Caffeine injection raises brain tryptophan level,but does not stimulate the rate of serotonin synthesis in rat brain

Acute caffeine injection (100 mg/kg) elevates brain levels of tryptophan (TRP), serotonin (5HT), and 5-hydroxyindoleacetic acid (5HIAA). Experiments were performed to determine if the increases in 5HT and 5HIAA result from a stimulation of the rate of 5HT synthesis. Both the rate of 5-hydroxytryptophan (5HTP) accumulation following NSD-1015 injection, and the rate of ³H-5-hydroxyindole synthesis from ³H-tryptophan were measured $\text{in vivo}$ following caffeine administration and found to be normal. Tryptophan hydroxylase activity, as measured $\text{in vitro}$ in brain homogenates, was also unaffected by caffeine. The results suggest that the elevations in brain 5HT and 5HIAA levels produced by caffeine do $\text{not}$ reflect enhanced 5HT synthesis, despite significant elevations in brain TRP level. Some other mechanism(s) must therefore be responsible for these elevations in brain 5-hydroxyindole levels.     

Serotonin action on short-circuit current and ion transport across isolated rabbit ileal mucosa.

Serotonin has previously been implicated as the cause of the diarrhea associated with carcinoid syndrome and the amine has been shown by others to be an intestinal secretagogue in preparations of intestinal loops $\text{in}$$\text{vivo}$. In the present paper the action of serotonin on isolated segments of rabbit ileal mucosa stripped of muscle layers was studied $\text{in}$$\text{vitro}$. Serotonin (10^?4M) caused an abrupt significant rise in short-circuit current (Isc) across the mucosal epithelial cell layer but this effect was transient. No change was observed in tissue conductance. In this preparation, serotonin did not alter ²²Na, ³⁶Cl or residual ion fluxes across the mucosa. High blood serotonin levels for a period of several days also did not alter ion fluxes or Isc in isolated rabbit ileum. Therefore, it is concluded that serotonin must cause its secretory activity observed $\text{in}$$\text{vivo}$ by some mechanism other than a direct action on epithelial cell transport mechanisms.     

Acute effects of aspartame on large neutral amino acids and monoamines in rat brain

The dipeptide aspartame (APM; aspartylphenylalanine methylester), an artificial sweetener, was studied $\text{in vivo}$ for its ability to influence brain levels of the large neutral amino acids and the rates of hydroxylation of the aromatic amino acids. The administration by gavage of APM (200 mg/kg) caused large increments in blood and brain levels of phenylalanine and tyrosine by 60 minutes. Brain tryptophan level was occasionally reduced significantly, but the brain levels of the branched-chain amino acids were always unaffected. Smaller doses (50, 100 mg/kg) also raised blood and brain tyrosine and phenylalanine, but did not reduce brain tryptophan levels. At the highest dose (200 mg/kg), APM gavage caused an insignificant increase in dopa accumulation (after NSD-1015), and a modest reduction in 5-hydroxytryptophan accumulation. No changes in the brain levels of serotonin, 5-hydroxyindoleacetic acid, dopamine, dihydroxyphenylacetic acid, homovanillic acid, or norepinephrine were produced by APM administration (200 mg/kg). These results thus indicate that APM, even when administered in amounts that cause large increments in brain tyrosine and phenylalanine, produce minimal effects on the rates of formation of monoamine transmitters.     

(2R,5R)-6-heptyne-2,5-diamine,an extremely potent inhibitor of mammalian ornithine decarboxylase

It was previously shown that 5-hexyne-1,4-diamine is a potent enzyme-activated irreversible inhibitor of mammalian ornithine decarboxylase. However this compound has secondary pharmacological effects owing to its $\text{in vivo}$ oxidation to 4-aminohex-5-ynoic acid, an irreversible inhibitor of 4-aminobutyrate aminotransferase. The first step of this oxidation is catalysed by mitochondrial monoamine oxidase. The monomethyl and dimethyl analogues of 5-hexyne-1,4-diamine, i.e. 6-heptyne-2,5-diamine and 2-methyl-6-heptyne-2,5-diamine, which cannot be substrate of monoamine oxidase, were tested as selective irreversible inhibitors of ornithine decarboxylase. Our results demonstrate that (2R,5R)-6-heptyne-2,5-diamine is greater than 10 times more potent, both $\text{in vitro}$ and $\text{in vivo}$, than α-difluoromethylornithine, the most widely used irreversible inhibitor of this enzyme.     

