PubMed Health⌕ Search

Biomedical subjects

D M Shasby

Publications and source records attributed to D M Shasby.

At least 19 recordsLinked to original sources
PubMed2000

Histamine alters endothelial barrier function at cell-cell and cell-matrix sites.

To determine how histamine regulates endothelial barrier function through an integrative cytoskeletal network, we mathematically modeled the resistance across an endothelial cell-covered electrode as a function of cell-cell, cell-matrix, and transcellular resistances. Based on this approach, histamine initiated a rapid decrease in transendothelial resistance predominantly through decreases in cell-cell resistance in confluent cultured human umbilical vein endothelial cells (HUVECs). Restoration of resistance was characterized by initially increasing cell-matrix resistance, with later increases in cell-cell resistance. Thus histamine disrupts barrier function by specifically disrupting cell-cell adhesion and restores barrier function in part through direct effects on cell-matrix adhesion. To validate the precision of our technique, histamine increased the resistance in subconfluent HUVECs in which there was no cell-cell contact. Exposure of confluent monolayers to an antibody against cadherin-5 caused a predominant decrease in cell-cell resistance, whereas the resistance was unaffected by the antibody to cadherin-5 in subconfluent cells. Furthermore, we observed an increase predominantly in cell-cell resistance in ECV304 cells that were transfected with a plasmid containing a glucocorticoid-inducible promoter controlling expression of E-cadherin. Transmission electron microscopy confirmed tens of nanometer displacements between adjacent cells at a time point in which histamine maximally decreased cell-cell resistance.

Cadherins↗
PubMed2000

Mechanisms regulating endothelial cell barrier function.

Endothelium forms a physical barrier that separates blood from tissue. Communication between blood and tissue occurs through the delivery of molecules and circulating substances across the endothelial barrier by directed transport either through or between cells. Inflammation promotes macromolecular transport by decreasing cell-cell and cell-matrix adhesion and increasing centripetally directed tension, resulting in the formation of intercellular gaps. Inflammation may also increase the selected transport of macromolecules through cells. Significant progress has been made in understanding the molecular and cellular mechanisms that account for constitutive endothelial cell barrier function and also the mechanisms activated during inflammation that reduce barrier function. Current concepts of mechanisms regulating endothelial cell barrier function were presented in a symposium at the 2000 Experimental Biology Conference and are reviewed here.

Animals↗
PubMed2000

Receptor-dependent activation of store-operated calcium entry increases endothelial cell permeability.

The present study evaluated the necessity of store-operated Ca(2+) entry in mediating thrombin-induced 20-kDa myosin light chain (MLC(20)) phosphorylation and increased permeability in bovine pulmonary artery endothelial cells (BPAECs). Thrombin (7 U/ml) and thapsigargin (1 microM) activated Ca(2+) entry through a common pathway in confluent BPAECs. Similar increases in MLC(20) phosphorylation were observed 5 min after thrombin and thapsigargin challenge, although thrombin produced a sustained increase in MLC(20) phosphorylation that was not observed in response to thapsigargin. Neither agonist increased MLC(20) phosphorylation when Ca(2+) influx was inhibited. Thrombin and thapsigargin induced inter-endothelial cell gap formation and increased FITC-dextran (molecular radii 23 A) transfer across confluent BPAEC monolayers. Activation of store-operated Ca(2+) entry was required for thapsigargin and thrombin receptor-activating peptide to increase permeability, demonstrating that activation of store-operated Ca(2+) entry is coupled with MLC(20) phosphorylation and is associated with intercellular gap formation and increased barrier transport of macromolecules. Unlike thrombin receptor-activating peptide, thrombin increased permeability without activation of store-operated Ca(2+) entry, suggesting that it partly disrupts the endothelial barrier through a proteolytic mechanism independent of Ca(2+) signaling.

Animals↗
PubMed1999

Histamine alters cadherin-mediated sites of endothelial adhesion.

