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We described the production and characterization of antibodies against three important groups of neuro-active haptens, e.g., neurotransmitters and biogenic amines. First, from the tryptophane metabolic pathway: tryptamine, serotonin, 5-hydroxy-indole acetic acid, and melatonin. Secondly, the tyrosine metabolic pathway: tyramine, dopamine, dihydroxyphenyl acetic acid, and norepinephrine. Thirdly, antibodies against excitatory and inhibitory neurotransmitters: glycine, glutamate, glutamine, and GABA. Immunogenic conjugates were prepared after linking haptens to carrier proteins. Most antibodies displayed high specificity against corresponding neuro-active haptens conjugated in vitro and in situ in biological specimens, but not to closely related conjugated metabolites, precursors, pharmaceuticals, agonists, antagonists, or free neuro-active haptens. Conjugated norepinephrine was highly tolerant in different animal species and produced incidentally a short specific antibody response.

The binding of [3H]5-hydroxytryptamine (5-HT, serotonin) to cerebellar membranes was examined after preincubation of [3H]5-HT in the presence or absence of ascorbate. The tissue preparation was identical in all experiments and consisted of rat cerebellar homog-enates in Tris-HCl buffer with 0.1% ascorbate. Cerebellar membranes were used because of their low density of 5-HT, binding sites. In the presence of ascorbate during a 4-h preincubation period, minimal specific binding of 2 nM [3H]5-HT is detected. Similar results are obtained with equimolar concentrations of other antioxidants (bu-tylated hydroxytoluene, sodium dithionite, and sodium metabisulfite). Apparent specific binding increases 14-fold following a 4-h preincubation of [3H]5-HT in the absence of ascorbate. The increase in apparent specific [3H]5-HT binding is time-dependent and plateaus after 4–6 h of preincubation. When ascorbate is present during the 4-h preincubation, Scatchard analysis of [3H]5-HT binding reveals a KD value of 3.0 ± 0.3 nM and a Bmax value of 1.9 ± 0.2 pmol/g tissue. When ascorbate is absent during the preincubation, the KD is essentially un changed at 3.6 ± 0.1 nM but the Bmax is significantly increased to 36.5 ± 7 pmol/g tissue. Drug competition studies reveal that the apparent specific “[3H]5-HT binding” in the absence of ascorbate appears to be displaced by nanomolar concentrations of hydroxylated tryptamines (5-HT, bufotenine) but not by nonhydroxy-lated tryptamines (5-methoxytryptamine, tryptamine). HPLC analysis demonstrates that [3H]5-HT is essentially destroyed by a 4-h incubation at 22°C in the absence of ascorbate. We conclude that oxidation of [3H]5-HT prior to the addition of membrane homogenates leads to the detection of an artifactual high-affinity and saturable “[3H]5-HT binding site” that appears to have a distinct pharmacological profile. The capacity of other ligands to compete for this binding site is related to their potential as antioxidants and not to their affinity for a novel membrane recognition site labeled by [3H]5-HT.

There is a lack of radioactive probes, particularly radioiodinated probes, for the direct labeling of serotonin-1B (5-HT1B) and serotonin-ID (5-HT1D) binding sites. Serotonin-0-carboxymethylglycyltyrosinamide (S-CM-GTNH2) was shown previously to be specific for these two subtypes; we, therefore, linked a 125I to its tyrosine residue. Biochemical and pharmacological properties of S-CM-G[125I]TNH2-binding sites were studied by quantitative au-toradiography on rat and guinea pig brain sections. S-CM-G[I25I]TNH2 binding is saturable and reversible with a KD value of 1.3 nM in the rat and 6.4 nM in the guinea pig. Binding is heterogeneous, paralleling the anatomical distribution of 5-HT1B sites in the rat and of 5-HTD sites in the guinea pig. The binding of 0.02 nM S-CM-G[125I]TNH2 was inhibited by low concentrations of 5-HT, S-CM-GTNH2, CGS 12066 B, 5-methoxytryptamine, and tryptamine in both species. Propranolol inhibited the radioligand binding with a greater affinity in the rat than in the guinea pig. Conversely, 8-hydroxy-2-(di-n-propylamino)tetralin inhibited S-CM-G[125I]TNH2 binding with a greater affinity in the guinea pig than in the rat. Other competitors, specific for 5-HT,c, 5-HT2, 5-HT3, and adrenergic receptors, inhibited S-CM-G[125I]TNH2 binding in rat and guinea pig substantia nigra and in other labeled structures known to contain these receptors, but only at high concentrations. S-CM-G[I25I]TNH2 is then a useful new probe for the direct study of 5-HT1B and 5-HT1D binding sites.

The kinetics of serotonin N-acetyltransferase (NAT) from the lateral eye of Rana perezi have been characterized. NAT from ocular tissue reached maximal activity at a phosphate buffer concentration of 250 mM and a pH of 6.5. Reaction linearity was highly conserved within the homogenate fraction range tested (0.033-0.33). The time course of ocular NAT reaction showed a high linearity at 25 and 35 degrees C. Km and Vmax estimations for acetyl-CoA at a 10 mM tryptamine concentration were 63.3 microM and 4.42 nmol/h per eye, respectively. Regardless of the acceptor amine (tryptamine or serotonin), the Km was not affected by the acetyl-CoA concentration (50 or 250 microM), whereas the Vmax was significantly increased at a 250 microM acetyl-CoA concentration. Ocular NAT showed a higher affinity for serotonin (Km = 20.7 microM) than for tryptamine (Km = 48-60 microM); Vmax, however, was similar for both substrates. Acetyl-CoA does not protect ocular NAT; in contrast, the use of EGTA (greater than or equal to 4 mM) in the assay is essential to protect the enzyme because NAT in ocular crude homogenate shows rapid inactivation. This result suggests that intracellular calcium levels are involved in the NAT inactivation mechanisms in frog ocular tissue.

5-Hydroxytryptamine (5-HT) showed a biphasic effect on the dissociation rate of [3H]imipramine from human platelet membranes: At low concentrations (EC50, approximately 2.5 microM), 5-HT stimulated the rate, as expected for mutually exclusive binding of 5-HT and imipramine; at higher concentrations (EC50, approximately 40 microM), 5-HT reduced this stimulated rate, a result consistent with 5-HT binding at a site that is physically distinct from both the imipramine binding site and the 5-HT transport recognition site of the 5-HT carrier. This modulatory effect could be mimicked by tryptamine, was saturable and independent of Na+ concentration, and could also be demonstrated for detergent-solubilized carriers. Monophasic association kinetics for [3H]imipramine binding were found. Heat stability experiments showed biphasic thermal inactivation curves. These results are consistent with [3H]imipramine binding to two classes of binding sites at the 5-HT carrier on human platelet membranes, with affinities three- to fivefold different. 5-HT can convert the lower-affinity state into the higher-affinity state.

