AM Fluorescence Research Articles

Cerebromicrovascular endothelial cells are resistant to l-glutamate

Cerebral microvascular endothelial cells (CMVECs) have recently been implicated as targets of excitotoxic injury by l-glutamate (l-glut) or N-methyl-d-aspartate (NMDA) in vitro. However, high levels of l-glut do not compromise the function of the blood-brain barrier in vivo. We sought to determine whether primary cultures of rat and piglet CMVECs or cerebral microvascular pericytes (CMVPCs) are indeed sensitive to l-glut or NMDA. Viability was unaffected by 8-h exposure to 1-10 mM l-glut or NMDA in CMVECs or CMVPCs isolated from both species. Furthermore, neither 1 mM l-glut nor NMDA augmented cell death induced by 12-h oxygen-glucose deprivation in rat CMVECs or by 8-h medium withdrawal in CMVPCs. Additionally, transendothelial electrical resistance of rat CMVEC-astrocyte cocultures or piglet CMVEC cultures were not compromised by up to 24-h exposure to 1 mM l-glut or NMDA. The Ca(2+) ionophore calcimycin (5 microM), but not l-glut (1 mM), increased intracellular Ca(2+) levels in rat CMVECs and CMVPCs assessed with fluo-4 AM fluorescence and confocal microscopy. CMVEC-dependent pial arteriolar vasodilation to hypercapnia and bradykinin was unaffected by intracarotid infusion of l-glut in anesthetized piglets by closed cranial window/intravital microscopy. We conclude that cerebral microvascular cells are insensitive and resistant to glutamatergic stimuli in accordance with their in vivo role as regulators of potentially neurotoxic amino acids across the blood-brain barrier.

Plasmin Potentiates Synaptic N-Methyl-D-aspartate Receptor Function in Hippocampal Neurons through Activation of Protease-activated Receptor-1

Protease-activated receptor-1 (PAR1) is activated by a number of serine proteases, including plasmin. Both PAR1 and plasminogen, the precursor of plasmin, are expressed in the central nervous system. In this study we examined the effects of plasmin in astrocyte and neuronal cultures as well as in hippocampal slices. We find that plasmin evokes an increase in both phosphoinositide hydrolysis (EC(50) 64 nm) and Fura-2/AM fluorescence (195 +/- 6.7% above base line, EC(50) 65 nm) in cortical cultured murine astrocytes. Plasmin also activates extracellular signal-regulated kinase (ERK1/2) within cultured astrocytes. The plasmin-induced rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the increase in phospho-ERK1/2 levels were diminished in PAR1(-/-) astrocytes and were blocked by 1 microm BMS-200261, a selective PAR1 antagonist. However, plasmin had no detectable effect on ERK1/2 or [Ca(2+)](i) signaling in primary cultured hippocampal neurons or in CA1 pyramidal cells in hippocampal slices. Plasmin (100-200 nm) application potentiated the N-methyl-D-aspartate (NMDA) receptor-dependent component of miniature excitatory postsynaptic currents recorded from CA1 pyramidal neurons but had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- or gamma-aminobutyric acid receptor-mediated synaptic currents. Plasmin also increased NMDA-induced whole cell receptor currents recorded from CA1 pyramidal cells (2.5 +/- 0.3-fold potentiation over control). This effect was blocked by BMS-200261 (1 microm; 1.02 +/- 0.09-fold potentiation over control). These data suggest that plasmin may serve as an endogenous PAR1 activator that can increase [Ca(2+)](i) in astrocytes and potentiate NMDA receptor synaptic currents in CA1 pyramidal neurons.

