Fura Red Research Articles

Nifuroxazide induces the apoptosis of human non‑small cell lung cancer cells through the endoplasmic reticulum stress PERK signaling pathway.

The aim of the present study was to investigate the molecular mechanism of nifuroxazide (NFZ) in the induction of apoptosis of NCI-H1299 human non-small cell lung cancer (NSCLC) cells through the reactive oxygen species (ROS)/Ca2+/protein kinase R-like ER kinase (PERK)-activating transcription factor 4 (ATF4)-DNA damage inducible transcript 3 (CHOP) signaling pathway. Morphological changes of cells were observed by microscopy, and the apoptosis and intracellular ROS levels of cells were observed by inverted fluorescence microscopy. Cell viability after the addition of the PERK inhibitor, GSK2606414, were detected by Cell Counting Kit-8 assay. Annexin V-FITC was used to detect cell apoptosis, Brite 670 was used to detect intracellular ROS and Fura Red AM was used to detect Ca2+ content. Western blotting was used to detect PERK, phosphorylated (P)-PERK, ATF4, CHOP, P-Janus kinase 2 and P-signal transducer and activator of transcription 3 expression levels. Compared with the dimethyl sulfoxide control group, NFZ inhibited the survival activity in the H1299 NSCLC cell line, in a time- and dose-dependent manner. However, GSK2606414 inhibited the NFZ-induced apoptosis of H1299 cells. GSK2606414 also inhibited the increase in ROS and Ca2+ in H1299 cells induced by NFZ. Western blotting results demonstrated that NFZ significantly increased the expression levels of P-PERK, ATF4 and CHOP, whereas GSK2606414 significantly reduced the NFZ-induced increase in these protein expression levels. In conclusion, NFZ may induce the apoptosis of H1299 NSCLC cells through the ROS/Ca2+/PERK-ATF4-CHOP signaling pathway.

On calcium fluorophore’s impact in platelet signaling studies

Observation of calcium signaling in platelets - blood cells designed to be involved in stopping bleeding and forming blood clots - is an important part of fundamental research in hemostasis. However, such a study is possible only with the use of calcium fluorophores - small molecules that penetrate the platelet membrane due to their hydrophobic -AM part, which is then hydrolyzed by cytosol esterases. In this work, we consider the phenomenon of inhomogeneous loading of calcium fluorophores into platelets.We used platelets from healthy adult donors loaded with various fluorescent probes (CalBryte590, DiOC6 (3), Fura Red, Fluo-4 and CellTracker Violet BMQC) and immobilized on antibodies to CD31 in parallel plane flow chambers. Total internal reflection fluorescence (TIRF) microscopy was used for observations.We demonstrated that all studied probes are loaded heterogeneously, with 30% platelets being loaded with a probe 2-6 times higher than the population median value. Using the CalBryte590 probe as an example, we have shown that a decrease in the incubation temperature, the addition of Pluronic 127 to the incubation medium, or membrane cholesterol depletion significantly reduces the heterogeneity of the probe distribution in the population. By looking at platelet activation from the surface, we have shown that the probability of experiencing strong activation, as measured by the intensity of calcium oscillations, correlates with the amount of probe in the platelet.Thus, we conclude that the type of fluorophore used and the conditions of its loading into platelets can significantly affect the results of experiments on the observation of calcium signaling in platelets.

Optogenetic control of cancer cell survival in ChR2‐transfected HeLa cells

Optogenetics is a molecular biological technique involving transfection of cells with photosensitive proteins and the subsequent study of their biological effects. The aim of this study was to evaluate the effect of blue light on the survival of HeLa cells, transfected with channelrhodopsin-2 (ChR2). HeLa wild-type cells were transfected with a plasmid that contained the gene for ChR2. Transfection and channel function were evaluated by real-time polymerase chain reaction (RT-PCR), fluorescence imaging using green fluorescent protein (GFP) and flow cytometry for intracellular calcium changes using a Fura Red probe. We developed a platform for optogenetic stimulation for use within the cell culture incubator. Different stimulation procedures using blue light (467nm) were applied for up to 24h. Cell survival was determined by flow cytometry using propidium iodide and rhodamine probes. Change in cell survival showed a statistically significant (p<0.05) inverse association with the frequency and time of application of the light stimulus. This change seemed to be associated with the ChR2 cis-trans-isomerization cycle. Cell death was associated with high concentrations of calcium in the cytoplasm and stimulation intervals less than the period of isomerization. It is possible to transfect HeLa cells with ChR2 and control their survival under blue light stimulation. We suggest that this practice should be considered in the future development of optogenetic systems in biological or biomedical research.

