Naunyn-Sehmiedeberg's Arch. Pharmacol. 287, 1--10 (1975) 9 by Springer-Verlag 1975
Studies on the Synthesis and Subeellular Distribution of Dopamine in the Rat Adrenal Medulla B e r t i l W a l d e c k , S t u a r t g . S n i d e r * , R o g e r B r o w n * * , a n d A r v i d Carlsson Department of Pharmacology, University of G6teborg Received September 23/Accepted December 5, 1974
Summary. Rats received intravenous injections of aH-tyrosine and were killed at various time intervals thereafter, aH-dopamine (DA) in the adrenals reached a maximum within 1.5 rain after the administration of 3H-tyrosine. From the 15th rain it disappeared with an apparent half life of 90 rain. 3H-Adrenaline (A) ~H-noradrenatine(NA) increased much more slowly and reached a plateau 120--240rain after the injection. The approximate synthesis rate of adrenal A-t-NA, calculated from the specific activity curves, ranged from 9.3 to 2.2 nmoles/ h per kg b.w. The highest value was noted the frrs~ few minutes, the lowest 1--2 hrs after the administration of 3H-tyrosine. In some experiments subcellular fractionation of the adrenals was performed. In untreated animals the amount of DA and A-l-HA recovered from the supernatant fraction was about 10 and 8 per cent, respectively, of the total amount recovered from the supernatant and particulate fractions. In the adrenals of animals receiving aH-tyrosine 3.75 or 60 rain beforehand these figures were significantly elevated whereas the DA and A-t-HA of the particulate fraction did not deviate significantly from control values. The specific activities of aH-DA were the same in the supernatant and particulate fractions within 3.75 rain after the injection of 3H-tyrosine. Key words: Dopamine -- Adrenomedullary Hormones -- Synthesis -- Rat Adrenals -- Subcellular Distribution. I n earlier studies we h a v e p r e s e n t e d evidence s u p p o r t i n g t h e v i e w t h a t in t h e r a t a d r e n a l m e d u l l a d o p a m i n e (DA) m a i n l y , if n o t e n t i r e l y , serves as a n i n t e r m e d i a t e in t h e s y n t h e s i s o f n o r a d r e n a l i n e (NA) a n d a d r e n a l i n e (A) (Snider a n d Carlsson, 1972; Carlsson et al., 1973b). I n t h e s e s studies, using t h e r a t e of d i s a p p e a r a n c e of D A a f t e r i n h i b i t i o n of t y r o s i n e h y d r o x y l a s e , t h e s y n t h e s i s r a t e o f a d r e n a l D A was e s t i m a t e d t o a b o u t 3 n m o l e s / h - kg -1. This figure is in g o o d a g r e e m e n t w i t h t h e t u r n o v e r r a t e which can be c a l c u l a t e d f r o m t h e d e c a y curve of r a d i o a c t i v e A in t h e r a t a d r e n a l s ( U d e n f r i e n d et al., 1953). F r o m t h e a c c u m u l a t i o n of 3 H - D o p a a f t e r a d m i n i s t r a t i o n of 3 t I - t y r o s i n e d u r i n g i n h i b i t i o n o f D o p a d e e a r b o x y l a s e , H e m p e l a n d M/~nnl (1967) c a l c u l a t e d a s y n t h e s i s r a t e for D o p a in t h e cat a d r e n a l s of a b o u t 0.02 ~xg/
Send oHprint requests to: B. Waldeck, AB Draeo, Fack S-221 01 Ltmd, Sweden. * Present address: Neurological Institute, 710 West 168th Street, New York, N. Y. 10032, U.S.A. ** Present address: National Institutes of Mental Health, Neuropsychology, Building 9, room 1 N 107, 9000 Rockville Pike, Bethesda, Maryland 20014, U.S.A. 1
Naun~m-Schmiedeberg's Arch. Pharmacol., Vol. 287
2
B. Waldeck et al.
r a i n . k g -1 (6 n m o l e s / h 9 kg-1). A f t e r a n i n t r a v e n o u s i n j e c t i o n of SH-tyrosine t o r a t , 3 H - ( A ~ - N A ) a p p e a r e d to a c c u m u l a t e for m o r e t h a n 2 hrs (Persson a n d W a l d e c k , 1970). H o w e v e r , a d e t a i l e d s t u d y on t h e d y n a m i c s of t h e a d r e n a l c a t e c h o l a m i n e s y n t h e s i s has so f a r n o t been p r e s e n t e d . V e r y few s t u d i e s h a v e so far b e e n p e r f o r m e d on t h e subcellular d i s t r i b u t i o n o f a d r e n a l DA. E a d e (1958) r e p o r t e d t h a t in t h e b o v i n e a d r e n a l m e d u l l a a b o u t t w o - t h i r d s of t h e D A was r e c o v e r e d f r o m t h e " l a r g e - g r a n u l e " fraction, w i t h m o s t of t h e r e m a i n d e r in t h e s u p e r n a t a n t . T h e d i s t r i b u t i o n was v e r y similar to t h a t of t h e t o t a l c a t e c h o l a m i n e s , of w h i c h D A c o n s t i t u t e d a b o u t 1 ~ I n t h e sheep a d r e n a l L i s h a j k o (1968) made similar observations. I n e x p e r i m e n t s on i s o l a t e d b o v i n e a d r e n o m e d u l l a r y granules Carlsson et al. (1963) o b s e r v e d t h a t D A , like H A a n d A, is t a k e n u p b y t h e granules b y a n A T P - M g 2 + - d e p e n d e n t m e c h a n i s m , which can be b l o c k e d b y reserpine. I n vivo, r e s e r p i n e has been f o u n d t o cause a r a p i d decrease o f D A in r a t a d r e n a l s (Snider a n d Carlsson, 1972). I n t h e p r e s e n t i n v e s t i g a t i o n t h e s y n t h e s i s of a d r e n o m e d u l l a r y c a t e c h o l a m i n e s , p a r t i c u l a r l y D A , h a s b e e n s t u d i e d following a pulse i n j e c t i o n of 3H-tyrosine. I n some e x p e r i m e n t s t h e subcellular d i s t r i b u t i o n of b o t h u n l a b e l l e d a n d l a b e l l e d c a t e c h o l a m i n e s was i n v e s t i g a t e d in o r d e r t o g a i n i n s i g h t i n t o t h e d y n a m i c s of t h e s t o r a g e m e c h a n i s m .
Material and Methods Male Sprague-Dawley rats, weighing about 250 g, were used. They were kept in the animal quarters for at least 48 hrs before use and allowed food pellets and water ad libitum. Experimental animals were handled as carefully as possible. In order to minimize the influence of diurnal variations all experiments were performed between 8 and 11 a.m. Intravenous injections were given into a tail vein with the animals briefly immobilized in a small cylindrical cage. Other injections were given intraperiteneally. Following drug treatment, rats were sacrificed by means of decapitation or concentrated chloroform vapor. Comparison between decapitation and chloroform revealed no significant difference in amine values. The adrenals were rapidly removed and extracted in 0.4 N perehlorie acid. In experiments where the subeellular distribution was studied, the adrenals were placed directly in 3 ml of 0.3 M sucrose at 0--4~ and homogenized with a teflon pestle in a glass homogenizer (for details see Itillarp, 1958). The pestle speed and homogenization procedure were the same in all experiments. The homogenate was centrifuged at 4~ and 20000 • g for 20 min. The supernatant and sediment were separated, and perehloric acid, 10~ EDTA, and 5 % ~a2S205 were added to each so that a final volume of 10.3 ml of 0.4 N perehloric acid, 0.2~ EDTA, and 0.05~ ~a2S205 resulted. After homogenization the protein precipitate was removed by centrifugation. Separation of unlabelled amines from the extracts was performed according to the method of Atack and Magnusson (1970). DA was assayed spectrofluorimetrically (Ataek 1973), and the A + N A was determined by native fluorescence against a hTA standard. Labelled amines were separated from the perchlorie acid extracts by a combination of alumina and Dowex 50 columns (Persson and Waldeck, 1968).