The presence of the so-called trace amines 2-phenylethylamine, m-tyramine, p-tyramine, m-octopamine, p-octopamine and tryptamine in the mammalian central nervous system has been known for several decades. Despite much initial interest, these amines have largely been thought of as little more than metabolic by-products. The recent description of a family of mammalian trace amine receptors has, however, seen a resurgence of interest in the physiological role of this class of compounds. Although the trace amines are well documented to cause amphetamine-like effects, such responses only occur at concentrations multiple orders of magnitude above normal physiological levels. As such, it seems unlikely that these responses reflect the true physiological role of the trace amines. In this article previous studies showing responses to physiologically relevant concentrations of trace amines are reviewed, along with those showing a reciprocal relationship between trace amine levels and fluctuations in basal monoaminergic tone. On the basis of these studies it is hypothesized that the trace amines function as endogenous neuromodulators of classical monoamine neurotransmitters. These effects are seen as an altered neuronal sensitivity to monoamine neurotransmitters, with no change in neuronal excitability in the absence of neurotransmitter.

Tryptamine dose-dependently increased phosphoinositide (PI) hydrolysis by approximately fourfold in primary cultures of rat cerebellar granule cells (EC50 = 56 microM). The PI response stimulated by tryptamine was dependent on the presence of extracellular Ca2+ and Na+. Tryptamine-induced PI breakdown could be partially inhibited by pretreatment with 4 beta-phorbol 12-myristate 13-acetate but not pertussis toxin. The presence of tryptamine markedly attenuated PI responses induced by norepinephrine (NE) and carbachol, with no apparent effect on the responses to 5-hydroxytryptamine and glutamate. The inhibition of NE- and carbachol-induced PI turnover by tryptamine was dose dependent with IC50 values of approximately 0.4 and approximately 2.5 mM, respectively. Pretreatment of cells with tryptamine (0.5 mM) also attenuated NE- and carbachol-induced PI turnover, but failed to affect 5-hydroxytryptamine- and glutamate-induced responses. Furthermore, ketanserin, atropine, and prazosin did not have any effect on inositol phosphate formation induced by tryptamine. These observations indicate that tryptamine markedly increased Ca(2+)- and Na(+)-dependent PI turnover in cerebellar neurons and selectively inhibited NE- and carbachol-induced PI hydrolysis.

The kinetics and distribution of [3H]tryptamine binding sites in human brain were investigated. Specific [3H]tryptamine binding in frontal cortex was of nanomolar affinity, reversible, saturable, and best fit to a single-site model. A heterogeneous distribution for this binding site was demonstrated, with the highest density observed in hippocampus, thalamus ≫ caudate nucleus, frontal cortex, pons, temporal cortex > globus pallidus/putamen, cerebellum. The similarities in kinetics and distribution of the [3H]tryptamine binding site in human and rat brain indicate that these two binding sites represent homologous structures. However, the present displacement studies using various ligands (indoleamines and other tryptophan metabolites, phenylethylamines, and miscellaneous drugs) and salts (Na+, K+, Ca2+, Mg2+, Cu2+) indicate stereospecific displacement as well as a rank-order potency profile that is different from that reported for the rat [3H]tryptamine binding site. This suggests the presence of distinct species-dependent [3H]tryptamine binding site subtypes. Taken together with the documented electrophysiological and behavioral evidence of tryptamine-mediated effects in the rat and the recent report of a significant loss of these binding sites in human portal systemic encephalopathy, as well as the present demonstration of an effect of guanine nucleotides on [3H]-tryptamine binding affinity, these findings suggest that these binding sites might be functional receptors. The implied role of tryptamine in neuropsychiatric disorders is supported by this demonstration of a receptor for [3H]-tryptamine in human brain.

The distribution of [3H]tryptamine binding sites, in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy (HE) and an equal number of age-matched control subjects free from hepatic, neurological, or psychiatric disorder, was investigated. Scatchard analysis demonstrated a heterogeneous distribution for this binding site, with the highest density being observed in hippocampus >> frontal cortex = caudate nucleus > temporal cortex = cerebellum. When comparing [3H]tryptamine binding site densities in control brain tissue with that in brain tissue from patients with HE, significant decreases in densities were observed in the frontal cortex (by 56%, p < 0.001), hippocampus (by 43%, p < 0.001), and caudate nucleus (by 41%, p < 0.01) of the HE group. Binding site affinities were within normal limits. The findings of decreased densities of [3H]tryptamine binding sites taken in conjunction with previous reports of increased CSF and brain tryptamine concentrations in HE suggest a pathogenic role for this neuroactive amine in HE resulting from chronic liver failure.

We have recently characterized a novel oxidation product of serotonin (5-hydroxytryptamine, 5-HT), tryptamine-4,5-dione, which increases 5-HT efflux from striatum and hippocampus and causes selective neuronal death. Exposure of striatal synaptosomes or the major brain guanine nucleotide-binding regulatory proteins Gi and Go to [3H]tryptamine-4,5-dione resulted in the radiolabeling of a major band with an apparent molecular mass equivalent to that of the alpha subunits of Gi and Go (approximately 40,000). The binding of [35S]guanosine-5'-O-(3-thiotriphosphate) ([35S]GTP-gamma-S) to Gi and Go and pertussis toxin-catalyzed [32P]ADP-ribosylation of the G protein alpha subunits were both inhibited in a dose-dependent manner by tryptamine-4,5-dione. Thus, neurotoxins such as tryptamine-4,5-dione may exert their effects through specific interactions with G proteins.

— Monoamine oxidase (MAO) activity against tryptamine was compared in a number of continuous rodent lines, including neuroblastoma, hepatoma, melanoma, nephroma, sarcoma and L cells. Activities against tryptamine varied over 300-fold in homogenates of different lines, being highest in hepatoma line MH1C1 and lowest in a neuroblastoma line lacking hypoxanthine phosphoribosyltransferase (HPRT) activity. The amount, but not the type, of MAO activity varied with the stage of growth in homogenates of neuroblastoma and hepatoma cells. Measurements of succinate-cytochrome c reductase (SCCR), another mitochondrial enzyme, also showed 20-fold variations between lines, being highest in neuroblastoma line N1E-115 and lowest in hepatoma line MH1C1; SCCR and MAO activities appeared to be regulated independently. The relative proportions of the A and B types of MAO activity were determined in homogenates and living cultures. Clorgyline inhibition of tryptamine deamination in homogenates indicated that in all lines except MH1C1, greater than 95% of the MAO activity was of the A type. In MH1C1 homogenates, using clorgyline or deprenyl, 40–70% of the activity appeared to be of the A type and 30-60% of the B type. In cultures of neuroblastoma N1E-115 cells, deamination of tryptamine and dopamine was sensitive to inhibition by low concentrations of clorgyline, indicating that the A type of activity is present intracellularly. as in homogenates. In MH1C1 hepatoma cultures, tryptamine deamination showed a biphasic sensitivity to clorgyline. We interpret this to mean that A and B types of MAO activity occur together in living hepatoma cells.

