Effect of the antidepressant maprotiline on calcium movement and the viability of renal tubular cells

In Madin-Darby canine kidney (MDCK) cells, the effect of maprotiline, an antidepressant, on intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured using fura-2. Maprotiline (>2.5 µM) caused a rapid rise of [Ca²⁺]_i in a concentration-dependent manner (EC₅₀ 200 µM). Maprotiline-induced [Ca²⁺]_i rise was reduced by removal of extracellular Ca²⁺ or by addition of La³⁺, but was not altered by voltage-gated Ca²⁺-channel blockers. Maprotiline-induced Mn²⁺ influx-associated fura-2 fluorescence quench directly suggests that maprotiline caused Ca²⁺ influx. In Ca²⁺-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase, caused a monophasic [Ca²⁺]_i rise, after which the increasing effect of maprotiline on [Ca²⁺]_i was nearly abolished; also, pretreatment with maprotiline reduced a portion of thapsigargin-induced [Ca²⁺]_i rise. U73122, an inhibitor of phospholipase C, abolished [Ca²⁺]_i rise induced by ATP (but not by maprotiline). Overnight incubation with 1–10 µM maprotiline enhanced cell viability, but 20–50 µM maprotiline decreased it. These findings suggest that maprotiline rapidly increases [Ca²⁺]_i in renal tubular cells by stimulating both extracellular Ca²⁺ influx and intracellular Ca²⁺ release, and may modulate cell proliferation in a concentration-dependent manner.     

Effect of calmidazolium on [Ca2+]i and viability in human hepatoma cells

The effect of calmidazolium on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability has not been explored in human hepatoma cells. This study examined whether calmidazolium altered [Ca²⁺]_i and caused cell death in HA59T cells. [Ca²⁺]_i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Calmidazolium at concentrations ≥1 μM increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 1.5 μM. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Calmidazolium induced Mn²⁺ quench of fura-2 fluorescence implicating Ca²⁺ influx. The Ca²⁺ influx was insensitive to L-type Ca²⁺ entry blockers, but was inhibited partly by enhancing or inhibiting protein kinase C activity. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), calmidazolium-induced [Ca²⁺]_i rises were largely inhibited; and conversely, calmidazolium pretreatment totally suppressed thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change calmidazolium-induced [Ca²⁺]_i rises. At concentrations between 1 and 15 μM, calmidazolium induced apoptosis-mediated cell death. Collectively, in HA59T hepatoma cells, calmidazolium induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca²⁺ influx via protein kinase C-regulated Ca²⁺ entry pathway. Calmidazolium caused cytotoxicity via apoptosis.     

2-Benzyloxybenzaldehyde inhibits formyl peptide-stimulated increase in intracellular Ca2+ in neutrophils mainly by blocking Ca2+ entry

2-Benzyloxybenzaldehyde (CCY1a) inhibited the formyl-Met-Leu-Phe (fMLP)-induced elevation of cytosolic [Ca²⁺] ([Ca²⁺]_i) in rat neutrophils. The late plateau phase, but not the initial Ca²⁺ spike, of the fMLP-induced [Ca²⁺]_i change was inhibited by CCY1a. In the absence of external Ca²⁺, CCY1a had no appreciable effect on either the fMLP- or cyclopiazonic acid (CPA)-induced [Ca²⁺]_i elevation. CCY1a failed to inhibit [Ca²⁺]_i changes induced by N-ethylmaleimide, GEA3162, ionomycin or sphingosine, but slightly inhibited the Ca²⁺ signals elicited by ATP or interleukin-8 (IL-8). In a classical Ca²⁺ readdition protocol, addition of CCY1a after cell activation strongly inhibited the [Ca²⁺]_i response to fMLP, whilst that to CPA was only slightly reduced. CCY1a nearly abrogated the fMLP-stimulated Mn²⁺ influx but was less effective on the CPA-induced response. CCY1a attenuated the levels of tyrosine-phosphorylated bands in the 70–85 kDa molecular mass range. CCY1a had no effect on the basal [Ca²⁺]_i level, the pharmacologically isolated plasma membrane Ca²⁺-ATPase activity or on the mitochondrial membrane potential. Thus, CCY1a blocks fMLP-induced Ca²⁺ entry into neutrophils probably by blocking the relevant Ca²⁺ channel directly or, alternatively, indirectly through the attenuation of tyrosine phosphorylation of some cellular proteins.     

