Harold D. Snoddy

Mechanisms of effects of d-Fenfluramine on brain serotonin metabolism in rats: Uptake inhibition versus release

d-Fenfluramine is an anorectic drug believed to act by enhancement on serotonergic function in the brain. d-Fenfluramine (or the racemate) releases serotonin through a carrier-dependent mechanism, and serotonin release is the mechanism usually thought to produce its serotonergic effects. However, d-fenfluramine also inhibits serotonin uptake in vitro, and serotonin uptake inhibition is sometimes suggested to contribute to its mechanism of anorectic activity. Neurochemical experiments were done to examine serotonin release and serotonin uptake inhibition as mechanisms of action of d-fenfluramine in rats and to compare d-fenfluramine to fluoxetine, a serotonin uptake inhibitor. d-Fenfluramine decreased serotonin concentration in rat brain as early as 1 hr; at 1 hr 5-hydroxyindoleacetic acid (5HIAA) concentration was slightly increased, but at later times 5HIAA was also decreased. Fluoxetine, in contrast, did not change serotonin concentration in whole brain but decreased 5HIAA concentration at all time points. At all time intervals studied, the 5HIAA/serotonin ratio was increased by d-fenfluramine (and by Ro 4-1284, a nonspecific serotonin releaser) but was decreased by fluoxetine, a serotonin uptake inhibitor. No decrease in 5HIAA concentration or in the 5HIAA/serotonin ratio was apparent at any time or after any dose of d-fenfluramine studied. The possibility that doses of d-fenfluramine below those needed for serotonin release might inhibit serotonin uptake was tested by determining whether d-fenfluramine could block the acute depletion of brain serotonin by p-chloroamphetamine, or the long-term neurotoxic effect of p-chloroamphetamine on brain serotonin neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of (8β)-8-[(Methylthio)methyl]-6-propylergoline on dopaminergic function and brain dopamine turnover in rats

Abstract (8β)-8-[(Methylthio)methyl]-6-propylergoline induced contralateral turning in rats with nigrostriatal lesions, lowered serum prolactin in reserpinized rats, and caused stereotyped hyperactivity. In addition to these functional effects typical of dopaminergic agonists, (8β)-8-[(methylthio)methyl]-6-propylergoline decreased dopamine turnover in rat brain. Decreased turnover was indicated by a diminished depletion of dopamine content after inhibition of its synthesis by α-methyltyrosine and by a decreased steady state concentration of the dopamine metabolite, 3, 4-dihydroxyphenylacetic acid (DOPAC). DOPAC concentration in whole brain was decreased after doses of (8β)-8-[(methylthio)methyl]-6-propylergoline as low as 0.003 mg/kg, and the lowering of DOPAC persisted for up to 16 hrs. after a 0.3 mg/kg dose. (8β)-8-[(Methylthio)-methyl]-6-propylergoline had less effect than a structurally-related compound, lergotrile, on 3-methoxy-4-hydroxy-phenyl-ethyleneglycol sulfate levels in whole brain and did not affect 5-hydroxy-indoleacetic acid levels over a dose range from .01–10 mg/kg. The behavioral and neuroendocrine effects of this new ergoline compound and its reduction of dopamine turnover in rat brain indicate that it is a potent dopamine receptor agonist in vivo .

Serotonin Receptor Subtypes Involved in the Elevation of Serum Corticosterone Concentration in Rats by Direct- and Indirect-Acting Serotonin Agonists

The serum corticosterone concentration in rats was increased by injection of quipazine, a relatively nonselective serotonin (5-hydroxytryptamine; 5-HT) agonist, or 8-hydroxy-2-(di-n- propylamino)tetralin (8-OH-DPAT), a serotonin agonist selective for the 5-HT1A subtype of receptor. The quipazine-induced increase in serum corticosterone was antagonized by 17 different serotonin antagonists; of these, MDL 11939, pirenperone, setoperone, mianserin, LY 281067, ketanserin, ritanserin and clozapine have relatively selective affinity for the 5-HT2 subtype of receptor. The 8-OH-DPAT-induced increase in serum corticosterone was not antagonized by metergoline, the most potent antagonist of the quipazine effect, but was antagonized by pindolol or penbutolol, 5-HT1A receptor antagonists. Pindolol did not block the effect of quipazine. The results support earlier evidence that serum corticosterone concentration in rats can be increased by activation of either 5-HT1A or 5-HT2 receptors. Indirect-acting serotonin agonists - fluoxetine, L-5-hydroxytryptophan and p-chloroamphetamine - also increased serum corticosterone concentrations. The increases elicited by those agents, which earlier had been reported not to be blocked by metergoline pretreatment, also were not blocked by pretreatment with pindolol or with the combination of metergoline and pindolol. Thus, an involvement of a specific serotonin receptor subtype in the actions of these indirect agonists has not been established.

