Naunyn-Schmiedeberg's Arch Pharmacol (1984) 327:119 - 123

Naunyn-Schmiedeberg'8

Archives of Pharmacology �9 Springer-Verlag 1984

Effect of triamterene on tyrosine hydroxylase activity

Pablo Steinberg and Modesto Carlos Rubio

Cfitedra de Farmacologia, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956, RA-1113 Buenos Aires, Argentina

Summary. Triamterene, which is structurally similar to the natural cofactor of tyrosine hydroxylase, (6R)-L-erythro- 5,6,7,8-tetrahydrobiopterin, inhibited tyrosine hydroxylase in rat adrenal gland homogenates and was found to be a competitive inhibitor of the synthetic cofactor 6,7-dimethyl- 5,6,7,8-tetrahydrobiopterin. When triamterene (30 mg/kg i.p.) was administered to rats, a significant decrease in the adrenal, whole brain and kidney enzyme activities was observed after 90 rain; if the drug was given orally, the diuretic affected only adrenal tyrosine hydroxylase. In both cases the drug decreased potassium excretion to 1/5 of con- trol values and increased the excretion of sodium. Catecholamine levels in atria, kidneys, adrenal glands and whole brain were not affected in acute experiments. Chronic treatment (triamterene 30 mg/kg p.o. twice daily during 4days) inhibited tyrosine hydroxylase and decreased catecholamine levels in the kidneys. Low potassium excre- tion was observed throughout the 4 days of treatment. In these chronic experiments the inhibition of the adrenal enzyme seen in acute treatments was not observed. This recovery cannot be explained by in increase in the adrenal biopterin levels. It could be mediated by an induction of the adrenal tyrosine hydroxylase.

Key words: Triamterene - Tyrosine hydroxylase activity - Biopterin - Catecholamines

Introduction

Tyrosine hydroxylase, an enzyme found in catecholamin- ergic neurons and the adrenal medulla, catalyzes the first step in the biosynthesis of catecholamines: the hydroxyla- tion of tyrosine. The enzyme requires (6R)-L-erythro-5,6,7,8- tetrahydrobiopterin as cofactor and L-tyrosine and molecu- lar oxygen as substrates to yield quinonoid-7,8-dihydro- biopterin, dihydroxyphenylalanine and water.

Triamterene (2,4,7-triamino-6-phenyl pteridine) is a diuretic that increases the excretion of sodium and depresses that of potassium.

Triamterene and (6R)-L-erythro-5,6,7,8-tetrahydrobi- opterin are structurally related. The purpose of our study was to determine the effect of triamterene on tyrosine hy- droxylase activity of different tissues, the possible variations of their cateeholamine levels after acute and chronic treat-

Send offprint requests to P. Steinberg at the above address

ment with the diuretic and the relationship between these possible actions and the effects on electrolyte excretion.

Materials and methods

Triamterene treatments. Male Wistar rats (160-240 g body weight) were used. Triamterene (30 mg/kg) was suspended in 1% w/v carboxymethylcellulose. In acute treatments the rats fasted for 18 h before as well as during the experiments but were allowed to drink water freely; the diuretic or the vehicle were administered orally or intraperitonealIy at 9.00 a.m. and the animals were decapitated after 90 or 270 min. In chronic treatments the rats fasted for 3 h before each administration; the diuretic or the vehicle were given orally twice daily (at 9.00 a.m. and 7.00 p.m.) during 4 days and the animals were decapitated 90 rain or 15 h after the last drug administration.

Assay of electrolyte excretion. The animals received an overload of 5 ml of water per 100 g body weight by means of a stomach tube at 9.00 a.m. Ten minutes later, triamterene 30 mg/kg or the vehicle were administered either orally or intraperitoneally. The rats were placed in metabolism cages, one animal per cage, and urine was collected for 4.5 h at 90 rain intervals. During chronic treatments the rats received the water overload once daily (at 9.00 a.m., 10 min before drug administration) during 4 days and were placed in metabolism cages. Urine was collected during the next 90 rain. In each sample sodium and/or potassium were determined by flame photometry.

