Fluorescence Video Microscopy Research Articles

Characterization of transient platelet contacts on a polyvinyl alcohol hydrogel by video microscopy

Acridine orange labelled, washed human platelets were counted and tracked on polyvinyl alcohol (PVA), heparin–PVA and polyethylene (PE)-coated coverslips with a view to understand why transient contact on the PVA hydrogels lead to elevated platelet activation and consumption relative to polyethylene. Over the 4 min of initial contact that was studied, platelet adhesion was higher on PE than on PVA or heparin–PVA at both 40 and 200 s -1, as expected, regardless of whether the surfaces were pre-treated with albumin or fibrinogen. Not all platelets appearing to make contact with the surface, actually attached. For example, less than 2% of the platelets contacting albumin pre-treated PVA (at 40 s -1) remained adherent at the end of the initial 60 s observation time, while the corresponding number for PE was greater than 9%. A greater fraction of the platelets remained adherent at the higher shear rate or with fibrinogen pre-treatment, but the difference between PVA and PE remained similar: for example, with fibrinogen pretreatment at 200 s -1, ∼25% of the platelet contacts resulted in adhesion on PVA while 66% did so on PE. While net platelet adhesion was less for the hydrogels, than for PE, the total number of contacts (adherents+non-adherents) were more comparable and unexpectedly higher for albumin pre-treatment than for fibrinogen. Net platelet adhesion is but one component of the total platelet interaction with a material surface. Fluorescent video microscopy has been shown to be a useful, albeit not unequivocal, method for assessing the platelets that make contact with but do not adhere to a surface.

Periodicity of λ DNA Motions during Field Inversion Electrophoresis in Dilute Hydroxyethyl Cellulose Visualized by High-Speed Video Fluorescence Microscopy

Using high-speed video fluorescence microscopy, we have imaged λ double-stranded (ds) DNA during both direct current (DC) and field inversion electrophoresis in dilute linear hydroxyethyl cellulose (HEC) using moderately high electric fields without the use of viscosity modifiers. The λ ds-DNA extension−contraction cycle resulting from entanglement with HEC molecules shows more periodicity with field inversion than with DC electric fields. The periodicity of the extension−contraction cycle is modulation depth- and field inversion frequency dependent. The extension−contraction kinetics of λ ds-DNA are similar in alternating and DC electric fields. We assert that modification of DNA−polymer entanglement lifetimes increases DNA motion periodicity during field inversion electrophoresis.

Increased Dermal Lymphatic Density in the Human Leg Compared with the Forearm

During orthostasis, capillary filtration pressure is higher in the leg than in the arm due to the effect of gravity. We investigated the possibility that the lymphatic network in leg skin might be adapted to cope with a greater fluid load. The dermal lymphatics of the forearm and lower leg were studied in white-skinned and brown-skinned men using fluorescence video microscopy. From video print lymphangiograms the following were determined: lymphatic length density at a series of radii from the centre of the lymphangiogram (LDr); maximum lymphatic density (LDmax); total length of lymphatic vessel (LL); maximum spread of lymphatic vessel; number and size of lymphatic rings (continuous circuits of vessel); and vessel diameter. There were no differences between the two racial types, but clear differences between the arm and leg. In the leg, mean (± SD) peak LDr(25.13 ± 5.65 cm−1), LDmax(32.95 ± 6.89 cm−1), LL (40.17 ± 27.42 cm), and spread (1.39 ± 0.08 cm) were all significantly higher than in the arm (18.03 ± 5.48 cm−1, 23.91 ± 7.21 cm−1, 11.76 ± 5.47 cm, and 1.00 ± 0.05 cm respectively,P≤ 0.001). In the region of maximum lymphatic density there were 2.4 times more rings in the leg than in the arm, ring size being similar. Vessel image diameters were 89.6 ± 26.3 μm (leg) and 94.8 ± 10.9 μm (arm). Leg skin thus has a denser and more extensive lymphatic network than arm skin. This supports the hypothesis that lymph transport capacity in the leg is enhanced to compensate for a greater gravitational fluid loading.

