Conversion of C14-S-adenosylmethionine to C14-formaldehyde and condensation with indoleamines: a side reaction in N-methyltransferase assay in blood

Incubation of N-methylserotonin (NMS) with erythrocyte hemolyzates and C¹⁴-S-adenosylmethionine (C¹⁴-SAM) leads to the formation of an unidentified product rather than the expected bufotenin. It is shown that this compound is a condensation product of NMS with C¹⁴-formaldehyde formed enzymatically from C¹⁴-SAM. Incubation of rabbit lung homogenates however, leads to formation of the expected product. Previous reports of N-methyltransferase activity in blood and perhaps other tissues should be re-evaluated in light of these findings, if rigorous proof of identity was not carried out.     

Substance P degrading systems of rat parotid and hypothalamus

Inactivation of substance P and its C-terminal hexapeptide analog [p-Glu⁶]substance P6–11 was studied in rat parotid and hypothalamic slices. It was found that in the parotid slice system the decay of substance P induced K⁺ release occurs concurrently with a decrease in the biologically active concentration of the peptide in the medium. The inactivation was further studied using [p-Glu⁶]substance P6–11 as substrate in the parotid and in the hypothalamic slice systems. In both tissue preparations the hexapeptide is degraded to small peptide fragments by metalloendopeptidase. Separation of the peptide fragments by high performance liquid chromatography and determination of their amino acid composition showed that in the hypothalamic slice system the major cleavage of the hexapeptide analog occurs between Phe⁸-Gly⁹ with minor cleavage sites between Phe⁷-Phe⁸ and Gly⁹-Leu¹⁰. In the rat parotid slice system the major cleavage occurs between Gly⁹-Leu¹⁰ with a minor cleavage site between Phe⁷-Phe⁸. The degradation of the hexapeptide analog in the hypothalamic system was inhibited 77% and 67% by treatment with 1 mM p-chloromercuriphenylsulfonate and p-chloromercuribenzoate, respectively, whereas in the parotid system these reagents inhibited the degradation of the hexapeptide only by 15% and 8%. These results may indicate that different proteases in the parotid and hypothalamus are involved in degradation of substance P. Kinetic studies, including the use of various inhibitors as well as competition by the peptide hormones somatostatin, LHRH, TRH and Leu-enkephalin-NH₂, revealed that in both tissues the hexapeptide analog is a preferred substrate for degradation by protease of considerable specificity towards the C-terminal sequence of substance P. It is suggested that this metalloendopeptidase may be important in the termination of the substance P response.     

The presence of free and conjugated bufotenin in normal human urine

The N,N-dimethylated derivative of serotonin, bufotenin , is excreted into normal human urine as a free amine. Conjugation of bufotenin is, however, possible because of the phenolic hydroxyl group on the molecule. In the present study the urinary excretion of free and conjugated bufotenin of ten healthy, drug free subjects was examined. Acid as well as enzymatic hydrolysis was used to liberate the amine from its conjugate. Quantification was achieved by isotope dilution mass fragmentography. Of total urinary bufotenin a relatively constant amount, 59.9 - 69.0%, was excreted in conjugated form. The conjugate was tentatively identified as a glucuronide.     

Effects of Indoleamines and Short Photoperiods on the Encystment of Gonyaulax polyedra

At a temperature of 15^oC, Gonyaulax polyedra responds to short days (light ≤ 10 h) by transition to the stage of a resting cyst. At 20^oC, even an lightdark (LD) cycle of 6:18 is incapable of inducing this process. In otherwise cyst-inducing conditions (15^oC; 10 h of light per day), an interruption of the scotophase by 2 h of light (LDLD 8:2:2:12 or 2:2:8:12) prevented encystment. Cyst induction is, therefore, initiated by a photoperiodic mechanism rather than by light deficiency. In Gonyaulax, photoperiodism may be mediated by the action of indoleamines. Melatonin, which exhibits a circadian rhythmicity in this organism, leads to encystment when given 1 h before lights-off in LD 11:13 at 15^oC, i.e., under otherwise noninducing conditions. Again, at 20^oC, melatonin is inefficient. Some analogues of melatonin, in particular, 5-methoxytryptamine and N,N-dimethyl-5-methoxytryptamine, and, at high concentrations, their respective precursors, serotonin and bufotenin, are capable of inducing cyst formation at 20^oC and in LD 12:12, whereas A'-acetyl-serotonin does not show this effect.     

