Mutations in monoamine oxidase (MAO) genes in mice lead to hypersensitivity to serotonin-enhancing drugs: implications for drug side effects in humans.

A possible side effect of serotonin-enhancing drugs is the serotonin syndrome, which can be lethal. Here we examined possible hypersensitivity to two such drugs, the serotonin precursor 5-hydroxy-L-tryptophan (5-HTP) and the atypical opioid tramadol, in mice lacking the genes for both monoamine oxidase A (MAOA) and MAOB. MAOA/B-knockout (KO) mice displayed baseline serotonin syndrome behaviors, and these behavioral responses were highly exaggerated following 5-HTP or tramadol versus baseline and wild-type (WT) littermates. Compared with MAOA/B-WT mice, baseline tissue serotonin levels were increased ∼2.6-3.9-fold in MAOA/B-KO mice. Following 5-HTP, serotonin levels were further increased ∼4.5-6.2-fold in MAOA/B-KO mice. These exaggerated responses are in line with the exaggerated responses following serotonin-enhancing drugs that we previously observed in mice lacking the serotonin transporter (SERT). These findings provide a second genetic mouse model suggestive of possible human vulnerability to the serotonin syndrome in individuals with lesser-expressing MAO or SERT polymorphisms that confer serotonergic system changes.

Genetic variation in the serotonin transporter promoter region affects serotonin uptake in human blood platelets.

The human serotonin transporter (5-HTT), encoded by a single gene on chromosome 17q11.2, is expressed in brain and blood cells. 5-HTT is implicated in mood and anxiety regulation, and is where antidepressant and antianxiety drugs initially act in the brain. A 5-HTT-linked promoter region (5-HTTLPR) insertion/deletion polymorphism with long (l) and short (s) forms affects transporter expression and function. The s variant reduced 5-HTT gene transcription in a reporter gene construct and human lymphoblasts, resulting in reduced transporter levels and 5-HT uptake, acting as a dominant allele. In this study, we investigated the expression and function of 5-HTT in platelets from healthy male volunteers. The l variant was associated with more rapid initial platelet 5-HT uptake (Vmax), the index of platelet 5-HTT function most clearly heritable, while the s allele was dominant. The 5-HTTLPR genotype had no effect on platelet [3H]paroxetine binding (Bmax), affinity for [3H]5-HT or [3H]paroxetine, or 5-HT content. The 5-HT uptake findings support a functional difference in the two 5-HTTLPR variants, reinforcing their attractiveness as candidate genes in neuropsychiatric research.

Behavioral, physiological and neuroendocrine responses in healthy volunteers to m-chlorophenylpiperazine (m-CPP) with and without ondansetron pretreatment.

Several serotonin3 (5-HT3) antagonists have been shown to attenuate the anxiogenic effects of the serotonergic agent, m-chlorophenylpiperazine (m-CPP), in animal models, but little data regarding possible effects of 5-HT3 antagonists on responses to m-CPP are available from studies in humans. Therefore, we studied the behavioral, physiological and neuroendocrine responses of 12 healthy volunteers to i.v. administered placebo and m-CPP (0.08 mg/kg), with and without i.v. pretreatment with the selective 5-HT3 antagonist, ondansetron (0.15 mg/kg). Compared to placebo, m-CPP given alone significantly increased ratings of anxiety and several other behavioral measures. m-CPP also produced statistically significant increases in temperature, systolic and diastolic blood pressure, heart rate, and in plasma concentrations of adrenocorticotropic hormone, cortisol, prolactin and norepinephrine. Responses to ondansetron given alone were no different from those of placebo. Pretreatment with ondansetron did not affect peak behavioral responses to m-CPP, but was associated with a significantly earlier return to baseline levels of ratings of anxiety and functional deficit as well as a summary measure of overall behavioral effects. Following ondansetron pretreatment, the increases produced by m-CPP in systolic and diastolic blood pressure and heart rate were no longer significantly different from placebo. Ondansetron pretreatment significantly reduced their plasma cortisol response to m-CPP without affecting the other plasma hormone responses. Plasma concentrations of m-CPP were unaffected by ondansetron pretreatment. These findings suggest that in normal human subjects some behavioral, cardiovascular and neuroendocrine effects of m-CPP may be partially modulated by 5-HT3 receptor-mediated mechanisms.

Regional brain expression of serotonin transporter mRNA and its regulation by reuptake inhibiting antidepressants.