Dose-dependent increase of transcapillary diffusion of sodium fluorescein after histamine microinjections.

To study the dose-dependent effects of histamine on capillary permeability in human skin, using the microinjection technique. Eight healthy volunteers (2 w, 6 m; mean age 33 years) were included in the study. On two separate occasions, glass microcannulas with a tip diameter of 7 to 9 microns were inserted into the subepidermal layer of the skin at the distal medial tibia surface of each lower limb with a micromanipulator. In each subject, 0.5 microliter of 3 different concentrations of histamine solution (0.1/1000, 0.01/1000 and 0.001/1000) were injected and compared to the solvent (0.9% NaCl). Transcapillary diffusion of intravenously administered Na-fluorescein was assessed simultaneously using two fluorescence videomicroscopy systems. Off-line video densitometry was performed in an area of 0.56 mm2 around the injection sites and fluorescence light intensities were measured in arbitrary units (AU) at 10, 30, 60, 120 and 600 s after dye appearance. Compared to the solvent histamine microinjections resulted in a dose-dependent increase of mean fluorescence light intensities (FLI). Whereas mean FLI for the 0.001/1000 histamine injection was only significantly elevated 10 min after dye appearance (p < 0.05) an increase of mean FLI was already observed 10 s after dye appearance following the 0.1/1000 histamine injection (p < 0.05), which was more pronounced at later time points (p < 0.001). Mean FLI's for the 0.01/1000 histamine solution were in between and resulted in significantly elevated values 1 min to 10 min after dye appearance (p < 0.05). We conclude that the microinjection technique together with fluorescence videomicroscopy described previously [6] is able to document a dose-dependent effect of histamine microinjections on skin capillary permeability. The technique may facilitate to determine appropriate dosages not only of histamine in order to test the effect of antagonists on human skin capillary permeability.

Calcium waves in colonic myocytes produced by mechanical and receptor-mediated stimulation.

The mechanisms underlying intracellular Ca2+ waves induced by either mechanical or receptor-mediated stimulation of myocytes isolated from the longitudinal muscle layer of the rabbit distal colon were compared using fura 2 and fluorescence videomicroscopy. Light focal mechanical deformation of the plasma membrane or focal application of substance P resulted in localized intracellular Ca2+ concentration ([Ca2+]i) transients that propagated throughout the cell. In both cases, the Ca2+ response consisted of a transient peak response followed by a delayed-phase response. Substance P-mediated [Ca2+]i responses involved generation of inositol 1,4, 5-trisphosphate and release of Ca2+ from thapsigargin-sensitive stores, whereas mechanically induced responses were partially (29%) dependent on La3+-sensitive influx of extracellular Ca2+ and partially on release of intracellular Ca2+ from thapsigargin-insensitive stores gated by ryanodine receptors. The delayed-phase response in both cases was dependent on extracellular Ca2+. However, although the response to substance P was sensitive to La3+, that after mechanical stimulation was not. In the later case, the underlying mechanism may involve capacitative Ca2+ entry channels that are activated after mechanical stimulation but not by substance P.

Taxol suppresses dynamics of individual microtubules in living human tumor cells.

Microtubules are intrinsically dynamic polymers, and their dynamics play a crucial role in mitotic spindle assembly, the mitotic checkpoint, and chromosome movement. We hypothesized that, in living cells, suppression of microtubule dynamics is responsible for the ability of taxol to inhibit mitotic progression and cell proliferation. Using quantitative fluorescence video microscopy, we examined the effects of taxol (30-100 nM) on the dynamics of individual microtubules in two living human tumor cell lines: Caov-3 ovarian adenocarcinoma cells and A-498 kidney carcinoma cells. Taxol accumulated more in Caov-3 cells than in A-498 cells. At equivalent intracellular taxol concentrations, dynamic instability was inhibited similarly in the two cell lines. Microtubule shortening rates were inhibited in Caov-3 cells and in A-498 cells by 32 and 26%, growing rates were inhibited by 24 and 18%, and dynamicity was inhibited by 31 and 63%, respectively. All mitotic spindles were abnormal, and many interphase cells became multinucleate (Caov-3, 30%; A-498, 58%). Taxol blocked cell cycle progress at the metaphase/anaphase transition and inhibited cell proliferation. The results indicate that suppression of microtubule dynamics by taxol deleteriously affects the ability of cancer cells to properly assemble a mitotic spindle, pass the metaphase/anaphase checkpoint, and produce progeny.

