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The microiontophoretic administration of putative neuromodulators (acetylcholine, norepinephrine, dopamine, 2-phenylethylamine, tryptamine, histamine) triggered firing or inhibited on-going activity in isolated frog sciatic nerves.
We have reported that in rats with D-galactosamine-induced liver injury, the serum level of indoleacetic acid (IAA), a metabolite of tryptophan (TRP), increases before the increase in serum transaminase activity. To determine whether this IAA is derived from hepatocytes, isolated hepatocytes were treated with D-galactosamine and loaded with TRP, and the changes in TRP and IAA levels in the culture supernatant of the isolated hepatocytes were measured at various time intervals. As a result, IAA level in the culture supernatant of hepatocytes treated with D-galactosamine and loaded with TRP significantly increased in a time-dependent manner. This indicates that in D-galactosamine-induced liver injury, a metabolic pathway which produces IAA from TRP through tryptamine is present in hepatocytes.
The aim of the present study was to further characterise the noradrenaline and 5-hydroxytryptamine [5-HT] transporters in rat lungs by examining the efflux of noradrenaline and 5-HT, respectively. Lungs from rats were isolated and perfused via the pulmonary artery. After loading the tissue with 3H-5-HT or 3H-noradrenaline the efflux of the relevant amine from the lungs was examined for 15-25 min. The rate constant for efflux of 3H-5-HT increased by 81% when Na+ ions were removed from the perfusion solution; increased gradually when a selective 5-HT transporter inhibitor, 200 nM citalopram, was added to the perfusion solution for the final 6 min of efflux; and increased markedly and rapidly when substrates of the 5-HT transporter, tryptamine (18 microM) and 7-methyltryptamine (12 microM), were added for the final 6 min of efflux. These effects of the substrates were abolished by 1 microM citalopram, but were not significantly affected by 1 microM desipramine, a selective uptake, inhibitor. On the other hand, the previously described substrate-induced increase in the rate of efflux of noradrenaline was significantly reduced by desipramine but was unaffected by citalopram. The results show that efflux of 5-HT is mediated only by the 5-HT transporter, with no significant contribution of uptake1, and efflux of noradrenaline from rat lungs is mediated only by uptake1 and not by the 5-HT transporter. The effects of dopamine on the efflux of noradrenaline over a concentration range of 100-600 nM were investigated and the results showed that 50% of the maximal increase in the rate of efflux occurred at a concentration of 275 nM. This value did not differ from the Km for uptake of dopamine. This result implies that the only factor affecting the substrate-induced increase in noradrenaline efflux is the affinity of the substrate for uptake1. The efflux of noradrenaline was also examined in the absence and presence of two concentrations of desipramine (0.35 and 1.5 microM). Analysis of these results showed that uptake1 contributed approximately 81% and diffusion 19% to the total efflux of noradrenaline and that 90% of the total noradrenaline efflux was subject to reuptake by uptake1 into the pulmonary endothelial cells.
In vertebrates, the nocturnal rise in pineal organ and retinal melatonin synthesis results from the increase in the activity of the serotonin N-acetyltransferase (NAT), a cAMP-dependent enzyme. In the fish pineal organ in culture, light and temperature act in a similar manner on cAMP content and NAT activity. It is not known whether the effects of temperature are mediated through cAMP or through modifications of NAT kinetics. The present study was designed: (1) to find out whether NAT activity from pineal organ homogenates is similar to NAT activity from pineal organs in culture, with regard to variations in temperature, and (2) to compare NAT activity from the pineal organ and the retina. Pineal organ and retinal NAT activity increased linearly with protein concentrations. Higher activities were obtained with 0.2 mol/l of phosphate buffer, pH 6. Higher molarity or a higher pH induced a decrease in retinal and pineal organ NAT activity: retinal NAT was more sensitive than pineal organ NAT to changes in molarity, whereas the opposite held true as far as pH was concerned. Pineal organ and retinal NAT obeyed the Michaelis-Menten equation with respect to increasing concentrations of acetyl-coenzyme A. With increasing concentrations of tryptamine: (1) pineal organ NAT activity increased in a manner suggesting positive co-operativity, (2) retinal NAT displayed, after an initial increase, inhibition by substrate. The kinetics of the reactions were temperature dependent. Maximal activities were reached at 18/20 degrees C in the pineal organ and at 37 degrees C in the retina. The present study is the first to describe the optimum conditions for the assay of NAT activity in homogenates from the retina of fish and from the pineal organ of poikilotherms, and also the first to compare some characteristics of NAT activity from these two analogous organs. Our results suggest that the effects of temperature on melatonin production are mediated, at least in part, through modifications of NAT kinetics. Future studies will aim to clarify whether the activities measured in the pineal organ and retinal homogenates reflect the presence of one or of several enzymes.
