Fura-2 Fluorescence Ratio Research Articles

Cellular and physiological approaches to evaluate the chelating effect of Chlorella on metal ion stressed lymphocytes.

Chlorella is a green alga consumed as dietary food supplement in pulverized form. In addition to its high nutritional value, it is reported as an excellent detoxifying agent. The pulverized Chlorella is partially soluble in water and insoluble portion has been reported for removal of mercury, cadmium and radioactive strontium from body. Chlorella contains a variety of metal-binding functional groups such as carboxyl, amino, phosphoryl, hydroxyl and carbonyl groups, which has high affinity towards various metal ions. The present study was envisaged to evaluate the chelating effect of water soluble fraction of Chlorella powder (AqCH) on metal ions. Fura-2 fluorescence ratio (F340/F380) was measured by fluorescence spectrometer (FS) after the exposure of chloride salt of metals viz., strontium, cobalt, barium, cesium, thallium and mercury to lymphocytes. Pretreatment of AqCH (0.1-20mg mL-1) was given to evaluate the attenuating effect on fura-2 fluorescence ratio induced by metal ions. The intracellular levels of these metal ions were analyzed by atomic absorption spectrophotometer (AAS) and fluorescence microscopy (FM). Pretreatment with AqCH significantly attenuated the metal induced fluorescence ratio in dose-dependent manner. The results of AAS and FM were found in coherence with fura-2 fluorescence ratio which emphasized that AqCH significantly prevented the metal ions internalization. The present study suggests AqCH chelates with these metal ions and prevents its interaction with cells thereby reducing the intracellular mobilization of Ca2+.

Impact of Maternal Vitamin D Receptor (VDR) Gene Polymorphisms on Spontaneous Preterm Birth (Egyptian Case-Control Study)

Diverse effects of Ba^(2+) and Co^(2+) on fura-2 fluorescence ratio signals (FR, F350/F380) were examined in cultured bovine aortic endothelial cells (BAECs). ATP (3 μM) induced FR oscillations in normal Ca^(2+)-containing solutions. Co^(2+), but not Ba^(2+), inhibited ATP-induced FR oscillations. In Ca^(2+)-free solutions, 3 μM ATP only elicited a single FR transient. When Ba^(2+) replaced Ca^(2+) in the perfusate, 3 μM ATP provoked a distinct FR transient. The maximal rate of rise of the signal was greater than seen without Ba^(2+) and the FR increased and maintained to a high steady level. When external Ca^(2+) was replaced by Co^(2+), ATP-induced FR transient oscillated with a long recovery time constant. In a survey of the single fluorescence at excitation wavelength 350 nm (F350), Co^(2+) decreased the intensity in the presence of ATP and Ca^(2+). Ba^(2+), however, increased F350 and FR as well, especially in Ca^(2+)-free solutions. The results suggest that ATP-induced Ca^(2+) fluctuation is balanced by Ca^(2+) release from internal stores and influx from external Ca^(2+). Ba2+ seems to be permeable through Ca^(2+) channels and competes with Ca^(2+) for the dye. Co^(2+) blocks Ca^(2+) channels but does not inhibit ATP-mediated Ca^(2+) release in cells.

Terbutaline, forskolin and cAMP reduce secretion of aqueous humour in the isolated bovine eye.

In order to elucidate involvement of cyclic AMP and intracellular Ca2+,[Ca2+]i, in the modulation of aqueous humour formation (AHF), we studied the effects of terbutaline, forskolin and 8-Br-cAMP in the isolated bovine eye. We also studied the interaction of cAMP on calcium signaling in cultured ciliary epithelial (CE) cells. Drug effects on AHF were measured by fluorescein dilution. Drug effects on [Ca2+]i were studied by the fura-2 fluorescence ratio technique. Terbutaline (100 nmol-100 M), forskolin (30 nM-100 M) or 8-Br-cAMP (100 nM- 10 μM), administered in the arterial perfusate produced significant reductions in AHF. The AH reducing effect of terbutaline was blocked by a selective inhibitor of protein kinase A (KT-5720). ATP (100 M) caused a rapid, transient (peak) increase in [Ca2+]i followed by a sustained plateau phase lasting more than 5 minutes. Preincubation of the cells (6 min) with terbutaline, forskolin or 8-Br-cAMP significantly reduced the peak calcium response to ATP. The sustained plateau phase of the response, on the other hand, was augmented by each of the agents. KT-5720 partially reversed the inhibitory effect of terbutaline on the peak and totally inhibited its effect on the plateau phase. These data indicate: (a) that AHF in the bovine eye can be manipulated through cyclic AMP, operating via protein kinase A, (b) that protein kinase A can affect [Ca2+]i homeostasis, (c) that calcium release from the intracellular store, not the entry, affects AHF, and (d) that interaction of [Ca2+]i with cAMP plays a role in modulating AH secretion.

