MONOAMINES AS POSSIBLE MEDIATORS IN THE REGULATION OF FAST AXOPLASMIC FLOW

—The effect of drugs which have been shown to alter monoamines in the CNS on the rate of fast axoplasmic flow of [³H]leucine labelled material in the rat sciatic nerve was examined. Drugs which deplete monoamines led to a decrease while those which increase the level of monoamines led to an increase in the rate of fast axoplasmic flow. Monoaminergic drugs were also found to alter the amount of [³H]leucine incorporated into spinal cord.     

Effect of antimediator preparations on the intercellular relations in early embryos of the sea urchin]

The dated treatment of the early embryos of an irregular (flat) sea urchin Scaphechinus mirabilis by neuropharmacological drugs (anti-neurotransmitters) during one of the first four cleavage divisions results in the impairment of intercellular connections and leads to the formation of twin embryos, dwarf embryos, embryos of the dumb-bell shape etc. In the experiments with some of the drugs under study such developmental abnormalities were not seen or were expressed much more weakly when serotonin or bufotenin (N,N-dimethylserotonin) were added to the medium. A suggestion is put forward that the early embryos possess an intracellular mechanism participating in the interaction between the cells and operating via endogenous monoamines, primarily serotonin.     

Effects of psychotropic drugs on neurotransmitters in man

Primary biochemical profiles of antidepressants and neuroleptics are summarized in comparison to their actual effects on monoamine neurotransmitters in humans during the time period when clinical response emerges. Even the most biochemically specific of these drugs produces effects on at least two monoamines by three to four weeks. Interestingly, taking into account relative changes in dopamine and serotonin metabolites in cerebrospinal fluid relates better to the primary biochemical action(s) of each drug than do absolute changes. Moreover, monoamine changes after drugs are in the opposite direction to those after ECT, suggesting that balance among rather than shifts in single neurotransmitters is the relevant target of major psychotropic drugs.     

Heat and other physiological stress-induced analgesia: Catecholamine mediated and naloxone reversible response

Acute environmental heat (40±2°C) and other physiological stressful situations increased the pain threshold to radiant heat in rats and mice. Naloxone pretreatment or chronic exposure to stress antagonised this response. After pretreatment with catecholamine depleters, α-methyl-p-tyrosine, reserpine or with adrenoceptor blockers, haloperidol and chlorpromazine, the stress-induced analgesic effect was abolished. Cyproheptadine, a serotonin antagonist, also blocked this response. The results suggest the role of brain monoamines in stress-mediated analgesia.     

Hyperforin inhibits vesicular uptake of monoamines by dissipating pH gradient across synaptic vesicle membrane

Extracts of Hypericum perforatum (St. John's wort) have antidepressant properties in depressed patients and exert antidepressant-like action in laboratory animals. The phloroglucinol derivative hyperforin has become a topic of interest, as this Hypericum component is a potent inhibitor of monoamines reuptake. The molecular mechanism by which hyperforin inhibits monoamines uptake is yet unclear. In the present study we try to clarify the mechanism by which hyperforin inhibits the synaptic vesicle transport of monoamines. The pH gradient across the synaptic vesicle membrane, induced by vacuolar type H(+)-ATPase, is the major driving force for vesicular monoamines uptake and storage. We suggest that hyperforin, like the protonophore FCCP, dissipates an existing Delta pH generated by an efflux of inwardly pumped protons. Proton transport was measured by acridine orange fluorescence quenching. Adding Mg-ATP to a medium containing 130 mM KCl and synaptic vesicles caused an immediate decrease in fluorescence of acridine orange and the addition of 1 microM FCCP abolished this effect. H(+)-ATPase dependent proton pumping was inhibited by hyperforin in a dose dependent manner (IC(50) = 1.9 x 10(-7) M). Hyperforin acted similarly to the protonophore FCCP, abolishing the ATP induced fluorescence quenching (IC(50) = 4.3 x 10(-7) M). Hyperforin and FCCP had similar potencies for inhibiting rat brain synaptosomal uptake of [3H]monoamines as well as vesicular monoamine uptake. The efflux of [3H]5HT from synaptic vesicles was sensitive to both drugs, thus 50% of preloaded [3H]5HT was released in the presence of 2.1 x 10(-7) M FCCP and 4 x 10(-7) M hyperforin. The effect of hyperforin on the pH gradient in synaptic vesicle membrane may explain its inhibitory effect on monoamines uptake, but could only partially explain its antidepressant properties.     

