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Reduction in the level of immunotitratable dopamine β-hydroxylase after chronic administration of l -DOPA or α-methyldopa
Neuroscience Vol. 4, pp. 1381 to 1389 0 Pergamon Press Ltd. 1979. Printed in Great
0306-4522/79/0901-1381102.M)/O Britain
REDUCTION IN THE LEVEL OF IMMUNOTITRATABLE DOPAMINE /GHYDROXYLASE AFTER CHRONIC ADMINISTRATION OF L-DOPA OR ccMETHYLDOPA ANNE J. CULVENOR~and B. JARROTT University of Melbourne, Clinical Pharmacology and Therapeutics Unit, Austin Hospital, Heidelberg, Victoria 3084, Australia
Abstract-The effect of chronic administration for 7 days of L-DOPA (1.0 g/kg/day) or t_-a-methyldopa (0.4g/kg/day) on the activity of dopamine /I-hydroxylase in rat adrenal glands and heart was studied. Both drugs reduced adrenal dopamine /?-hydroxylase activity by 35x, whereas only a-methyldopa administration lowered enzyme activity in the heart. The reductions were not due to the presence of substances inhibiting enzyme activity. Immunotitration of rat adrenal supernatants with a specific antibody raised to dopamine #I-hydroxylase purified from bovine adrenal medulla showed that the reductions in adrenal enzyme activity following L-DOPA and a-methyldopa were due to decreases in the amount of dopamine /I-hydroxylase protein. It is unlikely that these reductions in enzyme protein represent a generalized reduction in protein turnover as chronic administration of L-DOPA
did not alter the turnover of trichloracetic acid-precipitable soluble protein in adrenals or the heart. Chronic administration of L-DOPA also reduced tyrosine hydroxylase activity in adrenals and the heart and aromatic L-amino acid decarboxylase activity in the heart only. In contrast, heart tyrosine hydroxylase activity was slightly elevated by chronic administration of a-methyldopa, but this dosage regimen considerably reduced both adrenal and heart aromatic L-amino acid decarboxylase activity and slightly decreased the activity of the cytoplasmic marker, lactate dehydrogenase, in adrenal glands. The results are consistent with previous proposals that catecholamine levels may contribute to the regulation of the total amount of dopamine B-hydroxylase in the peripheral nervous system and that the amounts of catecholamine synthesizing and degradative enzymes are regulated in response to transmitter demand.
DOPAMINE /I-hydroxylase (dopamine fi-mono-oxygenase, EC 1.14.17.1) which catalyses the final step in the biosynthesis of noradrenaline from tyrosine in the adrenal medulla and noradrenergic neurons (LEON, LEVENBERG& KAUFMAN, 1960) is subject to long-term regulation of its levels by neuronal, hormonal and pharmacological influences. Although increased impulse traffic in the nerves innervating the adrenal glands and peripheral noradrenergic neurons increases the rate of synthesis of dopamine fl-hydroxylase and hence its steady-state level (MOLINOFF, BRIMIJOIN, WEINSHILBOUM& AXELROD, 1970; CIARANELLO,WWTEN & AXELROD, 1975; GAGNON, OITEN & THOENEN, 1976) complete abolition of nerve impulse activity by surgical decentralization of ganglia or section of the splanchnic nerve produces very little, if any, decline in the amount of adrenal dopamine fi-hydroxylase (CIARANELLO, WOOTEN & AXELROD, 1976). This suggests that basal nerve impulse activity to the adrenals does not greatly contribute to maintaining steady-state levels of the enzyme. In contrast, hypophysectomy markedly reduces the amount of dopamine /I-hydroxylase in adrenal glands (CIARANELLOet aZ., 1975). It has been proposed that
’ TO whom correspondence
should
be addressed.
ACTH may regulate steady-state levels of the enzyme by controlling the output of adrenal cortical glucocorticoids which inhibit dopamine /I-hydroxylase degradation (CIARANELU) ef al., 1976). The level of catecholamines in adrenal medullary cells and noradrenergic neurons may also contribute to regulation of the amount of dopamine p-hydroxylase. Administration of drugs which interfere with the reserpine-induced depletion of catecholamines from ganglia also blocks the reserpine mediated induction of dopamine p-hydroxylase in stellate ganglia (MOLINOFF,BRIMIJOIN& AXELROD,1972). Chronic administration of L-DOPA or dopamine lowers the activities of both dopamine B-hydroxylase (MOLINOFF et al., 1972) and tyrosine hydroxylase (DAIRMAN & UDENFRIEND, 1972) in rat heart, an effect which, it has been suggested, reflects reduced amounts of the enzymes. The present study has examined the effects of chronic administration of L-DOPA and a-methyldopa, a structural analogue of L-DOPA, on dopamine P-hydroxylase activity in rat adrenals and heart. The effect of these drugs on the amount of dopamine /I-hydroxylase protein in adrenal glands has been determined by immunotitration with a specific antibody raised to the enzyme. In addition, the activities of the other two enzymes of noradrenaline synthesis
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ANNE
J. CULVENORand B.
and of a cellular cytoplasmic enzyme marker, lactate dehydrogenase (EC 1.1.1.27), in adrenals and heart have been measured. EXPERIMENTAL Atlimals
PROCEDURES
and tissue preparation
Groups of male Sprague-Dawley rats (1.50-200 g) were given subcutaneous injections either of 1000 mg/kg L-DOPA or 4~mg/kg L-~-methyldopa or saline, once daily for 7 consecutive days. Drugs were suspended in saline by homogenization before injection. Animals were killed by stunning and decapitation 5 h after the final injection and adrenal glands and hearts were removed rapidly, Pairs of adrenal glands were homogenized in a KontesDual1 all-glass homogenizer in 1 ml of ice-cold 0.005 M Tris HCI. pH 7.2. 0.15 M sodium chloride, 0.2% (v/v) Triton X-100, 0.20:: bovine serum albumin. Preliminary experiments showed that the use of this medium for homogenization of tissues from both control and drug-treated animals completely solubilized dopamine /?-hydroxylase activity. Hearts were perfused with 5 ml of ice cold saline to remove blood and homogenized in a Polytron PT-IO (Kinematica) in 5 voi of the same medium used for adrenal glands. Homogenates were centrifuged at 27,OOOg for 30min at 3 ‘C and each supernatant divided into two aliquots. One aliquot was dialysed for 16 h at 4°C against two changes of 100 volumes of 0.005 M potassium phosphate, IO pM copper sulphate, pH 7.2 and used for the assay of dopamine [&hydroxylase activity. The second aliquot was dialysed against 0.1 M imidazole acetate, 10% (v/v) glycerol, pH 7.2 and used for the assay of tyrosine hydroxylase, aromatic L-amino acid decarboxylase and lactate dehydrogenase activities.