Reaction of tryptophan, tryptophan methyl ester and tryptamine with methylglyoxal (a physiological alpha-oxoaldehyde), which resulted in beta-carboline formation, showed that this type of nonenzymatic (Maillard) reaction could spontaneously occur in living organisms or during commercial or domestic food processing and storage.
A highly efficient ion-pair extraction technique for the isolation of tryptamine, 5-hydroxytryptamine, and their mono- and N,N-dimethylated derivatives from aqueous media is described. The technique has been used to isolate nanogram quantities of both N, N-dimethyltryptamine and 5-hydroxy-N, N-dimethyltryptamine from large volumes of urine. A rapid and efficient normal-phase liquid chromatographic procedure has also been developed for the subsequent purification of indolealkylamines isolated from urine. The methods described have been used in the measurement of the urinary excretion of 5-hydroxy-N, N-dimethyltryptamine. Analyses were performed by liquid chromatography using a cation-exchange column with online fluorescence detection. Further characterization was achieved by stop-flow spectroscopic analysis of the LC eluant.
An assay is described for the enzyme tryptophan decarboxylase from plant cell suspension cultures. It is based on the fluorometric detection of tryptamine by HPLC on a LiChrosorb RP-8 Select B column. Tryptophan decarboxylase from Catharanthus roseus was induced by transferring 14-day-old cells into an induction medium. Optimum activity was found 2 days after transfer, the increase being 5- to 10-fold. When kept at -15 degrees C the crude enzyme lost half its activity in about 7 days. The rate of the decarboxylation reaction was linear for at least 3 h at 35 degrees C.
A sensitive, rapid, and economical method has been developed for determination of serotonin N-acetyltransferase activity from a variety of enzyme sources. The assay is based upon separation and detection of N-acetyltryptamine formed from tryptamine and acetyl coenzyme A, by means of high-performance liquid chromatography with either electrochemical or fluorometric detection. The limit of sensitivity with both detection methods is less than 20 pmol of N-acetyltryptamine formed per sample. A method for synthesis of N-acetyltryptamine, used as an external standard in the assay, is described.
In order to investigate the mode of interaction between the N-quarternized cytosine base and the aromatic amino acid, the crystal structure of the 3-methyl-cytidine-5'-monophosphate:tryptamine complex was analyzed by X-ray diffraction. The complex crystals were stabilized by extensive hydrogen bond formations in which eight independent water molecules per complex pair participated. A prominent stacking interaction, characterized by a parallel alignment of both rings with a separation distance of ca. 3.4 A, was observed between the cytosine base and the indole ring. Combining the present results with X-ray crystallographic data on the adenine--and guanine--aromatic amino acid interactions, we summarize the structural characteristics observed in the stacking interaction of the N-quarternized nucleic acid base with the aromatic amino acid and discuss their biological implications, especially in connection with the significance of N-protonation of nucleic acid base for selective recognition by protein.
The gene for strictosidine synthase (str1), the enzyme which catalyzes the stereospecific condensation of tryptamine and secologanin to form the key indole alkaloid 3 alpha(S)-strictosidine has been isolated from genomic libraries prepared from Rauvolfia serpentina (India) and from Rauvolfia mannii (West Africa). The gene, str1, contained no introns and showed 100% nucleotide sequence homology over 1180 bp, encompassing the entire reading frame, between the two species. Transcription of the R. serpentina gene was found to start 81 nucleotides upstream from the AUG (26 nucleotides downstream from the TATA box). Transient expression assays in Nicotiana plumbaginifolia protoplasts of the R. serpentina str1 5'-noncoding region fused to the beta-glucuronidase reporter gene revealed promoter activity equivalent to 4 +/- 2% of that of 35 S CaMV promoter control. A series of truncated segments of the str1 promoter region indicated the presence of three areas of slight, but reproducible, negative control. Gel retardation assays demonstrated that several regions of the 5'-flanking sequences specifically bound nuclear protein from R. serpentina and that at least one region does not bind R. mannii nuclear protein. A survey of the expression of str1 in the R. serpentina plant suggested that strictosidine synthase poly(A)+ RNA was present predominantly, but not exclusively, in the root. This result correlated well with the distribution of both enzyme activity and indole alkaloids which were also predominant in the root, but, in general, distributed throughout the shrub.