Specific binding of an immunoreactive and biologically active 125I-labeled N(1)acylated substance P derivative to parotid cells

A biologically active ¹²⁵I-substance P derivative (I¹²⁵-BH-substance P), prepared by conjugation of substance P with [^125I]Bolton-Hunter reagent, binds specifically to isolated rat parotid cells. The Kd is 4 nM for I-BH-substance P, 5 nM for substance P, 0.18 μM for substance P octa(4–11)peptide, and 1.6 μM for substance P [pyroglutamyl⁶]hexa(6–11)peptide. Substance P free acid and substance P penta(7–11)peptide are much weaker competitors and the C-terminal tri(9–11)peptide has no effect at 30 μM. The binding is also inhibited by 1 μM physalaemin, eledoisin and substance P methyl ester, but not by unrelated peptides. The selective inhibition of the binding by the biologically active analogs and fragments of substance P indicates that the ¹²⁵I-labeled N(1)acylated substance P derivative may interact with a substance P receptor on parotid cells.     

Analgesic effect of antagonists of substance P

[D-Pro²,D-Phe⁷,D-Trp⁹]-SP and [D-Pro²,D-Trp^7,9]-SP havebeen shown to be antagonists of substance P. The hindlimb scratching syndrome of mice, known to be caused by substance P was absent when these peptides were injected into substance P-treated mice. Substance P shortens “tail withdrawal time” from hot water; the two peptides greatly prolonged tail withdrawal time. Antidromic stimulation of the saphenous nerve (rat), known to release substance P and to induce vasodilatation plasma extravasation, was also greatly inhibited by [D-Pro²,D-Phe⁷,D-Trp⁹]-SP. These peptides presumably cause anti-nociceptor effects (analgesia) by inhibition of substance P at receptors and favor the concept that substance P is a sensory neurotransmitter of nociceptive messages.     

DETECTION AND EVALUATION OF A NOVEL SEXUAL PHEROMONE THAT INDUCES SEXUAL CELL DMSION OF CLOSTERIUM EHRENBERGII (CHLOROPHYTA)1

Mating type-plus (mt⁺; NIES-228) cells of Closterium ehrenbergii undergo a division to form gamete-shaped cells. This cell division is induced by a substance produced by mating type-minus (mt^?; NIES-229) cells. Light and the presence of mt⁺ cells enhanced production of the substance. The active substance is heat labile and has an apparent molecular mass of 20 kDa. From these results, we conclude that the substance is a novel, proteinaceous sexual pheromone involved in reproduction of Closterium ehrenbergii.     

Effects of the unilateral nigral modulation of substance P transmission on the activity of the two nigro-striatal dopaminergic pathways.

The effects of the unilateral nigral application of substance P, 4–11 substance P, LHRH and of antibodies against substance P on the release of ³H-dopamine (³H-DA) in the two caudate nuclei (CN) and the two substantia nigrae (SN) were examined in halothane anaesthesized cats. The animals were implanted with four push-pull cannulae and ³H-DA was estimated in successive superfusate fractions during the continuous delivery of ³H-tyrosine. The unilateral nigral application of substance P or of 4–11 substance P resulted not only in a stimulation of ³H-DA release in the ipsilateral CN, but also in a reduction of the ³H-transmitter release from dendrites in the SN. As previously reported, the unilateral nigral application of antibodies against substance P induced opposite effects i.e. a decreased release of ³H-DA from nerve terminals associated to an increase of the ³H-amine release from dendrites. Surprisingly, in contrast to that previously observed during the unilateral nigral application of dopaminergic glycinergic or GABAergic agonists and antagonists neither substance P (or 4–11 substance P) nor antibodies against substance P affected the release of ³H-DA from nerve terminlas and dendrites of the contraletaral dopaminergic neurons. LHRH, a similar sized peptide was without effect on ipsilateral as well as on contralateral dopaminergic.     

