Luteinizing hormone inhibits conversion of pregnenolone to progesterone in luteal cells from rats on day 19 of pregnancy.
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We have previously reported that intrabursal ovarian administration of LH at the end of pregnancy in rats induces a decrease in luteal progesterone (P4) synthesis and an increase in P4 metabolism. However, whether this local luteolytic effect of LH is exerted directly on luteal cells or on other structures, such as follicular or stromal cells, to modify luteal function is unknown. The aim of the present study was to determine the effect of LH on isolated luteal cells obtained on Day 19 of pregnancy. Incubation of luteal cells with 1, 10, 100, or 1000 ng/ml of ovine LH (oLH) for 6 h did not modify basal P4 production. The addition to the culture medium of 22(R)-hydroxycholesterol (22R-HC, 10 microgram/ml), a membrane-permeable P4 precursor, or pregnenolone (10(-2) microM) induced a significant increase in P4 accumulation in the medium in relation to the control value. When luteal cells were preincubated for 2 h with oLH, a significant (p < 0.01) reduction in the 22R-HC- or pregnenolone-stimulated P4 accumulation was observed. Incubation of luteal cells with dibutyryl cAMP (1 mM, a cAMP analogue) plus isobutylmethylxanthine (1 mM, a phosphodiesterase inhibitor) also inhibited pregnenolone-stimulated P4 accumulation. Incubation with an inositol triphosphate synthesis inhibitor, neomycin (1 mM), or an inhibitor of intracellular Ca2+ mobilization, (8,9-N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate (1 mM), did not prevent the decrease in pregnenolone-stimulated P4 secretion induced by oLH. It was concluded that the luteolytic action of LH in late pregnancy is due, at least in part, to a direct action on the luteal cells and that an increase in intracellular cAMP level might mediate this effect. (+info)
8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate actions on calcium dynamics in cultured vascular smooth muscle cells.
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AIM: To study 8-(N,N-Diethylamino)n-octyl-3,4,5-trimethoxybenzoate (TMB), a potent Ca(2+)-antagonist, actions on cellular calcium dynamics in vascular smooth muscle cell (VSMC) cultures. METHODS: A7r5 VSMC were cultured with Fura-2 measurements of intracellular Ca2+ concentration, [Ca2+]i. RESULTS: TMB reduced [Ca2+]i from control levels and blocked [Ca2+]i increase caused by norepinephrine (NE) and 2,5-di (t-butyl)-1,4-benzohydroquinone (BHQ). [Ca2+]i reduction by TMB was further enhanced by ryanodine. CONCLUSION: TMB is an effective agent for blocking the [Ca2+]i increase caused by NE and BHQ and for enhancing the [Ca2+]i reduction caused by ryanodine. (+info)
Actions of 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate in vascular smooth muscle cell cultures.
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AIM: To study the effects of 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate (TMB-8) on vascular smooth muscle (VSM) cells A7r5. METHODS: The effects of TMB-8 were investigated in A7r5 cell cultures with 45CaCl2. RESULTS: TMB-8 reduced the intracellular free Ca2+ concentration, [Ca2+]i in a Ca(2+)-free medium and blocked Ca2+ entry from the extracellular site in a regular Ca2+ medium. The equilibrated total cellular binding of Ca2+ was increased by TMB-8 whereas 45Ca2+ entry activated by both NE and KCl was inhibited. However, the NE-activated Ca2+ entry was not blocked by TMB-8 if TMB-8 was added together with 45Ca2+ at a later time instead of by pretreatment. Similar to actions of NE and KCl, depletion of Ca2+ from sarcoplasmic reticulum (SR) would also activate Ca2+ entry, which was blocked by TMB-8. When TMB-8 was rinsed out alone or together with NE after pretreatment with NE plus TMB-8 in VSM cells, the inhibitory effect of TMB-8 was not affected. CONCLUSION: TMB-8 not only blocks Ca2+ entry from the extracellular site, but also enhances Ca2+ uptake into SR which, indirectly inhibits Ca2+ entry from the extracellular site. (+info)
Glutamate regulates IP3-type and CICR stores in the avian cochlear nucleus.