Studies on the Synthesis and Subcellular Distribution The 3H-content of each fraction was determined in a liquid scintillation counter after freeze-drying (Waldeck 1968). In some experiments blood was collected in plastic tubes containing ~n EDTA solution, after which the specific activity of 3H-tyrosine in plasma was determined (Carlsson et al., 1973a), tyrosine being determined according to Waalkes and Udenfriend (1957). Further experimental details are given in the text. The following drugs were used: a-propyldopacetamide (H22/54), and cr tyrosine methylester (H44/68) (AB H~ssle, ~Slndal), L-tyrosine, ring-3,5aH, specific activity about 47 Ci/mmole, and L-Dopa, ring-2,5,63H, specific activity, 1 Ci/mmole (The Radiochemical Centre, Amersham). The radiochemical purity was regularly checked by radiopaper chromatography in isopropanol/2N tiC1, 65/35, v/v. No 3H-Dopa was detected in the att-tyrosine, i.e. it contributed with less than about 1 ~ of the total activity. The 3H-Dopa contained some 5 % radioactive impurities, presumably condensation products with acetaldehyde (Waldeck, 1973). Since the specific activity of the all-labelled precursors were different and administration was made on a weight basis (5 ~g/kg b.w.) the specific activity of plasma tyrosine and DA and A + N A in the adrenals have been expressed as fmoles of substance derived from the labelled precursor per nmole total of the corresponding compound. When aH-tyrosine was used, corrections were made for the loss of one atom ~H per molecule in the calculations of aH-catecholamine specific activity. Results
A. Accumulation o/aH-Catecholamines in the Adrenals Following Injection o/aH-Tyrosine R a t s r e c e i v e d i n t r a v e n o u s injections of 3H-tyrosine, 5 ~g/kg, a n d were killed a t v a r i o u s t i m e i n t e r v a l s t h e r e a f t e r . L a b e l l e d a n d u n l a b e l l e d D A a n d A ~ ~ A in t h e a d r e n a l s a n d t y r o s i n e in t h e p l a s m a were determined.
Table 1. Tyrosine in plasma and catecholamines in the ~drenals at various time intervals after the intravenous injection of a trace amount of aH-tyrosine (el. Fig. 1). Shown are the means, s.e.m, and the number of experiments Time intervals in minutes
Tyrosine in plasma /~g/ml Dopamine in adrenals nmoles/kg b.w. Adrenaline ~noradrenaline in adrenals nmoles/kg b.w. 1"
1.5
3.75
7.5
15
30
60
120
240
16.8 • 1.63 (3) 5.15 J= 0.25 (3) 575 ~ 50 (3)
t9.4 • 0.68 (7) 6.50 • 0.75 (7) 590 • 65 (6)
20.6 =]=1.81 (6) 6.45 =~ 0.65 (6) 655 =~ 55 (6)
16.7 • 0.69 (5) 6.35 • 0.85 (7) 575 • 45 (7)
16.6 =L1.72 (4) 6.40 =~ 0.95 (6) 610 i 80 (6)
13.6 =~ 2.01 (5) 5.90 ~ 0.70 (7) 605 • 100 (6)
11.9 i 1.43 (4) 5.65 :t= 0.35 (6) 530 • 45 (6)
18.4 • 1.56 (6) 6.85 • 0.85 (6) 555 • 65 (6)
B. Waldeck et al. 367•
ILl r Z
N 2o0. (/1 m
(,D
p,.
150.
"6 100. ~100 m
< "~
50.
"6 E
.e
0"
15 ' 30 '
6"0
120
f,, 24O
180 rain.
:Fig. 1. Accumulation of ~H-catecholamines in the adrenals following injection of stI-tyrosine. Rats received intravenous injections of 8It-tyrosine, 5 ~zg/kg, and were killed at various time intervals thereafter. Shown are the specific activities of tyrosine in plasma o, and dopamine., and adrenaline-knoradrenaline . in the adrenals expressed as fmoles material derived from ~H-tyrosine per nmole total of the respective substance. The data are the means ~= s.e.m, of 3--7 rats analyzed individually (see Table 1). The adrenaline-~-noradrenaline values have been multiplied by 100 There were no significant changes in the levels of endogenous DA and A + NA in the adrenals (Table 1). From 7.5 to 120 rain after the injection, plasma tyrosine decreased from 20.6 to 11.9 ~zg/ml (P < 0.001), which appears to be below the range of normal values given in the literature (Zigmond and Wurtman, 1970; cf. also Munro, 1974). I t then returned to 18.4 ~zg/ml during the next 120 min (P < 0.005). This value was not significantly different from the values observed 1.5 to 7.5 rain after the injection of labelled tyrosine. The specific activities of tyrosine, DA and A + NA are shown in Fig. 1. The time course of plasma tyrosine specific activity appeared to be polyphasic as revealed b y a semilogarithmic plot. The specific activity of DA in the adrenals reached a m a x i m u m value within 1.5 rain after the administration of 3H-tyrosine. I t remained, with slight variation, at this level for the next 14 min while crossing the plasma 3tt-tyrosine specific activity curve (about 12 min after injection), and then started to decrease with an apparent half-life of 90 min. The DA curve, as it appears in Fig. l, suggests the existence of 2 peaks, 1 at 1.5 rain and 1 at 15 min. However, more data are necessary to establish their existence. The specific activity of A + NA increased much more
Studies on the Synthesis and Subcellul~r Distribution
5
slowly and appeared to approach a plateau 120 to 240 rain after the injection. After 240 rain the specific activity of DA was still 18 times higher than that of A + NA. Since the A Jr NA curve is magnified 100 • and the endogenous level of these amines is about 100 times that of DA (Table 1), Fig. i also shows e.g. the approximate time at which the amount of 3tt-(A + HA) equals that of 3H-DA. This occurs about 70 rain after the administration of the precursor. An attempt was made to calculate the rate of adrenal eatecholamine synthesis from the activity curves according to the equation A +HA=
3H- (A + NA) • D A ~H- DA
where A + 57A stands for the total amount newly formed A + NA during a given time interval, 3H-(A + HA) for the amount formed from 3H-tyrosine during the same time interval, and aH-DA/DA for the corresponding specific activity of DA (for details see e.g. Hempel and Ms 1967). Table 2 shows the result. The estimated synthesis rate appeared to be dependent on the time interval chosen for calculation, resulting in considerably higher values at early as compared with late time intervals. Table 2. Synthesis rate of adrenal adrenaline (A) + noradrenaline (NA) calculated from the specific activity curves in Fig. 1 (see text) Time interval in minu~es A +NA nmoles/h per kg b.w.
3.75--7.5 7.5--15
15--30
30--60
60--120
120--240
9.3
3.6
3.6
2.2
2.6
5.7
B. E]/ect o/Tyrosine-Hydroxylase Inhibitors on the Accumulation o/3H-Catecholamines in the Adrenals Following Injection o/3H-Tyrosine In other experiments H22/54, 500 mg/kg, was given 15 rain or It44/68, 400 mg/kg, 30 rain before the i.v. administration of 5 ~zg/kg aH-tyrosine. The animals were killed 3.75 rain after the injection. The specific activity of DA and A + NA in the adrenals are shown in Table 3 together with 3.75 mid values from Fig. 1 (controls). The specific activity of DA was less than 10 ~ of the control value. The level of radioactivity in the A + NA fraction was insignificant and very close to the background, in animals given a synthesis inhibitor. In contrast, aH-(A + NA) activity in the corresponding controls was about 7 times the background level.