α1-adrenergic drugs exhibit affinity to a thapsigargin-sensitive binding site and interfere with the intracellular Ca2+ homeostasis in human erythroleukemia cells

Even though the erythroleukemia cell lines K562 and HEL do not express α1-adrenoceptors, some α1-adrenergic drugs influence both survival and differentiation of these cell lines. Since Ca²⁺ is closely related to cellular homeostasis, we examined the capacity of α1-adrenergic drugs to modulate the intracellular Ca²⁺ content in K562 cells. Because of morphological alterations of mitochondria following α1-adrenergic agonist treatment, we also scrutinized mitochondrial functions. In order to visualize the non-adrenoceptor binding site(s) of α1-adrenergic drugs in erythroleukemia cells, we evaluated the application of the fluorescent α1-adrenergic antagonist BODIPY® FL-Prazosin. We discovered that the α1-adrenergic agonists naphazoline, oxymetazoline and also the α1-adrenergic antagonist benoxathian are able to raise the intracellular Ca²⁺-content in K562 cells. Furthermore, we demonstrate that naphazoline treatment induces ROS-formation as well as an increase in Δψm in K562 cells. Using BODIPY® FL-Prazosin we were able to visualize the non-adrenoceptor binding site(s) of α1-adrenergic drugs in erythroleukemia cells. Interestingly, the SERCA-inhibitor thapsigargin appears to interfere with the binding of BODIPY® FL-Prazosin.Our data suggest that the effects of α1-adrenergic drugs on erythroleukemia cells are mediated by a thapsigargin sensitive binding site, which controls the fate of erythroleukemia cells towards differentiation, senescence and cell death through modulation of intracellular Ca²⁺.     

Functional significance of alpha-stimulation and alpha-blockade on responses to cardiac nerve stimulation in anesthetized dogs

Experiments were conducted to determine if α-stimulants could inhibit responses to sympathetic nerve stimulation $\text{via}$ a feedback inhibition loop mediated by prejunctional α-receptors. Responses to cardiac nerve stimulation in anesthetized dogs were compared before, during the peak effect of a drug infusion, and during a second drug infusion subsequent to the administration of phentolamine (5 mg/kg i.v.). The drugs infused were norepinephrine, phenylephrine, clonidine, naphazoline - all α-stimulants - and guanethidine. All drugs caused marked elevations of blood pressure, an indication of α-stimulation, but only guanethidine caused significant blockade of responses to sympathetic nerve stimulation. In addition, phentolamine, an α-receptor blocker, and desipramine, an inhibitor of amine uptake, did not potentiate responses to sympathetic nerve stimulation. These results do not support the hypothesis that sympathetic nerves are under a functionally significant feedback loop mediated by α-receptors.     

The effects of biogenic monoamines and related agonists and antagonists on the isolated,perfused branchial heart of sepia officinalis L. (cephalopoda)

1. The chronotropic and inotropic effects of biogenic monoamines were examined on the isolated and perfused branchial heart of the common cuttlefish Sepia officinalis (L.).2. Adrenaline, noradrenaline and dopamine caused concentration-dependent increases in pressure amplitude, dopamine being 100-fold less potent than adrenaline and noradrenaline. The catecholamines hardly affected frequency.3. Octopamine, histamine and GABA did not influence normal heartbeat.4. Serotonin was either ineffective or produced variable responses, whereby the efficacy of the substance was not graded to the concentrations applied. It is supposed that serotonin is not involved in branchial heart regulation.5. The actions of different adrenergic agonists and antagonists indicate the presence of a myocardial adrenoceptor which closely resembles the α₁-type.     