Assay of tyrosine hydroxylase activity in adrenal gland homogenates. The adrenal glands were homogenized in 150 mM KC1 in a 1/50 (w/w) ratio. Triamterene added to the incubation medium was dissolved in dimethylsulphoxide. In the control tubes the activity of the enzyme was determined in the presence of this solvent. The method employed for the determination of tyrosine hydroxylase was the one described by Waymire et al. (1971), in the presence of a subsaturating concentration, 0.1 mM, of 6,7-dimethyl- 5,6,7,8-tetrahydrobiopterin (DMPH4) and a saturating con- centration, 0.1 mM, of [1-14C]-L-tyrosine (10.52 mCi/mmol, New England Nuclear Co., Boston, MA, USA). When the maximal enzyme activity in adrenal gland homogenates from control and chronically treated rats was measured, the assay was performed with saturant concentrations of DMPH4 and [1-14C]-L-tyrosine, 1 mM and 0.1 mM re- spectively.

120

To study enzyme kinetics, the adrenal gland homogenate was dialyzed overnight against 2 1 of cold 150 mM KC1 5o- solution containing 2 mM 2-mercaptoethanol; in this way tissue catecholamines, which could inhibit the hydroxylation 4o- of tyrosine in the presence of low concentrations of DMPH4, ,- were eliminated. Tyrosine hydroxylase activity was 0 determined in the supernatant obtained after centrifugation (10 min, 10,000 rpm) of the adrenal gland preparation, with .7 0 .1mM [1J4C]L-tyrosine, different concentrations of z 2o- DM PH4 (between 0.1 and 1.0 mM) and 1 mM triamterene dissolved in dimethylsulphoxide, or this solvent alone in the case of the control tubes.

Assay of tyrosine hydroxylase activity in intact tissues. After decapitation the intact tissues (brain, adrenal glands and kidneys) were quickly removed, weighed, chopped (pieces weighed 2 0 - 30 mg) and incubated in glass tubes for 20 min at 37~ in Krebs solution with the following composition (mM): NaC1, 118; KC1, 4.7; CaCI2, 2.6; MgCI2, 1.2; NaH2PO4, 1.0; NaHCO 3, 25.0; glucose, 11.1 ; EDTA, 0.004; ascorbic acid, 0.11 and [l-14-C]-L-tyrosine (58.1 mCi/mmol), 0.01. The final volume was of 0.4 ml. Blanks were obtained by adding 2.0 mM 3-I-tyrosine. In order to trap the 14CO2 formed in the decarboxylation step of the assay, a plastic well containing a folded paper imbibed in 0.2 ml of solubilizer Protosol (New England Nuclear Co., USA) was suspended from a rubber stopper. The reaction was stopped with 0.4 ml of 10% w/v trichloroacetic acid; after 1 h of continuous shaking the plastic wells are removed, wiped with absorbent tissue and placed in 5 ml of toluene scintillation fluid of the following composition: 0.6 g 1,4 bis 2-(5-phenyl-oxazolyl) benzene (POPOP); 4 g 2,5-diphenyloxazole (PPO) and 5 ml ethanol per liter of toluene. The samples were counted by liquid scintillation spectrometry.

Tissue content of endogenous catecholamines. The tissues were homogenized in 5 ml of ice cold 0.4 N HC104 contain- ing EDTA (1 mg/ml) and Na2SO3 (1 mg/ml). The super- natant obtained from centrifugation (5 min, 5,000 rpm) was adjusted to pH 8.2 and passed through an alumina column which was eluted with 3 ml 0.2 N acetic acid. For the meas- urement of catecholamines the fluorometric assay described by Laverty and Taylor (1968) was carried out in 1 ml of the eluate.

