The existence of a local 5-hydroxytryptaminergic system in peripheral arteries

BACKGROUND AND PURPOSE: 5-HT is a vasoconstrictor exhibiting enhanced effects in systemic arteries from subjects with cardiovascular disease. The effect of endogenous 5-HT on arteries is controversial, because the concentration of free circulating 5-HT is low and a 5-hydroxytryptaminergic system has not been identified in peripheral arteries. We hypothesized that a local 5-hydroxytryptaminergic system (including 5-HT synthesis, metabolism, uptake and release) with physiological function exists in peripheral arteries. EXPERIMENTAL APPROACH: The presence of key components of a 5-hydroxytryptaminergic system in rat aorta and superior mesenteric artery was examined using western blot analyses, immunohistochemistry and immunocytochemistry. The function of the rate-limiting enzyme in 5-HT biosynthesis, tryptophan hydroxylase (TPH), and 5-HT transporter was tested by measuring enzyme activity and 5-HT uptake, respectively. Isometric contraction of arterial strips was used to demonstrate the function of released endogenous 5-HT in arterial tissues. KEY RESULTS: mRNA for TPH-1 was present in arteries, with low levels of TPH protein and TPH activity. Expression and function of MAO A (5-HT metabolizing enzyme) was supported by immunohistochemistry, western analyses and the elevation of concentrations of 5-hydroxyindoleacetic acid (5-HT metabolite) after exposure to exogenous 5-HT. The 5-HT transporter was localized to the plasma membrane of freshly isolated aortic smooth muscle cells. Peripheral arteries actively took up 5-HT in a time-dependent and 5-HT transporter-dependent manner. The 5-HT transporter substrate, (+)-fenfluramine, released endogenous 5-HT from peripheral arteries, which potentiated noradrenaline-induced arterial contraction. CONCLUSIONS AND IMPLICATIONS: This study revealed the existence of a local 5-hydroxytryptaminergic system in peripheral arteries.     

Characterization of acute N-ethyl-3,4-methylenedioxyamphetamine (MDE) action on the central serotonergic system

The effect of N-ethyl-3,4-methylenedioxyamphetamine (MDE) on the central serotonergic system was studied. Within 1 hr after administration of MDE (10 mg/kg), the concentration of 5-hydroxytryptamine (5-HT) and the activity of tryptophan hydroxylase (TPH) had declined significantly in the hippocampus but returned to control within 12 hr. Hippocampal 5-hydroxyindoleacetic acid (5-HIAA) content decreased within 2 hr, rebounded to 22% above control by 12 hr, and returned to control by 24 hr. Blockade of the 5-HT uptake carrier with fluoxetine (10 mg/kg) prevented or attenuated the MDE-induced changes in 5-HT content and TPH activity, except for neostriatal TPH activity which remained unresponsive to the fluoxetine treatment. The MDE-induced decline in TPH activity could be reversed by incubating the TPH preparation with dithiothreitol and Fe2+ under nitrogen for 24 hr. This suggests that the loss in TPH activity induced by MDE results from an alteration of the oxidation-reduction state of a sulfhydryl group located on the enzyme. The inhibition of monoamine oxidase (MAO) by the administration of pargyline (75 mg/kg) failed to protect the neostriatal TPH activity from the MDE-induced decline while potentiating the MDE-induced decrease in cortical TPH activity. This suggests that H2O2 generated by MAO in vivo is not responsible for oxidation of the sulfhydryl site located on TPH during the MDE treatment.     

