Search

Additional file 1.

Additional file 2. Luciferase activity was not inhibited by miR-124. Luciferase assay using a portion of the Rhes mRNA 3′UTR and miRNAs. HEK293 cells were co-transfected with a reporter vector without the insert (Control) or with Rhes 3′UTR (Rhes) and 50 nM miRNA mimics (miR-sc or miR-124). At 24 h after transfection, the cells were lysed, and the luciferase activity of the cell lysates was measured. The activity was normalized to that of a control transfected with miR-sc. Data are presented as the mean ± SEM, n = 5.

Nowadays, a lot of food ingredients are marketed as dietary supplements for health. Because the effectiveness and mechanisms of these compounds have not been fully characterized, they might have unknown functions. Therefore, we investigated the effect of several food ingredients (Bergamottin, Chrysin, L-Citrulline and β-Carotene) known as health foods on adipocyte differentiation by using 3T3-L1 preadipocytes. In this study, we found that Bergamottin, a furanocoumarin isolated from grapefruit juice, promotes adipocyte differentiation. In addition, Bergamottin increases the expression of adiponectin, an anti-inflammatory adipokine, and peroxisome proliferator activated receptor γ (PPARγ), a nuclear receptor regulating adipocyte differentiation. Furthermore, the anti-inflammatory activity of Bergamottin was demonstrated by its inhibition of the activation of nuclear factor-κB (NF-κB), an inflammatory transcription factor. Stimulation of mature 3T3-L1 adipocytes by tumor necrosis factor-α (TNF-α) decreased the expression of the endogeneous NF-κB inhibitor, IκBα. Treatment with Bergamottin further decreased the TNF-α-induced change in IκBα expression, suggesting that Bergamottin mediated the inhibition of NF-κB activation. In addition, Bergamottin decreased the TNF-α-induced increase in the mRNA levels of pro-inflammatory adipokines, monocyte chemoattractant protein-1 and interleukin-6. Taken together, our results show that Bergamottin treatment could inhibit inflammatory activity through promoting adipocyte differentiation, which in turn suggests that Bergamottin has the potential to minimize the risk factors of metabolic syndrome.

St. John's wort (SJW), or Hypericum perforatum, is a perennial herb that has been used in the treatment of depression in several countries. Though its therapeutic effect on depression has been extensively studied, its influence on metabolic syndrome is yet to be fully characterized. Therefore, we investigated the effect of SJW extract on adipocyte differentiation and its anti-inflammatory effects by using 3T3-L1 preadipocytes. Oil Red O staining indicated that SJW promotes adipocyte differentiation, while immunoblots indicated that SJW increases the expression of peroxisome proliferator activated receptor γ (PPARγ), a nuclear receptor regulating adipocyte differentiation, and adiponectin, an anti-inflammatory adipokine. Furthermore, the anti-inflammatory activity of SJW was demonstrated by its inhibition of the activation of nuclear factor-κB (NF-κB), an inflammatory transcription factor. Stimulation of mature 3T3-L1 adipocytes by tumor necrosis factor-α (TNF-α) decreased the expression of the NF-κB inhibitor IκBα, and increased its phosphorylation. Treatment with SJW further decreased the TNF-α-induced perturbation in IκBα expression and phosphorylation, which indicated that SJW mediated the inhibition of NF-κB activation. In addition, SJW decreased the TNF-α-induced increase in the mRNA levels of pro-inflammatory adipokines, interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). Collectively, our results indicate that SJW treatment could promote adipocyte differentiation probably through its anti-inflammatory activity, which in turn suggests that SJW has the potential to minimize the risk factors of metabolic syndrome.

St. John’s wort (SJW), or Hypericum perforatum, is a perennial herb that is used for the treatment of depression in several countries. However, its effects in other common conditions, such as the metabolic syndrome, have not been elucidated. Some studies have investigated the effects of SJW extract on differentiation of adipocytes, inflammation, and glucose metabolism. In this review, we summarize these studies and show the following: SJW enhances differentiation of adipocytes via activation of peroxisome proliferator-activated receptor gamma and increases expression of adiponectin; SJW exerts anti-inflammatory effects by inhibiting expression of pro-inflammatory adipokines; and SJW improves glucose metabolism by increasing insulin sensitivity and insulin secretion. Although further investigation is necessary, SJW may help to attenuate the pathologic process associated with the metabolic syndrome.