Mechanisms of AM404-induced [Ca]i rise and death in human osteosarcoma cells

The effect of N-(4-hydroxyphenyl) arachidonoyl-ethanolamide (AM404), a drug commonly used to inhibit the anandamide transporter, on intracellular free Ca²⁺ levels ([Ca²⁺]_i) and viability was studied in human MG63 osteosarcoma cells using the fluorescent dyes fura-2 and WST-1, respectively. AM404 at concentrations ≥5 μM increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 60 μM. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. AM404 induced Mn²⁺ quench of fura-2 fluorescence implicating Ca²⁺ influx. The Ca²⁺ influx was sensitive to La³⁺, Ni²⁺, nifedipine and verapamil. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), AM404-induced [Ca²⁺]_i rise was abolished; and conversely, AM404 pretreatment totally inhibited thapsigargin-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 did not change AM404-induced [Ca²⁺]_i rise. At concentrations between 10 and 200 μM, AM404 killed cells in a concentration-dependent manner presumably by inducing apoptotic cell death. The cytotoxic effect of 50 μM AM404 was partly reversed by prechelating cytosolic Ca²⁺ with BAPTA/AM. Collectively, in MG63 cells, AM404 induced [Ca²⁺]_i rise by causing Ca²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca²⁺ influx via L-type Ca²⁺ channels. AM404 caused cytotoxicity which was possibly mediated by apoptosis.     

Mechanisms of carvedilol-induced [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> rises and death in human hepatoma cells

The effect of the cardiovascular drug carvedilol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability has not been explored in human hepatoma cells. This study examined whether carvedilol altered [Ca²⁺]_i and caused cell death in HA59T cells. [Ca²⁺]_i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Carvedilol at concentrations ≥1 μM increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 20 μM. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Carvedilol induced Mn²⁺ quench of fura-2 fluorescence, implicating Ca²⁺ influx. The Ca²⁺ influx was sensitive to La³⁺, econazole, nifedipine, and SKF96365. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), carvedilol-induced [Ca²⁺]_i rises were abolished; and conversely, carvedilol pretreatment inhibited a major part of thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change carvedilol-induced [Ca²⁺]_i rises. At concentrations between 1 and 50 μM, carvedilol killed cells in a concentration-dependent manner. The cytotoxic effect of 1 μM (but not 30 μM) carvedilol was fully reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Apoptosis was induced by 30 (but not 1) μM carvedilol. Collectively, in HA59T hepatoma cells, carvedilol induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum in a phospholipase-C-independent manner and Ca²⁺ influx via store-operated Ca²⁺ channels. Carvedilol-caused cytotoxicity was mediated by Ca²⁺ and apoptosis in a concentration-dependent manner.     

In vitro vasodilator mechanisms of the indole alkaloids rhynchophylline and isorhynchophylline,isolated from the hook of<Emphasis Type="Italic"> Uncaria rhynchophylla</Emphasis> (Miquel)

Rhynchophylline (Rhy) and isorhynchophylline (Isorhy), indole alkaloids from Uncaria hooks, reportedly exert hypotensive and vasodilatory effects, but the mechanism of action is unclear. We therefore investigated the relaxant effects of these two isomeric alkaloids in rat arteries in vitro, in particular in respect of the various functional Ca²⁺ pathways. Both Rhy and Isorhy relaxed aortic rings precontracted with phenylephrine (PE, 1 µM) in a dose-dependent manner (3–300 µM). Removal of endothelium and preincubation with L-NAME (300 µM) slightly inhibited but did not prevent the relaxant response. These results indicate that Rhy and Isorhy act largely in an endothelium-independent manner. Unlike nicardipine, both alkaloids not only inhibited the contraction induced by 60 mM KCl (IC₅₀ 20–30 µM), but also that induced by PE and U46619, albeit to a lesser extent (IC₅₀ 100 and 200 µM, respectively). These results suggest that Rhy and Isorhy may act via multiple Ca²⁺ pathways. In contrast to their inhibitory effects on KCl-induced and receptor-mediated contractions, where both isomers were comparably potent, Rhy was more potent than Isorhy at higher concentrations (>100 µM) in inhibiting both caffeine (25 mM)- and cyclopiazonic acid (CPA, 30 µM)-induced contractions. Similar results observed with caffeine in Ca²⁺-containing medium were also observed in Ca²⁺-free medium. However, 0.1–0.3 µM nicardipine (which completely inhibited KCl-induced contraction) had no significant inhibitory effect on CPA-induced contractions. Taken together, these results indicate discrimination between these two isomers with respect to Ca²⁺-induced Ca²⁺ release and non-L-type Ca²⁺ channel, but not for IP₃-induced Ca²⁺ release and L-type Ca²⁺ channels. Similar relaxant responses to KCl- and caffeine-induced contractions were seen when these two alkaloids were tested on the smaller mesenteric and renal arteries. In conclusion, the vasodilatory effects of Rhy and Isorhy are largely endothelium independent and are mediated by L-type Ca²⁺ channels. At higher concentrations, they also affect other Ca²⁺-handling pathways, although to a lesser extent. While there is no discrimination between the two isomers with respect to the contraction induced by KCl or agonists (PE and U46619), differential effects between Rhy and Isorhy were seen on caffeine- and CPA-induced contractions.     

