Chun-Peng Liu

Effect of timosaponin A-III, from Anemarrhenae asphodeloides Bunge (Liliaceae), on calcium mobilization in vascular endothelial and smooth muscle cells and on vascular tension

The effects of timosaponin A-III (TA-III), from Rhizoma Anemarrhenae, on Ca(2+) mobilization in vascular endothelial cells and smooth muscle cells and on vascular tension have been explored. TA-III increased intracellular Ca(2+) concentrations ([Ca(2+)](i)) in endothelials cells at a concentration larger than 5 microM with an EC(50) of 15 microM, and increased [Ca(2+)](i) in smooth muscle cells at a concentration larger than 1 microM with an EC(50) of 8 microM. Within 5 min, the [Ca(2+)](i) signal was composed of a gradual rise, and the speed of rising depended on the concentration of TA-III. The [Ca(2+)](i) signal was abolished by removing extracellular Ca(2+) and was recovered after reintroduction of Ca(2+). The TA-III-induced [Ca(2+)](i) increases in smooth muscle cells were partly inhibited by 10 microM nifedipine or 50 microM La(3+), but was insensitive to 10 microM verapamil and diltiazem. TA-III (10-100 microM) inhibited 0.3 microM phenylephrine-induced vascular contraction, which was abolished by pretreatment with 100 microM N(omega)-nitro-L-arginine (L-NNA) or by denuding the aorta. TA-III also increased [Ca(2+)](i) in renal tubular cells with an EC(50) of 8 microM. Collectively, the results show for the first time that TA-III causes [Ca(2+)](i) increases in the vascular system. TA-III acted by causing Ca(2+) influx without releasing intracellular Ca(2+). TA-III induced relaxation of phenylephrine-induced vascular contraction via inducing release of nitric oxide from endothelial cells.

AM-404 elevates renal intracellular Ca2+, questioning its selectivity as a pharmacological tool for investigating the anandamide transporter

The effect of N-(4-hydroxyphenyl)-arachidonamide (AM-404), a drug commonly used to inhibit the anandamide transporter, on intracellular free Ca(2+) levels ([Ca(2+)](i)) was studied in Madin Darby canine kidney (MDCK) cells. [Ca(2+)](i) was measured using fura-2 as a Ca(2+) indicator. Between 2 and 40 microM, AM-404 increased [Ca(2+)](i) in a concentration-dependent fashion with an EC(50) value of 20 microM. Removal of extracellular Ca(2+) abolished the [Ca(2+)](i) signals. The [Ca(2+)](i) increase was nearly abrogated by 10 microM La(3+), but was insensitive to 50 microM Ni(2+) and 10 microM of nifedipine, nimodipine, nicardipine, and verapamil. At a concentration that did not increase [Ca(2+)](i), AM-404 (1 microM) did not alter the [Ca(2+)](i) increases induced by 10 microM ATP and 1 microM bradykinin. AM-404 (5 microM) also increased [Ca(2+)](i) in Chang liver cells, PC3 human prostate cancer cells, BFTC human bladder cancer cells, and MG63 human osteoblast-like cells. Together, this study shows for the first time that AM-404 at concentrations commonly used to inhibit the anandamide transporter in various systems induced an increase in [Ca(2+)](i) in different cell types. The [Ca(2+)](i) increase was solely due to extracellular Ca(2+) influx. Thus caution must be exercised in using AM-404 as a selective inhibitor of the anandamide transporter.

Gossypol, a component in cottonseed, induced increases in cytosolic Ca2+ levels in Chang liver cells

