Intracellular Free Calcium Concentration Research Articles

Effect of losartan on intracellular free calcium concentration and improved aortic elasticity in spontaneously hypertensive rat

Objective To investigate the effects of losartan on aortic elasticity and remodeling in spontaneously hypertensive rats (spontaneously hypertensive rats SHR). Methods WKY (Wistar - Kyoto ) rats with normal blood pressure and 16 weeks spontaneously hypertensive rats were randomly divided into WKY control group, SHR control group, high dose losartan group (SHR + HL), low doses losartan group (SHR + LL). Each group has six animals which were given normal diet for 24 weeks. Losartan which was dissolved in 10 ml physiological saline was filling in stomach, other groups were filling with physiological saline. Tail arterial blood pressure, kidney tissues calcium concentration, renal small artery hydroxyproline content was measured and small arteries wall thickness of Glomerularwas detected, and the ratio of thickness and inner diameter (MT/LR) in kidney pathological were observed. Results The calcium concentration of SHR group in kidney tissues was [(18.42±2.34)μmol/g], kidney small artery hydroxyproline content was [(8.26±2.02)mg/g], which were greater than WKY group [(11.83±1.98)μmol/g,(5.16±0.98)mg/g] (t＝3.116,3.258,P＜0.05), but the two treatment groups were less than SHR group (t＝2.946,P＜0.05), the difference was significant. Small arteries wall thickness of Glomerular was [(5.25±1.13)μm], the ratio of thickness and inner diameter (MT/LR) was [(9.57±1.78)%], which were greater than WKY group[(4.03±0.16)μm ,(7.12±1.35)%](t＝2.836,3.425,P＜0.05), but the wall thickness of two treatment groups were [(7.64±1.29)%,(7.85±1.32)%], the two treatment groups were less than SHR group (t＝3.512,3.648,P＜0.05). Conclusions Losartanmay inhibit intracellular calcium overload, reduce fibrosis degree and improve renal arteriole resistance and reverse the renal arteriole reconstruction of SHR rats. Key words: Losartan/PD; Hypertension/ME/PA; Kidney/ME; Calcium/ME; Renal artery/DE

Gap junction channel protein connexin40 modulates rabbit pulmonary microvascular endothelial cells permeability in traumatic acute lung injury

Objective To investigate the influence of traumatic acute lung injury (ALI) rabbit serum on gap junction channel ( GJC ) function and connexin40 ( Cx40) expression, and the correlation of GJC and Cx40 with rabbit pulmonary microvascular endothelial cells permeability. Methods Cultured pulmonary microvessel endothelial cells were divided into three groups, control ( Gcontrol) , injured serum (Gserum) , and blocker agent ( Gblocker). GJC function was assessed by scrape-loading and dye transfer techniques. Pulmonary microvessel endothelial cells permeability was measured by Evans blue-labeled albumin transfer, and the expression of Cx40 was measured by Western blotting. Intracellular free calcium concentration was measured by fluo-3am. Results Injured serum decreased GJC function and the level of Cx40,which was aggravated by the GJC blocker (control 726 293. 2 ± 83 684. 1, serum 397 798. 1 ± 87 145. 7, blocker 316 977. 6 ±76 325. 9,n=4,P ＜0. 05). Similarly, pulmonary microvascular endothelial cells permeability was increased significantly in Gserum and Gblocker as compared with Gcontrol (P ＜0. 05). After treatment with injured serum in combination with gap junction blocker in vitro, intracellular free calcium concentration was increased [control (494. 80 ±41.94) nmol/L, serum (569. 80 ±6. 70) nmol/L, blocker (158. 80 ±13. 09) nmol/L, P＜ 0.05]. Conclusion Traumatic ALI rabbit serum inhibits the Cx40 expression and down-regulates GJC function, which contributes to the increase of pulmonary microvascular endothelial cells permeability after ALI This mechanism may participate in the increase of pulmonary vascular permeability and promote the morbidity of ALI after trauma. Key words: Acute lung injury; Connexin40; Pulmonary permeability

Exposure to an Environmental Neurotoxicant Hastens the Onset of Amyotrophic Lateral Sclerosis-Like Phenotype in Human Cu2+/Zn2+ Superoxide Dismutase 1 G93A Mice: Glutamate-Mediated Excitotoxicity