Abstract 14409: TRPA1 Channels Are a Source of Calcium-driven Cellular Mechano-arrhythmogenicity

Introduction: Pathologic changes in myocardial mechanics and hemodynamic load result in arrhythmias via mechanically-induced changes in electrophysiology or intracellular Ca 2+ (‘mechano-arrhythmogenicity’). While molecular mechanisms driving mechano-arrhythmogenicity are poorly defined, they are associated with disease-related alterations in voltage-Ca 2+ dynamics. Objective: Define mechanisms of mechano-arrhythmogenicity during alterations in voltage-Ca 2+ dynamics in rabbit ventricular myocytes. Methods: Rabbit (♀, NZW) LV myocytes were transiently stretched (8-16% change in sarcomere length, 100ms) during diastole or late repolarisation in control or during K ATP channel activation (pinacidil). Drugs were used to buffer Ca 2+ (BAPTA), stabilise RyR (dantrolene), non-selectively block stretch-activated channels (streptomycin), or specifically block (HC-030031) or activate (AITC) mechano-sensitive TRPA1 channels. Voltage-Ca 2+ dynamics were simultaneously monitored with fluorescent dyes (di-4-ANBDQPQ, Fluo-5F) and a single camera-optical splitter system and diastolic Ca 2+ was measured using Fura Red. Results: Pinacidil caused greater shortening of the AP than Ca 2+ transient (-144±17 vs -74±11ms; n =24 cells, N =7 rabbits; p &lt;0.001) with no change in cell stiffness or contractility. Stretch during pinacidil application caused arrhythmias in both diastole and late repolarisation (8 and 10% of stretches; n =46, N =5), which voltage-Ca 2+ imaging revealed were Ca 2+ -driven. Arrhythmias were reduced with BAPTA (3 and 0% of stretches; p &lt;0.05), streptomycin (4 and 2%; p &lt;0.05), and HC-030031(2 and 1%; p &lt;0.01), while dantrolene had no effect ( n =40, N =5 for each). Stretch in diastole during AITC application also caused arrhythmias (15%; p &lt;0.001), which were blocked by HC-030031 (4%; p &lt;0.001) or BAPTA (3%; p &lt;0.001; n =40, N =5 each). Both AITC and pinacidil caused an increase in diastolic Ca 2+ (112±29 and 78±29% of control; p&lt;0.05), which was reduced by HC-030031 with AITC (26±24%; p &lt;0.05), but not with pinacidil ( n =25, N =5 each). Conclusions: TRPA1 activation increases mechano-arrhythmogenicity via a Ca 2+ -driven mechanism and may represent a novel anti-arrhythmic target in pathologies involving altered cardiac mechanics.

Nitric oxide production by glomerular podocytes

Nitric Oxide (NO), a potent vasodilator and vital signaling molecule, has been shown to contribute to the regulation of glomerular ultrafiltration. However, whether changes in NO occur in podocytes during the pathogenesis of salt-sensitive hypertension has not yet been thoroughly examined. We showed here that podocytes produce NO, and further hypothesized that hypertensive animals would exhibit reduced NO production in these cells in response to various paracrine factors, which might contribute to the damage of glomeruli filtration barrier and development of proteinuria. To test this, we isolated glomeruli from the kidneys of Dahl salt-sensitive (SS) rats fed a low salt (LS; 0.4% NaCl) or high salt (HS; 4% NaCl, 3 weeks) diets and loaded podocytes with either a combination of NO and Ca2+ fluorophores (DAF-FM and Fura Red, respectively) or DAF-FM alone. Changes in fluorescence were observed with confocal microscopy in response to adenosine triphosphate (ATP), angiotensin II (Ang II), and hydrogen peroxide (H2O2). Application of Ang II resulted in activation of both NO and intracellular calcium ([Ca2+]i) transients. In contrast, ATP promoted [Ca2+]i transients, but did not have any effects on NO production. SS rats fed a HS diet for 3 weeks demonstrated impaired NO production: the response to Ang II or H2O2 in podocytes of glomeruli isolated from SS rats fed a HS diet was significantly reduced compared to rats fed a LS diet. Therefore, glomerular podocytes from hypertensive rats showed a diminished NO release in response to Ang II or oxidative stress, suggesting that podocytic NO signaling is dysfunctional in this condition and likely contributes to the development of kidney injury.