The trace amines phenylethylamine, tryptamine, p-tyramine and m-tyramine have been measured in the striatum of both control and MAO-treated rats. Dose-response and time-response studies have been carried out with clorgyline and deprenyl, inhibitors which preferentially inhibit the A and B forms of MAO, respectively, and with tranylcypromine and phenylethylhydrazine, which are used clinically in the treatment of depression. Phenylethylamine was increased by 1 mg/kg of deprenyl, but was unaffected by clorgyline at doses up to 50 mg/kg, while the tyramines and tryptamine were increased by low doses of clorgyline, but were increased only by much greater doses of deprenyl than those required to affect phenylethylamine. Phenylethylamine is oxidized by the B form of MAO, but tryptamine and the tyramines appear to be oxidized by both A and B MAO. The observed proportional increases in trace amine levels are much greater than those observed for the classical neurotransmitters, noradrenaline, dopamine and 5-hydroxytryptamine. As these increases are differential, selective manipulation of trace amine concentrations is possible.

A high-affinity (Kd= 5.9 nM) specific binding site for [3H]harmaline was detected in membranes from rat and bovine brains. Studies of the regional and subcellular distributions of this binding indicated its close association with monoamine oxidase type A activity (MAO A) measured with [3H]serotonin ([3H]5-HT) as the substrate. Maximal binding capacity and MAO A activity were found in mitochondrial enriched fractions. Mitochondria of synaptosomal or extra-synaptosomal origin exhibited very similar properties with respect to [3H]harmaline binding characteristics and MAO A activity. Among psychoactive drugs, only monoamine oxidase inhibitors (MAO I) prevented the specific binding of [3H]harmaline. Logit-log inhibition curves of binding by MAO I gave only one slope which was not significantly different from 1.0, suggesting the existence of only 1 category of specific sites for [3H]harmaline in the membrane preparations from rat and bovine brains. Consistent with the preferential inhibition of MAO A by harmaline, other MAO I of this class, i.e. clorgyline and Lilly 51641, were 102-2 × 103 times more efficient than deprenyl and pargyline, two inhibitors of MAO type B, in displacing [3H]harmaline from its specific binding site. Ki and IC50 values for the inhibition of [3H]harmaline binding by MAO I and MAO substrates (tryptamine, 5-HT, norepinephrine) were almost identical with those characterizing their action on MAO A activity with [3H]5-HT as the substrate. In conclusion, the specific binding site for [3H]harmaline exhibited all the expected properties of the active site of MAO A. Like the technique of precipitation with a specific antibody, binding of [3H]harmaline should be of great help for studying the structural characteristics of the active site of MAO A and determining the number of MAO molecules in tissues under various physiological conditions.

— Six endogenous substrates of monoamine oxidase (EC 1.4.3.4) (serotonin, l-norepinephrine, dopamine, tyramine, tryptamine and β-phenethylamine) were used separately and in pairs with human brain mitochondrial extracts. Apparent K1 values were obtained from experiments in which only 1 of 2 substrates was isotopically labelled, and these values were compared with experimental Km values. β-Phenethylamine appears to be metabolized at enzyme active sites independent from those which bind serotonin. The substrate l-norepinephrine competes with serotonin for an enzyme site, but also may be catalysed at an additional site which is independent of serotonin binding. Experiments in which [14C]tryptamine was combined with [3E]serotonin indicated that tryptamine is a much more potent inhibitor of serotonin oxidation than was predicted from Km values. It is suggested that the competition among substrates of MA0 which is observed in uitro may have relevance to in uiuo mechanisms for control of biogenic amine concentrations.

—In rat brain extract tryptamine is converted to 1,2,3,4-tetrahydro-β-carboline (THβJC) and N-methyltryptamine to 2-methyl-THβC in the presence of 5-methyltetrahydrofolic acid. We believe this occurs through enzymatic conversion of 5-methyltetrahydrofolic acid to formaldehyde and tetrahydrofolic acid, followed by spontaneous condensation of the radioactive formaldehyde with the substrate tryptamine (Donaldson & Keresztesy, 1961). The final products of the reactions have been identified by both thin layer chromatography and mass spectrophotometry. Subcellular fractionation shows more than 90 per cent of the formaldehyde-forming enzyme activity to be in the cytosol. Specific activities in fractions from 16 discrete regions of the brain and CNS range from 210·2 ± pmol of THβC/mg protein/h in corpus striatum to 62·9 ± 3·6 pmol of THβC/mg protein/h in corpus callosum.

5-Methoxytryptoline potently inhibits [3H]-imipramine binding to membranes from the cerebral cortex and platelets. Since 5-methoxytryptoline, which appears to occur endogenously with particularly high levels in the human pineal gland, also inhibits 5-hydroxy-tryptamine (5-HT, serotonin) uptake, it should be considered as a putative endogenous ligand modulating 5-HT transport. As the 5-HT transporter complex comprises the imipramine and the substrate recognition sites, which interact allosterically, it was essential to define the mechanism of inhibition of [3H]imipramine binding by 5-methoxytryptoline. Human platelets show an active and saturable uptake of 5-HT and tryptamine. The uptake of both substrates appears to be mediated by the same carrier and it is inhibited by 5-methoxytryptoline at submicromolar concentrations. 5-HT and tryptamine inhibit [3H]imipramine binding in human platelets with a Hill slope for inhibition close to unity and IC70 values of 3,265 and 3,475 nM, respectively. This inhibition is, however, not competitive because both 5-HT and tryptamine significantly decrease the rate of [3H]imipramine-receptor dissociation. Although 5-methoxytryptoline potently inhibits [3H]imipramine binding (IC50= 44 nM) in human platelets with a Hill slope of unity, it does not affect the receptor-ligand dissociation rate of [3H]imipramine even at concentrations up to 100 μM. The present experiments show that 5-methoxytryptoline, in spite of its chemical similarity to the indoleamine transporter substrates, interacts with the imipramine receptor through a mechanism of competitive inhibition. This conclusion is supported by a selective effect of 5-methoxytryptoline on the Kd of [3H]imipramine binding. These data are compatible with the hypothesis that 5-methoxytryptoline may be an endogenous modulator that interacts competitively with the imipramine receptor associated with the 5-HT uptake complex.

Tryptophan uptake, hydroxylation, and decarboxylation in isolated synaptosomes were studied to assess how their properties may determine the rate of serotonin synthesis in the presynaptic nerve terminals of the brain. Simultaneous measurements of the rates of uptake, hydroxylation, and decarboxylation in the presence and absence of various inhibitors showed that tryptophan hydroxylase is rate-limiting for serotonin synthesis in this model system. There was significant direct decarboxylation of tryptophan to tryptamine. Measurement of tryptophan hydroxylase flux with varying internal concentrations of tryptophan allowed the determination of the Km of tryptophan hydroxylase in synaptosomes for tryptophan of 120 +/- 15 microM. Depolarisation of synaptosomes with veratridine caused both a reduction in the internal tryptophan concentration and an apparent activation of tryptophan hydroxylase. This activation did not occur in the absence of Ca2+ or in the presence of trifluoperazine. Synaptosomal serotonin synthesis and brain stem-soluble tryptophan hydroxylase were inhibited by low concentrations of noradrenaline or dopamine. Dibutyryl cyclic AMP, glucagon, insulin, and vasopressin were observed to have no effect on tryptophan uptake or hydroxylation in synaptosomes.

