Kenzo Kudo

Effects of ginseng saponins on responses induced by various receptor stimuli

We investigated the effects of four ginseng saponins, ginsenoside-Rb1, -Rg2, -Rg3 and -Ro, on the responses induced by receptor stimulation of various stimuli. Ginsenoside-Rg2 (1-100 microM) reduced the secretions of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine and gamma-aminobutyric acid but not by angiotensin II, bradykinin, histamine and neurotensin. In guinea-pig, the ginsenoside also diminished the nicotine-induced secretion of catecholamines from the adrenal chromaffin cells, but it did not affect the muscarine- and the histamine-induced ileum contractions. On the other hand, ginsenoside-Rg3 (1-100 microM) reduced not only the acetylcholine-, the gamma-aminobutyric acid- and the neurotensin-induced secretions but also, at a higher concentration (100 microM), the angiotensin II-, the bradykinin- and the histamine-induced secretions from the bovine chromaffin cells. Furthermore, the saponin (3-100 microM) significantly inhibited the muscarine- and the histamine-induced ileum contractions of the guinea-pig. Ginsenoside-Rb1 and -Ro had no marked effect on their responses. These results strongly suggest that ginsenoside-Rg2 is a potent selective blocker of nicotinic acetylcholine and gamma-aminobutyric acid receptors (ionotropic receptors) and ginsenoside-Rg3 is not only a blocker of ionotropic receptors but also an antagonist of muscarinic or histamine receptors.

Properties of ginseng saponin inhibition of catecholamine secretion in bovine adrenal chromaffin cells

To investigate the relationship between the inhibitory effects of ginseng saponins (ginsenosides) on acetylcholine-evoked secretion of catecholamines and the structures of ginsenosides, we examined the effects of ginsenoside-Rg3 and -Rh2, which are panaxadiol saponins, 20(R)- and 20(S)-ginsenoside-Rg2, which are epimers involving the hydroxyl group at C-20 of sapogenin, and other plant saponins on the acetylcholine-evoked secretion of catecholamines from cultured bovine adrenal chromaffin cells. The ginsenoside-Rg3 (1-100 microM) and -Rh2 (10-100 microM) greatly reduced the acetylcholine-evoked secretion in a concentration-dependent manner comparable to that of ginsenoside-Rg2, a panaxatriol saponin, which was the most potent inhibitor in our previous study. 20(R)- and 20(S)-ginsenoside-Rg2 (1-100 microM) similarly reduced the acetylcholine-evoked secretion. In contrast, saikosaponin-a, glycyrrhizin and the cardiac glycosides (100 nM-100 microM), digitoxin and digoxin, had no significant inhibitory effect on catecholamine secretion. Saikosaponin-c (10-100 microM), however, had an inhibitory effect, which was less than that of ginsenoside-Rg2 and -Rg3. These results strongly suggest that the inhibitory effects of ginsenosides on the acetylcholine-evoked secretion of catecholamines from bovine adrenal chromaffin cells are a unique property of ginseng. Further, the relationship between the inhibitory effects and the structures of ginsenosides is discussed.

Characterization of ginseng saponin ginsenoside-Rg3 inhibition of catecholamine secretion in bovine adrenal chromaffin cells

Since ginsenoside-Rg(3), one of the panaxadiol saponins isolated from the ginseng root, significantly inhibited the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh), the properties of ginsenoside-Rg(3) inhibition were investigated. Although ginsenoside-Rg(3) inhibited the secretion evoked by ACh in a concentration-dependent manner, it affected the secretion stimulated by high K(+) or veratridine, an activator of the voltage-sensitive Ca(2+) or Na(+) channels, only slightly. The ACh-induced Na(+) and Ca(2+) influxes into the cells were also reduced by ginsenoside-Rg(3). The inhibitory effect of this saponin on the secretion of catecholamines was not altered by increasing the external concentration of ACh or Ca(2+). The ACh-evoked secretion of catecholamines was completely restored in cells that were preincubated with 10 microM ginsenoside-Rg(3) and then incubated without the saponin, whereas secretion was not completely restored in cells that were preincubated with 30 microM of this compound. Above 30 microM ginsenoside-Rg(3) increased the fluorescence anisotropy of diphenylhexatriene in the cells. Furthermore, the inhibitory effect of ginsenoside-Rg(3) at 30 microM on the ACh-evoked secretion of catecholamines was dependent upon the preincubation time, but this was not the case at 10 microM. These results strongly suggest that ginsenoside-Rg(3) blocks the nicotinic ACh receptor-operated cation channels, inhibits Na(+) influx through the channels, and consequently reduces both Ca(2+) influx and catecholamine secretion in bovine adrenal chromaffin cells. In addition to this action, the ginsenoside at higher concentrations modulates the fluidity of the plasma membrane, which probably contributes to the observed reduction in the secretion of catecholamines.

