Intracellular Ca Release Research Articles

Enhanced ROS Generation Mediated by Alzheimer's Disease Presenilin Regulation of InsP3R Ca2+ Signaling

Familial Alzheimer's disease (FAD) is caused by mutations in amyloid precursor protein and presenilins (PS1, PS2). Many FAD-linked PS mutations affect intracellular calcium (Ca(2+)) homeostasis by proximal mechanisms independent of amyloid production by dramatically enhancing gating of the inositol trisphosphate receptor (InsP(3)R) intracellular Ca(2+) release channel by a gain-of-function effect that mirrors genetics of FAD and is independent of secretase activity. Electrophysiological recordings of InsP(3)R in FAD patient B cells, cortical neurons of asymptomatic PS1-AD mice, and other cells revealed they have higher occupancy in a high open probability burst mode, resulting in enhanced Ca(2+) signaling. Exaggerated Ca(2+) signaling through this mechanism results in enhanced generation of reactive oxygen species, believed to be an important component in AD pathogenesis. Exaggerated Ca(2+) signaling through InsP(3)R-PS interaction is a disease specific and robust proximal mechanism in AD that may contribute to the pathology of AD by enhanced generation of reactive oxygen species.

Malignant hyperthermia in India

Malignant hyperthermia in India

Lateral diffusion of inositol 1,4,5‐trisphosphate receptor type 1 in Purkinje cells is regulated by calcium and actin filaments

Inositol 1,4,5-trisphosphate receptor type 1 (IP(3) R1) is an intracellular Ca(2+) release channel that plays crucial roles in the functions of Purkinje cells. The dynamics of IP(3) R1 on the endoplasmic reticulum membrane and the distribution of IP(3) R1 in neurons are thought to be important for the spatial regulation of Ca(2+) release. In this study, we analyzed the lateral diffusion of IP(3) R1 in Purkinje cells in cerebellar slice cultures using fluorescence recovery after photobleaching. In the dendrites of Purkinje cells, IP(3) R1 showed lateral diffusion with an effective diffusion constant of approximately 0.30 μm(2) /s, and the diffusion of IP(3) R1 was negatively regulated by actin filaments. We found that actin filaments were also involved in the regulation of IP(3) R1 diffusion in the spine of Purkinje cells. Glutamate or quisqualic acid stimulation, which activates glutamate receptors and leads to a Ca(2+) transient in Purkinje cells, decreased the diffusion of IP(3) R1 and increased the density of actin in spines. These findings indicate that the neuronal activity-dependent augmentation of actin contributes to the stabilization of IP(3) R1 in spines.

Distinct roles for non-muscle myosin II isoforms in mouse hepatic stellate cells

Upon liver injury, hepatic stellate cells (HSCs) undergo dramatic morphological and functional changes including migration and contraction. In the present study, we investigated the role of myosin II isoforms in the development of the contractile phenotype of mouse HSCs, which are considered therapeutic targets to decrease portal hypertension and fibrosis. We characterized the expression of myosin IIA and IIB in primary mouse HSCs and addressed their function by gene knock-down using isoform-specific siRNAs. We found that myosin IIA and IIB are differentially expressed and localized and have clearly different functions in HSCs. Myosin IIA is mainly located in the subcortical area of quiescent HSCs and at α-SMA-containing stress fibres after activation, while myosin IIB is located in the cytoplasm and at the edge of protrusions of quiescent HSCs, at stress fibres of activated cells, and at the leading edge of lamellipodia. Knock-down of myosin IIA in HSCs influences cell size and shape, results in the disruption of stress fibres and in a decrease of focal adhesions, and inhibits contractility and intra-cellular Ca(2+) release but increases cell migration. Myosin IIB contributes to the extension of lamellipodia and cell spreading but has no direct role in stress fibres and focal adhesion formation, contraction, or intra-cellular Ca(2+) signalling. In mouse HSCs, myosin IIA and IIB clearly fulfil distinct roles. Our results provide an insight into the contractile machinery of HSCs, that could be important in the search for new molecules to treat portal hypertension.