Modulation of membrane K+ conductance in T-lymphocytes by substance P via a GTP-binding protein

Summary Modulation of the voltage-gated K⁺ conductance in T-lymphocytes by substance P was examined. Whole-cell recordings from Jurkat E6-1 human T-lymphocytes revealed two components of substance P action on the outward K⁺ current: (i) dose- and time-dependent reduction of current peak amplitude; and (ii) acceleration of the current inactivation rate. This action was blocked by substituting Cs⁺ for K⁺ in the recording pipette and by the substance P antagonist, [d-Arg¹,d-Phe⁵,d-Trp^7.9, Leu¹¹]-substance P. As indicated by conductance-voltage relationship, the reduction in current peak amplitude as a result of substance P application was not due to a shift of the voltage dependence of the channel. Raising intracellular free calcium concentration from 2 to 200nm reversed the reduction, induced by substance P, in current peak amplitude and disclosed an apparent desensitization towards the neuropeptide action. The treatment, however, did not reverse substance P-induced acceleration of the rate of current decay. Intracellular administration of hydrolysis-resistant guanosine triphosphate (to persistently activate GTP-binding protein) and guanosine diphosphate (to competitively inhibit GTP—binding proteins) analogues mimicked and inhibited substance P-induced reduction of K⁺ conductance, respectively. The data demonstrate a modulation of T-lymphocyte K⁺ channels by substance P and substantiate a possible role for GTP-binding proteins in this modulation.     

Intrathecal substance P augments morphine-induced antinociception: Possible relevance in the production of substance P N-terminal fragments

The present study sought to examine the mechanism of substance P to modulate the antinociceptive action of intrathecal (i.t.) morphine in paw-licking/biting response evoked by subcutaneous injection of capsaicin into the plantar surface of the hindpaw in mice. The i.t. injection of morphine inhibited capsaicin-induced licking/biting response in a dose-dependent manner. Substance P (25 and 50 pmol) injected i.t. alone did not alter capsaicin-induced nociception, whereas substance P at a higher dose of 100 pmol significantly reduced the capsaicin response. Western blots showed the constitutive expression of endopeptidase-24.11 in the dorsal and ventral parts of lumbar spinal cord of mice. The N-terminal fragment of substance P (1–7), which is known as a major product of substance P by endopeptidase-24.11, was more effective than substance P on capsaicin-induced nociception. Combination treatment with substance P (50 pmol) and morphine at a subthreshold dose enhanced the antinociceptive effect of morphine. The enhanced effect of the combination of substance P with morphine was reduced significantly by co-administration of phosphoramidon, an inhibitor of endopeptidase-24.11. Administration of d-isomer of substance P (1–7), [d-Pro², d-Phe⁷]substance P (1–7), an inhibitor of [³H] substance P (1–7) binding, or antisera against substance P (1–7) reversed the enhanced antinociceptive effect by co-administration of substance P and morphine. Taken together these data suggest that morphine-induced antinociception may be enhanced through substance P (1–7) formed by the enzymatic degradation of i.t. injected substance P in the spinal cord.     

Isolation of a New Exopolygalacturonase Producing Digalacturonic Acid from Pectic Acid

Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena such as blood clotting, wound healing, apoptosis, and cell differentiation. Streptomyces lavendulae Y-200, isolated from soil, produced a substance that inhibited transglutaminases. The inhibitory substance was purified from the cultured medium by procedures of acid precipitation, deoxyribonuclease treatment, and gel filtration chromatography. The partially purified sample was dark brown. The inhibitory activity was stable under acidic, alkaline, and high temperature conditions, and resistant to the treatment with proteinases such as trypsin and Pronase. The molecular weight of the inhibitory substance was estimated to be between 10⁴ and 10⁵ from its permeability through ultrafilter membranes. The acid hydrolysate of the inhibitory substance contained amino acids and sugars. The inhibitory substance inhibited both calcium-dependent and calcium-independent transglutaminases in a competitive manner with a glutamine substrate. The extent of inhibition caused by the calcium-dependent transglutaminase increased with increasing calcium concentration. The results obtained here may help identify a novel regulatory substance of transglutaminase in biological systems.     

