Journal of Neurochrmtstry. 1976. V d 26. pp. 585-5x9 Pergamon Press. Printed in Great Britain
~-HYDROXY-L-TRYPTOPHANDECARBOXYLASE ACTIVITY: MICROASSAY AND DISTRIBUTION IN DISCRETE RAT BRAIN NUCLEI J. M. SAAVEDRA Section of Pharmacology, Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, M D 20014, U S A . (Received 24 March 1975. Accepted 26 August 1975)
Abstract-5-Hydroxy-~-tryptophan decarboxylase (EC 4.1.1.28) activity was measured in discrete regions of the rat brain by means of a sensitive micromethod lhat allows the quantitation of enzyme activity in pg amounts of brain tissue. A good correlation was obtained between serotonin levels and 5-hydroxy-~-tryptophandecarboxylase activity in all the brain regions examined. Wide differences in enzyme activity were found in the different brain nuclei, with a 20-fold difference in activity between the most active region (the nucleus raphe dorsalis) and the least active regions (the corpus callosum and the optic tract).
THERECENT development of a sensitive enzymatic-isotopic micromethod for the measurement of serotonin (SAAVEDRA et al., 1973) has made it possible to develop a microassay for 5-hydroxy-~-tryptophandecarboxylase (EC 4.1.1.28) activity. The assay is specific and more sensitive than the methods currently in use (KUNTZMAN et al., 1961; MCCAMAN et al., 1965; SNYDER& AXELROD, 1969). The assay is based on the enzymatic conversion of 5-hydroxytryptophan to melatonin, as follows:
al., 1973). A radioactive label is introduced into the final product by transferring the [3H]methyl group from [3H]methyl-S-adenosyl-~-methionine to N-acelylserotonin by the use of a partially purified hydroxyindole-0-methyl-transferasc (SAAVEDRA et al., 1973), followed by isolation of [3H]melatonin by extraction with an organic solvent. Under the conditions of the assay for serotonin. L3Hlmelatonin is the only radioactive product isolated in detectable amounts (SAAWRA et al., 1973).
5-hydroxytryptophan 5-hydroxy+-tryptophan decarboxylase (EC 4.1.1.28) serotonin N-acetyltransferase (EC 2.3.1.5) Acetyl coenzyme A N-acetylserotonin Hydroxyindole-0methyltransferase (EC 2.1.1.4) [3H]methyl-S-adenosyl-
L-methionine 5-Hydroxytryptophan is decarboxylated by the tissue decarboxylase lo serotonin, and serotonin is acetylated enzymatically with rat liver N-acetyltransferase and acetyl coenzyme A (acyl-CoA) (SAAVEDRA et Abbrrviutions used: 5-HTP
=
5-hydroxy-~-tryptophan. 585
MATERIALS A N D METHODS Animals. Sprague-Dawley male rats, weighing 20C-250 g were obtained from Zivic-Miller Laboratories (Allison Park, Fa.). All rats were housed in groups of eight under diurnal lighting conditions, with lights on from 6 a.m. to 6 p.m Materials. Pyridoxal phosphate was purchased from Calbiochem (Los Angeles, CA). 5-Hydroxy-~-tryptophan (5-HTP) was obtained from Sigma (St. Louis, MO.). Acetyl coenzyme A (acyl-CoA), sodium salt, 80% pure, was purchased from Aldrich Chemical Co. (Cedar Knolls, N.J.). Serotonin, N-acetylserotonin and melatonin were obtained from Regis Chemical Co. (Chicago, Ill.). N-Methyl-N-2propynylbenzylamine hydrochloride (pargyline) was a gift from Abbott Laboratories (North Chicago, Ill.). 1.-3-(3,4 Dihydroxyphenyl)-2-hydrazino-2-methylpropionicacid (carbidopa). was a gift from Merck. Sharp and Dohme Research Laboratories, Rahway, N.J. [3H]methyl-S-adenosylL-methionine, specific activity 8.3 Ci/mmol, was purchased from New England Nuclear Corp. (Boston, MA.). Tissue preparation and dissection. The animals were killed by decapitation at 9 a.m., and the brains were immediately removed and frozen on dry ice. Frontal sections of 300 pm thickness were cut in a cryostat at - 10°C. The different brain areas and nuclei were removed as previously et al., 1974a, b, c), with stainless steel described (PALKOVITS needles, under stereo-microscopic control. Two to four pellets (2C-lo0 pg protein) were taken from each nucleus. All
586
J. M. SAAVEDRA
determinations were performed on tissue from single ani- tetraacetic acid (EDTA) (0.1 mu), and rncrcdptoethanol (1 mM). The conditions for storage of the rat liver N-acetylmals. for 5-hydroxy-~-tryptophan-decarboxylaseactivity. transferase are essential for the stability of the enzyme. Immediately after dissection, the tissue pellets were blown Failure to add preservatives resulted in a 50% fall in endirectly onto micropestles and homogenized in micro- zyme activity within 15 days. The partially purified N-acetyltransferase from rat liver homogenizers (Micrometric Instrument CO.. Cleveland, in this assay also contained decarboxylase activity Ohio), containing ice-cold 0.02 M-sodium phosphate buffer, et al., 1974a,h). It was therefore essential to M-pyridoxal phosphate. Thirty to (SAAVEDRA p~ 8.2, and 5 x 150 p~ of buffer were used, depending on the brain area obtain a complete inhibition of decarboxylation, by addition of the decarboxylase inhibitor carbidopa, before this dissected. Five pl of the homogenate were removed for protein preparation was added to the system. The acetyl coenzyme A should be dissolved in determination (LOWRYet al., 1951), and the remainder was transferred to 0.4ml conic plastic tubes (A. H. Thomas 0.1 mM-HCI and stored at - 15°C. Under these conditions, Co., Philadelphia, PA.) and centrifuged for 30min at the compound was stable for at least one month. [3H]MethyI-S-adnosyl-~-methionine was stored at 4°C. 30,ooOg at 4'C. The three step enzymatic assay was performed as fol- Repeated freezing and thawing procedures should be avoided. lows: The addition of 10pg of nonradioactive melatonin as (1) Synthesis of serotonin. After centrifugation, 5 pl ahquots of the supernatant fluid were transferred to 15ml a carrier prevents losses of product during the extraction glass stoppered tubes. Five pl of a solution containing and evaporation procedures. The frozen sections should he stored at -10°C until 5-hydroxytryptophan, the monoamine oxidase