Mice expressing the human Cu(2+)/Zn(2+) superoxide dismutase 1 (hSOD1) gene mutation (hSOD1(G93A); G93A) were exposed to methylmercury (MeHg) at concentrations that did not cause overt motor dysfunction. We hypothesized that low concentrations of MeHg could hasten development of the amyotrophic lateral sclerosis (ALS)-like phenotype in G93A mice. MeHg (1 or 3 ppm/day in drinking water) concentration-dependently accelerated the onset of rotarod failure in G93A, but not wild-type, mice. At the time of rotarod failure, MeHg increased Fluo-4 fluorescence (free intracellular calcium concentration [Ca(2+)](i)) in soma of brainstem-hypoglossal nucleus. These motor neurons control intrinsic and some extrinsic tongue function and exhibit vulnerability in bulbar-onset ALS. The α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)/kainic acid receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione reduced [Ca(2+)](i) in all G93A mice, irrespective of MeHg treatment. N-acetyl spermine, which antagonizes Ca(2+)-permeable AMPA receptors, further reduced [Ca(2+)](i) more effectively in MeHg-treated than untreated G93A mice, suggesting that MeHg-treated mice have a greater Ca(2+)-permeable AMPA receptor contribution. The non-Ca(2+) divalent cation chelator N,N,N',N'-tetrakis(pyridylmethyl)ethylenediamine reduced Fluo-4 fluorescence in all G93A mice; FluoZin-(Zn(2+) indicator) fluorescence was increased in all MeHg-treated mice. Thus in G93A mice Zn(2+) apparently contributed measurably to the MeHg-induced effect. This is the initial demonstration of accelerated onset of ALS-like phenotype in a genetically susceptible organism by exposure to low concentrations of an environmental neurotoxicant. Increased [Ca(2+)](i) induced by the G93A-MeHg interaction apparently was associated with Ca(2+)-permeable AMPA receptors and may contribute to the hastened development of ALS-like phenotypes by subjecting motor neurons to excessive elevation of [Ca(2+)](i), leading to excitotoxic cell death.

Histamine Elevates Free Intracellular Calcium in Mouse Retinal Dopaminergic Cells via H1-Receptors

Previously, retinopetal axons containing histamine and dopaminergic neurons expressing histamine H(1)-receptor had been localized in mouse retinas using anatomic techniques. The goal of these experiments was to demonstrate that these receptors are functional. Dopaminergic cells were acutely isolated from retinas of transgenic mice expressing red fluorescent protein under control of the tyrosine hydroxylase promoter and loaded with the calcium indicator Fura-2. Under control conditions, there were spontaneous oscillations in the levels of free intracellular calcium in dopaminergic cells. These oscillations were abolished in nominally calcium-free extracellular medium and in 1 μM tetrodotoxin, findings suggesting that the oscillations were mediated by calcium entry across the plasma membrane in response to sodium-dependent action potentials. Histamine increased the mean free intracellular calcium in the dopaminergic cells by increasing the frequency and/or amplitude of the calcium oscillations. The effects of histamine were dose-dependent and reached maximum at 5 μM. With this dose, there was a 65% increase in the mean free intracellular calcium concentration. The histamine H(1)-receptor antagonist, pyrilamine, blocked the effects of 5 μM histamine when applied at 50 μM. The selective histamine H(1)-receptor agonists, 2-(3-trifluoromethylphenyl) histamine and methylhistaprodifen significantly increased mean free intracellular calcium when applied at 5 μM. Histamine released from retinopetal axons in the mouse retina can elevate intracellular calcium levels in the perikarya of dopaminergic cells via the activation of histamine H(1)-receptors.

Tanshinone IIa Alleviates the Biochemical Changes Associated with Hypoxic Ischemic Brain Damage in a Rat Model

This study aimed to investigate the effects of Tanshinone IIa (TanIIa) on the biochemical changes associated with hypoxic ischemic brain damage (HIBD) in a rat model. Neonatal SD rats were randomized into normal control, HIBD and TanIIa + HIBD groups. At different time points after HIBD, TanIIa was given at 1 µg/g. The intracellular free calcium concentration and the expression of phospho-NR1 S897 was determined. The intracellular free calcium concentration in the HIBD group was significantly increased. The induction of intracellular free calcium concentration in the TanIIa + HIBD group was less than that in the HIBD group. Large amounts of phospho-NR1 S897 positive cells were distributed in the cortex in the normal control group; the number of phospho-NR1 S897 positive cells in the ipsilateral cortex was dramatically decreased at 24 h after HIBD. Both the number of phospho-NR1 S897 positive cells and the FITC fluorescent density in the HIBD + TanIIa group were less than those in the normal control group at every time point after HIBD, but more than those in the HIBD group. TanIIa alleviated the down-regulation of phospho-NR1 S897 and the elevated intracellular free calcium concentration in the cerebral cortex in the HIBD model. TanIIa could exert a neuroprotective effect through affecting NMDA receptor expression, inhibiting calcium transportation and decreasing the intracellular free calcium concentration.