Function of myocardial alpha-adrenoceptors

Myocardial α-adrenoceptors are similar to vascular α-adrenoceptors; therefore the drugs which are used to study myocardial α-adrenoceptors can affect the heart indirectly by acting on vascular α-adrenoceptors. High concentrations of α-adrenoceptor stimulant and α-adrenoceptor blocking drugs can exert cardiac effects that do not result from action on α-adrenoceptors; therefore relatively low concentrations of these drugs must be used to obtain specific effects.An α-adrenoceptor mediated positive inotropic effect has been observed in relatively slow beating isolated heart preparations; it is not associated with shortening of the duration of systole or an increase in myocardial cyclic AMP concentration and is probably accompanied by an increase in Ca²⁺ influx. Usually α-adrenoceptor stimulation has no effect on heart rate.Myocardial α-adrenoceptor mediated ventricular arrhythmias have been caused in animals by high concentrations of catecholamines, and a transient increase in α-adrenoceptor concentration has been found in ischemic myocardium. We do not know how myocardial α-adrenoceptor stimulation causes arrhythmias. In isolated heart preparations low concentrations of epinephrine and norepinephrine prolong refractory period and action potential duration by α-adrenoceptor stimulation, and higher concentrations of the catecholamines shorten refractory period and action potential duration by α-adrenoceptor stimulation. In isolated specialized conducting fibers low concentrations of catecholamines reduce automaticity by α-adrenoceptor stimulation and higher concentrations increase automaticity by β-adrenoceptor stimulation. In partially depolorized ventricle preparations α-adrenoceptor stimulation has been reported both to depress and to restore electrical and mechanical activity. Clearly, much remains to be done before we understand α-adrenoceptor mediated cardiac effects.     

Biogenic amines in brain structures of rats of different zoo-social rank during immobilization stress]

The content of monoamines and their metabolites in different parts of the brain: mesencephalic reticular formation, locus coeruleus, sensomotor cortex was studied by high-performance liquid chromatography in rats with different zoo-social position. Content of dopamine and serotonin in the brain structures studied was found to be different in dominants and subdominants. Maximal changes of monoamines under immobilization stress were observed in dominants.     

Hyperforin depletes synaptic vesicles content and induces compartmental redistribution of nerve ending monoamines

Hyperforin, a phloroglucinol derivative found in Hypericum perforatum (St. John's wort) extracts has antidepressant properties in depressed patients. Hyperforin has a unique pharmacological profile and it inhibits uptake of biogenic monoamines as well as amino acid transmitters. We have recently showed that the monoamines uptake inhibition exerted by hyperforin is related to its ability to dissipate the pH gradient across the synaptic vesicle membrane thereby interfering with vesicular monoamines storage. In the present study we demonstrate that hyperforin induces dose-dependent efflux of preloaded [3H]5HT and [3H]DA from rat brain slices. Moreover, we show that hyperforin attenuates depolarization- dependent release of monoamines, while increasing monoamine release by amphetamine or fenfluramine. It is also demonstrated that preincubation of brain slices with reserpine is associated with dose- dependent blunting of efflux due to hyperforin. Our data indicate that hyperforin-induced efflux of [3H]5HT and [3H]DA reflect elevated cytoplasmic concentrations of the two monoamines secondary to the depletion of the synaptic vesicle content and the compartmental redistribution of nerve ending monoamines.     

Uptake of catecholamines and penetration of trypan blue after blood-brain barrier lesions

Summary Blood-brain barrier lesions were produced on rabbits which had been depleted of their endogenous monoamines with a large dose of reserpine. After the lesion, catecholamines and the blood-brain barrier indicator dye trypan blue were injected. After freeze-drying, the cellular distribution of the injected substances was observed in the fluorescence microscope.It was found that, in the injured areas, the monoamines and trypan blue had penetrated into the brain parenchyma, where the monoamines were taken up and concentrated in nerve terminals. Trypan blue was found diffusely in the neuropil, while the nerve cell bodies and axons exhibited no fluorescence of trypan blue. On the control side, this type of fluorescence of catecholamines or trypan blue could not be detected.The lesions applied seem to be quite specific for the blood-brain barrier, as an active and energy-dependent uptake of catecholamines could be demonstrated in central monoamine nerve terminals. Thus the results also show that these terminals have the same reserpine-resistant membrane pump in vivo as earlier demonstrated for peripheral adrenergic neurons, and for central neurons in vitro.This investigation has been supported by research grants (B 66-158 and B 66-257) from the Swedish Medical Research Council and by a Public Health Service Research Grant (NB 05236-02) from the National Institute of Neurological Diseases and Blindness. For generous supplies of drugs we thank the Swedish Ciba, Stockholm, Sweden for reserpine (Serpasil^®), the Swedish Pfizer, Stockholm, Sweden for nialamide (Niamid^®) and Hoechst Anilin AB, Göteborg, Sweden for -methylnoradrenaline (Corbasil^®).     