Identification of Farnesyl Pyrophosphate and N-Arachidonylglycine as Endogenous Ligands for GPR92

A series of small compounds acting at the orphan G protein-coupled receptor GPR92 were screened using a signaling pathway-specific reporter assay system. Lipid-derived molecules including farnesyl pyrophosphate (FPP), N-arachidonylglycine (NAG), and lysophosphatidic acid were found to activate GPR92. FPP and lysophosphatidic acid were able to activate both G(q/11)- and G(s)-mediated signaling pathways, whereas NAG activated only the G(q/11)-mediated signaling pathway. Computer-simulated modeling combined with site-directed mutagenesis of GPR92 indicated that Thr(97), Gly(98), Phe(101), and Arg(267) of GPR92 are responsible for the interaction of GPR92 with FPP and NAG. Reverse transcription-PCR analysis revealed that GPR92 mRNA is highly expressed in the dorsal root ganglia (DRG) but faint in other brain regions. Peripheral tissues including, spleen, stomach, small intestine, and kidney also expressed GPR92 mRNA. Immunohistochemical analysis revealed that GPR92 is largely co-localized with TRPV1, a nonspecific cation channel that responds to noxious heat, in mouse and human DRG. FPP and NAG increased intracellular Ca(2+) levels in cultured DRG neurons. These results suggest that FPP and NAG play a role in the sensory nervous system through activation of GPR92.

Complexation of amine free radicals during the radiation-induced chain oxidation reaction of N,N,N′,N′-tetramethyl-4,4′-diaminodiphenylmethane

Complexation of free radicals derived from the amine in the chain redox reaction in γ-irradiated solid PVC films doped with N,N,N′-N′-tetramethyl-4,4′-diaminodiphenylmethane (Am) and CBr4 was studied by means of the ESR and luminescence techniques. The effect of concentration of the additives Am and CBr4 on the quenching of fluorescence of Am· radicals was examined. The data were rationalized in terms of the Stern-Volmer formalism for static quenching, which results from the formation of nonfluorescing ground-state charge-transfer complexes between the radical Am·, as well as its dimeric form Am 2 · , and CBr4. The association constants of the complexes were determined. The mechanism of radiation-induced chain oxidation of Am involving these complexes is discussed.

Identification of five human novel genes associated with cell proliferation by cell-based screening from an expressed cDNA ORF library

The development of functional profiling technologies provides opportunity for high-throughput functional genomics studies. We describe a cell-based screening system to identify novel human genes associated with cell proliferation. The method integrates luciferase reporter gene activity, fluorescence stain, automated microscopy and cellular phenotype assays. We successfully used the system to screen 409 novel human genes cloned by our lab and found that 27 genes significantly up-regulated promoter-Renilla luciferase reporter plasmid (pRL) activity. Among them, five genes, TRAF3IP3, ZNF306, ZNF250, SGOL1, and ZNF434, were determined through morphological observation, calcein AM fluorescence stain, MTT assay and cell cycle analysis to be associated with cell proliferation. Furthermore, we showed that the gene TRAF3IP3, which initially was identified to specifically interact with TRAF3, stimulated cell growth by modulating the c-Jun N-terminal kinase (JNK) pathway, and RNAi of TRAF3IP3 confirmed that the effect was physiological and necessary. In summary, we integrated a rapid and efficient system for screening novel growth regulatory genes. Using the new screening system we identified five genes associated with cell proliferation for the first time.

The influence of dihydropyridines calcium antagonists on 5-HT-induced intracellular calcium signal

Confocal laser scanning microscopy (CLSM) was applied to detect the intracellular [Ca2+] variety of fluorescent intension, with Fluo-3/AM fluorescence loaded in SFSMC. The results show that 10 μmol/L Lacidipine can reduce the frequence which 10 μmol/L 5-HT induced [Ca2+] spark in SFSMC of calcium over loading to 50%, and amplitude to 50% or so. We can draw a conclusion that dihydropyridines calcium antagonists lacidipine can antagonize the release of intracellular [Ca2+] which 5-HT-induced in dose dependent manner.