BDNF stimulates Ca 2+ oscillation frequency in melanotrope cells of Xenopus laevis: Contribution of IP3-receptor-mediated release of intracellular Ca 2+ to gene expression

Pituitary melanotrope cells of the amphibian Xenopus laevis are neuroendocrine cells regulating the animal’s skin color adaptation through secretion of α-melanophore-stimulating hormone (α-MSH). To fulfill this function optimally, the melanotrope cell undergoes plastic changes in structure and secretory activity in response to changed background light conditions. Xenopus melanotrope cells display Ca 2+ oscillations that are thought to drive α-MSH secretion and gene expression. They also produce brain-derived neurotrophic factor (BDNF), which stimulates in an autocrine way the biosynthesis of the α-MSH precursor, pro-opiomelanocortin (POMC). We have used this physiological adaptation mechanism as a model to investigate the role of BDNF in the regulation of Ca 2+ kinetics and Ca 2+-dependent gene expression. By dynamic video imaging of isolated cultured melanotropes we demonstrated that BDNF caused a dose-dependent increase in Ca 2+ oscillation frequency up to 64.7 ± 2.3% of control level. BDNF also induced a transient Ca 2+ peak in Ca 2+-free medium, which was absent when calcium stores were blocked by thapsigargin and 2-aminoethoxydiphenyl borate, indicating that BDNF stimulates acute release of Ca 2+ from IP 3-sensitive intracellular Ca 2+ stores. Moreover, we show that thapsigargin inhibits the expression of BDNF transcript IV (by 61.1 ± 28.8%) but does not affect POMC transcript. We conclude that BDNF mobilizes Ca 2+ from IP 3-sensitive intracellular Ca 2+ stores and propose the possibility that the resulting Ca 2+ oscillations selectively stimulate expression of the BDNF gene.

Mechanisms of vasorelaxation induced by Ziziphora clinopodioides Lam. (Lamiaceae) extract in rat thoracic aorta

Ziziphora clinopodioides Lam. (ZC) is widely used in Uyghur folk medicine for the treatment of hypertension diseases in Xinjiang, an autonomous region of China. To provide pharmacological basis for this traditional use, we explored the vasodilating effects of ZC and investigated the underlying mechanisms. Activity of hexane (ZCHE), dichloromethane (ZCDE) and aqueous (ZCAE) extracts of ZC were evaluated on isolated rat aortic rings pre-contracted with phenylephrine (PE) or high KCl. The mechanisms were evaluated on ZCDE, the most potent extract. ZCDE-induced relaxation in endothelium-intact aortic rings pre-contracted with phenylephrine (PE, 10(-6) M) or high KCl (6×10(-2) M), with respective EC(50) values of 0.27±0.03 and 0.34±0.04 g/l. Mechanic removal of the endothelium did not significantly modify ZCDE-induced relaxation. In endothelium-denuded aorta pre-contracted with PE (10(-6) M), the vasorelaxant effect of ZCDE was significantly decreased by 4-amino-pyridine (10(-3) M), but not by glibenclamide (10(-4) M), iberiotoxin (3×10(-8) M) and thapsigargin (10(-7) M). In Ca(2+) free solution, ZCDE significantly inhibited extracellular Ca(2+)-induced contraction in high KCl and PE pre-contracted rings. Additionally ZDCE inhibited the intracellular Ca(2+) release sensitive to PE (10(-6) M). The results demonstrate that ZDCE exhibits endothelium-independent vasodilating properties that are mediated by inhibition of extracellular Ca(2+) influx through voltage- and receptor-operated Ca(2+) channels (VDDCs and ROCCs), by inhibition of Ca(2+) release from intracellular stores, and also by the opening of voltage-dependent K(+) channels.

