H1 and PAR2 receptors enhance delivery of immune-competent cells and molecules by interrupting E-cadherin adhesion in epithelia.

The lung's epithelial surface is at the same time vitally exchanging gas with the environment and acting as a barrier that protects the organism from the environment. We hypothesized that activation of epithelial-cell G-protein-coupled receptors for immune-defense molecules would temporarily interrupt cadherin-dependent cell-cell adhesion of epithelial cells and thereby focally and temporarily compromise the epithelial barrier to facilitate delivery of other immune molecules and cells to challenged sites. Activation of type 1 histamine or type 2 PAR receptors on the basolateral surface of primary airway epithelial cells or L-cells expressing E-cadherin interrupted cadherin adhesion and caused approximately a 50% decrease in the epithelial barrier for 2-3 minutes.

Compromised E-cadherin adhesion and epithelial barrier function with activation of G protein-coupled receptors is rescued by Y-to-F mutations in beta-catenin.

Activation of the type 1 histamine (H1) or the type 2 protease-activated (PAR-2) G protein-coupled receptors interrupts E-cadherin adhesion and decreases the transepithelial resistance (TER) of epithelium. Several reports suggest that cadherin adhesive function depends on the association of cadherin with beta-catenin and that this association is regulated by phosphorylation of tyrosines in beta-catenin. We tested the hypothesis that loss of cadherin adhesion and compromise of TER on activation of the H1 or PAR-2 receptor is due to phosphorylation of tyrosines in beta-catenin.

Paracellular permeability restricts airway epithelial responses to selectively allow activation by mediators at the basolateral surface.

Respiratory pathogens and toxins often assault the lung from the airway lumen. Airway epithelia may initiate and amplify inflammation in response to these attacks, but under certain conditions confinement of inflammation to the airway lumen may be beneficial to the host. Accordingly, we hypothesized that airway epithelial polarity allows different responses to basolateral vs apical stimuli that may modulate inflammation.

Cell-cell adhesion in lung endothelium.

Homotypic cell-cell adhesion is essential for tissue and organ development, remodeling, regeneration, and physiological function. Whereas a significant number of homotypic cell-cell adhesion molecules have been identified, much more is known about those concentrated in epithelia than in endothelia. Among the endothelial cell-cell adhesion molecules, very little is known that is specific to endothelium in the pulmonary and bronchial circulations.

PAR2 activation interrupts E-cadherin adhesion and compromises the airway epithelial barrier: protective effect of beta-agonists.

The airway epithelium is an important barrier between the environment and subepithelial tissues. The epithelium is also divided into functionally restricted apical and basolateral domains, and this restriction is dependent on the elements of the barrier. The protease-activated receptor-2 (PAR2) receptor is expressed in airway epithelium, and its activation initiates multiple effects including enhanced airway inflammation and reactivity.

Histamine selectively interrupts VE-cadherin adhesion independently of capacitive calcium entry.

Histamine is an important agent of innate immunity, transiently increasing the flux of immune-competent molecules from the vascular space to the tissues and then allowing rapid restoration of the integrity of the endothelial barrier. In previous work we found that histamine alters the endothelial barrier by disrupting cell-cell adhesion and identified VE-cadherin as an essential participant in this process. The previous work did not determine whether histamine directly interrupted VE-cadherin adhesion, whether the effects of histamine were selective for cadherin adhesion, or whether capacitive calcium flux across the cell membrane was necessary for the effects of histamine on cell-cell adhesion.

Histamine stimulates phosphorylation of adherens junction proteins and alters their link to vimentin.

Histamine increases microvascular permeability by creating small transitory (100-400 nm) gaps between adjacent endothelial cells at sites of vascular endothelial (VE)-cadherin-based adhesion. We examined the effects of histamine on the proteins within the VE-cadherin-based adherens junction in primary human umbilical vein endothelial cells. VE-cadherin is linked not only by beta- and alpha-catenin to cortical actin but also by gamma-catenin to the intermediate filament vimentin.

Human recombinant activated protein C in meningococcal sepsis.

A 19-year-old woman presented with purpura fulminans and septic shock; subsequently, progressive coagulopathy, widespread purpura fulminans associated with meningococcemia, severe shock, respiratory, and renal failure developed. This clinical course was associated with depletion of functional protein C levels to < 5%. We describe her clinical course and therapy with human recombinant activated protein C.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.