ATP acting on P2Y receptors triggers calcium mobilization in primary cultures of rat neurohypophysial astrocytes (pituicytes).

The effect of adenosine triphosphate (ATP) on the intracellular Ca2+ concentration ([Ca2+]i) of cultured neurohypophysial astrocytes (pituicytes) was studied by fluorescence videomicroscopy. ATP evoked a [Ca2+]i increase, which was dose dependent in the 2.5-50 microM range (EC50=4.3 microM). The ATP-evoked [Ca2+]i rise was not modified during the first minute following the removal of external Ca2+. Application of 500 nM thapsigargin inhibited the ATP-dependent [Ca2+]i increase. Caffeine (10 mM) and ryanodine (1 microM) did not affect the ATP-induced [Ca2+]i rise. The pituicytes responded to various P2 purinoceptor agonists with the following order of potency: ATP=ATP[gamma-S]=2-MeSATP>/=ADP, where ATP[gamma-S] is adenosine 5'-O-(3-thiotriphosphate) and 2-MeSATP is 2-methylthio-adenosine-5'-triphosphate. Adenosine, AMP, alpha, beta-methylene adenosine-5'-triphosphate (alpha,beta-MeATP), beta, gamma methylene adenosine-5'-triphosphate (beta,gamma-MeATP) and uridine 5'-triphosphate (UTP) were ineffective. The P2 purinoceptor antagonists blocked the ATP-evoked [Ca2+]i increase with the following selectivity: RB-2>suramin>PPADS, where RB-2 is Reactive Blue 2 and PPADS is pyridoxal-phosphate-6-azophenyl-2', 4'-disulphonic acid. The ATP-evoked [Ca2+]i increase was substantially blocked by pertussis toxin treatment, suggesting that it might be mediated by a pertussis-toxin-sensitive G protein. The phospholipase C (PLC) inhibitor U-73122 (0.5 microM) abolished the ATP-evoked [Ca2+]i rise, whereas its inactive stereoisomer U-73343 (0.5 microM) remained ineffective. Our results indicate that, in rat cultured pituicytes, ATP stimulation induces an increase in [Ca2+]i due to PLC-mediated release from intracellular stores through activation of a pertussis-toxin-sensitive, G-protein-linked P2Y receptor.

Single-polymer dynamics in steady shear flow.

The conformational dynamics of individual, flexible polymers in steady shear flow were directly observed by the use of video fluorescence microscopy. The probability distribution for the molecular extension was determined as a function of shear rate, gamma;, for two different polymer relaxation times, tau. In contrast to the behavior in pure elongational flow, the average polymer extension in shear flow does not display a sharp coil-stretch transition. Large, aperiodic temporal fluctuations were observed, consistent with end-over-end tumbling of the molecule. The rate of these fluctuations (relative to the relaxation rate) increased as the Weissenberg number, gamma;tau, was increased.

Influence of local haemodynamics on leucocyte rolling and chemoattractant-induced firm adhesion in microvessels of the rat mesentery.