Surface-Enhanced Raman Spectroscopy as a Tool for Detecting Ca2+ Mobilizing Second Messengers in Cell Extracts

Understanding of calcium signaling pathways in cells is essential for elucidating the mechanisms of both normal cell function and cancer development. Calcium messengers play the crucial role for intracellular Ca(2+) release. We propose a new approach to detecting the calcium second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) in cell extracts using surface-enhanced Raman spectroscopy (SERS). Currently available radioreceptor binding and enzymatic assays require extensive sample preparation and take more than 12 h. With a SERS sensor, NAADP can be detected in less than 1 min without any special sample preparation. To the best of our knowledge, this is the first demonstration of using SERS for calcium signaling applications.

Intracellular Ca2+ and TRPM5‐mediated membrane depolarization produce ATP secretion from taste receptor cells

ATP is a transmitter secreted from taste bud receptor (Type II) cells through ATP-permeable gap junction hemichannels most probably composed of pannexin 1. The elevation of intracellular Ca(2+) and membrane depolarization are both believed to be involved in transmitter secretion from receptor cells, but their specific roles have not been fully elucidated. In the present study, we show that taste-evoked ATP secretion from mouse vallate receptor cells is evoked by the combination of intracellular Ca(2+) release and membrane depolarization. Unexpectedly, ATP secretion is not blocked by tetrodotoxin, indicating that transmitter release from these cells still takes place in the absence of action potentials. Taste-evoked ATP secretion is absent in receptor cells isolated from TRPM5 knockout mice or in taste cells from wild type mice where current through TRPM5 channels has been eliminated. These findings suggest that membrane voltage initiated by TRPM5 channels is required for ATP secretion during taste reception. Nonetheless, even in the absence of TRPM5 channel activity, ATP release could be triggered by depolarizing cells with KCl. Collectively, the findings indicate that taste-evoked elevation of intracellular Ca(2+) has a dual role: (1) Ca(2+) opens TRPM5 channels to depolarize receptor cells and (2) Ca(2+) plus membrane depolarization opens ATP-permeable gap junction hemichannels.

Anemoside A3-induced Relaxation in Rat Renal Arteries: Role of Endothelium and Ca2+Channel Inhibition

Anemoside A(3), a lupane-type triterpenoid saponin, exists in the roots of Pulsatilla chinensis, but its pharmacological properties are largely unknown. The present study aimed to investigate the mechanisms underlying anemoside A(3)-induced relaxation in rat renal arteries. Changes of isometric force were determined on arteries with a myograph. Anemoside A(3) caused concentration-dependent relaxation in precontracted aortas, mesenteric, left coronary, and renal arteries. Removal of endothelium or treatment with charybdotoxin plus apamin slightly but significantly attenuated the relaxation in renal arteries. TEA(+) inhibited the relaxation caused by anemoside A(3) in renal arteries with and without endothelium while glibenclamide, BaCl(2), or capsaicin had no effect on it. Anemoside A(3) produced less relaxation in rings contracted by 60 mM KCl compared with rings contracted by receptor-dependent constrictors. It further inhibited contractions induced by Ca(2+) influx through nifedipine-sensitive voltage-gated Ca(2+) channels, nifedipine-insensitive receptor-operated Ca(2+) channels, and by intracellular Ca(2+) release. Pretreatment with nifedipine attenuated anemoside A(3)-induced relaxation. Taken together, the present results indicate that anemoside A(3) produces relaxation in rat renal arteries through multiple mechanisms. The release of CTX/apamin-sensitive endothelium-derived hyperpolarizing factor, stimulation of TEA(+)-sensitive K(+) channel, and inhibition of Ca(2+) influx jointly contribute to the relaxation.

