Modulation Of Intracellular Ca2 Research Articles

Drugs That Inhibit The Sarcoplasmic Reticulum Ca2+ ATPase (SERCA) and Prevention of Breast Cancer Cell Proliferation

Breast cancer is the most common cancer in women and a leading cause of death worldwide. Pharmacological modulation of intracellular Ca2+ homeostasis has been proposed as a potential target for therapeutic intervention for breast cancer. In this regard, thapsigargin and cyclopiazonic acid inhibit the Ca2+ ATPase of sarcoplasmic reticulum (SERCA) that pumps Ca2+ into the endoplasmic reticulum (ER) intracellular Ca2+ stores and display high efficacy for killing breast cancer cells. However, these “classic” SERCA blockers are highly toxic and also kill normal cells, thereby preventing use for anticancer therapy. We have determined that new drugs in the benzothiazepine (BZT) family (K201, CGP37157), as well as agents like pimozide and sipatrigine can inhibit SERCA through a novel Ca2+ dependent mechanism. These agents would not affect Ca2+ sequestration in ER when cytosolic Ca2+ levels are high, but potently inhibit this process when cytosolic Ca2+ approaches resting cell levels, which could help prevent ER overload under pathological conditions (ischemia) and provide cellular protection in normal tissues. Therefore, we tested the effectiveness of these drugs to inhibit growth of two breast cancer cell lines, MCF‐7 and MDA‐231, in culture. Our preliminary studies indicate that K201 inhibits MCF‐7 breast cancer cell growth and synergizes with anti‐cancer drugs to increase their effectiveness in killing these cells; thus, MCF‐7 cells are not “protected” by the decreased ER Ca2+ load provided by the SERCA inhibitors; in fact these drugs make them less viable. Building upon this data, we compared the effects of a “classic” SERCA blocker (thapsigargin) to the Ca2+ dependent SERCA blockers above on MCF‐7 and MDA‐231 breast cancer cells. We determined that all SERCA blockers effectively disrupted the metabolic activity of MCF‐7 cells but were much less effective in MDA‐231. In MCF‐7 cells, SERCA blockers also increased the effects of chemotherapeutic drugs (cisplatin, doxorubicin). The results indicate that targeting SERCA has potential to interfere with cancer cell growth. However, our results also suggest that resistance to chemotherapy in MDA‐231 may relate to these cancer cells becoming less dependent on intracellular Ca2+ signaling. We have also determined some of the structural characteristics that are required for BZT to block SERCA. Finally, we also found that drugs reported to do both, protection of normal cells in ischemia and inhibition of cancer cell proliferation, can also display significant SERCA blocking ability. These agents may serve as potential templates for developing new anti‐cancer drugs that target intracellular Ca2+ homeostasisSupport or Funding InformationAPS Frontiers in Physiology Research Fellowship to MLSIUSOM Team Development Award to SAT and JAC

Editorial: Roles of Ion Channels in Immune Cells

Editorial: Roles of Ion Channels in Immune Cells

Oxytocin Stimulates Extracellular Ca2+ Influx Through TRPV2 Channels in Hypothalamic Neurons to Exert Its Anxiolytic Effects.

There is growing interest in anxiolytic and pro-social effects of the neuropeptide oxytocin (OXT), but the underlying intraneuronal mechanisms are largely unknown. Here we examined OXT-mediated anxiolysis in the hypothalamic paraventricular nucleus (PVN) of rats and effects of OXT administration on signaling events in hypothalamic primary and immortalized cells. In vivo, the application of SKF96365 prevented the anxiolytic activity of OXT in the PVN, suggesting that changes in intracellular Ca(2+) mediate the acute OXT behavioral effects. In vitro, mainly in the neurons with autonomous Ca(2+) oscillations, OXT increased intracellular Ca(2+) concentration and oscillation amplitude. Pharmacological intervention revealed OXT-dependent changes in Ca(2+) signaling that required activation of transient receptor potential vanilloid type-2 channel (TRPV2), mediated by phosphoinositide 3-kinase. TRPV2 induced the activation of the anxiolytic mitogen-activated protein kinase kinase (MEK1/2). In situ, immunohistochemistry revealed co-localization of TRPV2 and OXT in the PVN. Thus, functional and pharmacological analyses identified TRPV2 as a mediator of anxiolytic effects of OXT, conveying the OXT signal to MEK1/2 via modulation of intracellular Ca(2+).