Effects of various ginseng saponins on 5-hydroxytryptamine release and aggregation in human platelets

The effects of various ginseng saponins isolated from red ginseng roots, on aggregation and 5-hydroxytryptamine release (5-HT) human platelets have been investigated. Among the six saponins tested, only ginsenoside Rg1 inhibited adrenaline- and thrombin-induced platelet aggregation and 5-HT release dose-dependently, at concentrations of 5 to 500 micrograms ML-1. Ginsenoside Rg1 had no effect on adrenaline- and thrombin-induced arachidonic acid release and diacylglycerol production. But it did reduce the elevation of cytosolic free calcium concentration (Ca2+)i shown in the second phase induced by adrenaline and thrombin, at concentrations of 10 to 500 micrograms mL-1. Those data suggest that the inhibitory effects of ginsenoside Rg1 on 5-HT release from, and aggregation of, platelets might be due to the reduction of (Ca2+)i elevation at the second phase induced by adrenaline and thrombin. The results suggest that ginsenoside Rg1 in red ginseng roots may be active as a drug in the treatment of artheroscleorosis and thrombosis.     

Direct central effects of acute methylenedioxymethamphetamine on serotonergic neurons

Acute peripheral administration of either the (+) or (-) stereoisomer of methylenedioxymethamphetamine (MDMA) to rats results in a rapid loss of tryptophan hydroxylase (TPH) activity in several brain regions. This decline in enzyme activity precedes a decrease in serotonin (5-HT) concentrations in the same areas. An initial rise in the concentration of 5-hydroxyindole acetic acid after drug administration suggests that an increase in the turnover of 5-HT is an early event in the development of these changes. Unsuccessful attempts to reproduce the in vivo effects of MDMA on TPH activity using in vitro preparations such as cortical slices or the mouse mastocytoma cell line, P-815, suggested a requirement for an intact neuronal system or metabolism of the drug. Injection of MDMA directly into several brain regions also had no effect on TPH activity or 5-HT concentrations. However, when brain concentrations of MDMA were maintained using a constant i.c.v. infusion, TPH activity declined as observed following peripheral administration. The results, therefore, indicate that the acute effect of MDMA on 5-HT synthesis is a direct central effect of the drug which may be triggered by a sustained increase in transmitter turnover.     

Increases in serotonergic neuronal activity following intracerebroventricular administration of AF64A in rats

Changes in the serotonergic nervous system after the intracerebroventricular (i.c.v.) administration of ethylcholine aziridinium (AF64A, 3 nmol/each ventricle) were studied in rats. Two weeks after the infusion of AF64A, the levels of 5-HT and 5-HIAA in microdialysed cerebrospinal fluid (CSF), the levels of total 5-HT and 5-HIAA, the density of serotonin uptake sites and the activities of tryptophan hydroxylase (TPH) and monoamine oxidase (MAO) in various brain regions were determined. After AF64A administration, the concentrations of 5-HT in lateral ventricle were increased and the levels of 5-HIAA were decreased. However, the hippocampal levels of total 5-HT were decreased without changes in the levels of 5-HIAA and the hippocampal turnover rates of 5-HT increased. Also, the density of uptake sites of serotonin ([³H]citalopram binding sites) was decreased in the various brain. The activities of TPH were increased in striatum and frontal cortex and the activity of MAO was also increased in striatum. These results indicate that AF64A induces an increase in serotonergic neuronal activity and decreased densities of 5-HT uptake sites which may affect the change in the other parameters of serotonergic neuronal activities. Furthermore, these results suggest that the impaired cholinergic neuronal activity induces the alteration in the serotonergic nervous activities.     

Effect of monoamine oxidase A and B inhibition on the uptake and metabolism of serotonin within serotonergic neurons of rat brain