Calcium Sensitive Fluorescent Dyes Fluo-4 and Fura Red under Pressure: Behaviour of Fluorescence and Buffer Properties under Hydrostatic Pressures up to 200 MPa.

The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (1 nM to 25.6 μM) and a specified dye concentration (12 μM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye molecules is determined to -17.8 ml mol-1 for Fluo-4 and -21.3 ml mol-1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence.

Abstract 106: Signaling Mechanisms Affecting Nitric Oxide Production in Glomerular Podocytes

While Nitric Oxide (NO), a potent vasodilator and vital signaling molecule, has been shown to contribute to the regulation of glomerular ultrafiltration, its role in podocytes during the pathogenesis of salt-sensitive hypertension has not yet been thoroughly examined. Recent studies have demonstrated that the deficiency of eNOS (the enzyme responsible for synthesizing NO) exacerbates renal injury and accelerates development of proteinuria and glomerulosclerosis. Considering this, we hypothesized that the podocytes of hypertensive animals would exhibit reduced NO production in response to various paracrine factors and this directly contributes to proteinuria. To test this, we isolated glomeruli from the kidneys of Dahl salt-sensitive (SS) rats fed either a high salt (HS; 4% NaCl, 3 weeks) or low salt (LS; 0.4% NaCl) diet and loaded podocytes with a combination of NO and Ca 2+ ionophores (DAF-FM and Fura Red, respectively). Changes in fluorescence were observed with the use of confocal microscopy in response to adenosine triphosphate (ATP), angiotensin II (Ang II), and H 2 O 2 . Application of Ang II or H 2 O 2 resulted in activation of both NO and [Ca 2+ ] i ; fluorescent transients which were significantly elevated in the soma and foot processes of podocytes of LS fed rats. In contrast, ATP specifically activated only changes in [Ca 2+ ] i , but did not have any effects on NO production. Ang II treatment also caused hypertrophy of the podocytes, whereas ATP had no effect on cell volume (41.1±7.7 vs. 0.1±3.6% increase for Ang II and ATP, respectively; P &lt;0.05). Collectively, our results show that in contrast to [Ca 2+ ] i , which is modulated by all studied paracrine molecules, NO is produced by podocytes only in response to Ang II and H 2 O 2 , but not ATP. SS rats fed a HS diet for 3 weeks demonstrated impaired NO production; the response to Ang II or H 2 O 2 on HS contained only 23.7±6.6 and 43.4±28.4% of total effects on LS, respectively ( P &lt;0.05). Therefore, when fed a HS diet, SS podocytes had an impaired NO response to Ang II or oxidative stress, suggesting that NO signaling is dysfunctional and likely contributes to the development of kidney injury.

Neural depolarization triggers Mg2+ influx in rat hippocampal neurons

Homeostasis of magnesium ion (Mg2+) plays key roles in healthy neuronal functions, and deficiency of Mg2+ is involved in various neuronal diseases. In neurons, we have reported that excitotoxicity induced by excitatory neurotransmitter glutamate increases intracellular Mg2+ concentration ([Mg2+]i). However, it has not been revealed whether neuronal activity under physiological condition modulates [Mg2+]i. The aim of this study is to explore the direct relationship between neural activity and [Mg2+]i dynamics. In rat primary-dissociated hippocampal neurons, the [Mg2+]i and [Ca2+]i dynamics were simultaneously visualized with a highly selective fluorescent Mg2+ probe, KMG-104, and a fluorescent Ca2+ probe, Fura Red, respectively. [Mg2+]i increase concomitant with neural activity by direct current stimulation was observed in neurons plated on an indium-tin oxide (ITO) glass electrode, which enables fluorescent imaging during neural stimulation. The neural activity-dependent [Mg2+]i increase was also detected in neurons whose excitability was enhanced by the treatment of a voltage-gated K+ channel blocker, tetraethylammonium (TEA) at the timings of spontaneous Ca2+ increase. Furthermore, the [Mg2+]i increase was abolished in Mg2+-free extracellular medium, indicating [Mg2+]i increase is due to Mg2+ influx induced by neural activity. The direct neuronal depolarization by veratridine, a Na+ channel opener, induced [Mg2+]i increase, and this [Mg2+]i increase was suppressed by the pretreatment of a non-specific Mg2+ channel inhibitor, 2-aminoethoxydiphenyl borate (2-APB). Overall, activity-dependent [Mg2+]i increase results from Mg2+ influx through 2-APB-sensitive channels in rat hippocampal neurons.