Medicinal effect and its JP2/RyR2-based mechanism of Smilax glabra flavonoids on angiotensin II-induced hypertrophy model of cardiomyocytes

Ethnopharmacological relevanceRhizome and root of Smilax glabra Roxb (Liliaceae family) is a widely used traditional Chinese medicine (TCM) named Tu-fu-ling (TFL) for cardiac disease therapy. The TFL flavonoids (TFLF) has been extracted and proven to possess the anti-cardiac hypertrophy effect in our previous reports. Such effect could be mediated by the modulation of intracellular Ca2+ flux in myocardial cells, in which junctophilin-2 (JP2) and ryanodine receptor 2 (RyR2) play an important role. However, its mechanism of the anti-cardiac hypertrophy effect remains unclarified. Materials and methods2μmol/L Ang II was applied to induce hypertrophy model of rat primary cardiomyocytes. After treatment of TFLF at 0.25, 0.5 and 1.0mg/ml, the cell size was microscopic measured, and the protein and mRNA expressions of JP2 and RyR2 in cardiomyocytes were estimated by immunofluorescence imaging, ELISA and real-time PCR assay. ResultsObvious hypertrophy of cardiomyocytes was induced by Ang II but reversed by TFLF from 0.5 to 1.0mg/ml. The protein and mRNA expressions of JP2 and RyR2 in cardiomyocytes were also inhibited by Ang II but restored by TFLF at its dose range. Such effect of TFLF was exerted at a dose dependent manner, which was even better than that of verapamil. ConclusionsOur findings may evidence the correlation between JP2/RyR2 and myocardiac hypertrophy, and indicate the JP2/RyR2-mediated anti-hypertrophy mechanism of TFLF for the first time. It deserves to be developed as a promising TCM candidate of new drug for myocardial hypertrophy treatment.

Androgen receptor-mediated non-genomic regulation of prostate cancer cell proliferation.

Androgen receptor (AR)-mediated signaling is necessary for prostate cancer cell proliferation and an important target for therapeutic drug development. Canonically, AR signals through a genomic or transcriptional pathway, involving the translocation of androgen-bound AR to the nucleus, its binding to cognate androgen response elements on promoter, with ensuing modulation of target gene expression, leading to cell proliferation. However, prostate cancer cells can show dose-dependent proliferation responses to androgen within minutes, without the need for genomic AR signaling. This proliferation response known as the non-genomic AR signaling is mediated by cytoplasmic AR, which facilitates the activation of kinase-signaling cascades, including the Ras-Raf-1, phosphatidyl-inositol 3-kinase (PI3K)/Akt and protein kinase C (PKC), which in turn converge on mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) activation, leading to cell proliferation. Further, since activated ERK may also phosphorylate AR and its coactivators, the non-genomic AR signaling may enhance AR genomic activity. Non-genomic AR signaling may occur in an ERK-independent manner, via activation of mammalian target of rapamycin (mTOR) pathway, or modulation of intracellular Ca2+ concentration through plasma membrane G protein-coupled receptors (GPCRs). These data suggest that therapeutic strategies aimed at preventing AR nuclear translocation and genomic AR signaling alone may not completely abrogate AR signaling. Thus, elucidation of mechanisms that underlie non-genomic AR signaling may identify potential mechanisms of resistance to current anti-androgens and help developing novel therapies that abolish all AR signaling in prostate cancer.