B. Waldeck et al.
6
Table 3. Effect of tyrosine hydroxylase inhibitors on the accumulation of aH-catecholamines in the adrenals as measured 3.75 rain after the intravenous administration of 5 ~g/kg 3H-tyrosine. ~-Propyldopacetamide (H22/54), 500 mg/kg, or a-methyltyrosine methylester (H44/68), 400 mg/kg, was given 15 and 30 min, respectively, beforehand. The result, expressed as fmoles aH-amine/nmole total of the corresponding amine, is shown together with that obtained at the same time interval without drug treatment (control, from Fig. 1). Shown are the mean 4- s.e.m. and the number of experiments Control
H22/54
H44/68
3H-Dopamine 78 4- 1.4(7) 4.4 4- 0.5(2) 7.4 4- 0.1(2) aH-(Adrenaline-[-noradrenaline) 0.13 ~ 0.014(7) 0.03 =~ 0.026(2) 0.01 =[: 0.003(2)
C. Accumulation o/8H-Catecholamines in the Adrenals Following Injection o/3H-Dopa 3H-Dopa, 5 ~g/kg, was g i v e n i n t r a v e n o u s l y to rats. T h e animals were killed 15 or 120 rain later. ~H-DA a n d 3H-(A -[- NA), b u t n o t t h e e n d o g e n o u s levels of t h e s e amines, were m e a s u r e d . Specific activities were t h e n c a l c u l a t e d on t h e basis o f t h e endogenous levels shown in T a b l e 1. T h e result, t o g e t h e r w i t h t h e v a l u e s o b t a i n e d a t t h e corresponding t i m e i n t e r v a l s a f t e r ~H-tyrosine (from Fig. 1), is shown in T a b l e 4. T h e n e t y i e l d o f 3 H - c a t e c h o l a m i n e s f r o m 8H-Dopa was 2 - - 4 t i m e s higher t h a n t h a t o b t a i n e d f r o m a n e q u i m o l a r dose of 3H-tyrosine. Thus, 1 ~ c o n t a m i n a t i o n of 3H-tyrosine w i t h a I t - D o p a will influence t h e r e s u l t by at most 2--4 ~
Table 4. Accumulation of aH-catecholamines in the adrenals following the intravenous injection of 5 ~zg/kg 3H-Dopa or 3H-tyrosine. The animals were killed 15 or 120 min after the injection. The result is presented as fmoles aH-amine/nmole total of the corresponding amine. Shown are the means • s.e.m, and the number of expriments 15 min 8H'D~ amine
/ from ( from all-(Adrenaline 4- / from noradrenaline) / from
3H-Dopa aH-tyrosine a 3H-Dopa 3H-tyrosinea
217 • 83(4) 88 4- 9(7) 0.60 4- 0.17(4) 0.30 4- 0.04(7)
120 min 122 4- 7(4) 33 =[=4(6) 1.49 4- 0.43(4) 0.87 4- 0.08(6)
a Data from Fig. 1.
D. Subcellular Distribution o/Labelled and Unlabelled Catecholamines in the Adrenals at Various Time Intervals Following Injection of 3H-Tyrosine R a t s r e c e i v e d i n t r a v e n o u s injections of 3H-tyrosine, 5 ~zg/kg, a n d were killed 3.75 or 60 m i n later. Control a n i m a l s r e c e i v e d no injections a n d
Studies on the Synthesis and Subcellular Distribution Table 5. Subceltular distribution of unlabelled and labelled eateeholamines in the adrenals at various time intervals after injection of 5 y.g/kg ~H-tyrosine. Control animals (0 rain) received no injections. Shown are the means • s.e.m, of 4 experimental groups, each comprising 2 rats. S ----supernatant, P = particulate fraction Unlabelled amine, nmoles/kg
Dopamine
rain
S
P
S N+-----P • 100
0
0.34 4- 0.02 0.70*** • 0.51"* •
3.28 =t=0.14 3.79 ~=0.14 3.41 =1=0.44
9.5 • 0.4 15.3~ 4- 1.7 13.3a 4-1.1
44 4-2 75* 6 63* • 3
519 4-9 509 ~7 550***+ • 10
7.9 !0.5 14.7~ 4-i.I 10.2a 4- 0.3
3.75 60 Adrenaline qnoradrenMine
0 3.75 60
Significantly different from
0 rain, P = b 3.75 min, P * 0 rain, P < ** 0 rain, P < *** 0 rain, P < ***+ 3.75 rain, P
all-labelled amine, fmoles/nmole total
S
P
--
--
71 =~15 44 4-1
72 • 56 4-5
--
--
0.11 ~0.01 0.44 b :L 0.08
0.12 i0.02 0.53 b 4- 0.05
0.014 = 0.014 0.005 0.01 0.025 < 0.025
SIP
1.01 • 0.20 0.80 4- 0.08
1.00
4- 0.09 0.80 4- O.O7
Mann-Whitney U-test Student's t-test
were n o t immobilized. Subcellular f r a e t i o n a t i o n of t h e adrenals was performed as described i n m e t h o d s a n d D A a n d A + NA, labelled a n d unlabelled, were d e t e r m i n e d i n t h e soluble a n d p a r t i c u l a t e fractions. I n t h e adrenals of u n t r e a t e d a n i m a l s t h e a m o u n t of D A recovered from t h e s u p e r n a t a n t fraction was a b o u t 1 0 % of t h e t o t a l a m o u n t recovered from t h e s u p e r n a t a n t a n d p a r t i c u l a t e fractions (Table 5). The corresponding figure for A + N A t e n d e d to be slightly lower, i.e. 8 ~ I n t h e adrenals of a n i m a l s receiving H-I-tyrosine 3.75 or 60 m i n before death, t h e a m o u n t s of u n l a b e l l e d D A a n d A + N A recovered from the s u p e r n a t a n t fraction were significantly elevated as compared to u n t r e a t e d control animals, whereas t h e D A a n d A + N A of the p a r t i c u l a t e fraction did n o t d e v i a t e significantly from c o n t r o l values. T h e specific activities of aH-DA were t h e same i n t h e s u p e r n a t a n t a n d p a r t i c u l a t e fractions as early as 3.75 rain after t h e i n j e c t i o n of 3tI-tyrosine. This was t r u e also of A + NA, e v e n t h o u g h t h e specific
8
B. Waldeck et al.
activities of the fl-hydroxylated catecholamines were much lower at the time intervals investigated. Discussion The approximately plateau-shaped specific activity curve of 3~-DA between 1 and 15 min after the injection of aH-tyrosine, its crossing the plasma 3H-tyrosine specific activity curve taking place during the latter part of this period, is not exactlyin agreement with a precursor-product relationship. I t should be emphasized that the specific activities for adrenomedullary 8H-tyrosine and 3H-Dopa, which were not measured, must show a (presumably slight) time lag in relation to the plasma 3H-tyrosine curve, and thus their crossings the aH-DA curve will take place even later, leading, if anything, to a certain further deviation from the ideal precursor-product relationship. The question arises whether this deviation is real or artifactual. One possible explanation would be the presence of traces of ~H-Dopa in the 3H-tyrosine (cf. Waldeck, 1971). Radiopaper chromatography indicated a contamination by 1 ~ aH-Dopa or less. This would have influenced the result by at most 40/0. Moreover, when the tyrosine hydroxylase was inhibited the specific activity of aH-DA 3.75 rain after the administration of aH-tyrosine was less than 10~ of the control value.