The phospholipase C inhibitor U-73122 inhibits Ca2+ release from the intracellular sarcoplasmic reticulum Ca2+ store by inhibiting Ca2+ pumps in smooth muscle

Background and purpose:

The sarcoplasmic reticulum (SR) releases Ca²⁺ via inositol 1,4,5-trisphosphate receptors (IP₃R) in response to IP₃-generating agonists. Ca²⁺ release subsequently propagates as Ca²⁺ waves. To clarify the role of IP₃ production in wave generation, the contribution of a key enzyme in the production of IP₃ was examined using a phosphoinositide-specific phospholipase C (PI-PLC) inhibitor, U-73122.

Experimental approach:

Single colonic myocytes were voltage-clamped in whole-cell configuration and cytosolic Ca²⁺ concentration ([Ca²⁺]_cyto) measured using fluo-3. SR Ca²⁺ release was evoked either by activation of IP₃Rs (by carbachol or photolysis of caged IP₃) or ryanodine receptors (RyRs; by caffeine).

Key results:

U-73122 inhibited carbachol-evoked [Ca²⁺]_cyto transients. The drug also inhibited [Ca²⁺]_cyto increases, evoked by direct IP₃R activation (by photolysis of caged IP₃) and RyR activation (by caffeine), which do not require PI-PLC activation. U-73122 also increased steady-state [Ca²⁺]_cyto and slowed the rate of Ca²⁺ removal from the cytoplasm. An inactive analogue of U-73122, U-73343, was without effect on either IP₃R- or RyR-mediated Ca²⁺ release.

Conclusions and implications:

U-73122 inhibited carbachol-evoked [Ca²⁺]_cyto increases. However, the drug also reduced Ca²⁺ release when evoked by direct activation of IP₃R or RyR, slowed Ca²⁺ removal and increased steady-state [Ca²⁺]_cyto. These results suggest U-73122 reduces IP₃-evoked Ca²⁺ transients by inhibiting the SR Ca²⁺ pump to deplete the SR of Ca²⁺ rather than by inhibiting PI-PLC.     

Functional and morphological properties of pericytes in suburothelial venules of the mouse bladder

Background and Purpose

In suburothelial venules of rat bladder, pericytes (perivascular cells) develop spontaneous Ca²⁺ transients, which may drive the smooth muscle wall to generate spontaneous venular constrictions. We aimed to further explore the morphological and functional characteristics of pericytes in the mouse bladder.

Experimental Approach

The morphological features of pericytes were investigated by electron microscopy and fluorescence immunohistochemistry. Changes in diameters of suburothelial venules were measured using video microscopy, while intracellular Ca²⁺ dynamics were visualized using Fluo-4 fluorescence Ca²⁺ imaging.

Key Results

A network of α-smooth muscle actin immunoreactive pericytes surrounded venules in the mouse bladder suburothelium. Scanning electron microscopy revealed that this network of stellate-shaped pericytes covered the venules, while transmission electron microscopy demonstrated that the venular wall consisted of endothelium and adjacent pericytes, lacking an intermediate smooth muscle layer. Pericytes exhibited spontaneous Ca²⁺ transients, which were accompanied by phasic venular constrictions. Nicardipine (1 μM) disrupted the synchrony of spontaneous Ca²⁺ transients in pericytes and reduced their associated constrictions. Residual asynchronous Ca²⁺ transients were suppressed by cyclopiazonic acid (10 μM), 2-aminoethoxydiphenyl borate (10 μM), U-73122 (1 μM), oligomycin (1 μM) and {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 (10 μM), but unaffected by ryanodine (100 μM) or YM-244769 (1 μM), suggesting that pericyte Ca²⁺ transients rely on Ca²⁺ release from the endoplasmic reticulum via the InsP₃ receptor and also require Ca²⁺ influx through store-operated Ca²⁺ channels.