Regional expression and antidepressant drug-induced regulation of mRNA encoding the serotonin (5-HT) transporter were studied in rat brain. While 5-HT transporter mRNA is abundantly expressed in the midbrain raphe complex, lower concentrations were also found in frontal cortex, hippocampus, and neostriatum using a combination of reverse transcriptase-polymerase chain reaction (RT-PCR), Southern hybridization, and sequence analysis. Long-term administration of antidepressants which inhibit 5-HT reuptake, but not monoamine oxidase inhibitors or 5-HT receptor agonists, decrease 5-HT transporter mRNA steady-state concentrations. Based on these observations, we conclude that (1) mRNA coding for the 5-HT transporter is present in several brain areas associated with ascending HT pathways, and (2) chronic treatment with reuptake inhibiting antidepressants may be associated with regulation of the 5-HT transporter at the level of gene expression which may contribute to the neuroadaptive mechanisms that likely underlie their therapeutic efficacy.

Fluoxetine modulates G protein alpha s, alpha q, and alpha 12 subunit mRNA expression in rat brain.

Signal-transducing G proteins are central to the coordination of receptor-effector communication. We have explored the effects of long-term fluoxetine administration of G alpha s, G alpha i1, G alpha i2, G alpha o, G alpha q and G alpha 12 mRNA expression in various rat brain regions using reverse transcriptase-polymerase chain reaction (RT-PCR)-mediated cross-species partial cDNA cloning. Northern blot analysis, and RNase protection assay techniques. Fluoxetine decreased G alpha s mRNA in midbrain, while mRNA expression of the novel G protein alpha subunits, G alpha q and G alpha 12, was increased in neostriatum and frontal cortex. We conclude that in addition to post-translational modification, regulation of G protein function by antidepressant drugs may occur at the level of gene expression.

Differential effects of long-term lithium and carbamazepine administration on Gs alpha and Gi alpha protein in rat brain.

The G protein subunits, Gs alpha, Gi alpha, and Go alpha, have been quantitated in various rat brain regions using enzyme-linked immunosorbent assay (ELISA) techniques. Chronic (3-week) treatment with carbamazepine decreased Gs alpha in several brain regions reaching significance in the neostriatum, while chronic lithium treatment had no unequivocal effect. Lithium significantly increased Gi alpha in the hypothalamus and hippocampus, whereas carbamazepine decreased Gi alpha in the frontal cortex. Both treatments had no consistent effects on Go alpha. We conclude that long-term treatment with lithium and carbamazepine exerts differential effects on G protein alpha subunits, and that this modification of signal transduction represents a potential mechanism of antibipolar drug-induced neural plasticity.

Regulation of G proteins by chronic antidepressant drug treatment in rat brain: tricyclics but not clorgyline increase Go alpha subunits.

The effect of long-term (3-week) administration of various antidepressant drugs on the steady-state concentrations of G protein alpha subunits, Gs alpha, Gi alpha, and Go alpha, has been investigated in rat brain using an enzyme-linked immunosorbent assay. Tricyclic antidepressants and clorgyline decreased Gs alpha and, to a lesser extent, Gi alpha in several brain regions, while Go alpha was increased by tricyclics but not clorgyline. We conclude that long-term treatment with antidepressant drugs exerts differential effects on G protein alpha subunits, and that antidepressant efficacy may potentially be based on functional modifications of signal transduction.

Long-term administration of m-chlorophenylpiperazine to rats does not alter functional sensitivity of either pre-synaptic or postsynaptic 5-HT1A receptors.

Administration of the selective 5-HT(1A) agonist, 8-hydroxy-2(di-n-propylamino)tetralin (8-OHDPAT ), to rats produced increases in food intake in freely fed animals and decreases in food intake in food-deprived animals. Acute pre-treatment with various doses of m-chlorophenylpiperazine (m-CPP, another 5-HT agonist) attenuated 8-OHDPAT-induced increases in food intake in the free-feeding paradigm and enhanced 8- OHDPAT-induced decreases in food intake in the food-deprived paradigm. In the two paradigms, however, neither increases nor decreases in food intake induced by 8-OHDPAT were altered in animals following long- term (21-day) treatment with m-CPP versus saline when animals were challenged with 8-OHDPAT 48 h after the last dose of m-CPP. These findings suggest that long-term m-CPP treatment does not alter the functional sensitivity of 5-HT(1A) receptors located pre-synaptically that mediate hyperphagia or 5-HT(1A) receptors located post-synaptically that mediate decreases in food intake by induction of the serotonin behavioural syndrome.

