STIMULATION OF BRAIN SEROTONIN SYNTHESIS BY DIBUTYRYL-CYCLIC AMP IN RATS

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Cyclic AMP and dibutyryl-cyclic AMP, a derivative of cyclic AMP resistant to phosphodiesterase inactivation, were injected into the lateral ventricles of rats. These nucleotides did not change the level of brain 5-HT but increased the brain level of
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  Journal of Neurochemistry, 1971, Vol. 18, pp. zyxwvut 191 to 1196. Pergamon Press. Printed in Great Britain. zyx STIMULATION OF BRAIN SEROTONIN SYNTHESIS BY DIBUTYRYL-CYCLIC AMP IN RATS A. TAGLIAMONTE, . TAGLIAMONTE, . FORN, J. PEREZ-CRUET, . KRISHNA nd Laboratory of Chemical Pharmacology, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014 zyx  Received 9 September 1970. Accepted 7 October 1970) Abstract-Cyclic AMP and dibutyryl-cyclic AMP, a derivative of cyclic AMP resistant to phosphodiesterase inactivation, were injected into the lateral ventricles of rats. These nucleotides did not change the level of brain 5-HT but increased the brain level of its principal metabolite, 5-hydroxyindoleacetic acid. Cyclic AMP was less potent than dibutyryl-cyclic AMP. Butyrate and 5’-AMP were inactive. The effect of dibutyryl cyclic AMP on 5-HT metabolism was studied both zyxwv n vivo and in vitro. The rate of synthesis of 5-HT was measured by the rate of accumulation of 5-hydroxyindoleacetic acid after the transport of this acid out of the brain was blocked with probenecid. The rate of synthesis of brain 5-HT increased from 038 pg/g/h in control rats to 065 pg/g/h after dibutyryl-cyclic AMP. In addition cyclic AMP and dibutyryl-cyclic AMP markedly increased brain tryptophan, while AMP was inactive. Since brain tryptophan hydroxylase has a K,,, for its substrate that is much higher than the concentrations of tryptophan normally present in the brain, it is likely that the increase in the rate of synthesis of brain 5-HT is secondary to the cyclic AMP induced increase in the levels of brain tryptophan. In uitro studies revealed that dibutyryl-cyclic AMP increased the uptake of radioactive labelled tryptophan into slices of rat brain stem and the formation of 5-HT and 5-hydroxyindoleacetic acid. BRAIN denyl cyclase and phosphodiesterase, which catalyse the formation and the inactivation of 3’,5’-adenosine monophosphate (cyclic AMP) are concentrated in the synaptosomal fraction of the tissue homogenates (DE ROBERTIS, ODRIGUEZ E LORES RNAIZ, LBERICI, UTCHER nd SUTHERLAND, 967; WEISS nd COSTA, 968). Thus, the cyclic AMP system is presumably located within the nerve endings where monoamine-containing vesicles are also present (DE ROBERTIS, 964), although the possibility that it is also present postsynaptically is not excluded. These considerations and the fact that cyclic AMP mediates enzyme synthesis (WICKS, 969; DE COM- BRUGHE, PERLMAN, ARMUS nd PASTAN, 969) and activation (SUTHERLAND, OBISON and BUTCHER, 968 zyxwv   ROBISON, UTCHER nd SUTHERLAND, 968) in various biological systems and stimulates melatonin and 5-HT synthesis in the pineal gland in vitro (SHEIN nd WURTMAN, 969; KLEIN, ERG, WELLER nd GLINSMANN, 970~; LEIN, BERG nd WELLER, 970b) prompted us to study the effects of cyclic AMP on the rate of synthesis of brain monoamines. We now report that cyclic AMP and N602’- dibutyryl-cyclic AMP (dibutyryl cyclic AMP) stimulate the synthesis of brain 5-HT in rats. Cyclic AMP is less potent than