In vitro synthesis of monoamine oxidase of rat liver outer mitochondrial membrane

Monoamine oxidase, an intrinsic protein of outer mitochondrial membrane, was purified from bovine liver and rabbit antibody against the enzyme was prepared. The antibody could react with the monoamine oxidase of rat liver mitochondria. When rat liver RNA was translated $\text{in}\text{vitro}$ using rabbit reticulocyte lysate and monoamine oxidase peptide in the translation products was immunoprecipitated by the antibody, the peptide was detected in the products programmed by the messenger RNA's from total and free polysomes but not that from bound polysomes. The enzyme synthesized $\text{in}\text{vitro}$ had the same apparent molecular size as the mature protein in outer mitochondrial membrane.     

Modulators of monoamine oxidase in plasma

Addition of small amounts of plasma activated the deamination of tryptamine by platelet monoamine oxidase (MAO). At higher concentrations, plasma inhibited the deamination instead. The inhibition was increased with increasing amounts of plasma added. The inhibition was uncompetitive in nature, partially reversed by prior ultrafiltration of the plasma through PM30 membranes and completely reversed by protein precipitation of plasma with perchloric acid. Addition of high amounts of plasma $\text{in}$$\text{vitro}$ also inhibited the activity of bovine striatal MAO. The inhibition of striatal deamination of tryptamine by plasma was noncompetitive in nature, completely reversed by ultrafiltration through PM30 membranes and partially reversed by perchloric acid treatment. The inhibition of striatal deamination of serotonin was noncompetitive in nature, not reversed by ultrafiltration but completely reversed by perchloric acid treatment. The pattern of inhibition of platelet or striatal MAO by plasma was different from that induced by addition of bovine serum albumin (BSA). Low concentrations of BSA added $\text{in}$$\text{vitro}$ activated the deamination of tryptamine or serotonin by platelet or striatal MAO by decreasing the K_m, while higher concentrations also decreased the V_max. The presence of protein, non-albumin circulating modulators of platelet or striatal MAO in plasma is discussed.     

Effect of ascorbic acid on neurochemical, behavioral, and physiological systems mediated by catecholamines

Ascorbic acid, at concentrations below that normally present in the brain, inhibited the dopamine-sensitive adenylate cyclase $\text{in}$$\text{vitro}$. Ascorbate had no effect on the norepinephrine-sensitive adenylate cyclase. To study the $\text{in}$$\text{vivo}$ effect of ascorbic acid on central dopaminergic systems, mice (C57 B1/6J) were injected with pharmacological doses (2 g/kg) of ascorbate, which produced a significant elevation in brain ascorbate concentration. Injecting the mice with ascorbate (2 g/kg) blocked the amphetamine-induced (15 mg/kg) increase in stereotype behavior which has been reported to be mediated by dopaminergic neural systems. Ascorbate had no effect on the amphetamine-induced locomotor activity thought to be mediated by norepinephrine systems. Ascorbate (1 g/kg) attenuated apmorphine-induced hypothermia in this same strain of mice. This demonstrates the specific neurochemical, physiological, and behavioral alterations in dopaminergic systems produced by ascorbic acid and suggests possible therapeutic uses for ascorbate in conditions involving functional dopamine excess.     