Tissue hyperaemia, oedema formation and leucocyte accumulation are characteristic features of the inflammatory process referable to changes at the microcirculatory level. Here, we used intravital fluorescence video microscopy to assess relationships among haemodynamical parameters, leucocyte rolling, and chemoattractant-induced firm adhesion in small venules (13-24 microM) of the rat mesentery. The rolling leucocyte flux in these vessels was directly proportional to the total leucocyte flux (r = 0.76, P < 0.001), which in turn closely correlated to the venular blood flow (r = 0.77, P < 0.001). Consequently, the rolling to total leucocyte flux fraction, averaging 39 +/- 15%, did not vary with the blood flow and showed no correlation to either blood flow velocity (r = -0.15, P = 0.42) or wall shear rate (r = -0.06, P = 0.77), indicating that the extent of leucocyte rolling is not primarily dependent on the fluid viscous drag at physiological blood flow rates in vivo. Stimulation of the mesentery with the chemoattractant fMLP (10(-6) M) induced firm adhesion of rolling leucocytes. It was found that the number of adherent leucocytes in individual vessels was directly related to the rolling leucocyte flux (r = 0.78, P < 0.001) and hence to the venular blood flow (r = 0.47, P < 0.05), while there was no correlation to the wall shear rate (r = 0.27, P = 0.24). The dependence of the firm adhesive response on the blood flow level and the delivery rate of leucocytes was confirmed at the whole organ level. Thus, leucocyte accumulation in rat skin lesions was markedly enhanced when a vasodilator was co-administered with the chemotactic stimulus compared with chemotactic stimulation alone. The data indicate that, within a physiological blood flow range, the leucocyte response to chemotactic stimulation is largely independent of the prevailing hydrodynamic shear forces. Instead, manifestation of the firm adhesive response, because of its dependence on the preceding rolling interaction, is clearly related to the blood flow level in the microvessels, which emphasizes the significance of tissue hyperaemia in inflammation.

Disruption of dynamic cell surface architecture of NIH3T3 fibroblasts by the N-terminal domains of moesin and ezrin: in vivo imaging with GFP fusion proteins.

Lamellipodia, filopodia, microspikes and retraction fibers are characteristic features of a dynamic and continuously changing cell surface architecture and moesin, ezrin and radixin are thought to function in these microextensions as reversible links between plasma membrane proteins and actin microfilaments. Full-length and truncated domains of the three proteins were fused to green fluorescent protein (GFP), expressed in NIH3T3 cells, and distribution and behaviour of cells were analysed by using digitally enhanced differential interference contrast (DIC) and fluorescence video microscopy. The amino-terminal (N-)domains of all three proteins localize to the plasma membrane and fluorescence recordings parallel the dynamic changes in cell surface morphology observed by DIC microscopy of cultured cells. Expression of this domain, however, significantly affects cell surface architecture by the formation of abnormally long and fragile filopodia that poorly attach and retract abnormally. Even more striking are abundant irregular, branched and motionless membraneous structures that accumulate during retraction of lamellipodia. These are devoid of actin, endogenous moesin, ezrin and radixin, but contain the GFP-labeled domain. While a large proportion of endogenous proteins can be extracted with non-ionic detergents as in untransfected control cells, >90% of N-moesin and >60% of N-ezrin and N-radixin remain insoluble. The minimal size of the domain of moesin required for membrane localization and change in behavior includes residues 1-320. Deletions of amino acid residues from either end result in diffuse intracellular distribution, but also in normal cell behavior. Expression of GFP-fusions of full-length moesin or its carboxy-terminal domain has no effect on cell behavior during the observation period of 6-8 hours. The data suggest that, in the absence of the carboxy-terminal domain, N-moesin, -ezrin and -radixin interact tightly with the plasma membrane and interfere with normal functions of endogeneous proteins mainly during retraction.