Post-conditioning protects cardiomyocytes from apoptosis via PKCε-interacting with calcium-sensing receptors to inhibit endo(sarco)plasmic reticulum–mitochondria crosstalk

The intracellular Ca(2+) concentration ([Ca(2+)](i)) is increased during cardiac ischemia/reperfusion injury (IRI), leading to endo(sarco)plasmic reticulum (ER) stress. Persistent ER stress, such as with the accumulation of [Ca(2+)](i), results in apoptosis. Ischemic post-conditioning (PC) can protect cardiomyocytes from IRI by reducing the [Ca(2+)](i) via protein kinase C (PKC). The calcium-sensing receptor (CaR), a G protein-coupled receptor, causes the production of inositol phosphate (IP(3)) to increase the release of intracellular Ca(2+) from the ER. This process can be negatively regulated by PKC through the phosphorylation of Thr-888 of the CaR. This study tested the hypothesis that PC prevents cardiomyocyte apoptosis by reducing the [Ca(2+)](i) through an interaction of PKC with CaR to alleviate [Ca(2+)](ER) depletion and [Ca(2+)](m) elevation by the ER-mitochondrial associated membrane (MAM). Cardiomyocytes were post-conditioned after 3 h of ischemia by three cycles of 5 min of reperfusion and 5 min of re-ischemia before 6 h of reperfusion. During PC, PKC(epsilon) translocated to the cell membrane and interacted with CaR. While PC led to a significant decrease in [Ca(2+)](i), the [Ca(2+)](ER) was not reduced and [Ca(2+)](m) was not increased in the PC and GdCl(3)-PC groups. Furthermore, there was no evident psi(m) collapse during PC compared with ischemia/reperfusion (I/R) or PKC inhibitor groups, as evaluated by laser confocal scanning microscopy. The apoptotic rates detected by TUNEL and Hoechst33342 were lower in PC and GdCl(3)-PC groups than those in I/R and PKC inhibitor groups. Apoptotic proteins, including m-calpain, BAP31, and caspase-12, were significantly increased in the I/R and PKC inhibitor groups. These results suggested that PKC(epsilon) interacting with CaR protected post-conditioned cardiomyocytes from programmed cell death by inhibiting disruption of the mitochondria by the ER as well as preventing calcium-induced signaling of the apoptotic pathway.

Primary cilium-dependent mechanosensing is mediated by adenylyl cyclase 6 and cyclic AMP in bone cells.

Primary cilia are chemosensing and mechanosensing organelles that regulate remarkably diverse processes in a variety of cells. We previously showed that primary cilia play a role in mediating mechanosensing in bone cells through an unknown mechanism that does not involve extracellular Ca(2+)-dependent intracellular Ca(2+) release, which has been implicated in all other cells that transduce mechanical signals via the cilium. Here, we identify a molecular mechanism linking primary cilia and bone cell mechanotransduction that involves adenylyl cyclase 6 (AC6) and cAMP. Intracellular cAMP was quantified in MLO-Y4 cells exposed to dynamic flow, and AC6 and primary cilia were inhibited using RNA interference. When exposed to flow, cells rapidly (<2 min) and transiently decreased cAMP production in a primary cilium-dependent manner. RT-PCR revealed differential expression of the membrane-bound isoforms of adenylyl cyclase, while immunostaining revealed one, AC6, preferentially localized to the cilium. Further studies showed that decreases in cAMP in response to flow were dependent on AC6 and Gd(3+)-sensitive channels but not intracellular Ca(2+) release and that this response mediated flow-induced COX-2 gene expression. The signaling events identified provide important details of a novel early mechanosensing mechanism in bone and advances our understanding of how signal transduction occurs at the primary cilium.

Agmatine‐mediated rat mesenteric artery relaxation via NO synthesis

AimTo determine membrane receptor‐dependent or independent mechanisms of agmatine (decarboxylated arginine) relaxation of rat mesenteric arteries.BackgroundIn spite of excess levels of intracellular L‐arginine, exogenous L‐arginine still activates cellular synthesis of nitric oxide (NO) in a phenomenon called “arginine paradox”. Our previously published data (PNAS, 104, 9982, 2007) show that L‐arginine or agmatine may be initiating the NO synthesis via receptor binding and release of intracellular Ca+2 in endothelial cells. Here we have tested the same mechanism in isolated rat mesenteric arteries.MethodsThe individual 2nd order mesenteric arteriolar segments from rats (250–300 g male, Sprague‐Dawley) were cannulated at both ends in a vessel chamber that was continuously perfused intraluminally and pre‐constricted with norepinephrine (2 μM) in modified Krebs buffer at 37°C. For internal diameter measurements, the vessel chamber was mounted on the stage of a microscope fitted with a video camera leading to video caliper. Mounted vessels were allowed to stabilize for 60 min before initiating experiment.ResultsThe data show that agmatine completely relaxed the vessel with an EC50 of 58.75 ± 12.1 μM (n=4) and this relaxation could be significantly inhibited with nitric oxide synthase inhibitor, L‐NAME (0.5 mM) with EC50 of 315 μM (n=1).ConclusionAgmatine dose‐dependently relaxed the rat mesenteric artery and NO mediated the relaxation process.This work was supported in part by NIH grant SC1HL095101