Altered levels of β-endorphin fragments after chronic morphine treatment of guinea-pig ileum invitro and invivo

The isolated myenteric plexus-longitudinal muscle of the guinea-pig ilem (GPI) was used as testsystem to study the influence of chronic morphine treatment on the levels of enkephalins, β-endorphin and some of its fragments. The peptides were assayed by means of a combination of high pressure liquid chromatography and radioimmunoassays. It was found that the levels of methionine- and leucine-enkephalin and β-endorphin were not altered by chronic morphine treatment of guinea-pigs $\text{in}$$\text{vivo}$ nor in GPI exposed to morphine $\text{in}$$\text{vitro}$. However, the levels of some β-endorphin fragments i.c. γ-endorphin and des-tyrosine-γ-endorphin were elevated after morphine treatment $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ respectively. It is suggested that β-endorphin and its fragments are involved in homeostatic processes during development of opiate tolerance.     

Differences in aminoacylation in vivo and in vitro of lysine isoaccepting tRNAs from virus-transformed cells

When murine sarcoma virus-transformed cells are labeled with [³H]lysine $\text{in}\text{vivo}$ for various periods, 5 of 6 isoaccepting lysine tRNAs separable by RPC-5 chromatography are aminoacylated in 1 hr to the same extent that they are aminoacylated $\text{in}\text{vitro}$. The sixth isoacceptor, tRNA₆^Lys, is not aminoacylated $\text{in}\text{vivo}$ to a measurable extent in 1 hr, although it is present in the tRNA prepared from the cells. All six isoacceptors are aminoacylated with [³H]lysine $\text{in}\text{vivo}$ when the labeling period is 2 or 3 hr. These results further show that $\text{in}\text{vitro}$ correlations of the amount of tRNA₄^Lys with cell division accurately reflect the situation $\text{in}\text{vivo}$. Results of differential centrifugation indicate that tRNA₆^Lys occurs in mitochondria.     

A peptide-like inhibitor of N-methyltransferase in rabbit brain

After pretreatment with pheniprazine, rabbits were administered C-14-tryptamine i.v. and the lung was assayed for the N-methylated derivatives. Unoxidized tryptamine was present, but no N-methyl or N, N-dimethyltryptamine was found in this tissue, which contains high levels of N-methyltransferase. It appears that the indolamine-N-methyltransfer reaction is inhibited in the intact tissue. Our investigation of the possible inhibitory mechanism has led to the purification and characterization of a dialysable factor which inhibits the enzyme $\text{in}$$\text{vitro}$. The factor, which is present in most tissues, was purified from newborn rabbit brain. It is present in two forms, one having approximate mol. wt. 1,500 and one mol. wt. 1,300. Both were inactivated by crystalline trypsin. The 1,300 form was digested by carboxypeptidase A to a smaller, but still active form. It is suggested that these peptides may control $\text{in}$$\text{vivo}$ the activity of the non-specific N-methyltransferase against tryptamine and serotonin.     

OKY-1581: A selective inhibitor of thromboxane synthesis invivo and invitro

OKY-1581 is an effective inhibitor of thromboxane synthesis $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. The generation of thromboxane B₂ (TxB₂), prostaglandin E (PGE) and prostaglandin F (PGF) was measured following clotting and during platelet aggregation induced by collagen. The presence of OKY 1581 either $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$ caused a reduction in TxB₂ generation during clotting and platelet aggregation with a concomitant increase in PGE and PGF. The effect could be observed two hours after oral or subcutaneous administration of 5 to 100 mg per rabbit and lasted for 24 to 48 hours. The reduction in TxB₂ was not accompanied by an inhibition of clotting or platelet aggregation. OKY-1581 appears to be a suitable agent for studying the role of TxB₂ in atherosclerosis.     

Deamination of 5-methoxytryptamine, serotonin and phenylethylamine by rat MAO in vitro and in vivo.