The effects of inhibiting monoamine oxidase (MAO) A and B on metabolism and uptake of serotonin (5-HT) in serotonergic synaptosomes were studied. To avoid contamination by extrasynaptosomal MAO, synaptosomes were separated from other components of rat brains by discontinuous sucrose density gradient centrifugation. Kinetic analysis of 5-HT uptake demonstrated that 5-HT was selectively transported into serotonergic synaptosomes through the high affinity 5-HT uptake process. Selectivity of the uptake and subsequent deamination of 5-HT within serotonergic synaptosomes were confirmed using selective and non-selective 5-HT and norepinephrine (NE) uptake inhibitors. MAO inhibitor analysis of 5-HT deamination occurring within serotonergic synaptosomes indicated that, at physiologically relevant concentrations of 5-HT, MAO A deaminates 5-HT, maintaining a low cytoplasmic concentration of 5-HT. When the cytoplasmic concentration of 5-HT is increased above physiologically relevant levels by the inhibition of MAO A, MAO B becomes active. [( 14)C] 5-HT uptake into synaptosomes was reduced by decreasing the V( max) of [(14)C] 5-HT uptake. One mechanism for a decrease in the V(max) could be the increase in the cytoplasmic concentration of 5-HT.     

人参皂苷Rb_1、Rg_1对许旺细胞NGF表达的影响

目的:研究人参皂苷Rb1和Rg1影响许旺细胞神经生长因子(NGF)的表达程度。方法:分离纯化成人新鲜离体神经许旺细胞,置于人参皂苷条件培养基孵育48h;采用间接免疫荧光法标记细胞,流式细胞仪检测许旺细胞NGF表达率。结果:人参皂苷Rb1和Rg1各实验组许旺细胞表达NGF的百分率较对照组显著升高(P<0.05);人参皂苷Rb1与Rg1组之间许旺细胞表达NGF的百分率无显著差异。结论:人参皂苷Rb1和Rg1可以通过促进许旺细胞合成分泌NGF而具有潜在加速周围神经损伤修复的作用。     

Ginsenoside Rb1 Modulates Level of Monoamine Neurotransmitters in Mice Frontal Cortex and Cerebellum in Response to Immobilization Stress

Cerebral monoamines play important roles as neurotransmitters that are associated with various stressful stimuli. Some components such as ginsenosides (triterpenoidal glycosides derived from the Ginseng Radix) may interact with monoamine systems. The aim of this study was to determine whether ginsenoside Rb1 can modulate levels of the monoamines such as dihydroxyphenylalanine (DOPA), dopamine (DA), norepinephrine (NE), epinephrine (EP), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydorxytryptamine (5-HT), 5-hydroxindole-3-acetic acid (5-HIAA), and 5-hydroxytryptophan (5-HTP) in mice frontal cortex and cerebellum in response to immobilization stress. Mice were treated with ginsenoside Rb1 (10 mg/kg, oral) before a single 30 min immobilization stress. Acute immobilization stress resulted in elevation of monoamine levels in frontal cortex and cerebellum. Pretreatment with ginsenoside Rb1 attenuated the stress-induced changes in the levels of monoamines in each region. The present findings showed the anti-stress potential of ginsenoside Rb1 in relation to regulation effects on the cerebral monoaminergic systems. Therefore, the ginsenoside Rb1 may be a useful candidate for treating several brain symptoms related with stress.     

N-propargylbenzylamine, a major metabolite of pargyline, is a potent inhibitor of monoamine oxidase type B in rats in vivo: a comparison with deprenyl.