Phosphatidylinositol 3,5 bisphosphate increases contractility and intracellular calcium in cardiac muscle by directly activating the cardiac ryanodine receptor.

We have previously reported that treatment of left ventricular cardiac muscle strips with phosphatidylinositol 3,5 bisphosphate (PI(3,5)P2) during field stimulation enhanced the magnitude of isometric force, the rate of force development, and the area associated with the contractile waveforms. In addition, PI(3,5)P2 caused an increase in intracellular calcium (Ca) in cardiac myocytes. These data suggested that PI(3,5)P2 increased contractile force by promoting extracellular Ca entry or by facilitating intracellular Ca release. To elucidate the mechanism responsible for the effects of PI(3,5) on intracellular Ca, we have performed ratiometric Ca imaging in isolated adult mouse cardiac myocytes. We have found that PI(3,5)P2 induced a dramatic increase in intracellular Ca levels, which was inhibited by preincubation with ryanodine. To confirm that PI(3,5)P2 was acting on RyR2 we performed electrophysiological and biochemical studies. PI(3,5)P2 increased [3H]ryanodine binding and this effect was prevented by preincubation with an antibody against PI(3,5)P2. Furthermore, single RyR2 channels reconstituted in lipid bilayers were activated by PI(3,5)P2 in a dose‐dependent manner, indicating that PI(3,5)P2 binds directly to RyR2. We provide inaugural evidence that PI(3,5)P2 regulates cardiac contractility via modulation of intracellular Ca by direct binding and activation of the RyR2 channel.

Estrogen receptor-mediated enhancement of venous relaxation in female rat: Implications in sex-related differences in varicose veins