Dopamine @-hydroxyiase activity in 50-100~1 of dialysed adrenal supernatants was measured by the method of NAGATSU & UDENFRIEND(1972), using a saturating concentration of tyramine (20 mM) and N-ethylmaleimide (30 mht) to neutralize endogenous inhibitors of the enzyme. Dopamine ~-hydroxyia~ activity in 20-40 141of dialysed heart supernatants was measured by the method of MOLI~VQFF,WEINSHILBOUM& AXELROD (1971) mine (1 mM) as substrate
and copper
using tyra-
sulphate
(LOOP(M), a concentration which was determined by preliminary titration experiments to neutralize endogenous enzyme inhibitors in the heart. Tyrosine hydroxylase activity was assayed by a slight modification of the method of WAYMIRE, BJUR & WEINER(1971) using 18 PM L-[i-*4C]tyrosine and 1rnM 6,7-d~methyi-5,6,7,8_tetrahydropterin hydrochloride in a final volume of 200 ~1. Aromatic L-amino acid decarboxylase activity was assayed by the method of LAMPRECHT & COYLE(1972), using a substrate concentration of 300 /IM L-[*4f]-DOPA in a final volume of 500$ Lactate dehydrogenase activity was assayed by the method of DL~YSENS & AMESZ(1Y57). All enzyme assays were linear with respect to protein concentration and incubation time. Protein was assayed in tissue extracts during purification by the method of LOWRY,ROSEBROUGH, FARR & RANDALL (1951).using bovine serum albumin as a standard. Pur$cation
of dopamine
P-hydroxylase
Dopamine B-hydroxylase was purified from bovine adrenal medulla by the method of RUSK,THOMAS, KINDLER & U~ENFRIEND (1974) and subjected to a final gel filtration
JARROTT
step as follows in order to remove contaminating proteins. The eiuate from the concanavalin A-Sepharose column was then concentrated by ultrafiltration and applied to a Sepharose 4B column (2.5 x 65 cm) equilibrated with 0.05 M potassium phosphate, pli 7.4. Dopamine r?-hydroxylase was eluted from the column with equihbration buffer at a flow rate of 9.6ml per h and fractions containing maximal enzyme activity were pooled, concentrated by uhrafihration and stored at -20°C. Polyacrylamide gel electrophoresis of 200 pg of the purified fraction was performed by standard methods (RAYMOND. 1964) and revealed a single protein band. Preparation
ofspecijic
u&serum
to dapamine
p-hydroxylase
The purified dopamine ~-hydroxylase (700 pg in 0.5 ml) was emulsified with an equal volume of complete Freund’s adjuvant and injected intramuscularly into each limb of two rabbits. Booster injections were given every 3 weeks. using decreasing amounts of antigen (700, 54 and 4Opg protein, respectively). Twelve weeks after the initial immunization, the rabbits were exsanguinated by cardiac puncture and serum was collected and stored at -20°C in 1ml aliquots. Ouchterlony double immun~iffusion of the antiserum was performed using standard methods (KABAT& MEYER, 1961). lt~ma~otiira~ion
of adrenal
dopamine
~-hydro.~y~s~
The experimental design of the immunotitration was based on the method of CHRISTENSON,DAIRMAN & UDENFRIEND(1972) for titration of aromatic t-amino acid decarboxylase, as described previously (CIJLVENOR & JARROT~. 1979). Dialysed adrenal supernatants from six control or drugtreated animals were pooled and immunotitration of dopamine b-hydroxylase was carried out by the method of CULVENOR & JARROTT (1979). Ahquots of lOO$ of pooled supernatants were incubated in duplicate with O-25 pl antiserum in a final volume of 475 ~1. After centrifugation of the solutions at 27.OOOgfor 30min. enzyme activity was measured in 4OOrd of supernatant. Sfatisrics
Student’s r-test was used to determine the significance of the difference between means. The measure of variation used throughout this study is the standard error of the mean. The standard errors of the slopes and equivalence points of the immunotitration curves were estimated by standard methods (ARMITAGE, 1971). Maier~a~s
spec. act. 59 mCi/ S-(methyl- “‘C]Adenosylmethionine, mmol, was obtained from ICN Pharmaceuticals (U.S.A.) and L-[I-r4C]tyrosine, spec. act. 54.6 mCi/mmol, from New England Nuclear (Boston, U.S.A.). L-3,4,-dihydroxyphenyl[i-t4C]alanine, spec. act. 23.5 mCi/mmoL, L-f4,53H]leucine, spec. act. 55 Ci/mmol and L-[U-*4C]leucine. spec. act, 348 mCi/mmol were purchased from the Radiochemical Centre. Amersham (U.K.). 6,7-Dimethyl-5.6,7,8tetrahydropterin hydrochloride, B grade, was obtained from Calbiochem (U.S.A.) and concanavalin A-Sepharose and Sepharose 4B were from Pharmacia South Seas (North Ryde, Australia). L-cr-Methyldopa was obtained from Merck, Sharp and Dohme (Sydney, Australia). All other reagents were of analytical grade.
FIG. 1. Ouchterlony double immunodiffusion. Plates were developed for 48 h at 25°C. (A) The centre well contained 15 pg purified bovine adrenal medullary dopamine b-hydroxylase. The outer wells contained: 1 o’clock, 10 pi of rabbit I antiserum to dopamine B-hydroxylase; 3 o’clock, IO pl of non-immune rabbit serum; 5 o’clock, 10 ~1 of antiserum to purified hog kidney aromatic L-amino acid decarboxylase; 8 o’clock, no addition; 10 o’clock, 10~1 of rabbit 2 antiserum to dopamine /?-hydroxylase. (B) The centre well contained 10 fi of rabbit 2 antiserum to dopamine &hydroxylase. The outer wells contained: 2 o’clock and 10 o’clock, 10~1 of soluble lysate from chromaffin granules; 11 o’clock, 10 pl of concentrated eluate from the concanavalin A-Sepharose column; 5 o’clock and 7 o’clock, no additions.
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Effect of L-DOPA or a-methyldopa on dopamine /?-hydroxylase RESULTS
Specificity of the antiserum Double immunodiffusion was used to test the specificity of the serum obtained from rabbits immunized with purified bovine adrenal medullary dopamine /3-hydroxylase. Single precipitin lines were observed when the antisera obtained from both rabbits were diffused against purified dopamine /3-hydroxylase (Fig. 1A) or against a crude tissue extract containing the enzyme (Fig. 1B). No lines were observed after diffusion of the antigen against non-immune serum or antiserum prepared against another enzyme of catecholamine biosynthesis, aromatic L-amino acid decarboxylase (Fig. 1A). Dopamine /I-hydroxylase activity in adrenal supernatants could be completely removed from solution after immunotitration with a sufficient amount of the antiserum to dopamine fi-hydroxylase. Tyrosine hydroxylase and aromatic L-amino acid decarboxylase activities in rat adrenal and heart extracts were not inhibited by the antiserum. Effect of chronic administration of L-DOPA and a-methyldopa on dopamine b-hydroxylase activity in adrenal glands and heart Chronic administration of L-DOPA (1000 mg/kg per day) or a-methyldopa (400 mg/kg per day) for 7 consecutive days resulted in a significant reduction of approx 35% in dopamine /?-hydroxylase activity measured in dialysed adrenal supernatants (Table 1). Heart enzyme activity was also significantly lowered by about 25% after chronic administration of cc-methyldopa, but not after L-DOPA Fable 1). To establish that the drug-induced reductions in dopamine fi-hydroxylase activity were not due to inhibitory substances in the dialysed supernatants, equal volumes of supernatants from control and drugtreated animals were mixed and assayed. The enzyme TABLE
activities were additive. In other experiments, purified bovine adrenal medullary dopamine /?-hydroxylase was added to supernatants from control and drugtreated animals. The recovery of enzyme activity was 9CrlOO% in all cases. Immunotitration of adrenal dopamine /Lhydroxylase Following immunotitration of dialysed adrenal supernatants from both control and drug-treated rats with the specific rabbit antiserum to dopamine bhydroxylase, significant reductions were found in the equivalence point of adrenal dopamine fl-hydroxylase after chronic administration of L-DOPA (1000 mg/kg) or a-methyldopa (400 mg/kg) (Fig. 2). Thus, in a typical experiment, 51 pl of antiserum was required to remove enzyme activity in dialysed adrenal supernatants from control rats, whereas only 40~1 of the same antiserum completely precipitated enzyme activity in the supernatants of L-DOPA-treated animals, expressed per adrenal pair (Fig. 2A). In a separate series of experiments, typical volumes of antiserum which completely removed dopamine /I-hydroxylase activity from the supernatants of control and a-methyldopa-treated rats were 60 and 47 ~1, respectively (Fig. 2B). These reductions in equivalence point indicated that chronic administration of L-DOPA and a-methyldopa lowered the amount of dopamine /%hydroxylase protein in the dialysed adrenal supernatants. For both drugs, the ratio (drug:control) of equivalence points was very similar to the ratio (drug:control) of dopamine /Ghydroxylase activities in the initial supernatants (Table 2). EfSect of chronic administration of L-DOPA a-methyldopa on other enzymes
Chronic administration of L-DOPA (1000 mg/kg per day) for 7 days reduced both adrenal and cardiac tyrosine hydroxylase activity by 15-25x (Table 3). In contrast, cc-methyldopa administration (400 mg/kg per
1. EFFECT OF CHRONICADMINISTRATION OF L-DOPA DOPAMINE B-HYDR~~~LASE
Tissue Adrenals (pair)
Heart
Treatment
and
AND
CCMETHYLDOPA
ON
ACTIVITY
Dopamine /Lhydroxylase activity (nmol octopamine formed/h/organ)
Control L-DOPA Control a-Methyldopa
1120 5 740 + 1810 k 1200 f
Control L-DOPA Control a-Methyldopa
15.30 & 0.69 (5) 15.27 + 1.20(6) 16.10 & 1.30(6) 12.12 k 0.62; (6)
llO(7) 20t (7) 140(6) 60* (6)
Percentage of control
66.1 66.3
99.8 75.3
Rats were injected as described in the legend to Fig. 2 and killed 5 h after the final injection. Dopamine B-hydroxylase activity was measured in dialysed supernatants of tissues as described in Experimental Procedures. Each value shown is the mean f standard error and the number of animals used is shown in parentheses after each value. *P c 0.02, tP < 0.001.