Localized Ca2+ uncaging induces Ca2+ release through IP3R in smooth muscle

Our previous study indicated that there are two types of Ca2+ release events seen in intact mouse bladder tissue. In this study our aim is to investigate the mechanism that underlies the phenomena of Ca2+ release in smooth muscle. Single cells were isolated and tissue segments were prepared by cutting the detrusor into 0.1 cm x 0.5 cm strips running along the axis from the neck to the fundus. Single cells and intact tissue strips were co-loaded with the Ca2+ indicator and caged Ca2+ by incubation with 10 micromol/L Fluo-4 AM and DMNP-EDTA-AM. Fluo-4 AM fluorescence was detected by laser scanning confocal microscopy, and local uncaging of DMNP-EGTA was achieved by brief exposure to the output of a diode-pumped, Ti:sapphire laser tuned to 730 nm. Local uncaging of caged Ca2+ was able to trigger Ca2+ release events in both single cells and tissue strips from mouse bladder. The Ca2+ release events could not be blocked by ryanodine alone, but the property of the Ca2+ release was markedly altered. Surprisingly, in the presence of ryanodine, Xestospongin C completely inhibited the Ca2+ release events both in single cell and tissue experiments. (1) Two photon flash photolysis (TPFP) triggers Ca2+ induced Ca2+ release. This process involves release through type 2 ryanodine receptor channels; (2) TPFP results in the release of Ca2+ through inositol 1,4,5-trisphosphate receptors in the absence of phospholipase C activation.

Antiangiogenic Effect of Rosiglitazone Is Mediated via Peroxisome Proliferator-activated Receptor γ-activated Maxi-K Channel Opening in Human Umbilical Vein Endothelial Cells

Recent evidence shows that peroxisome proliferator-activated receptor gamma (PPARgamma) ligands induce the antiangiogenic effect in endothelial cells and tumors. In the present study, we elucidated the involvement of maxi-K channel activation in the antiangiogenic effect of rosiglitazone, a well known PPARgamma ligand in human umbilical vein endothelial cells. We found that the antiangiogenic effects of rosiglitazone were reversed by either bisphenol A diaglycidyl ether, a PPARgamma antagonist, or iberiotoxin, a maxi-K channel blocker. Knockdown of maxi-K channel expression also reversed the antiangiogenic effects. Iberiotoxin reversed the rosiglitazone-induced hyperpolarization while having no effect on the endogenous PPARgamma activation, suggesting that rosiglitazone activates maxi-K channel via PPARgamma. In the rosiglitazone-induced antiangiogenic process, endothelial nitric-oxide synthase-Ser1179 phosphorylation and NO production were significantly elevated, and treatment with the NOS inhibitor N(G)-monomethyl-L-arginine acetate abolished the antiangiogenic and apoptotic effects of rosiglitazone, indicating NO as a key mediator of the rosiglitazone actions. In conclusion, rosiglitazone significantly inhibited VEGF165-induced angiogenesis by a proapoptotic mechanism via PPARgamma-mediated NO production, followed by maxi-K channel opening.

Impaired endothelin-induced vasoconstriction in coronary arteries of Zucker obese rats is associated with uncoupling of [Ca2+]isignaling

Although insulin resistance (IR) is a major risk factor for coronary artery disease, little is known about the regulation of coronary vascular tone in IR by endothelin-1 (ET-1). We examined ET-1 and PGF(2alpha)-induced vasoconstriction in isolated small coronary arteries (SCAs; approximately 250 microM) of Zucker obese (ZO) rats and control Zucker lean (ZL) rats. ET-1 response was assessed in the absence and presence of endothelin type A (ET(A); BQ-123), type B (ET(B); BQ-788), or both receptor inhibitors. ZO arteries displayed reduced contraction to ET-1 compared with ZL arteries. In contrast, PGF(2alpha) elicited similar vasoconstriction in both groups. ET(A) inhibition diminished the ET-1 response in both groups. ET(B) inhibition alone or in combination with ET(A) blockade, however, restored the ET-1 response in ZO arteries to the level of ZL arteries. Similarly, inhibition of endothelial nitric oxide (NO) synthase with N(omega)-nitro-l-arginine methyl ester (l-NAME) enhanced the contraction to ET-1 and abolished the difference between ZO and ZL arteries. In vascular smooth muscle cells from ZO, ET-1-induced elevation of myoplasmic intracellular free calcium concentration ([Ca2+]i) (measured by fluo-4 AM fluorescence), and maximal contractions were diminished compared with ZL, both in the presence and absence of l-NAME. However, increases in [Ca2+]i elicited similar contractions of the vascular smooth muscle cells in both groups. Analysis of protein and total RNA from SCA of ZO and ZL revealed equal expression of ET-1 and the ET(A) and ET(B) receptors. Thus coronary arteries from ZO rats exhibit reduced ET-1-induced vasoconstriction resulting from increased ET(B)-mediated generation of NO and diminished elevation of myoplasmic [Ca2+]i.