The metabolism of alpha,alpha,beta,beta- tetradeutero -N,N -dimethyltryptamine ( D4DMT ) in rat brain in vivo as a function of time and dose was examined. Quantification of D4DMT and its respective deutero-metabolites was accomplished using gas chromatographic/mass spectrometric/selected ion monitoring/isotope dilution techniques. The results of this study indicate that D4DMT is metabolized to the corresponding deutero-N-methyltryptamine, tryptamine, 1,2,3,4-tetrahydro-beta-carboline, and 2-methyl-1, 2,3,4-tetrahydro-beta-carboline in rat brain. The subcellular distribution of D4DMT and the aforementioned metabolites is also reported.
beta-Carboline alkaloids are derived as a result of condensation between indoleamine (e.g. tryptamine) and short-chain carboxylic acid (e.g. pyruvic acid) or aldehyde (e.g. acetaldehyde), a reaction that occurs readily at room temperature. These compounds have been found endogenously in human and animal tissues and may be formed as a byproduct of secondary metabolism: their endogenous functions however, are not well understood. Indoles and tryptophan derivatives exhibit antioxidative actions by scavenging free radicals and forming resonance stabilized indolyl radicals. Harmane and related compounds exhibited concentration-dependent inhibition of lipid peroxidation (measured as thiobarbiturate reactive products) in a hepatic microsomal preparation incubated with either enzymatic dependent (Fe3+ ADP/NADPH) or non-enzymatic dependent (Fe3+ ADP/dihydroxyfumarate) oxygen radical producing systems. Alkaloids with hydroxyl substitution and a partially desaturated pyridyl ring were found to have the highest antioxidative potencies. Substitution of a hydroxyl group by a methoxyl group at the 6-position resulted in a decrease of greater than 10-fold in the antioxidative activities. Harmane showed high efficacy in an enzymatic system but low efficacy in a non-enzymatic system. The antioxidative effects of harmane in the former system may be attributed to its ability to inhibit oxidative enzymes in the microsomal system. These results suggest that beta-carbolines may also serve as endogenous antioxidants.
Cell-free preparations from superior and inferior colliculi of very young rats (1-3 days old) contained adenylate cyclase systems which were highly responsive to serotonin. The response to serotonin declined markedly during early development and was very low at maturity. Adenylate cyclase activity in the 10,000 times g particulate fraction from colliculi of newborn rats was significantly stimulated by 0.05 muM serotonin. Half-maximal activation was produced with less than 1 muM serotonin. Maximal stimulation of collicular adenylate cyclase was about 80% above basal enzyme activity and occurred with approximately 50 muM serotonin. Tryptamine and several derivatives of serotonin produced responses which were comparable to that obtained with serotonin; 5-methoxytryptamine was uniformly the most active compound tested. Norepinephrine or dopamine produced much smaller increases in adenylate cyclase activity. Stimulation of collicular adenylate cyclase by serotonin was significantly but incompletely blocked by serotonin antagonists, including d-lysergic acid diethylamide (d-LSD), 2-bromo-d-lysergic acid diethylamide, methysergide, 1-methyl-8 beta-carbobenzyloxy-aminomethyl-10 alpha-ergoline and cyproheptadine. Chlorpromazine also produced partial blockade. In contrast, l-lysergic acid diethylamide, haloperidol, propranolol, phenoxybenzamine and morphine were ineffective as serotonin blocking agents. Of the compounds which produced a partial blockage of serotonin action, d-LSD, cyproheptadine and chlorpromazine were themselves capable of stimulating adenylate cyclase activity. These results are consisent with the existence of multiple receptors in rat brain which are capable of interacting with indoleamines.