Analysis of the Effect of β-Endorphin-Like Peptide Pentarphin on Intracellular Ca²⁺ Concentration in Early Mouse Embryos Using Fluorescent Probes.

At the early stages of mammalian embryo development, Ca(2+) influences the processes of cleavage and differentiation. An important role in these processes is played by peptide growth factors. One of them, pentarphin, is a peptide growth factor similar to the central part of β-endorphin molecule and a fragment 369-373 of IgG heavy chain. We used fluorescent Ca(2+) indicators Fluo-3 AM and Fura Red AM. Pentarphin added to embryos in a concentration of 0.1 μM 1.5-2 fold increased intercellular Ca(2+) level in comparison with the initial level.

A novel, rapid method to quantify intraplatelet calcium dynamics by ratiometric flow cytometry.

Cytosolic free calcium ions represent important second-messengers in platelets. Therefore, quantitative measurement of intraplatelet calcium provides a popular and very sensitive tool to evaluate platelet activation and reactivity. Current protocols for determination of intracellular calcium concentrations in platelets have a number of limitations. Cuvette-based methods do not allow measurement of calcium flux in complex systems, such as whole blood, and therefore require isolation steps that potentially interfere with platelet activation. Flow cytometry has the potential to overcome this limitation, but to date the application of calibrated, quantitative readout of calcium kinetics has only been described for Indo-1. As excitation of Indo-1 requires a laser in the ultraviolet range, such measurements cannot be performed with a standard flow cytometer. Here, we describe a novel, rapid calibration method for ratiometric calcium measurement in platelets using both Ar+-laser excited fluorescence dyes Fluo-4 and Fura Red. We provide appropriate equations that allow rapid quantification of intraplatelet calcium fluxes by measurement of only two standardisation buffers. We demonstrate that this method allows quantitative calcium measurement in platelet rich plasma as well as in whole blood. Further, we show that this method prevents artefacts due to platelet aggregate formation and is therefore an ideal tool to determine basal and agonist induced calcium kinetics.

Ratiometric analysis of fura red by flow cytometry: a technique for monitoring intracellular calcium flux in primary cell subsets.

Calcium flux is a rapid and sensitive measure of cell activation whose utility could be enhanced with better techniques for data extraction. We describe a technique to monitor calcium flux by flow cytometry, measuring Fura Red calcium dye by ratiometric analysis. This technique has several advantages: 1) using a single calcium dye provides an additional channel for surface marker characterization, 2) allows robust detection of calcium flux by minority cell populations within a heterogeneous population of primary T cells and monocytes 3) can measure total calcium flux and additionally, the proportion of responding cells, 4) can be applied to studying the effects of drug treatment, simultaneously stimulating and monitoring untreated and drug treated cells. Using chemokine receptor activation as an example, we highlight the utility of this assay, demonstrating that only cells expressing a specific chemokine receptor are activated by cognate chemokine ligand. Furthermore, we describe a technique for simultaneously stimulating and monitoring calcium flux in vehicle and drug treated cells, demonstrating the effects of the Gαi inhibitor, pertussis toxin (PTX), on chemokine stimulated calcium flux. The described real time calcium flux assay provides a robust platform for characterizing cell activation within primary cells, and offers a more accurate technique for studying the effect of drug treatment on receptor activation in a heterogeneous population of primary cells.