—Monoamine oxidase (MAO) activity in the brains of 15 suicides, of whom 8 were alcoholics, was compared to a control material of 20 individuals without known mental disorder. At autopsy 13 different parts of the brain were macroscopicaily dissected out and the MAO activity in the samples estimated with β-phenylethylamine and tryptamine as substrates. The MAO activity in all parts of the brain investigated was found to be significantly lower in the alcoholic suicides as compared to the controls, while there was no significant difference between the non-alcoholic suicides and controls. Different variables which might have influenced the MAO activity were investigated. There was no significant correlation between age and tryptamine-oxidizing activity, but a positive correlation between age and β-phenylethylamine-oxidizing activity was found. There was also a significant difference between the series in the time lapse between death and autopsy and in the time during which the dead body was kept at room temperature. However, neither of these variables could explain the differences between the series. The results thus demonstrate a connection between low MAO activity in the brain and suicidal behaviour among alcoholics.

Antisera were raised against tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-methoxytryptophan, and 5-methoxytryptamine, by conjugating each molecule to bovine serum albumin and to human serum albumin via glutaraldehyde, in such a way as to preserve the original part. Antibody specificity was tested with the enzyme-linked immunosorbent assay method. The specificity of each anti-indolealkylamine-glutaraldehyde antibody was established with competition experiments by using an adsorbed immunogenic conjugate and indolealkylamines either free or conjugated with poly-L-lysine. The nonconjugated compounds were poorly recognized. In the same way, the nonreduced conjugates always appeared less immunoreactive than the reduced ones. Calculated from the specificity study of each antiserum, the cross-reactivity ratios were found to be smallest for the most immunoreactive conjugates. Thus, a specific immune response was defined for each compound belonging to the same metabolic pathway.

Tryptophan, 5-hydroxyindoleacetic acid (5HIAA), and indoleacetic acid (IAA) were measured in rat cerebrospinal fluid (CSF) using high-performance liquid chromatography with fluorometric detection in order to study brain indoleamine metabolism. Previously we have shown that IAA in CSF is derived from tryptamine in the CNS. In this study our results indicated that the synthesis of both 5-hydroxytryptamine (5HT) and tryptamine varied with changes in brain tryptophan. After a tryptophan load, tryptamine synthesis increased much more than 5HT synthesis; under these circumstances, it can be of the same order of magnitude as 5HT synthesis. Studies with treatments that cause release of 5HT (reserpine and cold stress) indicated that tryptamine is not released with 5HT and is probably not stored in vesicles. Use of selective monoamine oxidase inhibitors suggested that results obtained in vitvo concerning the substrates for monoamine oxidase A and B apply in vivo. Thus, 5HT was acted on preferentially by the A enzyme, and tryptamine by the B enzyme. Measurement of IAA in brain, CSF, and plasma before and after probenecid indicated that there are active transport systems moving IAA from CSF and brain to blood, and from brain to CSF. Our data suggest that measurements of CSF IAA are a simple and convenient way of studying CNS tryptamine metabolism and should be applied clinically.

— With a modification of the spectrophotofluorometric (SPF) method of HESS & UDENFRIEND (1959) (J. Pharmac. exp. Ther.127, 175-177), brain tryptamine levels in the rat (20.9 ng/g) and guinea-pig (20.7 ng/g) were found to be less than those in the dog (32.1 ng/g) and cat (52.2 ng/g). Regional distribution studies in the dog and cat showed that tryptamine was present in all major brain regions with highest concentrations in the spinal cord. Blood levels of tryptamine in the guinea-pig, dog and cat (6-7 ng/ml) were lower than brain levels. Pargyline significantly increased brain tryptamine in both the dog and cat; whereas, isocarboxazid (after 4 h) increased brain tryptamine levels in the dog but decreased brain levels in the cat. Reserpine (0.5-1.0 mg/kg per day for 1-4 days) did not significantly decrease brain, spinal cord or blood tryptamine levels in the dog. Spinal cord transection did not decrease tryptamine levels below the lesion in the chronic spinal dog.

The possible existence of tryptamine-containing neurons originating in the midbrain raphe is suggested by several reports of tryptamine-mediated responses to electrical stimulation of the raphe nuclei. To assess this hypothesis, we have investigated the effects of electrolytic lesions of the median and dorsal raphe nuclei on striatal, hypothalamic, and hippocampal concentrations of tryptamine, 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid. In addition, the rat striatal tryptophan concentrations were also determined. No changes in the concentrations of tryptamine were observed at 1 or 2 weeks after lesioning the dorsal and median raphe nuclei, at which time the other 5-hydroxyindoles were markedly reduced; furthermore, no reductions were observed in tryptamine concentrations in the striatum, hypothalamus, or hippocampus of rats pretreated with a monoamine oxidase inhibitor. The only change observed in these rats was a limited increase in striatal tryptamine and tryptophan observed at 1 day after lesioning. The results indicate that tryptamine concentration is independent of the integrity of 5-HT-containing neurons of the midbrain raphe nuclei. Furthermore, if tryptamine-containing neurons that have terminal projections to the striatum, hypothalamus, and hippocampus exist, their cell bodies are located in regions outside the dorsal and median raphe nuclei. Another possibility could be that tryptamine is located in glial cells.

— Indoleamine-N-methyltransferase (INMT) activity in brain and other tissues from various species was investigated. Using conventional radiochemical assay techniques it was found that apparent INMT activity in brain was linear with time and concentration of protein, indoleamine substrate and methyl donor (S-adenosylmethionine). However, examination of the reaction products by means of exhaustive thin-layer chromatographic analysis failed to reveal evidence of significant N-methylation of tryptamine or N-methyltryptamine by S-adenosylmethionine. By contrast, with other tissues, notably rabbit lung. N-methylation of indoleamine was reproducibly demonstrable. The significance of these findings with reference to the transmethylation hypothesis of schizophrenia is discussed.

Tryptamine was degraded by incubation with rat brain homogenate to an unknown product. The reaction was stimulated by the nonionic detergents Triton X-100 and Lubrol PX and less by the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonate (CHAPS). The same results were obtained with pig brain and bovine brain. The monoamine oxidase inhibitor pargyline inhibited the reaction strongly, indicating the participation of the enzyme on the reaction. Addition of 17,000 g supernatant from rat brain homogenate increased the formation effectively whereas phospholipids or chloroform/methanol (7:3) extract from the 17,000 g supernatant showed only little or no effect. Chromatographic and electrophoretic properties as well as the chemical reaction of the product with specific reagents suggest that the compound consists of an indole part and an amino acid part. The product could be identified by fast atom bombardment mass spectrometry and by comparison with the synthetic substance (4R)-2-(3-indolylmethyl)-1,3-thiazolidine-4-carboxylic acid. It is formed by the enzymatic oxidation of tryptamine producing indole-3-acetaldehyde which spontaneously cyclizes with free L-cysteine from the tissue. The results suggest that the reaction of biogenic aldehydes with brain macromolecules may proceed via an analogous reaction.