We tested the hypothesis that histamine alters the focal apposition of endothelial cells by acting on sites of cadherin-mediated cell-cell adhesion. Focal apposition was measured as the impedance of a cell-covered electrode, which was partitioned into a cell-matrix resistance, a cell-cell resistance, and membrane capacitance. Histamine causes an immediate, short-lived decrease in the impedance of an electrode covered with human umbilical vein endothelial (HUVE) cells. ECV304 cells are a line of spontaneously transformed HUVE cells that do not express the endothelial cadherin, cadherin-5. Histamine increased ECV304 cell calcium to 600 nM. Histamine did not increase myosin light chain phosphorylation of control or transfected ECV304 cells. ECV304 cells transfected with either E-cadherin or cadherin-5 on a dexamethasone-responsive plasmid (pLKneo) increased their cell-cell resistance when stimulated with dexamethasone, whereas ECV304 cells transfected with pLKneo-lacZ did not. Histamine did not affect the impedance of ECV304 cells transfected with pLKneo-lacZ. In contrast, histamine decreased the cell-cell resistance of ECV304 cells transfected with either pLKneo-E-cadherin or pLKneo-cadherin-5. From these data, we conclude that histamine acts on sites of cadherin-mediated cell-cell apposition.

Animals↗
PubMed1998

cAMP protects endothelial barrier function independent of inhibiting MLC20-dependent tension development.

Exposure of cultured human umbilical vein endothelial cells to the cAMP agonists theophylline and forskolin decreased constitutive isometric tension of a confluent monolayer inoculated on a collagen membrane, but it did not prevent increased tension in cells exposed to thrombin. The inability of cAMP agonists to prevent tension development correlated with an inability of cAMP stimulation to prevent increased 20-kDa myosin light chain (MLC20) phosphorylation in response to thrombin. Although cAMP did not prevent tension development or increased MLC20 phosphorylation, cAMP attenuated the effect of thrombin on transendothelial electrical resistance across a confluent monolayer inoculated on a gold microelectrode. Activation of cAMP-dependent signal transduction did not prevent a decline in resistance in thrombin-treated cells, but it more promptly restored transendothelial resistance to initial basal levels (10 min) compared with thrombin only (60 min). ML-7, an MLC kinase antagonist, at doses that attenuate increased MLC20 phosphorylation and tension development, did not prevent a decline in resistance in thrombin-treated cells. Yet, ML-7 also restored transendothelial resistance more rapidly than thrombin alone (20 min) but at a slower rate than cAMP. These data demonstrate that activation of cAMP-dependent signal transduction protects barrier function independent of inhibition of MLC20-dependent tension development.

Cells, Cultured↗
PubMed1997

Isometric tension of cultured endothelial cells: new technical aspects.

In this paper new technical aspects are discussed in the measurement of the low amount of force typically expressed in cultured endothelial cells. We illustrate how potential background noises interfere with signal acquisition. We present a new generation prototype that measures isometric tension in vitro in multiple samples and in more than on isometric vector. We report that thrombin increases isometric tension in at least two separate vectors that are directed in opposite directions. We also report that phorbol ester dibutyrate can randomly mediate a false relaxation (anisotropic contraction) in cultured PPAEC, when the force vector is directed opposite to the referenced isometric vector of the transducer. In contrast, stimulation of cultured HUVEC with the cAMP agonists, theophylline and forskolin, decreased isometric force in both vectors. Thus direction of the force vector needs to be considered when interpreting isometric tension in cultured endothelial cells.

Animals↗
PubMed1997

Thrombin inhibits myosin light chain dephosphorylation in endothelial cells.

