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5-hydrotrtryptamine-O-sulfate: an alternate route of serotonin inactivation in brain
SHORT COMMUNICATIONS
309
5-Hydroxytryptamine-O-sulfate: an alternate route of serotonin inactivation in brain Hidaka et al. 4 have reported an enzyme system, 5-HT-sulfokinase (or sulfotransferase) in rabbit brain capable of sulfation of 5-hydroxytryptamine (5-HT) by the active sulfate of 3'-phosphoadenosine-5'-phosphosulfate (PAPS). It was postulated .~ that the enzymic O-sulfation of cerebral 5-HT may occur in vivo thus serving as a route of inactivation of 5-HT. However, K o r f and Sebens 7 failed to detect any evidence for cerebral sulfation of 5-HT in vivo even in animals pretreated with monoamine oxidase inhibitors. They, nevertheless, did not reject the possibility of the synthesis of cerebral 5-HT-O-sulfate and suggested that it was either produced in undetectably small amounts or that it was rapidly metabolized. Our experiments were designed to obtain information regarding the formation, rate of synthesis, and efflux of cerebral 5-HT-O-sulfate. 5-HT-O-sulfate was synthesized by the chlorosulfonic acid catalyzed sulfonation of 5-HT with 35S concentrated sulfuric acid (22 mCi total activity) and was freed of sulfate by barium carbonate and of 5-HT by passing it through a column of Amberlite CG-50 (H +) or Dowex-50-H ~ (ref. 6). The effluent containing 5-HT-O-sulfate was adjusted to pH 7.0 and passed through a column of Dowex-2. After washing the column with carbonate-free water, the 5-HT-O-sulfate was obtained by applying aliquots as a strip across Whatman No. 1 paper 1 and developing a descending chromatogram in i s o p r o p a n o l : 6 N NH4OH (20:5, v/v) for 18 h. I s o p r o p a n o l : 6 N NH4OH (20:3, v/v) in our hands did not separate 5-HT-O-SO3 from sulfate discreetly enough to exclude contamination from the latter. Elution of the strip corresponding to the 5-HT-O-sulfate marker with water will yield pure labeled 5-HT-O-sulfate (45 #Ci/mg or 1.2 mCi/mmole specific activity). Labeled 5-hydroxyindoleacetic acid (5-HIAA) was prepared from [2-14C]5-HT by enzymic synthesis lz. Male Sprague-Dawley rats (250-350 g) were used throughout the experiments and were fasted overnight. The animals were divided into 4 groups. The first and second group of rats were controls; the third and fourth group of animals received 80 mg/kg pargyline (Abbott Laboratory, Chicago, II1.). Two hours later the animals in the second and fourth groups were administered 200 mg/kg probenecid (Merck, Sharp, and Dohme Research Laboratory, Rahway, N.J.). Both drugs were injected intraperitoneally. At the end of the third hour 10/zl of a solution of 3~S-labeled 5-HTO-sulfate, or [~4C]5-H1AA, was delivered under light ether anesthesia through each of two holes bilaterally placed in the skull I mm from both the sagittal and coronal sutures about the depth of 1 mm into the cortex ~0. The animals were killed I h later; the brains were removed at 4 °C and homogenized separately with 0.1 N HCI. After centrifuging at 15,000 × g for 10 rain at 3 °C precipitates were resuspended in 3 ml 0.1 N HCI and centrifuged again. The supernatants were combined and adjusted to about pH 7.2. Aliquots of 0.2 ml were taken for scintillation counting to measure the total radioactivity. The remainder of the supernatants were passed through a column of Amberlite CG-50 (H +) resin 3. The effluents from the Amberlite columns were passed through Dowex-2 columns. Brain Research, 44 (1972) 309-312
310
SHORT COMMUNICATIONS