ANNE
J. CULVENOR
(Al
400
0
Saline
l
L - Dopo
300
500 I
(B! 0
Saline
. o- Methyldopo
and B. JARROTT ered heart enzyme activity by approx 30%, with no significant effect on the adrenal enzyme (Table 3). Both adrenal and heart aromatic L-amino acid decarboxylase activity were markedly reduced to 20--30?,<, of control values by x-methyldopa administration (Table 3). Mixing experiments showed that the enzyme activities in supernatants from all groups of animals were additive. In order to assess whether the reductions in enzyme activity were due to non-specific effects on tissues, the activity of a cellular cytoplasmic marker, lactate dehydrogenase, was measured. L-DOPA administration did not significantly alter adrenal or heart lactate dehydrogenase activities (Table 3) and in addition. tissue mean wet weights of control and L-DOPA-treated animals were not significantly different. Heart lactate dehydrogenase activity was not changed after cc-methyldopa administration. but the chronic drug treatment did produce a drop of about 25% in the activity of the cellular cytoplasmic marker in adrenal glands (Table 3). However, the mean adrenal wet weight in animals treated chronically with 2-methyldopa was not significantly different from control values. Turnover qj’trichloracetic
acid-precipitahle
soluble pro-
tein
Vdume of antisewn Dopomhe
p-
to
hydroxylose, $
FIG. 2. Immunotitration of adrenal dopamine ,%hydroxylase. Rats were injected subcutaneously once daily for 7 consecutive days with (A)‘lOOOmg/kg L-DOPA or saline; (B) 400 mg/kg a-methyldopa or saline and killed 5 h after the final injection. Dialysed adrenal supernatants from six rats per treatment group were pooled for each group and the control supernatants were diluted with phosphatebuffered saline to contain the same enzyme activity per unit volume as the pooled supernatant from the drugtreated group. Immunotitration using 100 ~1 of these supernatants was performed as described in Experimental Procedures. Enzyme activity remaining in the supernatants after removal of immunoprecipitates is shown on the ordinate in nmol octopamine formed/h/adrenal pair and the volume of antiserum in ~1 is shown on the abscissa. Each point is the mean of duplicate incubations. Linear regression analysis was used to obtain lines of best fit and equivalence points are indicated by the intersection of the extrapolated lines with the abscissa. day) for the same length of time did not alter adrenal enzyme activity and resulted in an increase of 28% in heart tyrosine hydroxylase (Table 3). Mixing experiments showed that enzyme activities in dialysed supematants from different treatment groups were additive. The effects of drug administration on aromatic L-amino acid decarboxylase differed from those on tyrosine hydroxylase. L-DOPA administration low-
The relative rate of soluble protein turnover was measured by estimating the ratio of tritium to carbon-f4 incorporated into trichloracetic acid-precipitable soluble protein after short- and long-term exposure of animals to L-[3H]leucine and L-[*4C]leucine, respectively (CULVENOR& JARROTT. 1979). The shortterm incorporation of tritium gives an estimate of the rate of protein synthesis, whereas the long-term carbon-14 incorporated into protein is a measure of the rate of protein degradation. The ratio of tritium to carbon-14 incorporated is therefore an estimate of the relative protein turnover. Chronic administration of L-DOPA did not alter soluble protein turnover in either adrenals or heart. Although there was increased incorporation of both tritium and carbon-14 into the adrenals of rats pretreated chronically with L-DOPA, the isotopic ratio was not changed (Table 4).
DISCUSSION Previous studies have reported that simultaneous administration of L-DOPA or monoamine oxidase inhibitors with reserpine blocks both the catecholamine depletion and the induction of dopamine B-hydroxylase in peripheral noradrenergic neurons normally produced by reserpine (MOLINOFF et al., 1972). Moreover, chronic administration of L-DOPA or dopamine lowers dopamine b-hydroxylase activity in rat heart (MOLINOFF et al., 1972). Although the amount of dopamine /I-hydroxylase protein was not measured directly in the latter study, MOLINOFF et al. (1972) implied that the level of the enzyme was lowered after
1387
Effect of L-DOPA or a-methyldopa on dopamine /3-hydroxylase TABLE2. EFFECTOF CHRONICADMINISTRATION OF L-DOPA AND GI-METHYLDOPA ON ADRENALDOPAMINE b-HYDROXYLASE ACTWITY AND ENZYME PROTEIN
Drug
Enzyme activity ratio (Drug:Controi)
Equivalence point ratio (Drug:Control)
0.71 0.73
0.78 0.77
L-DOPA a-Methyldopa
Rats were injected as described in the legend to Fig. 2 and killed 5 h after the final injection. Adrenal dopamine /?-hydroxylase activity and equivalence point (a measure of enzyme protein) were determined on the same dialysed supematants as described in Experimental Procedures. Each equivalence point ratio shown is computed from individual values of the equivalence point (see the legend of Fig. 2 for further details) and each .enzyme activity ratio is estimated from the mean of six or seven animals.
giving L-DOPA or dopamine and suggested that catecholamine levels might be important in regulating the synthesis of neuronal dopamine /?-hydroxylase. The present study has shown that dopamine j-hydroxylase activity in adrenal glands is also reduced after chronic administration of L-DOPA or a-methyldopa. Furthermore, it has now been demonstrated directly that this reduction in enzyme activity appears to be due entirely to a decrease in the amount of immunotitratable enzyme protein. Chronic administration of L-DOPA did not alter dopamine b-hydroxylase activity in noradrenergic nerve terminals of rat heart in the present study, a result which differed from a previous report in which a small reduction in heart dopamine /I-hydroxylase activity was obtained (MOLINOFF et al., 1972). The reason for this discrepancy is not clear, but if intracellular levels of catecholamines are responsible for the effect, it is possible that the more frequent dosage schedule used in the study of MOLINOFF et al. (1972) (200 mg/kg L-DOPA intraperitoneally, every 8 h for 48 h) may have resulted in relatively higher intracellular catecholamine levels. It is interesting to note that chronic administration of a smaller dose of dopamine TABLE3. EFFECTOF CHRONIC
Tissue
Treatment
(100 mg/kg, also given intraperitoneally, every 8 h for 48 h) was even more effective than L-DOPA in reducing heart dopamine /I-hydroxylase activity (MOLINOFF et al., 1972). In contrast to the inability of even large chronic doses of L-DOPA to alter cardiac dopamine &hydroxylase activity in the present study, chronic administration of a-methyldopa at a 2.5-fold smaller dose than that of L-DOPA significantly reduced the activity of the heart enzyme. It is not clear whether this decrease reflects a reduction in enzyme protein, since even the assay used to measure cardiac dopamine jI-hydroxylase in the present study was not sufficiently sensitive to measure enzyme activity in the incubation mixture used for immunotitration. It is not possible to draw any conclusions about the relative effectiveness of L-DOPA and a-methyldopa in lowering cardiac and adrenal dopamine /I-hydroxylase activity, since only single doses of the chronically administered drugs were given and since the relative tissue levels and metabolism of the drugs were not examined in the present study. The effects of chronic administration of L-DOPA on the other enzymes of noradrenaline biosynthesis
ADMJNISTRATION OF L-DOPA AND a-METHyLDoPA IN ADRENAL GLANDS AND HEART
Tyrosine hydroxylase activity (nmol L-DOPA formed/h/organ)
Adrenals (pair)
Control L-DOPA Control a-Methyldopa
4.2 3.6 37.8 36.9
Heart
Control L-DOPA Control a-Methyldopa
0.59 + 0.45 + 1.23 f 1.56 k
f k f k
Aromatic L-amino acid decarboxylase activity (nmol CO2 formed/h/organ)
0.2 0.2; 2.2 1.5
279 241 321 61.8
0.04 o.osj 0.05 0.077
1141 k64 793 * 35% 711 * 12 194 k 8.413
k + k k
17 13 27 4.2$
ON ENZYME
ACTIVIN
Lactate dehydrogenase activity bmol NADH formed/h/organ) 146 f 164 f 15Ok 114 *
11 11 6 12$
7128 f 8064 f 5232 f 4728 k
480 528 264 312
Rats were injected as described in the legend to Fig. 2 and killed 5 h after the final injection. Enzyme activities were measured in dialysed supernatants of tissues as described in Experimental Procedures. Each value shown is the mean f standard error of six or seven animals. NADH, reduced nicotinamige adenine dinucleotide. ‘P < 0.05, tP < 0.005, JP < 0.002, @P < 0.001.
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ANNE 3. CULVENORand B. JARROTT TABLE 4. INCORPORATION OF RADIOACTIVITY INTO TRICHLORACET~C ACID-PRECIPITABLE SOLUBLE PROTEIN FROM CONTROL AND L-DOPA-TREATED RATS Radioactivity 3H Adrenals Control L-DOPA Heart Control L-DOPA
(dpm x IF/organ) 3H:‘4C
2.70 1_ 0.18 3.74 + 0.25.
18.43 & 0.68 19.44 + 0.87
0.26 + 0.02 0.40 + 0.03*
10.12 i 0.65 9.31 + 0.38
1.88 + 0.13 2.24 * 0.15
9.75 & 0.23 9.60 + 0.48
Rats were injected subcutaneously once daily for 7 consecutive days with saline or lOOOmg/kg L-DOPA. Intraperitoneal injections of 1OjKi L-[i4CJIeucine and 100 &i L-[3H]Ieucine were given 24 and 4 h, respectively, before death. Incorporation of radioactivity into trichloracetic acid-precipitable soluble protein was measured as described previously (CULVENOR& JARROTT,1979). Each value shown is the mean & standard error of the mean of three to five animals. *P < 0.05.