Histamine and thrombin increase myosin light-chain kinase-mediated phosphorylation of myosin light chain (MLC) in human umbilical vein endothelial cells (HUVEC). The increase in MLC phosphorylation caused by thrombin persists longer (330 min) than the increase caused by histamine (<5 min), although both increase cell calcium similarly. We hypothesized that some of the longer duration of the increase in MLC phosphorylation caused by thrombin was because of inhibition of myosin dephosphorylation by thrombin. Calyculin A, an inhibitor of type 1 and 2A protein phosphatases, caused a time-dependent increase in MLC phosphorylation in unstimulated HUVEC. As thrombin-stimulated phosphorylation approached its peak at 15 min, calyculin A caused progressively less of an increase in MLC phosphorylation in thrombin-stimulated HUVEC, and no increase at the peak of thrombin stimulation. In HUVEC in which cell calcium was maintained at 600 nM, thrombin increased MLC phosphorylation above the level caused by increased calcium alone at a time coinciding with the peak of thrombin stimulation. However, when phosphatase activity was already inhibited with calyculin A, thrombin did not further increase MLC phosphorylation in cells in which calcium was maintained at 600 nM calcium. Thrombin increases MLC phosphorylation in HUVEC not only by increasing cell calcium but also by inhibiting calyculin A-sensitive dephosphorylation of MLC.

Calcium↗
PubMed1997

Spirometric values in obese individuals. Effects of body position.

Obesity is increasingly prevalent. Earlier studies indicated that there was a significant but small difference in spirometric values between sitting and standing position in the normal population. It is not known if this is true for obese individuals. The recommendations of the American Thoracic Society (ATS) are to document if a spirometry is done in a sitting or standing position. We performed a study in which we compared sitting and standing spirometric values in obese individuals. Patients with a body mass index (BMI) > or = 30 kg/m2 who were referred for spirometry were invited to participate. All tests were done according to American Thoracic Society recommendations. We studied 50 subjects (32 females and 18 males; mean age 45 yr [SD +/- 14.4]). Age range was 20-71 years. Average BMI was 39 (SD +/- 7, range 30 to 65). Twenty-two did the first testing in the sitting position and 28 standing. There was a small but statistically significant difference between forced vital capacity (FVC) in the standing versus sitting position (Wilcoxen test, p < or = 0.05). There was no significant difference in FEV1 between sitting and standing. Our conclusion is that body position is not important when performing spirometry in persons with BMI > or = 30 kg/m2.

Adult↗
PubMed1996

Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces.

We examined the contribution of actin-myosin contraction to the modulation of human umbilical vein endothelial cell focal adhesion caused by histamine and thrombin. Focal adhesion was measured as the electrical resistance across a cultured monolayer grown on a microelectrode. Actin-myosin contraction was measured as isometric tension of cultured monolayers grown on a collagen gel. Histamine immediately decreased electrical resistance but returned to basal levels within 3-5 min. Histamine did not increase isometric tension. Thrombin also immediately decreased electrical resistance, but, however, resistance did not return to basal levels for 40-60 min. Thrombin also increased isometric tension, ML-7, an inhibitor of myosin light chain kinase, prevented increases in myosin light chain phosphorylation and increases in tension development in cells exposed to thrombin. ML-7 did not prevent a decline in electrical resistance in cells exposed to thrombin. Instead, ML-7 restored the electrical resistance to basal levels in a shorter period of time (20 min) than cells exposed to thrombin alone. Also, histamine subsequently increased electrical resistance to above basal levels, and thrombin initiated an increase in resistance during the time of peak tension development. Hence, histamine and thrombin modulate endothelial cell focal adhesion through centripetal and centrifugal forces.

Biophysical Phenomena↗
PubMed1995

Pyocyanin from Pseudomonas aeruginosa inhibits prostacyclin release from endothelial cells.

Pseudomonas aeruginosa pneumonia causes a vasculitis of small pulmonary arteries. While the fully developed lesion demonstrates vessel wall necrosis, the early lesion is remarkable for preservation of viable endothelium despite vessel wall invasion by bacteria. Pyocyanin, an exoproduct of P. aeruginosa, markedly inhibited prostacyclin production by pulmonary artery endothelial cells without causing cell lysis. Pyocyanin might after vascular homeostasis in the absence of cytolysis.