Biopterin levels in adrenal glands. After decapitation the ad- renal glands were immediately weighed and homogenized in 2.5 ml 0.1 N HC1. Aliquots (1.0 ml) were used for the determination of tissue biopterins, according to the method of Fukushima and Nixon (1980). It was consistently found that 9 0 - 9 5 % of total biopterin in the adrenal glands was present as tetrahydrobiopterin and this proportion remained unchanged after acute and chronic treatments.

Assay of triamterene levels. The total concentration of triamterene plus metabolites in tissues (brain, atria, kidneys, adrenals glands) and biological fluids (plasma and urine) was determined as described by Dayton et al. (1972) and Pruitt et al. (1977).

Statistics. All values are reported as mean +_ SEM. Statisti- cal analysis were carried out using Student's t-test.

Drugs. The following drugs were used: triamterene (Lab. Beta, Buenos Aires, Argentina); 6,7-dimethyl-5,6,7,8-tetra-

1

"1 S_

N L l.lO I 3.101 3.10

2

3.10 3

TRIAMTERENE (}JM)

Fig. l. Effect of triamterene on tyrosine hydroxylase activity. Ordinate: percent inhibition of tyrosine hydroxylase activity. Abscissa: gmolar concentration of triamterene. Tyrosine hydroxyl- ase activity was determined in the presence of DMPH4 (subsaturant concentration) and [1-14C]_L.tyrosine in homogenates of rat adrenal glands as the source of the enzyme. Mean values + SEM of at least 5 experiments per concentration are shown. * P < 0.05

hydrobiopterin (DMPH4) and 3-I-tyrosine (Sigma, St. Louis, MO, USA).

Results

A. Acute effects of triamterene

Triamterene inhibited tyrosine hydroxylase present in rat adrenal gland homogenates; this effect was statistically sig- nificant between 10 txM and 3 mM (Fig. 1). Control enzyme activities were 80.4+_9.3 nmol/g/h in the presence of dimethylsulphoxide and of 75.1 + 7.8 nmol/g/h in the ab- sence of this solvent. The diuretic was found to be an inhibi- tor of tyrosine hydroxylase, competitive with the synthetic cofactor DMPH4 (Fig. 2).

The next step was to test if the diuretic inhibited the enzyme in vivo. In acute treatments, 90 rain after i.p. admin- istration of 30 mg/kg triamterene, there was 88%, 45% and 30% inhibition of the enzyme present in adrenal glands, whole brain and kidney respectively; when the oral route was used, there was a significant inhibition (38%) of the adrenal tyrosine hydroxylase, but no effect was observed either on the brain or on the kidney enzyme (Fig. 3). Oral administration of 10 or 20 mg/kg triamterene had no effect on adrenal tyrosine hydroxylase. No significant differences were observed in the levels of endogenous catecholamines 90 min after acute treatment with triamterene (30 mg/kg) either intraperitoneally or orally (Fig. 4).

Figure 5 shows the effect of triamterene (30 mg/kg) on electrolyte excretion during 4.5 h after the diuretic adminis- tration. When the drug was given intraperitoneally, the natriuretic effect was observed earlier (after 90 min) than in the case of the oral administration (after 180 min). However, the potassium-sparing effect which reduced potassium excre- tion to 1/5 of the control value was the first to be seen in both cases. Two hundred and seventy minutes after triamterene 30 mg/kg orally, kidney tyrosine hydroxylase was not affected (n = 5; tyrosine hydroxylase activity: 1,458 +_ 372 dpm/g/20 min for the control group and 1,209 + 345 dpm/g/20 min for the treated group).