In vitro inhibition of rat small intestinal absorption by lipophilic organic cations

Cationic, lipid-soluble organic compounds may interfere with cation-mediated membrane transport processes. Thus, small intestinal absorption may be influenced by lipophilic organic cations. Therefore a series of arylalkylamines was studied in the concentration range from 0.5 to 20 mmol/l for their effect on the transport of various monosaccharides and leucine in the rat small intestine in vitro by means of the tissue accumulation technique. Whereas the monophenyl substituted monoamines (e.g. benzylamine, 2-phenylethylamine, 3-phenylpropylamine) did not show a significant effect on the active transport, the corresponding ω,ω-diphenyl derivatives exhibited a strong inhibition of the active transport of the sugars and the amino acid. These monoamines and drugs of similar structure (e.g. benzoctamine, diphenhydramine) exhibited a mixed or non-competitive type of inhibition which correlated quite well with their octanol-water partition coefficients. In contrast, di- or triamines (e.g. harmaline, imipramine, pyrilamine) revealed a rather pure competitive type of inhibition. These findings tentatively suggest a different mode of action on the active transport by lipid-soluble organic amines according to the molecular charge distribution. In addition, membrane vesicles were used to examine the effect of the different amines on the sucrase activity. Regarding the cation-dependent hydrolysis of sucrose, however, no distinct pattern developed.     

Liquid chromatographic determination of free and conjugated 3-methoxy-4-hydroxyphenylethylene glycol with wide-ranging substances related to monoamine metabolism

A simple and sensitive procedure was developed for the simultaneous determination of substances metabolically related to monoamine transmitters including 3-methoxy-4-hydroxy-phenylethylene glycol (MOPEG) in dissected brain regions of rats using high-performance liquid chromatography combined with electrochemical detection. The tissue sample was homogenized in HCl solution. The homogenate was divided into two portions, of which one was used for the assay of MOPEG after enzymatic hydrolysis with sulfatase. A butanol extraction process was performed on the remaining portion to obtain the sample of monoamine transmitters, precursor amino acids, and acidic metabolites. The monoamines and precursor amino acids were finally recovered in HCl solution, while the acidic metabolites shifted into the alkaline buffer from the organic layer. The basic and neutral substances were separated with a 0.1 M sodium citrate/citric acid buffer system (pH 4.0) containing 1% tetrahydrofuran, and the acidic ones with 0.075 M sodium citrate/citric acid buffer (pH 3.5) containing 1% tetrahydrofuran, 10% methanol, and 12% acetic acid. The steady-state concentrations of three monoamine transmitters (noradrenaline, dopamine, and 5-hydroxytryptamine) were determined together with their precursors and metabolites. Changes in the concentrations of these substances were examined for various drugs, of which the effects had been previously confirmed. The changes reflected putative drug effects and demonstrated that the procedure was applicable to the regional determination of monoamines and their metabolically related substances.     

Enhanced BDNF Signaling is Associated with an Antidepressant-like Behavioral Response and Changes in Brain Monoamines

1. Neurotrophins and serotonin have both been implicated in the pathophysiology of depression and in the mechanisms of antidepressant treatments. 2. Brain-derived neurotrophic factor (BDNF) influences the growth and plasticity of serotonergic (5-HT) neurons via the activation of trkB receptor. 3. Transgenic mice overexpressing the full-length trkB receptor (TrkB.TK+) and showing increased trkB activity in brain, and their wild type (WT) littermates, were injected with the antidepressant fluoxetine or saline, and analyzed behaviorally in the forced swimming test paradigm and biochemically for the concentrations of brain monoamines and their metabolites. 4. The TrkB.TK+ mice displayed increased latency to immobility in the forced swim test, suggesting resistance to behavioral despair. 5. Fluoxetine increased the latency to immobility in wild-type mice to a similar level as seen in the trkB.TK+ mice after saline treatment, but had no further behavioral effect in the swimming behavior of the trkB.TK+ mice. 6. Only minor differences in the levels of brain monoamines and their metabolites were observed between the transgenic and wild-type mice. 7. These data, together with other recent observations, suggest that trkB activation may play a critical role in the behavioral responses to antidepressant drugs in mice.     