Mechano-Chemo-Transduction in Rabbit Cardiomyocytes Mediated by no Signaling

Background: Increased mechanical stress under pathological conditions such as hypertension, infarction and fibrosis can cause arrhythmias and heart failure. However, little is known about Mechano-Chemo-Transduction (MCT) mechanisms that underlie heart disease development. We developed a novel Cell-in-Gel system to control mechanical loading on cardiomyocytes during excitation-contraction coupling. Here we investigate mechanical load effects on modulating Ca2+ signaling and contraction dynamics to test the hypothesis NO signaling mediates MCT.Methods: Freshly isolated adult rabbit ventricular myocytes were embedded in polyvinyl alcohol (10%) and crosslinked with tetravalent boronate-PEG (7.5%) to form a 3D viscoelastic hydrogel. The Cell-in-Gel system was continuously perfused with Tyrode's solution and electrically paced at 0.5 Hz. Cardiomyocyte contraction and Ca2+ transients were measured in-gel and compared with load-free cells in solution.Results: Cardiomyocytes contracting in-gel under mechanical load showed augmented systolic Ca2+ transients (Fura-2 fluorescence ratio 1.47 ± 0.07 in-gel vs 0.85 ± 0.03 load-free), revealing MCT transduces mechanical stress to increase intracellular Ca2+ release. NOS inhibition using 1 mM L-NAME reduced the in-gel Ca2+ transient by 33% (n=15), suggesting NO signaling mediates MCT. Cells in-gel showed 8% decrease in contraction (fractional shortening 13.5 ± 0.4% in-gel vs 14.7 ± 0.3% load-free). Cells in-gel treated with L-NAME demonstrated a further reduction in contraction (11.6 ± 0.8%) as a result of decreased MCT by NOS inhibition. Importantly, 12% of cells in-gel showed mechanical load-induced alternans, which was abolished with L-NAME treatment, suggesting NO signaling contributes to load-induced Ca2+ dysregulation.Conclusion: Our findings demonstrate MCT increases the systolic Ca2+ transient to enhance contractility in response to increased afterload, serving a compensatory mechanism to autoregulate contractility. MCT is attenuated by inhibiting NOS. These results suggest NOS mediates increased Ca2+ signaling in cardiomyocytes under mechanical load, providing a mechanistic basis for the Anrep effect.

Heavy metal chelator TPEN attenuates fura-2 fluorescence changes induced by cadmium, mercury and methylmercury.

Stimulation with heavy metals is known to induce calcium (Ca2+) mobilization in many cell types. Interference with the measurement of intracellular Ca2+ concentration by the heavy metals in cells loaded with Ca2+ indicator fura-2 is an ongoing problem. In this study, we analyzed the effect of heavy metals on the fura-2 fluorescence ratio in human SH-SY5Y neuroblastoma cells by using TPEN, a specific cell-permeable heavy metal chelator. Manganese chloride (30–300 µM) did not cause significant changes in the fura-2 fluorescence ratio. A high concentration (300 µM) of lead acetate induced a slight elevation in the fura-2 fluorescence ratio. In contrast, stimulation with cadmium chloride, mercury chloride or MeHg (3–30 µM) elicited an apparent elevation of the fura-2 fluorescence ratio in a dose-dependent manner. In cells stimulated with 10 or 30 µM cadmium chloride, the addition of TPEN decreased the elevated fura-2 fluorescence ratio to basal levels. In cells stimulated with mercury or MeHg, the addition of TPEN significantly decreased the elevation of the fura-2 fluorescence ratio induced by lower concentrations (10 µM) of mercury or MeHg, but not by higher concentrations (30 µM). Pretreatment with Ca2+ channel blockers, such as verapamil, 2-APB or lanthanum chloride, resulted in different effects on the fura-2 fluorescence ratio. Our study provides a characterization of the effects of several heavy metals on the mobilization of divalent cations and the toxicity of heavy metals to neuronal cells.