The Regulatory Pathways of Nitric Oxide Production in Glomeruli Podocytes

Nitric Oxide (NO) is a major paracrine and autocrine signaling molecule. In the kidney, it can be released by the glomerulus and regulates microvascular hemodynamics in concert with antagonistic vasoactive substances such as angiotensin II (Ang II). Strong changes in glomerular NO production, which can be triggered by inflammation, oxidative stress or other factors, can lead to glomerular epithelial cells (podocytes) damage and subsequent alteration of glomerular filtration rate resulting in severe proteinuria. Because NO production is primarily Ca2+-dependent, we focused on the studies of the role of Ca2+ handling and NO production in response to different paracrine factors in the podocytes. The podocytes of freshly isolated glomeruli from the kidneys of Dahl salt-sensitive rats were labeled with a combination of NO and intracellular Ca2+ ionophores (DAF-FM and Fura Red, respectively) to monitor changes in fluorescence with the use of confocal microscopy. Paracrine molecules (ATP, Ang II, ET-1 and H2O2) were applied to the glomeruli to determine the effects on Ca2+ handling and NO release. To determine the specificity of the responses, control experiments were performed using a NO production blocker, L-NAME, which resulted in an attenuation of NO production. Both Ang II and H2O2 significantly increase NO production. In contrast, ATP and ET-1 have not affected NO level in glomeruli. These effects seemed to be localized to the podocyte foot processes, where they may be involved in altering glomerular permeability.

Detecting calcium in cardiac muscle: fluorescence to dye for.

THE IMPORTANCE OF Ca 2 as a second messenger in general and as a mediator between electrical excitation and cellular contraction in the heart is well accepted. Accordingly, numerous Ca 2 -sensitive fluorescent dyes are available nowadays, rendering the choice of the appropriate dye for a particular application a challenge. It appears to be important to provide calibrated Ca 2 signals rather than fluorescence transients, especially when comparing data between studies. In case calibrated signals cannot be provided, then the time course of fluorescence transients should be interpreted with caution and such signals should be referred to as fluorescence transients rather than Ca 2 transients. Additionally, we discuss how major properties of the fluorescence dyes should be considered with a special emphasis on their Ca 2 dissociation properties. Furthermore, we highlight putative risks of tripping, especially in the context of multicellular recordings, and how to circumvent them. As an ubiquitous intracellular messenger, Ca 2 ions play an important role in almost all living cells (4). A particularly dynamic role has been assigned to Ca 2 in muscle cells because here they not only control gene expression and other slower signaling processes but serve as a direct transducer of the electrical excitation of the plasma membrane into the activation of the physiological response of the cell, the contraction, by activation of the contractile machinery (5). Almost 50 years ago, Jobsis and O’Connor (10) reported the measurements of intracellular Ca 2 transients in skeletal muscle using the Ca 2 -sensitive dye murexide, which changes its absorbance upon binding of Ca 2 ions. Later, these absorbance dyes were improved and an important member of this family, arsenazo III, was widely used to study intracellular Ca 2 in

Effect of induced subclinical hypocalcemia on physiological responses and neutrophil function in dairy cows

The objectives were to study the effects of induced subclinical hypocalcemia [SCH, blood ionized Ca (iCa2+) <1.0mM, without recumbency] on physiological responses and function of immune cells in dairy cows. Ten nonpregnant, nonlactating Holstein cows were blocked by lactation and assigned randomly to a normocalcemic (NC; intravenous infusion of 0.9% NaCl i.v. plus 43g of oral Ca, as Ca sulfate and Ca chloride, at −1 and 11h) or an induced SCH [SCHI, 5% ethylene glycol tetraacetic acid (EGTA), a selective iCa2+ chelator, intravenous infusion] treatment for 24h, using a crossover design. The sequence of treatments was either NC–SCHI or SCHI–NC, with a 6-d washout period. Ionized Ca was evaluated before, hourly during the infusion period, and at 48 and 72h, to monitor concentrations and adjust the rate of infusion, maintaining blood iCa2+ <1.0mM in SCHI throughout the 24-h infusion period. Additional measurements included heart and respiratory rates, rectal temperature, dry matter intake, rumen contractions, whole-blood pH, concentrations of glucose and K in whole blood, concentrations of total Ca, Mg, nonesterified fatty acids, β-hydroxybutyrate, and insulin in plasma, and urinary excretion of Ca. Total and differential leukocyte count in blood was also performed. The concentration of cytosolic iCa2+ in neutrophils and lymphocytes was quantified and neutrophil function was assayed in vitro. Infusion of a 5% EGTA solution successfully induced SCH in all SCHI cows, resulting in decreased blood iCa2+ concentrations throughout the 24-h treatment period (0.77±0.01 vs. 1.26±0.01mM iCa2+). Induction of SCH reduced dry matter intake on the day of infusion (5.3±0.8 vs. 9.1±0.8kg/d) and rumen contractions (1.9±0.2 vs. 2.7±0.2 contractions/2min) for the last 12h of infusion. Cows in SCHI had decreased plasma insulin concentration (1.44±0.23 vs. 2.32±0.23ng/mL) evident between 6 and 18h after the beginning of the infusion, accompanied by increased concentrations of glucose (4.40±0.04 vs. 4.17±0.04mM). Plasma nonesterified fatty acids concentration was greater for SCHI than NC cows (0.110±0.019 vs. 0.061±0.014mM). Neutrophils of cows in SCHI had a faster decrease in cytosolic iCa2+ after stimulation with ionomycin (9.9±1.0 vs. 13.6±1.4 Fluo-4:Fura Red post-end ratio) in vitro. Furthermore, induction of SCH reduced the percentage of neutrophils undergoing phagocytosis (22.1±2.1 vs. 29.3±2.1%) and reduced the oxidative burst response after incubation of pathogenic bacteria (16.1±1.7 vs. 24.2±1.7%). Subclinical hypocalcemia compromised appetite, altered metabolism, and impaired function of immune cells in dairy cows.

