphate cofactor is presented. These observations indicate that a regulator molecule may have multiple effects on an enzyme system varying not only with dosage but with time of administration.
Norepinephrine suppresses the activity of rat liver tyrosine transaminase in vitro and in vivo (l-5). This neuromediator, by forming a complex with the pyridoxal-5’phosphate cofactor, appears to decrease the rate of apoenzyme synthesis (4, 5). In addition, treatment with norepinephrine suppressed the daily enzyme rhythm by abolishing the evening peak in tyrosine transaminase activity while daytime, basal activity was not altered (5). These observations suggested that the effects of norepinephrine on the hepatic tyrosine transaminase system vary with time of day. This communication reports that norepinephrine has a biphasic, time-related effect on hepatic tyrosine transaminase activity. MATERIALS
AND
used and were fasted for the duration of each experiment. The efficacy of adrenalectomy was established by the inability of randomly chosen rats to survive for 1 week on tap water, and by inspection of the retroperitoneal space at the time of death. Purina Chow and 1% NaCl solution were provided ad Zibitum except during experimentation. Hypophysectomized rats were maintained on 1% saline, oranges, and Purina Chow and used 1 week after surgery. At the termination of each experiment rats were killed by a blow to the head, and livers were immediately removed and frozen. After homogenization in isotonic KC1 solution, livers were assayed for tyrosine transaminase activity by a modification of the method of Diamondstone (8). (-) Arterenol bitartrate (norepinephrine) was obtained from Calbiochem. Cycloheximide and actinomycin D were generous gifts of Harry B. Wood, Chief, Drug Development Branch, NIH. Pyridoxine hydrochloride (Nutritional Biochemicals Corp.) was dissolved in water and brought to neutral pH before administration. Propranalol was purchased as Inderal from Ayerst Lab. Phenoxybenzamine was generously donated by Harry Green, Head, Neurobiochemistry Section at Smith, Kline and French Labs.
METHODS
Female, adrenalectomized, Sprague-Dawley, 160-180-g rats were housed in clear plastic cages and exposed to a lighting schedule, with lights on from 5 A.M. to 7 P.M. Since glucocorticoids (6) and various amino acids (7) induce tyrosine transaminase, only adrenalectomized rats were 614
BIPHASIC
EFFECTS
TABLE
RESULTS
Treatment with norepinephrine resulted in a biphasic response of tyrosine transaminase activity (Fig. 1). Enzyme activity increased for the first hour after injection but subsequently fell below control levels at 3 hr. By 4 hr, activity was returning to baseline levels. To preclude the possibility that one, or both, of the effects of norepinephrine was mediated by growth hormone secretion (9, lo), the experiment was also performed in hypophysectomized rats. The results were similar (Fig. 1). To elucidate the nature of this unusual time course, the elevation and depression of enzyme activity were characterized separately. Norepinephrine over the range of 1.0 to 4.0 mg/kg caused a 50-70 % increase in tyrosine transaminase activity in 1 hr (Table I). Treatment with higher doses resulted in a significant mortality rate. This
FIG. 1. Tyrosine transaminase activity after norepinephrine. Rats were treated with norepinephrine, 1.0 mg/kg subcutaneously at time zero (10 A.M.), and groups of six to eight were sacrificed at the indicated times. Results are expressed as mean f SE (vertical bars). Solid line represents adrenalectomized animals, and dashed line represents hypophysectomized rats. Control groups differ from l- and 3-hr groups at p < .Ol.
615
OF NOREPINEPHRINE I
ELEVATION OF TYHOSINE TRANS~MIN~SE ACTIVITY 1 HR AFTER NOREPINEPHRINE ADMINISTRATION” Dose (w/kg) 0 0.5 1.0 2.0 4.0
Tyrosine transaminase activity (pm&s product/g/hr)
67.6 84.5 88.3 89.7 102.6
f dz zt -L &
2.5 7.7 2.6b 3.5b 2.9*
a Groups of eight rats were treated with the indicated doses of norepinephrine subcutaneously in a volume of 1 ml and were killed 1 hr later. Results are expressed as mean f SE. b Differs from control at p < ,001.