The method of recovery of 5-HT-O-sulfate from the brain of experimental animals essentially followed the same method as described above for the purification of synthetic 5-HT-O-sulfate. The radioactivity in the strips corresponding to the markers was measured by cutting them into the vials containing the fluors and Counting them by liquid scintillation. The markers were identified with Ehrlich's reagent ~. sulfate was detected by rhodizonate reagent 14 lntracerebrally administered 5-HT-O-~5SO3 (1.3 × 104 disint./min or 0.13 c~g) is rapidly eliminated from the brain. Pargyline had no effect on its retention, whereas probenecid with pargyline reduced the rate of efltux by 35 ~ . Probenecid is known to arrest the efflux of endogenous 5-HIAAtL Time studies on the efflux of the 5-HTO-sulfate from the brain of control animals yielded a Tt/2 of 18 rain (k~ - 0.037 min-~), lntracerebrally administered 5-HIAA (5.3 x 104 disint./min or 0.32 # g ) h a d a ke of 0.026 min-L However, the rate ofefflux for endogenous 5-HIAA is k .... 0.014 min -~ (ref. IlL It is known that 5-HT-O-sulfate. like 5-HT. is unable to cross the 'blood-brain' barrier t3. Exogenous 5-HT-O-sulfate cannot penetrate the cellular mere branes and. therefore, it remained only in the extracellular space. 5-HT-O-sutfate synthesized within the cells is also removed at a rapid rate. k~ = 0.028 rain -J. My observations do not indicate that 5-HT-O-sulfate is a way of storage for 5-HT. as suggested by others 5. but rather a terminal way of eliminating 5-HT. Next we have examined the synthesis of cerebral 5-HT-O-sutfate in vivo. A group of rats received pargyline and probenecid, as described above, and 1 h after probenecid treatment, intraperitoneal radioactive sodium sulfate (10 mCi/kg). The animals (3 in each group) were killed I. 2 or 3 h after the injection o f laSS]sulfate. Chromatographic analysis of the region corresponding to 5-HT-O-sullate revealed the presence of radioactivity. This area was cut out and eluted. Unlabeled 5-HT-Osulfate was added to this eluate. After lyophilizing the eluate, the residue was taken up in 0.5 ml water. One-half of this solution (Table ID) was rechromatographed. TABLE I RADIOACTIVITY RECOVERED BY DESCENDING CHROMATOGRAPHY IN
5-HT-O-SO3REGION
R~
Total disint, min recovered Experiment
0.02 (su(fatej
A: Authentic a~SO4 = 400,000 disint./min spotted B: Authentic 5-HT-O-a~SO3 5,000 disint./min spotted C: Extract of brain after i.p.
393,000
3~SO 4 D : Spot from 5-HT-O-zsSOz in exp. C: eluted,rechromatographed E: Eluatc from cxp. C: Spot 5-HT-O-asSOs acid hydrolyzcd
0.41 ,5-HIAA)
-
90 610,000
0.48 ~J.70 .~5-HT-O-SOaJ (5-HT)
39 3940
1200
5531
-
2800
-
52
-
Brain Research, 44 (1972) 309-312
300 2,000
60
SHORT COMMUNICATIONS
3l l
while the other half (Table 1E) was submitted to acid hydrolysis with 70 ~ perchloric acidL This technique is only reliable if the supernatant of brain homogenates is lyophilized and the residue dissolved in water to achieve a concentration of 1.0-1.5 #g/ml of 5-HT, Otherwise the analysis of dilute solutions leads to an uncertainty which is larger in magnitude than the expected difference. Following acid hydrolysis most of the radioactivity appeared in the area corresponding to free sulfate (Table 1). These results were obtained from animals sacrificed at the first hour after administration of intraperitoneal sodium [35S]sulfate. The second and third hour values of the radioactivity recovered in the area corresponding to the 5-HT conjugate were 3695 and 3644 disint./min respectively, representing 6-4 ng of 5-HT in the labeled conjugate for the initial 3 h. These values do not reveal the actual pool size of 5-HT conjugate. Further demonstration of the synthesis of 5-HT-O-sulfate was achieved by recovery of the 14C-labeled conjugate from the brain of rats given intravenous DL-[z4C]5-HTP 1 h after their pretreatment with pargyline and