Evaluating Cellular Repair Potential - Lessons from Skeletal Muscle

The ability to repair disruptions in the plasma membrane is critical for maintaining cellular integrity and health; this is especially true for cells that routinely experience membrane damage as a result of activity such as skeletal muscle. Dysferlinopathies are a class of late onset muscle wasting diseases associated with the absence or truncation of the protein dysferlin. Dysferlin is postulated to have a role in membrane repair and the absence of dysferlin in both humans and animal models is associated with muscle damage. We are developing techniques to evaluate the repair potential of dysferlin null skeletal muscle fibers and myotubes using fluorescence assays of dye-leakage and calcium activity. In isolated muscle fibers or myotubes containing fluorescent dye and subjected to IR laser wounding, both the rate and duration of dye-leakage are measured by confocal microscopy. Upon wounding repair-competent fibers a “puff” of dye is released at the wound site and dye leakage ceases within 60 seconds. Under conditions where resealing is inhibited (i.e. low extracellular [Ca2+]) a large amount of dye is lost; leakage continues for >10 minutes. Changes in intracellular [Ca2+]free are measured using a combination of the calcium indicator dyes fluo-4 and Fura Red. Upon wounding, a rise in [Ca2+]free is observed at the wound site and either returns to the pre-wound level following successful resealing, or in the absence of resealing remains high and spreads through the entire fiber or mytube. Dysferlin null muscle, is repair competent but may differ in the developed calcium load, contractile response, and wounding threshold compared to dysferlin positive muscle. Since our overall goal is to develop interventions that boost cellular repair potential, quantifiable metrics of this complicated cellular process are required when testing the efficacy of repair-targeted treatments.

ATP Releasing Connexin 30 Hemichannels Mediate Flow-Induced Calcium Signaling in the Collecting Duct

ATP in the renal tubular fluid is an important regulator of salt and water reabsorption via purinergic calcium signaling that involves the P2Y2 receptor, ENaC, and AQP2. Recently, we have shown that connexin (Cx) 30 hemichannels are localized to the non-junctional apical membrane of cells in the distal nephron-collecting duct (CD) and release ATP into the tubular fluid upon mechanical stimuli, leading to reduced salt and water reabsorption. Cx30−/− mice show salt-dependent elevations in BP and impaired pressure-natriuresis. Thus, we hypothesized that increased tubular flow rate leads to Cx30-dependent purinergic intracellular calcium ([Ca2+]i) signaling in the CD. Cortical CDs (CCDs) from wild type and Cx30−/− mice were freshly dissected and microperfused in vitro. Using confocal fluorescence imaging and the calcium-sensitive fluorophore pair Fluo-4 and Fura Red, we found that increasing tubular flow rate from 2 to 20 nl/min caused a significant 2.1-fold elevation in [Ca2+]i in wild type CCDs. This response was blunted in Cx30−/− CCDs ([Ca2+]i increased only 1.2-fold, p < 0.0001 vs. WT, n = 6 each). To further test our hypothesis we performed CD [Ca2+]i imaging in intact mouse kidneys in vivo using multiphoton microscopy and micropuncture delivery of the calcium-sensitive fluorophore Rhod-2. We found intrinsic, spontaneous [Ca2+]i oscillations in free-flowing CDs of wild type but not Cx30−/− mice. The [Ca2+]i oscillations were sensitive also to P2-receptor inhibition by suramin. Taken together, these data confirm that mechanosensitive Cx30 hemichannels mediate tubular ATP release and purinergic calcium signaling in the CD which mechanism plays an important role in the regulation of CD salt and water reabsorption.