Development and movements of extraembryonic cells in the Indian meal moth, Plodia interpunctella Hübner (Lepidoptera: Pyralidae)

Summary Time-lapse video microscopy, fluorescence microscopy, and electron microscopy provide complementary perspectives on the genesis and movements of the serosa, amnion, and yolk cells of the Indian meal moth, Plodia interpunctella Hübner (Lepidoptera: Pyralidae). Prospective serosal cells undergo a nuclear division, round up and detach from the adjoining cells of the germ band. They reattach only to other serosal cells, creating a free migrating edge at the boundary between the germ band and the serosa. The serosa engages in epiboly, spreads to enclose the embryo, attaches to the vitelline envelope, and secretes materials onto the vitelline envelope on the inner side of the egg shell. The amnion derives from the dorsal edge of the germ band; at this time the germ band is a cell sheet composed of columnar cells (palisade-type). Amniotic cells change briefly into spindle-shaped, motile cells that dissociate from the germ band. They cross the dorsal rim of the germ band, then flatten and form an epithelium. The amnion engages in epiboly and spreads across most of the embryo, starting approximately 1h after the serosa does. A lamina is secreted between the amnion and the embryo. By mid-embryogenesis, the amnion reaches over the dorsal region, including some yolk cells. The lamina and amnion loosen from the embryo during blastokinesis, the stage after germ band shortening. The yolk endoplasm partitions into yolk cells between 1 to 2h after other cells appear. Yolk cells are highly motile during early embryogenesis, then gradually slow and engage in cell shape changes without locomotion. They tend to flatten and ruffle during blastokinesis. By the end of blastokinesis, they form stable membrane contacts, creating a cell sheet. These large cells exhibit fountanoid and centripetal flows when they are motile.

Carcinoembryonic antigen facilitates experimental metastasis through a mechanism that does not involve adhesion to liver cells.

Carcinoembryonic antigen (CEA) injected intravenously into athymic nude mice increases the ability of weakly metastatic human colorectal carcinoma (CRC) cells to colonize liver in an experimental metastasis assay. Since CEA acts as an intercellular adhesion molecule in vitro, several investigators have postulated that this facilitation of experimental metastasis may be mediated through adhesion between CEA on CRC and CEA-binding proteins on Kupffer or other cells lining the hepatic sinusoid. The present work tested this postulate both by intravital fluorescence videomicroscopy in vivo and in adhesion assays in vitro to enriched populations of Kupffer cells and hepatic sinusoidal endothelial cells (SEC). The data indicate that CEA expression does not effect adhesion to enriched Kupffer cells or SEC in vitro. These data suggest that CEA enhances liver colonization through another mechanism, possibly one that involves modulation of the hepatic response to tumor cell implantation.

Alpha4beta1 and alpha5beta1 control cell migration on fibronectin by differentially regulating cell speed and motile cell phenotype.

Computer-aided time lapse fluorescence videomicroscopy was used to study single cell migration behavior of human aortic endothelial cells on fibronectin coated substrates of varying protein surface density. The role of receptors alpha5beta1, alpha(v)beta3, and alpha4beta1 in mediating cell adhesion and migration on fibronectin was characterized using integrin specific monoclonal antibodies. Matrix density had a direct effect on controlling the proportion of migrating cells and the directional persistence of cell movement (p<0.01). While there was relatively little influence of fibronectin surface density on absolute migration speed, the ability of endothelial cells to disperse over a surface, as measured by the dispersion coefficient, was biphasic with respect to the surface density of this matrix protein (p<0.005). Both cell speed and the proportion of migrating cells was controlled by alpha4beta1 (p<0.01). However, alpha5beta1 selectively regulated the transformation of stationary cells to those exhibiting motile behavior (p<0.05). Migratory responses on fibronectin were not influenced by blockade of the alpha(v)beta3 receptor. It is noteworthy that cell surface adhesive receptors which control commitment to a motile phenotype are not necessarily the same as those that control migration speed.

Response of flexible polymers to a sudden elongational flow

Individual polymers at thermal equilibrium were exposed to an elongational flow producing a high strain rate, and their dynamics were recorded with video fluorescence microscopy. The flow was turned on suddenly so that the entire evolution of molecular conformation could be observed without initial perturbations. The rate of stretching of individual molecules is highly variable and depends on the molecular conformation that develops during stretching. This variability is due to a dependence of the dynamics on the initial, random equilibrium conformation of the polymer coil. The increasing appearance at high strain rates of slowly unraveling hairpin folds is an example of nonergodic dynamics, which can occur when a statistical mechanical system is subjected to nonadiabatic, or "sudden," external forces.