Another source of error might arise from a deviation from steady-state conditions. This possibility cannot be excluded. Even though care was taken to handle the animals gently, a brief immobilisation of the rats for the intravenous injection of aH-tyrosine could not be avoided. A moderate increase in DA and fl-hydroxylated catecholamines in the supernatant fraction was seen 3.75 and 60 rain after the injection of 3H-tyrosine without any concomitant change in the catecholamines of the particulate fraction. We cannot exclude the possibility that this change was induced by a brief neurogenic stimulation of the adrenal medulla. I f this is so, the effect is different from that of a more prolonged stimulation: a marked increase in total DA (Snider and Carlsson, 1972) without any significant change in subcellular distribution (unpubhshed data obtained after stimulation by means of insulin or physostigmine). Another sign of deviation from steady-state conditions was the decrease in plasma tyrosine level. This decrease appeared to be greater than that which can be explained by diurnal variations (cf. Zigmond and Wurtman, 1970). A third possibility would be that adrenomedullary DA does not exist in one homogeneous pool. In support of this it was found t h a t the major fraction of the adrenal DA occurred in a particulate fraction and that
Studies on the Synthesis and Subcellular Distribution incorporation into this fraction took place rapidly. Thus we have to consider the possibility t h a t DA exists in at least two pools in the adrenomedullary cells, a small pool in the cytoplasm where dopa decarboxylase is believed to occur and a large granular pool. The 3H-DA curve would then be composed of at least two different curves, one of which would equilibrate very rapidly with the plasma 3H-tyrosine specific activity. From the 15th rain after the injection of 3tt-tyrosine onwards, aH-DA disappeared with an apparent half life of 90 rain. This figure is in agreem e n t with the half life of adrenal DA following synthesis inhibition (Snider and Carlsson, 1972). These data do not exclude the existence of a small DA pool with a shorter half life. The radioactivity of the fl-hydroxylated catecholamines (A ~-NA) increased much more slowly t h a n t h a t of DA. I t should be possible to calculate the rate of synthesis of the fi-hydroxylated catecholamines from the activity curves ofDA as well usA-t- NA, provided t h a t 1. steadystate conditions prevailed, 2. DA existed in one homogeneous pool, and 3. initial losses of A ~- NA, e.g. through release, could be neglected. As a criterion of acceptable conditions the cMculated synthesis rate should be independent of the time interval chosen for the calculation. When this approach was tried, however, the calculated synthesis rate was found to decrease with increasing time intervals, indicating t h a t at least one of the criteria mentioned above was not fulfilled. Nevertheless, it is interesting to note t h a t the values obtained at the earlier intervals (see Table 2) were somewhat higher and those obtained at the longer intervals were somewhat lower t h a n those obtained b y Carlsson et al. (1973b), basing their calculation on the monoexponential decline of DA after t r e a t m e n t with cr Acknowledgements. This study has been supported by grants from the Swedish Medical Research Council (04X-155). For generous supply of tI 22/54 and tI 44/68 we thank AB tti~ssle, MSlndal. The excellent technical assistance by Mrs. Lena LSfberg is gratefully acknowledged. References Atack, C. V.: The determination of dopamine by a modification of the dihydroxyin. dole fluorimetric assay. Brit. J. Pharmacol. 48, 699--714 (1973) Atack, C. V., Magnusson, T,: Individual elution of noradrenaline (together with adrenaline), dopamine, 5-hydroxy~ryptamine and histamine from a single, strong cation exchange column, by means of mineral acid-organic solvent mixtures. J. Pharm. Pharmacol. 22, 625--627 (1970) Carlsson, A., Hillarp, N.-A, WMdeck, B.: Analysis of the Mg++-ATP dependent storage mechanism in the amine granules of the adrenal medulla. Acta physiol. stand. 59, Suppl. 215 (1963) Carlsson, A., Magnusson, T., Svensson, T. It., Waldeck, B. : Effect of ethanol on the metabolism of brain cateeholamines. Psychopharmacologia (Bert.) 80, 27--36 (1973a)
10
:B. Waldeek et al.
Carlsson, A., Snider, S. R., Almgren, 0., Lindqvist, M. : The neurogenic short-term control of catecholamine synthesis and release in the sympatho-adrenal system, as reflected in the levels of endogenous dopamine and fl-hydoxylated catecholamines. In: Frontiers in Catecholamine Research, Proceedings of the Third International Catecholamine Symposium, Strasbourg, E. Usdin and S. Snyder, eds. New York" Pergamon Press, 1973b Eade, N. : The distribution of the catecholamines in homogenates of the bovine adrenal medulla. J. Physiol. (Lond.) 141, 183--192 (1958) ttempe], K., M/innl, F. K. : •ber die Bildung yon tt-3-Dopa aus H-3-Tyrosin und die Bestimmung der Dopa-5leubildungsrate in der Nebenniere des Huhnes und der Katze unter in vivo-Bedingungen. Naunyn-Schmiedebergs Arch. Pharmak. 257, 391--408 (1967) Hillarp, N.-)~.: Isolation and some biochemical properties of the catecholamine granules in the cow adrenal medulla. Acta physiol, scand. 48, 82--96 (1958) Lishajko, F. : Occurrence and some properties of dopamine containing granules in the sheep adrenal. Acts physiol, scand. 72, 255--256 (1968) Munro, H. N.: Control of plasm~ amino acid concentrations. In: Aromatic amino acids in the brain, Ciba Foundation Symposium 22, pp. 5--18. G.E.W. Wolstenhohne and D.W. Fitzsimons, eds. Amsterdam: Associated Scientific Publishers 1974 Persson, T., Waldeck, B. : The use of 3H-Dopa for studying cerebral catecholamine metabolism. Acta pharmacol. (Kbh.) 26, 363--372 (1968) Persson, T., Waldeck, ]3." Some problems encountered in attempting to estimate catecholamine turnover using labelled tyrosine. J. Pharm. 1)harmacol. 22, 473--478 (1970) Snider, S. R., Carlsson, A. : The adrenal dopamine as an indicator of adrenomedulI~ry hormone biosynthesis. N~unyn-Schmiedeberg's Arch. Pharmacol. ~75, 347--357 (1972) Udcnfl-iend, S., Cooper, J. R., Clark, C. T., Baer, J. E.: Rate of turnover of epinephrine in the adrenal medulla. Science 117, 663--665 (1953) Waalkes, T. P., Udenfriend, S. : A fluorimetric method for the estimation of tyrosine in plasma and tissue. J. Lab. clin. Med. 50, 733--736 (1957) Waldeck, B. : Preparation of aqueous samples for liquid scintillation counting with the aid of freeze-drying. Acta physiol, stand. 78, 9--10 A (1968) Waldeck, B.: 3H-Dopa in aH-tyrosine with high specific activity: A serious complication in the study of catecholamine metabolism. J. Pharm. Pharmacol. 28, 64--65 (1971) Waldeck, ]3. : On the use of ethanol as a scavenger in storing labelled Dopa and 5-hydroxytryptamine in aqueous solutions, d. Pharm. Pharmacol. 25, 502--503 (1973) Zigmond, M. J., Wurtman, R. J. : Daily rhythm in the accumulation of brain catecholamines synthesized from circulating 3tt-tyrosine. d. Pharmacol. exp. Ther. 172, 416--422 (1970)