its dibutyryl derivative. G. L. GESSA MATERIALS AND METHODS Chemicals. N602’-Dibutyryl adenosine-3‘,5’-cyclic phosphate; adenosine 3’,5’-cyclic phosphate and adenosine-5’-monophosphate were purchased from CalBiochem; probenecid from Merck and Co.; ~[G-~Hltryptophan G) (3.4 Ci/mmol) from Amersham/Searle. Experiments in vivo. Male Sprague-Dawley rats weighing 2W250 g were used. Since cyclic AMP and its derivatives do not cross the blood-brain barrier (GESSA, RISHNA, ORN, TAGLIAMONTE nd Abbreviations used: 5-HIAA, 5-hydroxyindoleacetic acid. 1191  1192 A. TAGLIAMONTE, . TAGLIAMONTE, . FORN, . PEREZ-CRUET, . KRISHNA nd G. L. GESSA BRODIE, 970), they were injected into the cerebrospinal fluid. Rats were lightly anaesthetized with ether, rapidly fixed in a stereotaxis apparatus and injected into a lateral ventricle by a 25 gauge needle inserted through the skull. The needle was rapidly removed and the rats recovered from anaesthesia in about zyxwvut   min. The volumes of the injections were 5 zyxw l and the pH 7.4. The animals were killed by decapitation and the brains were quickly removed, frozen in solid CO, and stored at -20°C until analysed. Blood collected in centrifuge tubes containing heparin was immediately centrifuged and the plasma stored at -20°C until analysed. Simultaneous determinations of 5-HT and 5-hydroxy- indoleacetic acid (5-HIAA) were done in each whole brain. Each brain was homogenized in 10 z l z .1 N-HCI containing zyxwvu 3 (w/v) ascorbic acid. Proteins were denatured by addition of 1 ml of a 10 (w/v) ZnSO, solution followed by 1 ml of 1 N-NaOH. The mixtures were then centrifuged at 10 OOO for 30 min. 5-HIAA extraction. The supernatant layer was saturated with solid NaCI, acidified with 1 nil of 1 N-HCI and washed with zyxwvu 5 ml benzene to remove indoleacetic acid, which interferes with the fluores- cence assay of 5-HIAA.Then 5-HIAA was extracted into 10 ml of freshly distilled n-butylacetate.The n-butylacetate extract containing 5-HIAA was washed with 5 ml of a 0.1 N-HCI solution saturated with NaCl and the 5-HIAA was re-extracted into 2 ml of 0.1 M-EDTA, pH 7. The 5-HIAA was measured in an Aminco Bowman spectrophotofluorometer; 330 and 385 nm were respectively the excitation and fluorescence maxima. 5-HT extraction. After the n-butylacetate had been removed, the remaining aqueous phase was adjusted to pH 8 with ml of 1 M-K~CO~ nd ml 0.5 M-borate buffer, pH 10. The 5-HT was ex- tracted into 15 ml of redistilled n-butanol saturated with NaCI. Two volumes of heptane were added to the butanol phase and the 5-HT was returned into 2 ml of 1.0 N-HCI. 5-HT was then assayed fluorometrically. according to BOGDANSKI, LETSCHER, RODIE nd UDENFRIEND 1956). Tryptophan zyxwvut nd tyrosine. These were extracted from brain and plasma, according to the method of COSTA, PAND. GROPPETTI, LGERI nd NEFF 1968). Tryptophan was converted to norharman and assayed fluorometrically by the method of DENKLA nd DEWEY 1967). Tyrosine was assayed fluorometrically according to the method of UDENFRIEND, EISEACH nd BRODIE 1958). The rate of synthesis of brain 5-HT was calculated from the rate of accumulation of 5-HIAA after its transport out of the brain was blocked with probenecid (NEFF, TOZER nd BRODIE, 967). Experiments in vitro. Male Sprague-Dawley rats (200-250 g were killed by decapitation in a cold room (30 C), their brains removed, the brain stems separated and rapidly immersed in ice-cold Krebs-Ringer buffer