Conclusions and Implications

The pericytes in mouse bladder can generate spontaneous Ca²⁺ transients and contractions, and thus have a fundamental role in promoting spontaneous constrictions of suburothelial venules.     

Oxidized Low-density Lipoprotein- and Lysophosphatidylcholine-induced Ca2+ Mobilization in Human Endothelial Cells

The effects of oxidized low-density lipoprotein (OxLDL) and its major lipid constituent lysophosphatidylcholine (LPC) on Ca²⁺ entry were investigated in cultured human umbilical endothelial cells (HUVECs) using fura-2 fluorescence and patch-clamp methods. OxLDL or LPC increased intracellular Ca²⁺ concentration ([Ca²⁺]_i), and the increase of [Ca²⁺]_i by OxLDL or by LPC was inhibited by La³⁺ or heparin. LPC failed to increase [Ca²⁺]_i in the presence of an antioxidant tempol. In addition, store-operated Ca²⁺ entry (SOC), which was evoked by intracellular Ca²⁺ store depletion in Ca²⁺-free solution using the sarcoplasmic reticulum Ca²⁺ pump blocker, 2, 5-di-t-butyl-1, 4-benzohydroquinone (BHQ), was further enhanced by OxLDL or by LPC. Increased SOC by OxLDL or by LPC was inhibited by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. In voltage-clamped cells, OxLDL or LPC increased [Ca²⁺]_i and simultaneously activated non-selective cation (NSC) currents. LPC-induced NSC currents were inhibited by 2-APB, La³⁺ or {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, and NSC currents were not activated by LPC in the presence of tempol. Furthermore, in voltage-clamped HUVECs, OxLDL enhanced SOC and evoked outward currents simultaneously. Clamping intracellular Ca²⁺ to 1 µM activated large-conductance Ca²⁺-activated K⁺ (BK_Ca) current spontaneously, and this activated BK_Ca current was further enhanced by OxLDL or by LPC. From these results, we concluded that OxLDL or its main component LPC activates Ca²⁺-permeable Ca²⁺-activated NSC current and BK_Ca current simultaneously, thereby increasing SOC.     

KB-R7943, an inhibitor of the reverse Na+ /Ca2+ exchanger, blocks N-methyl-D-aspartate receptor and inhibits mitochondrial complex I

BACKGROUND AND PURPOSE

An isothiourea derivative (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methane sulfonate (KB-R7943), a widely used inhibitor of the reverse Na⁺/Ca²⁺ exchanger (NCX_rev), was instrumental in establishing the role of NCX_rev in glutamate-induced Ca²⁺ deregulation in neurons. Here, the effects of KB-R7943 on N-methyl-D-aspartate (NMDA) receptors and mitochondrial complex I were tested.

EXPERIMENTAL APPROACH

Fluorescence microscopy, electrophysiological patch-clamp techniques and cellular respirometry with Seahorse XF24 analyzer were used with cultured hippocampal neurons; membrane potential imaging, respirometry and Ca²⁺ flux measurements were made in isolated rat brain mitochondria.

KEY RESULTS

KB-R7943 inhibited NCX_rev with IC₅₀= 5.7 ± 2.1 µM, blocked NMDAR-mediated ion currents, and inhibited NMDA-induced increase in cytosolic Ca²⁺ with IC₅₀= 13.4 ± 3.6 µM but accelerated calcium deregulation and mitochondrial depolarization in glutamate-treated neurons. KB-R7943 depolarized mitochondria in a Ca²⁺-independent manner. Stimulation of NMDA receptors caused NAD(P)H oxidation that was coupled or uncoupled from ATP synthesis depending on the presence of Ca²⁺ in the bath solution. KB-R7943, or rotenone, increased NAD(P)H autofluorescence under resting conditions and suppressed NAD(P)H oxidation following glutamate application. KB-R7943 inhibited 2,4-dinitrophenol-stimulated respiration of cultured neurons with IC₅₀= 11.4 ± 2.4 µM. With isolated brain mitochondria, KB-R7943 inhibited respiration, depolarized organelles and suppressed Ca²⁺ uptake when mitochondria oxidized complex I substrates but was ineffective when mitochondria were supplied with succinate, a complex II substrate.