inhibitor pargyline, and albumin, in 0.02 M-sodium phosphate buffer, analyzed. Under the conditions of the assay, no loss of pH 8.3, were added, and the tubes were incubated for enzyme activity was detected when the tissues were ana15 min at 37°C. Final concentrations in the incubation lyzed during the first 48 h after killing. When homogenized mixture were: 5-hydroxytryptophan 0-33mM, pargylinc and stored in 5 x lo-' pyridoxal phosphate, the enzyme activity remained unchanged in the homogenates for at 1 mM and albumin 0.057/,. least 48 h if stored at - 10°C. The reaction was terminated by adding 5 111 of 0.5 mMTissue blanks should be assessed independently for all carbidopa in 0.1 N-HCI, to give a total vol of 15pL and tissues or brain areas examined, due to small but variable the tubes were placed in an ice bath. amounts of endogenous serotonin which are measured by (2) Conversion of serotonin to N-acetyl serotonin. 15 pl of a solution consisting of 143 parts of 1 M-sodium phos- the assay. Serotonin standards should also be carried phate buffer, pH 7.9, and 0.5 parts of 1 N-NaOH, was ad- through the procedure for all tissues or areas examined, to ded to each of the samples. Then, 5 pI of a solution consist- rule out the existence of inhibitors of the reaction, and ing of one part of partially purified rat liver N-acetyltranscorrect for any possible variables due to changes in endoet al., 1973) and one part of acetyl coen- genous S-adenosyl-L-methionine concentrations. ferase (SAAVEDRA zyme A (4mg/ml in 0.1 mM-HCI) was added, and the tubes The amount of [3H]melatonin formed in the reaction were incubated for 30min at 37°C. is proportional to the amount of serotonin formed by the (3) Conwrsion of N-acetyl serotonin to [3H]melatonin. tissue decarboxylase, up to 10 mg of serotonin. When dealWithout terminating the incubation above, 5 pl of a mix- ing with tissues rich in decarboxylase activity (e.g. raphe ture containing two parts of a partially purified hydroxyin- nuclei) care must be taken to dilute the samples or reduce dole-0-methyltransferase preparation (SAAVFDRAet al., the incubation time, so that no more than l o n g of sero1973), two parts of 0.2 M-sodium phosphate buffer, pH 7.9, tonin are formed (see Results). For the assay of regions and one part of [3H]methyl-S-adenosyI-methionine with low decarboxylase activity, the incubation time of the (0.0567 mmol) was added to each tube. After 10 min in decarboxylation reaction can be prolonged up to 90 min incubation, the reaction was stopped with 0 5 ml of 0.5 M(see Results). borate buffer, pH 10, and lop1 of a 1 mglml solution of melatonin in ethanol were added. RESULTS The radioactive product was extracted into 6 ml of Enzymatic assay. The influence of pH on the 5-HTP toluene by shaking in a Vortex mixer for 30s. Five ml of decarboxylase activity, and buffer, pyridoxal phosthe organic phase were then transferred to a scintillation phate a n d substrate concentrations required for maxicounting vial and dried overnight in a chromatography mal enzyme activity have been studied. The enzyme oven at 80°C. The radioactivity of the samples was determined by has a pH optimum between 8.1 and 8.5, and no differliquid scintillation spectroscopy, after addition of 1 ml of ences in activity were found when Tris-HCl or phosethanol and 10 ml of phosphor (40 ml of scintillant (Liqui- phate buffer were used, from 20 t o 50mM. Pyridoxal fluor, New England Nuclear Corp.) per liter of toluene). phosphate stimulated the 5 - H T P decarboxylase acTissue blanks were made by placing the entire assay tivity only 2-fold, and a concentration of pyridoxal mixture on ice during the first incubation, prior to the phosphate of 5 x M gave maximal enzyme addition of the 0.1N-HCI solution containing carbidopa. activity. The blanks were then incubated as the rest of the tubes. Maximal rates of enzyme activity were observed Standards were prepared by addition of 1-3ng of serowith 0.3 ~ M - ~ - H T aPn,d no substrate inhibition was tonin to sets of blanks. Precautions. After partial purification by ammonium sul- detected with concentrations u p t o 0.5 mM-5-HTP. Under the conditions of the assay, the decarboxylafate fractionation and dialysis, the N-acetyltransferase from rat liver was divided into aliquots and stored at -15"C, tion of 5-hydroxytryptophan by brain tissue samples after addition of ethylene gycol(lO% v/v), ethylenediamine- was found to be linear up t o 90min of incubation
5-HTP decarboxylase microassay and distribution in brain
587
/
INCUBATIONTIME (MINUTES1 SEROTONIN ng
FIG.1. Effect or incubation time on the 5-HTP decarboxylase reaction. Whole brain homogenates containing 5 pg
of protein were prepared as described in Methods and incubated for different time intervals for 5-HTP decarboxylase activity. Under the conditions utilized, the reaction was found to be linear up to 90 min. (Fig. 1) and over a wide range of tissue concentrations (Fig. 2). When exogenous serotonin was added to samples of tissue supernatant, the amount of [3H]melatonin formed in the reactions was proportional to the amount of serotonin added to the reaction mixture, up to 5 n g (Fig. 3). Table 1 represents a typical assay for 5-hydroxy-~tryptophan decarboxylase activity in brain. Tissues were diluted so that not more than 10ng of serotonin were formed. A 15 min incubation time for the decarboxylase reaction was routinely used (see Table 1). Distribution of 5-hydroxy-~-tryptophandecarboxylase. The distribution of 5-hydroxy-~-tryptophandecarboxylase activity in discrete brain regions is shown in Table 2. A 20-fold difference in decarboxylase
9r
It/'
ov ' 3' 1.5
I
6
I
9
rg PROTEIN
FIG. 2. Enzyme dilution curve. Homogenates from the nucleus raphe dorsalis were prepared as described in Methods and incubated for 5-HTP decarboxylase activity for 15 min. Enzymatic activity is linearly related to the amount of tissue assayed.
