IP3 constricts cerebral arteries via IP3 receptor-mediated TRPC3 channel activation and independently of sarcoplasmic reticulum Ca2+ release.

Vasoconstrictors that bind to phospholipase C-coupled receptors elevate inositol-1,4,5-trisphosphate (IP(3)). IP(3) is generally considered to elevate intracellular Ca(2+) concentration ([Ca(2+)](i)) in arterial myocytes and induce vasoconstriction via a single mechanism: by activating sarcoplasmic reticulum (SR)-localized IP(3) receptors, leading to intracellular Ca(2+) release. We show that IP(3) also stimulates vasoconstriction via a SR Ca(2+) release-independent mechanism. In isolated cerebral artery myocytes and arteries in which SR Ca(2+) was depleted to abolish Ca(2+) release (measured using D1ER, a fluorescence resonance energy transfer-based SR Ca(2+) indicator), IP(3) activated 15 pS sarcolemmal cation channels, generated a whole-cell cation current (I(Cat)) caused by Na(+) influx, induced membrane depolarization, elevated [Ca(2+)](i), and stimulated vasoconstriction. The IP(3)-induced I(Cat) and [Ca(2+)](i) elevation were attenuated by cation channel (Gd(3+), 2-APB) and IP(3) receptor (xestospongin C, heparin, 2-APB) blockers. TRPC3 (canonical transient receptor potential 3) channel knockdown with short hairpin RNA and diltiazem and nimodipine, voltage-dependent Ca(2+) channel blockers, reduced the SR Ca(2+) release-independent, IP(3)-induced [Ca(2+)](i) elevation and vasoconstriction. In pressurized arteries, SR Ca(2+) depletion did not alter IP(3)-induced constriction at 20 mm Hg but reduced IP(3)-induced constriction by approximately 39% at 60 mm Hg. [Ca(2+)](i) elevations and constrictions induced by endothelin-1, a phospholipase C-coupled receptor agonist, were both attenuated by TRPC3 knockdown and xestospongin C in SR Ca(2+)-depleted arteries. In summary, we describe a novel mechanism of IP(3)-induced vasoconstriction that does not occur as a result of SR Ca(2+) release but because of IP(3) receptor-dependent I(Cat) activation that requires TRPC3 channels. The resulting membrane depolarization activates voltage-dependent Ca(2+) channels, leading to a myocyte [Ca(2+)](i) elevation, and vasoconstriction.

Regulation of cell motility by tyrosine phosphorylated villin.

Temporal and spatial regulation of the actin cytoskeleton is vital for cell migration. Here, we show that an epithelial cell actin-binding protein, villin, plays a crucial role in this process. Overexpression of villin in doxycyline-regulated HeLa cells enhanced cell migration. Villin-induced cell migration was modestly augmented by growth factors. In contrast, tyrosine phosphorylation of villin and villin-induced cell migration was significantly inhibited by the src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) as well as by overexpression of a dominant negative mutant of c-src. These data suggest that phosphorylation of villin by c-src is involved in the actin cytoskeleton remodeling necessary for cell migration. We have previously shown that villin is tyrosine phosphorylated at four major sites. To further investigate the role of tyrosine phosphorylated villin in cell migration, we used phosphorylation site mutants (tyrosine to phenylalanine or tyrosine to glutamic acid) in HeLa cells. We determined that tyrosine phosphorylation at residues 60, 81, and 256 of human villin played an essential role in cell migration as well as in the reorganization of the actin cytoskeleton. Collectively, these studies define how biophysical events such as cell migration are actuated by biochemical signaling pathways involving tyrosine phosphorylation of actin binding proteins, in this case villin.

Lung ischemia: a model for endothelial mechanotransduction.

Endothelial cells in vivo are constantly exposed to shear associated with blood flow and altered shear stress elicits cellular responses (mechanotransduction). This review describes the role of shear sensors and signal transducers in these events. The major focus is the response to removal of shear as occurs when blood flow is compromised (i.e., ischemia). Pulmonary ischemia studied with the isolated murine lung or flow adapted pulmonary microvascular endothelial cells in vitro results in endothelial generation of reactive oxygen species (ROS) and NO. The response requires caveolae and is initiated by endothelial cell depolarization via K(ATP) channel closure followed by activation of NADPH oxidase (NOX2) and NO synthase (eNOS), signaling through MAP kinases, and endothelial cell proliferation. These physiological mediators can promote vasodilation and angiogenesis as compensation for decreased tissue perfusion.

Mechanical stretch decreases migration of alveolar epithelial cells through mechanisms involving Rac1 and Tiam1.

