Probe Indo-1 Research Articles

Cytosolic Ca2+ gradients and mitochondrial Ca2+ uptake in resting muscle fibers: A model analysis

Calcium ions (Ca2+) enter mitochondria via the mitochondrial Ca2+ uniporter, driven by electrical and concentration gradients. In this regard, transgenic mouse models, such as calsequestrin knockout (CSQ-KO) mice, with higher mitochondrial Ca2+ concentrations ([Ca2+]mito), should display higher cytosolic Ca2+ concentrations ([Ca2+]cyto). However, repeated measurements of [Ca2+]cyto in quiescent CSQ-KO fibers never showed a difference between WT and CSQ-KO. Starting from the consideration that fluorescent Ca2+ probes (Fura-2 and Indo-1) measure averaged global cytosolic concentrations, in this report we explored the role of local Ca2+ concentrations (i.e., Ca2+ microdomains) in regulating mitochondrial Ca2+ in resting cells, using a multicompartmental diffusional Ca2+ model. Progressively including the inward and outward fluxes of sarcoplasmic reticulum (SR), extracellular space, and mitochondria, we explored their contribution to the local Ca2+ distribution within the cell.The model predicts Ca2+ concentration gradients with hot spots or microdomains even at rest, minor but similar to those of evoked Ca2+ release. Due to their specific localization close to Ca2+ release units (CRU), mitochondria could take up Ca2+ directly from high-concentration microdomains, thus sensibly raising [Ca2+]mito, despite minor, possibly undetectable, modifications of the average [Ca2+]cyto.

Space and Time Resolved Detection of Platelet Activation and von Willebrand Factor Conformational Changes in Deep Suspensions

Tracking cells and proteins' phenotypic changes in deep suspensions is critical for the direct imaging of blood-related phenomena in in vitro replica of cardiovascular systems and blood-handling devices. This paper introduces fluorescence imaging techniques for space and time resolved detection of platelet activation, von Willebrand factor (VWF) conformational changes, and VWF-platelet interaction in deep suspensions. Labeled VWF, platelets, and VWF-platelet strands are suspended in deep cuvettes, illuminated, and imaged with a high-sensitivity EM-CCD camera, allowing detection using an exposure time of 1 ms. In-house postprocessing algorithms identify and track the moving signals. Recombinant VWF-eGFP (rVWF-eGFP) and VWF labeled with an FITC-conjugated polyclonal antibody are employed. Anti-P-Selectin FITC-conjugated antibodies and the calcium-sensitive probe Indo-1 are used to detect activated platelets. A positive correlation between the mean number of platelets detected per image and the percentage of activated platelets determined through flow cytometry is obtained, validating the technique. An increase in the number of rVWF-eGFP signals upon exposure to shear stress demonstrates the technique's ability to detect breakup of self-aggregates. VWF globular and unfolded conformations and self-aggregation are also observed. The ability to track the size and shape of VWF-platelet strands in space and time provides means to detect pro- and antithrombotic processes.

ENHANCED CYTOTOXICITY OF РHOTOEXCITED FULLERENE C60 AND CISPLATIN COMBINATION AGAINST D RUGRESISTANT LEUKEMIC CELLS

To evaluate the viability of leukemic cells sensitive (L1210S) and resistant (L1210R) to cisplatin, ROS production and free cytosolic Ca(2+) concentration under treatment with cisplatin or its combination with photoexcited fullerene C60. Cell viability was assessed by the MTT reduction assay. Light-emitting diode lamp (2.45 J/cm(2)) was used for photoexcitation of intracellular accumulated fullerene C60. Free cytosolic calcium concentration ([Ca(2+)]i) and ROS production in cells were estimated with the use of fluorescent probes Indo-1 and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), respectively. It is shown that viability of L1210R cells wasn't changed under treatment with cisplatin in concentration range 0.1-10 μg/ml. 50% and 30% decrease of L1210S cells were observed after 24 h of incubation with cisplatin at concentrations 5 and 1 μg/ml, respectively. Intensification of extranuclear cytotoxic effects (ROS production and [Ca(2+)]i increase) after treatment with 1 μg/ml was detected in L1210S, but not in L1210R cells. The most strongly pronounced increase of ROS production and [Ca(2+)]i in both L1210 cell lines was revealed in dynamics after combined treatment with cisplatin (1 μg/ml) and photoexcited fullerene C60 (10(-5) M) and was followed by decreased viability of not only L1210S, but of L1210R cells as well.. Combined treatment with photoexcited C60 and cisplatin allowed to decrease effective concentration of cisplatin against parental L1210 cells and to increase sensibility of resistant cells to the drug.