FIG.3. Linearity of recovery of exogenous serotonin from brain tissue. Serotonin standards were prepared and added to aliquots of brain supernatant (20 pg protein each) as described under Methods. Results are expressed in c.p.m. after substraction of the blank (400 c.p.m.). Average values were 6025 c.p.m. per ng of serotonin.
activity was found between the most active (nucleus raphe dorsalis) and the least active (corpus callosum, optic tract) regions. DISCUSSION
A single enzyme, aromatic-L-aminoacid decarboxylase, is currently regarded as responsible for the decarboxylation of several aminoacids, including 3,4dihydroxyphenylalanine (DOPA) (EC 4.1.1.26, dopa decarboxylase) and 5-hydroxytryptophan (5-HTP) (EC 4.1.1.28, 5-hydroxy-L-tryptophan decarboxylase) (LOVENBERG et al., 1962; CHRISTENSON et al., 1972; LANCASTER et al., 1972; HOKFELTet al., 1973). Although there have been claims for the existence of more than one aromatic amino acid decarboxylase, this question is not pertinent to the present topic. The formation of serotonin in the rat brain after administration of exogenous 5-HTP is decreased in animals with lesions of the raphe system, indicating that the 5-HTP decarboxylation occurs predominantly in 5-HT containing nerve endings (KORFet al., 1974). In studies on serotonin metabolism, therefore, it is necessafy to utilize 5-hydroxytryptophan instead of DOPA as a substrate, and to use optimal assay conditions for the 5-hydroxy-~-tryptophandecarboxylase reaction (SIMSet al., 1973). In combination with microdissecting techniques, the method allows the determination of 5-hydroxy-~tryptophan decarboxylase activity in discrete areas of the rat brain. The results are in general agreement with earlier biochemical determinations of 5-hydroxy-~-tryptophan decarboxylase activity in various regions of the rabbit (MCCAMAN et al., 1965), cat (KUNTZMAN et al., 1961), and rat brain (SIMS& BLOOM, 1973). Recently, HOKFELT et a!. (1973) reported the localization of DOPA-5HTP decarboxylase activity in the rat brain by means of immunohistological techniques,
J. M. SAAVEDRA
588
TABLE1. TYPICAL ASSAY pg protein
Tissue Whole brain Nucleus raphe dorsal15
per assay 21.
208 5* 5* 2 0.5
5-HYDROXY-L-TRYPTOPHANDECARBOXYI-ASE ACTIVITY
Radioactivity in blanks (c.p.m.)
Radioactivity in 1 ng 5-HT standard (c.p.m. over blanks)
Radioactivity in cxperiments (c.p.m. blanks)
405 440 805
6,080 6,100 5,992
20,055 z0,m 37,950
800
6,055
6,010 6, I 3n 6,300 6.010 6,060
650 410
0-1' 01'
5
Median eminence
FOR
285 250 505
39,400 1s.m 4,010 805 690 8,245
5-HTP dccarboxylase acthit?. Pmol cerntonin'ug protein per 11 cd incuh,itim 3.56 3.83 28.75 2954 29.80 29 70 29 00 26.06 6.17
Tissues were prepared as described in Methods, and incubated 15 min for 5-HTP decarboxylase activity. 5-HTP decarboxylase activity is measured indirectly through conversion of the decarboxylation product, serotonin. to its derivative melatonin, as described in Methods. * Duplicate samples.