Aggregation of alpha-synuclein (alpha-syn) is believed to play a critical role in the pathogenesis of disorders such as dementia with Lewy bodies and Parkinson's disease. The function of alpha-syn remains unclear, although several lines of evidence suggest that alpha-syn is involved in synaptic vesicle trafficking probably via lipid binding. Moreover, interactions with cholesterol and lipids have been shown to be involved in alpha-syn aggregation. In this context, the main objective of this study was to determine if statins--cholesterol synthesis inhibitors--might interfere with alpha-syn accumulation in cellular models. For this purpose, we studied the effects of lovastatin, simvastatin, and pravastatin on the accumulation of alpha-syn in a stably transfected neuronal cell line and in primary human neurons. Statins reduced the levels of alpha-syn accumulation in the detergent insoluble fraction of the transfected cells. This was accompanied by a redistribution of alpha-syn in caveolar fractions, a reduction in oxidized alpha-syn, and enhanced neurite outgrowth. In contrast, supplementation of the media with cholesterol increased alpha-syn aggregation in detergent insoluble fractions of transfected cells and was accompanied by reduced neurite outgrowth. Taken together, these results suggest that regulation of cholesterol levels with cholesterol inhibitors might be a novel approach for the treatment of Parkinson's disease.

While microarray studies are generating novel insights into the etiology of major psychiatric disorders, the validation of microarray-identified candidate genes and their role in the causality of these disorders has been less often studied. We have previously demonstrated, by microarray, up-regulation of SELENBP1 in the brain and blood of patients with schizophrenia. The main aim of the current study was to validate this finding using quantitative real-time PCR (QPCR) in an independent brain cohort that included patients with bipolar disorder. Our sample consisted of mRNAs from the dorsolateral prefrontal cortex (dlPFC) of 34 schizophrenic patients, 33 bipolar disorder patients (including 20 with psychotic history), and 34 normal control subjects. QPCR was employed to assess gene expression changes, with C(T) values analyzed using an ANCOVA approach. The results demonstrated that SELENBP1 mRNA was upregulated in schizophrenic brains versus controls (P = 0.046) and, in addition, that SELENBP1 gene expression was strongly positively correlated with presence of psychosis across diagnoses (P < 0.001, increased by 12%). Based on these findings, we conclude that elevated SELENBP1 is a possibly consistent feature in the schizophrenic brain and that this finding could underlie some commonalities of psychosis across the boundaries of diagnoses. Future studies should exploit DNA-based methods and molecular investigations on the role of SELENBP1 in order to gain insights into the nature of its influence on schizophrenia and psychotic symptoms.

DJ-1 is a multifunctional protein whose loss of function by gene mutations may play a causative role for familial Parkinson's disease (PD). A recent study has shown that the expression of this molecule is upregulated in both brains and cerebrospinal fluids (CSF) in various neurological disorders, including sporadic PD, Alzheimer's disease (AD) and stroke, raising a possibility that DJ-1 could be a potential biomarker for these diseases. In this context, the main objective of the present study was to determine if DJ-1 was increased in the plasma of PD patients. For this purpose, blood plasma samples collected from sporadic PD patients, dementia with Lewy bodies (DLB) and healthy age-matched controls were analyzed by immunoblotting and enzyme-linked immunosorbent assay. The results showed that the plasma DJ-1 levels in PD (n=104) were higher than those in control (n=80) (p

Paxillin is a substrate of the Src tyrosine onco-kinase and is involved in cell transformation, cell spreading, migration, and cancer development mediated through the mitogen-activated protein kinase signaling cascades. Here, we showed that paxillin plays a key role in skin cell transformation induced by epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). To investigate the mechanism of paxillin's role in cell transformation, we established a paxillin knockdown stably transfected cell line by introducing small interfering RNA-paxillin (si-paxillin). The si-paxillin cells displayed a dramatic suppression of cell proliferation and anchorage-independent cell transformation induced by EGF or TPA compared with si-mock control cells. In si-paxillin cells, decreased activator protein-1 (AP-1)–dependent luciferase activity corresponded with suppressed AP-1 DNA binding activity. Importantly, knockdown of paxillin inhibited EGF- or TPA-induced c-Jun phosphorylation at Ser63 and Ser73. Furthermore, total c-Jun protein level was dramatically decreased in si-paxillin cells and was dependent on serum deprivation time. The down-regulation of c-Jun was restored in si-paxillin cells by treatment with the proteasome inhibitor lactacystin but not by the lysosome inhibitor leupeptin. These results clearly provided evidence that paxillin regulates c-Jun protein level and plays a key role in cell transformation most likely through the regulation of c-Jun stability. (Cancer Res 2006; 66(11): 5968-74)