Caveolae are involved in stretch-induced Ca2+ signaling in pulmonary hypertension

Introduction Vascular smooth muscle cells are submitted to stretch forces exerted by the blood pressure. Pulmonary arteries can transduce a mechanical stimulus of stretch into a biological response of contraction, a mechanism called myogenic tone, which involves plasma membrane Ca 2+ stretch-activated channels (SAC). As membrane plasticity and shape are important for the SAC activity, we investigate the involvement of caveolae in the Ca 2+ and contraction response to stretch of pulmonary arterial smooth muscle cells (PA-SMC). Methods Isometric contractions were performed in pulmonary arterial vessels from normoxic rats (Nx rats) and rats with a pulmonary hypertension induced by a chronic hypoxia of 3 weeks (CH rats). Inward currents from SAC were recorded on freshly isolated PA-SMC after a negative pressure applied by a patch-clamp pipette and Ca 2+ variations were simultaneously recorded with the fluorescent probe indo-1. Sarcoplasmic reticulum Ca 2+ was measured by a confocal microscope with the fluo-5N probe. Finally, a pharmacological approach using methyl-β-cyclodextrin (MβCD, a caveolae disrupter) coupled with different immunolabelings of caveolin-1 and sarcoplasmic Ca 2+ stores were used to investigate the role of caveolae. Results We show that caveolae are present and caveolin-1 expressed in PA-SMC from both normal and pulmonary hypertensive rats. Isolated PA-SMC and pulmonary arteries exhibit a higher SAC activity, Ca 2+ response and contraction to stretch in CH rats than in Nx rats. These responses are reduced by MβCD only in CH rats. In the absence of extracellular calcium, a stretch induces a Ca 2+ and contraction response only in CH rats but not in Nx rats. This phenomenon involves a Ca 2+ release from the sarcoplasmic reticulum and is fully inhibited by MβCD. Conclusion Caveolae are important for stretch-induced calcium response in CH rats via a new subcellular organization between caveolae and intracellular calcium stores.

Dynamic buffering of mitochondrial Ca2+ during Ca2+ uptake and Na+-induced Ca2+ release

In cardiac mitochondria, matrix free Ca(2+) ([Ca(2+)]m) is primarily regulated by Ca(2+) uptake and release via the Ca(2+) uniporter (CU) and Na(+)/Ca(2+) exchanger (NCE) as well as by Ca(2+) buffering. Although experimental and computational studies on the CU and NCE dynamics exist, it is not well understood how matrix Ca(2+) buffering affects these dynamics under various Ca(2+) uptake and release conditions, and whether this influences the stoichiometry of the NCE. To elucidate the role of matrix Ca(2+) buffering on the uptake and release of Ca(2+), we monitored Ca(2+) dynamics in isolated mitochondria by measuring both the extra-matrix free [Ca(2+)] ([Ca(2+)]e) and [Ca(2+)]m. A detailed protocol was developed and freshly isolated mitochondria from guinea pig hearts were exposed to five different [CaCl2] followed by ruthenium red and six different [NaCl]. By using the fluorescent probe indo-1, [Ca(2+)]e and [Ca(2+)]m were spectrofluorometrically quantified, and the stoichiometry of the NCE was determined. In addition, we measured NADH, membrane potential, matrix volume and matrix pH to monitor Ca(2+)-induced changes in mitochondrial bioenergetics. Our [Ca(2+)]e and [Ca(2+)]m measurements demonstrate that Ca(2+) uptake and release do not show reciprocal Ca(2+) dynamics in the extra-matrix and matrix compartments. This salient finding is likely caused by a dynamic Ca(2+) buffering system in the matrix compartment. The Na(+)- induced Ca(2+) release demonstrates an electrogenic exchange via the NCE by excluding an electroneutral exchange. Mitochondrial bioenergetics were only transiently affected by Ca(2+) uptake in the presence of large amounts of CaCl2, but not by Na(+)- induced Ca(2+) release.