Type IV pilus protein PilA of Pseudomonas aeruginosa modulates calcium signaling through binding the calcium-modulating cyclophilin ligand

The lungs are a major site of Pseudomonas aeruginosa infection in patients with compromised immune systems. We have shown that a large number of cells of the P. aeruginosa wild-type PAO1 strain invade cultured human bronchial epithelial cells (BEAS-2B). In the present study, we investigated whether P. aeruginosa pilus protein PilA might modulate cellular functions by binding to unknown factor(s) in human host cells. Using a yeast two-hybrid screen, we showed that the calcium-modulating cyclophilin ligand (CAMLG), which is involved in Ca(2+) signaling, was the major host cell PilA binding protein. Overexpression of the pilA gene in BEAS-2B cells resulted in a significant increase in cytoplasmic Ca(2+) and consequent upregulation of the activity of the nuclear factor of activated T cells, followed by induction of cyclooxygenase 2 gene expression. Infection of BEAS-2B cells with the P. aeruginosa wild-type strain, but not with the pilA gene knockout strain (ΔpilA), caused a significant increase in intracellular Ca(2+) concentration in infected cells. Therefore, we propose a novel bacterial strategy for PilA modulation of intracellular Ca(2+) signaling through intracellular PilA-CAMLG interaction.

Permeant calcium ion feed-through regulation of single inositol 1,4,5-trisphosphate receptor channel gating

The ubiquitous inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) Ca2+ release channel plays a central role in the generation and modulation of intracellular Ca2+ signals, and is intricately regulated by multiple mechanisms including cytoplasmic ligand (InsP3, free Ca2+, free ATP4−) binding, posttranslational modifications, and interactions with cytoplasmic and endoplasmic reticulum (ER) luminal proteins. However, regulation of InsP3R channel activity by free Ca2+ in the ER lumen ([Ca2+]ER) remains poorly understood because of limitations of Ca2+ flux measurements and imaging techniques. Here, we used nuclear patch-clamp experiments in excised luminal-side-out configuration with perfusion solution exchange to study the effects of [Ca2+]ER on homotetrameric rat type 3 InsP3R channel activity. In optimal [Ca2+]i and subsaturating [InsP3], jumps of [Ca2+]ER from 70 nM to 300 µM reduced channel activity significantly. This inhibition was abrogated by saturating InsP3 but restored when [Ca2+]ER was raised to 1.1 mM. In suboptimal [Ca2+]i, jumps of [Ca2+]ER (70 nM to 300 µM) enhanced channel activity. Thus, [Ca2+]ER effects on channel activity exhibited a biphasic dependence on [Ca2+]i. In addition, the effect of high [Ca2+]ER was attenuated when a voltage was applied to oppose Ca2+ flux through the channel. These observations can be accounted for by Ca2+ flux driven through the open InsP3R channel by [Ca2+]ER, raising local [Ca2+]i around the channel to regulate its activity through its cytoplasmic regulatory Ca2+-binding sites. Importantly, [Ca2+]ER regulation of InsP3R channel activity depended on cytoplasmic Ca2+-buffering conditions: it was more pronounced when [Ca2+]i was weakly buffered but completely abolished in strong Ca2+-buffering conditions. With strong cytoplasmic buffering and Ca2+ flux sufficiently reduced by applied voltage, both activation and inhibition of InsP3R channel gating by physiological levels of [Ca2+]ER were completely abolished. Collectively, these results rule out Ca2+ regulation of channel activity by direct binding to the luminal aspect of the channel.

A data-driven model of a modal gated ion channel: The inositol 1,4,5-trisphosphate receptor in insect Sf9 cells

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) channel is crucial for the generation and modulation of intracellular Ca2+ signals in animal cells. To gain insight into the complicated ligand regulation of this ubiquitous channel, we constructed a simple quantitative continuous-time Markov-chain model from the data. Our model accounts for most experimentally observed gating behaviors of single native IP3R channels from insect Sf9 cells. Ligand (Ca2+ and IP3) dependencies of channel activity established six main ligand-bound channel complexes, where a complex consists of one or more states with the same ligand stoichiometry and open or closed conformation. Channel gating in three distinct modes added one complex and indicated that three complexes gate in multiple modes. This also restricted the connectivity between channel complexes. Finally, latencies of channel responses to abrupt ligand concentration changes defined a model with specific network topology between 9 closed and 3 open states. The model with 28 parameters can closely reproduce the equilibrium gating statistics for all three gating modes over a broad range of ligand concentrations. It also captures the major features of channel response latency distributions. The model can generate falsifiable predictions of IP3R channel gating behaviors and provide insights to both guide future experiment development and improve IP3R channel gating analysis. Maximum likelihood estimates of the model parameters and of the parameters in the De Young–Keizer model yield strong statistical evidence in favor of our model. Our method is simple and easily applicable to the dynamics of other ion channels and molecules.