probenecid according to the amount and schedule described in the other experiments. The radioactive 5-HT-O-sulfate was isolated and identified by chromatographic analysis while the 5-HT in the conjugate was determined by the difference in the 5-HT values obtained before and after acid hydrolysis7. The amount of 5-HT was assessed through measuring the fluorophore, a product of condensation of 5-HT with O-phthalaldehydes. The results reveal (Table I1) that the recovered 5-HT-O-sulfate represents about 8 ~ of the 5-HT pool in pargyline treated rats. The label in [14C]5-HT-O-sulfatecorresponds to about 4 ng of radioactive 5-HT which is consistent with the value calculated from the 35S-labeled conjugate. Assuming that probenecid completely inhibited the efflux of endogenous 5-HT-O-sulfate, the approximate rate of its synthesis is calculated to be about 25 ng/g/h. In rats treated with pargyline (80 mg/kg) the level of 5-HT/g of brain increased from about 0.8 to 1.6/~g/g between 2 and 24 h. At 24 h the specific activity of 5-HT has decreased to about 5 ~ over the 2-h value9. This represents an efflux of approximately 60 ng/g/h of 5-HT from the brain of pargyline treated animals. This amount of 5-HT stoichiometrically approximates the 5-HT recoverable in the cerebral 5-HTO-SO3 in the pargyline and probenecid pretreated rats. In control animals the conT A B L E 11 SYNTHESIS OF CEREBRAL [~IC]5-HT-O-SOa AFTER I.V. INJECTION OF [ 1 4 C ] 5 - H T P PROBENECID TREATED RATS
Time (h)
1 2
5-HT
IN PARGYLINE AND
5-HT-O-S03
pg/g
disint./min//tg
ng/g
disint./min/l¢g*
0.63 ~ 0.05** 0.81 :k 0.06
3980 2117
50 :k 2** 75 ::k 2
3654 2230
* Calculated as 5-HT. Each value represents an average o f 4 animals in duplicate determination. See text for experimental conditions. ** S.E.
Brain Research, 44 (1972) 309-312
312
SHORT COMMUNICATIONS
v e r s i o n o f 5 - H T to 5 - H I A A is t h e m a j o r r o u t e f o r the r e m o v a l o f 5 - H T since t h e r a t e of synthesis o f 5 - H I A A o u t s t r i p s the s u l f a t i o n o f 5 - H T , D u r i n g t h e i n h i b i t i o n ol" cerebral monoamine
o x i d a s e activity, h o w e v e r , t h e s y n t h e s i s o f 5 - H T - O - S O ~ m a y
b e c o m e a c o n t r o l l i n g m e c h a n i s m f o r the r e m o v a l o f 5 - H T . T h u s . t h e e n z y m a t i c sulr a t i o n o f 5 - H T c o u l d be v i e w e d as a r e g u l a t o r y r e s p o n s e to e l e v a t e d levels o f 5 - H T in t h e brain. T h i s s h o u l d be k e p t in m i n d w h e n d e t e r m i n a t i o n s f o r the c o n c e n t r a t i o n s of various cerebral metabolites of 5-HT are made. T h e s e o b s e r v a t i o n s reveal a n o t h e r m o d a l i t y o f i n t r a c e l l u l a r c o n t r o l o f 5 - H T levels in the brain. Neurochemical Research Laborator.v. Department o f Psychiatry, College o f Medicine, University o f h~wa. Iowa City, Iowa 52240 (U.S.A.,
E. MARTIN GAL
1 AIRAKSlNEr~.M. M., Studies of the metabolism and antidiuretic action of 5-hydroxytryptamine and the effect of the mode of administration, Ann. Med. exp. Biol. Fern., 41, Suppl. 4 (19637 t-91. 2 DAVIS, V. E.. HuEE. J. A., ANO BROWN. H.. Free and conjugated serotonin excretion in carcinoid syndrome, Clin. chin,. Acta, 13 (1966) 390-402. 3 G~L, E. M.. HEATER, R. D., AM3 M1LLARD.S. A., Studies on the metabolism of 5-hydroxytryptamine fserotoninl. VI. Hydroxylation and amines in cold-stressed reserpinised rats. Proc. Soc. exp. Biol. ( N. Y.), 128 (1968) 412-415, 4 HIDAKA. H.. NAGATSU.T.. TAKEYA, K., MATSUMOTO,S. H., AND YAGL K., Inactivation of serotonin by sulfotransferase system, J. Pharmacol. exp. TheE., 166 (1969) 272-275. 5 HIDAKA. H., NAGATSU. T.. AND YAGI. K.. Occurrence of a serotonin sulphotransferase in the brain, J. Neurochem.. 