increase in enzyme activity could be due to the activation of preexistent tyrosine transaminase molecules or to the accumulation of increased enzyme protein. To distinguish between these alternatives, rats were treated with norepinephrine and/or the protein synthesis inhibitor cycloheximide (11). Abolition of the increase in tyrosine transaminase activity by inhibition of protein synthesis suggests that norepinephrine induces enzyme (Table II). To determine whether on-going RNA synthesis is necessary for tyrosine transaminase induction by norepinephrine, rats were treated with actinomycin D (12). Inhibition of RNA synthesis did not prevent the rise in tyrosine transaminase activity after norepinephrine (Table II), suggesting that the neuromediator increases tyrosine transaminase activity through a process independent of RNA synthesis. Tyrosine transaminase has been reported to respond to cyclic AMP in fetal liver explants (13) and the rat fetus in utero (14). Since cyclic AMP is associated with the b-receptor (15), the possible role of /3receptor mediation was examined. Treatment with the P-receptor blocking agent, propranalol, had no effect on norepinephrine induction (Table III). Ergotamine tartrate, which specifically blocks hepatic P-receptor activity (16), was also without effect on induction. In addition, a-receptor blockade with phenoxybenzamine failed to alter the enzyme rise after norepinephrine. The decline of enzyme activity at 3 hr was related to the initial level of enzyme
616
BLACK
AND
activity: the higher the control level, the greater the decrease. It was of interest, therefore, to compare the effect of norepinephrine during the day when enzyme activity is low, with its action at night when enzyme activity is high. Maximal depression of enzyme activity occurred during the evening when tyrosine transaminase was at peak levels (Table IV) with resultant suppression of the enzyme rhythm. To further investigate this difference in norepinephrine effect with time of day, a time course was performed during the evening. Since enzyme activity rises to a sharp peak at night, norepinephrine was administered at various times before normal peak activity. All rats were sacrificed at 11 P.M. In the evening, the suppressive effects of the amine were apparent, with greatest depression of enzyme activity 4 hr after injection (Fig. 2). In contrast to daytime injection (Fig. l), no induction was observed 1 hr after the nightTABLE
II
EFFECT OF INHIBITION OF PROTEIN OR RNA SYNTHESIS ON ELEVATION OF TYROSINE TRANSAMINASE ACTIVITY BY NOREPINEPHRINE" Tyrosine transaminase Group
Experiment 1 Control Norepinephrine Cycloheximide Norepinephrine + Cycloheximide Experiment 2 Control Norepinephrine Actinomycin D Norepinephrine + Actinomycin D
activity &nmles product/g/hr)
77.8 137.9 69.8 74.1
f f f *
7.7 12.5* 6.6 7.9
85.4 130.6 92.9 116.4
f f f f
3.6 9.5 3.9 9.5c
a In Experiment 1 groups of eight rats were treated with norepinephrine, 1.0 mg/kg subcutaneously at 10 A.M. and/or cycloheximide, 50 mg/kg intraperitoneally. In Experiment 2 rats were treated in a similar fashion with norepinephrine and/or actinomycin D, 1.5 mg/kg intraperitoneally 15 min earlier. Controls were treated at appropriate times with saline. Results are expressed as mean f SE. * Differs from control and group treated with both drugs at p < .Ol. c Does not differ significantly from group treated with norepinephrine alone.
AXELROD TABLE
III
ROLES OF CC- AND &RECEPTORS IN NOREPINEPHRINE INDUCTION OF TYROSINE TRANSAMINaSE" Tyrosine transaminase Group
Experiment 1 Control Norepinephrine Phenoxybenzamine Propranalol Norepinephrine + Phenoxybenzamine Norepinephrine + Propranalol Experiment 2 Control Norepinephrine Ergotamine Norepinephrine + Ergotamine
activity (pm&s/ product/g/hr)
61.8 99.3 64.2 68.5 123.5
f It f zt f
3.9 4.23 8.3 6.3 ll.l*
105.6 f
8.7*
92.9 144.9 87.2 128.7
12.1 10.4c 12.3 9.e
i f f f
a Groups of eight rats were treated with norepinephrine as in Fig. 2 and/or in Experiment 1 with either phenoxybenzamine or propranalol, 10 mg/kg intraperitoneally 1 hr earlier. In Experiment 2 groups were treated with norepinephrine as above and/or with ergotamine tartrate, 1.0 mg/kg intraperitoneally 30 min earlier. Rats were killed 1 hr after norepinephrine injections. Results are expressed as mean f SE. b Differs from control at p < ,001. e Differs from control at p < .Ol.
time administration of norepinephrine. This indicates that depression of enzyme activity by norepinephrine is independent of prior induction. Previous work has demonstrated that norepinephrine inhibits tyrosine transaminase activity in vitro by competing with apoenzyme for the pyridoxald’-phosphate cofactor (4). If norepinephrine suppresses enzyme activity in vivo through combination with cofactor, the neuromediator should block the previously described induction (17) of enzyme by pyridoxine. This was found to be the case (Table V). Complex formation between norepii,ephrine and pyridoxal-5’-phosphate increases in a hyperbolic fashion with time, reaching a pleateau in 1 hr (4), the time after which tyrosine transaminase activity begins declining after injection of norepinephrine. To establish the period of time, if any, during
BIPHASIC TABLE SUPPRESSION
EFFECTS
TYROSINE
TRANSAMINASE RHYTHM NOREPINEPHRINE~ Tyrosine
which tyrosine transaminase is refractory to the stimulatory effects of norepinephrine, the amine was administered at hourly (Fig. 3) or two-hourly (Fig. 4) intervals.
IV
OF THE DAILY
BY
transaminase activity product/g/hr
qmles/
TABLE
DWi-eastafter
Day
2 P.M.
“i%
.