Calcimimetic R-568 and Its Enantiomer S-568 Increase Nitric Oxide Release in Human Endothelial Cells

BackgroundCalcimimetics, such as R-568, are thought to activate G protein-linked Ca2+-sensing receptor (CaSR) by allosterically increasing the affinity of the receptor for Ca2+ allowing for efficient control of uremic hyperparathyroidism. Several recent studies suggest they possess additional vascular actions. Although it has been postulated that calcimimetics may have a direct effect on CaSR in the blood vessels, further studies are needed to elucidate their vascular CaSR-dependent versus CaSR-independent effects.Methodology/Principal FindingsFocusing on human umbilical vein endothelial cells (HUVECs), we studied the CaSR expression and distribution by Immunofluorescence and Western Blot analysis. CaSR function was evaluated by measuring the potential effect of calcimimetic R-568 and its enantiomer S-568 upon the modulation of intracellular Ca2+ levels (using a single cell approach and FURA-2AM), in the presence or absence of Calhex-231, a negative modulator of CaSR. To address their potential vascular functions, we also evaluated R- and S-568-stimulated enzymatic release of Nitric Oxide (NO) by DAF-2DA, by Nitric Oxide Synthase (NOS) radiometric assay (both in HUVECs and in Human Aortic Endothelial Cells) and by measuring eNOS-ser1177 phosphorylation levels (Immunoblotting). We show that, although the CaSR protein was expressed in HUVECs, it was mainly distributed in cytoplasm while the functional CaSR dimers, usually localized on the plasma membrane, were absent. In addition, regardless of the presence or absence of Calhex-231, both R- and S-568 significantly increased intracellular Ca2+ levels by mobilization of Ca2+ from intracellular stores, which in turn augmented NO release by a time- and Ca2+-dependent increase in eNOS-ser1177 phosphorylation levels.Conclusions/SignificanceTaken together, these data indicate that in human endothelium there is no stereoselectivity in the responses to calcimimetics and that CaSR is probably not involved in the action of R- and S-568. This suggests an additional mechanism in support of the CaSR-independent role of calcimimetics as vasculotrope agents.

Update on vascular endothelial Ca2+signalling: A tale of ion channels, pumps and transporters

A monolayer of endothelial cells (ECs) lines the lumen of blood vessels and forms a multifunctional transducing organ that mediates a plethora of cardiovascular processes. The activation of ECs from as state of quiescence is, therefore, regarded among the early events leading to the onset and progression of potentially lethal diseases, such as hypertension, myocardial infarction, brain stroke, and tumor. Intracellular Ca(2+) signals have long been know to play a central role in the complex network of signaling pathways regulating the endothelial functions. Notably, recent work has outlined how any change in the pattern of expression of endothelial channels, transporters and pumps involved in the modulation of intracellular Ca(2+) levels may dramatically affect whole body homeostasis. Vascular ECs may react to both mechanical and chemical stimuli by generating a variety of intracellular Ca(2+) signals, ranging from brief, localized Ca(2+) pulses to prolonged Ca(2+) oscillations engulfing the whole cytoplasm. The well-defined spatiotemporal profile of the subcellular Ca(2+) signals elicited in ECs by specific extracellular inputs depends on the interaction between Ca(2+) releasing channels, which are located both on the plasma membrane and in a number of intracellular organelles, and Ca(2+) removing systems. The present article aims to summarize both the past and recent literature in the field to provide a clear-cut picture of our current knowledge on the molecular nature and the role played by the components of the Ca(2+) machinery in vascular ECs under both physiological and pathological conditions.