Pretreatment of rats with clorgyline, a selective inhibitor of MAO-A, significantly inhibited the $\text{in vivo}$ deamination of intraventricularly administered serotonin (5-HT) and 5-methoxytryptamine (5-MT), but not phenylethylamine (PEA). Pretreatment with d, l-deprenyl, a selective inhibitor of MAO-B, significantly inhibited the $\text{in vivo}$ deamination of all three substrates. Brain and liver homogenates from rats pretreated with clorgyline showed a decreased ability to deaminate ($\text{in vitro}$) 5-MT and 5-HT, but not PEA. Homogenates from animals pretreated with d,l-deprenyl showed a decreased capacity to deaminate PEA, but not 5-MT or 5-HT. Clorgyline, when added to brain and liver homogenates, selectively blocked the deamination of 5-MT and 5-HT, but not PEA, whereas, d,l-deprenyl blocked the deamination of PEA without affecting that of 5-MT or 5-HT. In addition, 5-MT was found to be 100 X more potent than PEA at inhibiting the $\text{in vitro}$ deamination of 5-HT. These findings suggest that 5-MT and 5-HT are favored substrates for MAO-A $\text{in vitro}$ and $\text{in vivo}$. However, $\text{in vivo}$, significant amounts of 5-MT and 5-HT can also be deaminated by MAO-B.     

The effects of narcotic analgesics and narcotic antagonists on ganglionic transmission in the rat.

The effects of narcotic analgesics, narcotic-antagonist analgesics and narcotic antagonists on ganglionic transmission in the superior cervical ganglia of the rat were studied $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. $\text{In}$$\text{vivo}$ administration of morphine, meperidine, methadone, pentazocine or naltrexone blocked ganglionic transmission. Levorphanol, cyclazocine, nalorphine and naloxone had no effect on ganglionic transmission in this procedure. $\text{In}$$\text{vitro}$ studies confirmed the $\text{in}$$\text{vivo}$ results with the exception of levorphanol, cyclazocine and nalorphine, which were also found to block ganglionic transmission $\text{in}$$\text{vitro}$. In both preparations, naloxone did not antagonize the effect of morphine, suggesting that the effects of morphine and the other opiates were nonspecific. Similar potency of $\text{d}$- and $\text{l}$-isomers of pentazocine and cyclazocine support this conclusion. The observation that naltrexone blocked ganglionic transmission, but the other pure narcotic antagonist, naloxone, was inactive is somewhat unique to this test procedure and possibly significant.     

An organ culture system for study of fetal lung development

Fetal rat lungs placed in $\text{in}$$\text{vitro}$ organ culture at 15.5 days gestation grow significantly based on accumulation of DNA and protein. In the experimental system described, DNA accumulated rapidly during the first three days in culture and increased from 4.8 to 15.6 micrograms per lung culture. Protein content increased more slowly and reached a value more than double the initial value after six days in the culture system. Glycogen accumulated in the tissue during the first six days in culture and was depleted during the subsequent culture period, a pattern strikingly similar to that observed during lung development $\text{in}$$\text{vivo}$. Phospholipid accumulation was biphasic with respect to time with an inflection point at about the sixth day of culture. The phosphatidylcholine species synthesized in the culture system $\text{in}$$\text{vitro}$ were similar to those produced $\text{in}$$\text{vivo}$ in fetal lung at 21 days gestation.     

Unscheduled DNA synthesis in isolated hepatic nuclei after treatment of rats with methylnitrosourea in vivo

Administration of the carcinogenic methylating agent, methylnitrosourea, to rats caused a significant increase in endogenous DNA synthesis assayed subsequently in isolated hepatic nuclei $\text{in}\text{vitro}$. DNA synthesis was related directly to the dose of carcinogen and inversely to the interval between treatment and isolation of nuclei. This synthesis appears to represent the continuation $\text{in}\text{vitro}$ of unscheduled, reparative DNA synthesis initiated in damaged cells $\text{in}\text{vivo}$.     