Black tea is one of the most popular beverages worldwide and especially in Western nations. Theaflavins, a mixture of theaflavin (TF-1), theaflavin-3-gallate (TF-2a), theaflavin-3'-gallate (TF-2b), and theaflavin-3,3'-digallate (TF-3) are the major components of black tea. Among these black tea components, theaflavin is generally considered to be the more effective component for the inhibition of carcinogenesis. Recently, TF-3 has been shown to have an antiproliferative effect on tumor cells, but the mechanism is not clear. In this study, we showed that TF-3-induced internalization and downregulation of the epidermal growth factor receptor (EGFR). These results suggested that TF-3 induces EGFR endocytosis and degradation. We further showed that TF-3 stimulated EGFR ubiquitination and tyrosine kinase activation. Interestingly, TF-3-induced EGFR downregulation is inhibited by the proteasome inhibitor, MG132, but not by the EGFR-specific receptor tyrosine kinase inhibitor, AG1478. Furthermore, pretreatment with TF-3 inhibited EGF-induced EGFR autophosphorylation, ERKs phosphorylation and AP-1 activation in JB6 Cl41 cells. In addition, TF-3 inhibited EGF-induced anchorage-independent cell transformation. Overall, our results indicate that TF-3 might exert chemopreventive effects through the downregulation of the EGFR.

Ginkgo biloba extract (GBE) has been used clinically for improving peripheral vascular diseases in France and Germany and is ingested widely as a herbal medicine in some countries. However, accurate information about its safety as an herbal medicine has not been sufficiently established. To address this issue, we examined the effect of GBE on hepatic drug metabolizing enzymes and their influence on hypotensive drug in rats. Male rats were fed either a control diet or diet containing GBE (0.5% w/w) for 4 weeks. The feeding of a GBE diet did not change the serum transaminase activity, but increased the liver weight and the phospholipid concentration in the liver. In addition, the GBE diet markedly increased the content of cytochrome P-450 (CYP), and the activity of glutathione S-transferase in the liver. Furthermore, the GBE diet markedly induced levels of CYP2B1/2, CYP3A1 and CYP3A2 mRNA in the liver. The levels of CYP1A1, CYP1A2, CYP2E1, CYP2C11 and CYP4A1 were unchanged. The feeding of GBE for 4 weeks significantly reduced the hypotensive effect of nicardipine that was reported to be metabolized by CYP3A2 in rats. These findings suggest that GBE reduces the therapeutic potency of the Ca2+ channel blocker, nicardipine, via enhancement of cytochrome P-450 expression.

1. The role of ATP in the regulatory volume decrease (RVD) after hypotonic cell swelling was examined in cultured endothelial cells isolated from the rat caudal artery. 2. Hypotonic stress increased [Ca2+]i in addition to increasing the overflow of ATP and cell volume. The hypotonicity induced increase in [Ca2+]i was prevented by pyridoxalphosphate-6-azophenyl-1-2',4'-disulphonic acid (PPADS; a P2 purinoceptor antagonist), U-73122 (a phospholipase C inhibitor) and thapsigargin (a Ca2+ pump inhibitor). However, the hypotonicity induced increase in cell volume was potentiated by PPADS, U-73122 and thapsigargin. 3. Similar changes were observed in cells treated with 2-methylthioATP, a P2Y purinoceptor agonist, but not by alpha,beta-methylene ATP, a P2X purinoceptor agonist. Thus, it appears that the responses observed following hypotonic stress are mediated by activation of P2Y purinoceptors. 4. On the basis of these findings, it is suggested that ATP, which is released by hypotonicity, may participate in the RVD as a substantial regulator or initiator via P2 purinoceptor-induced increases in [Ca2+]i.

Contractile response to angiotensin II (AngII) of vascular smooth muscle was compared between 12-week-old, stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto rats (WKY). AngII induced phasic and tonic contraction in denuded aortic ring preparation. The phasic contraction was concentration-dependent (AngII 10(-9) to 10(-6) mol/L) and similar in both strains. However, the relaxation after phasic contraction was significantly attenuated in SHRSP compared with that in WKY. To examine the recovery of contractile responses to the repeated stimulation, AngII was applied three times at 20- and 60-min intervals. The first maximal contraction was similar in both strains, but the response to the second stimulation was significantly reduced in SHRSP, compared with all three responses in WKY. These results suggested that Ca2+-sequestration into the Ca2+ store is delayed in SHRSP. Cyclopiazonic acid (10(-5) mol/L), an intracellular Ca2+-pump inhibitor, decreased spontaneous relaxation and increased the sustained contraction in WKY, whereas it did not affect the contraction in SHRSP. Insulin, which modulates tonic contraction by facilitating Ca2+-extrusion, was applied at peak contraction by AngII. It enhanced relaxation after phasic contraction in a concentration-dependent manner in SHRSP, but it did not affect the relaxation in WKY. These results suggest that increased sustained contraction observed in SHRSP reflects at least partly the impaired Ca2+-pump activity leading to hypertension.