In an effort to explore the contribution of the metabolites of pargyline towards the in vivo inhibition of monoamine oxidase (MAO), the effects of pargyline and its major metabolites on the production and metabolism of a number of biogenic amines were studied in rats. The administration of pargyline gave rise to three major ethyl acetate extractable metabolites: benzylamine, N-methylbenzylamine and N-propargylbenzylamine (NPB). Only NPB demonstrated in vivo monoamine oxidase inhibitory properties at an acute dose of 30 mg kg-1. The acute effects of pargyline, NPB, and deprenyl on urine and brain concentrations of a number of biogenic amines (phenylethylamine (PEA), m- and p-tyramine, noradrenaline (NA), dopamine, and 5-hydroxytryptamine (5-HT) and their metabolites were evaluated. Increased urine and brain concentrations of PEA were considered to represent in vivo inhibition of type B MAO while decreased concentrations of NA and 5-HT metabolites were regarded as indicators of an in vivo inhibition of MAO type A. NPB, like deprenyl and pargyline, significantly increased urine and brain PEA while only pargyline reduced 5-HT metabolism, suggesting that the metabolism of pargyline to NPB may contribute towards the MAO type B inhibitory effects of pargyline in vivo. Since the therapeutic benefits of MAO inhibitors in clinical practice usually require some period of chronic treatment, the chronic effects of repeated 14 daily doses of the above MAO inhibitors on central and peripheral biogenic amines were evaluated at the following times: during treatment, one day and five days after termination of treatment. The biochemical changes observed during the course of chronic NPB, pargyline and deprenyl treatments generally follow the expected in vitro characteristics of these drugs, but the detailed changes observed suggest clear differences. For example, the in vivo effect of pargyline on urine 5-hydroxyindoleacetic acid excretion was considerably weaker than its effect on the excretion of NA and dopamine metabolites. These changes are opposite to the in vitro effects of pargyline on 5-HT, dopamine and NA oxidative deamination. Inhibitions of the metabolism of all the amines studied were clearly observed during chronic MAOI treatments, but these effects were less evident five days after the end of treatment, suggesting an almost normal metabolism of biogenic amines. It is concluded that while MAO inhibitors may be the primary compound responsible for MAO inhibition, the effects of their metabolites in some cases may also play equally important roles in the regulation of monoamines both in the periphery and the brain.(ABSTRACT TRUNCATED AT 400 WORDS)     

人参皂苷Rg1保护心血管和中枢神经系统的药理活性研究进展

人参皂苷Rg1是人参和三七的主要有效成分之一。本文主要对近5年来人参皂苷Rg1在心血管和中枢神经系统的药理活性研究进展及其可能的作用机制进行了综述。     

Ginsenoside Rb3 ameliorates myocardial ischemia-reperfusion injury in rats

Context: Panax ginseng C. A. Mey (Araliaceae) has been widely used in clinic for treatment of cardiovascular diseases in China. Ginsenoside Rb3 is the main chemical component of Panax ginseng.

Objective: The aim of this study was to evaluate the effect of ginsenoside Rb3 on myocardial ischemia-reperfusion injury in rats.

Methods: Sprague–Dawley rats were orally treated with Rb3 (5, 10 or 20?mg/kg) daily for 3 days followed by subjecting to left anterior descending coronary artery ligation for 30?min and reperfusion for 24?h.

Results: This study showed that ginsenoside Rb3 treatment resulted in a reduction in myocardial infarct size. Ginsenoside Rb3 significantly attenuated the changes of creatine kinase activity and lactate dehydrogenase activity. The cardioprotective effect of ginsenoside Rb3 was further confirmed by histopathological examination. Ginsenoside Rb3 alleviated the increase of malondialdehyde content and the decrease of superoxide dismutase activity in left ventricle. Treatment with ginsenoside Rb3 also decreased plasma endothelin and angiotensin II levels.

Conclusion: These findings suggested that ginsenoside Rb3 possesses the effect against myocardial IR injury and the underlying mechanism is related to its antioxidant activity and microcirculatory improvement.     

Effects of ginsenoside Rb1 on central cholinergic metabolism

Ginsenosides, the saponins of ginseng, are bioactive ingredients which exert many beneficial effects. One ginsenoside, Rb1, extracted from North American ginseng (Panax quinquefolium L.), partially prevents the memory deficits induced by a cholinergic agent (scopolamine) in rats. In vitro studies show that Rb1 has no effect on quinuclidinyl benzylate binding or on acetylcholinesterase activity, but facilitates the release of acetylcholine (ACh) from hippocampal slices. The increase in ACh release is associated with an increased uptake of choline into nerve endings; however, calcium influx is unaltered. The ability of Rb1 to prevent memory deficits may be related to facilitation of ACh metabolism in the central nervous system.     