Animals↗
PubMed1995

Ionomycin and PDBU increase MDCK monolayer permeability independently of myosin light chain phosphorylation.

It has been hypothesized that modulation of epithelial paracellular permeability may be mediated by initiation of contraction of a band of actin and myosin located at the tight junction. Phosphorylation of myosin light chain (MLC) is an important determinant of actomyosin contraction. We asked if ionomycin (iono) and phorbol 12,13-dibutyrate (PDBU), which increase paracellular permeability of Madin-Darby canine kidney (MDCK) cell monolayers, increased MLC phosphorylation in MDCK cells. MDCK cell MLC was constitutively phosphorylated by myosin light chain kinase (MLCK), and after PDBU and iono > 99% of MLC continued to be phosphorylated by MLCK. Neither iono or PDBU, nor the combination of iono and PDBU, increased MLC phosphorylation. In contrast, the phosphatase inhibitor okadaic acid did increase MLC phosphorylation. Adenosine 3',5'-cyclic monophosphate (cAMP) and forskolin decreased MLC phosphorylation in control MDCK cells and in cells exposed to iono and PDBU. In contrast, cAMP and forskolin did not blunt the decrease in transepithelial resistance caused by iono and PDBU. Iono and PDBU increase MDCK monolayer permeability independently of an increase in MLC phosphorylation.

Animals↗
PubMed1993

Purification of dystrophin-related protein (utrophin) from lung and its identification in pulmonary artery endothelial cells.

Dystrophin-related protein (DRP or utrophin) is an autosomal homologue of dystrophin, a membrane cytoskeletal protein encoded by the Duchenne muscular dystrophy gene. In contrast to dystrophin which is predominantly expressed in muscle, DRP is expressed in various tissues. Here we report the purification and biochemical characterization of DRP from lung which shows the highest levels of DRP expression among adult tissues. DRP was purified from the high alkaline extract of lung membranes using heparin-agarose column chromatography followed by anti-DRP immunoaffinity column chromatography. DRP was expressed in the cultured pulmonary artery endothelial cells. Expression of DRP in endothelial cells could explain its abundant expression in lung. In analogy to dystrophin of muscle cells, DRP could be playing an important role in the mechanical stress mechanisms of endothelial cells.

Animals↗
PubMed1993

Lidocaine and dextran sulfate inhibit leukocyte accumulation but not postischemic contractile dysfunction in a canine model.

Leukocytes have been implicated as a possible factor in the pathogenesis of postischemic contractile dysfunction, probably through the release of oxygen free radicals. Lidocaine and dextran sulfate are known to inhibit leukocyte adherence to endothelial cells in vitro and in vivo. In an acute open-chest canine model both agents were found to inhibit the augmented accumulation of indium-111-labeled leukocytes in briefly ischemic and subsequently reperfused myocardium. Pharmacologic inhibition of leukocyte accumulation by lidocaine and dextran sulfate, however, was not associated with improvement in postischemic contractile dysfunction.

Animals↗
PubMed1993

Role of myosin light-chain phosphorylation in endothelial cell retraction.

Endothelial cells retract centripetally when they are exposed to histamine and when extracellular calcium is chelated. This centripetal retraction implies that a centripetal tension must be expressed in the cells. We asked whether phosphorylation of the light chain of myosin (MLC) was important for the retraction to occur, and, by inference, expression of the tension. In human umbilical vein endothelial (HUVE) cells and in porcine pulmonary artery endothelial (PPAE) cells tryptic peptide maps indicated that MLC was phosphorylated by myosin light-chain kinase (MLCK). Activity of MLCK is inhibited by ML-9, a kinase inhibitor with relative specificity for MLCK, and when MLCK is phosphorylated by the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent kinase. Pretreatment of HUVE cells or PPAE cells with ML-9 or forskolin-aminophylline (to increase cell cAMP) reduced basal MLC phosphorylation and prevented an expected increase in MLC phosphorylation following exposure of HUVE cells to histamine. Pretreatment of HUVE cells with ML-9 or forskolin-aminophylline prevented HUVE cell retraction (measured as an increase in permeability of a monolayer of HUVE cells) in response to histamine. Pretreatment of PPAE cells with ML-9 or forskolin-aminophylline prevented PPAE cell retraction in response to chelation of extracellular calcium. These data support the hypothesis that phosphorylation of MLC is an important component of endothelial cell retraction.