CONCLUSIONS AND IMPLICATIONS

KB-R7943, in addition to NCX_rev, blocked NMDA receptors in cultured hippocampal neurons and inhibited complex I in the mitochondrial respiratory chain. These findings are critical for the correct interpretation of experimental results obtained with KB-R7943 and a better understanding of its neuroprotective action.     

Effects of SEA0400 and KB-R7943 on Na+/Ca2+ exchange current and L-type Ca2+ current in canine ventricular cardiomyocytes

SEA0400 and KB-R7943 are compounds synthesised to block transsarcolemmal Na⁺/Ca²⁺ exchange current (I_Na/Ca); however, they Have also been shown to inhibit L-type Ca²⁺ current (I_Ca). The potential value of these compounds depends critically on their relative selectivity for I_Na/Ca over I_Ca. In the present work, therefore, the concentration-dependent effects of SEA0400 and KB-R7943 on I_Na/Ca and I_Ca were studied and compared in canine ventricular cardiomyocytes using the whole-cell configuration of the patch clamp technique. SEA0400 and KB-R7943 decreased I_Na/Ca in a concentration-dependent manner, having EC₅₀ values of 111±43 nM and 3.35±0.82 M, when suppressing inward currents, while the respective EC₅₀ values were estimated at 108±18 nM and 4.74±0.69 M in the case of outward current block. SEA0400 and KB-R7943 also blocked I_Ca, having comparable EC₅₀ values (3.6 M and 3.2 M, respectively). At higher concentrations (10 M) both drugs accelerated inactivation of I_Ca, retarded recovery from inactivation and shifted the voltage dependence of inactivation towards more negative voltages. The voltage dependence of activation was slightly modified by SEA0400, but not by KB-R7943. Based on the relatively good selectivity of submicromolar concentrations of SEA0400—but not KB-R7943—for I_Na/Ca over I_Ca, SEA0400 appears to be a suitable tool to study the role of I_Na/Ca in Ca²⁺ handling in canine cardiac cells. At concentrations higher than 1 M, however, I_Ca is progressively suppressed by the compound.     

Effect of bisphenol A on Ca2+ fluxes and viability in Madin-Darby canine renal tubular cells

The effect of the environmental contaminant, bisphenol A, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in Madin-Darby canine kidney (MDCK) cells is unclear. This study explored whether bisphenol A changed basal [Ca²⁺]_i levels in suspended MDCK cells by using fura-2 as a Ca²⁺-sensitive fluorescent dye. Bisphenol A, at concentrations between 50 and 300 µM, increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced, partly, by removing extracellular Ca²⁺. Bisphenol A induced Mn²⁺ influx, leading to quenching of fura-2 fluorescence, suggesting Ca²⁺ influx. This Ca²⁺ influx was inhibited by phospholipase A2 inhibitor aristolochic acid, store-operated Ca²⁺ channel blockers nifedipine and SK&F96365, and protein kinase C inhibitor GF109203X. In Ca²⁺-free medium, pretreatment with the mitochondrial uncoupler, carbonylcyanide m-chlorophenylhydrazone (CCCP), and the endoplasmic reticulum Ca²⁺ pump inhibitors, thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ), inhibited bisphenol A–induced Ca²⁺ release. Conversely, pretreatment with bisphenol A abolished thapsigargin (or BHQ)- and CCCP-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished bisphenol-induced [Ca²⁺]_i rise. Bisphenol A caused a concentration-dependent decrease in cell viability via apoptosis in a Ca²⁺-independent manner. Collectively, in MDCK cells, bisphenol A induced [Ca²⁺]_i rises by causing phospholipase C–dependent Ca²⁺ release from the endoplasmic reticulum and mitochondria and Ca²⁺ influx via phospholipase A2–, protein kinase C–sensitive, store-operated Ca²⁺ channels.     

Effect of diallyl disulfide on Ca movement and viability in PC3 human prostate cancer cells

The effect of diallyl disulfide (DADS) on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells is unclear. This study explored whether DADS changed [Ca²⁺]_i in PC3 cells by using fura-2. DADS at 50-1000 μM increased [Ca²⁺]_i in a concentration-dependent manner. The signal was reduced by removing Ca²⁺. DADS-induced Ca²⁺ influx was not inhibited by nifedipine, econazole, SK&F96365, and protein kinase C modulators; but was inhibited by aristolochic acid. In Ca²⁺-free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) nearly abolished DADS-induced [Ca²⁺]_i rise. Incubation with DADS inhibited thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 did not alter DADS-induced [Ca²⁺]_i rise. At 500-1000 μM, DADS killed cells in a concentration-dependent manner. The cytotoxic effect of DADS was partly reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Propidium iodide staining suggests that DADS (500 μM) induced apoptosis in a Ca²⁺-independent manner. Annexin V/PI staining further shows that 10 μM and 500 μM DADS both evoked apoptosis. DADS also increased reactive oxygen species (ROS) production. Collectively, in PC3 cells, DADS induced [Ca²⁺]_i rise probably by causing phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via phospholipase A₂-sensitive channels. DADS induced Ca²⁺-dependent cell death, ROS production, and Ca²⁺-independent apoptosis.     