and concluded that this enzyme corresponded with lase and tryptophan hydroxylase in these regions the distribution of cell bodies containing either dopa- supports the hypothesis of the local synthesis of seromine or SHTP, and that it was not localized in other tonin and its involvement in neuroendocrine regulaneuronal or extraneuronal systems. tion. The results also indicate a high degree of localizaWith the exception of the raphe nuclei, the area tion for 5-hydroxy-~-tryptophandecarboxylase ac- postrema is the region of the rat brain which has tivity in the rat brain, with a good correlation be- the highest 5-hydroxy-~-tryptophandecarboxylase actween enzyme activity and serotonin levels (PALKO- tivity. This observation confirms earlier reports (McVITS ef ul., 19746: SAAVEDRA et a/., 1974a, b) in the CAMAN e t a/., 1965) and supports the view that seroregions examined. tonin plays a significant role in the physiology of this The presence of serotonin and related enzymes in structure. certain brain areas is of special interest. Relatively high The existence of 5-hydroxy-~-tryptophandecarboxlevels of serotonin have been reported for some hypo- ylase activity in the locus coeruleus is worthy of note. thalamic nuclei (nucleus suprachiasmatis, nucleus Serotonin has also been detected in this structure arcuatus), and for the median eminence (SAAVEDRA (PALKOVITS e t a/.. 19720) as well as in other areas et a/., 1974b). Recently, tryptophan hydroxylase has of the brain stem rich in catecholamine containing also been found in these areas (KIZER et a[., 1975). cell bodies (SAAVEDRA,unpublished observation). The coexistence of 5-hydroxy-~-tryptophandecarboxy- Thus, innervation of these catecholamine rich areas by serotoninergic fibers seems probable. The present technique allows the precise biochemiTARLF 2. DISTRIBUTION OF 5-HYDROXY-L-TRYPTOPHAN cal quantitation of 5-hydroxy-~-tryptophandecarboxDECARROXYLASE ACTIVITY IN DISCRETE AREAS AND NUCLEI OF THE RAT BRAIN ylase activity in p g amounts of brain tissue. Together with the microassay for serotonin and tryptophan 5-HTP dccarboxylase actlvny hydroxylase, it offers a valuable tool for the study (pmo1:pg protein per of serotonin metabolism in discrete areas of the brain. Area h of incubation k s.t M Number of samnles ~~
~
~
Nucleus raphc dorsalis* h'uclrui centralis superior' Area postrema' Substantla nigra' Nucleus suprachiasmalist Caudate+ Locus cocruleus: Nucleus arcua tm: Median eminencc Tuberculum o l f a c t u r t u m ~ Cerebellum: Nucleus seplalis medialis: Medial forebrain bundls: Parietal cortext tfippnc.mpuc: Cingulatc cortex: Optic tract: Corpus callosum: -
4-0
(12)
15.6 t 2.1 13.8 i 1.8 13-1 k 2.0 1i.n rt 1.7 9 9 k 1-0 8.9 ? 2.1 6 2 f 0.7 5.3 f 1-2 4 9 f 0.9 4.7 0.8 4.3 i 0 7 3.8 ? 0 4 2.8 0.2 2.7 i 0.3 2.6 0.4 1.6 i 0-5 1.1 i 0-3
(11) (9)
27.0
*
*
+
(6)
(11)
(8) (6) (9) (I1)
(I11 (8) (12) (81 (8) ( 61 (12)
(6) (6)
Tissues werc dissected and analyzed as explained in Material and Methods. Each sample represents tissue from one rat. assayed individually (0024.1 mg protein). Diflerent vol of homogenate were used to produce amounts of serotonin in the linear range of the assay: *150 PI, T50 pl. Incubation time for 5-HTP-decarboxylase activity was I5 min.
REFERENCES J. G., DAIRMAN W. & UNDENFRIEND S. (1972) CHRISTENSON Proc. nafn. Acad. Sci., U.S.A. 69, 343-347. HOKFELT T., FUXEL. & GOLDSTEIN M. (1973) Bruin Rex 53, 175180. KIZERJ. S., ZIVINJ. A.. SAAVEDRA J. M. & BROWNSTEIN M. (1975) J . Neurochem. 24, 779-785. KORFJ., VENEMAK. & POSTEMA F. (1974) J . Neurochem. 23, 249-252. KUNTZMAN R., SHOREP. A., BCK~DANSKI D. & BRODIEB. B. (1961) J . Neurochem. 6, 226232. LANCASTER G. A. & SOURKES T. L. (1972) C m . J . Biochem. 50, 791-797. LOVENBERG W., WEISSBACH H. & UDENFRIENDS. (1962) J . hiol. Chem. 237, 89-93. LOWRY0. H., ROSEBROUGH N. J.. FARRA . L. & RANDALL R . J. (1951) 3.biol. Chem. 193, 265 275.
5-IIT'P decarboxylase microassay and distribution in brain
MCCAMAN R. E., MCCAMAN M. W., HUNTJ. M. & SMITH M. S. (1965) J . Neurochem. 12, 15-23. PALKOVITS M., BROWNSTEIN M., SAAVEDRA J. M. & AXELROD J. (1974a) Brain Res. 77, 137-149. PALKOVITS M., BROWNSTEIN M. & SAAVEDRA J. M. (19746) Brain Res. 80, 237-249. PALKOMTS M., SAAVEDRA J. M., KOBAYASHI R. M. & M. (1974~)Brain Rex 79, 443450. BROWNSTEIN SAAVEDRAJ. M., BROWNSTEINM. & AXELRODJ. (1973) J. Pharmac. exp. Ther. 186, 508-515.
589
SAAVEDKA J. M., BROWNSTEIN M. & PALKOVITS M. (1974aj Brain Res. 79, 437441. SAAVEDRA J. M., PALKOVITS M., BKOWNSTEIN M. & AXELROD J. (1974b) Bruin Res. 77, 157-165. SIMSK. L. & BLWM F. E. (1973) Brain Res. 49, 165S175. SIMSK. L., DAVIS G. A. & BLOOMF. E. (1973) J , Neurochem. 20, 449464. SYNDERS. H. & AXELRODJ. (1969) Biocheni. Pharmac. 13, 805-806.