Spatial Regulation of Platelet Aggregation and Physiology Under Flow

Abstract 3300 Background:Hemostasis is an important physiologic process that requires the aggregation of platelets at distinct sites of vascular injury to promote clot formation and prevent blood loss. Platelet response to soluble agonists and shear stress has been studied extensively, but little is known of how microenvironmental geometry affects platelet function. As platelets must quickly adhere to, aggregate, and initiate coagulation only at the affected areas, spatial cues must at some level regulate this process. This aspect of spatial regulation has been investigated under static conditions by our group and others (Kita et al., 2011; Van de Walle et al., 2012). Understanding this aspect of platelet function is vital for better understanding the process of hemostasis and pathophysiological conditions such as thrombosis. Here, we directly examine how spatial cues affect platelet aggregation and physiology under variable shear conditions by flowing heparinized whole blood over micropatterned collagen in a microfluidic channel. This system allows us to assess platelet aggregate morphology under different geometric constraints and shear rates, as well as evaluate platelet physiology at the single cell level by measuring calcium signaling using fluorogenic dyes. Results:A microfluidic channel was bonded to a glass coverslip stamped with FITC-conjugated Type I collagen using a novel technique combining microcontact printing and the stamp-stick bonding technique (Satyanarayana et al., 2005). Before flowing, each chamber was incubated with 1% bovine serum albumin (BSA) blocking solution for 1.5 hours. Whole heparinized blood was then flowed through the chamber at shear rates of 100, 1000, and 10000 s−1. Platelets were labeled with Fura Red, and time lapse confocal imaging was performed for 10 minutes to monitor the aggregation of platelets at the start of flow. The flow chambers were then flushed with Tyrode’s buffer with 0.1% BSA using the same experimental shear rates until the chamber was cleared of red blood cells. Image analysis was conducted using ImageJ (to calculate the percentage of platelet coverage on the collagen stamps at different shear rates.Platelets initially adhere to the distal edge of the collagen micropatterns for all shear rates (Fig. 1), indicating that platelets may require a priming region before forming a stable adhesion. As shear rate increased, platelet coverage of the collagen stamps decreased. However, aggregates also grew without conforming to the geometric constraints imposed by the collagen micropatterns more frequently at those higher shear rates (Fig. 1). Though platelet tethers generally aligned in the direction of the flow, increased tether lengths could be seen when platelets were exposed to higher shear, which may explain why platelets were able to span larger gaps and aggregate in a less spatially constrained manner at high shear rates. Image analysis shows that 51.5% of the collagen was covered by platelet aggregates for a shear rate of 100 s−1 with some platelets forming tethers to span gaps (Fig. 2). When the shear rate was increased to 1000 s−1, platelet coverage of the collagen microstamp drastically dropped to 18.5% (Fig. 2). At a pathophysiological shear rate of 10000 s−1, the percentage of collagen covered by platelets dropped further to 12.8% and adopted a linear shape, although a large portion of the aggregate can be seen spanning gaps between the collagen microstamp (Fig. 1 and 2). Conclusions and Ongoing Efforts:Ours is the first reported study of the spatial regulation of platelet aggregation under variable shear in a microfluidic channel. We have found that platelets are more spatially regulated under low shear conditions compared to high shear, which has implications for thrombosis and other clotting disorders. Future studies will incorporate the simultaneous use of ratiometric fluorgenic calcium signaling dyes to investigate the role of spatial regulation in Ca2+ signaling. Finally, we have developed a method to culture endothelial cells around a collagen micropattern to study this spatial regulation of platelet function under more physiological conditions (Fig. 3). [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Imaging intracellular calcium signals in intact kidney tissue