Effects of flavonoids isolated from scutellariae radix on the production of tissue-type plasminogen activator and plasminogen activator inhibitor-1 induced by thrombin and thrombin receptor agonist peptide in cultured human umbilical vein endothelial cells.

The effects of different flavonoids isolated from the roots of Scutellaria baicalensis Georgi on the production of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) induced by thrombin and thrombin receptor agonist peptide, Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe, have been examined in cultured human umbilical vein endothelial cells (HUVECs). Thrombin and thrombin receptor agonist peptide induced production of both t-PA and PAI-1 and the elevation of intracellular free calcium concentration ([Ca2+]i). Baicalein isolated from Scutellariae Radix dose-dependently inhibited PAI-1 production induced by thrombin and thrombin receptor agonist peptide; its concentrations for 50% inhibition (IC50) were 6.8 and 3.5 microM, respectively. Other flavonoids had no effect. In contrast, flavonoids isolated from Scutellariae Radix had no effect on production of t-PA induced by thrombin and thrombin receptor agonist peptide. Baicalein inhibited the elevation of [Ca2+]i induced by thrombin and thrombin receptor agonist peptide and, at a concentration of 1000 microM, slightly increased t-PA production. These findings suggest that the mechanism by which baicalein inhibits PAI-1 production induced by thrombin and thrombin receptor agonist peptide might be by reduction of [Ca2+]i elevation. The results suggest that baicalein in Scutellariae Radix might be active as a drug in the treatment of arteriosclerosis and thrombosis.

Extract of Paris polyphylla Simth protects cardiomyocytes from anoxia-reoxia injury through inhibition of calcium overload

To assess any direct effect of extract of Paris polyphylla Simth (EPPS), a Chinese plant, on a cardiomyocyte subject to ischemia-reperfusion injury and to further elucidate its protective effect against myocardium ischemia on the cellular level. Neonatal rat cardiomyocytes were isolated and subjected to an anoxia-reoxia injury simulating the ischemia-reperfusion injury in vivo in the presence or absence of EPPS or diltizem, a positive control. The lactate dehydrogenase (LDH) activities in culture supernatants and cell viabilities were analyzed using the enzymatic reaction kinetics monitoring-method and MTT method, respectively. Free intracellular calcium concentrations and activities of Na(+)-K(+) ATPase and Ca(2+) ATPase in cells were also measured with laser confocal microscopy and the inorganic phosphorus-transformation method, respectively. In cardiomyocytes subject to anoxia-reoxia injury, EPPS at 50-400 mg/L showed a concentration-dependent inhibition on LDH leakage and maintenance of cell viability, and the effect was significant at 275 and 400 mg/L (both P<0.01). In addition, EPPS at 275 and 400 mg/L significantly inhibited the increase in intracellular free calcium (both P<0.01) as well as decreased the activities of Na(+)-K(+) ATPase and Ca(2+) ATPase (P<0.01, P<0.05). EPPS prevents anoxia-reoxia injury in neonatal rat cardiomyocytes in vitro by preservation of Na(+)-K(+) ATPase and Ca(2+) ATPase activities and inhibition of calcium overload. The direct protective effect on cardiomyocytes may be one of the key mechanisms that underlie the potential therapeutic benefit of EPPS against myocardium ischemia.