Luteinizing hormone (LH) stimulates both intracellular calcium ion ([Ca2+]i) mobilization and transmembrane cation influx in single ovarian (granulosa) cells: recruitment as a cellular mechanism of LH-[Ca2+]i dose response.

The gonadotropic hormones, LH and FSH, activate adenylyl cyclase in their respective target cells and thereby initiate many biochemical responses. In addition to stimulating cAMP production, both LH and FSH promote agonist-specific increases in the cytoplasmic concentration of free calcium ions ([Ca2+]i) in gonadal cells. Here, we have applied single cell fluorescence video microscopy with the Ca2+-sensitive dye fura-2 to investigate the mechanism(s) by which LH induces a rise in the [Ca2+]i in individual (swine) granulosa cells collected from single Graafian follicles. Stimulation with LH induced a rapid onset, biphasic, spike- and plateau-like [Ca2+]i signal in responsive granulosa cells. The cellular mechanisms mediating this biphasic LH-stimulated increase in [Ca2+]i were examined by external Ca2+ removal and via the manganese (Mn2+) quench technique, which showed that LH triggers initial intracellular Ca2+ mobilization followed by delayed transmembrane Ca2+ influx. Single cell Ca2+ assessment of the LH dose-response mechanism(s) revealed that higher concentrations of LH progressively recruit a larger number of responding individual granulosa cells. Further analyses disclosed a marked [Ca2+]i response heterogeneity among individual granulosa cells harvested from the same Graafian follicle. In addition, the percentage of cells responding to LH [but not to an alternative putative agonist of the phospholipase C (PLC) pathway, viz. endothelin-1] with a biphasic [Ca2+]i rise increased with maturational development of the follicle. Pretreatment of granulosa cells with a specific PLC inhibitor, U-73122 (but not with its inactive congener U-73343), significantly reduced the percentage of cells responding to a LH challenge from 78% to 25% (P < 0.0001) and prolonged the time required to achieve a half-maximal value of the [Ca2+]i transient, viz. from 22 +/- 1.5 sec (n = 27 cells) to 39 +/- 7.2 sec (n = 12 cells; P = 0.002). In cell population studies, LH stimulated in a concentration- and time-dependent manner the accumulation of inositol phosphate in porcine granulosa cells. In summary, the present single cell investigations in mature granulosa cells demonstrate that LH drives initial intracellular Ca2+ mobilization followed by transmembrane divalent cation influx. The PLC inhibitor U-73122 antagonizes this action of LH. By analyzing [Ca2+]i responses in individual living granulosa cells, we further show that, despite within-follicle diversity, the LH dose biphasic [Ca2+]i response arises via the recruitment of a larger number of responding gonadal cells rather than by increased [Ca2+]i signal amplitude. Finally, the percentage of individual LH (but not endothelin-1)-responding granulosa cells increases with follicular maturation. Collectively, these data highlight the potential importance of the LH-stimulatable, PLC-transduced [Ca2+]i signaling mechanism in the later stages of granulosa cell differentiation.

Modulation of basal and postischemic leukocyte-endothelial adherence by nitric oxide.