Ionophorous functions of phosphatidic acid in the plant cell

Effects of phosphatidic acid (PA), a product of phospholipase D activity, on Ca 2+ and H + transport were investigated in membrane vesicles obtained from roots and coleoptiles of maize ( Zea mays L.). Calcium flows were measured with fluorescent probes indo-1 and chlorotetracycline loaded into the vesicles and added to the incubation medium, respectively. Phosphatidic acid (50-500 µ M) was found to induce downhill flow of Ca 2+ along the concentration gradient into the plasma membrane vesicles and endomembrane vesicles (tono- plast and endoplasmic reticulum). Protonophorous functions of PA were probed with acridine orange. First, the ionic H + gradient was created on the tonoplast vesicles by means of H + -ATPase activation with Mg-ATP addi- tion. Then, the vesicles were treated with 25-100 µ M PA, which induced the release of protons from tonoplast vesicles and dissipation of the proton gradient. Thus, PA could function as an ionophore and was able to transfer Ca 2+ and H + across plant cell membranes along concentration gradients of these ions. The role of PA in mech- anisms of intracellular signaling in plants is discussed.

Novel glycosaminoglycan mimetic (RGTA, RGD120) contributes to enhance skeletal muscle satellite cell fusion by increasing intracellular Ca2+ and calpain activity

Glycosaminoglycans (GAG) are classes of molecules that play an important role in cellular processes. The use of GAG mimetics called regenerating agent (RGTA) represents a tool to investigate the effect of GAG moiety on cellular behavior. A first member of the RGTA family (RG1192), a dextran polymers with defined amounts of sulfate, carboxymethyl, as well as hydrophobic groups (benzylamide), was shown to stimulate skeletal muscle repair after damage and myoblast differentiation. To obtain a comprehensive insight into the mechanism of action of GAG mimetics, we investigated the effect on myoblast differentiation of a novel RGTA, named RGD120, which was devoid of hydrophobic substitution and had ionic charge similar to heparin. Myoblasts isolated from adult rat skeletal muscles and grown in primary cultures were used in this study. We found that chronic treatment with RGD120 increased the growth of adult myoblasts and induced their precocious fusion into myotubes in vitro. It also partially overcame the inhibitory effect of the calpain inhibitor N-acetyl-leu-leu-norleucinal (ALLN) on these events. Western blot and zymography analyses revealed that milli calpain was slightly increased by RGD120 chronic treatment. In addition, using fluorescent probes (Indo-1 and Boc-leu-met-MAC), we demonstrated that RGD120 added to prefusing myoblast cultures accelerates myoblast fusion into myotubes, induced an increase of cytosolic free calcium concentration, and concomitantly an increase of intracellular calpain protease activity. Altogether, these results suggested that the efficiency of RGD120 in stimulating myogenesis might be in part explained through its effect on calcium mobilization as well as on the calpain amount and activity.

A functional comparison of the venom of three Australian jellyfish— Chironex fleckeri, Chiropsalmus sp., and Carybdea xaymacana—on cytosolic Ca 2+, haemolysis and Artemia sp. lethality

Cnidarian venoms produce a wide spectrum of envenoming syndromes in humans ranging from minor local irritation to death. Here, the effects of Chironex fleckeri, Chiropsalmus sp., and Carybdea xaymacana venoms on ventricular myocyte cytosolic Ca 2+, haemolysis and Artemia sp. lethality are compared for the first time. All three venoms caused a large, irreversible elevation of cytosolic Ca 2+ in myocytes as measured using the Ca 2+ sensitive fluorescent probe Indo-1. The L-type Ca 2+ channel antagonist verapamil had no effect on Ca 2+ influx whilst La 3+, a non-specific channel and pore blocker, inhibited the effect. Haemolytic activity was observed for all venoms, with C. xaymacana venom displaying the greatest activity. These activities are consistent with the presence of a pore-forming toxin existing in the venoms which has been demonstrated by transmission electron microscopy in the case of C. fleckeri. The venom of C. fleckeri was found to be more lethal against Artemia sp. than the venom of the other species, consistent with the order of known human toxicities. This suggests that the observed lytic effects may not underlie the lethal effects of the venom, and raises the question of how such potent activities are dealt with by envenomed humans.