~-HYDROXY-L-TRYPTOPHANDECARBOXYLASE ACTIVITY: MICROASSAY AND DISTRIBUTION IN DISCRETE RAT BRAIN NUCLEI J. M. SAAVEDRA Section of Pharmacology, Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, M D 20014, U S A . (Received 24 March 1975. Accepted 26 August 1975)
Abstract-5-Hydroxy-~-tryptophan decarboxylase (EC 4.1.1.28) activity was measured in discrete regions of the rat brain by means of a sensitive micromethod lhat allows the quantitation of enzyme activity in pg amounts of brain tissue. A good correlation was obtained between serotonin levels and 5-hydroxy-~-tryptophandecarboxylase activity in all the brain regions examined. Wide differences in enzyme activity were found in the different brain nuclei, with a 20-fold difference in activity between the most active region (the nucleus raphe dorsalis) and the least active regions (the corpus callosum and the optic tract).
THERECENT development of a sensitive enzymatic-isotopic micromethod for the measurement of serotonin (SAAVEDRA et al., 1973) has made it possible to develop a microassay for 5-hydroxy-~-tryptophandecarboxylase (EC 4.1.1.28) activity. The assay is specific and more sensitive than the methods currently in use (KUNTZMAN et al., 1961; MCCAMAN et al., 1965; SNYDER& AXELROD, 1969). The assay is based on the enzymatic conversion of 5-hydroxytryptophan to melatonin, as follows:
al., 1973). A radioactive label is introduced into the final product by transferring the [3H]methyl group from [3H]methyl-S-adenosyl-~-methionine to N-acelylserotonin by the use of a partially purified hydroxyindole-0-methyl-transferasc (SAAVEDRA et al., 1973), followed by isolation of [3H]melatonin by extraction with an organic solvent. Under the conditions of the assay for serotonin. L3Hlmelatonin is the only radioactive product isolated in detectable amounts (SAAWRA et al., 1973).
5-hydroxytryptophan 5-hydroxy+-tryptophan decarboxylase (EC 4.1.1.28) serotonin N-acetyltransferase (EC 2.3.1.5) Acetyl coenzyme A N-acetylserotonin Hydroxyindole-0methyltransferase (EC 2.1.1.4) [3H]methyl-S-adenosyl-
L-methionine 5-Hydroxytryptophan is decarboxylated by the tissue decarboxylase lo serotonin, and serotonin is acetylated enzymatically with rat liver N-acetyltransferase and acetyl coenzyme A (acyl-CoA) (SAAVEDRA et Abbrrviutions used: 5-HTP
=
5-hydroxy-~-tryptophan. 585
MATERIALS A N D METHODS Animals. Sprague-Dawley male rats, weighing 20C-250 g were obtained from Zivic-Miller Laboratories (Allison Park, Fa.). All rats were housed in groups of eight under diurnal lighting conditions, with lights on from 6 a.m. to 6 p.m Materials. Pyridoxal phosphate was purchased from Calbiochem (Los Angeles, CA). 5-Hydroxy-~-tryptophan (5-HTP) was obtained from Sigma (St. Louis, MO.). Acetyl coenzyme A (acyl-CoA), sodium salt, 80% pure, was purchased from Aldrich Chemical Co. (Cedar Knolls, N.J.). Serotonin, N-acetylserotonin and melatonin were obtained from Regis Chemical Co. (Chicago, Ill.). N-Methyl-N-2propynylbenzylamine hydrochloride (pargyline) was a gift from Abbott Laboratories (North Chicago, Ill.). 1.-3-(3,4 Dihydroxyphenyl)-2-hydrazino-2-methylpropionicacid (carbidopa). was a gift from Merck. Sharp and Dohme Research Laboratories, Rahway, N.J. [3H]methyl-S-adenosylL-methionine, specific activity 8.3 Ci/mmol, was purchased from New England Nuclear Corp. (Boston, MA.). Tissue preparation and dissection. The animals were killed by decapitation at 9 a.m., and the brains were immediately removed and frozen on dry ice. Frontal sections of 300 pm thickness were cut in a cryostat at - 10°C. The different brain areas and nuclei were removed as previously et al., 1974a, b, c), with stainless steel described (PALKOVITS needles, under stereo-microscopic control. Two to four pellets (2C-lo0 pg protein) were taken from each nucleus. All
586
J. M. SAAVEDRA
determinations were performed on tissue from single ani- tetraacetic acid (EDTA) (0.1 mu), and rncrcdptoethanol (1 mM). The conditions for storage of the rat liver N-acetylmals. for 5-hydroxy-~-tryptophan-decarboxylaseactivity. transferase are essential for the stability of the enzyme. Immediately after dissection, the tissue pellets were blown Failure to add preservatives resulted in a 50% fall in endirectly onto micropestles and homogenized in micro- zyme activity within 15 days. The partially purified N-acetyltransferase from rat liver homogenizers (Micrometric Instrument CO.. Cleveland, in this assay also contained decarboxylase activity Ohio), containing ice-cold 0.02 M-sodium phosphate buffer, et al., 1974a,h). It was therefore essential to M-pyridoxal phosphate. Thirty to (SAAVEDRA p~ 8.2, and 5 x 150 p~ of buffer were used, depending on the brain area obtain a complete inhibition of decarboxylation, by addition of the decarboxylase inhibitor carbidopa, before this dissected. Five pl of the homogenate were removed for protein preparation was added to the system. The acetyl coenzyme A should be dissolved in determination (LOWRYet al., 1951), and the remainder was transferred to 0.4ml conic plastic tubes (A. H. Thomas 0.1 mM-HCI and stored at - 15°C. Under these conditions, Co., Philadelphia, PA.) and centrifuged for 30min at the compound was stable for at least one month. [3H]MethyI-S-adnosyl-~-methionine was stored at 4°C. 30,ooOg at 4'C. The three step enzymatic assay was performed as fol- Repeated freezing and thawing procedures should be avoided. lows: The addition of 10pg of nonradioactive melatonin as (1) Synthesis of serotonin. After centrifugation, 5 pl ahquots of the supernatant fluid were transferred to 15ml a carrier prevents losses of product during the extraction glass stoppered tubes. Five pl of a solution containing and evaporation procedures. The frozen sections should he stored at -10°C until 5-hydroxytryptophan, the monoamine oxidase inhibitor pargyline, and albumin, in 0.02 M-sodium phosphate buffer, analyzed. Under the conditions of the assay, no loss of pH 8.3, were added, and the tubes were incubated for enzyme activity was detected when the tissues were ana15 min at 37°C. Final concentrations in the incubation lyzed during the first 48 h after killing. When homogenized mixture were: 5-hydroxytryptophan 0-33mM, pargylinc and stored in 5 x lo-' pyridoxal phosphate, the enzyme activity remained unchanged in the homogenates for at 1 mM and albumin 0.057/,. least 48 h if stored at - 10°C. The reaction was terminated by adding 5 111 of 0.5 mMTissue blanks should be assessed independently for all carbidopa in 0.1 N-HCI, to give a total vol of 15pL and tissues or brain areas examined, due to small but variable the tubes were placed in an ice bath. amounts of endogenous serotonin which are measured by (2) Conversion of serotonin to N-acetyl serotonin. 