A Data Driven Approach to Learning the Kinetics of Single Molecules: A Case Study of Single Inositol 1,4,5-Trisphosphate Receptor Channel

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) channel plays a central role in the generation and modulation of intracellular Ca2+ signals in animal cells. To gain insight into the crucial intricate ligand regulation of this ubiquitous channel, we constructed the simplest quantitative continuous-time Markov-chain model using a transparent iterative approach, which can be adopted to Markov chain models in general. Our model accounts for all experimentally observed gating behaviors of single native IP3R channels from insect Sf9 cells. Ligand (Ca2+ and IP3) dependencies of channel open probability (PO) established six main ligand-bound channel complexes, where a complex consists of one or more states with the same ligand stoichiometry and open or closed conformation. Channel gating in three distinct modes added one additional complex and indicated that three of the complexes can gate in two different modes. This also restricted the connectivity between channel complexes. Finally, channel responses to abrupt ligand concentration changes defined a model with 9 closed states and 3 open states, and its network topology. The model with 24 parameters can closely reproduce the equilibrium PO and channel gating statistics for all three gating modes for a broad range of ligand concentrations. It also captures the major features of channel response latency distributions. The model can generate falsifiable predictions of IP3R channel gating behaviors not yet explored, and provide insights to both guide future experiment development and improve IP3R channel gating analysis.

Modulation of intracellular Ca2+ levels in chromaffin cells by nanoelectropulses

Exposing chromaffin cells to a single 5ns, 5 MV/m pulse causes Ca2+ influx and a rapid, transient rise in intracellular calcium concentration ([Ca2+]i). A comparison of responses at room temperature versus 37°C revealed no effect of temperature on the magnitude of the increase in [Ca2+]i. The Ca2+ transient, however, was shortened in duration almost twofold at 37°C, indicating that the rate of recovery was temperature-sensitive. Temperature also affected the interval required for a second pulse to elicit another maximal rise in [Ca2+]i, which was shorter at the higher temperature. In addition, a second pulse applied 5s after the first pulse was sufficient to cause cells at room temperature to become refractory to subsequent stimulation. At 37°C, cells became refractory after 5 pulses regardless of whether pulse delivery was at low (1 and 10Hz) or high (1kHz) rates. When refractory, cells showed no signs of swelling or uptake of the impermeant dye YO-PRO-1. These results demonstrate that temperature plays a role in determining how chromaffin cells respond to and become refractory to nanoelectropulses. They also indicate that despite the ultra-short duration of the pulses, pronounced effects on cell excitability result from the application of only very few pulses.

Calcium signaling around Mitochondria Associated Membranes (MAMs)

Calcium (Ca2+) homeostasis is fundamental for cell metabolism, proliferation, differentiation, and cell death. Elevation in intracellular Ca2+ concentration is dependent either on Ca2+ influx from the extracellular space through the plasma membrane, or on Ca2+ release from intracellular Ca2+ stores, such as the endoplasmic/sarcoplasmic reticulum (ER/SR). Mitochondria are also major components of calcium signalling, capable of modulating both the amplitude and the spatio-temporal patterns of Ca2+ signals. Recent studies revealed zones of close contact between the ER and mitochondria called MAMs (Mitochondria Associated Membranes) crucial for a correct communication between the two organelles, including the selective transmission of physiological and pathological Ca2+ signals from the ER to mitochondria. In this review, we summarize the most up-to-date findings on the modulation of intracellular Ca2+ release and Ca2+ uptake mechanisms. We also explore the tight interplay between ER- and mitochondria-mediated Ca2+ signalling, covering the structural and molecular properties of the zones of close contact between these two networks.

α1-adrenergic drugs exhibit affinity to a thapsigargin-sensitive binding site and interfere with the intracellular Ca 2+ homeostasis in human erythroleukemia cells

Even though the erythroleukemia cell lines K562 and HEL do not express α1-adrenoceptors, some α1-adrenergic drugs influence both survival and differentiation of these cell lines. Since Ca 2+ is closely related to cellular homeostasis, we examined the capacity of α1-adrenergic drugs to modulate the intracellular Ca 2+ content in K562 cells. Because of morphological alterations of mitochondria following α1-adrenergic agonist treatment, we also scrutinized mitochondrial functions. In order to visualize the non-adrenoceptor binding site(s) of α1-adrenergic drugs in erythroleukemia cells, we evaluated the application of the fluorescent α1-adrenergic antagonist BODIPY® FL-Prazosin. We discovered that the α1-adrenergic agonists naphazoline, oxymetazoline and also the α1-adrenergic antagonist benoxathian are able to raise the intracellular Ca 2+-content in K562 cells. Furthermore, we demonstrate that naphazoline treatment induces ROS-formation as well as an increase in Δψm in K562 cells. Using BODIPY® FL-Prazosin we were able to visualize the non-adrenoceptor binding site(s) of α1-adrenergic drugs in erythroleukemia cells. Interestingly, the SERCA-inhibitor thapsigargin appears to interfere with the binding of BODIPY® FL-Prazosin. Our data suggest that the effects of α1-adrenergic drugs on erythroleukemia cells are mediated by a thapsigargin sensitive binding site, which controls the fate of erythroleukemia cells towards differentiation, senescence and cell death through modulation of intracellular Ca 2+.