Mechanical ventilation can overdistend the lungs or generate shear forces in them during repetitive opening/closing, contributing to lung injury and inflammation in patients with acute respiratory distress syndrome (ARDS). Repair of the injured lung epithelium is important for restoring normal barrier and lung function. In the current study, we investigated the effects of cyclic mechanical strain (CS), constant distention strain (CD), and simulated positive end-expiratory pressure (PEEP) on activation of Rac1 and wound closure of rat primary alveolar type 2 (AT2) cells. Cyclic stretch inhibited the migration of wounded AT2 cells in a dose-dependent manner with no inhibition occurring with 5% CS, but significant inhibition with 10% and 15% CS. PEEP conditions were investigated by stretching AT2 cells to 15% maximum strain (at a frequency of 10 cycles/min) with relaxation to 10% strain. AT2 cells were also exposed to 20% CD. All three types of mechanical strain inhibited wound closure of AT2 cells compared with static controls. Since lamellipodial extensions in migrating cells at the wound edge were significantly smaller in stretched cells, we measured Rac1 activity and found it to be decreased in stretched cells. We also demonstrate that Tiam1, a Rac1-specific guanine nucleotide exchange factor, was expressed mainly in the cytosol of AT2 cells exposed to mechanical strain compared with membrane localization in static cells. Downregulation of Tiam1 with 100 microM NSC-23766 inhibited activation of Rac1 and migration of AT2 cells, suggesting its involvement in repair mechanisms of AT2 cells subjected to mechanical strain.

Localized elasticity measured in epithelial cells migrating at a wound edge using atomic force microscopy.

Restoration of lung homeostasis following injury requires efficient wound healing by the epithelium. The mechanisms of lung epithelial wound healing include cell spreading and migration into the wounded area and later cell proliferation. We hypothesized that mechanical properties of cells vary near the wound edge, and this may provide cues to direct cell migration. To investigate this hypothesis, we measured variations in the stiffness of migrating human bronchial epithelial cells (16HBE cells) approximately 2 h after applying a scratch wound. We used atomic force microscopy (AFM) in contact mode to measure the cell stiffness in 1.5-microm square regions at different locations relative to the wound edge. In regions far from the wound edge (>2.75 mm), there was substantial variation in the elastic modulus in specific cellular regions, but the median values measured from multiple fields were consistently lower than 5 kPa. At the wound edge, cell stiffness was significantly lower within the first 5 microm but increased significantly between 10 and 15 microm before decreasing again below the median values away from the wound edge. When cells were infected with an adenovirus expressing a dominant negative form of RhoA, cell stiffness was significantly decreased compared with cells infected with a control adenovirus. In addition, expression of dominant negative RhoA abrogated the peak increase in stiffness near the wound edge. These results suggest that cells near the wound edge undergo localized changes in cellular stiffness that may provide signals for cell spreading and migration.

High tidal volume mechanical ventilation with hyperoxia alters alveolar type II cell adhesion.

Patients with acute respiratory distress syndrome undergoing mechanical ventilation may be exposed to both high levels of stretch and high levels of oxygen. We hypothesized that the combination of high stretch and hyperoxia promotes loss of epithelial adhesion and impairs epithelial repair mechanisms necessary for restoration of barrier function. We utilized a model of high tidal volume mechanical ventilation (25 ml/kg) with hyperoxia (50% O(2)) in rats to investigate alveolar type II (AT2) cell adhesion and focal adhesion signaling. AT2 cells isolated from rats exposed to hyperoxia and high tidal volume mechanical ventilation (MVHO) exhibited significantly decreased cell adhesion and reduction in phosphotyrosyl levels of focal adhesion kinase (FAK) and paxillin compared with control rats, rats exposed to hyperoxia without ventilation (HO), or rats ventilated with normoxia (MV). MV alone increased phosphorylation of p130(Cas). RhoA activation was increased by MV, HO, and the combination of MV and HO. Treatment of MVHO cells with keratinocyte growth factor (KGF) for 1 h upon isolation reduced RhoA activity and restored attachment to control levels. Attachment and migration of control AT2 cells was significantly decreased by constitutively active RhoA or a kinase inactive form of FAK (FRNK), whereas expression of dominant negative RhoA in cells from MVHO-treated rats restored cell adhesion. Mechanical ventilation with hyperoxia promotes changes in focal adhesion proteins and RhoA in AT2 cells that may be deleterious for cell adhesion and migration.

Nitric oxide attenuates IGF-I-induced aortic smooth muscle cell motility by decreasing Rac1 activity: essential role of PTP-PEST and p130cas.