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Neurons of the avian cochlear nucleus, nucleus magnocellularis (NM), are activated by glutamate released from auditory nerve terminals. If this stimulation is removed, the intracellular calcium ion concentration ([Ca2+]i) of NM neurons rises and rapid atrophic changes ensue. We have been investigating mechanisms that regulate [Ca2+]i in these neurons based on the hypothesis that loss of Ca2+ homeostasis causes the cascade of cellular changes that results in neuronal atrophy and death. In the present study, video-enhanced fluorometry was used to monitor changes in [Ca2+]i stimulated by agents that mobilize Ca2+ from intracellular stores and to study the modulation of these responses by glutamate. Homobromoibotenic acid (HBI) was used to stimulate inositol trisphosphate (IP3)-sensitive stores, and caffeine was used to mobilize Ca2+ from Ca2+-induced Ca2+ release (CICR) stores. We provide data indicating that Ca2+ responses attributable to IP3- and CICR-sensitive stores are inhibited by glutamate, acting via a metabotropic glutamate receptor (mGluR). We also show that activation of C-kinase by a phorbol ester will reduce HBI-stimulated calcium responses. Although the protein kinase A accumulator, Sp-cAMPs, did not have an effect on HBI-induced responses. CICR-stimulated responses were not consistently attenuated by either the phorbol ester or the Sp-cAMPs. We have previously shown that glutamate attenuates voltage-dependent changes in [Ca2+]i. Coupled with the present findings, this suggests that in these neurons mGluRs serve to limit fluctuations in intracellular Ca2+ rather than increase [Ca2+]i. This system may play a role in protecting highly active neurons from calcium toxicity resulting in apoptosis. (+info)
Suppression of extracellular signals and cell proliferation by the black tea polyphenol, theaflavin-3,3'-digallate.
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Previous studies in our laboratory have shown that the major green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), suppressed autophosphorylation of epidermal growth factor (EGF) receptor induced by EGF in human A431 epidermoid carcinoma cells. In this study, we examined the inhibitory effects of black tea polyphenols, including theaflavin (TF-1), a mixture (TF-2) of theaflavin-3-gallate (TF-2a) and theaflavin-3'-gallate (TF-2b), theaflavin-3,3'-digallate (TF-3) and the thearubigin fraction on the autophosphorylation of the EGF and PDGF receptors in A431 cells and mouse NIH3T3 fibroblast cells, respectively. First, we examined the effects of these polyphenols on the proliferation of A431 and NIH3T3 cells. Both EGCG and TF-3 strongly inhibited the proliferation of A431 and NIH3T3 cells more than the other theaflavins did. In cultured cells with pre-treatment of tea polyphenol, TF-3 was stronger than EGCG on the reduction of EGF receptor and PDGF receptor autophosphorylation induced by EGF and PDGF, respectively. Other theaflavins slightly reduced the autophosphorylation of the EGF and PDGF receptors; furthermore, TF-3 could reduce autophosphorylation of the EGF receptor (or PDGF receptor) even with co-treatment with EGF (or PDGF) and TF-3, but EGCG was inactive under these conditions. In addition, TF-3 was stronger than EGCG in blocking EGF binding to its receptor. These results suggest that not only the green tea polyphenol, EGCG, but also the black tea polyphenol, TF-3, have an antiproliferative activity on tumor cells, and the molecular mechanisms of antiproliferation may block the growth factor binding to its receptor and thus suppress mitogenic signal transduction. (+info)
Calcium recruitment in renal vasculature: NE effects on blood flow and cytosolic calcium concentration.