Effect of Serotonin-Releasing Drugs on Serum Corticosterone Concentration in Rats

(+/-)p-Chloroamphetamine hydrochloride, at doses of 1-8 mg/kg i.p. in rats, caused a dose-related increase in serum corticosterone concentration. The increase occurred rapidly, within 30 min, and was over within 4 h. Evidence that the increase was mediated by serotonin release consisted of the following findings: (1) a similar increase did not occur with (+/-)o-chloroamphetamine or (+/-)p-chloro-alpha-methylbenzylamine, analogs of p-chloroamphetamine lacking its ability to deplete serotonin; (2) the increase was prevented by prior treatment with p-chlorophenylalanine, which reduced the brain stores of serotonin available for release by p-chloroamphetamine; (3) the increase was prevented by prior treatment with fluoxetine, an inhibitor of the uptake pump on serotonin neurons, which blocks serotonin release by p-chloroamphetamine, and (4) the increase was mimicked by fenfluramine and norfenfluramine, agents known to release brain serotonin in a manner similar to the action of p-chloroamphetamine. These findings strengthen earlier evidence that brain serotonin neurons have a stimulatory influence on pituitary-adrenocortical function in rats.

Importance of duration of drug action in the antagonism of p-chloroamphetamine depletion of brain serotonin-comparison of fluoxetine and chlorimipramine.

Abstract Fluoxetine inhibited both the rapid depletion of brain serotonin by p -chloroamphetamine (PCA) and the ultimate irreversible effects of PCA on brain serotonin neurons in rats; the differences between fluoxetine and chlorimipramine as PCA antagonists appeared to be related to the duration of uptake inhibition by these agents. Fluoxetine given along with PCA in a single dose prevented serotonin depletion at all times after PCA. Chlorimipramine antagonized serotonin depletion initially, but at later times there was little or no protection against PCA effects. The dose-dependence of the antagonism of PCA by fluoxetine did not vary greatly with the time of serotonin measurement after PCA. but with chlorimipramine the effectiveness of a given dose depended markedly on that time interval. Lengthening the pretreatment interval prior to PCA injection from 0 to 16 hr diminished the effectiveness of chlorimipramine but not fluoxetine as antagonists of serotonin depletion. The differences between fluoxetine and chlorimipramine may arise primarily because these compounds are metabolized by N -demethylation. The demethylated metabolite of fluoxetine was as potent and specific as fluoxetine itself as a serotonin uptake inhibitor both in vitro and in vivo , whereas the demethylated metabolite of chlorimipramine was less active than chlorimipramine as a serotonin uptake inhibitor and more active as a norepinephrine uptake inhibitor. Chlorimipramine was a more effective PCA antagonist when injected into mice in repeated doses or when injected into rats along with an inhibitor of liver microsomal enzymes. Thus, comparison of uptake inhibitors as antagonists of PCA is strongly influenced by the pharmacokinetics of the drugs involved.

Pharmacologic evidence for a serotonin neural pathway involved in hypothalamus-pituitary-adrenal function in rats.

Abstract The hypothesis that a serotonin neural pathway stimulates ACTH secretion in rats was supported by pharmacologic data. Fluoxetine, an inhibitor of serotonin reuptake, caused a dose-related elevation of plasma corticosterone levels in intact but not in hypophysectomized rats. The previously-reported elevation of plasma corticosterone by 5-hydroxytryptophan (5HTP) was confirmed and shown to be stereospecific, L-5HTP being much more active than D-5HTP. Simultaneous injection of subeffective doses of fluoxetine and L-5HTP caused marked elevation of plasma corticosterone. Fluoxetine pretreatment potentiated the elevation of plasma corticosterone by L-5HTP. Although the elevation of plasma corticosterone by fluoxetine was of short duration (perhaps due to compensatory reduction of serotonin release), the potentiation of the L-5HTP effect by fluoxetine lasted for more than 24 hrs as predicted by the duration of uptake inhibition by fluoxetine. The dose-response characteristics for corticosterone elevation and L-5HTP potentiation by fluoxetine were similar to those for serotonin uptake blockade.

The effects of quipazine on serotonin metabolism in rat brain.