Modulation of intracellular Na+ and Ca2+ induced by the cardiac Na+-Ca2+ exchanger in guinea pig ventricular myocytes.

The Na+-Ca2+ exchanger current was measured in single guinea pig ventricular myocytes, using the whole-cell voltage-clamp technique, and intracellular free calcium concentration ([Ca2+](i)) was monitored simultaneously with the fluorescent probe Indo-1 applied intracellularly through a perfused patch pipette. In external solutions, which have levels of Ca2+ (approximately 66 microM Ca2+) thought low enough to inhibit exchanger turnover, the removal of external Na+ (by replacement with Li+) induced both an outward shift of the holding current and an increase in [Ca2+](i), even though the recording pipette contained 30 mM bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), sufficient to completely block phasic contractions. The effects of Na+ removal were blocked either by the extracellular application of 2 mM Ni2+ or by chelating extracellular Ca2+ with 1 mM EGTA. In the presence of 10 microM Ryanodine, the effects of external Na+ substitution with Li(+) on both membrane current and [Ca2+](i) were attenuated markedly in amplitude and at a much slower time course. Reversal potentials were estimated by using ramp pulses and by defining exchange currents as the Ni2+-sensitive components. The experimental values of the reversal potential and [Ca2+](i) were used to calculate cytosolic Na+ ([Na+](i)) by assuming an exchanger stoichiometry of 3Na+ : 1Ca2+. These calculations suggested that in the nominal absence of external Ca2+ ( approximately 66 microM under our experimental conditions), the exchanger operates at -40 mV as though approximately 40 mM Na+ had accumulated in the vicinity of the intracellular binding sites. We conclude that under the conditions of low extracellular Ca2+ and high intracellular Ca2+ buffering, the Na+-Ca2+ exchanger can still generate sufficient Ca2+ influx on the removal of external Na+ to markedly increase cytosolic free Ca2+.

Sevoflurane preconditioning before moderate hypothermic ischemia protects against cytosolic [Ca(2+)] loading and myocardial damage in part via mitochondrial K(ATP) channels.

Brief sevoflurane exposure and washout (sevoflurane preconditioning [SPC]) before 30-min global ischemia at 37 degrees C is known to improve cardiac function, decrease cytosolic [Ca(2+)] loading, and reduce infarct size on reperfusion. It is not known if anesthetic preconditioning (APC) applies as well to hypothermic ischemia and reperfusion and if K(ATP) channels are involved. The authors examined in guinea pig isolated hearts the effect of sevoflurane exposure before 4-h global ischemia at 17 degrees C on cardiac function, cytosolic [Ca(2+)] loading, and infarct size. In addition they tested the potential role of the mitochondrial K(ATP) channel in eliciting the cardioprotection by SPC. Hearts were randomly assigned to (1) a nontreated hypothermic ischemia group (CON), (2) a group given 3.5 vol% sevoflurane for 15 min with a 15-min washout before hypothermic ischemia (SPC), and (3) an SPC group in which anesthetic exposure was bracketed with 200 microm 5-hydroxydecanoate (5-HD) from 5 min before until 5 min after sevoflurane (SPC + 5-HD). Cytosolic [Ca(2+)] was measured in the left ventricular (LV) free wall with the intracellularly loaded fluorescence probe indo-1. Initial reperfusion in CON hearts markedly increased systolic and diastolic [Ca(2+)] and reduced contractility (dLVP/dt(max)), relaxation (diastolic LVP, dLVP/dt(min)), myocardial oxygen consumption (MvO(2)), and cardiac efficiency. In SPC hearts, cytosolic [Ca(2+)] overloading (especially diastolic [Ca(2+)]) was decreased with increased myocardial [Ca(2+)] influx (d[Ca(2+)]/dt(max)) and efflux (d[Ca(2+)]/dt(min)), improved contractility, relaxation, coronary flow, MvO(2), cardiac efficiency, and decreased infarct size. In SPC + 5HD hearts, the reduction in infarct size was antagonized by 5-HD, but functional return was less affected by 5-HD. Anesthetic preconditioning occurs after long-term hypothermic ischemia, and the infarct size reduction is the result, in part, of mitochondrial K(ATP) channel opening.