0,15 j

v= o,lo %

0,05-

c E 3.1(~

E=

1.10 ~

g 10

1 / D M P H 4 ( 1 / m M )

Fig. 2. Kinetic study of adrenal tyros• hydroxylase. Reciprocal enzyme activities were plotted graphically against reciprocals of DMPH4 concentrations; the lines were fitted to the points by the method of least squares. Each point represents the mean of two determinations. Tyrosine hydroxylase activity was determined in the presence of [1-14C]-L-tyrosine, different concentrations of DMPH4 (between 0.1 and J .0 mM), adrenal gland homogenates prepared as described in Materials and methods as source of the enzyme, and 1 mM triamterene ( 0 ) dissolved in dimethylsulphoxide, or this solvent alone (O) in the case of the control tubes

o

0 I,I 3

adrenal brain kidney adrenal brain kidney

Fig. 3. Hydroxylation of tyrosine after administration of triamterene A intraperitoneally, B orally. Ordinates: hydroxylation of tyrosine (dpm of 14CO2 liberated per gram of tissue in 20 min). Ninety minutes after administration of 30 mg/kg triamterene (11) or vehicle ([~), the rats were sacrificed and the tissues were incubated for 20 rain in Krebs solution with [1J4C]-r~-tyrosine (see Materials and methods). Means values _+ SEM of at least 5 experiments per tissue are shown. *P < 0.05; **P < 0.0a

B. Chronic effects of triamterene

Chronic oral adminis t ra t ion (4 days) of t r iamterene 30 mg/ kg significantly decreased the hydroxyla t ion of tyrosine and the endogenous catecholamines in the kidney a l though the animals were killed 15 h after the last drug administrat ion. It did not affect the other tissues studied (Fig. 6). Kidney tyrosine hydroxylase was inhibited about 60% and the levels of catecholamines were reduced by about 55% when compared with controls. The inhibi t ion o f adrenal tyros• hydroxylase observed in acute experiments was not observed

121

1,4.106~ ; }

o

adrenal atria brain kidney adrenal atria brain kidney

Fig. 4. Levels of endogenous catecholamines after the administra- tion of triamterene A intraperitoneally, B orally. Ordinates. endogenous catecholamines (ng of noradrenatine plus adrenaline per gram of tissue). Ninety minutes after administration of 30 mg/kg triamterene (B) or vehicle ([Z), the rats were sacrificed and tissue catecholamines were quantified (see Materials and methods). Mean values ,+ SEM of 6 experiments per tissue are shown

E

0~90 90-180 IB0-270 0 - 9 0 90-180 180 -270 time interval (min,) time interval (rain,)

t I0 ~ I00

~ ~- .E 50

0 - 9 0 90 -180 180-270 0 - 9 0 90-180 180 270 t ime interval (rnin.) time interval (rain j

Fig. 5. Urinary potassium and sodium excretion after the adminis- tration of triamterene A--B orally, C- -D intraperitoneally. Ordinates: urinary potassium or sodium excretion (expressed as mEq/liter of urine). Abscissae: time intervals (expressed in minutes). The rats received an overload of 5 ml water per 100 g body weight by means of a stomach tube and 30 mg/kg triamterene (11) or vehicle ([2). They were then placed in metaboIism cages, one animal per cage, and urine was collected in 90 mix intervals during 4.5 h. Mean values _+ SEM of at least 5 experiments per time interval are shown. *P < 0.05; **P < 0.01

after the 4 days treatment. Even when the animals were sacrificed 90 min after the last adminis t ra t ion of t r iamterene no inhibit ion of the adrenal enzyme was seen (n = 6; tyrosine hydroxylase activity: 27,052 + 6,183 dpm/g/20 mix for the control group and 30,947 _+ 4,509 dpm/g/20 min for the treated group).

When the maximal enzyme activity was measured in adrenal gland homogenates of rats sacrificed 15 h after the last dose, there was a 23% increase in adrenal tyrosine hydroxylase activity (n = 6; tyros• hydroxylase activ- ity: 762__ 71nmol /g /h for the control group and 945 +_ i 03 nmol /g /h for the treated group; the difference was statistically significant at the P < 0.05 level, Student ' s t-test was applied), but adrenal biopter in levels were not affected (Table 1).