Quipazine, 2-(1-piperazinyl)-quinoline, is a drug that has been reported to stimulate serotonin receptors in brain. We therefore studied the effect of quipazine on several parameters of serotonin metabolism in rat brain. Quipazine caused a slight, dose-related elevation of serotonin levels and decrease in 5-hydroxyindoleacetic acid levels for 2–4 hrs after it was administered. The decrease in 5-hydroxyindoleacetic acid levels was probably due primarily to a depression of 5-hydroxyindole synthesis, since quipazine also decreased the rate of 5-hydroxytryptophan accumulation after NSD 1015, the rate of serotonin decline after α-propyldopacetamide, and the rate of 5-hydroxyindoleacetic acid accumulation after probenecid. The elevation of serotonin was probably due to weak inhibition of monoamine oxidase. Quipazine reversibly inhibited the oxidation of serotonin by rat brain monoamine oxidase invitro and protected against the irreversible inactivation of the enzyme invivo. Quipazine also was a potent inhibitor of serotonin uptake into brain synaptosomes invitro and attained concentrations in brain higher than the invitro IC50. However, quipazine did not prevent the depletion of brain serotonin by p-chloroamphetamine invivo. In addition to stimulating serotonin receptors in brain, quipazine may inhibit monoamine oxidase and serotonin reuptake invivo.

Feeding Schedule Alteration of Daily Rhythm in Tyrosine Alpha-Ketoglutarate Transaminase of Rat Liver

Liver tyrosine alpha-ketoglutarate transaminase has a daily rhythm such that in rats fed on an unrestricted basis the activity is highest at approximately 11:00 p.m. In contrast, rats fed only from 8:00 a.m. to noon show a markedly different rhythm in the enzyme, with maximum activity at 11:00 a.m. Controlling the time of food intake seems to be a useful means of studying the mechanism of the daily changes in this enzyme.

Comparison of norfluoxetine enantiomers as serotonin uptake inhibitors in vivo

Norfluoxetine, the N-desmethyl metabolite of fluoxetine, has been reported to resemble fluoxetine in being a potent and selective inhibitor of the serotonin uptake carrier. The enantiomers of norfluoxetine have now been compared as serotonin uptake inhibitors in vivo, based on their antagonism of p-chloroamphetamine-induced depletion of serotonin in brain and their lowering of concentrations of the metabolite of serotonin, 5-hydroxyindoleacetic acid (5-HIAA) in brain. In rats, S-norfluoxetine (ED50 3.8 mg/kg) was more potent than R-norfluoxetine (ED50 > 20 mg/kg) in blocking the depletion of serotonin by p-chloroamphetamine after intraperitoneal administration. The S enantiomer decreased concentrations of 5-HIAA in whole brain after doses of 2.5-20 mg/kg, whereas the R enantiomer did not. The concentrations of both enantiomers in brain increased in proportion to dose and the R enantiomer disappeared from the brain at a slightly slower rate than the S enantiomer. The relative inability of the R enantiomer to block the uptake of serotonin was therefore not a result of smaller concentrations of drug in the brain. In mice, S-norfluoxetine was also more potent than R-norfluoxetine in blocking depletion of serotonin by p-chloroamphetamine (ED50 values 0.82 and 8.3 mg/kg, respectively). Thus, in contrast to the relatively similar potencies of the enantiomers of fluoxetine in blocking the uptake of serotonin, the enantiomers of norfluoxetine have markedly different potencies as inhibitors of the uptake of serotonin.

Evaluation of nefopam as a monoamine uptake inhibitor in vivo in mice.

Nefopam antagonized 6-hydroxydopamine-induced depletion of heart norepinephrine in mice with an ED50 value of 12 mg/kg. Nefopam was ineffective in antagonizing p-chloroamphetamine-induced depletion of brain serotonin in our standard assay in mice, apparently due to a short duration of action. Brain concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) were decreased after a 32 mg/kg, i.p., dose of nefopam at 1 and 2 hr but not at 4 hr. When nefopam was injected simultaneously with p-chloroamphetamine, it prevented brain serotonin depletion initially, but by 6 hr the protective effect was essentially lost, suggesting that p-chloroamphetamine persisted in mouse brain longer than did nefopam. Nefopam caused a dose-related antagonism of brain serotonin depletion at 2 hr after injection of a low dose of p-chloroamphetamine hydrochloride (10 mg/kg, i.p.), with a calculated ED50 value of 11 mg/kg. The lowering of brain 5-HIAA concentration 2 hr after nefopam injection occurred after a 32 mg/kg dose but not after a 3 or 10 mg/kg dose. These data suggest that nefopam is effective as an inhibitor of norepinephrine and serotonin uptake at doses previously shown to be analgesic in mice, consistent with uptake inhibition being a postulated mechanism important in its analgesic effect. Nonetheless, nefopam is a relatively weak inhibitor of monoamine uptake with a short duration of action in mice.