Studies on the Synthesis and Subeellular Distribution of Dopamine in the Rat Adrenal Medulla B e r t i l W a l d e c k , S t u a r t g . S n i d e r * , R o g e r B r o w n * * , a n d A r v i d Carlsson Department of Pharmacology, University of G6teborg Received September 23/Accepted December 5, 1974
Summary. Rats received intravenous injections of aH-tyrosine and were killed at various time intervals thereafter, aH-dopamine (DA) in the adrenals reached a maximum within 1.5 rain after the administration of 3H-tyrosine. From the 15th rain it disappeared with an apparent half life of 90 rain. 3H-Adrenaline (A) ~H-noradrenatine(NA) increased much more slowly and reached a plateau 120--240rain after the injection. The approximate synthesis rate of adrenal A-t-NA, calculated from the specific activity curves, ranged from 9.3 to 2.2 nmoles/ h per kg b.w. The highest value was noted the frrs~ few minutes, the lowest 1--2 hrs after the administration of 3H-tyrosine. In some experiments subcellular fractionation of the adrenals was performed. In untreated animals the amount of DA and A-l-HA recovered from the supernatant fraction was about 10 and 8 per cent, respectively, of the total amount recovered from the supernatant and particulate fractions. In the adrenals of animals receiving aH-tyrosine 3.75 or 60 rain beforehand these figures were significantly elevated whereas the DA and A-t-HA of the particulate fraction did not deviate significantly from control values. The specific activities of aH-DA were the same in the supernatant and particulate fractions within 3.75 rain after the injection of 3H-tyrosine. Key words: Dopamine -- Adrenomedullary Hormones -- Synthesis -- Rat Adrenals -- Subcellular Distribution. I n earlier studies we h a v e p r e s e n t e d evidence s u p p o r t i n g t h e v i e w t h a t in t h e r a t a d r e n a l m e d u l l a d o p a m i n e (DA) m a i n l y , if n o t e n t i r e l y , serves as a n i n t e r m e d i a t e in t h e s y n t h e s i s o f n o r a d r e n a l i n e (NA) a n d a d r e n a l i n e (A) (Snider a n d Carlsson, 1972; Carlsson et al., 1973b). I n t h e s e s studies, using t h e r a t e of d i s a p p e a r a n c e of D A a f t e r i n h i b i t i o n of t y r o s i n e h y d r o x y l a s e , t h e s y n t h e s i s r a t e o f a d r e n a l D A was e s t i m a t e d t o a b o u t 3 n m o l e s / h - kg -1. This figure is in g o o d a g r e e m e n t w i t h t h e t u r n o v e r r a t e which can be c a l c u l a t e d f r o m t h e d e c a y curve of r a d i o a c t i v e A in t h e r a t a d r e n a l s ( U d e n f r i e n d et al., 1953). F r o m t h e a c c u m u l a t i o n of 3 H - D o p a a f t e r a d m i n i s t r a t i o n of 3 t I - t y r o s i n e d u r i n g i n h i b i t i o n o f D o p a d e e a r b o x y l a s e , H e m p e l a n d M/~nnl (1967) c a l c u l a t e d a s y n t h e s i s r a t e for D o p a in t h e cat a d r e n a l s of a b o u t 0.02 ~xg/
Send oHprint requests to: B. Waldeck, AB Draeo, Fack S-221 01 Ltmd, Sweden. * Present address: Neurological Institute, 710 West 168th Street, New York, N. Y. 10032, U.S.A. ** Present address: National Institutes of Mental Health, Neuropsychology, Building 9, room 1 N 107, 9000 Rockville Pike, Bethesda, Maryland 20014, U.S.A. 1
Naun~m-Schmiedeberg's Arch. Pharmacol., Vol. 287
2
B. Waldeck et al.
r a i n . k g -1 (6 n m o l e s / h 9 kg-1). A f t e r a n i n t r a v e n o u s i n j e c t i o n of SH-tyrosine t o r a t , 3 H - ( A ~ - N A ) a p p e a r e d to a c c u m u l a t e for m o r e t h a n 2 hrs (Persson a n d W a l d e c k , 1970). H o w e v e r , a d e t a i l e d s t u d y on t h e d y n a m i c s of t h e a d r e n a l c a t e c h o l a m i n e s y n t h e s i s has so f a r n o t been p r e s e n t e d . V e r y few s t u d i e s h a v e so far b e e n p e r f o r m e d on t h e subcellular d i s t r i b u t i o n o f a d r e n a l DA. E a d e (1958) r e p o r t e d t h a t in t h e b o v i n e a d r e n a l m e d u l l a a b o u t t w o - t h i r d s of t h e D A was r e c o v e r e d f r o m t h e " l a r g e - g r a n u l e " fraction, w i t h m o s t of t h e r e m a i n d e r in t h e s u p e r n a t a n t . T h e d i s t r i b u t i o n was v e r y similar to t h a t of t h e t o t a l c a t e c h o l a m i n e s , of w h i c h D A c o n s t i t u t e d a b o u t 1 ~ I n t h e sheep a d r e n a l L i s h a j k o (1968) made similar observations. I n e x p e r i m e n t s on i s o l a t e d b o v i n e a d r e n o m e d u l l a r y granules Carlsson et al. (1963) o b s e r v e d t h a t D A , like H A a n d A, is t a k e n u p b y t h e granules b y a n A T P - M g 2 + - d e p e n d e n t m e c h a n i s m , which can be b l o c k e d b y reserpine. I n vivo, r e s e r p i n e has been f o u n d t o cause a r a p i d decrease o f D A in r a t a d r e n a l s (Snider a n d Carlsson, 1972). I n t h e p r e s e n t i n v e s t i g a t i o n t h e s y n t h e s i s of a d r e n o m e d u l l a r y c a t e c h o l a m i n e s , p a r t i c u l a r l y D A , h a s b e e n s t u d i e d following a pulse i n j e c t i o n of 3H-tyrosine. I n some e x p e r i m e n t s t h e subcellular d i s t r i b u t i o n of b o t h u n l a b e l l e d a n d l a b e l l e d c a t e c h o l a m i n e s was i n v e s t i g a t e d in o r d e r t o g a i n i n s i g h t i n t o t h e d y n a m i c s of t h e s t o r a g e m e c h a n i s m .
Material and Methods Male Sprague-Dawley rats, weighing about 250 g, were used. They were kept in the animal quarters for at least 48 hrs before use and allowed food pellets and water ad libitum. Experimental animals were handled as carefully as possible. In order to minimize the influence of diurnal variations all experiments were performed between 8 and 11 a.m. Intravenous injections were given into a tail vein with the animals briefly immobilized in a small cylindrical cage. Other injections were given intraperiteneally. Following drug treatment, rats were sacrificed by means of decapitation or concentrated chloroform vapor. Comparison between decapitation and chloroform revealed no significant difference in amine values. The adrenals were rapidly removed and extracted in 0.4 N perehlorie acid. In experiments where the subeellular distribution was studied, the adrenals were placed directly in 3 ml of 0.3 M sucrose at 0--4~ and homogenized with a teflon pestle in a glass homogenizer (for details see Itillarp, 1958). The pestle speed and homogenization procedure were the same in all experiments. The homogenate was centrifuged at 4~ and 20000 • g for 20 min. The supernatant and sediment were separated, and perehloric acid, 10~ EDTA, and 5 % ~a2S205 were added to each so that a final volume of 10.3 ml of 0.4 N perehloric acid, 0.2~ EDTA, and 0.05~ ~a2S205 resulted. After homogenization the protein precipitate was removed by centrifugation. Separation of unlabelled amines from the extracts was performed according to the method of Atack and Magnusson (1970). DA was assayed spectrofluorimetrically (Ataek 1973), and the A + N A was determined by native fluorescence against a hTA standard. Labelled amines were separated from the perchlorie acid extracts by a combination of alumina and Dowex 50 columns (Persson and Waldeck, 1968).