mostly confirmed previous reports (DAIRMAN, CHRIS-
in adrenals or heart suggested that a non-specific decrease in protein turnover was not responsible for the FRIEND,1971; 1972; TARVER, BERKOWITZ & SPECTOR, decreased level of adrenal and possibly also, cardiac 1971; CULVENOR& JARROTT, 1979). Thus tyrosine hydopamine jY-hydroxylase protein. Althou~ the reladroxylase activity in adrenal glands and heart was tive rates of soluble protein synthesis and degradation lowered and aromatic L-amino acid decarboxylase ac- were increased in adrenal glands following chronic administration of L-DOPA, the overall turnover was tivity in adrenals remained unchanged after giving not altered. Moreover, the effects on synthesis and L-DOPA. degradation were the reverse of those which wouid Possible rnech~~~~~ by whjch the drug treatments be predicted to lower the amount of dopamine /Ia&ect enzyme levels hydroxylase protein. It is also pertinent to note that It is unlikely that the reduction in dopamine P-hy monoamine oxidase activity in peripheral organs indroxylase protein occurs via a neuronal mechanism creases with chronic L-DOPA treatment (TARVER et as the time-scale of the reduction in adrenal dopaa/., 1971; LYLE& 1978). mine /.Lhydroxylase activity following section of the Although chronic administration of I-methyldopa splanchnic nerve (10% reduction at 10 days, 30% lowers the relative rates of soluble protein synthesis reduction at 19 days after the operation ; CIARANELLO and degradation in heart, overall soluble protein turnet al., 1976) is not compatible with, and cannot be over is not altered by this drug treatment (CULVENOR & JARROTT, 1979). However, adrenal soluble protein invoked as an adequate explanation for, the 35% drop after only 7 days of drug administration observed in turnover is slightly reduced by 160//,after chronic adthe present study. An increased loss of dopamine ministration of a-methyldopa (CULVENOR & JARROTT, 1979). This may explain the drop of approx 2SP: in ~-hy~oxyla~ by exocytotic release from adrenals the activity of the cellular cytoplasmic marker, lactate and heart also seems to be an unlikely explanation since administration of either L-DOPA (WATANABE, dehydrogenase, in this tissue, although surprisingly, JUDY & CARDON, 1974) or a-methyldopa (BAUM, it is not reflected in any alteration in adrenal wet weights. However, a nonspecific effect of r-methylSHROPSHIRE& VARNER, 1972) acutely reduces the cendopa administration on overall protein turnover is tral nervous drive to peripheral tissues and since innot a sufficient explanation for the reduced dopamine creased exocytosis is usually associated with enhanced /I-hydroxylase level, since the activity of adrenal tyroimpulse traffic in preganglionic neurons rather than sine hydroxylase was not changed by this drug treatthe reverse. ment. In addition, unless dopamine P-hydroxylase It was important to establish that the drug-induced levels are altered by different mechanisms after reduction in dopamine /I-hydroxylase protein did not chronic administration of L-DOPA and a-methylmerely reflect a non-specific reduction in protein turndopa, it should be stressed that both drugs reduced over, in view of the reports that acute administration of L-DOPA disaggregates brain polysomes (WEISS, the amount of enzyme protein, whereas only cr-methyldopa has a small effect on soluble protein turnover RoEL, MUNRO & WURTMAN, 1974) and that high con(CULVENOR & JARROTT, 1979). Cardiac tyrosine hycentrations of L-DOPA retard protein synthesis in a droxylase activity was increased after chronic adminmouse leukaemic cell line by inhibiting the charging of transfer RNA and leucine (ROSMANN,SHEA,CASE istration of a-methyldopa. If this represents an induction of enzyme protein, it seems surprising that the & PIKE, 1971). The lack of any effect of chronic activity in adrenal giands was not altered in parallel. L-DOPA ad~nistration on soluble protein turnover TENSON &
UDENFRIEND, 1971;
DAIRMAN &
UDEN-
Effect of L-DOPA or a-methyldopa on dopamine jI-hydroxylase Since the reduction in the amount of adrenal dopamine @hydroxylase protein is therefore unlikely to he due to nonspecific depression of total protein turnover or increased exocytotic release, it may reflect either a decrease in the rate of synthesis of the enzyme, or an enhanced rate of degradation.
The present study has demonstrated that the amount of dopamine /?-hydroxylase protein in adrenal glands is significantly decreased after chronic administration of catecholamine precursors. The significance of this finding in relation to patients undergoing chronic therapy with L-DOPA or cc-methyldopa
1389
should be considered. Although the doses used in the present study are much Iarger on a weight per body weight basis than those used clinically, caution should be exercised when extrapolating from one species to another, since the rate of amino acid metabolism in rats has been reported to be four to five times faster than that in humans {WEI.%et a!., 1974). The possible bi~herni~~ ministration
and clinical implications of chronic adof catecholamine precursors need to be
assessed. Acknowledgemenr-This work was supported by a grant from the National Health and Medical Research Council of Australia.
REFERENCES ARMITAGEP. (1971) Statistical Methods in Medical Research, p. 159. Blackwell Scientific Publications, Oxford. BAUMT., SHROP~H~RE A. T & VARNERL. V. (1972) Contribution of the central nervous system to the action of several antiby~rtensive agents (methyldopa, hydral~ine and guaneth~dine). J. Phurmac. exp. Ther. 182, 135-144. &X~MANNH. B., SHEA M. B., CASE K. R. & PIKE G. Z. (1971) L-DOPA inhibits macromolecular synthesis. Pbysiol. Chem. & Physics. 3, 226-234. CHRISTENSONJ. G., DAIRMAN W. D. & UDENFRIEND S. (1972) On the identity of DOPA decarboxylase and 5-hydroxytryptophan decarboxylase. Proc. natn. Acad. Sci. U.S.A. 69, 343-347. CIARANELLO R. D., WHITENG. F. & AXELROD J. (1975) Regulation of dopamine B-hydroxylase in rat adrenal glands. J. 6ioI. Chem. 250, 3204-3211. CIA~NEL~ R. D., WCJC~TEN G. F. & AXELROD J. (1976) Regulation of rat adrenal dopamine /?-hydroxylase-II. Receptor interaction in the regulation of enzyme synthesis and degradation. Brain Res. 113, 349-362. CIJLVENORA. J. $ JARROTT B. (1979) Reduction of aromatic L-amino acid decarboxylase protein in rats after chronic administration of alpha-methyldopa. Molec. Pharmac. 15, 8698. DAIRMANW., CHRISTENSON J. G. & UDENFRIEND S. (1971) Decrease in liver aromatic L-amino acid decarboxylase produced by chronic administration of L-DOPA. Proc. n&n. Acad. Sci. U.S.A. 68, 2117-2120. DAIR~PAN W. & UDENFRIEND S. (1971) Decrease in adrenal tyrosine hydroxylase and increase in norepinephrine synthesis in rats given L-dopa. Science, N.Y. 171, 1022-1024. DAIRMANW. & UDENFRIENDS. (1972) Studies on the mechanism of r_-3,4-dihydroxyphenylalanine-induced decrease in tyrosine hydroxylase activity. Molec. Pharmac. 8, 291-299. DUYSENSL. N. M. & AMESZJ. (1957) Fluorescence spectraphotometry of reduced phosphopyridine nucleotide in intact cells in the near ultraviolet and visible region. Eiochim. biophys. Acta 24, 19-26. GAGNONC., OTTENU. & THOENENH. (1976) Increased synthesis of dopamine &hydroxylase in cultured rat adrenal medullae after in I.&Oadminis~ation of reserpine. J. ~euruchem. 27, 259-265. KABATE. A. & MEYERM. M. (1961) ~xpffimental ~mmunochemjstry. Charles C. Thomas, Illinois. LAMPRECHT F. & COYLEJ. T. (1972) Dopa decarboxylase in the developing rat brain. Brain Res. 41, 503-506. LEV~NE. Y., LEVENBERG B. SC KAUFMANS. (1960) The enzymatic conversion of 3,4_dihydroxyphenylethylamine to norepinephrine. J. biol. Chem. 235, 2080-2086. LOWRY 0. H., RO~EBROUGH N. Y., FARR A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 26.5-275. LYLESG. A. (1978) Effects of L-DOPA administration upon monoamine oxidase activity in rat tissues. Life Sci. 22, 603610. MOLINOFFP. B., BRIMIJOINS. % AXELRODJ. (1972) Induction of dopamine &hydroxylase and tyrosine hydroxylase in rat hearts and sympathetic ganglia. J. Pharmac. exp. Ther. 182, 116-129. MOLINOFFP. B., BRIMIJOINS., WEINSHILBOUM R. M. & AXELRODJ. (1970) Neurally mediated increase in dopamine fl-hydroxylase activity. Proc. natn. Acad. Sci. U.S.A. 66, 453458. MOLINOFFP. B., WEINSHILBOUM R. & AXELRODJ. (1971) A sensitive enzymatic assay for dopamine-~-hydroxyiase. J. Pharmac. exp. Ther. 178, 425-431. NAGATSUT. & UDENFRIENDS. (1972) Photometric assay of dopamine /?-hydroxylase activity in human blood. Clin. Chem. 18, 980-983. RAYMOND S. (1964) Acrylamide gel electrophoresis. Ann. N.Y. Acad. Sci. 121, 350-365. RUSH R. A., THOMASP. E., KINDLERS. H. & UDENFXIENDS. (1974) The interaction of dopamine B-hydroxylase with concanavalin A and its use in enzyme purification. Biochem. biophys. Res. Commun. 57, 1301-1305. TARvEa J. J., BERKOWITZB. & SPECTORS. (1971) Alterations in tyrosine hydroxylase and monoamine oxidase activity in blood vessels. Nature, Load. 231, 252-253. WATANABE A. M., JWDY W. V. & CARDONP. V. (1974) Effect of L-dopa on blood pressure and sympathetic nerve activity after decarboxylase inhibition in cats. J. Pharmac. exp. Ther. 1% 107-l 13. (Accepted 5 April 1979)
0306-4522/79/0901-1381102.M)/O Britain
REDUCTION IN THE LEVEL OF IMMUNOTITRATABLE DOPAMINE /GHYDROXYLASE AFTER CHRONIC ADMINISTRATION OF L-DOPA OR ccMETHYLDOPA ANNE J. CULVENOR~and B. JARROTT University of Melbourne, Clinical Pharmacology and Therapeutics Unit, Austin Hospital, Heidelberg, Victoria 3084, Australia
Abstract-The effect of chronic administration for 7 days of L-DOPA (1.0 g/kg/day) or t_-a-methyldopa (0.4g/kg/day) on the activity of dopamine /I-hydroxylase in rat adrenal glands and heart was studied. Both drugs reduced adrenal dopamine /?-hydroxylase activity by 35x, whereas only a-methyldopa administration lowered enzyme activity in the heart. The reductions were not due to the presence of substances inhibiting enzyme activity. Immunotitration of rat adrenal supernatants with a specific antibody raised to dopamine #I-hydroxylase purified from bovine adrenal medulla showed that the reductions in adrenal enzyme activity following L-DOPA and a-methyldopa were due to decreases in the amount of dopamine /I-hydroxylase protein. It is unlikely that these reductions in enzyme protein represent a generalized reduction in protein turnover as chronic administration of L-DOPA
did not alter the turnover of trichloracetic acid-precipitable soluble protein in adrenals or the heart. Chronic administration of L-DOPA also reduced tyrosine hydroxylase activity in adrenals and the heart and aromatic L-amino acid decarboxylase activity in the heart only. In contrast, heart tyrosine hydroxylase activity was slightly elevated by chronic administration of a-methyldopa, but this dosage regimen considerably reduced both adrenal and heart aromatic L-amino acid decarboxylase activity and slightly decreased the activity of the cytoplasmic marker, lactate dehydrogenase, in adrenal glands. The results are consistent with previous proposals that catecholamine levels may contribute to the regulation of the total amount of dopamine B-hydroxylase in the peripheral nervous system and that the amounts of catecholamine synthesizing and degradative enzymes are regulated in response to transmitter demand.