Recent studies indicate that leukocytes are important contributors to secondary vascular and parenchymal injury after cerebral ischemia. The present study was undertaken to define nitric oxide (NO)-based mechanisms that regulate leukocyte-endothelial interactions in the cerebral vasculature, how these mechanisms are affected by cerebral ischemia, and whether NO-based therapies can affect postischemic leukocyte dynamics. Leukocyte adherence to pial venules of anesthetized newborn piglets was quantified by in situ fluorescence videomicroscopy through closed cranial windows during basal conditions and during reperfusion after 9 minutes of asphyxia. Nitric oxide synthase (NOS) was inhibited by local window superfusion of L-nitroarginine; superfusion of sodium nitroprusside was used to donate NO. Local inhibition of NOS under resting conditions increased leukocyte-endothelial adherence 2.2-fold and 3.9-fold over baseline values after 1 hour and 2 hours, respectively; this response was completely blocked by cosuperfusion with L-arginine. Cosuperfusion of superoxide dismutase reversed L-nitroarginine-induced leukocyte adherence by 89% and 63% at these respective time points. The extent of acute leukocyte adherence elicited by NOS inhibition was similar in magnitude to that observed during the initial 2 hours of reperfusion after asphyxia. Leukocyte adherence was not additionally increased in asphyxic animals treated with L-nitroarginine. Sodium nitroprusside robustly inhibited asphyxia-induced leukocyte adherence back to control levels. NO exerts a tonic antiadherent effect in the cerebral microcirculation by inactivation of adherence-promoting superoxide radical formation. Cerebral ischemia is associated with an inhibition of NOS or lower levels of NO, which results in leukocyte-endothelial adherence that can be prevented by NO donors. The latter may be useful therapeutically to prevent the purported vascular and parenchymal dysfunction and injury caused by activated leukocytes in ischemic brain.

Pasteurella haemolytica A1-derived leukotoxin and endotoxin induce intracellular calcium elevation in bovine alveolar macrophages by different signaling pathways.

Leukotoxin and endotoxin derived from Pasteurella haemolytica serotype 1 are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Activation of bovine alveolar macrophages with endotoxin or leukotoxin results in the induction of cytokine gene expression, with different kinetics (H. S. Yoo, S. K. Maheswaran, G. Lin, E. L. Townsend, and T. R. Ames, Infect. Immun. 63:381-388, 1995; H. S. Yoo, B. S. Rajagopal, S. K. Maheswaran, and T. R. Ames, Microb. Pathog. 18:237-252, 1995). Furthermore, extracellular Ca2+ is required for leukotoxin-induced cytokine gene expression. However, the involvement of Ca2+ in endotoxin effects and the precise signaling mechanisms in the regulation of intracellular Ca2+ by leukotoxin and endotoxin are not known. In fura-2-acetoxymethyl ester-loaded alveolar macrophages, intracellular Ca2+ regulation by leukotoxin and endotoxin was studied by video fluorescence microscopy. Leukotoxin induced a sustained elevation of intracellular Ca2+ in a concentration-dependent fashion by influx of extracellular Ca2+ through voltage-gated channels. In the presence of fetal bovine serum, endotoxin elevated intracellular Ca2+ even in the absence of extracellular Ca2+. Leukotoxin-induced intracellular Ca2+ elevation was inhibited by pertussis toxin, inhibitors of phospholipases A2 and C, and the arachidonic acid analog 5,8,11,14-eicosatetraynoic acid. Intracellular Ca2+ elevation by endotoxin was inhibited by inhibitors of phospholipase C and protein tyrosine kinase, but not by pertussis toxin, or the arachidonic acid analog. To the best of our knowledge, this is the first report of Ca2+ signaling by leukotoxin through a G-protein-coupled mechanism involving activation of phospholipases A2 and C and release of arachidonic acid in bovine alveolar macrophages. Ca2+ signaling by endotoxin, on the other hand, involves activation of phospholipase C and requires tyrosine phosphorylation. The differences in the Ca2+ signaling mechanisms may underlie the reported temporal differences in gene expression during leukotoxin and endotoxin activation.

Visualizing the dynamics of T cell activation: intracellular adhesion molecule 1 migrates rapidly to the T cell/B cell interface and acts to sustain calcium levels.

