Effect of strain on actomyosin kinetics in isometric muscle fibers.

Investigations were conducted into the biochemical and mechanical states of cross-bridges during isometric muscle contraction. Rapid length steps (3 or 6 nm hs(-1)) were applied to rabbit psoas fibers, permeabilized and isometric, at either 12 degrees C or 20 degrees C. Fibers were activated by photolysis of P(3)-1-(2-nitrophenyl)-ethyl ester of ATP infused into rigor fibers at saturating Ca(2+). Sarcomere length, tension, and phosphate release were recorded-the latter using the MDCC-PBP fluorescent probe. A reduction in strain, induced by a rapid release step, produced a short-lived acceleration of phosphate release. Rates of the phosphate transient and that of phases 3 and 4 of tension recovery were unaffected by step size but were elevated at higher temperatures. In contrast the amplitude of the phosphate transient was smaller at 20 degrees C than 12 degrees C. The presence of 0.5 or 1.0 mM added ADP during a release step reduced both the rate of tension recovery and the poststep isometric tension. A kinetic scheme is presented to simulate the observed data and to precisely determine the rate constants for the elementary steps of the ATPase cycle.

Phenotype of the endothelium in the human term placenta.

The placental endothelium contributes to regulating transplacental exchange and maintaining the immunological maternofetal barrier. We characterized the endothelial phenotype in human normal term placentae with a panel of antibodies to endothelial antigens using a standardized immunofluorescence method. Placental endothelium strongly expressed vWF, PAL-E, H-antigen, thrombomodulin, PECAM-1, CD34, CD36, ICAM-1, CD44, thy-1, A10/33-1, VE-cadherin, caveolin-1 and HLA-G, whereas occludin, claudin-1, eNOS, angiotensin converting enzyme (ACE), ICAM-2, endoglin and integrin-alphathetabeta(3)were weakly expressed. PGI(2)synthase, tissue factor, E-selectin and VCAM-1 were not detected. Some antigens were heterogenously expressed along the vascular tree or within individual villi. Expression of ACE, eNOS, vWF, P-selectin, E-selectin, integrin alpha(v)beta(3)and endoglin was stronger in the maternal decidual vessels, while PECAM-1, CD44, thy-1 and caveolin-1 expression was stronger in fetal vessels. Some endothelial markers were present in trophoblasts and stroma. Endothelial proliferation was apparent in mature intermediate and terminal villi. There was limited inflammatory response to TNFalpha in explants, characterized by upregulation of vWF, P-selectin, PECAM-1 and CD44, downregulation of thrombomodulin, but no increase in ICAM-1 expression, nor induction of E-selectin, VCAM-1 or tissue factor. These patterns of heterogeneity, proliferative activity and inflammatory activation may underlie the specific physiological roles of the placental endothelium.

Capillary filtration is reduced in lungs adapted to chronic heart failure: morphological and haemodynamic correlates.

OBJECTIVE: To determine pulmonary capillary filtration in experimental chronic heart failure and to investigate some morphological and haemodynamic mechanisms that could account for reduced filtration in lungs adapted to chronic heart failure. METHODS: We studied pulmonary capillary filtration, vascular resistances and morphology in lungs from guinea-pigs adapted to chronic heart failure. Heart failure was induced by banding of the ascending aorta (n=66) or sham control operation (n=78) in guinea-pigs which were studied at 150+/-8 days post-operation. RESULTS: Reduced cardiac output, increased systemic vascular resistance and LV end diastolic pressure and increased LV and RV weight:body weight ratio (all P<0.05) indicated chronic heart failure at 5 months following aortic banding in guinea-pigs. Lung weight was increased (61%, P<0.05) in heart failure compared with controls, but lung water content was reduced (5.5%, P<0.05), a reversal of the pattern seen acutely. Studies in isolated perfused lungs demonstrated a reduced capillary filtration coefficient (0. 018+/-0.003 vs. 0.003+/-0.002 ml min(-1)mmHg(-1)g(-1), P<0.001), increased arterial (61%) and venous resistance (50%) in heart failure lungs, P<0.05. Wall thickness:lumen ratio was increased in small (<250 microm) pulmonary arterioles (0.15+/-0.02 vs. 0.08+/-0. 01) and venules (0.06+/-0.005 vs. 0.04+/-0.002) in heart failure, P<0.01. Alveolar septal volume fractions (35.2+/-5.1 vs. 23.1+/-2.7) and septal:air-space volume ratios (60.5+/-13.6 vs. 31.9+/-5.3) were also increased in heart failure, P<0.05. CONCLUSIONS: Pulmonary adaptation to chronic heart failure is associated with vascular and alveolar remodelling that contributes to increased vascular resistance and reduced capillary filtration. These changes are likely to be important in mediating resistance to pulmonary oedema in chronic heart failure.