The Spider Toxin GsMTx-4 Blocks TRPV4 Cation Channels Expressed in HEK-293 Cells

The transient receptor potential channel TRPV4 is a calcium conducting, osmosensitive cation channel and expressed in motoneurons and skeletal muscles. In mouse muscle fibers TRPV4 forms or contributes to a mechanosensitive cation channel. The molecular architecture of this channel is still unclear. Since mechanosensitive cation channels can be blocked by the peptide toxin GsMTx-4, we studied whether TRPV4 itself is sensitive to GsMTx-4. To this end a TRPV4-YFP construct was functionally expressed in HEK-293 cells. Intracellular Ca2+ concentrations were monitored with the fluorescent Ca2+ indicator Fura-2 and the data presented as Fura-2 fluorescence ratios after alternate excitation at 340 and 380 nm. Non-transfected cells (n=47) had an average resting ratio of 0.39 (range 0.38 and 0.42), while TRPV4 transfected cells (n=524) showed on average an increased fluorescence ratio of 1.11 ranging from 0.44 to 2.71. Application of 4a-PDD (5 µM), a known TRPV4 activator, increased the fluorescence ratios in the HEK cells to 1.72, 1.73 and 1.77 with a peak or plateau at 1-3 min (n=13-19 cells, each experiment). The effect of 4a-PDD could be reversed within about 10-30 min by application of the TRPV4 blocker HC-067047 (1 µM) and GsMTx-4 (5 µM). The final resting ratios were 0.77 for HC-067047 and 1.16 for GsMTx-4. At a concentration of 1 µM the toxin was almost ineffective. The results indicate that TRPV4 activity can be inhibited by GsMTx-4. However, the required concentration of the toxin is higher than those required for inhibition of TRPC6, TRPC1 and voltage-gated sodium channels. The results support the view that TRPV4 may be part of mechanosensitive cation channels that are sensitive to the channel blocker GsMTx-4.The work was supported by the Deutsche Duchenne Stiftung der aktion benni & co e. V.

Mechano-Chemotransduction in the Single Cardiac Myocyte Contracting in 3D Elastic Gel

Objective: Mechanical stress under pathological conditions such as hypertension, infarction and fibrosis can cause heart failure and arrhythmias. However, little is known about the mechanotransduction mechanisms that underlie heart disease development due to previous lack of practical techniques to control the mechanical stress on single myocytes necessary for investigating at cellular and molecular levels. Here we use this system to study the mechanical load effects on modulating myocyte Ca2+ signaling and contraction dynamics. Methods: Recently, we developed a novel Cell-in-Gel system that allows control of mechanical load on single rabbit myocytes during excitation-contraction coupling in a 3D elastic gel matrix composed of polyvinyl alcohol (PVA) and tetravalent boronate-PEG crosslinker. The mechanical load can be controlled by adjusting gel stiffness with different mixing ratios of PVA and crosslinker. Results: Myocyte contraction and calcium transients were measured in-gel and compared with load-free cells. Contracting cells in-gel showed a significantly lower fractional shortening (12.6 ± 0.9 in-gel vs. 18.2 ± 0.9 load-free, p<0.001), demonstrating a “knock-down factor” of 31% when the myocyte is pulling mechanical load. Contraction departure and return velocities were significantly slower in-gel than in the load-free state as expected. However, the systolic calcium transient was greater in-gel than load-free (Fura-2 fluorescence ratio peak height 1.47 ± 0.09 in-gel vs. 0.87 ± 0.04 load-free, p<0.0001), revealing the mechano-chemotransduction that translates external stress to intracellular Ca2+ increase. The calcium transient departure and return velocities were also significantly higher in-gel than load-free. Conclusions: Our newly-developed versatile Cell-in-Gel system provides a novel experimental method to control mechanical stress at the single cell level for investigating mechano-chemotransduction pathways in intact myocytes. The above experimental results are consistent with our modeling predictions, demonstrating the mechanical load effects on altering myocyte Ca2+ handling and contraction dynamics.

Type 1 Inositol (1,4,5)-Trisphosphate Receptor Activates Ryanodine Receptor 1 to Mediate Calcium Spark Signaling in Adult Mammalian Skeletal Muscle

Functional coupling between inositol (1,4,5)-trisphosphate receptor (IP(3)R) and ryanodine receptor (RyR) represents a critical component of intracellular Ca(2+) signaling in many excitable cells; however, the role of this mechanism in skeletal muscle remains elusive. In skeletal muscle, RyR-mediated Ca(2+) sparks are suppressed in resting conditions, whereas application of transient osmotic stress can trigger activation of Ca(2+) sparks that are restricted to the periphery of the fiber. Here we show that onset of these spatially confined Ca(2+) sparks involves interaction between activation of IP(3)R and RyR near the sarcolemmal membrane. Pharmacological prevention of IP(3) production or inhibition of IP(3)R channel activity abolishes stress-induced Ca(2+) sparks in skeletal muscle. Although genetic ablation of the type 2 IP(3)R does not appear to affect Ca(2+) sparks in skeletal muscle, specific silencing of the type 1 IP(3)R leads to ablation of stress-induced Ca(2+) sparks. Our data indicate that membrane-delimited signaling involving cross-talk between IP(3)R1 and RyR1 contributes to Ca(2+) spark activation in skeletal muscle.