16 (1969) 783-785. 6 KISFIIMOTO.Y., TAKAHASHI.N.. AND EGAMI. F.. Synthesis and properties of serotonin-O-sulfate. J. Biochem., 49 (1961) 43~440. 7 KORE, J.. AND SEVENS, J.B., Failure to detect 5-hydroxytryptamine-O-sulfate tn normal rat brain and after monoamine oxidase inhibition. J. Neurochem.. 17 (1970) 447-448. 8 MAICKEL,R. P.. COX. R.. JR., SAILLANT,J., AND MILLER, F. P.. A method for the determination of serotonin and norepinephrine in discrete areas of rat brain, Int. J. Pharmacol., 7 (1968) 275-281. 9 M~LLARD.S. A., COSTA, E.. AND G~,L, E. M., On the control of brain serotonin turnover by end product inhibition in the rat. Brain Research, 40 (1972) 545-551. 10 M~LLARD.S. A., ANt~ G~,L, E. M.. The contribution of 5-hydroxyindolepyruvic acid to cerebral 5-hydroxyindole metabolism, Int. J. Neurosci.. 1 (197t) 211-218. 11 NEFF. N. H.. TOZER, T. N.. AND BRODtE, B. B., Application of steady-state kinetics to studies of the transfer of 5-hydroxyindoleacetic acid from brain to plasma, J. Pharmacot. exp. Ther.. 158 t1967) 214-218. 12 RENSON,J., WEISSBACH,H.. AND UDENFRIEND,S., Studies on the biological activities of the aldehydes derived from norepinephrine, serotonin, tryptamine and histamine. J. Pharmacol. exp. Ther.. 143 (1964) 326-331. 13 ROSE. F. A., AND BLESZYNSKt. W. S.. The metabolism of 5-hydroxytryptamine-O-(S3~)-sulfate. Biochem, J.. 122 (1971) 601-602. 14 SCItNEIDER, J. J.. AND LEWBART. M. L., Enzymatic synthesis of steroid sulfates..L biol. Chem.. 222 (1956~ 787-794. (Accepted June 9th. 1972)
Brain Research, 44 (1972) 309-312
309
5-Hydroxytryptamine-O-sulfate: an alternate route of serotonin inactivation in brain Hidaka et al. 4 have reported an enzyme system, 5-HT-sulfokinase (or sulfotransferase) in rabbit brain capable of sulfation of 5-hydroxytryptamine (5-HT) by the active sulfate of 3'-phosphoadenosine-5'-phosphosulfate (PAPS). It was postulated .~ that the enzymic O-sulfation of cerebral 5-HT may occur in vivo thus serving as a route of inactivation of 5-HT. However, K o r f and Sebens 7 failed to detect any evidence for cerebral sulfation of 5-HT in vivo even in animals pretreated with monoamine oxidase inhibitors. They, nevertheless, did not reject the possibility of the synthesis of cerebral 5-HT-O-sulfate and suggested that it was either produced in undetectably small amounts or that it was rapidly metabolized. Our experiments were designed to obtain information regarding the formation, rate of synthesis, and efflux of cerebral 5-HT-O-sulfate. 5-HT-O-sulfate was synthesized by the chlorosulfonic acid catalyzed sulfonation of 5-HT with 35S concentrated sulfuric acid (22 mCi total activity) and was freed of sulfate by barium carbonate and of 5-HT by passing it through a column of Amberlite CG-50 (H +) or Dowex-50-H ~ (ref. 6). The effluent containing 5-HT-O-sulfate was adjusted to pH 7.0 and passed through a column of Dowex-2. After washing the column with carbonate-free water, the 5-HT-O-sulfate was obtained by applying aliquots as a strip across Whatman No. 1 paper 1 and developing a descending chromatogram in i s o p r o p a n o l : 6 N NH4OH (20:5, v/v) for 18 h. I s o p r o p a n o l : 6 N NH4OH (20:3, v/v) in our hands did not separate 5-HT-O-SO3 from sulfate discreetly enough to exclude contamination from the latter. Elution of the strip corresponding to the 5-HT-O-sulfate marker with water will yield pure labeled 5-HT-O-sulfate (45 #Ci/mg or 1.2 mCi/mmole specific activity). Labeled 5-hydroxyindoleacetic acid (5-HIAA) was prepared from [2-14C]5-HT by enzymic synthesis lz. Male Sprague-Dawley rats (250-350 g) were used throughout the experiments and were fasted overnight. The animals were divided