Expression of functional GABAA receptors in transfected L929 cells isolated by immunomagnetic bead separation.

Transient cotransfection of fibroblasts, with plasmids encoding individual GABAA receptor (GABAAR) subunits, has provided a model to characterize the pharmacological and kinetic properties of receptor subtype combinations. However, identifying transfected cells for electrophysiological recording is often difficult due to low transfection efficiencies. Selection of transfected cells has required cotransfection with a marker gene and fluorescence microscopic localization prior to recording. To circumvent these problems, two GABAAR subtypes combinations in transfected L929 cells were isolated with a novel biomagnetic separation system. Cell selection was accomplished by cotransfection with a plasmid (pHook-1) encoding a single-stranded cell surface antibody (sFv), which bound to ferromagnetic beads, coated with an antigen (phOx). Bead-covered cells were then magnetically separated from non-transfected cells. Bead-selected cells cotransfected with alpha 6, beta 3 and gamma 2L subtypes, expressed GABAAR currents in 95% (41/43) of cells recorded. Cells cotransfected with alpha 5, beta 3 and gamma 2L subtypes had an EC50 for GABA of 5.4 microM and a Hill slope of 1.4. Membrane patches from cells expressing the alpha 5 beta 3 gamma 2L isoform demonstrated single channel currents with a main conductance state of 23 pS. Magnetic bead immunoselection provides a purified population of transfected cells well suited for whole cell and single channel recording.

Applications of capillary electrophoresis/laser-induced fluorescence detection to groundwater migration studies.

Capillary electrophoresis has been applied to the determination of groundwater migration based on laser-induced fluorescence (LIF) detection and traditional spectrofluorimetry. Detection limits of injected dye-fluorescent whitening agent (tinopal) in the low ppt ranges have been accomplished with both a spectrofluorometer and with CE/LIF based on the HeCd laser. The real-world problem was the determination of groundwater migration between adjacent Resource Conservation and Recovery Act (RCRA) and Superfund sites. Fluorescent dyes were injected into wells and were discovered in monitoring wells by extracting pads that adsorbed the dye. The methodology based on CE/LIF exhibits increased specificity over existing methodology due to the separation and unique migration time of the dye. Additional studies were aimed at achieving sub-ppt levels in the water directly using solid-phase extraction (SPE) and field-amplified injection techniques.

Loreclezole enhances apparent desensitization of recombinant GABAA receptor currents.

Loreclezole is a newly developed antiepileptic drug which has been shown to act at a specific site on beta 2 or beta 3 GABAA receptor subtypes to enhance the peak whole-cell response to submaximal concentrations of GABA. Potentiation by loreclezole occurred with high affinity only at GABAA receptors containing a beta 2 or beta 3 subtype, not a beta 1 subtype. We have studied the effect of loreclezole on whole-cell currents from recombinant GABAA receptors transiently expressed in L929 fibroblasts and on currents from cultured mouse cortical neurons and have found a second, inhibitory action of loreclezole that was independent of the beta-subunit subtype composition of the receptor. Loreclezole, at concentrations above 6 microM, enhanced the degree and rate of apparent desensitization of the whole-cell current in a concentration-dependent manner. This effect was voltage-independent, non-competitive and increased with increasing GABA concentration. The increase in desensitization was not blocked by the benzodiazepine antagonist flumazenil and did not require the presence of a gamma subunit. Loreclezole acted at a novel inhibitory allosteric site to increase the apparent desensitization of the GABAA receptor, regardless of its subunit composition. This activity of loreclezole may have implications for its experimental or clinical use as an antiepileptic drug.

Single-channel currents from diethylpyrocarbonate-modified NMDA receptors in cultured rat brain cortical neurons.

The role of histidine residues in the function of N-methyl-D-aspartate (NMDA)-activated channels was tested with the histidine-modifying reagent diethylpyrocarbonate (DEP) applied to cells and membrane patches from rat brain cortical neurons in culture. Channels in excised outside-out patches that were treated with 3 mM DEP for 15-30 s (pH 6.5) showed an average 3.4-fold potentiation in steady state open probability when exposed to NMDA and glycine. Analysis of the underlying alterations in channel gating revealed no changes in the numbers of kinetic states: distributions of open intervals were fitted with three exponential components, and four components described the shut intervals, in both control and DEP-modified channels. However, the distribution of shut intervals was obviously different after DEP treatment, consistent with the single-channel current record. After modification, the proportion of long shut states was decreased while the time constants were largely unaffected. Burst kinetics reflected these effects with an increase in the average number of openings/burst from 1.5 (control) to 2.2 (DEP), and a decrease in the average interburst interval from 54.1 to 38.2 ms. These effects were most likely due to histidine modification because other reagents (n-acetylimidazole and 2,4,6-trinitrobenzene 1-sulfonic acid) that are specific for residues other than histidine failed to reproduce the effects of DEP, whereas hydroxylamine could restore channel open probability to control levels. In contrast to these effects on channel gating, DEP had no effect on average single-channel conductance or reversal potential under bi-ionic (Na+:Cs+) conditions. Inhibition by zinc was also unaffected by DEP. We propose a channel gating model in which transitions between single- and multi-opening burst modes give rise to the channel activity observed under steady state conditions. When adjusted to account for the effects of DEP, this model suggests that one or more extracellular histidine residues involved in channel gating are associated with a single kinetic state.