The effect of gossypol, a compound found in cottonseed, on intracellular free Ca2+ levels ([Ca2+](i)) in Chang liver cells were evaluated using fura-2 as a fluorescent Ca2+ indicator. Gossypol (0.2-5microM) increased [Ca2+](i) in a concentration-dependent manner with an EC(50) value of 1.5microM. The [Ca2+](i) response was composed of an initial rise and a slow decay to a sustained phase within 5min after drug application. Removal of extracellular Ca2+ markedly reduced the [Ca2+](i) signals by 80+/-2%. Preincubation with 0.1mM La3+ or 10microM nimodipine abolished the Ca2+ influx. Gossypol (5microM)-induced release of intracellular Ca2+ was reduced by 75% by pretreatment with 1microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+. Conversely, pretreatment with gossypol abolished thapsigargin-induced Ca2+ release. After pretreatment with 5microM gossypol in Ca2+-free medium for several min, addition of 3mM Ca2+ induced a [Ca2+](i) increase of a magnitude nine-fold greater than control. Gossypol (5microM)-induced Ca2+ release was not affected by inhibiting phospholipase C with 2microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Together, this study shows that gossypol induced significant [Ca2+](i) increases in Chang liver cells by releasing Ca2+ from intracellular pools in a phospholipase C-dissociated fashion and by causing La3+- and nimodipine-sensitive Ca2+ influx.

Effect of oleamide on Ca2+ signaling in human bladder cancer cells

Abstract The effect of oleamide, a sleep-inducing endogenous lipid in animal models, on intracellular free levels of Ca 2+ ([Ca 2+ ] i ) in non-excitable and excitable cells was examined by using fura-2 as a fluorescent dye. [Ca 2+ ] i in pheochromocytoma cells, renal tubular cells, osteoblast-like cells, and bladder cancer cells were increased on stimulation of 50 μM oleamide. The response in human bladder cancer cells (T24) was the greatest and was further explored. Oleamide (10–100 μM) increased [Ca 2+ ] i in a concentration-dependent fashion with an EC 50 of 50 μM. The [Ca 2+ ] i signal comprised an initial rise and a sustained plateau and was reduced by removing extracellular Ca 2+ by 85 ± 5%. After pre-treatment with 10–100 μM oleamide in Ca 2+ -free medium, addition of 3 mM Ca 2+ increased [Ca 2+ ] i in a manner dependent on the concentration of oleamide. The [Ca 2+ ] i increase induced by 50 μM oleamide was reduced by 100 μM La 3+ by 40%, but was not altered by 10 μM nifedipine, 10 μM verapamil, and 50 μM Ni 2+ . In Ca 2+ -free medium, pre-treatment with thapsigargin (1 μM), an endoplasmic reticulum Ca 2+ pump inhibitor, abolished 50 μM oleamide-induced [Ca 2+ ] i increases; conversely, pretreatment with 50 μM oleamide reduced 1 μM thapsigargin-induced [Ca 2+ ] i increases by 50 ± 3%. Suppression of the activity of phospholipase C with 2 μM U73122 failed to alter 50 μM oleamide-induced Ca 2+ release. Linoleamide (10–100 μM), another sleep-inducing lipid with a structure similar to that of oleamide, also induced an increase in [Ca 2+ ] i . Together, it was shown that oleamide induced significant [Ca 2+ ] i increases in cells by a phospholipase C-independent release of Ca 2+ from thapsigargin-sensitive stores and by inducing Ca 2+ entry.

Novel action of lignans isolated from Hernandia nymphaeifolia on Ca2+ signaling in renal tubular cells

The effect of five lignans, epi-aschantin, epi-magnolin, epi-yangambin, deoxypodophyllotoxin and yatein, isolated from Hernandia nymphaeifolia on Ca(2+) signaling in Madin-Darby canine kidney cells was examined using fura-2 as a Ca(2+) indicator. These lignans at concentrations between 10 and 100 microM increased [Ca(2+)](i) in a concentration-dependent manner. Removal of extracellular Ca(2+) abolished the Ca(2+) signals evoked by 50 microM of the lignans. La(3+)(50 microM) abolished the Ca(2+) signals induced by 100 microM of epi-aschantin, epi-magnolin and epi-yangambin, and 20 microM deoxypodophyllotoxin, but inhibited by 60% 50 microM yatein-induced responses. All five lignans (50-100 microM) inhibited by 42-65% thapsigargin-induced capacitative Ca(2+) entry, and inhibited by 23-61% thapsigargin-induced intracellular Ca(2+) release. Epi-yangambin (100 microM), epi-magnolin (100 microM), and epi-aschantin (100 microM) inhibited by 8-38% 10 microM ATP-induced Ca(2+) release. Trypan blue exclusion revealed that incubation with deoxypodophyllotoxin or yatein (but not the other lignans) decreased cell viability in a concentration-dependent manner. Together, the results suggest that, in renal tubular cells, these lignans exert multiple actions on Ca(2+) signaling. They caused Ca(2+) influx but reduced thapsigargin-induced capacitative Ca(2+) entry and also thapsigargin- and ATP-induced Ca(2+) release. Additionally, deoxypodophyllotoxin and yatein may be cytotoxic.