A greater incidence of varicose veins has been reported in premenopausal women than in men. We hypothesized that the sex differences in venous function reflect reduced constriction and enhanced venous dilation in women due to direct venous relaxation effects of estrogen on specific estrogen receptors (ER). Circular segments of inferior vena cava (IVC) from male and female Sprague-Dawley rats were suspended between two wires, and isometric contraction (in mg/mg tissue) to phenylephrine, angiotensin II (AngII), and 96 mM KCl was measured. To investigate sex differences in venous smooth muscle, Ca(2+) release from the intracellular stores, and Ca(2+) entry from the extracellular space, the transient phenylephrine contraction in 0 Ca(2+) Krebs was measured. Extracellular CaCl(2) (0.1, 0.3, 0.6, 1, 2.5 mM) was added, and the [Ca(2+)](e)-dependent contraction was measured. To investigate sex differences in venous endothelial function, acetylcholine-induced relaxation was measured. To test the role of specific ERs, the amount of venous tissue ERs was measured using Western blots, and the venous relaxation in response to 17beta-estradiol (E2, activator of most ERs), 4,4,'4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)-tris-phenol (PPT; ERalpha agonist), 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; ERbeta agonist), and ICI 182,780 (ERalpha/ERbeta antagonist, and G protein-coupled receptor 30 [GPR30] agonist) was measured in IVC segments nontreated or treated with the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). Phenylephrine caused concentration-dependent contraction that was less in female (max 104.2 +/- 16.2) than male IVC (172.4 +/- 20.4). AngII (10(-6))-induced contraction was also less in female (81.0 +/- 11.1) than male IVC (122.5 +/- 15.0). Phenylephrine contraction in 0 Ca(2+) Krebs was insignificantly less in female (4.8 +/- 1.8) than male IVC (7.2 +/- 1.7), suggesting little difference in the intracellular Ca(2+) release mechanism. In contrast, the [Ca(2+)](e)-dependent contraction was significantly reduced in female than male IVC. Also, contraction to membrane depolarization by 96 mM KCl, which stimulates Ca(2+) influx, was less in female (129.7 +/- 16.7) than male IVC (319.7 +/- 30.4), supporting sex differences in Ca(2+) entry. Acetylcholine relaxation was greater in female (max 80.6% +/- 4.1%) than male IVC (max 48.0% +/- 6.1%), suggesting sex differences in the endothelium-dependent relaxation pathway. Western blots revealed greater amounts of ERalpha, ERbeta, and GPR30 in female than male IVC. ER agonists caused concentration-dependent relaxation of phenylephrine contraction in female IVC. E2-induced relaxation (max 76.5% +/- 3.4%) was more than DPN (74.8% +/- 9.1%), PPT (71.4% +/- 12.5%), and ICI 182,780 (67.4% +/- 7.8%), and was similar in L-NAME-treated and nontreated IVC. The reduced alpha-adrenergic, AngII, depolarization-induced, and [Ca(2+)](e)-dependent venous contraction in female rats is consistent with sex differences in the Ca(2+) entry mechanisms, possibly due to enhanced endothelium-dependent vasodilation and increased ER expression/activity in female rats. E2/ER-mediated venous relaxation in female rats is not prevented by NOS blockade, suggesting activation of an NO-independent relaxation pathway. The decreased venous contraction and enhanced E2/ER-mediated venous relaxation would lead to more distensible veins in female rats.

Eicosapentaenoic acid-induced endothelium-dependent and -independent relaxation of sheep pulmonary artery

It is known that long chain polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), have beneficial effects on cardiovascular function including pulmonary hypertension. The purpose of the present study was to examine the mechanisms involved in EPA-induced relaxation of sheep isolated pulmonary artery by measuring isometric tension. Nitric oxide (NO) derived from constitutive nitric oxide synthase (cNOS) was measured by Greiss method in the presence of the inducible nitric oxide synthase (iNOS) selective inhibitor N-[[3-(aminomethyl) phenyl]methyl]-ethanimidamide, dihydrochloride (1400 W). EPA (10(-)(7)-10(-)(4)M) caused concentration-dependent relaxation of sheep pulmonary artery with a pD(2) of 5.56+/-0.09 and E(max) of 87.40+/-3.10% (n=9). N(G)-nitro-L-arginine methyl ester (L-NAME) 100 microM significantly attenuated (E(max) 41.95+/-6.70%; n=8) EPA-induced relaxation of endothelium intact arterial rings. Similarly, endothelium denudation markedly inhibited (E(max) 17.60+/-1.21%; n=4) EPA-induced relaxation. EPA (30 microM) significantly increased the cNOS-derived NO release (10.17+/-0.96; n=8 versus control 7.43+/-0.78 pmol/mg tissue wet wt./h; n=7) in endothelium intact vessels. However, EPA-stimulated NO release was markedly blunted by either 100 microM L-NAME (7.07+/-0.54 pmol/mg tissue wet wt./h; n=8) or endothelium removal (6.97+/-0.87 pmol/mg tissue wet wt./h; n=17). In endothelium-denuded K(+) (80 mM)-depolarized arterial rings, EPA (30 microM) significantly inhibited CaCl(2)-induced contractions (E(max) 42.77+/-5.90% versus control 94.78+/-9.82%; n=5). The fatty acid also inhibited nifedipine (1 microM)-insensitive 5-HT-induced contractions in this vessel (E(max) 70.57+/-4.88% versus control 161.50+/-17.46%; n=5). In conclusion, EPA relaxes sheep pulmonary artery primarily through endothelium-dependent NO release, and the residual endothelium-independent relaxation may result from inhibition of Ca(2+)-influx through L-type calcium channels, as well as 5-HT-stimulated intracellular Ca(2+) release.