Tipifarnib-Induced Apoptosis in Acute Myeloid Leukemia and Multiple Myeloma Cells Depends on Ca2+Influx through Plasma Membrane Ca2+Channels

A major contributing factor to the high mortality rate associated with acute myeloid leukemia and multiple myeloma is the development of resistance to chemotherapy. We have shown that the combination of tipifarnib, a nonpeptidomimetic farnesyltransferase inhibitor (FTI), with bortezomib, a proteosome inhibitor, promotes synergistic death and overcomes de novo drug resistance in acute myeloid leukemia cell lines. Experiments were undertaken to identify the molecular mechanisms by which tipifarnib produces cell death in acute myeloid leukemia and multiple myeloma cell lines (U937 and 8226, respectively). Tipifarnib, but not other FTIs tested [N-[4-[2(R)-amino-3-mercaptopropyl]amino-2-phenylbenzoyl]methionine methyl ester trifluoroacetate salt (FTI-277) and 2'-methyl-5-((((1-trityl-1H-imidazol-4-yl)methyl)amino)methyl)-[1,1'-biphenyl]-2-carboxylic acid (FTI-2153), promotes elevations in intracellular free-calcium concentrations ([Ca(2+)](i)) in both cell lines. These elevations in [Ca(2+)](i) were accompanied by highly dynamic plasmalemmal blebbing and frequently resulted in membrane lysis. The tipifarnib-induced elevations in [Ca(2+)](i) were not blocked by thapsigargin or ruthenium red, but were inhibited by application of Ca(2+)-free extracellular solution and by the Ca(2+) channel blockers Gd(3+) and La(3+). Conversely, 2-aminoethoxydiphenyl borate (2-APB) potentiated the tipifarnib-evoked [Ca(2+)](i) overload. Preventing Ca(2+) influx diminished tipifarnib-evoked cell death, whereas 2-APB potentiated this effect, demonstrating a link between tipifarnib-induced Ca(2+) influx and apoptosis. These data suggest that tipifarnib exerts its effects by acting on a membrane channel with pharmacological properties consistent with store-operated channels containing the Orai3 subunit. It is noteworthy that Orai3 transcripts were found to be expressed at lower levels in tipifarnib-resistant 8226/R5 cells. Our results indicate tipifarnib causes cell death via a novel mechanism involving activation of a plasma membrane Ca(2+) channel and intracellular Ca(2+) overload.

Lowered insulin-sensitive Ca2+ transport in cultured glomerular mesangial cells from spontaneously hypertensive rats.

Insulin-resistant state has been postulated in the pathogenesis of hypertension. To investigate the role of insulin in Ca2+ regulation of the glomerular mesangial cell (MC) in hypertension, the effect of insulin on Ca2+ transport and intracellular-free calcium concentration ([Ca2+]i) was measured in serially passaged cultured MC obtained from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Insulin produced a substantial increase in 45Ca uptake as well as [Ca2+]i in quiescent cultured MC from both strains. These stimulatory effects of insulin were completely inhibited by diltiazem, and partially inhibited by H-7, TMB-8 and 5-N,N (hexamethylene) amiloride (HMA), but were not inhibited by W-7 or trifluoroperazine. Although basal ([Ca2+]i) values were not different, insulin-sensitive 45Ca uptake of SHR MC was significantly less than that of WKY MC. Insulin-sensitive increase in ([Ca2+]i) of SHR MC was also less than that of WKY MC. We concluded that insulin increased 45Ca uptake, leading to an increase in ([Ca2+]i), presumably through the voltage-dependent Ca2+ channel, intracellular Ca2+ release and/or proteinkinase C-mediated mechanisms in cultured MC. Blunted response of insulin-sensitive Ca2+ uptake and ([Ca2+]i) in SHR MC suggested differential regulation of Ca2+ transport in response to insulin in the kidney with primary hypertension.

Protective effects of glutamine preconditioning on ischemia-reperfusion injury in rats

Hepatic ischemia-reperfusion injury is a common phenomenon in hepatic surgical procedures and can result in further severe damage. This study aimed to investigate the protective effects of glutamine preconditioning on hepatic ischemia-reperfusion injury in rats and its dose-dependency. Thirty-two healthy male Wistar rats were randomly divided into four groups (n=8 per group). One group received 0.9% NaCl (control) and the other three received glutamine (Gln groups) 4 hours before ischemia. The Gln groups were named GL, GM, and GH according to the glutamine dose. The liver was subjected to 1 hour of ischemia and 2 hours of reperfusion. Two hours later, the levels of alanine aminotransferase (ALT), intracellular free calcium (Ca2+), and activity of Na+/K+ adenosine triphosphatase (ATPase) and superoxide dismutase (SOD) were assessed, and liver tissue sections were examined under a microscope. The Gln and control groups differed in the concentration of intracellular free calcium (P<0.05), and the activity of Na+/K+ ATPase and SOD in the Gln groups was higher than in the control group (P<0.05). The ALT level was lower in the GM and GH groups than in the control group (P<0.05). The levels of Na+/K+ ATPase and SOD rose gradually with increasing glutamine dose (P<0.05), and the concentration of Ca2+ declined gradually with increasing glutamine dose (P<0.05). The degree of hepatocyte injury was milder in the Gln groups than in the control group. Glutamine preconditioning protected effectively against hepatic ischemia-reperfusion injury. These protective effects were related to the dose of glutamine and due to the reduction of intracellular calcium overload and the improvements in the activity of Na+/K+ ATPase and SOD.