人胰腺α-淀粉酶抑制剂高通量筛选模型的建立及其应用

【目的】针对人胰腺α-淀粉酶这个糖代谢途径中重要的靶蛋白,建立α-淀粉酶抑制剂高通量筛选模型。【方法】采用毕赤酵母表达系统克隆和表达人胰腺α-淀粉酶;利用酶的催化特性建立α-淀粉酶抑制剂筛选模型;应用该模型对放线菌发酵液冻干物进行高通量筛选;通过构建16S rRNA系统发育树分析阳性菌株的分类地位。【结果】成功克隆、表达了具催化活性的人胰腺α-淀粉酶;建立了α-淀粉酶抑制剂的筛选模型;对近2000株放线菌的发酵液冻干物进行高通量筛选,最终得到14株α-淀粉酶抑制剂产生菌株,且在分类学上具有丰富的菌种多样性。【结论】本研究建立的α-淀粉酶抑制剂高通量筛选模型具有很强的实用价值,可用于新型淀粉酶抑制剂类降糖药物的开发。     

Role of the monoaminergic system in the transmission of the effect of androgens on neurons of separate limbic structures of the rat brain

By means of the histochemical method intensity of monoamines fluorescence has been studied in 3-, 5-, 7-, 10-, 20-, 30- and 60-day-old intact and neonatally androgenized female rats. The neonatal androgenization increases fluorescent intensity of monoamines in the neuropil of the adjoining nucleus of the septum, of the nucleus in the terminal stripe bed and the caudate nucleus. This is especially evident on the 3d, 7th and 30th days. On the 5th day of the postnatal life the difference in fluorescent intensity of monoamines in the brain of control and test animals is statistically insignificant. Possible mechanisms responsible for the fluorescent intensity of monoamines and the role of the latter in transmitting the sex hormones effect to the neurons of the forebrain structures investigated are discussed.     

Monoamines in the C cells of the thyroid gland of callithricid primates

Summary The thyroid gland of three species of marmosets were studied with the Falck and Hillarp fluorescence technique for monoamines. Two types of fluorescent C cells were observed. The predominant type displayed the greenish (or blue) fluorescence characteristic of catecholamines. The other type displayed a yellowish fluorescence most probably due to 5-HT. The presence of monoamines in C cells now reported for the first time in Primates offers an interesting possibility for studying their presumed role in calcitonin secretion.     

Modification of d-amphetamine- or chlorpromazine-induced hypothermia by β-endorphin,MIF-I,and α-MSH: Mediation by the dopaminergic system

The effects of β-endorphin, MIF-I, and α-MSH on d-amphetamine- and CPZ-induced hypothermias in rats kept at 4°C were tested in three experimental groups: (a) intact; (b) rats with lesions of the olfactory tubercle; and (c) rats in which the link between the DA mesolimbic pathway and the striatum was disconnected. All drugs tested alone (except MIF-I) caused significant hypothermia. Pretreatment with CPZ, MIF-I, and α-MSH potentiated d-amphetamine-induced hypothermia in intact rats. Pretreatment with α-MSH potentiated CPZ-induced hypothermia. β-Endorphin partially blocked d-amphetamine-induced hypothermia, but did not interact with CPZ, MIF-I, or α-MSH. All potentiations were either reduced or disappeared in the incisioned rats. CPZ and α-MSH caused hypothermia in olfactory tubercle-lesioned rats. The results indicate that: (a) the DA mesolimbic pathway is involved in the hypothermic response of all drugs tested; (b) an intact feedback loop is required for the potentiation of the hypothermic response of CPZ on d-amphetamine, MIF-I on d-amphetamine, and α-MSH on d-amphetamine and CPZ; (c) β-endorphin acts as a partial blocker of d-amphetamine; MIF-I is a weak potentiator of d-amphetamine. α-MSH acts as a negative modulator of the DA system, most probably in the striatum.     

A comparative study of the neurochemical profiles of remoxipride, raclopride and metoclopramide activity]

Effects of intraperitoneal administration of remoxipride (2.4 mg/kg), raclopride (1.2 mg/kg) and metoclopramide (5 mg/kg) on the concentration of monoamines and metabolites in various brain regions, on the DA and serotonin biosynthesis in the striatum and nucleus accumbens, on the K(+)-stimulated DA release from the isolated striatum, on the extracellular levels of DA and metabolites in the striatum of freely moving rats were studied. Remoxipride and raclopride increase DA turnover, biosynthesis and DA release, studied both in vitro and in vivo. Metoclopramide was shown to be more effective in increasing DA turnover and biosynthesis, while exerted less activity in regard to increasing DA release in vivo and failed to affect release in vitro. Possible neurochemical mechanisms underlying pharmacological effects of these drugs are discussed.