. l1
product/ dhr
Control
15.9 f
10.4
Norepinephrine
j!j.4 f
4.7
30.5
norepinephrine rmoles/ product/ dhr
184.1 f17.4b 81.6 f11.3”
V
NOREPINEPHRINE BLOCKADE OF TYROSINE TRANSAMINASE INDUCTION BY PYRIDOXINE DURING THE DAY”
De,cfteea;se norepinephrine /.mmles/
617
OF NOREPINEPHRINE
Tyrosine transminase activity (fimoles product/g/hr)
Group
Control Pyridoxine Norepinephrine Pyridoxine + Norepinephrine
102.5
(1Groups of six rats were treated with either saline or norepinephrine, 1.0 mg/kg subcutaneously 4 hr before death. Rats were killed .- at 2 P.M. and 11 P.M. Results are expressed as mean f SE. b Differs from daytime control and from those treated with norepinephrine at night at p < 901. c Does not significantly differ from those treated with norepinephrine during the day.
68.1 154.2 54.2 70.3
f f f f
8.0 10.gb 5.5 6.7
n Groups of eight rats were injected with pyridoxine hydrochloride 109 mg/lOO g in a volume of 1 ml, intraperitoneally at 8 A.M. and 10 A.M. Norepinephrine, 1.0 mg/kg, was injected subcutaneously at 8 A.M. Controls were treated with saline at appropriate times. Rats were killed at noon. b Differs from all other groups at p < .OOl. MULTIPLE
120 r
NE INJECTIONS T
160
I
I
I
I
2
3
4
5
TIME
I
6
(hours)
FIG. 2. Effect of norepinephrine at night. Groups of six to eight adrenalectomized rats were injected with norepinephrine, 1.0 mg/kg subcutaneously at varying times before the normal enzyme peak (11 P.M.), e.g., the 4-hr group was treated at 7 P.M. All rats were killed at 11 P.M. Results are expressed as mean f SE.
‘1
2
0
TIME
3
(hours)
FIG. 3. Effect of norepinephrine injections every hour. Adrenalectomized rats were treated with norepinephrine, 1 mg/kg subcutaneously at 0, 1 and 2 hr (arrows). Groups of six to eight were sacrificed as indicated, e.g., those killed at 3 hr received three injections. Results are expressed as mean f SE.
618
BLACK
I
1
I
I
0
I
2
3
TIME
AND
(hours)
4. Effect of norepinephrine injections 2 hr apart. Adrenalectomized rats were treated with norepinephrine, 1 mg/kg subcutaneously at zero time. The dotted line represents those which received a second injection at 2 hr (see arrows). Results are expressed as mean f SE. FIG.
One hour after an initial injection of norepinephrine, enzyme is unresponsive to an additional exposure to the amine: not only is there no further increase, but enzyme activity actually falls from the induced level. However, 2 hr after injection, another injection of norepinephrine results in enzyme elevation, identifying a refractory period as restricted to the second hour after injection. DISCUSSION
Norepinephrine alters hepatic tyrosine transaminase activity in a biphasic fashion in the adrenalectomized rat. After an initial induction, enzyme activity is depressed below control values. The elevation of enzyme activity requires on-going protein synthesis but appears to be independent of RNA synthesis. 3’) 5’Cyclic AMP has been reported to induce tyrosine transaminase in fetal and neonatal rats. However, in the present experiments performed on adult, adrenalectomized rats neither (Y- nor /3blockade abolished induction by norepinephrine. Tyrosine transaminase activity displays a
AXELROD
circadian rhythm with peak levels at night and lowest values during the day. The magnitude of the depressive effect of norepinephrine varies with level of basal (control) enzyme activity. I\linimal depression occurred during the day when enzyme activity was lowest, while the greatest decline of enzyme activity resulted at night when this enzyme normally rises to peak levels. Pyridoxine induces tyrosine transaminase by increasing the rate of enzyme synthesis (5, IS), and norepinephrine blocks this induction (Table V). These data suggest that suppression of enzyme activity by norepinephrine occurs as a result of formation of a complex with the pyridoxald’-phosphate cofactor with consequent inhibition of tyrosine transaminase synthesis. The marked decrease in enzyme activity after norepinephrine at night, in the absence of an initial induction, demonstrates that these two phases of norepinephrine action are separate and independent. Variation of the effects of the neuromediator with time of administration suggests temporal alterations in the state of the tyrosine transaminase system. These observations suggest that in the interaction of a regulator with an enzyme system one must consider time of administration in addition to dose. During the biphasic time course of norepinephrine action there is a refractory period when a repeated administration fails to induce the enzyme. During the second hour after administration of the catecholamine enzyme activity falls and cannot be elevated by a second injection of norepinephrine. By 2 hr, however, norepinephrine again causes an inductive response. The magnitude of the inductive and suppressive effects of norepinephrine on this enzyme system may vary with experimental model dependent, for example, on local concentrations of pyridoxaW-phosphate. In addition, results may vary as a function of time after norepinephrine exposure. The data presented indicate that a regulator molecule may have both inhibitory and stimulatory effects on an enzyme system in vivo, either directly or through a number of mediators.
BIPHASIClEFFECTS ACKNOWLEDGMENTS The authors thank Mrs. Dorothy Rutherford for their assistance.
Helen Hunt and excellent technical
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OF NOREPINEPHRINE
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