Evidence for the presence of N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptor ligands in human amniotic fluid and fMLP receptor modulation by physiological labour

Objectives The presence of amniotic binding sites for N-formyl-methionyl-leucyl-phenylalanine (fMLP), an inflammatory peptide, and its ability to induce prostaglandin E 2 synthesis in the human amnion prompted us to investigate for: (1) the presence of fMLP receptor ligands (fMLPRL) in the amniotic fluid; (2) expression of the fMLP receptor in amniotic tissue; (3) the effect of amniotic fMLPRL on neutrophil cyclic AMP (cAMP) level and calcium concentration ([Ca 2+] i ) during physiological pregnancy and labour. Methods Binding assays were performed on neutrophils to determine the presence of fMLRL in the amniotic fluid at the 17th week of pregnancy, as well as at term, before and after the onset of labour. The expression of fMLP receptor mRNA was evaluated by RT-PCR, the cAMP level by a radiochemical assay, and the calcium concentration by Fura-2 AM fluorescence measurement. Results fMLPRLs were detectable in amniotic fluid throughout pregnancy, and their levels did not vary during gestation. Labour significantly increased both the amniotic fMLPRL level and the expression of fMLP receptor in amnion tissue. The increased amniotic fMLPRL concentration noted during labour significantly increased neutrophil cAMP level and [Ca 2+] i . Conclusions These findings demonstrate for the first time the presence of fMLP receptor ligands in amniotic fluid, and indicate a modulation of the fMLP system by the events of physiological labour.

Impaired Intracellular Ca2+Dynamics in Live Cardiomyocytes Revealed by Rapid Line Scan Confocal Microscopy

Altered intracellular Ca2+ dynamics are characteristically observed in cardiomyocytes from failing hearts. Studies of Ca2+ handling in myocytes predominantly use Fluo-3 AM, a visible light excitable Ca2+ chelating fluorescent dye in conjunction with rapid line-scanning confocal microscopy. However, Fluo-3 AM does not allow for traditional ratiometric determination of intracellular Ca2+ concentration and has required the use of mathematic correction factors with values obtained from separate procedures to convert Fluo-3 AM fluorescence to appropriate Ca2+ concentrations. This study describes methodology to directly measure intracellular Ca2+ levels using inactivated, Fluo-3-AM-loaded cardiomyocytes equilibrated with Ca2+ concentration standards. Titration of Ca2+ concentration exhibits a linear relationship to increasing Fluo-3 AM fluorescence intensity. Images obtained from individual myocyte confocal scans were recorded, average pixel intensity values were calculated, and a plot is generated relating the average pixel intensity to known Ca2+ concentrations. These standard plots can be used to convert transient Ca2+ fluorescence obtained with experimental cells to Ca2+ concentrations by linear regression analysis. Standards are determined on the same microscope used for acquisition of unknown Ca2+ concentrations, simplifying data interpretation and assuring accuracy of conversion values. This procedure eliminates additional equipment, ratiometric imaging, and mathematic correction factors and should be useful to investigators requiring a straightforward method for measuring Ca2+ concentrations in live cells using Ca2+-chelating dyes exhibiting variable fluorescence intensity.