Disruption of a Nuclear NFATc2 Protein Stabilization Loop Confers Breast and Pancreatic Cancer Growth Suppression by Zoledronic Acid

The aminobisphosphonate zoledronic acid has elicited significant attention due to its remarkable anti-tumoral activity, although its detailed mechanism of action remains unclear. Here, we demonstrate the existence of a nuclear GSK-3β-NFATc2 stabilization pathway that promotes breast and pancreatic cancer growth in vitro and in vivo and serves as a bona fide target of zoledronic acid. Specifically, the serine/threonine kinase GSK-3β stabilizes nuclear NFATc2 through phosphorylation of the serine-rich SP2 domain, thus protecting the transcription factor from E3-ubiquitin ligase HDM2-mediated proteolysis. Zoledronic acid disrupts this NFATc2 stabilization pathway through two mechanisms, namely GSK-3β inhibition and induction of HDM2 activity. Upon nuclear accumulation, HDM2 targets unphosphorylated NFATc2 for ubiquitination at acceptor lysine residues Lys-684/Lys-897 and hence labels the factor for subsequent proteasomal degradation. Conversely, mutagenesis-induced constitutive serine phosphorylation (Ser-215, Ser-219, and Ser-223) of the SP2 domain prevents NFATc2 from HDM2-mediated ubiquitination and degradation and consequently rescues cancer cells from growth suppression by zoledronic acid. In conclusion, this study demonstrates a critical role of the GSK-3β-HDM2 signaling loop in the regulation of NFATc2 protein stability and growth promotion and suggests that double targeting of this pathway is responsible, at least to a significant part, for the potent and reliable anti-tumoral effects of zoledronic acid.

Glucose Regulates Free Cytosolic Zn2+ Concentration, Slc39 (ZiP), and Metallothionein Gene Expression in Primary Pancreatic Islet β-Cells

Zn2+ is an important cofactor for insulin biosynthesis and storage in pancreatic β-cells. Correspondingly, polymorphisms in the SLC30A8 gene, encoding the secretory granule Zn2+ transporter ZnT8, are associated with type 2 diabetes risk. Using a genetically engineered (FRET)-based sensor (eCALWY-4), we show here that elevated glucose time-dependently increases free cytosolic Zn2+ ([Zn2+]cyt) in mouse pancreatic β-cells. These changes become highly significant (853 ± 96 pm versus 452 ± 42 pm, p < 0.001) after 24 h and are associated with increased expression of the Zn2+ importer family members Slc39a6, Slc39a7, and Slc39a8, and decreased expression of metallothionein 1 and 2. Arguing that altered expression of the above genes is not due to altered [Zn2+]cyt, elevation of extracellular (and intracellular) [Zn2+] failed to mimic the effects of high glucose. By contrast, increases in intracellular cAMP prompted by 3-isobutyl-1-methylxanthine and forskolin partially mimicked the effects of glucose on metallothionein, although not ZiP, gene expression. Modulation of intracellular Ca2+ and insulin secretion with pharmacological agents (tolbutamide and diazoxide) suggested a possible role for changes in these parameters in the regulation of Slc39a6 and Slc39a7 but not Slc39a8, nor metallothionein expression. In summary, 1) glucose induces increases in [Zn2+]cyt, which are then likely to facilitate the processing and/or the storage of insulin and its cocrystallization with Zn2+, and 2) these increases are associated with elevated expression of zinc importers. Conversely, a chronic increase in [Zn2+]cyt following sustained hyperglycemia may contribute to β-cell dysfunction and death in some forms of diabetes.