Angiotensin II inhibits interleukin-6 mRNA expression of LPS-stimulated macrophages through down-regulating calcium signaling

BackgroundThe renin-angiotensin system plays a key role in the regulation of blood pressure following hemorrhage and shock. Recent studies also suggest renin-angiotensin system regulates inflammatory mediator production although the amechanism is largely unknown. This purpose of the study was to examine the effect of angiotensin II on macrophage (MØ) IL-6 messenger RNA (mRNA) expression induced by lipopolysaccharide (LPS) and on the alterations in the calcium influx. MethodsJ774A.1 cells, a mouse MØ cell line, were exposed to E. coli LPS (1 or 10 μg/ml) in the presence of angiotensin II (10 nM to 1 μM). IL-6 mRNA expression was determined by the reverse transcription polymerase chain reaction technique. IL-6 protein production was measured by ELISA. To examine the involvement of calcium signaling in IL-6 mRNA expression, MØ were exposed to various calcium agonists and antagonists in the presence of LPS stimulation. Changes of intracellular [Ca2+] by LPS stimulation and angiotensin II treatment were determined by a fura-2 fluorescence ratio method. ResultsLPS stimulation increased MØ IL-6 mRNA expression, which was inhibited by Angiotensin II in a dose-dependent fashion. Both thapsigargin and A23187 augmented the IL-6 mRNA levels induced by LPS stimulation, but only thapsigargin was able to induce IL-6 mRNA directly. TMB-8 but not verapamil inhibited LPS-stimulated MØ IL-6 mRNA. Finally, angiotensin II significantly altered the changes in intracellular [Ca2+] levels induced by LPS stimulation by reducing both the peak and slope of calcium spikes. ConclusionsOur data show that calcium signaling is closely related to IL-6 mRNA expression. Angiotensin II inhibits IL-6 mRNA expression of LPS-stimulated MØ. The inhibitory effects of angiotensin II appear, at least in part, to be mediated through down regulating calcium dependent pathways.

Visualization of Subunit-Specific Delivery of Glutamate Receptors to Postsynaptic Membrane during Hippocampal Long-Term Potentiation

An increase in the number of AMPA-type glutamate receptors (AMPARs) is critical for long-term potentiation (LTP), synaptic plasticity regarded as a basal mechanism of learning and memory. However, when and how each type of AMPAR reaches the postsynaptic membrane remain unclear. We have developed experimental methods to form postsynaptic-like membrane (PSLM) on a glass surface to precisely visualize the location and movement of receptors. We observed fluorescence-labeled AMPAR subunits (GluA1-3) around PSLM with total internal reflection fluorescence microscopy. The increases of GluA1, 2, and 3 in PSLM showed different time courses after LTP induction. GluA1 increased first, and was exocytosed to the periphery of PSLM soon after LTP induction. GluA2 and GluA3 initially decreased, and then increased. Exocytosis of GluA2 and GluA3 occurred primarily in non-PSLM, and later than exocytosis of GluA1. Thus, GluA1-3 appear to increase in the postsynaptic membrane through distinct pathways during LTP.

N-Methyl-d-aspartate Receptor Mechanosensitivity Is Governed by C Terminus of NR2B Subunit

N-methyl-D-aspartate receptors (NMDARs), critical mediators of both physiologic and pathologic neurological signaling, have previously been shown to be sensitive to mechanical stretch through the loss of its native Mg(2+) block. However, the regulation of this mechanosensitivity has yet to be further explored. Furthermore, as it has become apparent that NMDAR-mediated signaling is dependent on specific NMDAR subtypes, as governed by the identity of the NR2 subunit, a crucial unanswered question is the role of subunit composition in observed NMDAR mechanosensitivity. Here, we used a recombinant system to assess the mechanosensitivity of specific subtypes and demonstrate that the mechanosensitive property is uniquely governed by the NR2B subunit. NR1/NR2B NMDARs displayed significant stretch sensitivity, whereas NR1/NR2A NMDARs did not respond to stretch. Furthermore, NR2B mechanosensitivity was regulated by PKC activity, because PKC inhibition reduced stretch responses in transfected HEK 293 cells and primary cortical neurons. Finally, using NR2B point mutations, we identified a PKC phosphorylation site, Ser-1323 on NR2B, as a unique critical regulator of stretch sensitivity. These data suggest that the selective mechanosensitivity of NR2B can significantly impact neuronal response to traumatic brain injury and illustrate that the mechanical tone of the neuron can be dynamically regulated by PKC activity.