Novel potent and selective inhibitors of inducible nitric oxide synthase.

We have identified two novel potent and selective inhibitors of inducible nitric oxide synthase, S-ethylisothiourea and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine. Ki values of 14.7 nM for S-ethylisothiourea and 4.2 nM for 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine were obtained with partially purified preparations of inducible nitric oxide synthase. These compounds demonstrate about 1000-fold greater potency than prototypical inhibitors, and the inhibitions are 10-40-fold more selective for murine inducible nitric oxide synthase, compared with the rat neuronal and bovine endothelial isoforms of nitric oxide synthase. These compounds also potently inhibit the nitric oxide synthase activity in intact J774 mouse macrophages. The inhibition is competitive with the substrate L-arginine and reversible in both enzymatic and intact cell assays. These potent and selective inhibitors of inducible nitric oxide synthase may have potential therapeutic applications in the treatment of inflammatory and autoimmune diseases.

The endothelial vesicular system in cardiac capillaries subjected to hypoxia.

The precise role of the numerous plasmalemmal vesicles in capillary endothelia is unclear. They undoubtedly play a part in macromolecular transport across the endothelium but, as serial section studies have shown the vast majority of vesicles to be attached to either the luminal or abluminal membrane, it is possible that transport is not their primary role. The suggestion that vesicles form a membrane reserve was explored in capillaries of isolated perfused rat hearts subjected to hypoxia, which has been reported to cause endothelial cell swelling. Luminal, "cytoplasmic," and abluminal vesicles were counted and their diameters measured in well-oxygenated and hypoxic hearts and a number of other endothelial cell parameters were measured. Total capillary and luminal cross-sectional area and luminal and abluminal membrane length decreased in hypoxia. There was no change in endothelial cell cross-sectional area despite an appearance of swelling. The numbers of abluminal and "cytoplasmic" vesicles did not change as a result of hypoxia. Luminal vesicles were, however, reduced in number but increased in size and density. The results are consistent both with the theory that the vesicles are a membrane reserve and that they may be present in order to expose membrane enzymes and receptors on the cell surface or internalise them in response to a variety of external stimuli.

Hypoxia induced disruption of the cardiac endothelial glycocalyx: implications for capillary permeability.

OBJECTIVE: The aim was to determine the effect of hypoxia on the ultrastructure of the endothelial glycocalyx of cardiac capillaries. METHODS: Isolated rat hearts were perfused with oxygenated or hypoxic Krebs solution for 30 min after equilibration with oxygenated medium. They were then perfused with a cationic marker (ruthenium red, lanthanum nitrate, or cationised ferritin) to delineate the cardiac endothelial glycocalyx in the electron microscope. With ruthenium red and lanthanum, perfusions were carried out both in the presence and absence of bovine serum albumin. Ferritin perfused hearts were used to quantify changes in the glycocalyx as a result of hypoxia and to measure the cross sectional area of the endothelial cells. RESULTS: In all the hearts perfused with well oxygenated solution, all three markers showed an even, electron dense layer on the luminal surface of the capillaries. With ruthenium red and lanthanum (but not with ferritin), the marker was occasionally observed throughout the length of the interendothelial clefts and on the albuminal surface. After 30 min hypoxic perfusion, both ruthenium red and lanthanum showed disruption and irregular clumping of the glycocalyx, with or without albumin. Ferritin, however, showed a sparse and uneven layer. Measurements of endothelial cell area showed that some cells from hypoxic hearts were swollen when compared with controls. Measurements of the percentage of luminal membrane covered by ferritin molecules showed a significant loss of glycocalyx in hypoxic hearts. There was, however, no correlation between loss of glycocalyx and endothelial cell swelling. CONCLUSIONS: The endothelial cell glycocalyx of continuous capillaries is sensitive to changes in PO2. The disruption of this surface coat may explain the reported increase in capillary permeability in hypoxia.