Studies on the Synthesis and Subcellular Distribution The 3H-content of each fraction was determined in a liquid scintillation counter after freeze-drying (Waldeck 1968). In some experiments blood was collected in plastic tubes containing ~n EDTA solution, after which the specific activity of 3H-tyrosine in plasma was determined (Carlsson et al., 1973a), tyrosine being determined according to Waalkes and Udenfriend (1957). Further experimental details are given in the text. The following drugs were used: a-propyldopacetamide (H22/54), and cr tyrosine methylester (H44/68) (AB H~ssle, ~Slndal), L-tyrosine, ring-3,5aH, specific activity about 47 Ci/mmole, and L-Dopa, ring-2,5,63H, specific activity, 1 Ci/mmole (The Radiochemical Centre, Amersham). The radiochemical purity was regularly checked by radiopaper chromatography in isopropanol/2N tiC1, 65/35, v/v. No 3H-Dopa was detected in the att-tyrosine, i.e. it contributed with less than about 1 ~ of the total activity. The 3H-Dopa contained some 5 % radioactive impurities, presumably condensation products with acetaldehyde (Waldeck, 1973). Since the specific activity of the all-labelled precursors were different and administration was made on a weight basis (5 ~g/kg b.w.) the specific activity of plasma tyrosine and DA and A + N A in the adrenals have been expressed as fmoles of substance derived from the labelled precursor per nmole total of the corresponding compound. When aH-tyrosine was used, corrections were made for the loss of one atom ~H per molecule in the calculations of aH-catecholamine specific activity. Results
A. Accumulation o/aH-Catecholamines in the Adrenals Following Injection o/aH-Tyrosine R a t s r e c e i v e d i n t r a v e n o u s injections of 3H-tyrosine, 5 ~g/kg, a n d were killed a t v a r i o u s t i m e i n t e r v a l s t h e r e a f t e r . L a b e l l e d a n d u n l a b e l l e d D A a n d A ~ ~ A in t h e a d r e n a l s a n d t y r o s i n e in t h e p l a s m a were determined.
Table 1. Tyrosine in plasma and catecholamines in the ~drenals at various time intervals after the intravenous injection of a trace amount of aH-tyrosine (el. Fig. 1). Shown are the means, s.e.m, and the number of experiments Time intervals in minutes
Tyrosine in plasma /~g/ml Dopamine in adrenals nmoles/kg b.w. Adrenaline ~noradrenaline in adrenals nmoles/kg b.w. 1"
1.5
3.75
7.5
15
30
60
120
240
16.8 • 1.63 (3) 5.15 J= 0.25 (3) 575 ~ 50 (3)
t9.4 • 0.68 (7) 6.50 • 0.75 (7) 590 • 65 (6)
20.6 =]=1.81 (6) 6.45 =~ 0.65 (6) 655 =~ 55 (6)
16.7 • 0.69 (5) 6.35 • 0.85 (7) 575 • 45 (7)
16.6 =L1.72 (4) 6.40 =~ 0.95 (6) 610 i 80 (6)
13.6 =~ 2.01 (5) 5.90 ~ 0.70 (7) 605 • 100 (6)
11.9 i 1.43 (4) 5.65 :t= 0.35 (6) 530 • 45 (6)
18.4 • 1.56 (6) 6.85 • 0.85 (6) 555 • 65 (6)
B. Waldeck et al. 367•
ILl r Z
N 2o0. (/1 m
(,D
p,.
150.
"6 100. ~100 m
< "~
50.
"6 E
.e
0"
15 ' 30 '
6"0
120
f,, 24O
180 rain.
:Fig. 1. Accumulation of ~H-catecholamines in the adrenals following injection of stI-tyrosine. Rats received intravenous injections of 8It-tyrosine, 5 ~zg/kg, and were killed at various time intervals thereafter. Shown are the specific activities of tyrosine in plasma o, and dopamine., and adrenaline-knoradrenaline . in the adrenals expressed as fmoles material derived from ~H-tyrosine per nmole total of the respective substance. The data are the means ~= s.e.m, of 3--7 rats analyzed individually (see Table 1). The adrenaline-~-noradrenaline values have been multiplied by 100 There were no significant changes in the levels of endogenous DA and A + NA in the adrenals (Table 1). From 7.5 to 120 rain after the injection, plasma tyrosine decreased from 20.6 to 11.9 ~zg/ml (P < 0.001), which appears to be below the range of normal values given in the literature (Zigmond and Wurtman, 1970; cf. also Munro, 1974). I t then returned to 18.4 ~zg/ml during the next 120 min (P < 0.005). This value was not significantly different from the values observed 1.5 to 7.5 rain after the injection of labelled tyrosine. The specific activities of tyrosine, DA and A + NA are shown in Fig. 1. The time course of plasma tyrosine specific activity appeared to be polyphasic as revealed b y a semilogarithmic plot. The specific activity of DA in the adrenals reached a m a x i m u m value within 1.5 rain after the administration of 3H-tyrosine. I t remained, with slight variation, at this level for the next 14 min while crossing the plasma 3tt-tyrosine specific activity curve (about 12 min after injection), and then started to decrease with an apparent half-life of 90 min. The DA curve, as it appears in Fig. l, suggests the existence of 2 peaks, 1 at 1.5 rain and 1 at 15 min. However, more data are necessary to establish their existence. The specific activity of A + NA increased much more
Studies on the Synthesis and Subcellul~r Distribution
5
slowly and appeared to approach a plateau 120 to 240 rain after the injection. After 240 rain the specific activity of DA was still 18 times higher than that of A + NA. Since the A Jr NA curve is magnified 100 • and the endogenous level of these amines is about 100 times that of DA (Table 1), Fig. i also shows e.g. the approximate time at which the amount of 3tt-(A + HA) equals that of 3H-DA. This occurs about 70 rain after the administration of the precursor. An attempt was made to calculate the rate of adrenal eatecholamine synthesis from the activity curves according to the equation A +HA=
3H- (A + NA) • D A ~H- DA
where A + 57A stands for the total amount newly formed A + NA during a given time interval, 3H-(A + HA) for the amount formed from 3H-tyrosine during the same time interval, and aH-DA/DA for the corresponding specific activity of DA (for details see e.g. Hempel and Ms 1967). Table 2 shows the result. The estimated synthesis rate appeared to be dependent on the time interval chosen for calculation, resulting in considerably higher values at early as compared with late time intervals. Table 2. Synthesis rate of adrenal adrenaline (A) + noradrenaline (NA) calculated from the specific activity curves in Fig. 1 (see text) Time interval in minu~es A +NA nmoles/h per kg b.w.
3.75--7.5 7.5--15
15--30
30--60
60--120
120--240
9.3
3.6
3.6
2.2
2.6
5.7
B. E]/ect o/Tyrosine-Hydroxylase Inhibitors on the Accumulation o/3H-Catecholamines in the Adrenals Following Injection o/3H-Tyrosine In other experiments H22/54, 500 mg/kg, was given 15 rain or It44/68, 400 mg/kg, 30 rain before the i.v. administration of 5 ~zg/kg aH-tyrosine. The animals were killed 3.75 rain after the injection. The specific activity of DA and A + NA in the adrenals are shown in Table 3 together with 3.75 mid values from Fig. 1 (controls). The specific activity of DA was less than 10 ~ of the control value. The level of radioactivity in the A + NA fraction was insignificant and very close to the background, in animals given a synthesis inhibitor. In contrast, aH-(A + NA) activity in the corresponding controls was about 7 times the background level.