Cyanide modulates on the cervical activity in neonatal rat brainstem-spinal cord preparation

Methylmercury (MeHg) is a widespread environmental toxicant with major actions on the central nervous system. Among the neurons reportedly affected in cases of Hg poisoning are motor neurons; however, the direct cellular effects of MeHg on motor neurons have not been reported. Ratiometric fluorescence imaging, using the Ca2+-sensitive fluorophore fura-2, was used to examine the effect of MeHg on Ca2+ homeostasis in primary cultures of mouse spinal motor neurons. In vitro MeHg exposure at concentrations (0.1–2 μM) known to affect other neurons in culture differentially, induced a biphasic rise in fura-2 fluorescence ratio indicating an increase in [Ca2+]i. The time-to-onset of these fura-2 fluorescence ratio changes was inversely correlated with MeHg concentration. TPEN (20 μM), a non-Ca2+, divalent cation chelator, reduced the amplitude of the increase in fura-2 fluorescence induced by MeHg in the first phase, indicating that both Ca2+ and non-Ca2+ divalent cations contribute to the MeHg-induced effect. When examining various Ca2+ entry pathways as possible targets contributing to Ca2+ influx, we found that excitatory amino acid receptor blockers MK-801 (15 μM), and AP-5 (100 μM)—both NMDA receptor-operated ion channel blockers, CNQX (20 μM), a non-NMDA receptor blocker, and the voltage-dependent Ca2+ channel blockers nifedipine (1 μM) and ω-conotoxin-GVIA (1 μM) all significantly delayed the development of increased Ca2+ caused by MeHg. The voltage-dependent Na+ channel blocker tetrodotoxin (TTX, 1 μM) did not alter the MeHg-induced increases in fura-2 fluorescence ratio. Thus, MeHg alters Ca2+ homeostasis in mouse spinal motor neurons through excitatory amino acid receptor-mediated pathways, and nifedipine and ω-conotoxin-GVIA-sensitive pathways. Spinal motor neurons are highly sensitive to this effect of acute exposure to MeHg.

Ca2+ entry pathways in mouse spinal motor neurons in culture following in vitro exposure to methylmercury

Methylmercury (MeHg) is a widespread environmental toxicant with major actions on the central nervous system. Among the neurons reportedly affected in cases of Hg poisoning are motor neurons; however, the direct cellular effects of MeHg on motor neurons have not been reported. Ratiometric fluorescence imaging, using the Ca2+-sensitive fluorophore fura-2, was used to examine the effect of MeHg on Ca2+ homeostasis in primary cultures of mouse spinal motor neurons. In vitro MeHg exposure at concentrations (0.1–2μM) known to affect other neurons in culture differentially, induced a biphasic rise in fura-2 fluorescence ratio indicating an increase in [Ca2+]i. The time-to-onset of these fura-2 fluorescence ratio changes was inversely correlated with MeHg concentration. TPEN (20μM), a non-Ca2+, divalent cation chelator, reduced the amplitude of the increase in fura-2 fluorescence induced by MeHg in the first phase, indicating that both Ca2+ and non-Ca2+ divalent cations contribute to the MeHg-induced effect. When examining various Ca2+ entry pathways as possible targets contributing to Ca2+ influx, we found that excitatory amino acid receptor blockers MK-801 (15μM), and AP-5 (100μM)—both NMDA receptor-operated ion channel blockers, CNQX (20μM), a non-NMDA receptor blocker, and the voltage-dependent Ca2+ channel blockers nifedipine (1μM) and ω-conotoxin-GVIA (1μM) all significantly delayed the development of increased Ca2+ caused by MeHg. The voltage-dependent Na+ channel blocker tetrodotoxin (TTX, 1μM) did not alter the MeHg-induced increases in fura-2 fluorescence ratio. Thus, MeHg alters Ca2+ homeostasis in mouse spinal motor neurons through excitatory amino acid receptor-mediated pathways, and nifedipine and ω-conotoxin-GVIA-sensitive pathways. Spinal motor neurons are highly sensitive to this effect of acute exposure to MeHg.