with the following composition: 119 mM-NaCI, 4.8 mM-KCI, 25 mM-NaHC03, 1.2 mM-KH,PO,, 2 mM-CaCI,, 2.4 mM-MgS0, and 0.1 glucose. The brain stems were sectioned sagitally in two equal halves. Each half was cut into 250 pm thick slices with a Sorval Tissue Sectioner and two halves from different animals were placed in an incubation tube containing 5 ml of Krebs- Ringer medium and various concentrations of dibutyryl cyclic AMP. The other halves of the two brain stems were placed in another tube and served as the paired controls. The tubes were preincubated at 37°C for 5 min, then 30 pCi of randomly labelled~[~H]tryptophan were added to give a final concentration of lo4 M. The tubes were gassed with a 95 02-5 C02 mixture, sealed and incubated at 37°C for various times. Labelled and unlabelled tryptophan and radioactive 5-HIAA were measured both in the incubation mediumand in the slices. When the content of the labelled compounds was measured in the slices, they were washed six times with 15 ml of cold Krebs-Ringer solution. Then 5 ml of 0.4 HCIO, containing 50 pg of 5-HT and 20 pg of 5-HIAA were added as cold carriers. The slices were homogenized with a glass homogenizer and tryptophan, 5-HT and 5-HIAA were assayed. Tryptophan and 5-HIAA were extracted as described above except that the n-butyryl acetate phase, containing labelled 5-HIAA, was washed four times, instead of once, with 0.1 N-HCI solution. Labelled 5-HT was extracted as described by COSTA t zyx l. (1968). RESULTS In animals receiving dibutyryl-cyclic AMP there was a dose-dependent increase in the level of brain 5-HIAA, but the concentration of brain 5-HT was not changed Table 1). Cyclic AMP, although less potent than its dibutyryl derivative, also in- creased 5-HIAA levels without modifying those of 5-HT. On the other hand, 5 -AMP and butyrate modified neither the 5-HT nor the 5-HIAA levels in brain. The presence of the cyclic AMP molecule thus is essential for the observed effect. The accumulation of 5-HIAA in the brain produced by cyclic AMP and its derivative was not related to the behavioural changes or to changes in body temperature induced by these nucleo- tides (GESSA zyxwvut t al. 1970). In fact these changes did not always occur in animals  Stimulation of 5-HT by cyclic AMP zyx 193 z ABLE -EFFECT OF THE INTRAVENTRICULAR INJECTION OF DIBUTYRYL-CYCLIC AMP AND CYCLIC AMP ON THE 5-HT AND 5-HIAA CONTENT OF THE RAT BRAIN Treatment zyxwvu   5-HT 5-HIAA zy   @g/g) MId Saline 21) 0.65 zyxw   .01 0.81 zyx   01 Dibutyryl-cyclic AMP 8) zyxwvut 00 pg 0.63 * 0 02 1.47 i .02 <0.001 Dibutyryl-cyclic AMP 8) zyxwvu 00 PLg 060 .01 1.30 003 <0.001 Dibutyryl-cyclic AMP 8) zyxwv 0 pg 0.69 0.03 1.20 03 <0.001 Cyclic AMP 8) 500 Pg 0.60 * 004 1.35 .06 <OW1 Butyrate 6) 100 Pg 0.71 .03 0.78 .02 n.s. AMP 500 pg 6) 0.68 i .03 0.76 rt 0.08 n.s. * Compounds were given 2 h before killing. Each value is the average S.E.M. of the number of determinations indicated in brackets, n.s. = not significant. TABLE EFFECT OF THE INTRAVENTRICULAR INJECTION OF DIBUTYRYL-CYCLIC AMP ON THE 5-HUA CONTENT OF RATS TREATED WITH PROBENECID Treatments* htraperitoneally In the lateral 5-HIAA P ventricle rglg None Saline 0.82 .01 None Dibutyrylcyclic AMP 1.30 .02 c 0.001 100 rglg Probenecid Saline 1.20 0.03 <0.001 250 mgk 250 mglkg 100 Pglg Probenecid Dibutytyl-cyclic AMP 2.20 04 < 0001 * Treatments were given at the same time 60 min prior to killing. Each value is the average S.E.M. of at least six determinations. TABLE .