Effect of NPC-14686 (Fmoc-L-homophenylalanine) on intracellular Ca2+ levels in human hepatoma cells.

The effect of NPC-14686, a potential anti-inflammatory drug, on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in HA22/VGH human hepatoma cells was explored by using fura-2 as a fluorescent Ca(2+) indicator. NPC-14686 at concentrations above 10 microM increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 100 microM. The Ca(2+) signal was reduced by removing extracellular Ca(2+) or by 10 microM nifedipine and was not changed by verapamil or diltiazem. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+) abolished 200 microM NPC-14686-induced Ca(2+) release; and conversely pretreatment with NPC-14686 abolished thapsigargin-induced Ca(2+) release. The Ca(2+) release induced by 200 microM NPC-14686 was not changed by inhibiting phospholipase C with 2 microM U73122. Together, the results suggest that in human hepatoma cells, NPC-14686 induced a [Ca(2+)](i) increase by causing store Ca(2+) release from the endoplasmic reticulum in an phospholipase C-independent manner, and by inducing nifedipine-sensitive Ca(2+) influx.

Effect of nordihydroguaiaretic acid on intracellular Ca2+ concentrations in C6 glioma cells

The effect of nordihydroguaiaretic acid (NDGA) on Ca(2+) signaling in C6 glioma cells has been investigated. NDGA (5-100 microM) increased [Ca(2+)]i concentration-dependently. The [Ca(2+)]i increase comprised an initial rise and an elevated phase over a time period of 4 min. Removal of extracellular Ca(2+) reduced NDGA-induced [Ca(2+)]i signals by 52+/-2%. After incubation of cells with NDGA in Ca(2+)-free medium for 4 min, addition of 3 mM CaCl2 induced a concentration-dependent increase in [Ca(2+)]i. NDGA (100 microM)-induced [Ca(2+)]i increases in Ca(2+)-containing medium was not changed by pretreatment with 10 microM nifedipine or verapamil. In Ca(2+)-free medium, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (1 microM) abolished 100 microM NDGA-induced [Ca(2+)]i increases. Inhibition of phospholipase C with 2 microM U73122 had little effect on 100 microM NDGA-induced Ca(2+) release. Several other lipoxygenase inhibitors had no effect on basal [Ca(2+)]i. Collectively, the results suggest that NDGA increased [Ca(2+)]i in glioma cells in a lipoxygenase-independent manner, by releasing Ca(2+) from the endoplasmic reticulum in a manner independent of phospholipase C activity and by causing Ca(2+) influx.

Effects of the antianginal drug fendiline on Ca2+ movement in hepatoma cells

This study investigated the effect of the anti-anginal drug, fendiline, on intracellular free Ca2+ levels ([Ca2+]i)inHA/ 22 human hepatoma cells by using fura-2 as a fluorescent Ca2+ dye. Fendiline (1-100 μM) increased [Ca2+]i with an EC50 of 25 μM. Removal of extracellular Ca2+ reduced the [Ca2+]i signals by 51-5%. Fendiline (10 μM)-induced Ca2+release was abolished by pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Inhibition of phospholipase C with 2 μM 1-(6-((17β 3 methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H pyrrole-2,5-dione (U73122) did not alter 10 μM fendilineinducedCa2+ release.Severalothercalmodulinantagonists, such as phenoxybenzamine (100-200 μM), trifluoperazine (5-50 μM),andfluphenazine N-chloroethane(2-100 μM), hadno effect on[Ca2+]i. Together, it wasfound that fendiline increased [Ca2+]i in human hepatoma cells by discharging Ca2+ from the endoplasmic reticulum in an inositol 1,4,5 trisphosphate-independent manner and by inducing Ca2+entry. This effect of fendiline does not appear to be via antagonism of calmodulin.