15 pl of a solution consisting of 143 parts of 1 M-sodium phos- the assay. Serotonin standards should also be carried phate buffer, pH 7.9, and 0.5 parts of 1 N-NaOH, was ad- through the procedure for all tissues or areas examined, to ded to each of the samples. Then, 5 pI of a solution consist- rule out the existence of inhibitors of the reaction, and ing of one part of partially purified rat liver N-acetyltranscorrect for any possible variables due to changes in endoet al., 1973) and one part of acetyl coen- genous S-adenosyl-L-methionine concentrations. ferase (SAAVEDRA zyme A (4mg/ml in 0.1 mM-HCI) was added, and the tubes The amount of [3H]melatonin formed in the reaction were incubated for 30min at 37°C. is proportional to the amount of serotonin formed by the (3) Conwrsion of N-acetyl serotonin to [3H]melatonin. tissue decarboxylase, up to 10 mg of serotonin. When dealWithout terminating the incubation above, 5 pl of a mix- ing with tissues rich in decarboxylase activity (e.g. raphe ture containing two parts of a partially purified hydroxyin- nuclei) care must be taken to dilute the samples or reduce dole-0-methyltransferase preparation (SAAVFDRAet al., the incubation time, so that no more than l o n g of sero1973), two parts of 0.2 M-sodium phosphate buffer, pH 7.9, tonin are formed (see Results). For the assay of regions and one part of [3H]methyl-S-adenosyI-methionine with low decarboxylase activity, the incubation time of the (0.0567 mmol) was added to each tube. After 10 min in decarboxylation reaction can be prolonged up to 90 min incubation, the reaction was stopped with 0 5 ml of 0.5 M(see Results). borate buffer, pH 10, and lop1 of a 1 mglml solution of melatonin in ethanol were added. RESULTS The radioactive product was extracted into 6 ml of Enzymatic assay. The influence of pH on the 5-HTP toluene by shaking in a Vortex mixer for 30s. Five ml of decarboxylase activity, and buffer, pyridoxal phosthe organic phase were then transferred to a scintillation phate a n d substrate concentrations required for maxicounting vial and dried overnight in a chromatography mal enzyme activity have been studied. The enzyme oven at 80°C. The radioactivity of the samples was determined by has a pH optimum between 8.1 and 8.5, and no differliquid scintillation spectroscopy, after addition of 1 ml of ences in activity were found when Tris-HCl or phosethanol and 10 ml of phosphor (40 ml of scintillant (Liqui- phate buffer were used, from 20 t o 50mM. Pyridoxal fluor, New England Nuclear Corp.) per liter of toluene). phosphate stimulated the 5 - H T P decarboxylase acTissue blanks were made by placing the entire assay tivity only 2-fold, and a concentration of pyridoxal mixture on ice during the first incubation, prior to the phosphate of 5 x M gave maximal enzyme addition of the 0.1N-HCI solution containing carbidopa. activity. The blanks were then incubated as the rest of the tubes. Maximal rates of enzyme activity were observed Standards were prepared by addition of 1-3ng of serowith 0.3 ~ M - ~ - H T aPn,d no substrate inhibition was tonin to sets of blanks. Precautions. After partial purification by ammonium sul- detected with concentrations u p t o 0.5 mM-5-HTP. Under the conditions of the assay, the decarboxylafate fractionation and dialysis, the N-acetyltransferase from rat liver was divided into aliquots and stored at -15"C, tion of 5-hydroxytryptophan by brain tissue samples after addition of ethylene gycol(lO% v/v), ethylenediamine- was found to be linear up t o 90min of incubation
5-HTP decarboxylase microassay and distribution in brain
587
/
INCUBATIONTIME (MINUTES1 SEROTONIN ng
FIG.1. Effect or incubation time on the 5-HTP decarboxylase reaction. Whole brain homogenates containing 5 pg
of protein were prepared as described in Methods and incubated for different time intervals for 5-HTP decarboxylase activity. Under the conditions utilized, the reaction was found to be linear up to 90 min. (Fig. 1) and over a wide range of tissue concentrations (Fig. 2). When exogenous serotonin was added to samples of tissue supernatant, the amount of [3H]melatonin formed in the reactions was proportional to the amount of serotonin added to the reaction mixture, up to 5 n g (Fig. 3). Table 1 represents a typical assay for 5-hydroxy-~tryptophan decarboxylase activity in brain. Tissues were diluted so that not more than 10ng of serotonin were formed. A 15 min incubation time for the decarboxylase reaction was routinely used (see Table 1). Distribution of 5-hydroxy-~-tryptophandecarboxylase. The distribution of 5-hydroxy-~-tryptophandecarboxylase activity in discrete brain regions is shown in Table 2. A 20-fold difference in decarboxylase
9r
It/'
ov ' 3' 1.5
I
6
I
9
rg PROTEIN
FIG. 2. Enzyme dilution curve. Homogenates from the nucleus raphe dorsalis were prepared as described in Methods and incubated for 5-HTP decarboxylase activity for 15 min. Enzymatic activity is linearly related to the amount of tissue assayed.