DOPAMINE /I-hydroxylase (dopamine fi-mono-oxygenase, EC 1.14.17.1) which catalyses the final step in the biosynthesis of noradrenaline from tyrosine in the adrenal medulla and noradrenergic neurons (LEON, LEVENBERG& KAUFMAN, 1960) is subject to long-term regulation of its levels by neuronal, hormonal and pharmacological influences. Although increased impulse traffic in the nerves innervating the adrenal glands and peripheral noradrenergic neurons increases the rate of synthesis of dopamine fl-hydroxylase and hence its steady-state level (MOLINOFF, BRIMIJOIN, WEINSHILBOUM& AXELROD, 1970; CIARANELLO,WWTEN & AXELROD, 1975; GAGNON, OITEN & THOENEN, 1976) complete abolition of nerve impulse activity by surgical decentralization of ganglia or section of the splanchnic nerve produces very little, if any, decline in the amount of adrenal dopamine fi-hydroxylase (CIARANELLO, WOOTEN & AXELROD, 1976). This suggests that basal nerve impulse activity to the adrenals does not greatly contribute to maintaining steady-state levels of the enzyme. In contrast, hypophysectomy markedly reduces the amount of dopamine /I-hydroxylase in adrenal glands (CIARANELLOet aZ., 1975). It has been proposed that
’ TO whom correspondence
should
be addressed.
ACTH may regulate steady-state levels of the enzyme by controlling the output of adrenal cortical glucocorticoids which inhibit dopamine /I-hydroxylase degradation (CIARANELU) ef al., 1976). The level of catecholamines in adrenal medullary cells and noradrenergic neurons may also contribute to regulation of the amount of dopamine p-hydroxylase. Administration of drugs which interfere with the reserpine-induced depletion of catecholamines from ganglia also blocks the reserpine mediated induction of dopamine p-hydroxylase in stellate ganglia (MOLINOFF,BRIMIJOIN& AXELROD,1972). Chronic administration of L-DOPA or dopamine lowers the activities of both dopamine B-hydroxylase (MOLINOFF et al., 1972) and tyrosine hydroxylase (DAIRMAN & UDENFRIEND, 1972) in rat heart, an effect which, it has been suggested, reflects reduced amounts of the enzymes. The present study has examined the effects of chronic administration of L-DOPA and a-methyldopa, a structural analogue of L-DOPA, on dopamine P-hydroxylase activity in rat adrenals and heart. The effect of these drugs on the amount of dopamine /I-hydroxylase protein in adrenal glands has been determined by immunotitration with a specific antibody raised to the enzyme. In addition, the activities of the other two enzymes of noradrenaline synthesis
1381
1382
ANNE
J. CULVENORand B.
and of a cellular cytoplasmic enzyme marker, lactate dehydrogenase (EC 1.1.1.27), in adrenals and heart have been measured. EXPERIMENTAL Atlimals
PROCEDURES
and tissue preparation
Groups of male Sprague-Dawley rats (1.50-200 g) were given subcutaneous injections either of 1000 mg/kg L-DOPA or 4~mg/kg L-~-methyldopa or saline, once daily for 7 consecutive days. Drugs were suspended in saline by homogenization before injection. Animals were killed by stunning and decapitation 5 h after the final injection and adrenal glands and hearts were removed rapidly, Pairs of adrenal glands were homogenized in a KontesDual1 all-glass homogenizer in 1 ml of ice-cold 0.005 M Tris HCI. pH 7.2. 0.15 M sodium chloride, 0.2% (v/v) Triton X-100, 0.20:: bovine serum albumin. Preliminary experiments showed that the use of this medium for homogenization of tissues from both control and drug-treated animals completely solubilized dopamine /?-hydroxylase activity. Hearts were perfused with 5 ml of ice cold saline to remove blood and homogenized in a Polytron PT-IO (Kinematica) in 5 voi of the same medium used for adrenal glands. Homogenates were centrifuged at 27,OOOg for 30min at 3 ‘C and each supernatant divided into two aliquots. One aliquot was dialysed for 16 h at 4°C against two changes of 100 volumes of 0.005 M potassium phosphate, IO pM copper sulphate, pH 7.2 and used for the assay of dopamine [&hydroxylase activity. The second aliquot was dialysed against 0.1 M imidazole acetate, 10% (v/v) glycerol, pH 7.2 and used for the assay of tyrosine hydroxylase, aromatic L-amino acid decarboxylase and lactate dehydrogenase activities.
Dopamine @-hydroxyiase activity in 50-100~1 of dialysed adrenal supernatants was measured by the method of NAGATSU & UDENFRIEND(1972), using a saturating concentration of tyramine (20 mM) and N-ethylmaleimide (30 mht) to neutralize endogenous inhibitors of the enzyme. Dopamine ~-hydroxyia~ activity in 20-40 141of dialysed heart supernatants was measured by the method of MOLI~VQFF,WEINSHILBOUM& AXELROD (1971) mine (1 mM) as substrate
and copper
using tyra-
sulphate
(LOOP(M), a concentration which was determined by preliminary titration experiments to neutralize endogenous enzyme inhibitors in the heart. Tyrosine hydroxylase activity was assayed by a slight modification of the method of WAYMIRE, BJUR & WEINER(1971) using 18 PM L-[i-*4C]tyrosine and 1rnM 6,7-d~methyi-5,6,7,8_tetrahydropterin hydrochloride in a final volume of 200 ~1. Aromatic L-amino acid decarboxylase activity was assayed by the method of LAMPRECHT & COYLE(1972), using a substrate concentration of 300 /IM L-[*4f]-DOPA in a final volume of 500$ Lactate dehydrogenase activity was assayed by the method of DL~YSENS & AMESZ(1Y57). All enzyme assays were linear with respect to protein concentration and incubation time. Protein was assayed in tissue extracts during purification by the method of LOWRY,ROSEBROUGH, FARR & RANDALL (1951).using bovine serum albumin as a standard. Pur$cation
of dopamine
P-hydroxylase
Dopamine B-hydroxylase was purified from bovine adrenal medulla by the method of RUSK,THOMAS, KINDLER & U~ENFRIEND (1974) and subjected to a final gel filtration
JARROTT
step as follows in order to remove contaminating proteins. The eiuate from the concanavalin A-Sepharose column was then concentrated by ultrafiltration and applied to a Sepharose 4B column (2.5 x 65 cm) equilibrated with 0.05 M potassium phosphate, pli 7.4. Dopamine r?-hydroxylase was eluted from the column with equihbration buffer at a flow rate of 9.6ml per h and fractions containing maximal enzyme activity were pooled, concentrated by uhrafihration and stored at -20°C. Polyacrylamide gel electrophoresis of 200 pg of the purified fraction was performed by standard methods (RAYMOND. 1964) and revealed a single protein band. Preparation
ofspecijic
u&serum
to dapamine
p-hydroxylase
The purified dopamine ~-hydroxylase (700 pg in 0.5 ml) was emulsified with an equal volume of complete Freund’s adjuvant and injected intramuscularly into each limb of two rabbits. Booster injections were given every 3 weeks. using decreasing amounts of antigen (700, 54 and 4Opg protein, respectively). Twelve weeks after the initial immunization, the rabbits were exsanguinated by cardiac puncture and serum was collected and stored at -20°C in 1ml aliquots. Ouchterlony double immun~iffusion of the antiserum was performed using standard methods (KABAT& MEYER, 1961). lt~ma~otiira~ion
of adrenal
dopamine
~-hydro.~y~s~
The experimental design of the immunotitration was based on the method of CHRISTENSON,DAIRMAN & UDENFRIEND(1972) for titration of aromatic t-amino acid decarboxylase, as described previously (CIJLVENOR & JARROT~. 1979). Dialysed adrenal supernatants from six control or drugtreated animals were pooled and immunotitration of dopamine b-hydroxylase was carried out by the method of CULVENOR & JARROTT (1979). Ahquots of lOO$ of pooled supernatants were incubated in duplicate with O-25 pl antiserum in a final volume of 475 ~1. After centrifugation of the solutions at 27.OOOgfor 30min. enzyme activity was measured in 4OOrd of supernatant. Sfatisrics
Student’s r-test was used to determine the significance of the difference between means. The measure of variation used throughout this study is the standard error of the mean. The standard errors of the slopes and equivalence points of the immunotitration curves were estimated by standard methods (ARMITAGE, 1971). Maier~a~s
spec. act. 59 mCi/ S-(methyl- “‘C]Adenosylmethionine, mmol, was obtained from ICN Pharmaceuticals (U.S.A.) and L-[I-r4C]tyrosine, spec. act. 54.6 mCi/mmol, from New England Nuclear (Boston, U.S.A.). L-3,4,-dihydroxyphenyl[i-t4C]alanine, spec. act. 23.5 mCi/mmoL, L-f4,53H]leucine, spec. act. 55 Ci/mmol and L-[U-*4C]leucine. spec. act, 348 mCi/mmol were purchased from the Radiochemical Centre. Amersham (U.K.). 6,7-Dimethyl-5.6,7,8tetrahydropterin hydrochloride, B grade, was obtained from Calbiochem (U.S.A.) and concanavalin A-Sepharose and Sepharose 4B were from Pharmacia South Seas (North Ryde, Australia). L-cr-Methyldopa was obtained from Merck, Sharp and Dohme (Sydney, Australia). All other reagents were of analytical grade.