We studied the enzyme monoamine oxidase (MAO) in isolated cerebral microvessels, and in mitochondria-enriched brain and liver preparations from six mammalian species, including human. We also studied MAO distribution in various tissues and in discrete brain regions of the rat. MAO was assessed by measuring the specific binding of [3H]pargyline, an irreversible MAO inhibitor, and the rates of oxidation of known MAO substrates: benzylamine, tyramine, tryptamine, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Molecular forms of MAO were examined by using specific MAO inhibitors, and by polyacrylamide gel electrophoresis after [3H]pargyline binding. In general, the liver from all species had higher MAO levels than the brain, with minor variation among species in their brain and liver MAO content. However, there were remarkable species differences in brain microvessel MAO, with rat microvessels having one of the highest MAO activity among all tissues, whereas MAO activities in brain microvessels from humans, mice, and guinea pigs were very low. In most rat tissues, including the brain, there was a preponderance of MAO-B over MAO-A. The only exceptions were the heart and skeletal muscle. Estimates of MAO half-life in rat brain microvessels, rat brain, and rat liver indicated that microvessel MAO had a higher turnover rate. The reasons underlying the remarkable enrichment of rat cerebral microvessels with MAO-B are unknown, but it is evident that there are marked species differences in brain capillary endothelium MAO activity. The biological significance of these findings vis a vis the role of MAO as a "biochemical blood-brain barrier" that protects the brain from circulating neurotoxins and biogenic amines should be investigated.

— A simple method for the separation of 5-hydroxyindoleacetic acid (5-HIAA) and 5-methoxyindoleacetic acid (5-MeOIAA) on columns of non-ionic polystyrene (Amberlite XAD-2) is described. Administration of 5-methoxy-N, N-dimethyl-tryptamine (5-MeODMT) 2 mg/kg i. p. to rats results in a sixfold increase in brain 5-MeOIAA within 15 min. This increase is blocked by the selective inhibitor of MAO-A, clorgyline, but not by the selective inhibitor of MAO-B, deprenyl, indicating that 5-MeODMT is deaminated almost entirely by MAO-A. The apparent 5-MeOIAA concentration in the brains of L-tryptophan loaded rats is not reduced by clorgyline and deprenyl, either singly or in combination, indicating that most of this fluorescence is due to other, unidentified substances. The apparent concentration of 5-HIAA in rat brain, minus 5-MeOIAA, is unaffected by deprenyl and reduced by clorgyline. However, clorgyline and deprenyl in combination reduced 5-HIAA values below those obtained with clorgyline alone. It is concluded that very little 5-MeODMT or other 5-methoxyindoleamines are formed endogenously in rat brain, and that the stereotyped syndrome of hyperactivity and tremors produced in rats by pretreatment with MAO inhibitors and L-tryptophan is dependent on the formation of an N-substituted derivative of 5-HT which is at least partly deaminated by MAO-B to 5-HIAA.

— The kinetic parameters of monoamine oxidase (MAO; E.C 1.4.3.4) and catechol-O-methyl-transferase (COMT; EC 2.1.1.6) were evaluated in extracts of adrenergic and non-adrenergic mouse neuroblastoma cells and in rat glioma cells. Using the naturally-occurring substrates tyramine, tryptamine, serotonin and norepinephrine, the affinity of MAO for a given substrate was independent of the presence of the catecholaminergic pathway or cell type used, with apparent Km values ranging from 8–14 μM for tryptamine to 510–580 μM for norepinephrine. The MAO activity in glioma cells was substantially greater than in either neuroblastoma clone, but Vmax values varied little with substrate among cell lines. Both the neuronal and glial COMT had a similar Km for I-norepinephrine (200μM); the corresponding Vmax values were also similar among the different cell lines, but represented only 2–10% of the maximal MAO activity. Neuroblastoma and glioma cells, when grown from early logarithmic to stationary phase, showed no significant changes in specific activity of either MAO or COMT. Growth of cells for 3 days with 1 mM-N6,O2′ -dibutyryl adenosine-3′,5′-cyclic monophosphate resulted in no marked change in either MAO or COMT activity. These results suggest that in neurons neither MAO nor COMT plays a major role in the type of transmitter inactivation that is analogous to that of acetylcholinesterase in cholinergic synapses. The occurrence of considerable MAO and acetylcholinesterase activities in glioma cells may indicate a role for these cells in neurotransmitter inactivation.

Dark-adapted retinas of mice (C57BL/6J) incubated in the dark in media containing 1 mM 3-isobutylmethylxanthine (IBMX) or 5 mM Co2+ accumulate cyclic AMP (cAMP). A portion of this pool is light sensitive, as light can prevent or reverse its accumulation. Similarly, tryptamine, serotonin, 5-methoxytryptamine, bufotenine, and 5-methoxydimethyltryptamine can block the accumulation of the light-sensitive pool of cAMP, whereas tryptophan, melatonin, N-acetylserotonin, 5-methoxytryptophol, and tetrahydro-beta-carbolines are inactive. The phenomenon is not seen with mutant mouse retinas (rd/rd), which lack most photoreceptors, but persists in abnormal retinas containing photoreceptors but with extensive neuronal depletion in the inner retina. Tryptamine also inhibits cAMP accumulation in either dark or light-adapted retinas exposed to forskolin alone but not in media containing high levels of forskolin plus 1 mM IBMX. There is some suggestion that serotonin 5-HT-2 antagonists can partially reverse the action of the tryptamines, but hitherto undescribed receptors may be involved. Current data suggest that photoreceptors are the target for the action of the tryptamines.

—The quantitative subcellular distribution of β-phenylethylamine, p-tyramine and tryptamine in rat brain was investigated using the mass spectrometric integrated ion current technique. More of the total cellular tryptamine was found to be associated with paniculate fractions than was the case for phenyiethylamine and p-tyramine but a significant amount of this tryptamine was found to be labile. Analysis of the particulate fractions indicated that each of the amines was localized predominantly in the crude P2 pellet and that the bulk of this was associated with the synaptosomal (P2B) fraction. Inhibition of monoamine oxidase systems with pargyline caused an increase in the level of all three amines in all fractions, but the increase was greater in the supernatant than in the combined particulate fractions. This treatment produced changes in the distribution of β-phenylethylamine and p-tyramine between the various particulate subcellular fractions but did not markedly alter the distribution of tryptamine between the same fractions.