FIG.3. Linearity of recovery of exogenous serotonin from brain tissue. Serotonin standards were prepared and added to aliquots of brain supernatant (20 pg protein each) as described under Methods. Results are expressed in c.p.m. after substraction of the blank (400 c.p.m.). Average values were 6025 c.p.m. per ng of serotonin.
activity was found between the most active (nucleus raphe dorsalis) and the least active (corpus callosum, optic tract) regions. DISCUSSION
A single enzyme, aromatic-L-aminoacid decarboxylase, is currently regarded as responsible for the decarboxylation of several aminoacids, including 3,4dihydroxyphenylalanine (DOPA) (EC 4.1.1.26, dopa decarboxylase) and 5-hydroxytryptophan (5-HTP) (EC 4.1.1.28, 5-hydroxy-L-tryptophan decarboxylase) (LOVENBERG et al., 1962; CHRISTENSON et al., 1972; LANCASTER et al., 1972; HOKFELTet al., 1973). Although there have been claims for the existence of more than one aromatic amino acid decarboxylase, this question is not pertinent to the present topic. The formation of serotonin in the rat brain after administration of exogenous 5-HTP is decreased in animals with lesions of the raphe system, indicating that the 5-HTP decarboxylation occurs predominantly in 5-HT containing nerve endings (KORFet al., 1974). In studies on serotonin metabolism, therefore, it is necessafy to utilize 5-hydroxytryptophan instead of DOPA as a substrate, and to use optimal assay conditions for the 5-hydroxy-~-tryptophandecarboxylase reaction (SIMSet al., 1973). In combination with microdissecting techniques, the method allows the determination of 5-hydroxy-~tryptophan decarboxylase activity in discrete areas of the rat brain. The results are in general agreement with earlier biochemical determinations of 5-hydroxy-~-tryptophan decarboxylase activity in various regions of the rabbit (MCCAMAN et al., 1965), cat (KUNTZMAN et al., 1961), and rat brain (SIMS& BLOOM, 1973). Recently, HOKFELT et a!. (1973) reported the localization of DOPA-5HTP decarboxylase activity in the rat brain by means of immunohistological techniques,
J. M. SAAVEDRA
588
TABLE1. TYPICAL ASSAY pg protein
Tissue Whole brain Nucleus raphe dorsal15
per assay 21.
208 5* 5* 2 0.5
5-HYDROXY-L-TRYPTOPHANDECARBOXYI-ASE ACTIVITY
Radioactivity in blanks (c.p.m.)
Radioactivity in 1 ng 5-HT standard (c.p.m. over blanks)
Radioactivity in cxperiments (c.p.m. blanks)
405 440 805
6,080 6,100 5,992
20,055 z0,m 37,950
800
6,055
6,010 6, I 3n 6,300 6.010 6,060
650 410
0-1' 01'
5
Median eminence
FOR
285 250 505
39,400 1s.m 4,010 805 690 8,245
5-HTP dccarboxylase acthit?. Pmol cerntonin'ug protein per 11 cd incuh,itim 3.56 3.83 28.75 2954 29.80 29 70 29 00 26.06 6.17
Tissues were prepared as described in Methods, and incubated 15 min for 5-HTP decarboxylase activity. 5-HTP decarboxylase activity is measured indirectly through conversion of the decarboxylation product, serotonin. to its derivative melatonin, as described in Methods. * Duplicate samples.