Activation of NADPH Oxidase 1 Increases Intracellular Calcium and Migration of Smooth Muscle Cells

Redox-dependent migration and proliferation of vascular smooth muscle cells (SMCs) are central events in the development of vascular proliferative diseases; however, the underlying intracellular signaling mechanisms are not fully understood. We tested the hypothesis that activation of Nox1 NADPH oxidase modulates intracellular calcium ([Ca(2+)](i)) levels. Using cultured SMCs from wild-type and Nox1 null mice, we confirmed that thrombin-dependent generation of reactive oxygen species requires Nox1. Thrombin rapidly increased [Ca(2+)](i), as measured by fura-2 fluorescence ratio imaging, in wild-type but not Nox1 null SMCs. The increase in [Ca(2+)](i) in wild-type SMCs was inhibited by antisense to Nox1 and restored by expression of Nox1 in Nox1 null SMCs. Investigation into potential mechanisms by which Nox1 modulates [Ca(2+)](i) showed that thrombin-induced inositol triphosphate generation and thapsigargin-induced intracellular calcium mobilization were similar in wild-type and Nox1 null SMCs. To examine the effects of Nox1 on Ca(2+) entry, cells were either bathed in Ca(2+)-free medium or exposed to dihydropyridines to block L-type Ca(2+) channel activity. Treatment with nifedipine or removal of extracellular Ca(2+) reduced the thrombin-mediated increase of [Ca(2+)](i) in wild-type SMCs, whereas the response in Nox1 null SMCs was unchanged. Sodium vanadate, an inhibitor of protein tyrosine phosphatases, restored the thrombin-induced increase of [Ca(2+)](i) in Nox1 null SMCs. Migration of SMCs was impaired with deficiency of Nox1 and restored with expression of Nox1 or the addition of sodium vanadate. In summary, we conclude that Nox1 NADPH oxidase modulates Ca(2+) mobilization in SMCs, in part through regulation of Ca(2+) influx, to thereby promote cell migration.

Decreased Liver Tumorigenesis in Sulfatase 2 (SULF2) Knockout Mice May Be Mediated Through Inhibition of the Epithelial-Mesenchymal Transition (EMT)

Background and Aim. The pathogenesis and progression of pancreatic cancer involves conversion of TGFβ suppressive signaling in early lesions to an unmasked, TGFβ proliferative signaling cascade in metastatic lesions. We have previously demonstrated that TGFβ proliferative signaling downregulates the tumor suppressor PTEN and induces motility of pancreatic cancer cells after ligand-receptor binding with subsequent increase in cytoplasmic free Ca2+ concentration ([Ca2+]cyt) and activation of PKCα and NF-κB. [Ca2+]cyt homeostasis is important to maintain cell functionality dysregulation of which contributes to tumorigenesis of pancreas. Transient receptor potential ion channels (TRPC) help regulate [Ca2+]cyt, and here we evaluated the role of TRPC channels in regulating TGFβ proliferative signaling. Methods. BxPc3 pancreatic cancer cells were treated with TGFβ (10 ng/mL), and TRPC and PTEN expression were analyzed by RT-PCR and/or Western blots. [Ca2+]cyt was assessed using Fura-2 fluorescence ratio digital imaging. Pharmacological reagents (TRPC inhibitors: 2-APB, SKF96365, and LaCl3) and siRNAs to TRPCs were used to determine the role of TRPCs on TGFβ-induced PTEN regulation and cell migration. Cell motility was assessed by Boyden chamber migration assay. Results. BxPc3 cells express TRPC1, 4, and 6 isoforms. TGFβ induced an increase in [Ca2+]cyt in Ca2+-free solutions and also induced an increase in [Ca2+]cyt when extracellular Ca2+ was restored. TGFβ-induced Ca2+ entry involves the activation of TRPC, as the TRPC inhibitors 2-APB, SKF96365, and LaCl3, were able to reduce peak [Ca2+]cyt by 80%, 40%, and 29%, respectively, and reversed TGFβ-induced PTEN downregulation. The 2-APB, SKF96365, and LaCl3 inhibitors also reduced TGFβinduced cell motility. Using siRNA, inhibition of TRPC1 but not other isoforms, successfully prevented TGFβ-induced cell motility. Conclusion. TRPC1 regulates TGFβ-induced downregulation of PTEN and pancreatic cell motility. This aspect of TGFβ regulation of PTEN and pancreatic cell motility might be exploited therapeutically to control pancreatic cancer metastasis.