11,12‐EET activated non‐selective cation channels in the cortical collecting duct

Patch-clamp experiments have identified a 30 pS non-selective (NS) cation channel in the apical membrane of the principal cell (PC) in the cortical collecting duct (CCD). This cation channel was inhibited by gadolinium, a non-specific TRP channel inhibitor, suggesting the possibility that the 30 pS NS cation channel may be a TRP channel. Moreover, single channel recordings demonstrated that the 30 pS NS cation channel was activated by 11,12-epoxyeicosatrienoic acid (11,12-EET), a CYP-epoxygenase-dependent metabolite of arachidonic acid, in both cell-attached and inside-out patches. To study the possibility that the 11,12-EET-sensitive cation channel may be involved in regulating Ca2+ influx in the CCD, we used fura-2 to measure the effect of 11,12-EET on the intracellular Ca2+. Additional 100 nM 11,12-EET significantly increased intracellular Ca2+ concentrations in PC of the rat CCD. Furthermore, removal of extracellular Ca2+ or applying gadolinium to the bath abolished the stimulatory effect of 11,12-EET on the intracellular Ca2+. We conclude that 11,12-EET activated 30 pS cation channels and that the 11,12-EET-sensitive cation channel may play a role in modulation of intracellular Ca2+ flux in the CCD.

Modulation of Intracellular Ca 2+ Concentration in Brain Microvascular Endothelial Cells in vitro by Acoustic Cavitation

Localized delivery of therapeutic agents through the blood–brain barrier (BBB) is a clinically significant task that remains challenging. Ultrasound (US) application after intravenous administration of microbubbles has been shown to generate localized BBB opening in animal models but the detailed mechanisms are not yet fully described. The current study investigates the effects of US-stimulated microbubbles on in vitro murine brain microvascular endothelial (bEnd.3) cells by monitoring sonoporation and changes in intracellular calcium concentration ([Ca 2+] i) using real-time fluorescence and high-speed brightfield microscopy. Cells seeded in microchannels were exposed to a single US pulse (1.25 MHz, 10 cycles, 0.24 MPa peak negative pressure) in the presence of Definity™ microbubbles and extracellular calcium concentration [Ca 2+] o = 0.9 mM. Disruption of the cell membrane was assessed using propidium iodide (PI) and change in the [Ca 2+] i was measured using fura-2. Cells adjacent to a microbubble exhibited immediate [Ca 2+] i changes after US pulse with and without PI uptake and the [Ca 2+] i changes were twice as large in cells with PI uptake. Cell viability assays showed that sonoporated cells could survive with modulation of [Ca 2+] i and uptake of PI. Cells located near sonoporated cells were observed to exhibit changes in [Ca 2+] i that were delayed from the time of US application and without PI uptake. These results demonstrate that US-stimulated microbubbles not only directly cause changes in [Ca 2+] i in brain endothelial cells in addition to sonoporation but also generate [Ca 2+] i transients in cells not directly interacting with microbubbles, thereby affecting cells in larger regions beyond the cells in contact with microbubbles. (E-mail: cxdeng@umich.edu)

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.

Optical Stimulation of Ca2+ Transients in Smooth Muscle Cells

The modulation of intracellular Ca2+ plays a huge role in controlling important cell functions such as cell division, signaling, contraction and cell death. To date, intracellular Ca2+ dynamics have mainly been investigated using electrophysiological measurement techniques such as patch clamp experiments often used in combination with fluorescence imaging techniques. To activate intracellular channels, drugs are often used. However, because of diffusion and mixing constraints there is little precision in the time course of channel activation and deactivation. In addition, the invasive nature of conventional whole cell patch clamp techniques disturbs the intracellular environment and may so alter channel behaviour.We report a non-invasive technique that induces controlled Ca2+ responses in isolated smooth muscle cells using a holosteric incoherent light source as a stimulus. Using a conventional epi-fluorescence microscope configuration, cells labeled with a Ca2+ fluorescent indicator (Fluo-3AM) were stimulated using the low intensity light (<1.5mW ≅ λ=488nm) and the resultant Ca2+ transients were visualized using a highly sensitive CCD camera. We will describe the cell stimulation protocol used and present data demonstrating the efficacy of this low cost and minimally invasive technique. We will also describe the investigation into the origin of the light induced Ca2+ responses.