122

1,5.1

[ �9 ~ 3.1 =m IJ

Z

H

adrenal brain kidney adrenal atria brain kidney

Fig. 6A, B. Hydroxylation of tyrosine and levels of endogenous catecholamines after chronic treatment with triamterene. Ordinates: A Hydroxylation of tyrosine (dpm of 14CO2 liberated per gram of tissue in 20 min). B Endogenous catecholamines (ng of noradrena- line + adrenaline per gram of tissue). The rats received an oral dose of 30 mg/kg triamterene (B) or vehicle ( S ) twice daily during 4 days. The hydroxylation of tyrosine and the levels of endogenous catecholamines were determined as described in Materials and methods. Mean values • SEM of at least 5 experiments per tissue are shown. * P < 0.05.

Table 1. Biopterin levels in adrenal glands after acute and chronic treatment with triamterene. In acute treatments triamterene 30 mg/ kg or the vehicle were administered orally and the rats were sacri- ficed after 90 min. In chronic experiments the rats received an oral dose of triamterene 30 mg/kg or the vehicle twice daily during 4 days and the animals were decapitated 15 h after the last drug administration. Biopterin measurements were carried out as described in Materials and methods. Mean values • SEM of 5 experiments per group and treatment are shown

Treatment Group Total biopterin (nmot/g)

Control Treated

Acute 6.30 • 1.28 6.17 _ 1.91 Chronic 6.41 • 1.69 6.25 • 1.55

Table 2. Urinary potassium excretion during chronic administration of triamterene. The rats received an oral dose of 30 mg/kg triamterene twice daily (at 9.00 a.m. and 7.00 p.m.) during 4 days. Each day, 10 min before drug administration they were given an overload of 5 ml water per 100 g body weight and were placed in metabolism cages, one animal per cage; urine was collected during 90 rain. Mean values • SEM of at least 5 experiments per day are shown. * P < 0.01

Time (h) K + (mEq/1)

0 10.7 + 2.6 24 2.2 • 0.9" 48 1.8 + 1.3" 72 2.3 • 1.1 * 96 2.0 _ 0.6"

Table 2 shows potass ium excretion during chronic treatments. The potass ium levels were reduced to abou t 20% of the control value during the whole treatment. The levels of t r iamterene plus metabol i tes in tissues and biological

Table 3. Endogenous levels of triamterene plus metabolites in tissues and biological fluids. In acute treatments triamterene 30 mg/kg was administered orally or intraperitoneally and the rats were sacrificed after 90 rain. In chronic treatments the animals received an oral dose of triamterene 30 mg/kg twice daily during 4 days and the rats were decapitated 15 h after the last dose. Drug measurements were carried out as described in Materials and methods. Mean values +__ SEM of 4 experiments per tissue or biological fluid are shown

Acute i.p. Acute p.o. Chronic p.o.

Adrenal gland (gg/g) 4.49 • 0.36 0.52 _ 0.11 0.50 _ 0.26 Atria (gg/g) 4.05 + 0.6i 0.43 +0.13 0.31_+0.20 Brain (gg/g) 2.47_ 1.12 0.43_ 0.19 0.45_ 0.17 Kidney (lag/g) 4.27 _ 1.41 0.95 _ 0.22 8.36 _ 2.89 Plasma (lag/ml) 4.57_ 0.87 0.39 • 0.11 - Urine @g/ml) 27.12_ 6.96 - -

fluids studied in acute and chronic t reatments can be seen in Table 3. The acute experiments showed that drug levels were 10 times higher when the diuretic is administered in- traperi toneally. When the oral route was used, there was 2 - 3 times more drug in the kidneys than in the other tissues studied. Af ter 4 days t rea tment there was a high concentra- t ion of the diuretic agent and its metaboli tes in the kidneys. The other tissues mainta ined the levels seen in acute experi- ments.