Clinical Outcome of Polypoid Lesion of Gallbladder 10 mm or Larger

Background and Aim. The pathogenesis and progression of pancreatic cancer involves conversion of TGFβ suppressive signaling in early lesions to an unmasked, TGFβ proliferative signaling cascade in metastatic lesions. We have previously demonstrated that TGFβ proliferative signaling downregulates the tumor suppressor PTEN and induces motility of pancreatic cancer cells after ligand-receptor binding with subsequent increase in cytoplasmic free Ca2+ concentration ([Ca2+]cyt) and activation of PKCα and NF-κB. [Ca2+]cyt homeostasis is important to maintain cell functionality dysregulation of which contributes to tumorigenesis of pancreas. Transient receptor potential ion channels (TRPC) help regulate [Ca2+]cyt, and here we evaluated the role of TRPC channels in regulating TGFβ proliferative signaling. Methods. BxPc3 pancreatic cancer cells were treated with TGFβ (10 ng/mL), and TRPC and PTEN expression were analyzed by RT-PCR and/or Western blots. [Ca2+]cyt was assessed using Fura-2 fluorescence ratio digital imaging. Pharmacological reagents (TRPC inhibitors: 2-APB, SKF96365, and LaCl3) and siRNAs to TRPCs were used to determine the role of TRPCs on TGFβ-induced PTEN regulation and cell migration. Cell motility was assessed by Boyden chamber migration assay. Results. BxPc3 cells express TRPC1, 4, and 6 isoforms. TGFβ induced an increase in [Ca2+]cyt in Ca2+-free solutions and also induced an increase in [Ca2+]cyt when extracellular Ca2+ was restored. TGFβ-induced Ca2+ entry involves the activation of TRPC, as the TRPC inhibitors 2-APB, SKF96365, and LaCl3, were able to reduce peak [Ca2+]cyt by 80%, 40%, and 29%, respectively, and reversed TGFβ-induced PTEN downregulation. The 2-APB, SKF96365, and LaCl3 inhibitors also reduced TGFβinduced cell motility. Using siRNA, inhibition of TRPC1 but not other isoforms, successfully prevented TGFβ-induced cell motility. Conclusion. TRPC1 regulates TGFβ-induced downregulation of PTEN and pancreatic cell motility. This aspect of TGFβ regulation of PTEN and pancreatic cell motility might be exploited therapeutically to control pancreatic cancer metastasis.

Esmolol cardioplegia: the cellular mechanism of diastolic arrest

Esmolol, an ultra-short-acting beta-blocker, acts as a cardioplegic agent at millimolar concentrations. We investigated the mechanism by which esmolol induces diastolic ventricular arrest. In unpaced Langendorff-perfused rat hearts, esmolol (0.03-3 mmol/L) had a profound negative inotropic effect resulting in diastolic arrest at 1 mmol/L and above. This inhibition of contraction was maintained during ventricular pacing. At 3 mmol/L, esmolol also abolished action potential conduction. To determine the cellular mechanism for the negative inotropism, we measured contraction (sarcomere shortening) and the calcium transient (fura-2 fluorescence ratio; Ca(tr)) in electrically-stimulated rat ventricular myocytes at 23 and 34 degrees C. The decrease in contraction (by 72% at 23 degrees C, from 0.16 +/- 0.01 to 0.04 +/- 0.01 microm, P < 0.001) was similar to that of isolated hearts and was caused by a large decrease in Ca(tr) (from 0.13 +/- 0.02 to 0.07 +/- 0.02, P < 0.001). There was no additional effect on myofilament Ca(2+) sensitivity. Esmolol's effects on contraction and Ca(tr) were not shared or altered by the beta-blocker, atenolol (1 mmol/L). Sarcoplasmic reticulum inhibition with thapsigargin did not alter the inhibitory effects of esmolol. Whole-cell voltage-clamp experiments revealed that esmolol inhibited the L-type calcium current (I(Ca,L)) and the fast sodium current (I(Na)), with IC(50) values of 0.45 +/- 0.05 and 0.17 +/- 0.025 mmol/L, respectively. Esmolol at millimolar concentrations causes diastolic ventricular arrest by two mechanisms: at 1 mmol/L (and below), the pronounced negative inotropic effect is due largely to inhibition of L-type Ca(2+) channels; additionally, higher concentrations prevent action potential conduction, probably due to the inhibition of fast Na(+) channels.

Nox1‐derived reactive oxygen species (ROS) mediate thrombin‐induced influx of calcium in vascular smooth muscle cells (SMC)