Single-Camera Multi-Parametric Optical Mapping During Local Excitation of Isolated Rat Heart

Simultaneous optical mapping of multiple electrophysiologically relevant parameters in living tissue is desirable for integrative exploration of mechanisms underlying organ function in norm and pathology. Current multi-parametric methods are technically challenging, usually involving several sensors, complex optical configurations/alignment and moving parts (e.g. mechanical shutters and filter wheels). This increases logistic and economical thresholds, preventing turn-key solutions and broader application. Therefore we developed a simple, affordable and effective method for single-sensor multi-parametric optical mapping.The developed system can measure membrane potential (Vm) and intracellular free calcium concentration ([Ca2+]i) using the following functional dyes: Di-4-ANBDQPQ (Vm), Fura-2 ([Ca2+]i). Four parameter (ratiometric Vm and ratiometric [Ca2+]i) configuration has been designed using light-emitting-diode (LED) excitation sources ranging from UV to red wavelengths. High-speed coordination between the LEDs and a 128x128, 16-bit electron-multiplied charge-coupled device (EMCCD) camera system (Cascade 128+; Photometrics) was achieved with inexpensive and versatile microcontroller-based electronics. Emission fluorescence from dyes was passed through off-the-shelf and custom multiband filters, a technology not widely explored in the context of optical mapping.Here, we present simultaneous ratiometric Vm and ratiometric [Ca2+]i imaging with Di-4-ANBDQPQ and Fura-2 in the rat whole-heart. For proof-of-principle application, we integrated this imaging technology with timed local electrical and mechanical stimulation to study mechano-electric coupling at the organ level.Combining an affordable off-the-shelf camera with optical filters and LEDs, single-sensor multi-parametric optical mapping can be practically implemented and applied to heart research. The moderate system complexity and component cost (less than 5% of camera cost) may lower the threshold to broader application of functional imaging and ease implementation of more complex optical mapping approaches such as panoramic multi-parametric optical mapping.

Cell Signaling of Angiotensin II on Vascular Tone: Novel Mechanisms

Angiotensin II (Ang II) is a pleiotropic hormone that influences the function of many cell types and regulates many organ systems. In the cardiovascular system, it is a potent vasoconstrictor that increases peripheral vascular resistance and elevates arterial pressure. It also promotes inflammation, hypertrophy, and fibrosis, which are important in vascular remodeling in cardiovascular diseases. The diverse actions of Ang II are mediated via AT(1) and AT(2) receptors, which couple to many signaling molecules, including small G proteins, phospholipases, mitogen-activated protein (MAP) kinases, phosphatases, tyrosine kinases, NADPH oxidase, and transcription factors. In general, acute Ang II stimulation induces vasoconstriction through changes in the intracellular free calcium concentration [Ca(2+)](i), whereas long-term stimulation leads to cell proliferation and proinflammatory responses. This review focuses on signaling processes of vasoconstriction and highlights some new mechanisms regulating the contractile machinery in controlling vasomotor tone by Ang II, including RhoA/Rho kinase, transient receptor potential (TRP) channels, reactive oxygen species, and arachidonic acid metabolites.