Controlled comparisons of clomipramine and fluoxetine in the treatment of obsessive-compulsive disorder. Behavioral and biological results.

Treatment with fluoxetine hydrochloride was compared with treatment with clomipramine hydrochloride in two groups of patients with obsessive-compulsive disorder using two different experimental designs. In the first group of 11 patients with obsessive-compulsive disorder studied using a randomized, double-blind, crossover design, treatment with fluoxetine for 10 weeks was found to produce therapeutic effects similar to treatment with clomipramine for 10 weeks. There were significantly fewer total side effects reported during fluoxetine than clomipramine treatment. Drug tapering and placebo substitution in the 4-week crossover interval phase led to substantial relapses in obsessive-compulsive disorder symptoms and depression. Furthermore, responses to the second drug took as long to occur as responses to the first drug, although both drugs are thought to act by a common mechanism, serotonin uptake inhibition. A second group of 21 patients with obsessive-compulsive disorder that had been previously stabilized on clomipramine treatment with at least partial benefit were crossed over to fluoxetine treatment in a double-blind fashion. After 10 weeks of fluoxetine administration, most patients manifested behavioral rating scores of obsessive-compulsive disorder and depressive symptoms that were comparable with precrossover ratings completed during clomipramine treatment. A significant exacerbation in obsessive-compulsive disorder and depression ratings as well as a similar lag in therapeutic efficacy were also noted in this second cohort of patients with obsessive-compulsive disorder. Platelet 5-HT concentrations were reduced 95% during both clomipramine and fluoxetine treatment periods. These results suggest that fluoxetine may represent a viable alternative to clomipramine in the treatment of obsessive-compulsive disorder, although further studies with larger sample sizes are needed.

Serotonin in human lumbar cerebrospinal fluid: a reassessment.

An inter-laboratory comparison study was carried out in order to ascertain mean levels of serotonin (5-HT) in human lumbar cerebrospinal fluid (CSF). Analyses were performed using high performance liquid chromatography (HPLC) coupled with either electrochemical (LC-EC) or fluorometric (LC-F) detection. With the detection limits obtained (7-8 pg/ml for LC-EC, 7-15 pg/ml for LC-F) 5-HT was not usually detected in human lumbar CSF. The findings indicate that the true mean concentration of CSF 5-HT is less than 10 pg/ml. This upper limit is substantially lower than all previous reports of 5-HT concentrations in normal human lumbar CSF. The extremely low concentrations of 5-HT present in CSF make it unlikely that CSF 5-HT will be of clinical utility in assessing central serotonergic function.

Comparative anxiogenic, neuroendocrine, and other physiologic effects of m-chlorophenylpiperazine given intravenously or orally to healthy volunteers.

The serotonin agonist m-chlorophenylpiperazine (m-CPP) had greater anxiogenic and other mood and cognitive effects when administered intravenously (0.1 mg/kg) rather than orally (0.5 mg/kg) to healthy subjects. Nonetheless, similar elevations in peak plasma cortisol and prolactin concentrations were obtained with the two dosage regimens, and temperature elevations were greater after oral m-CPP. Plateau phase plasma concentrations of m-CPP at the times of the maximum neuroendocrine responses to intravenous and oral m-CPP were similar. Since all rodent and nonhuman primate studies have used parenterally administered m-CPP, and previous clinical investigations using intravenous rather than oral m-CPP have yielded somewhat discrepant results, our normative data should be useful for comparing results across different human studies and across species.

Differential neuroendocrine responses to the 5-HT agonist m-chlorophenylpiperazine in Fawn-Hooded rats relative to Wistar and Sprague-Dawley rats.

The effect of various doses of the 5-HT agonist m-chlorophenylpiperazine (MCPP) on neuroendocrine function (prolactin and corticosterone responses) were compared in three different rat strains: Wistar, Sprague-Dawley (SD), and Fawn-Hooded (FH) rats. Administration of various doses of MCPP produced increases in plasma concentrations of prolactin and corticosterone in all three rat strains. The prolactin responses of FH rats to MCPP were significantly smaller than that of either Wistar or SD rats, while corticosterone responses were equivalent across all three strains. On the other hand, baseline concentrations of corticosterone, but not of prolactin, were significantly higher in FH animals relative to both Wistar and SD animals. There was no significant difference in either baseline hypothalamic concentrations of serotonin, 5-hydroxyindoleacetic acid, norepinephrine, or dopamine or brain concentrations of MCPP among these three rat strains. These findings support some other data indicating that FH rats, a strain with a peripheral platelet serotonin storage pool disorder, also possess alterations in some neuroendocrine functions which are modulated by serotonin.