— The distribution of some monoamines in the tissues of an echinoderm, the sunflower starfish, Pycnopodia helianthoides, has been investigated in order to ascertain whether monoamine levels are similar to those found in other Deuterostomia. Dopamine, noradrenaline and octopamine were present in the arm nerves at concentrations of 5954 ng/g, 2133 ng/g and 260 ng/g respectively. The octopamine/ noradrenaline ratio for the arm nerve was 0.12 and thus similar to the typical mammalian (deuteros-tome) ratio rather than the higher invertebrate (protostome) ratio. Trace amounts of p-tyramine, β-phenylethylamine were also present. 5-Hydroxytryptamine was not detected but tryptamine was present in high concentrations (1251 ng/g).

Serotonin (5-hydroxytryptamine, 5-HT; 0.5 micro M and above) stimulated the release of [3H]dopamine ([3H]DA) from particulate fractions of the carp (Cyprinus carpio) retina. The 5-HT effect was dose- and Ca2+-dependent, and was structurally specific. A similar response was not elicited by the other indoles (5,6-dihydroxytryptamine, 5,7-dihydroxytryptamine, 5-hydroxytryptophan, or 5-hydroxyindoleacetic acid) examined. An increase in [3H]DA release was elicited by addition of 5-HT agonists (5-methoxytryptamine, 5-methoxy-N,N-dimethyltryptamine, and tryptamine), but not antagonized by three 5-HT antagonists (metergolin, methysergide, and spiperone). Either DA alone or noradrenaline (0.5 mM) produced a large increase in [3H]DA release from the particulate fractions, but this action was Ca2+-independent. Further, no significant release of [3H] gamma-aminobutyric acid could be evoked by 5-HT (0.5 mM) under similar experimental conditions. Taken together, the present data suggest that 5-HT stimulates [3H]DA release from the fish retina through a specific receptor-mediated mechanism on dopaminergic terminals, but not through an exchange or nonspecific phenomenon.

— The effects of various drugs and lesions on rat brain 5-hydroxytryptamine and tryptamine were determined. Monoamine oxidase inhibition caused a proportionately greater increase in tryptamine than in 5-hydroxytryptamine, reserpine depleted 5-hydroxytryptamine but had no effect on tryptamine while p-chlorophenylalanine lowered 5-hydroxytryptamine but increased tryptamine. α-Methyl-p-tyrosine reduced striatal dopamine with no effect on either 5-hydroxytryptamine or tryptamine. Increasing brain tryptophan by amphetamine administration. 24 h food deprivation or giving L-tryptophan did not increase brain tryptamine. However a high dose of L-tryptophan (100 or 200mg/kg) together with a monoamine oxidase inhibitor caused a proportionately much greater increase in tryptamine than in 5-hydroxytryptamine. Raphe lesions reduced 5-hydroxytryptamine by 64 per cent and tryptamine by only 29 per cent while intraventricular 6-hydroxydopamine lowered striatal dopamine (56 per cent), had no effect on 5-hydroxytryptamine but reduced tryptamine by 24 per cent, suggesting that tryptamine can be formed in both 5-HT and catecholaminergic neurones. The results are discussed in relation to the formation, distribution, storage and possible transmitter function of tryptamine in rat brain.

1-Methyl-4-phenyl- 1,2,5,6 -tetrahydropyridine (MPTP) is a chemical that, after injection into experimental animals, including mice and monkeys, causes a degeneration of the nigrostriatal pathway. We carried out experiments designed to study the in vitro oxidation of MPTP by mouse brain mitochondrial preparations. MPTP was actively oxidized by the mitochondrial preparations, with Km and Vmax values very similar to those of benzyl amine, a typical substrate for MAO-B. MPTP was oxidized considerably better than many of its analogs, even those with relatively minor structural changes. Several monoamine oxidase inhibitors (MAOI) were potent inhibitors of MPTP oxidation, and there was a highly significant correlation between the capacity of the MAOI tested to inhibit MPTP oxidation and benzylamine oxidation. There was no correlation between the capacity of the MAOI to inhibit MPTP oxidation and their capacity to inhibit the oxidation of tryptamine, a substrate for MAO-A. In other experiments, MPTP was an excellent substrate for pure MAO-B, prepared from bovine liver. All of these data. combined with the fact that MAO-B inhibitors can protect against MPTP-induced dopami nergic neurotoxicity in vivo. point to an important role for MAO-B in MPTP metabolism in vivo.

— Rats were injected intracerebrally with labelled tryptamine, 5-hydroxytrypt-amine (5-HT) and norepinephrine (NE). The disappearance of the amines and their metabolites as a function of time was determined. Tryptamine disappeared very rapidly, with a half-life of 5 min in normal animals and of 45 min in rats treated with a monoamine oxidase (MAO) inhibitor. The level of radioactive 5-HT declined in two phases, with half-lives of 45 min and 3 h respectively. The 5-hydroxyindoleacetic acid (5-HIAA) that was formed disappeared with a half-life of approximately 1 h. After inhibition of monoamine oxidase, there was only a single phase of 5-HT disappearance (half-life of 4 h). Reserpine decreased and imipramine increased the amount of 5-HT remaining 4 h after injection. Of the NE injected, 12 per cent was converted to methoxyhydroxyphenylglycol sulphate (MHPGS), which disappeared with a half life of 3 h. Reserpine doubled the amount of methoxyhydroxyphenylglycol sulfate formed, but did not alter its rate of disappearance, its peak concentration occurring about 30 min after injection of NE in both control and reserpine-treated rats.

— In order to examine the possibility that acidic lipids can account for the binding of 5-hydroxy [3H]tryptamine (5-HT) to brain tissue, the binding to six acidic lipids was studied using an isobutanol-water partition method. With the exception of the polyphosphoinositides, all the acidic lipids examined bind saturably and with high affinity. The apparent dissociation constants of 5-HT to the acidic lipids were as follows: phosphatidylserine, 0.4 μM; phosphatidic acid, 0.6μM; diphosphoinositide, 0.8 μM; cerebroside sulfate, 1.4 μM; monophosphoinositide, 1.9 μM; and triphosphoinositide, 10 μM. The high affinity of these lipids to 5-HT raises the possibility of some role for them in serotonergic activity.

Seven L-amino acids (Trp, Arg, Lys, Met, Ile, Val, and Phe) partially (28-81%) reversed the inhibitory action of 1 microM gamma-aminobutyric acid (GABA) on t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to rat brain membranes, with EC50 values ranging from 5 to 120 mM. D-Trp, D-Arg, D-Lys, D-Met, D-Val, and D-Phe were approximately equipotent with their L-isomers. Tyramine, phenethylamine, and tryptamine, the decarboxylation products of the aromatic amino acids (Tyr, Phe, and Trp, respectively), reversed the inhibitory action of 1 microM GABA on [35S]TBPS binding more potently than the parent amino acids (EC50 values = 1.5-3.0 mM). Human hereditary amino acidemias involving Arg, Lys, Ile, Val, and Phe are associated with seizures, and these amino acids and/or their metabolites may block GABA-A receptors. Five other L-amino acids (ornithine, His, Glu, Pro, and Ala) as well as Gly and beta-Ala inhibited [35S]TBPS binding with IC50 values ranging from 0.1 to 37 mM, and these inhibitions were reversed by the GABA-A receptor blocker R 5135 in all cases. The inhibitory effects of L-ornithine, L-Ala, L-Glu, and L-Pro were stereospecific, because the corresponding D-isomers were considerably less inhibitory. L-His, D-His, and L-Glu gave incomplete (plateau) inhibitions. Human hereditary amino acidemias involving L-ornithine, His, Pro, Gly, and beta-Ala are also associated with seizures, and we speculate that these GABA-mimetic amino acids may desensitize GABA-A receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