Kisspeptin-10 elicits triphasic cytosolic calcium responses in immortalized GT1-7 GnRH neurones

Kisspeptins, which are alternatively called as metastin since they were originally identified as products of metastasis suppressor gene KiSS-1, are the natural ligands for the G protein-coupled receptor 54 (GPR54). Kisspeptins are the most potent activators of hypothalamic–pituitary–gonadal (HPG) axis reported to date. The pulsatile pattern of GnRH release, which results in the intermittent release of gonadotropic hormones from the pituitary, has a critical importance for reproductive function but the factors responsible from this release pattern are not known. Therefore, the pattern of kisspeptin-induced intracellular signaling and the role of PKC in the intracellular signaling cascade were investigated by fluorescence calcium imaging using the immortalized GnRH-secreting GT1-7 hypothalamic neurons. Kisspeptin-10 caused a triphasic change characterized by an initial small increase followed by a significant decrease and increase in intracellular free calcium concentrations ([Ca 2+] i ). The changes in [Ca 2+] i were significantly attenuated by pre-treatment with protein kinase C inhibitor. The compatibility of appeared mirrored-patterns of kisspeptin-10-induced changes in [Ca 2+] i concentrations in these neurons and GnRH secretion confirm the importance of intracellular calcium flux downstream from GPR54 through PKC signaling pathway.

Ca2+]isignaling vs. eNOS expression as determinants of NO output in uterine artery endothelium: relative roles in pregnancy adaptation and reversal by VEGF165

Pregnancy is a time of greatly increased uterine blood flow to meet the needs of the growing fetus. Increased uterine blood flow is also observed in the follicular phase of the ovarian cycle. Simultaneous fura-2 and 4,5-diaminofluoresceine (DAF-2) imaging reveals that cells of the uterine artery endothelium (UA Endo) from follicular phase ewes produce marginally more nitric oxide (NO) in response to ATP than those from luteal phase. However, this is paralleled by changes in NO in response to ionomycin, suggesting this is solely due to higher levels of endothelial nitric oxide synthase (eNOS) protein in the follicular phase. In contrast, UA Endo from pregnant ewes (P-UA Endo) produces substantially more NO (4.62-fold initial maximum rate, 2.56-fold overall NO production) in response to ATP, beyond that attributed to eNOS levels alone (2.07-fold initial maximum rate, 1.93-fold overall with ionomycin). The ATP-stimulated intracellular free calcium concentration ([Ca(2+)](i)) response in individual cells of P-UA Endo comprises an initial peak followed by transient [Ca(2+)](i) bursts that are limited in the luteal phase, not altered in the follicular phase, but are sustained in pregnancy and observed in more cells. Thus pregnancy adaptation of UA Endo NO output occurs beyond the level of eNOS expression and likely through associated [Ca(2+)](i) cell signaling changes. Preeclampsia is a condition of a lack of UA Endo adaptation and poor NO production/vasodilation and is associated with elevated placental VEGF(165). While treatment of luteal NP-UA Endo and P-UA Endo with VEGF(165) acutely stimulates a very modest [Ca(2+)](i) and NO response, subsequent stimulation of the same vessel with ATP results in a blunted [Ca(2+)](i) and an associated NO response, with P-UA Endo reverting to the response of luteal NP-UA Endo. This demonstrates the importance of adaptation of cell signaling over eNOS expression in pregnancy adaptation of uterine endothelial function and further implicates VEGF in the pathophysiology of preeclampsia.

Cardiotonic steroids attenuate ERK phosphorylation and generate cell cycle arrest to block human hepatoma cell growth

Recent studies revealed the potential of Na +/K +-ATPase as a target for anticancer therapy and showed additional modes of action of cardiotonic steroids (CSs), a diverse family of naturally derived compounds, as inhibitors of Na +/K +-ATPase. The results from epidemiological studies showed significantly lower mortality rates in cancer patients receiving CSs, which sparked interest in the anticancer properties of these drugs. The present study was designed to investigate the anticancer effect of CSs (ouabain or cinobufagin) and to elucidate the molecular mechanisms of CS activity in hepatoma cell lines (HepG2 and SMMC-7721). Ouabain and cinobufagin significantly inhibited cell proliferation by attenuating the phosphorylation of extracellular regulated kinase (ERK) and down-regulating the expression of C-myc. These CSs also induced cell apoptosis by increasing the concentration of intracellular free calcium ([Ca 2+] i ) and induced S phase cell cycle arrest by down-regulating the expression of Cyclin A, cyclin dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA) as well as up-regulating the expression of cyclin dependent kinase inhibitor 1A (p21 CIP1). Overexpression of ERK reversed the antiproliferation effect of ouabain or cinobufagin in HepG2 and SMMC-7721 cells. Currently, the first generation of CS-based anticancer drugs (UNBS1450 and Anvirzel) are in Phase I clinical trials. These data clearly support their potential use as cancer therapies.