FIG. 1. Ouchterlony double immunodiffusion. Plates were developed for 48 h at 25°C. (A) The centre well contained 15 pg purified bovine adrenal medullary dopamine b-hydroxylase. The outer wells contained: 1 o’clock, 10 pi of rabbit I antiserum to dopamine B-hydroxylase; 3 o’clock, IO pl of non-immune rabbit serum; 5 o’clock, 10 ~1 of antiserum to purified hog kidney aromatic L-amino acid decarboxylase; 8 o’clock, no addition; 10 o’clock, 10~1 of rabbit 2 antiserum to dopamine /?-hydroxylase. (B) The centre well contained 10 fi of rabbit 2 antiserum to dopamine &hydroxylase. The outer wells contained: 2 o’clock and 10 o’clock, 10~1 of soluble lysate from chromaffin granules; 11 o’clock, 10 pl of concentrated eluate from the concanavalin A-Sepharose column; 5 o’clock and 7 o’clock, no additions.
1383
1385
Effect of L-DOPA or a-methyldopa on dopamine /?-hydroxylase RESULTS
Specificity of the antiserum Double immunodiffusion was used to test the specificity of the serum obtained from rabbits immunized with purified bovine adrenal medullary dopamine /3-hydroxylase. Single precipitin lines were observed when the antisera obtained from both rabbits were diffused against purified dopamine /3-hydroxylase (Fig. 1A) or against a crude tissue extract containing the enzyme (Fig. 1B). No lines were observed after diffusion of the antigen against non-immune serum or antiserum prepared against another enzyme of catecholamine biosynthesis, aromatic L-amino acid decarboxylase (Fig. 1A). Dopamine /I-hydroxylase activity in adrenal supernatants could be completely removed from solution after immunotitration with a sufficient amount of the antiserum to dopamine fi-hydroxylase. Tyrosine hydroxylase and aromatic L-amino acid decarboxylase activities in rat adrenal and heart extracts were not inhibited by the antiserum. Effect of chronic administration of L-DOPA and a-methyldopa on dopamine b-hydroxylase activity in adrenal glands and heart Chronic administration of L-DOPA (1000 mg/kg per day) or a-methyldopa (400 mg/kg per day) for 7 consecutive days resulted in a significant reduction of approx 35% in dopamine /?-hydroxylase activity measured in dialysed adrenal supernatants (Table 1). Heart enzyme activity was also significantly lowered by about 25% after chronic administration of cc-methyldopa, but not after L-DOPA Fable 1). To establish that the drug-induced reductions in dopamine fi-hydroxylase activity were not due to inhibitory substances in the dialysed supernatants, equal volumes of supernatants from control and drugtreated animals were mixed and assayed. The enzyme TABLE
activities were additive. In other experiments, purified bovine adrenal medullary dopamine /?-hydroxylase was added to supernatants from control and drugtreated animals. The recovery of enzyme activity was 9CrlOO% in all cases. Immunotitration of adrenal dopamine /Lhydroxylase Following immunotitration of dialysed adrenal supernatants from both control and drug-treated rats with the specific rabbit antiserum to dopamine bhydroxylase, significant reductions were found in the equivalence point of adrenal dopamine fl-hydroxylase after chronic administration of L-DOPA (1000 mg/kg) or a-methyldopa (400 mg/kg) (Fig. 2). Thus, in a typical experiment, 51 pl of antiserum was required to remove enzyme activity in dialysed adrenal supernatants from control rats, whereas only 40~1 of the same antiserum completely precipitated enzyme activity in the supernatants of L-DOPA-treated animals, expressed per adrenal pair (Fig. 2A). In a separate series of experiments, typical volumes of antiserum which completely removed dopamine /I-hydroxylase activity from the supernatants of control and a-methyldopa-treated rats were 60 and 47 ~1, respectively (Fig. 2B). These reductions in equivalence point indicated that chronic administration of L-DOPA and a-methyldopa lowered the amount of dopamine /%hydroxylase protein in the dialysed adrenal supernatants. For both drugs, the ratio (drug:control) of equivalence points was very similar to the ratio (drug:control) of dopamine /Ghydroxylase activities in the initial supernatants (Table 2). EfSect of chronic administration of L-DOPA a-methyldopa on other enzymes
Chronic administration of L-DOPA (1000 mg/kg per day) for 7 days reduced both adrenal and cardiac tyrosine hydroxylase activity by 15-25x (Table 3). In contrast, cc-methyldopa administration (400 mg/kg per
1. EFFECT OF CHRONICADMINISTRATION OF L-DOPA DOPAMINE B-HYDR~~~LASE
Tissue Adrenals (pair)
Heart
Treatment
and
AND
CCMETHYLDOPA
ON
ACTIVITY
Dopamine /Lhydroxylase activity (nmol octopamine formed/h/organ)
Control L-DOPA Control a-Methyldopa
1120 5 740 + 1810 k 1200 f
Control L-DOPA Control a-Methyldopa
15.30 & 0.69 (5) 15.27 + 1.20(6) 16.10 & 1.30(6) 12.12 k 0.62; (6)
llO(7) 20t (7) 140(6) 60* (6)
Percentage of control
66.1 66.3
99.8 75.3
Rats were injected as described in the legend to Fig. 2 and killed 5 h after the final injection. Dopamine B-hydroxylase activity was measured in dialysed supernatants of tissues as described in Experimental Procedures. Each value shown is the mean f standard error and the number of animals used is shown in parentheses after each value. *P c 0.02, tP < 0.001.