and concluded that this enzyme corresponded with lase and tryptophan hydroxylase in these regions the distribution of cell bodies containing either dopa- supports the hypothesis of the local synthesis of seromine or SHTP, and that it was not localized in other tonin and its involvement in neuroendocrine regulaneuronal or extraneuronal systems. tion. The results also indicate a high degree of localizaWith the exception of the raphe nuclei, the area tion for 5-hydroxy-~-tryptophandecarboxylase ac- postrema is the region of the rat brain which has tivity in the rat brain, with a good correlation be- the highest 5-hydroxy-~-tryptophandecarboxylase actween enzyme activity and serotonin levels (PALKO- tivity. This observation confirms earlier reports (McVITS ef ul., 19746: SAAVEDRA et a/., 1974a, b) in the CAMAN e t a/., 1965) and supports the view that seroregions examined. tonin plays a significant role in the physiology of this The presence of serotonin and related enzymes in structure. certain brain areas is of special interest. Relatively high The existence of 5-hydroxy-~-tryptophandecarboxlevels of serotonin have been reported for some hypo- ylase activity in the locus coeruleus is worthy of note. thalamic nuclei (nucleus suprachiasmatis, nucleus Serotonin has also been detected in this structure arcuatus), and for the median eminence (SAAVEDRA (PALKOVITS e t a/.. 19720) as well as in other areas et a/., 1974b). Recently, tryptophan hydroxylase has of the brain stem rich in catecholamine containing also been found in these areas (KIZER et a[., 1975). cell bodies (SAAVEDRA,unpublished observation). The coexistence of 5-hydroxy-~-tryptophandecarboxy- Thus, innervation of these catecholamine rich areas by serotoninergic fibers seems probable. The present technique allows the precise biochemiTARLF 2. DISTRIBUTION OF 5-HYDROXY-L-TRYPTOPHAN cal quantitation of 5-hydroxy-~-tryptophandecarboxDECARROXYLASE ACTIVITY IN DISCRETE AREAS AND NUCLEI OF THE RAT BRAIN ylase activity in p g amounts of brain tissue. Together with the microassay for serotonin and tryptophan 5-HTP dccarboxylase actlvny hydroxylase, it offers a valuable tool for the study (pmo1:pg protein per of serotonin metabolism in discrete areas of the brain. Area h of incubation k s.t M Number of samnles ~~
~
~
Nucleus raphc dorsalis* h'uclrui centralis superior' Area postrema' Substantla nigra' Nucleus suprachiasmalist Caudate+ Locus cocruleus: Nucleus arcua tm: Median eminencc Tuberculum o l f a c t u r t u m ~ Cerebellum: Nucleus seplalis medialis: Medial forebrain bundls: Parietal cortext tfippnc.mpuc: Cingulatc cortex: Optic tract: Corpus callosum: -
4-0
(12)
15.6 t 2.1 13.8 i 1.8 13-1 k 2.0 1i.n rt 1.7 9 9 k 1-0 8.9 ? 2.1 6 2 f 0.7 5.3 f 1-2 4 9 f 0.9 4.7 0.8 4.3 i 0 7 3.8 ? 0 4 2.8 0.2 2.7 i 0.3 2.6 0.4 1.6 i 0-5 1.1 i 0-3
(11) (9)
27.0
*
*
+
(6)
(11)
(8) (6) (9) (I1)
(I11 (8) (12) (81 (8) ( 61 (12)
(6) (6)
Tissues werc dissected and analyzed as explained in Material and Methods. Each sample represents tissue from one rat. assayed individually (0024.1 mg protein). Diflerent vol of homogenate were used to produce amounts of serotonin in the linear range of the assay: *150 PI, T50 pl. Incubation time for 5-HTP-decarboxylase activity was I5 min.
REFERENCES J. G., DAIRMAN W. & UNDENFRIEND S. (1972) CHRISTENSON Proc. nafn. Acad. Sci., U.S.A. 69, 343-347. HOKFELT T., FUXEL. & GOLDSTEIN M. (1973) Bruin Rex 53, 175180. KIZERJ. S., ZIVINJ. A.. SAAVEDRA J. M. & BROWNSTEIN M. (1975) J . Neurochem. 24, 779-785. KORFJ., VENEMAK. & POSTEMA F. (1974) J . Neurochem. 23, 249-252. KUNTZMAN R., SHOREP. A., BCK~DANSKI D. & BRODIEB. B. (1961) J . Neurochem. 6, 226232. LANCASTER G. A. & SOURKES T. L. (1972) C m . J . Biochem. 50, 791-797. LOVENBERG W., WEISSBACH H. & UDENFRIENDS. (1962) J . hiol. Chem. 237, 89-93. LOWRY0. H., ROSEBROUGH N. J.. FARRA . L. & RANDALL R . J. (1951) 3.biol. Chem. 193, 265 275.
5-IIT'P decarboxylase microassay and distribution in brain
MCCAMAN R. E., MCCAMAN M. W., HUNTJ. M. & SMITH M. S. (1965) J . Neurochem. 12, 15-23. PALKOVITS M., BROWNSTEIN M., SAAVEDRA J. M. & AXELROD J. (1974a) Brain Res. 77, 137-149. PALKOVITS M., BROWNSTEIN M. & SAAVEDRA J. M. (19746) Brain Res. 80, 237-249. PALKOMTS M., SAAVEDRA J. M., KOBAYASHI R. M. & M. (1974~)Brain Rex 79, 443450. BROWNSTEIN SAAVEDRAJ. M., BROWNSTEINM. & AXELRODJ. (1973) J. Pharmac. exp. Ther. 186, 508-515.
589
SAAVEDKA J. M., BROWNSTEIN M. & PALKOVITS M. (1974aj Brain Res. 79, 437441. SAAVEDRA J. M., PALKOVITS M., BKOWNSTEIN M. & AXELROD J. (1974b) Bruin Res. 77, 157-165. SIMSK. L. & BLWM F. E. (1973) Brain Res. 49, 165S175. SIMSK. L., DAVIS G. A. & BLOOMF. E. (1973) J , Neurochem. 20, 449464. SYNDERS. H. & AXELRODJ. (1969) Biocheni. Pharmac. 13, 805-806.