Discussion

Because of the structural similarity between tr iamterene and (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin, the natural cofactor of tyrosine hydroxylase, we studied first of all the effect of the diuretic on the adrenal tyrosine hydroxylase. We found a significative inhibit ion, due to compet i t ion with the pteridine cofactor. The inhibi tory effect was also observed in intact tissue. Acute experiments ex vivo showed that the inhibi t ion was much greater when the drug was adminis tered intraper i toneal ly than when it was given orally. This result is in accordance with the 10 times higher levels of t r iamterene plus metabol i tes found in the different tissues when the int raper i toneal route was used, When the diuretic was given int raper i toneal ly the action on adrenal glands was more significant than that on bra in or kidney. Ninety minutes after an oral dose of tr iamterene, the drug levels were similar in adrenal glands and brain, but twice as much diuretic was found in the kidneys; the diuretic was effective in adrenal glands but it did not affect either the bra in or the kidney enzyme. Three explanat ions can be given: a) adrenal tyrosine hydroxylase might have different requirements i f compared with the bra in or the kidney enzyme; this has not been repor ted up to date; b) a heterogeneous dis t r ibut ion of t r iamterene in each tissue studied might occur; c) there could be differences in the tissue levels of biopterin. Fukush ima and Nixon (/980) studied the dis tr ibut ion o f biopter in in different rat tissues and repor ted that b iopter in levels were higher in adrenal glands (the level in the medul lary area, where hydroxyla t ion of tyros ine occurs, appears to be higher than that of the total gland) than in the kidneys, a l though the effective concentrat ion in the renal catecholaminergic nerve endings rests unknown.

N o effect on whole brain tyros• hydroxylase was observed after oral adminis t ra t ion o f the diuretic; however, we cannot exclude an inhibi tory effect of tr iamterene on

123

tyrosine hydroxylase in specific brain regions where the drug could be concentrated.

The possibility that the hyperkalemia and/or the hypo- natremia observed after the administration of triamterene were responsible for the effect observed on tyrosine hy- droxylase activity can be rejected because the sodium and potassium levels required to observe changes in the enzyme activity (Gutman and Segal 1972, 1973) are not achieved in our treatments.

It is interesting to note that although the acute adminis- tration of triamterene affects the biosynthesis of catechol- amines by inhibiting tyrosine hydroxylase in some tissues, it does not produce a fall in their levels of catecholamines. It is possible that the time period chosen (90 rain) was too short to observe depletion of noradrenaline and/or adrena- line. When tyrosine hydroxylase is inhibited the catecholamine depletion rate depends on neuronal uptake and activity (Bhatnagar and Moore 1971).

In the kidney chronic (4 days) but not acute treatments inhibited tyrosine hydroxylase and decreased the concentra- tion of eatecholamines. This is probably due to the fact that the diuretic does not reach an inhibitory concentration 1.5 h after a single administration (if compared with acute experi- ments there is a 9-fold increase in the triamterene levels after chronic administration of the drug). The inhibition of adrenal tyrosine hydroxylase seen in acute tests was not ob- served after chronic treatment. Metabolization of triamterene in the interval (15 h) between the last drug administration and the decapitation of the rats is not responsible for this rever- sion because: a) even when the rats were killed 90 min after the last dose of diuretic, no inhibition of the adrenal enzyme was noted; b) the levels of triamterene in adrenal glands were similar in acute and chronic treatments. It seems pos- sible that during those 4 days a compensatory mechanism has developed to increase tyrosine hydroxylase activity up to control values despite the presence of triamterene in the tissue. This compensation could be mediated by: a) an induc- tion of tyrosine hydroxylase; b) an activation of this enzyme; c) an increase in the levels of reduced biopterin; tetra- hydrobiopterin is found in subsaturating levels in mono- amine-containing cells (Lloyd and Weiner 1971 ;Weiner et al. 1972) and its concentration may be an important regulatory factor for tyrosine hydroxylase (Nagatsu 1981). After a chronic treatment with the diuretic an increase in the maximal tyrosine hydroxylase activity measured in adrenal gland homogenates in the presence of saturant concentra- tions of DMPH4 and L-tyrosine was observed, while adrenal biopterin levels were not affected. Our data suggest that the reversion of the adrenal enzyme inhibition could be mediated by an induction of tyrosine hydroxylase.