B. Waldeck et al.
6
Table 3. Effect of tyrosine hydroxylase inhibitors on the accumulation of aH-catecholamines in the adrenals as measured 3.75 rain after the intravenous administration of 5 ~g/kg 3H-tyrosine. ~-Propyldopacetamide (H22/54), 500 mg/kg, or a-methyltyrosine methylester (H44/68), 400 mg/kg, was given 15 and 30 min, respectively, beforehand. The result, expressed as fmoles aH-amine/nmole total of the corresponding amine, is shown together with that obtained at the same time interval without drug treatment (control, from Fig. 1). Shown are the mean 4- s.e.m. and the number of experiments Control
H22/54
H44/68
3H-Dopamine 78 4- 1.4(7) 4.4 4- 0.5(2) 7.4 4- 0.1(2) aH-(Adrenaline-[-noradrenaline) 0.13 ~ 0.014(7) 0.03 =~ 0.026(2) 0.01 =[: 0.003(2)
C. Accumulation o/8H-Catecholamines in the Adrenals Following Injection o/3H-Dopa 3H-Dopa, 5 ~g/kg, was g i v e n i n t r a v e n o u s l y to rats. T h e animals were killed 15 or 120 rain later. ~H-DA a n d 3H-(A -[- NA), b u t n o t t h e e n d o g e n o u s levels of t h e s e amines, were m e a s u r e d . Specific activities were t h e n c a l c u l a t e d on t h e basis o f t h e endogenous levels shown in T a b l e 1. T h e result, t o g e t h e r w i t h t h e v a l u e s o b t a i n e d a t t h e corresponding t i m e i n t e r v a l s a f t e r ~H-tyrosine (from Fig. 1), is shown in T a b l e 4. T h e n e t y i e l d o f 3 H - c a t e c h o l a m i n e s f r o m 8H-Dopa was 2 - - 4 t i m e s higher t h a n t h a t o b t a i n e d f r o m a n e q u i m o l a r dose of 3H-tyrosine. Thus, 1 ~ c o n t a m i n a t i o n of 3H-tyrosine w i t h a I t - D o p a will influence t h e r e s u l t by at most 2--4 ~
Table 4. Accumulation of aH-catecholamines in the adrenals following the intravenous injection of 5 ~zg/kg 3H-Dopa or 3H-tyrosine. The animals were killed 15 or 120 min after the injection. The result is presented as fmoles aH-amine/nmole total of the corresponding amine. Shown are the means • s.e.m, and the number of expriments 15 min 8H'D~ amine
/ from ( from all-(Adrenaline 4- / from noradrenaline) / from
3H-Dopa aH-tyrosine a 3H-Dopa 3H-tyrosinea
217 • 83(4) 88 4- 9(7) 0.60 4- 0.17(4) 0.30 4- 0.04(7)
120 min 122 4- 7(4) 33 =[=4(6) 1.49 4- 0.43(4) 0.87 4- 0.08(6)
a Data from Fig. 1.
D. Subcellular Distribution o/Labelled and Unlabelled Catecholamines in the Adrenals at Various Time Intervals Following Injection of 3H-Tyrosine R a t s r e c e i v e d i n t r a v e n o u s injections of 3H-tyrosine, 5 ~zg/kg, a n d were killed 3.75 or 60 m i n later. Control a n i m a l s r e c e i v e d no injections a n d
Studies on the Synthesis and Subcellular Distribution Table 5. Subceltular distribution of unlabelled and labelled eateeholamines in the adrenals at various time intervals after injection of 5 y.g/kg ~H-tyrosine. Control animals (0 rain) received no injections. Shown are the means • s.e.m, of 4 experimental groups, each comprising 2 rats. S ----supernatant, P = particulate fraction Unlabelled amine, nmoles/kg
Dopamine
rain
S
P
S N+-----P • 100
0
0.34 4- 0.02 0.70*** • 0.51"* •
3.28 =t=0.14 3.79 ~=0.14 3.41 =1=0.44
9.5 • 0.4 15.3~ 4- 1.7 13.3a 4-1.1
44 4-2 75* 6 63* • 3
519 4-9 509 ~7 550***+ • 10
7.9 !0.5 14.7~ 4-i.I 10.2a 4- 0.3
3.75 60 Adrenaline qnoradrenMine
0 3.75 60
Significantly different from
0 rain, P = b 3.75 min, P * 0 rain, P < ** 0 rain, P < *** 0 rain, P < ***+ 3.75 rain, P
all-labelled amine, fmoles/nmole total
S
P
--
--
71 =~15 44 4-1
72 • 56 4-5
--
--
0.11 ~0.01 0.44 b :L 0.08
0.12 i0.02 0.53 b 4- 0.05
0.014 = 0.014 0.005 0.01 0.025 < 0.025
SIP
1.01 • 0.20 0.80 4- 0.08
1.00
4- 0.09 0.80 4- O.O7
Mann-Whitney U-test Student's t-test
were n o t immobilized. Subcellular f r a e t i o n a t i o n of t h e adrenals was performed as described i n m e t h o d s a n d D A a n d A + NA, labelled a n d unlabelled, were d e t e r m i n e d i n t h e soluble a n d p a r t i c u l a t e fractions. I n t h e adrenals of u n t r e a t e d a n i m a l s t h e a m o u n t of D A recovered from t h e s u p e r n a t a n t fraction was a b o u t 1 0 % of t h e t o t a l a m o u n t recovered from t h e s u p e r n a t a n t a n d p a r t i c u l a t e fractions (Table 5). The corresponding figure for A + N A t e n d e d to be slightly lower, i.e. 8 ~ I n t h e adrenals of a n i m a l s receiving H-I-tyrosine 3.75 or 60 m i n before death, t h e a m o u n t s of u n l a b e l l e d D A a n d A + N A recovered from the s u p e r n a t a n t fraction were significantly elevated as compared to u n t r e a t e d control animals, whereas t h e D A a n d A + N A of the p a r t i c u l a t e fraction did n o t d e v i a t e significantly from c o n t r o l values. T h e specific activities of aH-DA were t h e same i n t h e s u p e r n a t a n t a n d p a r t i c u l a t e fractions as early as 3.75 rain after t h e i n j e c t i o n of 3tI-tyrosine. This was t r u e also of A + NA, e v e n t h o u g h t h e specific
8
B. Waldeck et al.
activities of the fl-hydroxylated catecholamines were much lower at the time intervals investigated. Discussion The approximately plateau-shaped specific activity curve of 3~-DA between 1 and 15 min after the injection of aH-tyrosine, its crossing the plasma 3H-tyrosine specific activity curve taking place during the latter part of this period, is not exactlyin agreement with a precursor-product relationship. I t should be emphasized that the specific activities for adrenomedullary 8H-tyrosine and 3H-Dopa, which were not measured, must show a (presumably slight) time lag in relation to the plasma 3H-tyrosine curve, and thus their crossings the aH-DA curve will take place even later, leading, if anything, to a certain further deviation from the ideal precursor-product relationship. The question arises whether this deviation is real or artifactual. One possible explanation would be the presence of traces of ~H-Dopa in the 3H-tyrosine (cf. Waldeck, 1971). Radiopaper chromatography indicated a contamination by 1 ~ aH-Dopa or less. This would have influenced the result by at most 40/0. Moreover, when the tyrosine hydroxylase was inhibited the specific activity of aH-DA 3.75 rain after the administration of aH-tyrosine was less than 10~ of the control value.