T cell recognition typically involves both the engagement of a specific T cell receptor with a peptide/major histocompatibility complex (MHC) and a number of accessory interactions. One of the most important interactions is between the integrin lymphocyte function-associated antigen 1 (LFA-1) on the T cell and intracellular adhesion molecule 1 (ICAM-1) on an antigen-presenting cell. By using fluorescence video microscopy and an ICAM-1 fused to a green fluorescent protein, we find that the elevation of intracellular calcium in the T cell that is characteristic of activation is followed almost immediately by the rapid accumulation of ICAM-1 on a B cell at a tight interface between the two cells. This increased density of ICAM-1 correlates with the sustained elevation of intracellular calcium in the T cell, known to be critical for activation. The use of peptide/MHC complexes and ICAM-1 on a supported lipid bilayer to stimulate T cells also indicates a major role for ICAM-1/LFA-1 in T cell activation but, surprisingly, not for adhesion, as even in the absence of ICAM-1 the morphological changes and adhesive characteristics of an activated T cell are seen in this system. We suggest that T cell antigen receptor-mediated recognition of a very small number of MHC/peptide complexes could trigger LFA-1/ICAM-1 clustering and avidity regulation, thus amplifying and stabilizing the production of second messengers.

Mechanically-induced calcium transients in DRG neurons are amiloride senstive

BACKGROUND: Mechanosensitive transduction mechanisms in visceral afferent neurons are an attractive target for drug development in gastrointestinal disorders involving visceral hypersensitivity. We have previously reported gadolinium-sensitive [Ca2+]~ transients induced by mechanical stimulation in 26 % of small size DRG neurons in primary culture (Soc Neurosci Abstr 23:1259,1997). The alpha subunit of the amiloridesensitive epithelial sodium channel (ENaC) displays mechanosensitivity when expressed in Xenopus oocytes and may function as a stretch-activated cation channel in mechanically responsive cells (Proc N Acad Sci 94: 1013, 1997). AIMS: a) To determine if mechanically induced [Ca2+]i transients in small DRG neurons are sensitive to amiloride, and b) whether expression of the ENaC subunits may underlie this sensitivity. METHODS: Video fluorescent microscopy was used to measure [Ca2+]i transients in fura-2=labeled adult rat lumbosacral DRG neurons growing in short term primary culture. Increases in [Ca2+]i were measured in response to mechanostimulation using a flame polished probe. Amiloride (10-1001aM) was administered by superfusion to test for sensitivity. RT-PCR was used to assay for the expression of various ENaC channel subunits in freshly dissociated and cultured DRG neurons. RESULTS: Low concentrations of amiloride (10/aM) did not alter either the resting level of [Ca2+] i (86 ± 7 nM (amiloride) vs 84 ± 7 nM (control)) or the amplitude of the mechanically induced [Ca2+]i transient (317 _+ 34 nM (amiloride) vs 316 ± 24 nM (control)). In contrast, high concentrations of amiloride (1001aM) completely abolished (5 out of 7) or significantly inhibited the mechanically induced [Ca2+] i transient while not affecting resting [Ca2+]~ levels. The ICso for amiloride was 271aM. RT-PCR analyses showed that cultured and freshly dissociated DRG neurons express the ct-ENaC subunit, but not the I 3or y-ENaC subunits. CONCLUSIONS: Based on these findings, we hypothesize that a percentage of small diameter DRG neurons express ENaC-like channels on their soma which are sensitive to gadolinium and amiloride, and that mediate Ca 2+ influx in response to mechanical stimuli. If expressed on the peripheral nerve terminals of DRG neurons, these channels could mediate the mechanosensitivity of small unrnyelinated afferent neurons. Supported by NIH grants DK 48351 and DK 40919 (EAM), DK 41004 (HER), and by DFG grant Gs9/I-1

Microcirculation research in experimental surgery

The microcirculation is the organ that provides the direct link between blood and tissue, and thereby between the whole organism and the single cell. Modern microcirculation research in experimental surgery is characterized by the use of high-resolution video fluorescence microscopy and quantitative computer-assisted image analysis coupled with the techniques of molecular biology and transgenic or knockout gene technology. These advances should improve knowledge about surgically relevant physiologic and pathophysiologic phenomena at the interface between blood and tissues and help us to understand the initial molecular mechanisms leading to organ dysfunction following inflammation, ischemia-reperfusion, and transplantation.