MODULATION OF ANAPHASE SPINDLE MICROTUBULE STRUCTURE IN STAMEN HAIR-CELLS OF TRADESCANTIA BY CALCIUM AND RELATED AGENTS

We have previously shown that changes in the intracellular free calcium concentration ([Ca2+]i) modulate the rate of anaphase chromosome motion in stamen hair cells of Tradescantia. Elevations between 0.8-1.0-mu-M accelerate motion, while increases above 2.0-mu-M or decreases below resting level inhibit motion. The related signaling agent GTP-gamma-S also accelerates motion, however, by a mechanism that does not appear to involve changes in [Ca2+]i. To explore further the mechanism by which Ca2+ and GTP-gamma-S regulate chromosome motion we have analyzed the direct effect of these agents on the structure of the spindle microtubules (MTs). First, we injected carboxyfluorescein-derivatized brain tubulin and allowed it to incorporate into spindle MTs. Then, during appropriate times of anaphase, we injected Ca2+ or related agents and monitored their effect on spindle MT fluorescence using a confocal laser scanning microscope. A high level of Ca2+ (10-mu-M), known to inhibit motion, causes extensive degradation of spindle MT structure. An intermediate level (2-mu-M), which slows but does not stop movement, produces a distinct decay of fluorescence. A level of Ca2+ (0.8-1-mu-M) known to accelerate motion, however, generates only a small change in which the kinetochore fibers appear less distinct, and the overall spindle fluorescence is more diffuse. The Ca2+ buffer EGTA, which transiently blocks motion, has no detectable effect on spindle structure. GTP-gamma-S, which enhances motion also has no discernible effect on spindle structure. The results with Ca2+ support the idea that the ion facilitates anaphase motion through depolymerization of kinetochore MTs. However, if the depolymerization is extensive then inhibition of motion ensues. GTP-gamma-S, on the other hand, accelerates motion without inducing a detectable effect on spindle MT structure.

Tanshinone IIA attenuates neuronal damage and the impairment of long-term potentiation induced by hydrogen peroxide

Tanshinone IIA (Tan IIA) is one of the key components of Salvia miltiorrhiza Bunge that has been widely used for various cardiovascular and cerebrovascular disorders in Asian countries. Many studies have reported that Tan IIA has antioxidative properties, but whether Tan IIA can rescue neurons from oxidative insult has never been reported. The present study was undertaken to evaluate the possible neuroprotective effects of Tan IIA on hydrogen peroxide (H(2)O(2))-induced oxidative stress in rats. H(2)O(2)-induced cytotoxicity was evaluated by the cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and flow cytometry with PI staining. Calcium imaging experiments were carried out to measure intracellular free calcium concentration. Western blotting was used to determine the expression of Bax and Bcl-2 protein. Electrophysiological studies in hippocampal slices were performed to investigate the effect of Tan IIA on synaptic function and cognitive impairment caused by H(2)O(2). It was found that pretreatment with Tan IIA protected primary rat cortical neurons against H(2)O(2)-induced cytotoxicity. Furthermore, Tan IIA markedly reduced the elevation of [Ca(2+)](i) evoked by H(2)O(2). Western blot analysis indicated that pretreatment with Tan IIA prevented the increase in Bax/Bcl-2 ratio induced by H(2)O(2). In addition, preincubation of Tan IIA 20 min prior to H(2)O(2) exposure could reverse H(2)O(2)-induced hippocampal LTP impairment, but without significant alteration in basal synaptic transmission and LTP induction. These findings demonstrate that Tan IIA might serve as a novel promising therapeutic agent for oxidative stress injury in neurodegenerative diseases.