Thrombin- and ionophore A23187-induced aggregation, secretion and 47-kDa protein phosphorylation in washed platelets from stroke-prone spontaneously hypertensive rats (SHRSP) were attenuated with the development of hypertension. In this study, the ultrastructure of platelets from SHRSP and Wistar Kyoto rats (WKY) at pre- and posthypertensive ages was analyzed in relation to physiological functions using electron microscopy. The platelet size of SHRSP at hypertensive ages (14 and 18 weeks old) was larger on average than that of age-matched WKY. However, the platelet granule size, number and distribution in SHRSP were normal and SHRSP platelets contained similar inclusion bodies to those seen in WKY platelets. At a prehypertensive age (3 weeks old), there was no difference in the morphological characteristics and the platelet size between SHRSP and WKY platelets. At a late hypertensive age (28 weeks old), the large size of SHRSP platelets at 14 weeks of age was normalized and there were no differences in the size between SHRSP and WKY. ADP-induced platelet aggregation measured by a turbidimetric method and electron microscopy was greatly reduced in SHRSP (18 weeks old) compared to WKY. These results suggest that hypofunction of SHRSP platelets at hypertensive and posthypertensive ages would not reflect changes in platelet ultrastructure, although there might be some relationship between hypofunction and larger platelet size at hypertensive ages.

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder caused by mutations in the dystrophin gene, which encodes a cytoskeletal protein, dystrophin. Creatine kinase (CK) is generally used as a blood-based biomarker for muscular disease including DMD, but it is not always reliable since it is easily affected by stress to the body, such as exercise. Therefore, more reliable biomarkers of muscular dystrophy have long been desired. MicroRNAs (miRNAs) are small, ∼22 nucleotide, noncoding RNAs which play important roles in the regulation of gene expression at the post-transcriptional level. Recently, it has been reported that miRNAs exist in blood. In this study, we hypothesized that the expression levels of specific serum circulating miRNAs may be useful to monitor the pathological progression of muscular diseases, and therefore explored the possibility of these miRNAs as new biomarkers for muscular diseases. To confirm this hypothesis, we quantified the expression levels of miRNAs in serum of the dystrophin-deficient muscular dystrophy mouse model, mdx, and the canine X-linked muscular dystrophy in Japan dog model (CXMD(J)), by real-time PCR. We found that the serum levels of several muscle-specific miRNAs (miR-1, miR-133a and miR-206) are increased in both mdx and CXMD(J). Interestingly, unlike CK levels, expression levels of these miRNAs in mdx serum are little influenced by exercise using treadmill. These results suggest that serum miRNAs are useful and reliable biomarkers for muscular dystrophy.

α-Synuclein (α-Syn) is a major component of protein inclusions known as Lewy bodies, which are hallmarks of synucleinopathies such as Parkinson's disease (PD). Theα-Syn gene is one of the familial PD-causing genes and is also associated with an increased risk of sporadic PD. Numerous studies usingα-Syn expressing transgenic animals have indicated thatα-Syn plays a critical role in the common pathogenesis of synucleinopathies.Drosophila melanogasterhas several advantages for modeling human neurodegenerative diseases and is widely used for studying their pathomechanisms and therapies. In fact,Drosophilamodels expressingα-Syn have already been established and proven to replicate several features of human PD. In this paper, we review the current research on synucleinopathies usingα-SynDrosophilamodels and, moreover, explore the possibilities of these models for comprehensive genetic analyses and large-scale drug screening towards elucidating the molecular pathogenesis and developing therapies for synucleinopathies.