The vasodilator mechanism of sulfur dioxide on isolated aortic rings of rats: Involvement of the K and Ca channels

The present study was designed to investigate vasodilator effect of endogenous gaseous sulfur dioxide (SO₂) and roles of Ca²⁺ channels and K⁺ channels in relaxations of SO₂ in isolated rat aortic rings. Isolated rat aortic rings were perfused in organ baths and changes in isometric tension were recorded. The results showed that: (1) SO₂ could relax isolated aortic rings contracted by norepinephrine in a dose-dependent manner (EC₅₀, 1247.38 ± 98.32 µM). The vasorelaxant effect of SO₂ at basal (110.34 ± 35.22 µM) and low concentrations (< 450 µM) was endothelium-dependent, while it was endothelium-independent at high concentrations (> 500 µM). (2) The vasorelaxation of 1500 µM SO₂ on both endothelium-intact and endothelium-denuded aortic rings was partially inhibited by nifedipine, an L-type calcium-channel blocker. (3) The vasoconstriction responses induced by CaCl₂ were inhibited by 1500 µM SO₂ on both endothelium-intact and endothelium-denuded aortic rings. (4) The initial fast vasoconstriction induced by intracellular Ca²⁺ release was enhanced by 1500 µM SO₂, but the sustained vasoconstriction evoked by extracellular Ca²⁺ influx was inhibited by 1500 µM SO₂. (5) Pretreated by 1500 µM SO₂, the vasoconstriction responses induced by norepinephrine or KCl were enhanced at low concentrations and inhibited at high concentrations. (6) The SO₂-induced vasorelaxation was partially inhibited by tetraethylammonium (TEA) and glibenclamide for both endothelium-intact and endothelium-denuded rings. For the endothelium-intact rings, the vasorelaxant effects induced by 30 and 300 µM SO₂ were partially inhibited by iberiotoxin. These results led to the conclusions that endogenous gaseous SO₂ could cause vasorelaxation on rat aortic rings in a dose-dependent manner. The vasorelaxant effects of SO₂ at basal and low concentrations were endothelium-dependent, which might be partly related to big-conductance Ca²⁺-activated K⁺ (BK_Ca) channel. The mechanism of SO₂-induced vasorelaxation at high concentrations was shown to be endothelium-independent, which might be related to ATP-sensitive K⁺ (K_ATP) channel and L-type calcium-channel as well as possible alterations in Ca-influx and release pathways.     

Epigallocatechin-3-gallate increases intracellular [Ca<Superscript>2+</Superscript>] in U87 cells mainly by influx of extracellular Ca<Superscript>2+</Superscript> and partly by release of intracellular stores

Green tea has been receiving considerable attention as a possible preventive agent against cancer and cardiovascular disease. Epigallocatechin-3-gallate (EGCG) is a major polyphenol component of green tea. Using digital calcium imaging and an assay for [³H]-inositol phosphates, we determined whether EGCG increases intracellular [Ca²⁺] ([Ca²⁺]_i) in non-excitable human astrocytoma U87 cells. EGCG induced concentration-dependent increases in [Ca²⁺]_i. The EGCG-induced [Ca²⁺]_i increases were reduced to 20.9% of control by removal of extracellular Ca²⁺. The increases were also inhibited markedly by treatment with the non-specific Ca²⁺ channel inhibitors cobalt (3 mM) for 3 min and lanthanum (1 mM) for 5 min. The increases were not significantly inhibited by treatment for 10 min with the L-type Ca²⁺ channel blocker nifedipine (100 nM). Treatment with the inhibitor of endoplasmic reticulum Ca²⁺-ATPase thapsigargin (1 µM) also significantly inhibited the EGCG-induced [Ca²⁺]_i increases. Treatment for 15 min with the phospholipase C (PLC) inhibitor neomycin (300 µM) attenuated the increases significantly, while the tyrosine kinase inhibitor genistein (30 µM) had no effect. EGCG increased [³H]-inositol phosphates formation via PLC activation. Treatment for 10 min with mefenamic acid (100 µM) and flufenamic acid (100 µM), derivatives of diphenylamine-2-carboxylate, blocked the EGCG-induced [Ca²⁺]_i increase in non-treated and thapsigargin-treated cells but indomethacin (100 µM) did not affect the increases. Collectively, these data suggest that EGCG increases [Ca²⁺]_i in non-excitable U87 cells mainly by eliciting influx of extracellular Ca²⁺ and partly by mobilizing intracellular Ca²⁺ stores by PLC activation. The EGCG-induced [Ca²⁺]_i influx is mediated mainly through channels sensitive to diphenylamine-2-carboxylate derivatives.     