Experiments have been performed on pentobarbitone anesthetized or decerebrated rats. The nature of the synchronous rhythmical activity which occurs in the inferior olive following the electrophoretic or systemic administration of harmaline, harmine, dihydro-beta-erythroidine and various other compounds, is described. Harmine was shown to reduce the late phase of biphasic unitary action potentials and to evoke massed synchronous rhythmical activity on which the units were superimposed. The beta-carboline was more effective than ACh or DL-homocysteate (DLH) in increasing cell discharge rates. Synchronized rhythmical activity was recorded more than 500 mum from the site of ejection of the rhythm-inducing drugs. Developed rhythmical activity reduced the size of antidromic field potentials, but antidromic invasion could reset the rhythm of submaximal rhythmical activity. The effects of ACh and DLH, glycine, GABA, NA, DA and 5-HT were tested on established rhythmical activity. Of these, 5-HT was the only compound which almost invariably antagonized the rhythm. A number of tryptamine derivatives and reported 5-HT antagonists, as well as parachlorophenylalanine, have been tested, but the results were largely inconclusive. The hypothesis is advanced that the drug-induced rhythm results from the inhibition of a tonic inhibitory serotonergic input. This antagonism releases an innate tendency of olivary cells to discharge both rhythmically and synchronously.
The fate of serotonin was studied in several tissues of the marine mollusc, Aplysia californica. When isolated nervous tissue was bathed in [3H]serotonin, two radioactive derivatives were formed; both appeared to be sugar conjugates: the first, possibly of glucuronic acid, and the second, of a more complex sugar moiety. When [3H]serotonin was injected directly into cell bodies of identified neurons, both serotonergic and non-serotonergic, only the conjugate which behaved as the glucuronide was formed. [3H]Serotonin was also converted only to this substance during incubation with isolated heart, kidney and hemolymph. Metabolic activity of the blood resided within cellular elements. No evidence of oxidative deamination was found in any tissue. In contrast to serotonin, however, [3H]tryptamine was readily oxidized to indoleacetic acid by nervous tissue.
It is demonstrated that the increased tryptaminuria which follows alcohol ingestion can be abolished by the concurrent ingestion of alkali even when the amount of alkali consumed is insufficient to increase urinary pH to 6.5 (above which pH a fall in urinary tryptamine may be anticipated, from previous studies, to occur). It is suggested that this increased tryptaminuria is largely if not wholly dependent on the metabolic acidosis induced by alcohol which the concurrent ingestion of alkali abolishes.
The midpontine decerebrate dog, immobilized with gallamine, was used to determine the changes in the transcallosally evoked potential (TEP) produced by intravenous infusions of various drugs. A total of 50 TEPs, recorded from the g. ectolateralis, was computer analyzed before, during and after administration of the drugs. Changes in the TEP were also correlated with changes in the EEG recorded from the g. ectolateralis. The EEG was analyzed by inspection and amplitude integration (electrogenesis). LSD (30 microng/kg) significantly depressed the TEP, and the effect persisted for at least 80 min. DMT (1 mg/kg) caused a significant and reversible increase in the amplitude of the TEP. LSD and DMT reduced the alpha activity of the EEG and enhanced the amplitude of the low-frequency waves. DMT produced a significant and LSD a marginal increase in electrogenesis. Tryptamine (10 and 20 mg/kg), mescaline (6 mg/kg), methoxamine (0.88 mg/kg) and apomorphine (5 mg/kg) had no significant effect on the TEP or EEG. These results suggest that depression of the TEP is not related to spinal reflex facilitation in the dog or hallucinogenic activity in man.
The effect of ergotamine on the isolated rat stomach and its influence on the response to ACh and 5-HT were investigated. The log dose-response curve of ergotamine was bell-shaped. Extension of the incubation time of ergotamine resulted in a parallel shift to the left of the curve. The response to ergotamine was inhibited by methyserigide and piperoxan. Incubation with ergotamine resulted in a decrease of the pD2-value of 5-HT together with a marked suppression of the maximum of the 5-HT curve. The response to ACh was affected in accordance with the prediction of an action of ACh and ergotamine on different receptors. The prolonged receptor stimulation by 5-HT or ACh resulted in a decrease of the apparent affinity towards their receptors. Incubation with ACh resulted in a parallel shift to the right of the 5-HT curve. However no inconsistency with the theoretical prediction of an action on separate receptors was observed with the ACh curve in the presence of 5-HT. It is concluded that ergotamine is a partial agonist on the D-tryptamine receptors of tbe isolated rat stomach. The marked decrease of the maximum of the 5-HT curve by ergotamine is probably caused by the slowly reversible character of its antagonism. The parallel shift to the left of the ergotamine curve with the extension of the incubation time and the persistence of its antagonism both are probably caused by a slow diffusion into and from the biophase.