-EFFECT OF THE INTRAVENTRICULAR INJECTION OF DIBUTYRYL-CYCLIC AMP ON THE TRYPTOPHAN LEVELS OF THE RAT BRAIN Treatment in zyxwvutsrq l Brain Brain intraventricularly tryptophan P tyrosine P pgig) pglml) Saline 4.45 0.06 11.91 0.34 Dibutyryl-cyclic AMP 11.17 rt 008 <0901 23.6 .80 <0401 AMP 500 pg 5.11 1.20 n.s. 12.14 .60 ns. 100 Pg Each value is the average .E.M. of at least six determinations, ns. = not significant. Animals were killed 2 h after treatments.  1194 A. TAGLIAMONTE, . TAGLIAMONTE, . FORN, PEREZ-CRUET, zyx   KRISHNA nd G L. CESSA z injected with cyclic AMP or with a dose of dibutyryl-cyclic AMP as low as 50 pg, and yet the brain levels of 5-HIAA were in all instances increased. These results suggested that the nucleotides either affect the transport of 5-HIAA from the brain or increase its rate of formation, causing a new steady state between the rate of formation and efflux of this metabolite. In order to clarify this, transport of 5-HIAA from the brain was blocked by the administration of probenecid (250 mg/kg intraperitoneally) and the rates of accumulation of 5-HIAA in brain were compared in rats injected with either dibutyryl-cyclic AMP or saline. Probenecid raised 5-HIAA level at a linear rate for 90 min, both in animals treated with saline and in those treated with dibutyryl-cyclic AMP. However, as shown in Table 2 the rate of 5-HIAA synthesis calculated from the rate of 5-HIAA accumulation during the first hour was 0.38 pg/g/h in control animals and 0.65 pg/g/h in animals treated with dibutyryl- cyclic AMP indicating that the rate of 5-HT synthesis is also increased by the same extent (NEFF et al. 1963). Since tryptophan hydroxylase, the rate-limiting step in 5-HT synthesis, has a K,,, for its substrate much higher than the concentration of tryptophan normally present in brain (JEQUIER, ORENBERG nd SJOERDSMA, 967 z   MCGEER, ETERS nd MCGEER, 968) the rate of synthesis of brain 5-HT is dependent upon the availability of the substrate as well as on the activity of the enzyme (GREEN, GREENBERG, RICKSON, AWYER nd ELLISON, 962). We therefore studied the effect of dibutyryl-cyclic AMP on the concentration of brain tryptophan. As shown in Table 3, dibutyryl-cyclic AMP increased the content of tryptophan in brain by about 140 per cent. The tyrosine level in brain was also increased by about 100 per cent. In order to clarify the mechanism by which dibutyryl-cyclic AMP increases brain tryptophan, ~[G-~HItryptophan zyxw 500 pCi/kg, 20 pg/kg) was injected intravenously to a group of 12 rats. Two hours later the animals were divided into two equal groups and injected intraventricularly with either saline or dibutyryl-cylic AMP (100 pg/rat), and specific activities of tryptophan in brain and in plasma were determined at 1 h after injection. In both groups of rats the specific activity of brain tryptophan was almost identical to that of plasma tryptophan (Table 4) indicating that the tissue amino acid is in equilibrium with the plasma amino acid and that the dibutyryl- cyclic AMP induced increase of brain tryptophan is probably due to increased transport of plasma tryptophan into the CNS. TABLE .