Lysosome-dependent Ca2+ release response to Fas activation in coronary arterial myocytes through NAADP: evidence from CD38 gene knockouts

Activation of the death receptor Fas has been implicated in the development of vascular injury or disease, but most studies have focused on its role in the regulation of cell apoptosis and growth. The present study was designed to examine the early response of coronary artery to Fas activation by its ligand, FasL. The hypothesis being tested is that CD38 signaling pathway mediates FasL-induced intracellular Ca(2+) release through nicotinic acid adenine dinucleotide phosphate (NAADP) in mouse coronary arterial myocytes (CAMs) and thereby produces vasoconstriction in coronary arteries. HPLC analysis demonstrated that FasL markedly increased NAADP production in CAMs from wild-type mice (CD38(+/+)) but not in cells from CD38 knockout (CD38(-/-)) mice. Using fluorescent Ca(2+) imaging analysis, we found that FasL (10 ng/ml) significantly increased Ca(2+) release from 142.5 +/- 22.5 nM at the basal level to 509.4 +/- 64.3 nM in CD38(+/+) CAMs but not in CD38(-/-) CAMs. However, direct delivery of NAADP, the CD38 metabolite, into CD38(-/-) CAMs still markedly increased Ca(2+) release, which could be significantly attenuated by a lysosomal function inhibitor, bafilomycin A1 (Baf), or a NAADP antagonist, pyridoxalphosphate-6-azophenyl-2-disulfonic acid. Confocal microscopy further demonstrated that FasL produced a typical two-phase Ca(2+) release with a local Ca(2+) burst from lysosomes, followed by a global Ca(2+) response in CD38(+/+) CAMs. In isolated perfused septal coronary arteries from CD38(+/+) mice, FasL was found to significantly increase U-46619-induced vasoconstriction from 29.2 +/- 7.3 to 63.2 +/- 10.3%, which was abolished by Baf (100 nM). These results strongly indicate that the early response of CAMs to FasL is to increase intracellular Ca(2+) levels and enhance the vascular reactivity through stimulation of NAADP production and lysosome-associated two-phase Ca(2+) release in coronary arteries.

ROS-Dependent Signaling Mechanisms for Hypoxic Ca2+Responses in Pulmonary Artery Myocytes

Hypoxic exposure causes pulmonary vasoconstriction, which serves as a critical physiologic process that ensures regional alveolar ventilation and pulmonary perfusion in the lungs, but may become an essential pathologic factor leading to pulmonary hypertension. Although the molecular mechanisms underlying hypoxic pulmonary vasoconstriction and associated pulmonary hypertension are uncertain, increasing evidence indicates that hypoxia can result in a significant increase in intracellular reactive oxygen species concentration ([ROS](i)) through the mitochondrial electron-transport chain in pulmonary artery smooth muscle cells (PASMCs). The increased mitochondrial ROS subsequently activate protein kinase C-epsilon (PKCepsilon) and NADPH oxidase (Nox), providing positive mechanisms that further increase [ROS](i). ROS may directly cause extracellular Ca(2+) influx by inhibiting voltage-dependent K(+) (K(V)) channels and opening of store-operated Ca(2+) (SOC) channels, as well as intracellular Ca(2+) release by activating ryanodine receptors (RyRs), leading to an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and associated contraction. In concert with ROS, PKCepsilon may also affect K(V) channels, SOC channels, and RyRs, contributing to hypoxic Ca(2+) and contractile responses in PASMCs.

Endothelium-independent vasorelaxant effect of dioclein, a new flavonoid isolated from Dioclea grandiflora, in the rat aorta.