Simultaneous determination of ginsenoside Rb(1) and Rg(1) in human plasma by liquid chromatography-mass spectrometry

A liquid chromatographic-mass spectrometric (LC/MS) method for the simultaneous determination of ginsenoside Rb(1) and Rg(1) in human plasma was developed. The method involved the protein precipitation followed by analysis of ginsenoside Rb(1) and Rg(1) in an Atlantis C(18) column with the gradient elution of acetonitrile and ammonium formate (10mM, pH 3.0) at a flow rate of 0.2 ml/min. The analytes were determined using electrospray negative ionization mass spectrometry in the selected ion monitoring mode. The standard curves for ginsenoside Rb(1) and Rg(1) were linear over the concentration range of 10.0-1000 ng/ml. The lower limit of quantification was 10.0 ng/ml using 100 microl plasma sample. The coefficient of variation of intra- and inter-day assays for ginsenoside Rb(1) and Rg(1) at three quality control levels ranged from 1.0 to 6.8% and 5.4 to 9.8%, respectively. Ginsenoside Rb(1) and Rg(1) were stable in blank human plasma at room temperature for 24h and following three freeze-thaw cycles.     

Ginsenoside Rb1 attenuates angiotensin II-induced abdominal aortic aneurysm through inactivation of the JNK and p38 signaling pathways

BackgroundAbdominal aortic aneurysm (AAA), a life-threatening vascular disease, accounts for approximately 10% of the morbidity in people over 65 years old. No satisfactory approach is available to treat AAA. Ginsenosides Rb1 and Rg1 are primary ingredients of Panax notoginseng for the treatment of cardiovascular diseases, but their impact on AAA is unknown.Methods and resultsAn AAA model was established using an Ang II infusion in ApoE^−/− mice. After continuous stimulation of Ang II for 28 days, suprarenal aortic aneurysms developed in 77% mice and 12% mice died suddenly due to AAA rupture. Administration of ginsenoside Rb1 (20 mg/kg/day), but not ginsenoside Rg1, significantly reduced the incidence and mortality of AAA. Ginsenoside Rb1 treatment dramatically suppressed Ang II-induced diameter enlargement, extracellular matrix degradation, matrix metalloproteinase (MMP) production, inflammatory cell infiltration, and vascular smooth muscle cell (VSMC) dysfunction. Mechanistic studies indicated that the protective effects of ginsenoside Rb1 were associated with the inactivation of JNK and p38 MAPK signaling pathways. A specific activator of JNK and p38, anisomycin, nearly abolished ginsenoside Rb1-driven suppression of MMP secretion by VSMCs.ConclusionsGinsenoside Rb1, as a potential anti-AAA agent, suppressed AAA through inhibiting the JNK and p38 signaling pathways.     

Estrogen desensitizes 5-HT(1A) receptors and reduces levels of G(z), G(i1) and G(i3) proteins in the hypothalamus

The present study investigated whether estrogen would desensitize hypothalamic serotonin(1A) (5-HT(1A)) receptors by examining the neuroendocrine response to 8-OH-DPAT, a 5-HT(1A) agonist. Rats were ovariectomized, allowed to recover for 5 days, then given 2 daily injections of estradiol benzoate or vehicle (10 microg/day, s.c.). Twenty-four hours after the second injection, rats were challenged with a sub-maximal dose of 8-OH-DPAT (50 microg/kg, sc) or saline 15 min prior to sacrifice. 8-OH-DPAT produced a significant increase in plasma oxytocin, ACTH and corticosterone levels in ovariectomized rats. While estrogen treatment for 2 days did not alter basal hormone levels, it did significantly reduce the magnitude of oxytocin, ACTH and corticosterone responses to 8-OH-DPAT. The reduction in hormone responses was accompanied by a significant reduction in hypothalamic levels of G(z), G(i1) and G(i3) proteins (by 50%, 30% and 50%, respectively). These findings suggest that a reduction in these G proteins may contribute to the mechanisms underlying estrogen-induced desensitization of 5-HT(1A) receptors. The desensitization of 5-HT(1A) receptors has been suggested to underlie the therapeutic effects of antidepressant 5-HT uptake inhibitors (SSRIs). Thus, the present results suggest that estrogen or estrogen-like substances in combination with SSRIs may prove effective in developing novel therapeutic strategies for neuropsychiatric disorders in women.     