ANNE
J. CULVENOR
(Al
400
0
Saline
l
L - Dopo
300
500 I
(B! 0
Saline
. o- Methyldopo
and B. JARROTT ered heart enzyme activity by approx 30%, with no significant effect on the adrenal enzyme (Table 3). Both adrenal and heart aromatic L-amino acid decarboxylase activity were markedly reduced to 20--30?,<, of control values by x-methyldopa administration (Table 3). Mixing experiments showed that the enzyme activities in supernatants from all groups of animals were additive. In order to assess whether the reductions in enzyme activity were due to non-specific effects on tissues, the activity of a cellular cytoplasmic marker, lactate dehydrogenase, was measured. L-DOPA administration did not significantly alter adrenal or heart lactate dehydrogenase activities (Table 3) and in addition. tissue mean wet weights of control and L-DOPA-treated animals were not significantly different. Heart lactate dehydrogenase activity was not changed after cc-methyldopa administration. but the chronic drug treatment did produce a drop of about 25% in the activity of the cellular cytoplasmic marker in adrenal glands (Table 3). However, the mean adrenal wet weight in animals treated chronically with 2-methyldopa was not significantly different from control values. Turnover qj’trichloracetic
acid-precipitahle
soluble pro-
tein
Vdume of antisewn Dopomhe
p-
to
hydroxylose, $
FIG. 2. Immunotitration of adrenal dopamine ,%hydroxylase. Rats were injected subcutaneously once daily for 7 consecutive days with (A)‘lOOOmg/kg L-DOPA or saline; (B) 400 mg/kg a-methyldopa or saline and killed 5 h after the final injection. Dialysed adrenal supernatants from six rats per treatment group were pooled for each group and the control supernatants were diluted with phosphatebuffered saline to contain the same enzyme activity per unit volume as the pooled supernatant from the drugtreated group. Immunotitration using 100 ~1 of these supernatants was performed as described in Experimental Procedures. Enzyme activity remaining in the supernatants after removal of immunoprecipitates is shown on the ordinate in nmol octopamine formed/h/adrenal pair and the volume of antiserum in ~1 is shown on the abscissa. Each point is the mean of duplicate incubations. Linear regression analysis was used to obtain lines of best fit and equivalence points are indicated by the intersection of the extrapolated lines with the abscissa. day) for the same length of time did not alter adrenal enzyme activity and resulted in an increase of 28% in heart tyrosine hydroxylase (Table 3). Mixing experiments showed that enzyme activities in dialysed supematants from different treatment groups were additive. The effects of drug administration on aromatic L-amino acid decarboxylase differed from those on tyrosine hydroxylase. L-DOPA administration low-
The relative rate of soluble protein turnover was measured by estimating the ratio of tritium to carbon-f4 incorporated into trichloracetic acid-precipitable soluble protein after short- and long-term exposure of animals to L-[3H]leucine and L-[*4C]leucine, respectively (CULVENOR& JARROTT. 1979). The shortterm incorporation of tritium gives an estimate of the rate of protein synthesis, whereas the long-term carbon-14 incorporated into protein is a measure of the rate of protein degradation. The ratio of tritium to carbon-14 incorporated is therefore an estimate of the relative protein turnover. Chronic administration of L-DOPA did not alter soluble protein turnover in either adrenals or heart. Although there was increased incorporation of both tritium and carbon-14 into the adrenals of rats pretreated chronically with L-DOPA, the isotopic ratio was not changed (Table 4).
DISCUSSION Previous studies have reported that simultaneous administration of L-DOPA or monoamine oxidase inhibitors with reserpine blocks both the catecholamine depletion and the induction of dopamine B-hydroxylase in peripheral noradrenergic neurons normally produced by reserpine (MOLINOFF et al., 1972). Moreover, chronic administration of L-DOPA or dopamine lowers dopamine b-hydroxylase activity in rat heart (MOLINOFF et al., 1972). Although the amount of dopamine /I-hydroxylase protein was not measured directly in the latter study, MOLINOFF et al. (1972) implied that the level of the enzyme was lowered after
1387
Effect of L-DOPA or a-methyldopa on dopamine /3-hydroxylase TABLE2. EFFECTOF CHRONICADMINISTRATION OF L-DOPA AND GI-METHYLDOPA ON ADRENALDOPAMINE b-HYDROXYLASE ACTWITY AND ENZYME PROTEIN
Drug
Enzyme activity ratio (Drug:Controi)
Equivalence point ratio (Drug:Control)
0.71 0.73
0.78 0.77
L-DOPA a-Methyldopa
Rats were injected as described in the legend to Fig. 2 and killed 5 h after the final injection. Adrenal dopamine /?-hydroxylase activity and equivalence point (a measure of enzyme protein) were determined on the same dialysed supematants as described in Experimental Procedures. Each equivalence point ratio shown is computed from individual values of the equivalence point (see the legend of Fig. 2 for further details) and each .enzyme activity ratio is estimated from the mean of six or seven animals.
giving L-DOPA or dopamine and suggested that catecholamine levels might be important in regulating the synthesis of neuronal dopamine /?-hydroxylase. The present study has shown that dopamine j-hydroxylase activity in adrenal glands is also reduced after chronic administration of L-DOPA or a-methyldopa. Furthermore, it has now been demonstrated directly that this reduction in enzyme activity appears to be due entirely to a decrease in the amount of immunotitratable enzyme protein. Chronic administration of L-DOPA did not alter dopamine b-hydroxylase activity in noradrenergic nerve terminals of rat heart in the present study, a result which differed from a previous report in which a small reduction in heart dopamine /I-hydroxylase activity was obtained (MOLINOFF et al., 1972). The reason for this discrepancy is not clear, but if intracellular levels of catecholamines are responsible for the effect, it is possible that the more frequent dosage schedule used in the study of MOLINOFF et al. (1972) (200 mg/kg L-DOPA intraperitoneally, every 8 h for 48 h) may have resulted in relatively higher intracellular catecholamine levels. It is interesting to note that chronic administration of a smaller dose of dopamine TABLE3. EFFECTOF CHRONIC
Tissue
Treatment
(100 mg/kg, also given intraperitoneally, every 8 h for 48 h) was even more effective than L-DOPA in reducing heart dopamine /I-hydroxylase activity (MOLINOFF et al., 1972). In contrast to the inability of even large chronic doses of L-DOPA to alter cardiac dopamine &hydroxylase activity in the present study, chronic administration of a-methyldopa at a 2.5-fold smaller dose than that of L-DOPA significantly reduced the activity of the heart enzyme. It is not clear whether this decrease reflects a reduction in enzyme protein, since even the assay used to measure cardiac dopamine jI-hydroxylase in the present study was not sufficiently sensitive to measure enzyme activity in the incubation mixture used for immunotitration. It is not possible to draw any conclusions about the relative effectiveness of L-DOPA and a-methyldopa in lowering cardiac and adrenal dopamine /I-hydroxylase activity, since only single doses of the chronically administered drugs were given and since the relative tissue levels and metabolism of the drugs were not examined in the present study. The effects of chronic administration of L-DOPA on the other enzymes of noradrenaline biosynthesis
ADMJNISTRATION OF L-DOPA AND a-METHyLDoPA IN ADRENAL GLANDS AND HEART
Tyrosine hydroxylase activity (nmol L-DOPA formed/h/organ)
Adrenals (pair)
Control L-DOPA Control a-Methyldopa
4.2 3.6 37.8 36.9
Heart
Control L-DOPA Control a-Methyldopa
0.59 + 0.45 + 1.23 f 1.56 k
f k f k
Aromatic L-amino acid decarboxylase activity (nmol CO2 formed/h/organ)
0.2 0.2; 2.2 1.5
279 241 321 61.8
0.04 o.osj 0.05 0.077
1141 k64 793 * 35% 711 * 12 194 k 8.413
k + k k
17 13 27 4.2$
ON ENZYME
ACTIVIN
Lactate dehydrogenase activity bmol NADH formed/h/organ) 146 f 164 f 15Ok 114 *
11 11 6 12$
7128 f 8064 f 5232 f 4728 k
480 528 264 312
Rats were injected as described in the legend to Fig. 2 and killed 5 h after the final injection. Enzyme activities were measured in dialysed supernatants of tissues as described in Experimental Procedures. Each value shown is the mean f standard error of six or seven animals. NADH, reduced nicotinamige adenine dinucleotide. ‘P < 0.05, tP < 0.005, JP < 0.002, @P < 0.001.
1388
ANNE 3. CULVENORand B. JARROTT TABLE 4. INCORPORATION OF RADIOACTIVITY INTO TRICHLORACET~C ACID-PRECIPITABLE SOLUBLE PROTEIN FROM CONTROL AND L-DOPA-TREATED RATS Radioactivity 3H Adrenals Control L-DOPA Heart Control L-DOPA
(dpm x IF/organ) 3H:‘4C
2.70 1_ 0.18 3.74 + 0.25.
18.43 & 0.68 19.44 + 0.87
0.26 + 0.02 0.40 + 0.03*
10.12 i 0.65 9.31 + 0.38
1.88 + 0.13 2.24 * 0.15
9.75 & 0.23 9.60 + 0.48
Rats were injected subcutaneously once daily for 7 consecutive days with saline or lOOOmg/kg L-DOPA. Intraperitoneal injections of 1OjKi L-[i4CJIeucine and 100 &i L-[3H]Ieucine were given 24 and 4 h, respectively, before death. Incorporation of radioactivity into trichloracetic acid-precipitable soluble protein was measured as described previously (CULVENOR& JARROTT,1979). Each value shown is the mean & standard error of the mean of three to five animals. *P < 0.05.