Tetrahydrofolic acid: An inhibitor of the methyltetrahydrofolic acid-mediated methylation of indolethylamines

Tetrahydrofolic acid exerts a product inhibition on the methyltetrahydrofolic acid-mediated methylation of indolethylamines. Kinetic studies showed that this inhibition was competitive with respect to methyltetrahydrofolic acid and non-competitive with respect to N-methylserotonin. Chromatographic separation of S-adenosylmethioniee-dependent indolethylamine N-methyltransferase and methyltetrahydrofolic acid-dependent methyltransferase from rabbit lung was obtained. There was no cross reaction of the two enzymes to tetrahydrofolic acid, S-adenosylhomocysteine, N,N-dimethyltryptamine or bufotenin.     

Incorporation of methanol into pectic substance

Others have shown that l-methionine is utilized in the biosynthesis of methyl ester groups in pectic substance. Methanol, like l-methionine, is used for methyl ester biosynthesis by detached parsley leaves (Petroselinum crispum). When a combination of methanol-³H and methanol-¹⁴C is given to parsley leaves, methanol recovered from pectic substance by alkaline hydrolysis has a ³H/¹⁴C ratio about one-fourth that of the mixture administered. Unlike l-methionine, methanol is oxidized prior to its utilization as a carbon source for methyl ester biosynthesis.     

Selective depletion of substance P-immunoreactive neurones in the transitional zone of the colon in piebald lethal mice

The distribution of substance P in the colon of piebald lethal (s¹/s¹) mice was studied by radioimmunoassay and immunohistochemistry. These animals inherit as a Mendelian recessive trait an aganglionic distal colon. In the region proximal to the aganglionic segment, there is an extensive transitional or hypoganglionic zone, in which the total number of nerve cells in the myenteric plexus is reduced, while those in the submucous plexus tend to be normal. Immunohistochemical studies indicate that substance P-immunoreactive neurones accounted for approx. 10% of the total number of normal myenteric neurones, but in the hypoganglionic region they accounted for about 5%, and this difference was statistically significant. By radioimmunoassay, the concentrations of substance P in both the aganglionic and the hypoganglionic regions of the colon were reduced compared with the corresponding segments in normal mice. However calculation of the mean substance P content per neurone revealed similar quantities (about 1 fmol) in both normal and s¹/s¹ mice. Substance P-immunoreactivity in the tissue extracts eluted in the same position as the synthetic peptide on ion exchange and gel filtration chromatography. It is suggested that a sub-population of substance P-immunoreactive neurones in the hypoganglionic zone is selectively depleted compared with other myenteric neurones. The factors involved remain to be elucidated, but this strain of mice could prove useful for studies of the mechanisms involved in differentiation and development of enteric peptidergic neurones.     

Immunohistochemical evidence for different pathways immunoreactive to substance P and calcitonin gene-related peptide (CGRP) in the guinea-pig stellate ganglion