— Biogenic amines and related enzymes were quantitatively measured in the pituitary gland of the rat. The sensitivity of the assays used allows the determinations to be performed in single pituitary lobes. Relatively high values of histamine and serotonin were found in all three lobes, with higher amounts in the posterior and intermediate lobes. Highest catecholamine concentrations were detected in the posterior lobe, and only very low amounts of dopamine were measured in the anterior lobe. Throughout the gland, norepinephrine concentrations were low, about one-tenth that of dopamine. Tryptamine could not be detected. High levels of A and B monoamine oxidase were found in all three pituitary lobes. Tyrosine hydroxylase activity was measured in the posterior and intermediate lobes, but was not detected in the anterior lobe. Tryptophan hydroxylase was present in all three pituitary lobes. A relatively low catechol-O-methyltransferase activity was found in the anterior lobe, and none was detected in the intermediate and posterior lobe. Choline acetyltransferase, dopamine-β-hydroxylase and phenylethanolamine-N-methyltransferase activities could not be detected.

— The dependence of rat brain tryptamine (TA) formation on tryptophan availability was investigated. Rat brain TA was found to accumulate linearly with respect to time over 90min following pargyline (75 mg/kg. i.p.). The in vivo rate of brain TA appearance was estimated to be 0.1 nmol/g/h. After tryptophan loading the accumulation of brain TA and 5-HT in pargyline-pretreated rats was linearly related to both brain and plasma tryptophan concentrations. Low doses of tryptophan (5–10 mg/kg. i.p.) produced statistically significant increments in brain TA formation. The findings of this study indicate that the in vivo formation of TA in rat brain is directly dependent upon brain and plasma tryptophan concentrations.

In vivo conversion of intracisternally administered [14C]tryptamine to [14C]N,N-dimethyl-tryptamine (DMT) in rat brain, even in the presence of an excess of substrate and methyl donor appeared to be insignificant, although enzymatically synthesized [14C]DMT was recovered readily after intracranial injection. Authenticity of [14C]DMT was demonstrated by cocrystallization with authentic DMT and oxalic acid to constant specific radioactivity after chromatographic separation of [14C]DMT. In rabbit lung, the apparent Km for S-adenosylmcthionine (SAMe) (29 μM) with indolethylamine-N-methyltransferase was found to be close to endogenous levels of SAMe (34 μM) that are not likely to saturate the enzyme normally. Nevertheless. large doses of L-methioninc or SAMe failed to increase the in vivo conversion of [14C]N-methyltryptamine to [14C]DMT in this tissue. The production of [14]DMT was instead markedly irihihitrd by this treatment. possibly due to an effect of S-adeno-sylhomocysteine. Our results fail to support the hypothesis that psychotropic effects of methionine or SAMe are due to increased accumulations of pharmacologically active methylated indoleamines.

— Slices from four areas of guinea-pig brain (hypothalamus, corpus striatum, median ponsmedulla and cerebral cortex) were incubated with concentrations of l-[3-14C]tryptophan varying from 20 to 300 μm. in the presence of 5 × 10-5m-pargyline. The formation of tryptamine and serotonin was studied. Serotonin synthesis reached its highest level at a concentration of approx 80 μm-tryptophan in all 4 brain areas. Activity was high in pons-medulla and hypothalamus but only about one third as high in corpus striatum and cortex. Tryptamine formation continued to increase within the concentration range of tryptophan used. Activity was high in corpus striatum where, at 300 μm-tryptophan, tryptamine formation exceeded serotonin formation, but was low in cortex and intermediate in pons-medulla and hypothalamus. The possible formation of kynuramine and 5-hydroxykynuramine was also investigated. No evidence was obtained for the formation of 5-hydroxykynuramine. Traces of radioactivity were found corresponding to kynuramine, but these were insufficient to establish its formation with certainty. If formed. the rate of kynuramine synthesis did not exceed 2% of the combined formation of serotonin and tryptamine.

— The expression of monoamine oxidase (MAO) in a variety of mammalian cells has been investigated employing tryptamine as substrate. The enzyme present in those cell lines having sufficient activity for detailed analysis exhibited a monophasic response to the inhibitor clorgyline. On this basis the cell lines examined were found to express only A, or only B, type activity. Hypoxanthineguanine phosphoribosyl transferase (HGPRT) deficient derivatives of both MAO type A, or MAOtype B, expressing cells were examined. The HGPRT status of the cells appeared to have little influence on the expression of the enzyme.

Cultured C6 rat glial cells preferentially deaminated 5-hydroxytryptamine, tryptamine, dopamine and tyramine in comparison to phenylethylamine and benzylamine. Deamination of all substrates was uniformly sensitive to inhibition by clorgyline and relatively insensitive to deprenyl. These data together with the observations of simple sigmoid curves for the inhibition of tyramine deamination by both inhibitors suggest that C6 glial cells contain mainly monoamine oxidase type A, which previously had been suggested to be primarily an intraneuronal MAO type. As these findings are in agreement with other studies of brain MA0 activity in mitochondria separated from neuronal vs glial cell preparations, they help explain why MA0 activity measured with some substrates may be little affected by lesions or by drugs producing nerve ending degeneration.

Brain mitochondria were prepared from rabbit and bovine cerebral cortex and the purity and intactness of the preparation assessed through the use of enzyme markers and electron microscopy. Enzymatic properties of monoamine oxidase were studied in the purified mitochondrial preparations which were essentially devoid of major contamination by other organelles, especially microsomes. Five substrates were used for characterization of the enzyme: dopamine, kynuramine, serotonin, tryptamine and tyramine. It was found that there was considerable substrate variation in the properties, but in general, the two species showed similar characteristics. The more pertinent findings were: (1) apparent Km values ranged from 1.1 ± 10−5m for tryptamine to 2.5 ± 10−4m for dopamine; (2) substrate specificity from Vmax values in decreasing order was tyramine > dopamine > kynuramine > serotonin > tryptamine for the bovine enzyme and tyramine > kynuramine > dopamine > serotonin > tryptamine for rabbit; (3) there appeared to be three distinct pH optima according to substrate: pH 7.5 for phenylethylamines, pH 8.2–8.5 for the indolylamines and pH 9.1 for kynuramine; and (4) the activity with tyramine was highly sensitive to increased oxygen tension while kynuramine showed no sensitivity. It is proposed that the properties of monoamine oxidase, a membrane-bound enzyme, might be influenced by the microenvironment and results are also discussed in terms of multiple forms or multiple activity sites on a single form.