Mechanoelectrical coupling enhances initiation and affects perpetuation of atrial fibrillation during acute atrial dilation

Acute atrial dilation increases the susceptibility to atrial fibrillation (AF). However, the mechanisms by which atrial stretch may contribute to the initiation and perpetuation of AF remain to be determined. The purpose of this study was to use a novel multiscale model of atrial electromechanics and mechanoelectrical feedback to test the hypothesis that acute stretch increases vulnerability to AF by heterogeneous activation of stretch-activated channels. Human atria were represented by a triangular mesh obtained from magnetic resonance imaging data. Atrial trabecular bundle structure was incorporated by varying thicknesses of the atrial wall. Atrial membrane behavior was modeled by the Courtemanche-Ramirez-Nattel model with the addition of a nonselective stretch-activated cation current (I(sac)). Mechanical behavior was modeled by a series elastic, a contractile, and a parallel elastic element in which contractile force was related to intracellular concentration of free calcium and sarcomere length. Acute atrial dilation was simulated by increasing stretch throughout the atrial wall. Stimulation near the pulmonary vein ostia at an interval of 600 ms induced AF at an overall stretch ratio of 1.10. Initiation and perpetuation of AF in our model were related to increased dispersion of effective refractory period, conduction slowing, and local conduction block, all related to heterogeneous activation of I(sac). Upon local contraction, mechanoelectrical coupling affects perpetuation of AF by temporarily changing local excitability. During acute atrial dilation, heterogeneous activation of I(sac) enhances initiation and can affect perpetuation of AF.

The Calcium-Sensing Receptor Mediates Hypoxia-Induced Proliferation of Rat Pulmonary Artery Smooth Muscle Cells Through MEK1/ERK1,2 and PI3K Pathways

Activation of the calcium-sensing receptor (CaSR) leads to an increase of intracellular calcium concentration and alteration of cellular activities. High level of intracellular calcium is involved in hypoxia-induced proliferation of pulmonary arterial smooth muscle cells (PASMCs). However, whether the CaSR is expressed in PAMSCs and is related to the hypoxia-induced proliferation of PASMCs is unclear. In this study, the expression and distribution of CaSRs were detected by RT-PCR, western blotting and immunofluorescence; the intracellular concentration of free calcium ([Ca(2+) ](i) ) was determined by confocal laser scanning microscopy; cell proliferation was tested using an MTT and BrdU incorporation assay; cell cycle analysis was carried out using a flow cytometric assay; and the expression of proliferating cell nuclear antigen (PCNA), extracellular signal-regulated protein kinase 1,2 (ERK1,2) and AKT were analysed by western blotting. We observed that both CaSR mRNA and protein were expressed in rat PASMCs. Lowering of oxygen from 21% to 2.5% led to increased [Ca(2+) ](i) and CaSR expression. This condition of hypoxia also stimulated PASMCs proliferation accompanying with increased phosphorylation of ERK1,2 and AKT. GdCl(3) (an agonist of CaSR) or NPS2390 (an antagonist of CaSR) amplified or weakened the effect of hypoxia, respectively. PD98059 (a MEK1 inhibitor) or LY294002 (a PI3K inhibitors) decreased the up-regulation of PCNA expression and the increase of the cell proliferation index induced by hypoxia and GdCl(3) in PASMCs. Our results suggest that CaSR is expressed in rat PASMCs, and that CaSR activation through MEK1/ERK1,2 and PI3 kinase pathways is involved in hypoxia-induced proliferation of PASMCs.

Constant TCR triggering suggests that the TCR expressed on intestinal intraepithelial γδ T cells is functional in vivo

Intestinal intraepithelial lymphocytes carrying the γδ TCR (γδ iIEL) are involved in the maintenance of epithelial integrity. γδ iIEL have an activated phenotype, characterized by CD69 expression and increased cell size compared with systemic T lymphocytes. As an additional activation marker, the majority of γδ iIEL express the CD8αα homodimer. However, our knowledge about cognate ligands for most γδ TCR remains fragmentary and recent advances show that γδ T cells including iIEL may be directly activated by cytokines or through NK-receptors, TLR and other pattern recognition receptors. We therefore asked whether the TCR of γδ iIEL was functional beyond its role during thymic selection. Using TcrdH2BeGFP (Tcrd, T-cell receptor δ locus; H2B, histone 2B) reporter mice to identify γδ T cells, we measured their intracellular free calcium concentration in response to TCR-crosslinking. In contrast to systemic γδ T cells, CD8αα(+) γδ iIEL showed high basal calcium levels and were refractory to TCR-dependent calcium-flux induction; however, they readily produced CC chemokine ligand 4 (CCL4) and IFN-γ upon TCR triggering in vitro. Notably, in vivo blocking of the γδ TCR with specific mAb led to a decrease of basal calcium levels in CD8αα(+) γδ iIEL. This suggests that the γδ TCR of CD8αα(+) γδ iIEL is constantly being triggered and therefore functional in vivo.