The colocalization of immunoreactivities to substance P and calcitonin gene-related peptide (CGRP) in nervous structures and their correlation with other peptidergic structures were studied in the stellate ganglion of the guinea pig by the application of double-labelling immunofluorescence. Three types of fibre were distinguished. (1) Substance P⁺/CGRP⁺ fibres, which sometimes displayed additional immunoreactivity for enkephalin, constituted a small fibre population of sensory origin, as deduced from retrograde labelling of substance P⁺/CGRP⁺ dorsal root ganglion cells. (2) Substance P⁺/CGRP^– fibres were more frequent; some formed baskets around non-catecholaminergic perikarya that were immunoreactive to vasoactive intestinal polypeptide (VIP). (3) CGRP⁺/substance P^– fibres were most frequent and were mainly distributed among tyrosine hydroxylase (TH)-immunoreactive cell bodies. The peptide content of fibre populations (2) and (3) did not correspond to that of sensory ganglion cells retrogradely labelled by tracer injection into the stellate ganglion. Therefore, these fibres are throught to arise from retrogradely labelled preganglionic sympathetic neurons of the spinal cord, in which transmitter levels may have been too low for immunohistochemical detection of substance P or CGRP. CGRP-immunoreactivity but no substance P-immunolabelling was observed in VIP-immunoreactive postganglionic neurons. Such cell bodies were TH-negative and were spared by substance P-immunolabelled fibre baskets. Retrograde tracing with Fast Blue indicated that the sweat glands in the glabrous skin of the forepaw were the targets of these neurons. The streptavidin-biotin-peroxidase method at the electron-microscope level demonstrated that immunoreactivity to substance P and CGRP was present in dense-cored vesicles of 50–130 nm diameter in varicosities of non-myelinated nerve fibres in the stellate ganglion. No statistically significant difference in size was observed between vesicles immunolabelled for substance P and CGRP. Immunoreactive varicosities formed axodendritic and axosomatic synaptic contacts, and unspecialized appositions to non-reactive neuronal dendrites, somata, and axon terminals. Many varicosities were partly exposed to the interstitial space. The findings provide evidence for different pathways utilizing substance P and/or CGRP in the guinea-pig stellate ganglion.     

Inhibition of lymphoma cell proliferation by supernatant from fibrosarcoma cultures: Preliminary evidence that the inhibitory material is prostaglandin E

Supernatants obtained from mouse fibrosarcoma cultures 48 hr after the addition of fresh medium contained dialyzable material which inhibited the proliferation of syngeneic lymphoma cells , as measured by ³H-thymidine incorporation. Three lines of evidence indicate that the supernatant inhibitory material is probably prostaglandin (PG) E. First, the supernatant and dialysate of the supernatant contained a substance with the same characteristics as PGE₁ or PGE₂ as detected by thin layer chromatography. Second, PGE₂-treatment of lymphoma cells mimicked the inhibition of proliferation observed with supernatant inhibitory substance. Third, indomethacin treatment of fibrosarcoma cultures reduced the amount of supernatant inhibitory substance present.     

Production of gibberellin-like substance in the embryo of barley during germination

Summary Embryos were excised from barley seeds, their homogenates were incubated with ficin, and their content in gibberellin-like substance was assayed by means of -amylase-producing activity, but no gibberellin-like substance could be detected.Embryos free from endosperm which were cultured for five days produced measurable amounts of gibberellin-like substance. This substance was not produced when a carbon source was absent from the medium, or when air was removed after 2 days of aerobic culture. CCC and Phosfon D (at the concentration of 2×10^-4 M and 0.8×10^-4 M, respectively) inhibited the formation of the gibberellin-like substance in cultured embryos without affecting their growth.Mevalonic acid could be used as a carbon source in the culture of the embryos. The formation of the gibberellin-like substance was in this case inhibited by 0.8×10^-4 M Phosfon D, but was not inhibited by 2×10^-3 M CCC.     

Pharmacological Evidence for the Involvement of Calciuml Calmodulin in Serotonin 5-HT3 Receptor-Mediated Cation Permeability in NG 108-15 Cells

Abstract: In NG 108–15 clonal cells, extracellular application of micromolar concentrations of serotonin [5-hydroxy-tryptamine (5-HT)] and substance P induces the opening of a cation permeability monitored by the influx of [¹⁴C]-guanidinium. The serotoninergic component of this cation permeability Is linked to 5-HT₃ receptor activation, whereas the substance P component probably involves an “N-terminal-dependent substance P receptor.” In this study, [¹⁴C]guanidinium influx triggered by 1 μM 5-HT plus 10 μM substance P was shown to be insensitive to tetrodotoxin, verapamil, diltiazem, nimodipine, and ω-conotoxin, as expected from a process independent of voltage-sensitive sodium and calcium channels. In contrast, [¹⁴C]guanidinium influx was inhibited by millimolar concentrations of extracellular calcium and by the chelation of intracellular calcium by bis-O-aminophenoxyethanetetraacetic acid. The inhibition by extracellular calcium apparently involved a competition between the divalent cation and [¹⁴C]guanidinium for the same channel. When NG 108–15 cells were exposed to X537A, an ionophore that specifically induces release of calcium from intracellular stores, [¹⁴C]guanidinium uptake was markedly increased even in the absence of 5-HT and/or substance P. Conversely, [¹⁴C]guanidinium influx due to the latter substances could be reversibly and dose-dependently blocked by various drugs that possess calmodulin-antagonizing properties. These results strongly suggest that the cation permeability opened by 5-HT and substance P in NG 108–15 cells involves a calcium/calmodulin-dependent process. However, as the phosphodiesterase inhibitor isobutylmethylxanthine, the nitric oxide synthase inhibitor A/monomethylarginine, the protein kinase C inhibitor staurosporine, and the protein kinase C activator 12-O-tetradeca-noylphorbol 13-acetate did not alter [¹⁴C]guanidinium influx in NG 108–15 cells exposed to 5-HT and substance P., this process probably does not involve the calcium-dependent nitric oxide pathway and protein kinase C activation.     