Monoamine oxidase (MAO) activity was studied in cultured skin fibroblasts from 10 Lesch-Nyhan patients, a Lesch-Nyhan variant and 11 controls matched for age. sex and race. Activity (predominantly type A) was measured in cell homogenates using tryptamine as the substrate. For each line activity varied with the conditions of culture. Activity increased 3-10 fold as cultures went from logarithmic to stationary phase of growth. When cultures were confluent, activity was lowered by frequent feedings or the use of fresh medium and serum. Activity for each line remained fairly stable during successive passages, but rose 3-8 fold as cultures became senescent. When comparing activity between control and Lesch-Nyhan lines, cells were cultured under standardized conditions. The mean value of MAO activity in Lesch-Nyhan lines was approximately one fourth of the mean activity in control lines (P < 0.012), In the control population, the distribution of activity appeared to be bimodal. Activities in the Lesch-Nyhan lines fell completely within the lower portion of the control distribution. Cells from a Lesch-Nyhan patient who lacked several of the neurologic symptoms of the disease (including self-mutilation) had an MAO activity 6 fold greater than the control mean. although his hypoxanthine phosphoribosyltransferase activity was

Serotonin has no obvious effect on basal cyclic AMP levels but reduces the forskolin-, isoproterenol-, and vasoactive intestinal peptide-induced stimulation of cyclic AMP levels in a dose-dependent manner. Serotonergic, cholinergic, muscarinic, α-adrenergic, and dopaminergic antagonists have no effect on the serotonin response. Topical Application of a serotonin/pargyline solution to the living eye causes desen-sitisation of the serotonin response in the iris-ciliary body, an observation confirming the presence of specific serotonergic receptors linked to adenylate cyclase. The 5-HT1A [5-hy-droxytryptamine (serotonin) type 1A] receptor agonists 8-hydroxy-2-(di-n-propylamino)tetralin and bujspirone mimic the serotonin response in reducing the forskolin-stimulated cyclic AMP levels, as do the indole derivatives 5-methoxy- tryptamine, 5-hydroxtryptophan, and tryptamine. However, the ineffectiveness of the 5-HT1A agonist ipsapirone and the inability of spiroxatrine to block the serotonin response show that classical 5-HT1A receptors are not involved. The serotonin response is blocked by pertussis toxin and is insensitive to the phosphodiesterase inhibitor theophylline, which indicates the involvement of an inhibitory guanine regulatory protein in the coupling of the serotonin receptor to the adenylate cyclase catalytic unit.

— Rats were given an intravenous injection of 100 μCi of l–[3H]tryptophan and were killed between 5 and 180 min post–injection. Brains were dissected into seven areas, and specific activities were determined for major components (tryptophan, 5–hydroxytryptophan. serotonin and 5–hydroxy–indoleacetic acid) and detectable minor components (tryptamine, 5–methoxytryptamine. 5–hydroxyindo–lepyruvic acid, and 5–hydroxytryptophol) of serotonergic metabolism. The minor components represented small amounts of the total radioactivity with respect to the four major components. Anomalies in the product–precursor relationships for 5–hydroxytryptophan and serotonin, as well as for serotonin and 5–hydroxyindoleactic acid, suggest intraneuronal compartmentation of serotonin.

— MAO of human brain and liver mitochondria was solubilized by a procedure that preserved the substrate and inhibitor selectivities of the original mitochondrial preparation. Techniques that are designed to separate proteins on the basis of molecular size or net surface charge did not yield a physical separation of enzymically active A and B forms, even in the presence of ionic detergents or with limited proteolysis. However, sulfhydryl inhibitors, inorganic salts, ionic detergents, heat treatment, and sonication all tended to cause selective inactivation of serotonin-metabolizing activity in solubilized preparations. Experiments with selectively inhibited (membrane-bound or solubilized) MAO supported the concept of at least two independent kinds of substrate binding site, only one of which metabolizes serotonin (A type) and another (B type) which has a very strong affinity for β-phenethylamine. l-Norepinephrine, tryptamine, dopamine, and tyramine could be classified as common substrates. The lowest Km values were found for tryptamine at A sites and for β-phenethylamine at B sites. Results of this study suggest that the different MAO sites could be part of the same large molecular complex, which may normally be embedded in the outer mitochondrial membrane so that A sites are more dependent on their lipid environment within this membrane.

— The affinities of 5-hydroxy-[3H]tryptamine (5-HT) for cerebroside sulfate, 1-phosphatidylinositol, 1-phosphatidylinositol 4-phosphate, 1-phosphatidylinositol 4,5-bisphosphate, phosphatidic acid, and phosphatidyl serine were determined in an aqueous medium. They were observed to be, in general, much lower than and poorly correlated with the values previously reported by Johnsonet al (1977a), measured in isobutanol. This suggested that these lipids probably are not 5-HT receptors and that drug affinities measured in organic media must be evaluated with caution.

Earlier experiments have shown that unilateral electrolytic lesions of the substantia nigra result in significant reductions in the rate of accumulation of rat striatal tryptamine. For elucidation of the type of neuronal degeneration that is associated with tryptamine depletion, the effects of intranigral injections of 6-hydroxydopamine or 5, 7-dihydroxytryptamine, which would affect, respectively, dopamine- or indoleamine-containing neurons, have been assessed. Nigral 6-hydroxydopamine lesions resulted in an ipsilateral reduction in the rate of accumulation of striatal tryptamine, but no changes were observed after nigral injections of 5, 7-dihydroxytryptamine. The present results suggest that decreases in the pargyline-induced accumulation of striatal tryptamine may be associated with lesions of the nigral dopamine-containing cell bodies. Alternatively, there may exist specific tryptamine-containing neurons that are damaged by 6-hydroxytryptamine and unaffected by 5, 7-dihydroxytryptamine.

Competition binding studies, subcellular distribution, and in vitro autoradiography were employed to compare the binding in rat brain of [3H]tryptamine with two radio-ligands for monoamine oxidase (MAO), [3H]pargyline, and [3H] 1 -methyl-4-phenyl-1,2,5,6-tetrahydropyridine ([3H]MPTP). The MAO inhibitors pargyline, clorgyline, and deprenyl all yielded biphasic competition curves versus [3H]tryptamine. At low concentrations, these drugs stimulated binding by protecting the radioligand from MAO oxidation; at considerably higher concentrations, they inhibited binding by direct competition at the [3H]tryptamine binding site. In subcellular distribution studies, [3H]tryptamine was localized preferentially to the synaptosomal fraction, whereas [3H]pargyline showed greater binding to the mitochondrial fraction. Equilibrium binding studies revealed that the potencies of a series of seven compounds at inhibiting [3H]tryptamine binding were completely different from their potencies at inhibiting [3H]MPTP binding. Finally, the au-toradiographic distribution of [3H]tryptamine binding in rat brain v/as different from that of [3H]MPTP and [3H]pargyline. We conclude that the [3H]tryptamine binding site in rat brain is not equivalent to MAO.