Reversal by bile acid on the inhibition of alpha-tocopherol absorption by retinoic acid.

This study explores several possible mechanisms by which dietary retinoic acid may cause the previously described reduced intestinal absorption of alpha-tocopherol. Measurement of fecal excretion of rats showed that dietary retinoic acid caused twice as much alpha-tocopherol to be excreted as when retinol was the source of vitamin A. Excretion was the same for free and esterified alpha-tocopherol, thus, the retinoic acid effect originally observed was not due to impaired hydrolysis of the ester. There was no effect of retinoic acid on triglyceride absorption. Collection and analysis of bile from rats fed either form of vitamin A showed no difference in bile volume or bile acid composition. The addition of 0.2% taurocholic acid to the diet, however, reversed the effect of retinoic acid on tocopherol absorption. In vitro studies of mixed micelles containing 3H-alpha-tocopherol and retinoic acid or retinol showed no difference in size due to the form of vitamin A in the micelles.

Reduced intestinal absorption of vitamin E by low dietary levels of retinoic acid in rats.

It was observed that rats fed a low dietary level of retinoic acid had markedly lower plasma concentrations of alpha-tocopherol than did rats fed the same amount of retinol. In this report, the possible mechanisms by which retinoic acid alters vitamin E metabolism has been investigated. Weanling male rats were fed a complete purified diet with either retinol or retinoic acid at 4 mg/kg diet; plasma and tissues were analyzed after 2-5 weeks. The plasma alpha-tocopherol concentration in rats ingesting retinoic acid was one-half that of rats ingesting retinol, and this difference also occurred in the liver and adipose tissue. Similar effects occurred in chicks. This low dietary level of retinoic acid had no effect on plasma triglyceride concentration, as has been reported for higher intakes, and plasma cholesterol and total lipids were also unaffected. Retinoic acid did not affect the rate of decrease in endogenous alpha-tocopherol in normal rats fed a vitamin E-free diet for 3 weeks. In rats with mesenteric lymph cannulas, dietary retinoic acid caused a reduced absorption of 3H-labeled alpha-tocopherol. In chicks fed retinoic acid, plasma and liver radioactivity 2.5 hours after an oral dose of 3H-alpha-tocopherol was one-fifth that of chicks fed retinol. More oxidation of alpha-tocopherol occurred during absorption in rats fed retinoic acid than in those fed retinol, as evidenced by more alpha-tocopherylquinone in the collected lymph. We postulate that dietary retinoic acid reduces the intestinal absorption of alpha-tocopherol and may also promote its oxidation.

Lipofuscin in vitamin E deficiency and the possible role of retinol.

This study was designed to determine if the vitamin A status of rats could affect the degree of lipofuscin formation in vitamin E deficient rats, inasmuch as an earlier report proposed a retinoyl complex in human brain lipofuscin pigment. Female rats were depleted of vitamin E from weaning while being maintained on different intakes of vitamin A (0, 0.8 and 8.0 mg/kg diet). The amount of lipofuscin present in the uterus was estimated at intervals between 2 and 8 months by visual observations, by histological fluorescence and by organic solvent extractable fluorescence. There was no difference in pigment deposition by any of the three criteria used, whether the animals were made retinol deficient and maintained on retinoic acid or were fed a low or high intake of retinol. Organic solvent extractable fluorescence was a poor indicator of the degree of pigment deposition in the uterus. It appears unlikely that retinol is a significant component of lipofuscin pigment in this tissue.

Simultaneous determination of alpha-tocopherol and retinol in plasma or red cells by high pressure liquid chromatography.

This paper describes a rapid, microprocedure for the simultaneous determination of alpha-tocopherol (vitamin E) and retinol (vitamin A) in plasma, and of alpha-tocopherol alone in red cells since cells do not contain retinol. A total lipid extract from 0.1 ml plasma or 0.125 ml red cells and containing internal standards of alpha-tocopheryl acetate and retinyl acetates is injected onto a high pressure liquid chromatography with a reverse phase column developed with methanol-water. An ultraviolet detector with 280-nm filter is used. The chromatogram is complete in 8 min and the alpha-tocopherol and retinol are quantitated by the peak height ratio method. Comparison of results with both plasma and red cells gave excellent agreement with conventional methods for these vitamins. The procedure should be particularly useful for clinical studies and nutrition surveys.