An aryl hydrocarbon receptor induces VEGF expression through ATF4 under glucose deprivation in HepG2.

BackgroundAryl hydrocarbon receptor (AhR) not only regulates drug-metabolizing enzyme expression but also regulates cancer malignancy. The steps to the development of malignancy include angiogenesis that is induced by tumor microenvironments, hypoxia, and nutrient deprivation. Vascular endothelial growth factor (VEGF) plays a central role in the angiogenesis of cancer cells, and it is induced by activating transcription factor 4 (ATF4).ResultsRecently, we identified that glucose deprivation induces AhR translocation into the nucleus and increases CYP1A1 and 1A2 expression in HepG2 cells. Here, we report that the AhR pathway induces VEGF expression in human hepatoblastoma HepG2 cells under glucose deprivation, which involves ATF4. ATF4 knockdown suppressed VEGF expression under glucose deprivation. Moreover, AhR knockdown suppressed VEGF and ATF4 expression under glucose deprivation at genetic and protein levels.ConclusionsThe AhR-VEGF pathway through ATF4 is a novel pathway in glucose-deprived liver cancer cells that is related to the microenvironment within a cancer tissue affecting liver cancer malignancy.

Inflammatory cytokines decrease the expression of nicotinic acetylcholine receptor during the cell maturation

It is known that the nervous system significantly attenuates systemic inflammatory responses through the parasympathetic nervous system. Furthermore, it has been reported that the alpha7 subunit of a nicotinic acetylcholine receptor is required for a cholinergic inhibition against cytokine synthesis in a macrophage. As antigen-presenting cells (APCs) play a central role in the generation of primary T cell responses and the maintenance of immunity, in this study, we investigated the expression level of nicotinic receptors of a p53-deficient APC cell line (JawsII) derived from a mouse bone marrow. We showed that stimulation of the JawsII cells with lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-α) led increase of CD80 and CD86 expression while diminishment of the surface nicotinic receptor. On the other hand, stimulation of nicotinic receptor had no effect on these phenomena. Furthermore, we examined the ability of the cells to release cytokine when stimulated with both nicotine and LPS and showed that the stimulation with LPS augmented the secretion of IL-1a, IL-1b, IL-6, and TNF-α. These results suggested that nicotinic stimulation had no effect on the diminishment of alpha7 nicotinic acetylcholine receptor on JawsII cells by LPS stimulation.

Inhibition by pregnenolone sulfate of nicotinic acetylcholine response in adrenal chromaffin cells

To evaluate whether pregnenolone sulfate, an abundant neurosteroid in the brain, modulates nicotinic receptor-mediated responses, the effect of pregnenolone sulfate on acetylcholine-induced catecholamine secretion was investigated in cultured bovine adrenal chromaffin cells. Pregnenolone sulfate inhibited acetylcholine-induced catecholamine secretion (IC(50): 27 microM). In addition, pregnenolone sulfate inhibited acetylcholine-induced Na(+) (IC(50): 12 microM) and Ca(2+) (IC(50): 20 microM) influxes. However, pregnenolone sulfate did not inhibit either catecholamine secretion or Ca(2+) influx stimulated by high K(+). Binding of [3H]nicotine to nicotinic receptors was not altered by pregnenolone sulfate. The inhibitory effect on the acetylcholine-induced secretion was insurmountable by increasing acetylcholine concentrations, but was enhanced by decreasing external Na(+) concentrations. These results suggest strongly that pregnenolone sulfate noncompetitively inhibits nicotinic receptor-operated ion channels, thereby suppressing Na(+) influx through the channels and, consequently, attenuates both Ca(2+) influx and catecholamine secretion. Our results further indicate that pregnenolone sulfate may modulate nicotinic receptor-mediated responses in the brain.