into 4 groups. The first and second group of rats were controls; the third and fourth group of animals received 80 mg/kg pargyline (Abbott Laboratory, Chicago, II1.). Two hours later the animals in the second and fourth groups were administered 200 mg/kg probenecid (Merck, Sharp, and Dohme Research Laboratory, Rahway, N.J.). Both drugs were injected intraperitoneally. At the end of the third hour 10/zl of a solution of 3~S-labeled 5-HTO-sulfate, or [~4C]5-H1AA, was delivered under light ether anesthesia through each of two holes bilaterally placed in the skull I mm from both the sagittal and coronal sutures about the depth of 1 mm into the cortex ~0. The animals were killed I h later; the brains were removed at 4 °C and homogenized separately with 0.1 N HCI. After centrifuging at 15,000 × g for 10 rain at 3 °C precipitates were resuspended in 3 ml 0.1 N HCI and centrifuged again. The supernatants were combined and adjusted to about pH 7.2. Aliquots of 0.2 ml were taken for scintillation counting to measure the total radioactivity. The remainder of the supernatants were passed through a column of Amberlite CG-50 (H +) resin 3. The effluents from the Amberlite columns were passed through Dowex-2 columns. Brain Research, 44 (1972) 309-312
310
SHORT COMMUNICATIONS
The method of recovery of 5-HT-O-sulfate from the brain of experimental animals essentially followed the same method as described above for the purification of synthetic 5-HT-O-sulfate. The radioactivity in the strips corresponding to the markers was measured by cutting them into the vials containing the fluors and Counting them by liquid scintillation. The markers were identified with Ehrlich's reagent ~. sulfate was detected by rhodizonate reagent 14 lntracerebrally administered 5-HT-O-~5SO3 (1.3 × 104 disint./min or 0.13 c~g) is rapidly eliminated from the brain. Pargyline had no effect on its retention, whereas probenecid with pargyline reduced the rate of efltux by 35 ~ . Probenecid is known to arrest the efflux of endogenous 5-HIAAtL Time studies on the efflux of the 5-HTO-sulfate from the brain of control animals yielded a Tt/2 of 18 rain (k~ - 0.037 min-~), lntracerebrally administered 5-HIAA (5.3 x 104 disint./min or 0.32 # g ) h a d a ke of 0.026 min-L However, the rate ofefflux for endogenous 5-HIAA is k .... 0.014 min -~ (ref. IlL It is known that 5-HT-O-sulfate. like 5-HT. is unable to cross the 'blood-brain' barrier t3. Exogenous 5-HT-O-sulfate cannot penetrate the cellular mere branes and. therefore, it remained only in the extracellular space. 5-HT-O-sutfate synthesized within the cells is also removed at a rapid rate. k~ = 0.028 rain -J. My observations do not indicate that 5-HT-O-sulfate is a way of storage for 5-HT. as suggested by others 5. but rather a terminal way of eliminating 5-HT. Next we have examined the synthesis of cerebral 5-HT-O-sutfate in vivo. A group of rats received pargyline and probenecid, as described above, and 1 h after probenecid treatment, intraperitoneal radioactive sodium sulfate (10 mCi/kg). The animals (3 in each group) were killed I. 2 or 3 h after the injection o f laSS]sulfate. Chromatographic analysis of the region corresponding to 5-HT-O-sullate revealed the presence of radioactivity. This area was cut out and eluted. Unlabeled 5-HT-Osulfate was added to this eluate. After lyophilizing the eluate, the residue was taken up in 0.5 ml water. One-half of this solution (Table ID) was rechromatographed. TABLE I RADIOACTIVITY RECOVERED BY DESCENDING CHROMATOGRAPHY IN
5-HT-O-SO3REGION
R~
Total disint, min recovered Experiment
0.02 (su(fatej
A: Authentic a~SO4 = 400,000 disint./min spotted B: Authentic 5-HT-O-a~SO3 5,000 disint./min spotted C: Extract of brain after i.p.