Mechanisms by which NADPH oxidase‐derived ROS modulate cell signaling pathways are poorly understood. Using SMC cultured from aorta of Nox1 null and littermate control (WT) mice, we tested the hypothesis that ROS generated by Nox1, a catalytic subunit of NADPH oxidase, modulate thrombin‐induced increases in intracellular calcium ([Ca2+]I). In WT SMC, thrombin (1 U/ml) rapidly increased [Ca2+]I with a peak of 1100±70 nM Ca2+ (n=89 cells), as measured by fura‐2 fluorescence ratio imaging, which was inhibited by antisense to Nox1. Similarly, thrombin‐induced increase in [Ca2+]I was markedly attenuated in Nox1 null SMC (620±70 nM Ca2+, n=114 cells, p&lt;0.05). Investigation into potential mechanisms by which Nox1‐derived ROS modulate [Ca2+]I showed that thrombin‐induced generation of inositol trisphosphate and the release of intracellular Ca2+by thapsigargin were similar in WT and Nox1 null SMC. In contrast, bathing cells in Ca2+‐free media significantly reduced the thrombin‐mediated increase of [Ca2+]I in WT SMC, whereas the response in Nox1 null SMC was unchanged. Sodium vanadate, an inhibitor of protein tyrosine phosphatases (PTPs), normalized the thrombin‐induced increase of [Ca2+]I in Nox1 null SMC. These data suggest that inhibition of PTPs by Nox1‐derived ROS mediate influx of extracellular Ca2+ in SMC, and identify a novel signaling mechanism that may have a role in vascular disease.

Misinterpretation of the effect of amlodipine on cytosolic calcium concentration with fura-2 fluorospectrometry

Due to its high sensitivity, reproducibility and reliability, fura-2 is one of the most preferred tools to quantify Ca2+ levels. Recently, using fura-2/AM, some calcium channel blockers (CCBs) have been reported to have inhibitory effects on intracellular Ca2+ signaling even in nonexcitable cells that do not possess voltage-dependent Ca2+ channels (VDCC). Some CCBs are known to be photosensitive. Since photosensitive chemicals often have autofluorescent property, it is likely that CCBs with autofluorescence interfere with the fluorescent dye probes for Ca2+, which may cause misinterpretation of the results. The aim of this study was to clarify the characteristics of various CCBs on Ca2+ fluorescent probes and to identify proper fluorescent probes for each CCB to allow reliable Ca2+ measurement using endothelial cells (ECs), which possess no VDCC. Thapsigargin (TG, Ca2+-ATPase inhibitor on endoplasmic reticulum) increased fura-2 F340/F380 ratio from 0.75+/-0.06 to 4.28+/-0.32, suggesting the increase in cytosolic Ca2+ concentrations in ECs. Verapamil, nifedipine and nicardipine did not affect TG-induced increase in fura-2 F340/F380 ratio; however, amlodipine dose dependently inhibited the rise of fura-2 ratio by 24% at 100 nM, by 55% at 1 microM and by 82% at 10 microM, respectively, in ECs. In ex vivo experiments without cells, due to the autofluorescence of amlodipine excited by ultraviolet (UV light), amlodipine itself boosted the intensity of F380 much more than it did that of F340, which results in a decrease in F340/F380 ratio of fura-2 fluorescence. Rhod-2/AM, a fluorescent probe of which the excitation wavelength is out of the excitation spectrum of amlodipine, showed that amlodipine did not affect TG-induced intracellular Ca2+ increase in ECs. When the effect of amlodipine on intracellular Ca2+ concentration is assessed, fura-2 fluorospectrometry should not be used due to fluorescent interaction between amlodipine and fura-2, and using rhod-2 is strongly recommended.

4α‐PDD induces Ca2+ influx in human corneal epithelial cells by activating TRPV4 channels

Abstract Purpose: Transient receptor potential (TRP) isoform expression is evident in human corneal epithelial cells (HCEC‐SV40). However, their role in maintaining corneal epithelial homeostasis is not fully understood. We probed for gene and protein expression as well as functional activity of the vanilloid subtype, TRPV4, in immortalized HCEC‐SV40 since they elicit Ca2+ dependent regulatory volume decrease (RVD) responses during exposure to a hypotonic challenge. Methods: RT‐PCR and Western blotting analyses identified TRPV4 gene and protein expression. Functional activity was assessed based on determining whether the TRPV4 selective agonist, 4α‐PDD, induced transients increases in intracellular Ca2+ concentration. Results: Single cell fluorescence imaging results showed that 4α‐PDD (3 μM) increased intracellular Ca2+ concentration. The fura2 fluorescence ratio (f340/f380) was 0.39 ± 0.03578 in the resting state (n = 5). After application of 4α‐PDD it increased to 0.904 ± 0.14363 (n = 5; p = 5.72077×10‐5). This increase was abolished by the TRP channel blocker ruthenium red or by Ca2+‐free Ringer's medium. Conclusions: In conclusion, there is functional TRPV4 expression in HCEC‐SV40. TRPV4 expression may provide an osmosensor role to induce RVD behavior during exposure to a hypotonic challenge since this response is mediated through intracellular Ca2+ transients.Supported in part DFG Pl 150/14‐1 and NIH, EY04795. CR: none