Misfolding and pathological aggregation of neuronal proteins has been proposed to play a critical role in the pathogenesis of neurodegenerative disorders. Alzheimer's disease (AD) and Parkinson's disease (PD) are frequent neurodegenerative diseases of the aging population. While progressive accumulation of amyloid beta protein (Abeta) oligomers has been identified as one of the central toxic events in AD, accumulation of alpha-synuclein (alpha-syn) resulting in the formation of oligomers and protofibrils has been linked to PD and Lewy body Disease (LBD). We have recently shown that Abeta promotes alpha-syn aggregation and toxic conversion in vivo, suggesting that abnormal interactions between misfolded proteins might contribute to disease pathogenesis. However the molecular characteristics and consequences of these interactions are not completely clear.In order to understand the molecular mechanisms involved in potential Abeta/alpha-syn interactions, immunoblot, molecular modeling, and in vitro studies with alpha-syn and Abeta were performed. We showed in vivo in the brains of patients with AD/PD and in transgenic mice, Abeta and alpha-synuclein co-immunoprecipitate and form complexes. Molecular modeling and simulations showed that Abeta binds alpha-syn monomers, homodimers, and trimers, forming hybrid ring-like pentamers. Interactions occurred between the N-terminus of Abeta and the N-terminus and C-terminus of alpha-syn. Interacting alpha-syn and Abeta dimers that dock on the membrane incorporated additional alpha-syn molecules, leading to the formation of more stable pentamers and hexamers that adopt a ring-like structure. Consistent with the simulations, under in vitro cell-free conditions, Abeta interacted with alpha-syn, forming hybrid pore-like oligomers. Moreover, cells expressing alpha-syn and treated with Abeta displayed increased current amplitudes and calcium influx consistent with the formation of cation channels.These results support the contention that Abeta directly interacts with alpha-syn and stabilized the formation of hybrid nanopores that alter neuronal activity and might contribute to the mechanisms of neurodegeneration in AD and PD. The broader implications of such hybrid interactions might be important to the pathogenesis of other disorders of protein misfolding.

Parkinson disease (PD) belongs to a heterogeneous group of neurodegenerative disorders with movement alterations, cognitive impairment, and alpha-synuclein accumulation in cortical and subcortical regions. Jointly, these disorders are denominated Lewy body disease. Mutations in the parkin gene are the most common cause of familial parkinsonism, and a growing number of studies have shown that stress factors associated with sporadic PD promote parkin accumulation in the insoluble fraction. alpha-Synuclein and parkin accumulation and mutations in these genes have been associated with familial PD. To investigate whether alpha-synuclein accumulation might be involved in the pathogenesis of these disorders by interfering with parkin solubility, synuclein-transfected neuronal cells were transduced with lentiviral vectors expressing parkin. Challenging neurons with proteasome inhibitors or amyloid-beta resulted in accumulation of insoluble parkin and, to a lesser extent, alpha-tubulin. Similarly to neurons in the brains of patients with Lewy body disease, in co-transduced cells alpha-synuclein and parkin colocalized and co-immunoprecipitated. These effects resulted in decreased parkin and alpha-tubulin ubiquitination, accumulation of insoluble parkin, and cytoskeletal alterations with reduced neurite outgrowth. Taken together, accumulation of alpha-synuclein might contribute to the pathogenesis of PD and other Lewy body diseases by promoting alterations in parkin and tubulin solubility, which in turn might compromise neural function by damaging the neuronal cytoskeleton. These studies provide a new perspective on the potential nature of pathogenic alpha-synuclein and parkin interactions in Parkinson disease.

We previously reported data regarding the mechanism of neoplastic transformation in JB6 Cl41 mouse skin epidermal cells. However, experimental in vitro models for studying neoplastic transformation of human cells could provide further insight into the mechanisms of human cancer development. In this study, we have established a neoplastic transformation model with HaCaT cells, a human keratinocyte cell line, and showed the usefulness of this cell line for studying the mechanisms of neoplastic transformation. Epidermal growth factor (EGF) treatment induced a dose-dependent anchorage-independent cell transformation in HaCaT cells. Furthermore, PD98059, a mitogen-activated protein (MAP) kinase/ERK kinase (MEK) inhibitor, or SP600125, c-Jun N-terminal kinase (JNK) inhibitor, decreased cell growth, EGF-induced DNA synthesis and transformation. Unlike observations in the JB6 mouse epidermal cell model, SB203580, a stress-activated protein kinase-2/p38 alpha and beta (p38) inhibitor, increased EGF-induced transformation in HaCaT cells. These results suggest that extracellular-signal regulated kinase (ERK), JNK, or p38 are implicated in EGF-induced neoplastic transformation of human cells.