Aminophylline↗
PubMed1993

The effect of histamine and cyclic adenosine monophosphate on myosin light chain phosphorylation in human umbilical vein endothelial cells.

Histamine causes adjacent endothelial cells to retract from each another. We examined phosphorylation of the 20-kD myosin light chain (MLC20) in human umbilical vein endothelial cells (HUVECs) exposed to histamine to determine if we could find evidence to support the hypothesis that retraction of these cells in response to histamine represents an actomyosin-initiated contraction of the endothelial cytoskeleton. We found that MLC20 in HUVECs was constitutively phosphorylated with approximately 0.2 mol phosphate/mol MLC20. Histamine increased MLC20 phosphorylation by 0.18 +/- 0.05 mol phosphate/mol MLC20. This peak increase in phosphorylation occurred 30 s after initiating histamine exposure, persisted through 90s, and returned to control levels by 5 min. Agents that increase HUVEC cAMP prevent cell retraction in response to histamine. An increase in HUVEC cAMP decreased MLC20 phosphorylation by 0.18 +/- 0.02 mol phosphate/mol MLC20 and prevented the increase in MLC20 phosphorylation after exposure to histamine. Tryptic peptide maps of phosphorylated myosin light chain indicated that myosin light chain kinase phosphorylated MLC20 in HUVECs under basal, cAMP-, and histamine-stimulated conditions. Phosphoaminoacid analysis of the monophosphorylated peptide indicated that, in contrast to smooth muscle cells, ser19 and thr18 monophosphorylation occurs in HUVECs. On the basis of our results, modulation of myosin light chain kinase activity may be an important regulatory step in the control of endothelial barrier function.

Cyclic AMP↗
PubMed1992

Insight into the nature and site of oxygen-centered free radical generation by endothelial cell monolayers using a novel spin trapping technique.

Spin trapping, a sensitive and specific means of detecting free radicals, is optimally performed on cell suspensions. This makes it unsuitable for the study of adherent endothelial cell monolayers because disrupting the monolayer to induce a cell suspension could introduce confounding factors. This problem was eliminated through the use of endothelial cells that were grown to confluence on microcarrier beads. Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the nature of free radical species generated by suspensions of microcarrier bead adherent porcine pulmonary endothelial cells under various forms of oxidant stress was examined. Exposure of these endothelial cells to paraquat resulted in the spin trapping of superoxide (.O2-). Endothelial cell incubation in the presence of either bolus or continuous fluxes of hydrogen peroxide (H2O2) yielded spin trap evidence of hydroxyl radical formation, which was preventable by pretreating the cells with deferoxamine. Chromium oxalate which eliminates extracellular electron paramagnetic resonance spectrometry (EPR) signals, prevented the detection of DMPO spin adducts generated by paraquat but not H2O2-treated endothelial cells. When endothelial cells were coincubated with PMA-stimulated monocytes evidence of both .O2- and hydroxyl radical production was detected, whereas with PMA-stimulated neutrophils only .O2- production could be confirmed. Neutrophil elastase, cathepsin G, and the combination of PMA and A23187 have previously been suggested to induce endothelial cell oxy-radical generation. However, exposure of endothelial cells to each of these agents did not yield DMPO spin adducts or cyanide-insensitive endothelial cell O2 consumption. These data indicate that endothelial cell exposure: to paraquat induces extracellular .O2- formation; to H2O2 leads to intracellular hydroxyl radical production; and to elastase, cathepsin G, or A23187/PMA does not appear to cause oxy-radical generation.

Animals↗