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This study provides new information about the relative importance of Ca2+ mobilization and entry in the renal vascular response to adrenoceptor activation. We measured renal blood flow (RBF) in Sprague-Dawley rats in vivo using electromagnetic flowmetry. We measured intracellular free Ca2+ concentration ([Ca2+]i) in isolated afferent arterioles utilizing ratiometric photometry of fura-2 fluorescence. Renal arterial injection of NE produced a transient decrease in RBF. The response was attenuated, in a dose-dependent manner, up to approximately 50% by nifedipine, an antagonist of L-type Ca2+ entry channels. Inhibition of Ca2+ mobilization by 3,4, 5-trimethoxybenzoic acid-8-(diethylamino)octyl ester (TMB-8) inhibited the renal vascular effects of NE in a dose-dependent manner, with maximal blockade of approximately 80%. No additional attenuation was observed when nifedipine and TMB-8 were administered together. In microdissected afferent arterioles, norepinephrine (NE; 10(-6) M) elicited an immediate square-shaped increase in [Ca2+]i, from 110 to 240 nM. This in vitro response was blocked by nifedipine (10(-6) M) and TMB-8 (10(-5) M) to a degree similar to that of the in vivo experiments. A nominally calcium-free solution blocked 80-90% of the [Ca2+]i response to NE. The increased [Ca2+]i elicited by depolarization with medium containing 50 mM KCl was totally blocked by nifedipine. In contrast, TMB-8 had no effect. Our results indicate that both Ca2+ entry and mobilization play important roles in the renal vascular Ca2+ and contractile response to adrenoceptor activation. The entry and mobilization mechanisms activated by NE may interact. That a calcium-free solution caused a larger inhibition of the NE effects on afferent arterioles than nifedipine suggests more than one Ca2+ entry pathway. (+info)
Gallates inhibit cytokine-induced nuclear translocation of NF-kappaB and expression of leukocyte adhesion molecules in vascular endothelial cells.
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Gallates (gallic acid esters) belong to the class of phenolic compounds, which are abundant in red wine. In this study, we show that gallates can inhibit cytokine-induced activation of nuclear factor kappaB (NF-kappaB) and thereby reduce expression of endothelial-leukocyte adhesion molecules in cultured human umbilical vein endothelial cells (HUVECs). Pretreatment of HUVECs with ethyl gallate (3 to 10 micromol/L) significantly suppressed interleukin-1alpha (IL-1alpha)- or tumor necrosis factor-alpha (TNF-alpha)- induced mRNA and cell-surface expression of vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and E-selectin, which was associated with reduced adhesion of leukocytes to HUVECs. Gel shift assays with the NF-kappaB consensus sequence showed the decreased densities of the shifted bands in gallate-treated HUVECs. Furthermore, gallate pretreatment inhibited cytokine-induced transcription of a fusion gene, which consisted of 4 repeats of the NF-kappaB consensus sequence and the luciferase reporter gene. Immunoblot analysis of nuclear extracts and whole-cell lysates demonstrated the decreased amounts of NF-kappaB p65 in nuclei but equal amounts of inhibitor-kappaBalpha (I-kappaBalpha) in whole-cell lysates of ethyl gallate-treated HUVECs. Incubation of the nuclear extracts from cytokine-activated HUVECs with ethyl gallate did not affect the NF-kappaB shifted bands induced by cytokines in gel shift assays. Taken together, these data demonstrate that ethyl gallate can inhibit cytokine-induced nuclear translocation of NF-kappaB p65 by way of a mechanism independent of I-kappaBalpha degradation and thereby suppress expression of VCAM-1, ICAM-1, and E-selectin, which was associated with reduced adhesion of leukocytes. These results in vitro demonstrate that gallates can exhibit anti-inflammatory properties by blocking activation of NF-kappaB and suggest that these natural compounds, abundant in red wine, may play important roles in the prevention of atherosclerosis and inflammatory responses in vivo. (+info)
8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate reduced [Ca2+]i elevation induced by histamine, serotonin, and glutamate in cultured calf basilar artery smooth muscle cells.
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AIM: To study the effects of 8-(N,N-diethylamino)-n-octyl-3,4,5- trimethoxybenzoate (TMB-8) on intracellular free calcium ([Ca2+]i) in cultured calf basilar artery smooth muscle cells. METHODS: [Ca2+]i was examined by a system of measurement of AR-CM-MIC, using Fura 2-AM as a fluorescent indicator. RESULTS: In the presence of extracellular Ca2+ 1.3 mmol.L-1, histamine (His), serotonin (5-HT), and sodium glutamate (Glu) markedly increased the [Ca2+]i which was attenuated by TMB-8. In Ca2+ free Hanks' solution containing egtazic acid 0.1 mmol.L-1, TMB-8 not only reduced the resting [Ca2+]i, but also inhibited the elevation of [Ca2+]i evoked by His and 5-HT. CONCLUSION: TMB-8 reduced the resting [Ca2+]i and attenuated His-, 5-HT-, and Glu-induced increases of [Ca2+]i in basilar artery smooth muscle cells. (+info)