A New Toxic Substance,Teleocidin, Produced by Streptomyces

The production and isolation of a new toxic substance, Teleocidin, and its biological properties were previously reported^1,2). Thereafter it has been found that an other strain of Streptomyces produced such specific toxic substance as Teleocidin in its cultured mycellium. Comparative tests of these two purified crystalline powders showed the new toxic substance resembles Teleocidin closely though differs in certain chemical properties. Therefore, the original Teleocidin is designated Teleocidin A, whereas that produced by a new strain of Streptomyces is named Teleocidin B, which had been tentatively called as the SK-toxic substance.

From the results of the chemical studies of Teleocidin B and its hydrogenated derivative, which was easily obtained as a crystalline form by the catalytic hydrogenation of Teleocidin B with Adam’s catalyst, molecular formula, C₂₈H_39~41N₃O₂ was postulated for Teleocidin B.

It was also recognized that an alcoholic hydroxyl, a lactam ring and a heterocyclic ring like indole or pyrrole structure existed as the functional groups of Teleocidin B.     

Structure-activity-relationships of certain hallucinogenic substances based on brain levels.

A structure-activity-relationship of a variety of behaviorally active and inactive compounds which are naturally occuring or closely related structurally to putative neurotransmitters has been assembled. For the first time comparisons of activity are based on actual brain levels instead of doses administered.A different and new SAR is obtained if minimal effective brain levels (MEBL), instead of administered doses, are used. Differences are due to the fate of the substance in the body and its availability to the CNS. Predictions of brain levels based on $\text{in}$$\text{vitro}$ data, blood levels and/or lipid solubility can be misleading.It is suggested that the “minimal effective brain level (MEBL)” or that concentration of a substance in the brain in moles/g at which time the first significant behavioral effect in a particular test situation can be detected, be used as the basis of comparison in SAR studies of behaviorally active substances. This is, of course, not the ultimate parameter which might be the number of molecules of a substance at a particular receptor, but it would eliminate a great number of “artifacts” which can be controlled with the present state of the art.The use of MEBLs will allow more valid correlations between behavioral activity and certain physico-chemical parameters of the compounds, and an attempt to use MEBLs was recently made by Houk and co-workers (53). Also, MEBLs in animals can serve as a better base for the interpretation of human studies and can make these behavioral studies in man more meaningful; for instance, the lack of behavioral activity in bufotenin in man observed recently (54) could perhaps be due to the fact that at the doses tested the compound did not reach the CNS in sufficient concentrations because of its extreme difficulty in crossing the BBB. Similarly, 3,4-dimethoxyphenylethylamine has been found to be inactive in man after oral administration (55); based on our studies (27) the compound would actually not be expected to be active by this route since it is too quickly metabolized and cannot reach the MEBL, thus, the behavioral activity of this compound in man remains unknown.Based on the criteria applied in this review, LSD and 5-methoxytryptamine are the most potent psychoactive substances followed by tryptamine (after MAO-inhibition) and pentamethoxyphenylethylamine. It is of interest to note that tryptamine and 5-methoxytryptamine are known to occur naturally in the mammalian brain. It remains to be determined what role, if any, these substances may play in the pathogenesis of abnormal behavior in man.