VEGF expression is regulated by HIF-1α and ARNT in 3D KYSE-70, esophageal cancer cell spheroids

In 3D cultured cell systems, the cells form 3D spheroids that mimic cancer cell spheroids in vivo. Cancer cells form cell spheroids as they grow. The in vivo spheroids do not contain a vascular network; therefore, oxygen and nutrition supplies are insufficient. Specifically, the cells in the core region of the cluster are exposed to higher stress levels than the cells in the outer spheroid layer. As a result, the cells in the spheroid are exposed to low nutrition and hypoxia conditions. To overcome these shortages, angiogenesis is induced in cancer spheroids in vivo. Vascular endothelial growth factor (VEGF) is an important molecule involved in angiogenesis. VEGF is secreted by cancer cells in vivo in response to stress conditions such as hypoxia. VEGF expression in cancer cells is mediated by hypoxia‐inducible factor 1α (HIF1α), which accumulates in cancer cells during hypoxia. In this report, we show that VEGF expression is regulated by HIF1α and that VEGF is secreted to the outside of the spheroid in vitro. Several investigators have reported that HIF1α forms a protein–protein complex with aryl hydrocarbon receptor translocator (ARNT). We report here that not only HIF1α but also ARNT regulates VEGF expression in 3D cancer spheroids. Our results suggest the utility of the in vitro 3D cancer spheroid model for investigating angiogenesis in cancerous tissues.

Effects of Interferons on Cortisol Production in Bovine Adrenal Fasciculata Cells Stimulated by Adrenocorticotropin

The effects of interferons (IFNs) IFN‐α, IFN‐β and IFN‐γ on the production of cortisol in bovine adrenal fasciculata cells have been investigated.

Serotonin suppresses β-casein expression via PTP1B activation in human mammary epithelial cells

Serotonin (5-hydroxytriptamine, 5-HT) has an important role in milk volume homeostasis within the mammary gland during lactation. We have previously shown that the expression of β-casein, a differentiation marker in mammary epithelial cells, is suppressed via 5-HT-mediated inhibition of signal transduction and activator of transcription 5 (STAT5) phosphorylation in the human mammary epithelial MCF-12A cell line. In addition, the reduction of β-casein in turn was associated with 5-HT7 receptor expression in the cells. The objective of this study was to determine the mechanisms underlying the 5-HT-mediated suppression of β-casein and STAT5 phosphorylation. The β-casein level and phosphorylated STAT5 (pSTAT5)/STAT5 ratio in the cells co-treated with 5-HT and a protein kinase A (PKA) inhibitor (KT5720) were significantly higher than those of cells treated with 5-HT alone. Exposure to 100 μM db-cAMP for 6 h significantly decreased the protein levels of β-casein and pSTAT5 and the pSTAT5/STAT5 ratio, and significantly increased PTP1B protein levels. In the cells co-treated with 5-HT and an extracellular signal-regulated kinase1/2 (ERK) inhibitor (FR180294) or Akt inhibitor (124005), the β-casein level and pSTAT5/STAT5 ratio were equal to those of cells treated with 5-HT alone. Treatment with 5-HT significantly induced PTP1B protein levels, whereas its increase was inhibited by KT5720. In addition, the PTP1B inhibitor sc-222227 increased the expression levels of β-casein and the pSTAT5/STAT5 ratio. Our observations indicate that PTP1B directly regulates STAT5 phosphorylation and that its activation via the cAMP/PKA pathway downstream of the 5-HT7 receptor is involved in the suppression of β-casein expression in MCF-12A cells.

Serotonin regulates β-casein expression via 5-HT7 receptors in human mammary epithelial MCF-12A cells.

We previously reported that serotonin (5-hydroxytryptamine; 5-HT) suppresses β-casein expression, a differentiation marker in mammary epithelial cells, via inhibition of the signal transducer and activator of transcription 5 (STAT5) phosphorylation in the human mammary epithelial cell line, MCF-12A. In this study, we investigated the expression pattern of the different 5-HT receptor subtypes in MCF-12A cells, and identified the receptors involved in 5-HT-mediated suppression of β-casein protein expression. β-Casein mRNA expression was inhibited by 30 µM 5-HT in a time-dependent manner. Treatment with 30 µM 5-HT for 72 h decreased β-casein protein levels and STAT5 phosphorylation (pSTAT5). The cells expressed four 5-HT receptors subtypes (5-HTR1D, 2B, 3A, and 7) at the mRNA and protein level, and their expression was elevated by prolactin (PRL) treatment. Additionally, the mRNA levels of 5-HTR1D and 5-HTR7 were significantly higher than the other 5-HT receptors in the cells. Tryptophan hydroxylase 1 mRNA was detectable in the cells in the absence of PRL, and PRL treatment significantly increased its expression. β-Casein and pSTAT5/STAT5 levels in the cells co-treated with 5-HT and a selective 5-HTR1D inhibitor, BRL15572, were equal to those observed in cells treated with 5-HT alone. However, in the cells co-treated with 5-HT and a selective 5-HTR7 inhibitor, SB269970, β-casein and pSTAT5/STAT5 levels increased in a SB269970 concentration-dependent manner. In conclusion, we showed that 5-HT regulates β-casein expression via 5-HTR7 in MCF-12A human mammary epithelial cells.