Effects of Infrasound on Changes of Intracellular Calcium Ion Concentration and on Expression of RyRs in Hippocampus of Rat Brain

In the present study, we investigated the molecular biological effect of infrasound on changes of intracellular free Ca2+ concentration ([Ca2+]i) and on expression of ryanodine receptors (RyRs) in the hippocampus of rats. 60 SD rats were randomized into ten groups and examined for [Ca2+]i and RyRs expression after exposure to 8Hz 90dB infrasound at days 1, 7, 14 and 21. Groups exposed to infrasound were exposed to 8Hz 90dB infrasound for 2 hours once daily. Acute cell separation, fluo-3/AM fluorescence technique and laser scanning confocal microscope (LSCM) were used for measurement of the [Ca2+]i level. There was no significant difference of [Ca2+]i at 1d and 7d infrasound exposure groups compared to control, but there was a significant incease at 14d group (P<0.01) and 21d group (P<0.05). Immunohistochemistry indicated the expression of RyRs in CA1 and CA3 pyramidal cells of the hippocampus showed no difference at 1d and 7d groups compared to control, whereas significant decrease was observed at 14d group (P<0.01) and 21d group (P<0.05). The data suggest that infrasound of 8Hz 90dB can induce [Ca2+]i increase and decrease the expression of RyRs in CA1 and CA3 regions of the hippocampus after exposure to infrasound for 14 and 21 days.

Intracellular Ca2+ measurements in live cells by rapid line scan confocal microscopy: simplified calibration methodology

Altered intracellular Ca2+ dynamics are characteristically observed in cardiomyocytes from failing hearts. Studies of Ca2+ handling in myocytes predominantly use Fluo-3 AM, a visible light excitable Ca2+ chelating fluorescent dye in conjunction with rapid line-scanning confocal microscopy. However, Fluo-3 AM does not allow for traditional ratiometric determination of intracellular Ca2+ concentration and has required use of mathematic correction factors with values obtained from separate procedures to convert Fluo-3 AM fluorescence to appropriate CA2+ concentrations. This study describes methodology to directly measure intracellular Ca2+ levels using inactivated, Fluo-3 AM loaded cardiomyocytes equilibrated with Ca2+ concentration standards. Titration of Ca2+ concentration exhibits a linear relationship to increasing Fluo-3 AM fluorescence intensity. Images obtained from individual myocyte confocal scans were recorded, average pixel intensity values were calculated, and a plot is generated relating the average pixel intensity to known Ca2+ concentrations. These standard plots can be used to convert transient Ca2+ fluorescence obtained with experimental cells to Ca2+ concentrations by linear regression analysis. Standards are determined on the same microscope used for acquisition of unknown Ca2+ concentrations, simplifying data interpretaton and assuring accuracy of conversion values. This procedure eliminates additional equipment, ratiometric imaging, and mathematic correction factors and should be used to investigators requiring a straightforward method for measuring Ca2+ concentrations in live cells using Ca2+-chelating dyes exhibiting variable fluorescence intensity.

Signal Amplification in Multichromophore Luminescence-Based Sensors

A method for signal amplification in the detection of vapors with luminescence-based sensors is described. Amplification involves energy transfer between two or more fluorescent chromophores in a carefully selected polymer matrix. A quantitative model has been derived that can be applied to any luminescence sensor comprising donor−acceptor pairs, and it can be generalized to multichromophore systems with n chromophores leading to n-fold signal amplification. Signal amplification has been demonstrated experimentally in the fluorescent sensing of dimethyl methylphosphonate (DMMP) using two dyes, 3-aminofluoranten (AM) and Nile Red (NR), in a hydrogen-bond acidic polymer matrix. The selected polymer matrix quenches the fluorescence of both dyes and shifts dye emission and absorption spectra relative to those of more inert matrixes. Upon DMMP sorption, the AM fluorescence shifts to the red at the same time that the NR absorption shifts to the blue, resulting in more band overlap and increased energy transfer ...

Spectroscopic characterization of trivalent f-element (Eu, Am) solid carbonates

Solubility studies show that trivalent f-element carbonate compounds are the predominant solids that primarily limit the soluble metal ion concentration under environmental conditions. We systematically investigated the spectroscopic characteristics of a series of solid f-element (Eu and 243Am) carbonates. Varying pH, ionic strength, and carbonate concentration results in the formation of M(OH) 3, MOHCO 3, M 2(CO 3) 3·nH 2O, and NaM(CO 3) 2·nH 2O, where M=Eu(III) or Am(III). Solids were characterized by FTIR, fluorescence, and EXAFS spectroscopies that determined and confirmed the coordination environment, and by their individual X-ray diffraction powder patterns. The number of coordinated crystal waters was determined to be 2–3 for Eu 2(CO 3) 3·nH 2O and 5 for NaEu(CO 3) 2·nH 2O using thermogravimetric/differential thermal analysis and fluorescence lifetime. We report studies of the fluorescence of Am(III) and the effect of carbonate coordination on the 5D 1→ 7F 1 transition.