Role of cyclic GMP‐dependent protein kinase in the contractile response to exogenous nitric oxide in rat cardiac myocytes

Nitric oxide (NO) can directly modulate cardiac contractility by accelerating relaxation and reducing diastolic tone. The intracellular mechanisms underlying these contractile effects are poorly understood. Here we investigate the role of cyclic GMP-dependent protein kinase (PKG) in the contractile response to exogenous NO in rat ventricular myocytes. Isolated ventricular myocytes were stimulated electrically and contractility was assessed by measuring cell shortening. Some cells were loaded with the fluorescent Ca(2+) probe indo-1 AM for simultaneous assessment of the intracellular Ca(2+) transient. The NO donor diethylamine NONOate (DEA/NO, 10 microM) significantly increased resting cell length, reduced twitch amplitude and accelerated time to 50 % relaxation (to 100.8 +/- 0.2, 83.7 +/- 3.0 and 88.9 +/- 3.7 % of control values, respectively). The contractile effects of DEA/NO occurred without significant changes in the amplitude or kinetics of the intracellular Ca(2+) transient, suggesting that the myofilament response to Ca(2+) was reduced. These effects were abolished by inhibition of either guanylyl cyclase (with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; ODQ, 10 microM) or PKG (with Rp-8-Br-cGMPs, 10 microM) suggesting that, at the concentration investigated, the effects of DEA/NO were mediated exclusively by PKG, following activation of guanylyl cyclase and elevation of cGMP. Direct activation of PKG with 8-pCPT-cGMP (10 microM) mimicked the effects of DEA/NO (resting cell length and time to 50 % relaxation were 100.6 +/- 0.1 and 90.5 +/- 1.5 % of control values, respectively).The reduced myofilament Ca(2+) responsiveness was not attributable to an intracellular acidosis since the small reduction in pH(i) induced by DEA/NO was found to be uncoupled from its contractile effects. However, hearts treated with DEA/NO (10 microM) showed a significant increase (1.4-fold; P < 0.01) in troponin I phosphorylation compared to control, untreated hearts. These results suggest that the reduction in myofilament Ca(2+) responsiveness produced by DEA/NO results from phosphorylation of troponin I by PKG.

The Creatine Kinase System Is Essential for Optimal Refill of the Sarcoplasmic Reticulum Ca2+ Store in Skeletal Muscle

Muscle function depends on an adequate ATP supply to sustain the energy consumption associated with Ca(2+) cycling and actomyosin sliding during contraction. In this regulation of energy homeostasis, the creatine kinase (CK) circuit for high energy phosphoryl transfer between ATP and phosphocreatine plays an important role. We earlier established a functional connection between the activity of the CK system and Ca(2+) homeostasis during depolarization and contractile activity of muscle. Here, we show how CK activity is coupled to the kinetics of spontaneous and electrically induced Ca(2+) transients in the sarcoplasm of myotubes. Using the UV ratiometric Ca(2+) probe Indo-1 and video-rate confocal microscopy in CK-proficient and -deficient cultured cells, we found that spontaneous and electrically induced transients were dependent on ryanodine-sensitive Ca(2+) release channels, sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase pumps, extracellular calcium, and functional mitochondria in both cell types. However, at increasing sarcoplasmic Ca(2+) load (induced by electrical stimulation at 0.1, 1, and 10 Hz), the Ca(2+) removal rate and the amount of Ca(2+) released per transient were gradually reduced in CK-deficient (but not wild-type) myotubes. We conclude that the CK/phosphocreatine circuit is essential for efficient delivery of ATP to the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase pumps and thereby directly influences sarcoplasmic reticulum refilling and the kinetics of the sarcoplasmic Ca(2+) signals.

Sevoflurane before or after ischemia improves contractile and metabolic function while reducing myoplasmic Ca(2+) loading in intact hearts.