Our results indicate that the potassium-sparing and natriuretic effects of triamterene are independent from the inhibition of kidney tyrosine hydroxylase by the diuretic: after the oral administration of the drug the reduction of potassium excretion is already observed during the first

90 rain, while the natriuretic effect appears after 180 rain. In both cases kidney tyrosine hydroxylase is not inhibited. Lower doses (10 or 20 mg/kg p.o.) do not affect the enzyme, whereas much lower doses are significantly active on electrolyte excretion in the rat (Lassen and Nielsen 1963; Wiebelhaus et al. (1965). Amiloride, structurally different from triamterene, also decreases potassium excretion but does not affect tyrosine hydroxylase present in adrenal gland homogenates (unpublished observations).

Acknowledgements. We thank Dr. F. J. E. Stefano for helpful advice and Dr. Claudia Garcia Bonelli for the biopterin measurements.

References

Bhatnagar RK, Moore KE (1971) Effects of electrical stimulation, ~-methyl-p-tyrosine and desmethylimipramine on the nor- epinephrine contents of neuronal cell bodies and terminals. J Pharmacol Exp Ther 178: 450- 463

Dayton PG, Pruitt AW, McNay JL, Steinhorst J (1972) Studies with triamterene, a substituted pteridine. Unusual brain to plasma ratio in mammals. Neuropharmacology 11 : 435-- 446

Fukushima T, Nixon JC (1980) Analysis of reduced forms of biopterin in biological tissues and fluids. Anal Bioehem 102:176-188

Gutman Y, Segal J (1972) Effect of calcium, potassium and sodium on adrenal tyrosine hydroxylase activity in vitro. Bioehem Pharmacol 21 : 2664- 2666

Gutman Y, Segal J (1973) Effect of calcium, potassium and sodium on tyrosine hydroxylase activity in different regions of the rat brain. Bioehem Pharmacol 22: 865 - 868

Lassen JB, Nielsen OE (1963) Investigations into the diuretic effect and elimination of triamterene. Acta Pharmaeol Toxicol 20:309-316

Laverty R, Taylor KM (1968) The fluorimetrie assay of catechol- amines and related compounds: improvements and extension to the hydroxyindole technique. Anal Biochem 22:269-279

Lloyd T, Weiner N (1971) Isolation and characterization of a tyrosine hydroxylase cofaetor from bovine adrenal medulla. Mol Pharmaeol 7: 569- 580

Nagatsu T (1981) Biopterin cofactor and regulation of monoamine- synthesizing mono-oxygenase. Trends Pharmacol Sci 2 (10): 276- 279

Pruitt AW, Winkel JS, Dayton PG (1977) Variations in the fate of triamterene. Clin Pharmacol Ther 21 : 610- 619

Waymire JC, Bjur R, Weiner N (1971) Assay of tyrosine hydroxylase by couple decarboxylation of DOPA from [l-14C]-L-tyrosine. Anal Biochem 43 : 588 - 600

Weiner N, Cloutier G, Bjur R, Pfeffer RI (1972) Modification of norepinephrine synthesis in intact tissue by drugs and during short term adrenergic nerve stimulation. Pharmacol Rev 24: 203 - 22 l

Wiebelhaus VD, Weinstock.J, Maass AR, Brennan FT, Sosnowski G, Larsen T (1965) The diuretic and natriuretic activity of triamterene and several related pteridines in the rat. J Pharmacol Exp Ther 149:397--403

Received January 9, 1984/Accepted May 24, 1984