Emodin inhibits ATP-induced IL-1β secretion,ROS production and phagocytosis attenuation in rat peritoneal macrophages via antagonizing P2X7 receptor

Context: Previous in vitro studies have demonstrated that emodin (1,3,8-trihydroxy-6-methyl-anthraquinone), an anthraquinone derivative from the rhizome of Rheum palmatum L., can inhibit the activation of P2X₇ receptors (P2X₇R) as a potential antagonist. However, the effects of emodin on P2X₇R-related inflammatory processes remain unclear.

Objective: This study aimed to investigate the effects of emodin on different inflammation responses of macrophages induced by ATP, the natural ligand of P2X₇R.

Materials and methods: Rat peritoneal macrophages were treated with millimolar ATP and emodin (0.1, 0.3,?1,?3,?10?µM) or brilliant blue G (BBG, 0.1,?1,?10?µM). Cytosolic Ca²⁺ concentration ([Ca²⁺]_c) was detected by fluorescent Ca²⁺ imaging. Interleukin-1β (IL-1β) release was measured by rat IL-1β ELISA kits. Reactive oxygen species (ROS) generation was examined by dihydroethidium (DHE) fluorescent staining. Phagocytic activity was tested by neutral red uptake assay.

Results: We found that the [Ca²⁺]_c increase evoked by ATP (5?mM) was inhibited by emodin, in a dose-dependent manner with IC₅₀ of 0.5?μM. Furthermore, emodin reduced the IL-1β release induced by ATP (2?mM) in lipopolysaccharide (LPS)-activated macrophages, with an IC₅₀ of 1.6?μM. Emodin also strongly suppressed the ROS production and phagocytosis attenuation triggered by ATP (1?mM), with IC₅₀ values of 1?μM and 0.7?μM, respectively. Besides, BBG, a specific antagonist of P2X₇R, exhibited similar suppressive effects on these inflammation responses.

Conclusion: These results showed the inhibitory effects of emodin on ATP-induced [Ca²⁺]_c increase, IL-1β release, ROS production and phagocytosis attenuation in rat peritoneal macrophages, by inhibiting the activation of P2X₇R.     

Effect of thymol on Ca homeostasis and viability in human glioblastoma cells

The effect of the natural essential oil thymol on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in human glioblastoma cells was examined. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Thymol at concentrations of 400–1000 μM induced a [Ca²⁺]_i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca²⁺. Thymol-induced Ca²⁺ signal was not altered by nifedipine, econazole, SK&F96365, and protein kinase C activator phorbol myristate acetate (PMA), but was inhibited by the protein kinase C inhibitor GF109203X. When extracellular Ca²⁺ was removed, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished thymol-induced [Ca²⁺]_i rise. Incubation with thymol also abolished thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished thymol-induced [Ca²⁺]_i rise. At concentrations of 200–800 μM, thymol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that thymol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. Collectively, in human glioblastoma cells, thymol induced a [Ca²⁺]_i rise by inducing phospholipase C- and protein kinase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via non store-operated Ca²⁺ channels. Thymol induced cell death that may involve apoptosis.     