Clotrimazole inhibits the recombinant human cardiac L-type Ca2+ channel alpha 1C subunit.

1. Clotrimazole (CLT) is an antimycotic agent with a potential role in the treatment of cancer. Whole-cell patch clamp recordings and Fura-2 AM fluorescence measurements were used to investigate the inhibition by CLT of recombinant human cardiac L-type Ca2+ channel alpha 1C subunits, stably expressed in human embryonic kidney (HEK 293) cells. 2. CLT (100 nmol l-1 to 25 mumol l-1) reduced Ca2+ channel currents in a concentration-dependent manner. Inhibition was neither use- or voltage-dependent. The effects of CLT were rapid and maximal effects were attained within 3 min. Application of CLT also caused an acceleration of apparent Ca2+ channel current inactivation. 3. Basal current density and the degree of inhibition due to CLT were not significantly altered by pretreating cells with 3 mmol l-1 1-aminobenzotriazole for 1 h, or by dialysing cells for 10 min with 2 mmol l-1 alpha-napthoflavone via the patch pipette, suggesting that the inhibitory action of CLT was not due to inhibition of cytochrome P-450. 4. CLT (10 mumol l-1) did not influence [Ca2+]i, as determined by Fura-2 AM fluorescence measurements. 5. Dialysing cells for 10 min with the non-specific serine/threonine kinase inhibitor H-7 (10 mumol l-1) was without effect on basal current density or on the inhibitory response to 10 mumol l-1 CLT, indicating that CLT is not acting via an indirect effect on these kinases. 6. These data suggest that CLT exerts a direct blocking effect on the alpha 1C subunit at therapeutic concentrations. This effect may explain the abbreviation of the action potential duration by CLT observed in cardiac myocytes.

A combination assay for simultaneous assessment of multiple signaling pathways

We have developed an assay in which modulation of two or more signaling pathways can be assessed concurrently by combining reporter gene systems with fluorescent probe technology. The validation of this method was achieved by indirect analysis of adenylyl cyclase activation with the use of a cyclic AMP response element (CRE)–luciferase reporter system in combination with the measurement of calcium mobilization by Calcium Green™-1 AM fluorescence on a fluorescent imaging plate reader. To demonstrate the utility of the method in studying the pharmacology of receptors that couple to more than one G protein, Chinese hamster ovary (CHO) cells, which stably expressed both the CRE-luciferase reporter gene and the human pituitary adenylyl cyclase-activating peptide (PACAP) receptor, were treated with PACAP 1-27 and 1-38. Calcium mobilization and the induction of adenylyl cyclase activity in response to each concentration of peptide were assessed in individuals wells. This assay may also be used to screen for ligands of two or more unrelated receptors simultaneously without compromising the assessment of either signaling pathway. To illustrate this point, Rat-1 fibroblasts, which expressed human α 1A receptors, were cocultured with CRE-luciferase CHO cells, which expressed human GLP-1 receptors. Calcium mobilization elicited by phenylephrine agonism of the α 1A receptor was assessed in the same assay as GLP-1-induced activation of adenylyl cyclase. The pEC 50 for each agonist was similar to that observed when the cell lines were not cocultured. The number of different receptors that can be screened per well is limited only by the ability to distinguish different reporter gene signals and fluorescent indicators.