We have investigated the endothelium-independent vasorelaxant effect of the new flavonoid dioclein (5,2',5'-trihydroxy-6-7-dimethoxyflavanone) in the rat aorta. In endothelium-denuded vessels, dioclein induced a concentration-dependent relaxation of aortic rings precontracted with noradrenaline (IC50 = 3.5+/-0.89 x 10(-4) M and KCl (IC50 = 5.2+/-1.2 x 10(-4) M). In the absence of extracellular calcium, dioclein reduced the contraction induced by noradrenaline (maximal reduction approximately 33%) but not that induced by caffeine. Dioclein also produced a concentration-dependent inhibition of the sustained contractions induced by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate in normal (IC50 = 4.0+/-0.2 x 10(-4) M) and Ca2+-free solution (IC50 = 4.0+/-0.3 x 10(-4) M). The results indicate that the endothelium-independent vasorelaxant effect of dioclein may be explained by inhibition of contractions dependent on activation of protein kinase C, voltage-dependent Ca2+ influx and on the release of intracellular Ca2+ stores sensitive to noradrenaline.

Intracellular Ca2+ release‐dependent inactivation of Ca2+ currents in thalamocortical relay neurons

Neuronal Ca(2+) channels are rapidly inactivated by a mechanism that is termed Ca(2+)-dependent inactivation (CDI). In this study we investigated the influence of intracellular Ca(2+) release on CDI of high-voltage-activated Ca(2+) channels in rat thalamocortical relay neurons by combining voltage-clamp, Ca(2+) imaging and immunological techniques. Double-pulse protocols revealed CDI, which depended on the length of the conditioning pulses. Caffeine caused a concentration-dependent increase in CDI that was accompanied by an increase in the duration of Ca(2+) transients. Inhibition of ryanodine receptors and endoplasmic Ca(2+) pumps (by thapsigargin or cyclopiazonic acid) resulted in a reduction of CDI. In contrast, inhibition of inositol 1,4,5-tris-phosphate receptors by intracellular application of 2-aminoethoxy diphenyl borate or heparin did not influence CDI. The block of transient receptor potential channels by extracellular application of 2-aminoethoxy diphenyl borate, however, resulted in a significant reduction of CDI. The central role of L-type Ca(2+) channels was emphasized by the near-complete block of CDI by nifedipine, an effect only surpassed when Ca(2+) was replaced by Ba(2+) and chelated by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid (BAPTA). Trains of action potential-like stimuli induced a strong reduction in high-voltage-activated Ca(2+) current amplitude, which was significantly reduced when intracellular Ca(2+) stores were made inoperative by thapsigargin or Ba(2+)/BAPTA. Western blotting revealed expression of L-type Ca(2+) channels in thalamic and hippocampal tissue but not liver tissue. In summary, these results suggest a cross-signalling between L-type Ca(2+) channels and ryanodine receptors that controls the amount of Ca(2+) influx during neuronal activity.

Fs-laser-induced Ca^2+ concentration change during membrane perforation for cell transfection

Fs-laser based opto-perforation is a gentle method for gene transfer into sensitive cells such as stem cells or primary cells. The high selectivity and the low damage to the cell lead to a high efficiency of transfection. However, there are side effects which induce stress to the cell due to the exchange of intra- and extracellular media as well as the disintegration of the structure of biomolecules resulting from the laser exposure. Moreover, the mechanisms of the optical transfection are still unclear. In this paper, we present our study on calcium (Ca(2+)) homeostasis during cell surgery, especially during laser induced membrane perforation. We show that the manipulation of cells can induce an increase in the cytosolic Ca(2+) concentration. This increase was not observed if the manipulation of the cells was performed in absence of the extracellular calcium indicating the importance of the Ca(2+) uptake. We found, that the uptake of extracellular Ca(2+) strongly depends on the repetition rate and the irradiation time of the laser pulses. The exposure for several seconds to kHz pulses even induces Ca(2+) induced Ca(2+) release. Dependent on the location of perforation, probably in the vicinity of an intracellular Ca(2+) stock, an instantaneous intracellular Ca(2+) release can be induced. Since Ca(2+) could be involved in negative side effect by cell surgery, we propose an application of the optoperforation technique in nominal Ca(2+)-free external solution.