393,000
3~SO 4 D : Spot from 5-HT-O-zsSOz in exp. C: eluted,rechromatographed E: Eluatc from cxp. C: Spot 5-HT-O-asSOs acid hydrolyzcd
0.41 ,5-HIAA)
-
90 610,000
0.48 ~J.70 .~5-HT-O-SOaJ (5-HT)
39 3940
1200
5531
-
2800
-
52
-
Brain Research, 44 (1972) 309-312
300 2,000
60
SHORT COMMUNICATIONS
3l l
while the other half (Table 1E) was submitted to acid hydrolysis with 70 ~ perchloric acidL This technique is only reliable if the supernatant of brain homogenates is lyophilized and the residue dissolved in water to achieve a concentration of 1.0-1.5 #g/ml of 5-HT, Otherwise the analysis of dilute solutions leads to an uncertainty which is larger in magnitude than the expected difference. Following acid hydrolysis most of the radioactivity appeared in the area corresponding to free sulfate (Table 1). These results were obtained from animals sacrificed at the first hour after administration of intraperitoneal sodium [35S]sulfate. The second and third hour values of the radioactivity recovered in the area corresponding to the 5-HT conjugate were 3695 and 3644 disint./min respectively, representing 6-4 ng of 5-HT in the labeled conjugate for the initial 3 h. These values do not reveal the actual pool size of 5-HT conjugate. Further demonstration of the synthesis of 5-HT-O-sulfate was achieved by recovery of the 14C-labeled conjugate from the brain of rats given intravenous DL-[z4C]5-HTP 1 h after their pretreatment with pargyline and probenecid according to the amount and schedule described in the other experiments. The radioactive 5-HT-O-sulfate was isolated and identified by chromatographic analysis while the 5-HT in the conjugate was determined by the difference in the 5-HT values obtained before and after acid hydrolysis7. The amount of 5-HT was assessed through measuring the fluorophore, a product of condensation of 5-HT with O-phthalaldehydes. The results reveal (Table I1) that the recovered 5-HT-O-sulfate represents about 8 ~ of the 5-HT pool in pargyline treated rats. The label in [14C]5-HT-O-sulfatecorresponds to about 4 ng of radioactive 5-HT which is consistent with the value calculated from the 35S-labeled conjugate. Assuming that probenecid completely inhibited the efflux of endogenous 5-HT-O-sulfate, the approximate rate of its synthesis is calculated to be about 25 ng/g/h. In rats treated with pargyline (80 mg/kg) the level of 5-HT/g of brain increased from about 0.8 to 1.6/~g/g between 2 and 24 h. At 24 h the specific activity of 5-HT has decreased to about 5 ~ over the 2-h value9. This represents an efflux of approximately 60 ng/g/h of 5-HT from the brain of pargyline treated animals. This amount of 5-HT stoichiometrically approximates the 5-HT recoverable in the cerebral 5-HTO-SO3 in the pargyline and probenecid pretreated rats. In control animals the conT A B L E 11 SYNTHESIS OF CEREBRAL [~IC]5-HT-O-SOa AFTER I.V. INJECTION OF [ 1 4 C ] 5 - H T P PROBENECID TREATED RATS
Time (h)
1 2
5-HT
IN PARGYLINE AND
5-HT-O-S03
pg/g
disint./min//tg
ng/g
disint./min/l¢g*
0.63 ~ 0.05** 0.81 :k 0.06
3980 2117
50 :k 2** 75 ::k 2
3654 2230
* Calculated as 5-HT. Each value represents an average o f 4 animals in duplicate determination. See text for experimental conditions. ** S.E.