In vivo [3H]ketanserin binding: effect of modifying synaptic serotonin levels

Most antidepressant therapies aim to increase the synaptic concentration of one or more of the monoamines. Synaptic monoamine levels are, however, extremely difficult to measure. In order to estimate synaptic levels of serotonin, an indirect method has been used. Since [³Hjketanserin is an antagonist at the 5HT₂ serotonin receptor, one would expect its in vivo binding to be inhibited by increased levels of synaptic serotonin. This hypothesis was tested in mice by measuring the effect of compounds which are considered to raise the synaptic concentration of serotonin. Directly acting agents such as quipazine, methysergide and mianserin inhibited [³H]ketanserin binding in vitro and in vivo. On the other hand, indirect agonists such as the monoamine oxidase inhibitors, pargyline and niamide, the serotonin uptake blockers, paroxetine and midalcipran, the serotonin releasers, p-chloroamphetamine and H75/12 and the serotonin precursor, 5-hydroxytryptophan had no effect on [³H]ketanserin binding in vivo. This was in spite of the fact that at doses used a very marked serotonin-induced behaviour was observed. In view of its insensitivity to changes in synaptic concentrations of serotonin, it is possible that the sites labelled in vivo by [³H]ketanserin are not innervated by the serotonin nerve terminals through which these indirect serotonin agonists act.     

Role of protein binding and pharmacokinetics in the lack of correlation between in vitro inhibition of PG-synthetase and in vivo antiinflammatory activity of a homologous series of nonsteroidal antiinflammatory compounds.

The antiinflammatory activity of a homologous series of α-alkyl substituted [4-(1-oxo-2-iso-indolinyl)-phenyl]-acetic acid has been assayed by some $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ tests.These compounds were shown to be particularly active in inhibiting prostaglandin biosynthesis from bovine seminal vesicles, and their potency was seen to increase as the size of the substituents in the side chain increased.The antiinflammatory activity $\text{in}$$\text{vivo}$ is not correlated with $\text{in}$$\text{vitro}$ inhibition of PG-synthetase. Discussion of the data takes into account the plasma protein binding and pharmacokinetics of these compounds.     

Identification of opiate receptor binding in intact animals.

After intravenous administration of ³H-naloxone to rats, particulate bound radioactivity accumulated in the brain is selectively associated with opiate receptor binding sites, providing a means of labeling the opiate receptor $\text{in vivo}$. The regional distribution of ³H-naloxone bound $\text{in vivo}$ closely parallels regional differences in opiate receptor binding $\text{in vitro}$ with highest levels in the corpus striatum, negligible receptor-associated binding in the cerebellum and intermediate levels in other regions. ³H-Naloxone binding $\text{in vivo}$ is saturable with the same total number of binding sites determined $\text{in vivo}$ as by $\text{in vitro}$ procedures. Nalorphine is markedly more potent than morphine in inhibiting ³H-naloxone binding $\text{in vivo}$ and non-opiates are ineffective. The half-life for dissociation of ³H-naloxone bound to particles $\text{in vivo}$ is the same as its dissociation rate after binding occurs $\text{in vitro}$, and sodium stabilizes ³H-naloxone bound $\text{in vivo}$ from initial rapid dissociation as predicted from the known properties of the opiate receptor $\text{in vitro}$.     

Specificity of serotoninergic involvement in the decrease of food intake induced by quipazine in the rat.

The effect of quipazine on brain monoamines and the significance of this interaction in its anorectic activity was studied in rats. At doses ranging from 2.5 to 10 mg/kg quipazine markedly reduced brain 5-hydroxyindolacetic acid concentrations without significant effects on steady-state levels of serotonin, noradrenaline and dopamine. Striatal levels of homovanillic acid were significantly reduced by 10 mg/kg of quipazine but not modified by a dose of 5 mg/kg. Quipazine counteracted the decrease of brain serotonin induced by fenfluramine but did not significantly modify the effect of 6-hydroxydopamine on brain nonadrenaline and dopamine. The decrease of food intake induced by 5 mg/kg of quipazine was completely prevented by pretreatment with methergoline but was not affected by an intraventricular injection of 6-hydroxydopamine or pretreatment with penfluridol, propranolol or phentolamine. The results indicate that at doses between 2.5 and 5 mg/kg quipazine specifically acts on brain serotonin and this interaction may be important for its anorectic activity.