Tryptophol-induced change in brain 5-hydroxytryptamine metabolism

The effects of tryptophol (TOL) on brain 5-hydroxytryptamine (5-HT) metabolism were studied. After TOL injection (200 mg/kg IP), brain 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) levels were increased, 5-HT synthesis rate was decreased, and monoamine oxidase (MAO) activity remained unchanged. Pretreatment of mice with p-chlorophenylalanine (PCPA), a potent inhibitor of 5-HT synthesis, did not affect the anticonvulsant action of TOL. These results suggest that alteration of 5-HT metabolism after TOL injection is not directly related to the anticonvulsant action of TOL.     

Tryptophan hydroxylase: a target for neuroendocrine disruption

Tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin (5-HT) synthesis, performs an essential role in the maintenance of serotonergic functions in the central nervous system (CNS), including regulation of the neuroendocrine system controlling reproduction. The results of recent studies in a teleost model of neuroendocrine disruption, Atlantic croaker, indicated that hypothalamic TPH is a major site of interference of hypothalamic-pituitary-gonadal function by environmental stressors. The effects of exposure to two different types of environmental stressors, low dissolved oxygen (hypoxia) and a polychlorinated biphenyl mixture (Aroclor 1254), on the stimulatory brain serotonergic system controlling reproductive neuroendocrine function in Atlantic croaker are reviewed. Exposure to both stressors produced decreases in TPH activity, which were accompanied by a fall in hypothalamic 5-HT and gonadotropin-releasing hormone (GnRH I) content in the preoptic-anterior hypothalamic area and were associated with reduction in luteinizing hormone (LH) secretion and gonadal development. Pharmacological restoration of hypothalamic 5-HT levels after exposure to both stressors also restored neuroendocrine and reproductive functions, indicating that the serotonergic system is an important site for hypoxia- and Aroclor 1254-induced inhibition of reproductive neuroendocrine functions. The mechanisms underlying downregulation of TPH activity by these stressors remain unclear but may involve alterations in hypothalamic antioxidant status. In support of this hypothesis, treatment with an antioxidant, vitamin E, was found to reverse the inhibitory effects of Aroclor 1254 on TPH activity. The results suggest that TPH is a major target for neuroendocrine disruption by diverse environmental stressors.     

Constitutive beta-glucosidases hydrolyzing ginsenoside Rb1 and Rb2 from human intestinal bacteria

When ginsenoside Rb1 and Rb2 were anaerobically incubated with human intestinal microflora, these ginsenosides were metabolized to 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol (compound K) and 20(S)-protopanaxadiol. Several kinds of intestinal bacteria hydrolyzed these ginsenosides. Eubacterium sp., Streptococcus sp. and Bifidobacterium sp., which more potently hydrolyzed gentiobiose than sophorose, metabolized ginsenoside Rb1 to compound K via ginsenoside Rd rather than gypenoside XVII. However, Fusobacterium K-60, which more potently hydrolyzed sophorose than gentiobiose, metabolized to compound K via gypenoside XVII. Ginsenoside Rb2 was also metabolized to compound K via ginsenoside Rd or compound O by human intestinal microflora. Eubacterium sp., Streptococcus sp. and Bifidobacterium sp. metabolized ginsenoside Rb2 to compound K via ginsenoside Rd rather than compound O. Fusobacterium K-60 metabolized ginsenoside Rb2 to compound K via compound O.