-sPECIFIC ACTMTY OF PLASMA AND BRAIN TRYPTOPHAN IN CONTROL AND DIBUTYRYL-CYCLIC AMP TREATED RATS Tryptophan specific activity specific activity Injection Plasma Brain Saline 800 zyxw   16 780 zyx   8 Dibutyryl-cyclic AMP 760 20 830 2 Each value is the average + S.E.M. of four experiments. L[~H]- Tryptophan (500 pCi/kg) was injected intravenously 3 h before killing. Saline or dibutyryl-cyclic AMP (100 at) were injected into the lateral ventricle 1 h before killing. Experiments in vitro. Incubation of ~[~Hltryptophan ith slices from rat brain  Stimulation of 5-HT by cyclic AMP 1195 z stems resulted in the incorporation of ~[~Hltryptophan n the tissue and the formation of 5-HT and 5-HIAA (Table zyxw  . The addition of dibutyryl-cyclic AMP (10-3~) o the incubating medium increased labelled 5-HT by 25 per cent in the slices, labelled 5- HIAA in the slices by 47 per cent and in the medium by 30 per cent. In addition, dibutyryl-cyclic AMP increased the accumulation of both labelled and unlabelled tryptophan by about zyxwv 0 per cent in the slices. The effects of dibutyryl-cyclic AMP were maximal after 90 min incubation. TABLE .-EFFECT OF DIBUTYRYL-CYCLIC AMP ON UPTAKE OF TRYPTOPHAN AND ON FORMATION OF 5-HT ND 5-HIAA BY BRAIN SLICES Tryptophan in the Serotonin in the 5-HIAA in the 5-HIAA in the slices slices slices medium Sample c.p.m. Pg c.p.m. c.p.m. c.p.m. Control 105,300 zyxwvu   170: 4.5 zyxwv   0.1 3406 63 1026 81 1308 97 Dibutyryl- 146,470 260 5.7 zyxw .1 4221 33: 1473 87: 1705 11: cyclic AMP Brain stem slices were incubated in Krebs-Ringer bicarbonate at 37°C for 90 min in presence of [3H]-tryptophan 1V M). Dibutyryl-cyclic AMP (lW3 M) was added 5 min before the beginning of the incubation. The values are expressed per 100 mg tissue and they are average .E.M. of four determinations. * zyxwvut   < 0.01. DISCUSSION These results indicate that dibutyryl-cyclic AMP and cyclic AMP increase the rate of synthesis of brain 5-HT. Dibutyryl-cyclic AMP is more potent than the parent nucleotide presumably because it either resists hydrolysis by phosphodiesterase or penetrates the cell membranes more easily (SUTHERLAND zyx t al., 1968). Although the precise mechanism by which dibutyryl-cyclic AMP enhances the synthesis of 5-HT has not yet been identified, evidence presented in this paper suggests that it is not due to enhanced enzyme synthesis but to an increase in brain levels of tryptophan caused by an increased transport of plasma tryptophan into the brain. The effect is not specific for tryptophan, since the nucleotide also increases brain tyrosine levels. Thus, the mechanism, by which dibutyryl-cyclic AMP stimulates 5-HT synthesis in brain differs from that by which it stimulates 5-HT and melatonin synthesis in the pineal gland in vitro, where it does not increase the intracellular accumulation of tryptophan (SHEIN nd WURTMAN, 969). In agreement with the view that dibutyryl-cyclic AMP enhances the uptake of tryptophan within the nerve tissue and this produces an increased formation and metabolism of 5-HT, dibutyryl-cyclic AMP stimulates the uptake of tryptophan by brain slices. Similarly, dibutyryl-cyclic AMP has been shown to increase the accumulation of D-amino isobutyric acid and cycloleucine by liver slices of rats (KEWS, WOODCOCK nd HARPER, 970). Since the alterations of 5-HT metabolism were obtained using relatively high concentrations of nucleotides, we do not know whether the observed effects are pharmacological or if they reflect the action of the endogenous cyclic AMP. It is significant, however, that the effects are caused by the cyclic AMP but not by 5 -AMP, indicating that cyclic AMP and not its metabolites are involved.
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