Ca(2+) loading occurs during myocardial reperfusion injury. Volatile anesthetics can reduce reperfusion injury. The authors tested whether sevoflurane administered before index ischemia in isolated hearts reduces myoplasmic diastolic and systolic [Ca(2+)] and improves function more so than when sevoflurane is administered on reperfusion. Four groups of guinea pig hearts were perfused with crystalloid solution (55 mmHg, 37 degrees C): (1) no treatment before 30 min global ischemia and 60 min reperfusion (CON); (2) 3.5 vol% sevoflurane administered for 10 min before ischemia (SBI); (3) 3.5 vol% sevoflurane administered for 10 min after ischemia (SAI); and (4) 3.5 vol% sevoflurane administered for 10 min before and after ischemia (SBAI). Phasic myoplasmic diastolic and systolic [Ca(2+)] were measured in the left ventricular free wall with the fluorescence probe indo-1. Ischemia increased diastolic [Ca(2+)] and diastolic left ventricular pressure (LVP). In CON hearts, initial reperfusion greatly increased diastolic [Ca2+] and systolic [Ca(2+)] and reduced contractility (systolic-diastolic LVP, dLVP/dt(max)), relaxation (diastolic LVP, dLVP/dt(min)), myocardial oxygen consumption (MvO(2)), and cardiac efficiency. SBI, SAI, and SBAI each reduced ventricular fibrillation, attenuated increases in systolic and systolic-diastolic [Ca(2+)], improved contractile and relaxation indices, and increased coronary flow, percent oxygen extraction, MvO(2), and cardiac efficiency during 60 min reperfusion compared with CON. SBI was more protective than SAI, and SBAI was generally more protective than SAI. Sevoflurane improves postischemic cardiac function while reducing Ca(2+) loading when it is administered before or after ischemia, but protection is better when it is administered before ischemia. Reduced Ca(2+) loading on reperfusion is likely a result of the anesthetic protective effect.

External Bioenergy Increases Intracellular Free Calcium Concentration and Reduces Cellular Response to Heat Stress

BackgroundExternal bioenergy (energy emitted from the body) can influence a variety of biological activities. It has been shown to enhance immunity, promote normal cell proliferation, increase tumor cell death, and...

Solubilized zona pellucida proteins and progesterone induce calcium influx and the acrosome reaction in capacitated dog spermatozoa.

Spermatozoa from the sperm-rich fractions of the semen of 6 beagle dogs were capacitated and the effect of both zona pellucida (ZP) proteins and progesterone on calcium flux and the acrosome reaction measured. Sperm calcium flux was determined using the dual wavelength calcium probe indo-1/AM (6 microM) in a flow cytometric assay (one ejaculate from each dog examined; n = 6). No calcium flux was observed in the negative control treatments (RPMI medium or DMSO). Both heat-solubilized bitch ZP proteins and progesterone caused a similar response characterized by a gradual but marked influx of calcium ions which was sustained over 2 min. Acrosomal status was assessed by indirect immunofluorescence using a specific monoclonal antibody following 1 hr incubation for each treatment (four ejaculates from each dog examined; n = 24). The level of acrosomal exocytosis was very high for samples treated with ZP proteins (70.3 +/- 2.1%) and progesterone (84.6 +/- 1.5%) and was significantly different from the respective controls (P < 0.001). Interestingly the patterns of calcium flux in response to both ZP proteins and progesterone were in contrast to the situation in other species studied to date raising the possibility that the mechanism for triggering the acrosome reaction may be different in dog spermatozoa. In addition the high degree of progesterone-induced acrosomal exocytosis compared to other species raises the probability that the majority of dog spermatozoa are already undergoing the acrosome reaction before they reach the egg ZP.

Membrane-Initiated Ca 2+ Signals Are Reshaped during Propagation to Subcellular Regions