Calcium (Ca2+) is an intracellular secondary messenger with multiple important physiological roles; disturbances in intracellular Ca2+ homeostasis have been implicated in the pathogenesis of various kidney diseases. We have recently demonstrated that multiphoton microscopy can be used to image a range of intracellular signals in ex vivo rat kidney preparations such as tissue slices [1, 2] and isolated perfused organs [3].Here, we demonstrate that the tissue slice model can be used to load established Ca2+ indicators into both tubular and endothelial cells in the kidney. Kidney slices (200 μm thick) were obtained from adult, male sprague dawley rat kidneys (following cervical dislocation) and maintained in a physiological saline solution, bubbled with 95% O2/5% CO2. Loading of exogenous Ca2+ sensitive dyes (Fluo4, Rhod‐2, Fura Red) was achieved ex vivo by using a re‐circulating perfusion system. Confocal microscopy was used to image Ca2+ loading in the renal medulla, and to record changes in signal in response to physiological stimuli, i.e. ATP, Angiotensin II.We believe that the application of these imaging techniques have much potential to enhance understanding of the roles of intracellular Ca2+ signalling in renal physiology and pathophysiology.Research supported by the Medical Research Council

Identification of signal transduction pathways involved in the formation of platelet subpopulations upon activation

Platelets are formed elements of blood. Upon activation, they externalize phosphatidylserine, thus accelerating membrane-dependent reactions of blood coagulation. Activated platelets form two subpopulations, only one of which expresses phosphatidylserine. This study aimed to identify signalling pathways responsible for this segregation. Gel-filtered platelets, intact or loaded with calcium-sensitive dyes, were activated and labelled with annexin V and antibodies, followed by flow cytometric analysis. Calcium Green and Fura Red dyes were compared, and only the latter was able to detect calcium level differences in the platelet subpopulations. Phosphatidylserine-positive platelets produced by thrombin had stably high intracellular calcium level; addition of convulxin increased and stabilized calcium level in the phosphatidylserine-negative subpopulation. PAR1 agonist SFLLRN also induced calcium rise and subpopulation formation, but the resulting platelets were not coated with alpha-granule proteins. Adenylatecyclase activator forskolin inhibited phosphatidylserine-positive platelets formation several-fold, while its inhibitor SQ22536 had no effect. This suggests that adenylatecyclase inactivation is necessary, but not rate-limiting, for subpopulation segregation. Inhibition of mitogen-activated protein kinase kinase (U0126) and glycoprotein IIb-IIIa (Monafram(®)) was without effect, whereas inhibitors of phosphatidylinositol 3-kinase (wortmannin) and Src tyrosine kinase (PP2) decreased the procoagulant subpopulation threefold. These data identify the principal signalling pathways controlling platelet heterogeneity.

Trpc2 depletion protects red blood cells from oxidative stress-induced hemolysis

Transient receptor potential (TRP) channels Trpc2 and Trpc3 are expressed on normal murine erythroid precursors, and erythropoietin stimulates an increase in intracellular calcium ([Ca(2+)](i)) through TRPC2 and TRPC3. Because modulation of [Ca(2+)](i) is an important signaling pathway in erythroid proliferation and differentiation, Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were utilized to explore the roles of these channels in erythropoiesis. Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were not anemic, and had similar red blood cell counts, hemoglobins, and reticulocyte counts as wild-type littermate controls. Although the erythropoietin-induced increase in [Ca(2+)](i) was reduced, these knockout mice showed no defects in red cell production. The major phenotypic difference at steady state was that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrit of red cells were significantly greater in Trpc2 and Trpc2/Trpc3 double knockout mice, and mean corpuscular hemoglobin concentration was significantly reduced. All hematological parameters in Trpc3 knockout mice were similar to controls. When exposed to phenylhydrazine, unlike the Trpc3 knockouts, Trpc2 and Trpc2/Trpc3 double knockout mice showed significant resistance to hemolysis. This was associated with a significant reduction in hydrogen peroxide-induced calcium influx in erythroblasts. Although erythropoietin-induced calcium influx through TRPC2 or TRPC3 is not critical for erythroid production, these data demonstrate that TRPC2 plays an important role in oxidative stress-induced hemolysis, which may be related to reduced calcium entry in red cells in the presence of Trpc2 depletion.