Brain Research, 44 (1972) 309-312
312
SHORT COMMUNICATIONS
v e r s i o n o f 5 - H T to 5 - H I A A is t h e m a j o r r o u t e f o r the r e m o v a l o f 5 - H T since t h e r a t e of synthesis o f 5 - H I A A o u t s t r i p s the s u l f a t i o n o f 5 - H T , D u r i n g t h e i n h i b i t i o n ol" cerebral monoamine
o x i d a s e activity, h o w e v e r , t h e s y n t h e s i s o f 5 - H T - O - S O ~ m a y
b e c o m e a c o n t r o l l i n g m e c h a n i s m f o r the r e m o v a l o f 5 - H T . T h u s . t h e e n z y m a t i c sulr a t i o n o f 5 - H T c o u l d be v i e w e d as a r e g u l a t o r y r e s p o n s e to e l e v a t e d levels o f 5 - H T in t h e brain. T h i s s h o u l d be k e p t in m i n d w h e n d e t e r m i n a t i o n s f o r the c o n c e n t r a t i o n s of various cerebral metabolites of 5-HT are made. T h e s e o b s e r v a t i o n s reveal a n o t h e r m o d a l i t y o f i n t r a c e l l u l a r c o n t r o l o f 5 - H T levels in the brain. Neurochemical Research Laborator.v. Department o f Psychiatry, College o f Medicine, University o f h~wa. Iowa City, Iowa 52240 (U.S.A.,
E. MARTIN GAL
1 AIRAKSlNEr~.M. M., Studies of the metabolism and antidiuretic action of 5-hydroxytryptamine and the effect of the mode of administration, Ann. Med. exp. Biol. Fern., 41, Suppl. 4 (19637 t-91. 2 DAVIS, V. E.. HuEE. J. A., ANO BROWN. H.. Free and conjugated serotonin excretion in carcinoid syndrome, Clin. chin,. Acta, 13 (1966) 390-402. 3 G~L, E. M.. HEATER, R. D., AM3 M1LLARD.S. A., Studies on the metabolism of 5-hydroxytryptamine fserotoninl. VI. Hydroxylation and amines in cold-stressed reserpinised rats. Proc. Soc. exp. Biol. ( N. Y.), 128 (1968) 412-415, 4 HIDAKA. H.. NAGATSU.T.. TAKEYA, K., MATSUMOTO,S. H., AND YAGL K., Inactivation of serotonin by sulfotransferase system, J. Pharmacol. exp. TheE., 166 (1969) 272-275. 5 HIDAKA. H., NAGATSU. T.. AND YAGI. K.. Occurrence of a serotonin sulphotransferase in the brain, J. Neurochem.. 16 (1969) 783-785. 6 KISFIIMOTO.Y., TAKAHASHI.N.. AND EGAMI. F.. Synthesis and properties of serotonin-O-sulfate. J. Biochem., 49 (1961) 43~440. 7 KORE, J.. AND SEVENS, J.B., Failure to detect 5-hydroxytryptamine-O-sulfate tn normal rat brain and after monoamine oxidase inhibition. J. Neurochem.. 17 (1970) 447-448. 8 MAICKEL,R. P.. COX. R.. JR., SAILLANT,J., AND MILLER, F. P.. A method for the determination of serotonin and norepinephrine in discrete areas of rat brain, Int. J. Pharmacol., 7 (1968) 275-281. 9 M~LLARD.S. A., COSTA, E.. AND G~,L, E. M., On the control of brain serotonin turnover by end product inhibition in the rat. Brain Research, 40 (1972) 545-551. 10 M~LLARD.S. A., ANt~ G~,L, E. M.. The contribution of 5-hydroxyindolepyruvic acid to cerebral 5-hydroxyindole metabolism, Int. J. Neurosci.. 1 (197t) 211-218. 11 NEFF. N. H.. TOZER, T. N.. AND BRODtE, B. B., Application of steady-state kinetics to studies of the transfer of 5-hydroxyindoleacetic acid from brain to plasma, J. Pharmacot. exp. Ther.. 158 t1967) 214-218. 12 RENSON,J., WEISSBACH,H.. AND UDENFRIEND,S., Studies on the biological activities of the aldehydes derived from norepinephrine, serotonin, tryptamine and histamine. J. Pharmacol. exp. Ther.. 143 (1964) 326-331. 13 ROSE. F. A., AND BLESZYNSKt. W. S.. The metabolism of 5-hydroxytryptamine-O-(S3~)-sulfate. Biochem, J.. 122 (1971) 601-602. 14 SCItNEIDER, J. J.. AND LEWBART. M. L., Enzymatic synthesis of steroid sulfates..L biol. Chem.. 222 (1956~ 787-794. (Accepted June 9th. 1972)
Brain Research, 44 (1972) 309-312