Another source of error might arise from a deviation from steady-state conditions. This possibility cannot be excluded. Even though care was taken to handle the animals gently, a brief immobilisation of the rats for the intravenous injection of aH-tyrosine could not be avoided. A moderate increase in DA and fl-hydroxylated catecholamines in the supernatant fraction was seen 3.75 and 60 rain after the injection of 3H-tyrosine without any concomitant change in the catecholamines of the particulate fraction. We cannot exclude the possibility that this change was induced by a brief neurogenic stimulation of the adrenal medulla. I f this is so, the effect is different from that of a more prolonged stimulation: a marked increase in total DA (Snider and Carlsson, 1972) without any significant change in subcellular distribution (unpubhshed data obtained after stimulation by means of insulin or physostigmine). Another sign of deviation from steady-state conditions was the decrease in plasma tyrosine level. This decrease appeared to be greater than that which can be explained by diurnal variations (cf. Zigmond and Wurtman, 1970). A third possibility would be that adrenomedullary DA does not exist in one homogeneous pool. In support of this it was found t h a t the major fraction of the adrenal DA occurred in a particulate fraction and that
Studies on the Synthesis and Subcellular Distribution incorporation into this fraction took place rapidly. Thus we have to consider the possibility t h a t DA exists in at least two pools in the adrenomedullary cells, a small pool in the cytoplasm where dopa decarboxylase is believed to occur and a large granular pool. The 3H-DA curve would then be composed of at least two different curves, one of which would equilibrate very rapidly with the plasma 3H-tyrosine specific activity. From the 15th rain after the injection of 3tt-tyrosine onwards, aH-DA disappeared with an apparent half life of 90 rain. This figure is in agreem e n t with the half life of adrenal DA following synthesis inhibition (Snider and Carlsson, 1972). These data do not exclude the existence of a small DA pool with a shorter half life. The radioactivity of the fl-hydroxylated catecholamines (A ~-NA) increased much more slowly t h a n t h a t of DA. I t should be possible to calculate the rate of synthesis of the fi-hydroxylated catecholamines from the activity curves ofDA as well usA-t- NA, provided t h a t 1. steadystate conditions prevailed, 2. DA existed in one homogeneous pool, and 3. initial losses of A ~- NA, e.g. through release, could be neglected. As a criterion of acceptable conditions the cMculated synthesis rate should be independent of the time interval chosen for the calculation. When this approach was tried, however, the calculated synthesis rate was found to decrease with increasing time intervals, indicating t h a t at least one of the criteria mentioned above was not fulfilled. Nevertheless, it is interesting to note t h a t the values obtained at the earlier intervals (see Table 2) were somewhat higher and those obtained at the longer intervals were somewhat lower t h a n those obtained b y Carlsson et al. (1973b), basing their calculation on the monoexponential decline of DA after t r e a t m e n t with cr Acknowledgements. This study has been supported by grants from the Swedish Medical Research Council (04X-155). For generous supply of tI 22/54 and tI 44/68 we thank AB tti~ssle, MSlndal. The excellent technical assistance by Mrs. Lena LSfberg is gratefully acknowledged. References Atack, C. V.: The determination of dopamine by a modification of the dihydroxyin. dole fluorimetric assay. Brit. J. Pharmacol. 48, 699--714 (1973) Atack, C. V., Magnusson, T,: Individual elution of noradrenaline (together with adrenaline), dopamine, 5-hydroxy~ryptamine and histamine from a single, strong cation exchange column, by means of mineral acid-organic solvent mixtures. J. Pharm. Pharmacol. 22, 625--627 (1970) Carlsson, A., Hillarp, N.-A, WMdeck, B.: Analysis of the Mg++-ATP dependent storage mechanism in the amine granules of the adrenal medulla. Acta physiol. stand. 59, Suppl. 215 (1963) Carlsson, A., Magnusson, T., Svensson, T. It., Waldeck, B. : Effect of ethanol on the metabolism of brain cateeholamines. Psychopharmacologia (Bert.) 80, 27--36 (1973a)
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Carlsson, A., Snider, S. R., Almgren, 0., Lindqvist, M. : The neurogenic short-term control of catecholamine synthesis and release in the sympatho-adrenal system, as reflected in the levels of endogenous dopamine and fl-hydoxylated catecholamines. In: Frontiers in Catecholamine Research, Proceedings of the Third International Catecholamine Symposium, Strasbourg, E. Usdin and S. Snyder, eds. New York" Pergamon Press, 1973b Eade, N. : The distribution of the catecholamines in homogenates of the bovine adrenal medulla. J. Physiol. (Lond.) 141, 183--192 (1958) ttempe], K., M/innl, F. K. : •ber die Bildung yon tt-3-Dopa aus H-3-Tyrosin und die Bestimmung der Dopa-5leubildungsrate in der Nebenniere des Huhnes und der Katze unter in vivo-Bedingungen. Naunyn-Schmiedebergs Arch. Pharmak. 257, 391--408 (1967) Hillarp, N.-)~.: Isolation and some biochemical properties of the catecholamine granules in the cow adrenal medulla. Acta physiol, scand. 48, 82--96 (1958) Lishajko, F. : Occurrence and some properties of dopamine containing granules in the sheep adrenal. Acts physiol, scand. 72, 255--256 (1968) Munro, H. N.: Control of plasm~ amino acid concentrations. In: Aromatic amino acids in the brain, Ciba Foundation Symposium 22, pp. 5--18. G.E.W. Wolstenhohne and D.W. Fitzsimons, eds. Amsterdam: Associated Scientific Publishers 1974 Persson, T., Waldeck, B. : The use of 3H-Dopa for studying cerebral catecholamine metabolism. Acta pharmacol. (Kbh.) 26, 363--372 (1968) Persson, T., Waldeck, ]3." Some problems encountered in attempting to estimate catecholamine turnover using labelled tyrosine. J. Pharm. 1)harmacol. 22, 473--478 (1970) Snider, S. R., Carlsson, A. : The adrenal dopamine as an indicator of adrenomedulI~ry hormone biosynthesis. N~unyn-Schmiedeberg's Arch. Pharmacol. ~75, 347--357 (1972) Udcnfl-iend, S., Cooper, J. R., Clark, C. T., Baer, J. E.: Rate of turnover of epinephrine in the adrenal medulla. Science 117, 663--665 (1953) Waalkes, T. P., Udenfriend, S. : A fluorimetric method for the estimation of tyrosine in plasma and tissue. J. Lab. clin. Med. 50, 733--736 (1957) Waldeck, B. : Preparation of aqueous samples for liquid scintillation counting with the aid of freeze-drying. Acta physiol, stand. 78, 9--10 A (1968) Waldeck, B.: 3H-Dopa in aH-tyrosine with high specific activity: A serious complication in the study of catecholamine metabolism. J. Pharm. Pharmacol. 28, 64--65 (1971) Waldeck, ]3. : On the use of ethanol as a scavenger in storing labelled Dopa and 5-hydroxytryptamine in aqueous solutions, d. Pharm. Pharmacol. 25, 502--503 (1973) Zigmond, M. J., Wurtman, R. J. : Daily rhythm in the accumulation of brain catecholamines synthesized from circulating 3tt-tyrosine. d. Pharmacol. exp. Ther. 172, 416--422 (1970)