mostly confirmed previous reports (DAIRMAN, CHRIS-
in adrenals or heart suggested that a non-specific decrease in protein turnover was not responsible for the FRIEND,1971; 1972; TARVER, BERKOWITZ & SPECTOR, decreased level of adrenal and possibly also, cardiac 1971; CULVENOR& JARROTT, 1979). Thus tyrosine hydopamine jY-hydroxylase protein. Althou~ the reladroxylase activity in adrenal glands and heart was tive rates of soluble protein synthesis and degradation lowered and aromatic L-amino acid decarboxylase ac- were increased in adrenal glands following chronic administration of L-DOPA, the overall turnover was tivity in adrenals remained unchanged after giving not altered. Moreover, the effects on synthesis and L-DOPA. degradation were the reverse of those which wouid Possible rnech~~~~~ by whjch the drug treatments be predicted to lower the amount of dopamine /Ia&ect enzyme levels hydroxylase protein. It is also pertinent to note that It is unlikely that the reduction in dopamine P-hy monoamine oxidase activity in peripheral organs indroxylase protein occurs via a neuronal mechanism creases with chronic L-DOPA treatment (TARVER et as the time-scale of the reduction in adrenal dopaa/., 1971; LYLE& 1978). mine /.Lhydroxylase activity following section of the Although chronic administration of I-methyldopa splanchnic nerve (10% reduction at 10 days, 30% lowers the relative rates of soluble protein synthesis reduction at 19 days after the operation ; CIARANELLO and degradation in heart, overall soluble protein turnet al., 1976) is not compatible with, and cannot be over is not altered by this drug treatment (CULVENOR & JARROTT, 1979). However, adrenal soluble protein invoked as an adequate explanation for, the 35% drop after only 7 days of drug administration observed in turnover is slightly reduced by 160//,after chronic adthe present study. An increased loss of dopamine ministration of a-methyldopa (CULVENOR & JARROTT, 1979). This may explain the drop of approx 2SP: in ~-hy~oxyla~ by exocytotic release from adrenals the activity of the cellular cytoplasmic marker, lactate and heart also seems to be an unlikely explanation since administration of either L-DOPA (WATANABE, dehydrogenase, in this tissue, although surprisingly, JUDY & CARDON, 1974) or a-methyldopa (BAUM, it is not reflected in any alteration in adrenal wet weights. However, a nonspecific effect of r-methylSHROPSHIRE& VARNER, 1972) acutely reduces the cendopa administration on overall protein turnover is tral nervous drive to peripheral tissues and since innot a sufficient explanation for the reduced dopamine creased exocytosis is usually associated with enhanced /I-hydroxylase level, since the activity of adrenal tyroimpulse traffic in preganglionic neurons rather than sine hydroxylase was not changed by this drug treatthe reverse. ment. In addition, unless dopamine P-hydroxylase It was important to establish that the drug-induced levels are altered by different mechanisms after reduction in dopamine /I-hydroxylase protein did not chronic administration of L-DOPA and a-methylmerely reflect a non-specific reduction in protein turndopa, it should be stressed that both drugs reduced over, in view of the reports that acute administration of L-DOPA disaggregates brain polysomes (WEISS, the amount of enzyme protein, whereas only cr-methyldopa has a small effect on soluble protein turnover RoEL, MUNRO & WURTMAN, 1974) and that high con(CULVENOR & JARROTT, 1979). Cardiac tyrosine hycentrations of L-DOPA retard protein synthesis in a droxylase activity was increased after chronic adminmouse leukaemic cell line by inhibiting the charging of transfer RNA and leucine (ROSMANN,SHEA,CASE istration of a-methyldopa. If this represents an induction of enzyme protein, it seems surprising that the & PIKE, 1971). The lack of any effect of chronic activity in adrenal giands was not altered in parallel. L-DOPA ad~nistration on soluble protein turnover TENSON &
UDENFRIEND, 1971;
DAIRMAN &
UDEN-
Effect of L-DOPA or a-methyldopa on dopamine jI-hydroxylase Since the reduction in the amount of adrenal dopamine @hydroxylase protein is therefore unlikely to he due to nonspecific depression of total protein turnover or increased exocytotic release, it may reflect either a decrease in the rate of synthesis of the enzyme, or an enhanced rate of degradation.
The present study has demonstrated that the amount of dopamine /?-hydroxylase protein in adrenal glands is significantly decreased after chronic administration of catecholamine precursors. The significance of this finding in relation to patients undergoing chronic therapy with L-DOPA or cc-methyldopa
1389
should be considered. Although the doses used in the present study are much Iarger on a weight per body weight basis than those used clinically, caution should be exercised when extrapolating from one species to another, since the rate of amino acid metabolism in rats has been reported to be four to five times faster than that in humans {WEI.%et a!., 1974). The possible bi~herni~~ ministration
and clinical implications of chronic adof catecholamine precursors need to be
assessed. Acknowledgemenr-This work was supported by a grant from the National Health and Medical Research Council of Australia.
REFERENCES ARMITAGEP. (1971) Statistical Methods in Medical Research, p. 159. Blackwell Scientific Publications, Oxford. BAUMT., SHROP~H~RE A. T & VARNERL. V. (1972) Contribution of the central nervous system to the action of several antiby~rtensive agents (methyldopa, hydral~ine and guaneth~dine). J. Phurmac. exp. Ther. 182, 135-144. &X~MANNH. B., SHEA M. B., CASE K. R. & PIKE G. Z. (1971) L-DOPA inhibits macromolecular synthesis. Pbysiol. Chem. & Physics. 3, 226-234. CHRISTENSONJ. G., DAIRMAN W. D. & UDENFRIEND S. (1972) On the identity of DOPA decarboxylase and 5-hydroxytryptophan decarboxylase. Proc. natn. Acad. Sci. U.S.A. 69, 343-347. CIARANELLO R. D., WHITENG. F. & AXELROD J. (1975) Regulation of dopamine B-hydroxylase in rat adrenal glands. J. 6ioI. Chem. 250, 3204-3211. CIA~NEL~ R. D., WCJC~TEN G. F. & AXELROD J. (1976) Regulation of rat adrenal dopamine /?-hydroxylase-II. Receptor interaction in the regulation of enzyme synthesis and degradation. Brain Res. 113, 349-362. CIJLVENORA. J. $ JARROTT B. (1979) Reduction of aromatic L-amino acid decarboxylase protein in rats after chronic administration of alpha-methyldopa. Molec. Pharmac. 15, 8698. DAIRMANW., CHRISTENSON J. G. & UDENFRIEND S. (1971) Decrease in liver aromatic L-amino acid decarboxylase produced by chronic administration of L-DOPA. Proc. n&n. Acad. Sci. U.S.A. 68, 2117-2120. DAIR~PAN W. & UDENFRIEND S. (1971) Decrease in adrenal tyrosine hydroxylase and increase in norepinephrine synthesis in rats given L-dopa. Science, N.Y. 171, 1022-1024. DAIRMANW. & UDENFRIENDS. (1972) Studies on the mechanism of r_-3,4-dihydroxyphenylalanine-induced decrease in tyrosine hydroxylase activity. Molec. Pharmac. 8, 291-299. DUYSENSL. N. M. & AMESZJ. (1957) Fluorescence spectraphotometry of reduced phosphopyridine nucleotide in intact cells in the near ultraviolet and visible region. Eiochim. biophys. Acta 24, 19-26. GAGNONC., OTTENU. & THOENENH. (1976) Increased synthesis of dopamine &hydroxylase in cultured rat adrenal medullae after in I.&Oadminis~ation of reserpine. J. ~euruchem. 27, 259-265. KABATE. A. & MEYERM. M. (1961) ~xpffimental ~mmunochemjstry. Charles C. Thomas, Illinois. LAMPRECHT F. & COYLEJ. T. (1972) Dopa decarboxylase in the developing rat brain. Brain Res. 41, 503-506. LEV~NE. Y., LEVENBERG B. SC KAUFMANS. (1960) The enzymatic conversion of 3,4_dihydroxyphenylethylamine to norepinephrine. J. biol. Chem. 235, 2080-2086. LOWRY 0. H., RO~EBROUGH N. Y., FARR A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 26.5-275. LYLESG. A. (1978) Effects of L-DOPA administration upon monoamine oxidase activity in rat tissues. Life Sci. 22, 603610. MOLINOFFP. B., BRIMIJOINS. % AXELRODJ. (1972) Induction of dopamine &hydroxylase and tyrosine hydroxylase in rat hearts and sympathetic ganglia. J. Pharmac. exp. Ther. 182, 116-129. MOLINOFFP. B., BRIMIJOINS., WEINSHILBOUM R. M. & AXELRODJ. (1970) Neurally mediated increase in dopamine fl-hydroxylase activity. Proc. natn. Acad. Sci. U.S.A. 66, 453458. MOLINOFFP. B., WEINSHILBOUM R. & AXELRODJ. (1971) A sensitive enzymatic assay for dopamine-~-hydroxyiase. J. Pharmac. exp. Ther. 178, 425-431. NAGATSUT. & UDENFRIENDS. (1972) Photometric assay of dopamine /?-hydroxylase activity in human blood. Clin. Chem. 18, 980-983. RAYMOND S. (1964) Acrylamide gel electrophoresis. Ann. N.Y. Acad. Sci. 121, 350-365. RUSH R. A., THOMASP. E., KINDLERS. H. & UDENFXIENDS. (1974) The interaction of dopamine B-hydroxylase with concanavalin A and its use in enzyme purification. Biochem. biophys. Res. Commun. 57, 1301-1305. TARvEa J. J., BERKOWITZB. & SPECTORS. (1971) Alterations in tyrosine hydroxylase and monoamine oxidase activity in blood vessels. Nature, Load. 231, 252-253. WATANABE A. M., JWDY W. V. & CARDONP. V. (1974) Effect of L-dopa on blood pressure and sympathetic nerve activity after decarboxylase inhibition in cats. J. Pharmac. exp. Ther. 1% 107-l 13. (Accepted 5 April 1979)