Chronic Administration of Nifedipine Induces Up-regulation of Functional Calcium Channels in Rat Myocardium

Previous studies from our laboratory demonstrated the up-regulation of cardiac dihydropyridine (DHP) receptors in rabbits chronically treated with nifedipine (NIFE). The goal of the present study was to further examine the functionality of this increased number of receptors by analysing different steps of excitation contraction coupling mechanism in adult rats chronically treated with NIFE (a single 10-mg oral dose/kg/day for 28 days). Ca2+channel density was assessed by specific binding at the DHP receptors with [methyl-3H]PN 200-110 in rat ventricular membranes. Chronic NIFE treatment produced up-regulation of Ca2+channels, being the maximal binding capacities 222±19 fmol/mg protein (n=14) and 310±21 fmol/mg protein (n=11) in untreated and treated animals, respectively (P<0.05). The functional consequences of this up-regulation of Ca2+channels were determined in isolated ventricular myocytes by measuring L -type Ca2+currents (ICa) with the whole-cell configuration of patch-clamp technique and by intracellular Ca2+(Ca2+i) transients estimated by the Indo-1/AM fluorescence ratio (410/482) simultaneously monitored with cell shortening. Peak ICadensity recorded at 0 mV was 32% greater in myocytes isolated from the treated group than in those obtained from the untreated group (−10.43±0.73 pA/pF (n=13) vs−7.10±0.59 pA/pF (n=12) P<0.05). Ca2+itransient amplitude and cell shortening, explored at 1 and 2 m m extracellular calcium ([Ca]0) were significantly higher in ventricular myocytes obtained fom NIFE-treated rats than in myocytes isolated from untreated animals. At 2 m m[Ca]0, the values of Ca2+itransient and shortening were 460±61 n m and 11±1% of resting length (L0) in myocytes from treated rats (n=9) and 212±22 n m and 5.3±0.5% of L0in myocytes from control rats (n=6, P<0.05). The results demonstrate an up-regulation of functionally-active cardiac Ca2+channels after NIFE treatment, and offer a possible explanation for a “withdrawal effect” at myocardial level after the suppression of the treatment with this drug.

Deformation and flow of red blood cells in a synthetic lattice: evidence for an active cytoskeleton

We introduce the use of microfabrication techniques to construct on a silicon wafer a synthetic capillary bed with 2.5- to 4-micron (mu)-wide channels. Establishment of a fluid pressure gradient allowed us to observe simultaneously using optical microscopy hundreds of cells flowing through the bed at physiological speeds. We find a large distribution of mobilities among red cells flowing through the structure; smaller channels provide a greater impedance to flow than larger ones, indicating that kinetic drag variations provide the origin of the distribution. The mobility of a particular cell is not correlated with the cell diameter but appears to be inversely correlated with intracellular calcium concentration of the cell, as determined by fluorescence of the calcium-binding dye fluo-3 AM. Also, we are able to use the parallel processing nature of our arrays to observe isolated events where the rigidity of the red cell seems to change suddenly over several orders of magnitude as it blocks a channel in the array.

Function associated molecules on nonspecific cytotoxic cells: Role in calcium signaling, redirected lysis, and modulation of cytotoxicity

Monoclonal antibodies (mAbs) specific for a putative antigen receptor (function associated molecule, FAM) on fish NCC were tested for their ability to initiate signal transduction responses. Anti-FAM hybridoma target cells were significantly lysed by NCC when tested by redirected lysis (RL). Redirected lysis was enhanced by 1-h pretreatment of the NCC with anti-FAM mAb; however, 18-h pretreatment produced significant inhibition. The effects of the calcium ionophore A23187 on RL were also examined: 10 −4 M but not 10 −5 M A23187 significantly increased RL. Purified NCC were treated with mAb and cellular DNA synthesis was determined. Significant increases in NCC incorporation of tritiated thymidine were measured 72 h after treatment with mAb. Experiments were also conducted to determine levels of free cytosolic calcium in NCC following mAb binding. Anti-FAM mAb binding produced a rapid rise in cytosolic calcium (200–500 n M) determined by Fura-2/AM fluorescence. Calcium ionophore A23187, PHA, and Con-A treatment of NCC also produced significantly increased [Ca 2+]i. Target cell binding by NCC caused a three-fold increase in [Ca 2+]i. These data suggest that triggering of the FAM may initiate a signal(s) that activates cytotoxicity, increases cytosolic free calcium concentrations, and initiates DNA synthesis.