Very little is known about the role of histone H3 phosphorylation in malignant transformation and cancer development. Here, we examine the function of H3 phosphorylation in cell transformation in vivo. Introduction of small interfering RNA-H3 into JB6 cells resulted in decreased epidermal growth factor (EGF)–induced cell transformation. In contrast, wild-type histone H3 (H3 WT)–overexpressing cells markedly stimulated EGF-induced cell transformation, whereas the H3 mutant S10A cells suppressed transformation. When H3 WT was overexpressed, EGF induction of c-fos and c-jun promoter activity was significantly increased compared with control cells but not in the H3 mutant S10A or S28A cells. In addition, activator protein-1 activity in H3 WT–overexpressing cells was markedly up-regulated by EGF in contrast to the H3 mutant S10A or S28A cells. These results indicate that the phosphorylation of histone H3 at Ser10 is an essential regulatory mechanism for EGF-induced neoplastic cell transformation.

Previously, no member of the mixed-lineage kinase (MLK) protein family was known to function as an oncogene. Here, we demonstrate that MLK-like mitogen-activated protein triple kinase (MLTK)-α, a member of the MLK family, induced neoplastic cell transformation and tumorigenesis in athymic nude mice. Introduction of small interference RNA (siRNA)-MLTK-α into MLTK-α-overexpressing cells dramatically suppressed cell transformation. Nuclear accumulation of the pHisG-MLTK-α fusion protein was observed after epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate treatment. Phosphorylation of downstream mitogen-activated protein kinase-targeted transcription factors including c-Myc, Elk-1, c-Jun, and activating transcription factor (ATF) 2 was also differentially enhanced in MLTK-α-overexpressing cells exposed to epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate stimulation compared with cells expressing mock vector or siRNA-MLTK-α. Very importantly, MLTK-α-overexpressing cells formed fibrosarcomas when injected s.c. into athymic nude mice, whereas almost no tumor formation was observed in mice that received injections of mock or siRNA-MLTK-α stably transfected cells. These results are the first to indicate that MLTK-α plays a key role in neoplastic cell transformation and cancer development.

It is generally agreed that the release of norepinephrine (NE) is inhibited by activation of prejunctional purinoceptor. We examined the pharmacological properties of purinoceptors on vascular sympathetic nerve terminals and the source of endogenous adenyl purines. Electrically (1 Hz) evoked NE-release was inhibited by not only P1-agonists but also P2-agonists. Although the inhibition induced by P2-agonists was blocked by P1-antagonists, P2-agonists-induced inhibition was not due to the breakdown to adenosine. Therefore, there may be a new class of purinoceptor that is activated by both P1- and P2-agonists and antagonized by P1-antagonists. Electrical stimulation at 8 Hz but not at 1 Hz evoked the release of adenyl purines such as ATP, ADP, AMP and adenosine, in addition to NE; and the purines-release was blocked by an alpha1-antagonist. Methoxamine, an alpha1-agonist, also evoked the release of purines. Electrically (1 Hz)-evoked NE-release was inhibited by methoxamine, and this inhibition was blocked by not only an alpha1-antagonist but also a P1-antagonist. Therefore, the activation of alpha1-adrenoceptor appeared to release purines, which in turn inhibited NE-release via prejunctional purinoceptors. From these results, it is suggested that the unique purinoceptor and the endogenous purines released from alpha1-adrenoceptor-sensitive sources participate in the antidromic transsynaptic modulation of vascular sympathetic neurotransmission.

We investigated the effects of dietary supplements on atria isolated from male Wistar rats. The examined supplements, which are increasingly used in Japan, those were Ginkgo biloba extract (GBE), catechins, isoflavones, sodium iron chlorophyllin and sodium copper chlorophyllin. GBE at 100-1000 microg/ml significantly increased the beat rate and the contractile force. Catechins at 1-100 microg/ml significantly potentiated the contractile force but did not effect the beat rates. However, isoflavones, sodium iron and sodium copper chlorophyllins did not change the contractile force or the beat rates. To identify the active ingredient of GBE, ginkgolide B, quercetin and amentoflavone on the atria were tested. Ginkgolide B weakened the contractile force. Quercetin potentiated the contractile force at only 30 microg/ml. Amentoflavone significantly increased the beat rate. From these findings, amentoflavone and quercetin were considered to be the principal ingredients of GBE producing the positive chronotropic and inotropic actions, respectively. In the case of catechins, (-)-epigallocatechin gallate (EGCg), one of the principal ingredients, produced inotropic actions. These findings suggest that there are some dietary supplements which affect cardiac function, such GBE and catechins.