Octyl Gallate Inhibits ATP-induced Intracellular Calcium Increase in PC12 Cells by Inhibiting Multiple Pathways

Phenolic compounds affect intracellular free Ca²⁺ concentration ([Ca²⁺]_i) signaling. The study examined whether the simple phenolic compound octyl gallate affects ATP-induced Ca²⁺ signaling in PC12 cells using fura-2-based digital Ca²⁺ imaging and whole-cell patch clamping. Treatment with ATP (100 µM) for 90 s induced increases in [Ca²⁺]_i in PC12 cells. Pretreatment with octyl gallate (100 nM to 20 µM) for 10 min inhibited the ATP-induced [Ca²⁺]_i response in a concentration-dependent manner (IC₅₀=2.84 µM). Treatment with octyl gallate (3 µM) for 10 min significantly inhibited the ATP-induced response following the removal of extracellular Ca²⁺ with nominally Ca²⁺-free HEPES HBSS or depletion of intracellular Ca²⁺ stores with thapsigargin (1 µM). Treatment for 10 min with the L-type Ca²⁺ channel antagonist nimodipine (1 µM) significantly inhibited the ATP-induced [Ca²⁺]_i increase, and treatment with octyl gallate further inhibited the ATP-induced response. Treatment with octyl gallate significantly inhibited the [Ca²⁺]_i increase induced by 50 mM KCl. Pretreatment with protein kinase C inhibitors staurosporin (100 nM) and GF109203X (300 nM), or the tyrosine kinase inhibitor genistein (50 µM) did not significantly affect the inhibitory effects of octyl gallate on the ATP-induced response. Treatment with octyl gallate markedly inhibited the ATP-induced currents. Therefore, we conclude that octyl gallate inhibits ATP-induced [Ca²⁺]_i increase in PC12 cells by inhibiting both non-selective P2X receptor-mediated influx of Ca²⁺ from extracellular space and P2Y receptor-induced release of Ca²⁺ from intracellular stores in protein kinase-independent manner. In addition, octyl gallate inhibits the ATP-induced Ca²⁺ responses by inhibiting the secondary activation of voltage-gated Ca²⁺ channels.     

Evidence for a distinct Ca2+ antagonist receptor for the novel benzothiazinone compound HOE 166

Summary The pharmacological and binding properties of the novel enantiomerically pure benzothiazinone (R)-(+)-3, 4-dihydro-2-isopropyl-4-methyl-2-[2-[4-[4-[2-(3,4,5-trimethoxyphenyl)-ethyl]-piperazinyl]-butoxyl-phenyl]-2H-1, 4-benzothiazine-3-one dihydrochloride (HOE 166), are described. HOE 166 stereoselectively inhibited KCl^– but not noradrenaline-induced contractions of guinea-pig pulmonary arteries, rabbit aorta, rat mesenteric artery preparations and k-strophantin-induced enhancement of guinea-pig papillary muscle contraction in a dose-dependent manner. KCl-induced smooth muscle contraction was inhibited by HOE 166 with IC₅₀-values of 70 nM (5–11 times less potent than nifedipine, 2–16 times more potent than verapamil), the respective S-(–)-enantiomer being 10-fold less potent. HOE 166 decreased the upstroke velocity of the slow action potential in partially depolarized guinea-pig papillary muscle at similar concentrations than nifedipine. To investigate possible interactions with the calcium channel, HOE 166 and its S-(–)-enantiomer were characterized by radioligand binding studies in heart, brain and skeletal muscle transverse-tubule membranes. HOE 166 was a 4–15 times more potent inhibitor of reversible (+)-[³H]PN200-110, (–)-[³H]desmethoxyverapamil and d-cis [³H]diltiazem binding compared to its pharmacologically less active (S)-(–)-enantiomer, with IC₅₀ values in the low nanomolar range. Extensive equilibrium and kinetic studies suggest that HOE 166 exerts its Ca²⁺-antagonistic effect by binding to a Ca²⁺-channel-associated drug receptor which is distinct from the 1,4-dihydropyridine, phenylalkylamine or benzothiazepine-selective domain. This HOE 166-selective site is, however, allosterically linked to the other sites of the Ca²⁺ antagonist receptor complex. We conclude that HOE 166 is a novel calcium antagonist.Send offprint requests to H. Glossmann     

赛庚啶对离体犬心肌肌质网Ca2+,Mg2+—ATP酶活性和45Ca2+摄取功能的影响

当赛庚啶浓度在8×10^-6mol/L～2×10^-4mol/L之间时,该药对正常犬心肌肌质网Ca²⁺,Mg²⁺—ATP酶活性几乎没有影响,仅在10^-3mol/L时对该酶活性才有一定的抑制作用(抑制率为39.85%,P<0.01)。正常犬心肌肌质网的⁴⁵Ca²⁺摄取过程有明显的时间依赖性,至第30 min,其⁴⁵Ca²⁺摄取量可达312.79±22.25 nmol/mg protein.赛庚啶对心肌肌质网的~(45)Ca²⁺摄取有一定的抑制作用,其IC₅₀为1.94×10^-4mol/L。