An important aspect of Ca 2+ signaling is the ability of cells to generate intracellular Ca 2+ waves. In this study we have analyzed the cellular and subcellular kinetics of Ca 2+ waves in a neuroendocrine transducer cell, the melanotrope of Xenopus laevis, using the ratiometric Ca 2+ probe indo-1 and video-rate UV confocal laser-scanning microscopy. The purpose of the present study was to investigate how local Ca 2+ changes contribute to a global Ca 2+ signal; subsequently we quantified how a Ca 2+ wave is kinetically reshaped as it is propagated through the cell. The combined kinetics of all subcellular Ca 2+ signals determined the shape of the total cellular Ca 2+ signal, but each subcellular contribution to the cellular signal was not constant in time. Near the plasma membrane, [Ca 2+] i increased and decreased rapidly, processes that can be described by a linear and exponential function, respectively. In more central parts of the cell slower kinetics were observed that were best described by a Hill equation. This reshaping of the Ca 2+ wave was modeled with an equation derived from a low-pass RC filter. We propose that the differences in spatial kinetics of the Ca 2+ signal serves as a mechanism by which the same cellular Ca 2+ signal carries different regulatory information to different subcellular regions of the cell, thus evoking differential cellular responses.

The triakontatetraneuropeptide TTN increases [Ca2+]i in rat astrocytes through activation of peripheral‐type benzodiazepine receptors

Astrocytes synthesize a series of regulatory peptides called endozepines, which act as endogenous ligands of benzodiazepine receptors. We have recently shown that one of these endozepines, the triakontatetraneuropeptide TTN, stimulates DNA synthesis in astroglial cells. The purpose of the present study was to determine the mechanism of action of TTN on cultured rat astrocytes. Binding of the peripheral-type benzodiazepine receptor ligand [3H]Ro5-4864 to intact astrocytes was displaced by TTN, whereas its C-terminal fragment (TTN[17-34], the octadecaneuropeptide ODN) did not compete for [3H]Ro5-4864 binding. Microfluorimetric measurement of cytosolic calcium concentrations ([Ca2+]i) with the fluorescent probe indo-1 showed that TTN (10(-10) to 10(-6) M) provokes a concentration-dependent increase in [Ca2+]i in cultured astrocytes. Simultaneous administration of TTN (10(-8) M) and Ro5-4864 (10(-5) M) induced an increase in [Ca2+]i similar to that obtained with Ro5-4864 alone. In contrast, the effects of TTN (10(-8) M) and ODN (10(-8) M) on [Ca2+]i were strictly additive. Chelation of extracellular Ca2+ by EGTA (6 mM) or blockage of Ca2+ channels with Ni2+ (2 mM) abrogated the stimulatory effect of TTN. The calcium influx evoked by TTN (10(-7) M) or by Ro5-4864 (10(-5) M) was not affected by the N- and T-type calcium channel blockers omega-conotoxin (10(-6) M) and mibefradil (10(-6) M), but was significantly reduced by the L-type calcium channel blocker nifedipine (10(-7) M). Patch-clamp studies showed that, at negative potentials, TTN (10(-7) M) induced a sustained depolarization. Reduction of the chloride concentration in the extracellular solution shifted the reversal potential from 0 mV to a positive potential. These data show that TTN, acting through peripheral-type benzodiazepine receptors, provokes chloride efflux, which in turn induces calcium influx via L-type calcium channels in rat astrocytes.

Plug flow cytometry extends analytical capabilities in cell adhesion and receptor pharmacology.

Plug flow cytometry is a recently developed system for the automated delivery of multiple small boluses or "plugs" of cells or particles to the flow cytometer for analysis. Important system features are that sample plugs are of precisely defined volume and that the sample vessel need not be pressurized. We describe how these features enable direct cell concentration determinations and novel ways to integrate flow cytometers with other analytical instruments. Adhesion assays employed human polymorphonuclear neutrophils (PMNs) loaded with Fura Red and Chinese hamster ovary (CHO) cells cotransfected with genes for green fluorescent protein (GFP) and human P-selectin. U937 cells expressing the human 7-transmembrane formyl peptide receptor were loaded with the fluorescent probe indo-1 for intracellular ionized calcium determinations. A computer-controlled syringe or peristaltic pump loaded the sample into a sample loop of the plug flow coupler, a reciprocating eight-port valve. When the valve position was switched, the plug of sample in the sample loop was transported to the flow cytometer by a pressure-driven fluid line. In stirred mixtures of PMNs and CHO cells, we used plug flow cytometry to directly quantify changes in concentrations of nonadherent singlet PMNs. This approach enabled accurate quantification of adherent PMNs in multicell aggregates. We constructed a novel plug flow interface between the flow cytometer and a cone-plate viscometer to enable real-time flow cytometric analysis of cell-cell adhesion under conditions of uniform shear. The High Throughput Pharmacology System (HTPS) is an instrument used for automated programming of complex pharmacological cell treatment protocols. It was interfaced via the plug flow coupling device to enable rapid (< 5 min) flow cytometric characterization of the intracellular calcium dose-response profile of U937 cells to formyl peptide. By facilitating the coupling of flow cytometers to other fluidics-based analytical instruments, plug flow cytometry has extended analytical capabilities in cell adhesion and pharmacological characterization of receptor-ligand interactions.

Na +-Dependent Ca 2+ Transport Modulates the Secretory Response to the Fc ϵ Receptor Stimulus of Mast Cells

Immunological stimulation of rat mucosal-type mast cells (RBL-2H3 line) by clustering of their Fc ϵ receptors (Fc ϵRI) causes a rapid and transient increase in free cytoplasmic Ca 2+ ion concentration ([Ca 2+] i) because of its release from intracellular stores. This is followed by a sustained elevated [Ca 2+] i, which is attained by Ca 2+ influx. Because an Fc ϵRI-induced increase in the membrane permeability for Na + ions has also been observed, and secretion is at least partially inhibited by lowering of extracellular sodium ion concentrations ([Na +] o), the operation of a Na +/Ca 2+ exchanger has been considered. We found significant coupling between the Ca 2+ and Na + ion gradients across plasma membranes of RBL-2H3 cells, which we investigated employing 23Na-NMR, 45Ca 2+, 85Sr 2+, and the Ca 2+-sensitive fluorescent probe indo-1. The reduction in extracellular Ca 2+ concentrations ([Ca 2+] o) provoked a [Na +] i increase, and a decrease in [Na +] o results in a Ca 2+ influx as well as an increase in [Ca 2+] i. Mediator secretion assays, monitoring the released β-hexosaminidase activity, showed in the presence of extracellular sodium a sigmoidal dependence on [Ca 2+] o. However, the secretion was not affected by varying [Ca 2+] o as [Na +] o was lowered to 0.4 mM, while it was almost completely inhibited at [Na +] o = 136 mM and [Ca 2+] o < 0.05 mM. Increasing [Na +] o caused the secretion to reach a minimum at [Na +] o = 20 mM, followed by a steady increase to its maximum value at 136 mM. A parallel [Na +] o dependence of the Ca 2+ fluxes was observed: Antigen stimulation at [Na +] o = 136 mM caused a pronounced Ca 2+ influx. At [Na +] o = 17 mM only a slight Ca 2+ efflux was detected, whereas at [Na +] o = 0.4 mM no Ca 2+ transport across the cell membrane could be observed. Our results clearly indicate that the [Na +] o dependence of the secretory response to Fc ϵRI stimulation is due to its influence on the [Ca 2+] i, which is mediated by a Na +-dependent Ca 2+ transport.

Measurement of intracellular calcium concentration and plasma membrane potential in human spermatozoa using flow cytometry.

We report 2 novel approaches using flow cytometry to measure intracellular calcium concentration and plasma membrane potential in human spermatozoa. Both approaches have the potential to measure different responses in subpopulations of cells, which is particularly useful when studying heterogeneous populations such as human spermatozoa. Intracellular calcium concentration ([Ca2+]i) was measured using the probe indo-1/AM. This allowed measurements to be made that were independent of variation in cell size and dye loading. It also enabled dead cells to be directly identified and excluded from the analyses without the need for counterstaining. Mean basal [Ca2+]i was determined as 50 nM (25-75 nM range) and, in response to the agonist progesterone (20 microM), this increased transiently to 195 nM (125-285 nM range) before declining to approximately half the maximal level within 2 minutes (values in parentheses correspond to the range of values typically found within a sperm population from 1 sample). These results are comparable with previously published data on whole sperm populations. Sperm membrane potential (VM) was assayed using the probe DiOC6(3). In carefully controlled experiments, a marked depolarization of the plasma membrane potential of capacitated spermatozoa was observed in response to progesterone (20 microM). Following in vitro capacitation, the sperm plasma membrane potential became hyperpolarized compared with the noncapacitated state. Therefore, this technique may be used to assay for sperm capacitation in vitro.