Recent data support the hypothesis that reactive oxygen species (ROS) play a central role in the initiation and progression of vascular diseases. An important vasoprotective function related to the regulation of ROS levels appears to be the antioxidant capacity of nitric oxide (NO). We previously reported that treatment with NO decreases phosphotyrosine levels of adapter protein p130(cas) by increasing protein tyrosine phosphatase-proline, glutamate, serine, and threonine sequence protein (PTP-PEST) activity, which leads to the suppression of agonist-induced H(2)O(2) elevation and motility in cultured rat aortic smooth muscle cells (SMCs). The present study was performed to investigate the hypotheses that 1) IGF-I increases the activity of the small GTPase Rac1 as well as H(2)O(2) levels and 2) NO suppresses IGF-I-induced H(2)O(2) elevation by decreasing Rac1 activity via increased PTP-PEST activity and dephosphorylation of p130(cas). We report that IGF-I induces phosphorylation of p130(cas) and activation of Rac1 and that NO attenuates these effects. The effects of NO are mimicked by the overexpression of PTP-PEST or dominant-negative (dn)-p130(cas) and antagonized by the expression of dn-PTP-PEST or p130(cas). We conclude that IGF-I induces rat aortic SMC motility by increasing phosphotyrosine levels of p130(cas) and activating Rac1 and that NO decreases motility by activating PTP-PEST, inducing dephosphorylating p130(cas), and decreasing Rac1 activity. Decreased Rac1 activity lowers intracellular H(2)O(2) levels, thus attenuating cell motility.

Cyclic mechanical strain increases reactive oxygen species production in pulmonary epithelial cells.

Overdistention of lung tissue during mechanical ventilation may be one of the factors that initiates ventilator-induced lung injury (VILI). We hypothesized that cyclic mechanical stretch (CMS) of the lung epithelium is involved in the early events of VILI through the production of reactive oxygen species (ROS). Cultures of an immortalized human airway epithelial cell line (16HBE), a human alveolar type II cell line (A549), and primary cultures of rat alveolar type II cells were cyclically stretched, and the production of superoxide (O2-) was measured by dihydroethidium fluorescence. CMS stimulated increased production of O2- after 2 h in each type of cell. 16HBE cells exhibited no significant stimulation of ROS before 2 h of CMS (20% strain, 30 cycles/min), and ROS production returned to control levels after 24 h. Oxidation of glutathione (GSH), a cellular antioxidant, increased with CMS as measured by a decrease in the ratio of the reduced GSH level to the oxidized GSH level. Strain levels of 10% did not increase O2- production in 16HBE cells, whereas 15, 20, and 30% significantly increased generation of O2-. Rotenone, a mitochondrial complex I inhibitor, partially abrogated the stretch-induced generation of O2- after 2 h CMS in 16HBE cells. NADPH oxidase activity was increased after 2 h of CMS, contributing to the production of O2-. Increased ROS production in lung epithelial cells in response to elevated stretch may contribute to the onset of VILI.

Continuous exposure of airway epithelial cells to hydrogen peroxide: protection by KGF.

Reactive oxygen species (ROS) increase permeability in the airway epithelium. Extended periods of oxidant exposure may be experienced by those suffering from chronic inflammation of the lungs, receiving supplemental oxygen, or living in areas with high levels of air pollution. We studied the effects of long-term, continuous exposure to hydrogen peroxide (H(2)O(2)) on the trans-epithelial electrical resistance (TER) across cultured monolayers of a transformed cell line of human bronchial epithelial cells, 16HBE14o- (16HBE). A TER perfusion system was employed to continuously monitor the TER without disturbing the tissue model. The TER decreased in a dose-dependent manner with increasing concentrations of H(2)O(2) (0.1, 0.5, and 1.0 mM), regardless of pre-incubation conditions. Cell cultures pre-treated with 50 ng/ml keratinocyte growth factor (KGF) showed a significant delay in oxidant-induced TER decreases caused by 0.1 mM H(2)O(2). Exposure to 0.1 mM H(2)O(2) for 350 min led to disruption of tight junction proteins, ZO-1 and occludin, but KGF treatment prevented this damage. The recovery of epithelial barrier function after exposure to oxidants was also studied. Tissue models exposed to 0.5 mM H(2)O(2) for 25 min showed complete recovery of TER after 20 h, independent of culture pre-treatment. In contrast, KGF pre-incubation enhanced the recovery of 16HBE cultures exposed for 50 min to 0.5 mM H(2)O(2).

Nitric oxide attenuates insulin- or IGF-I-stimulated aortic smooth muscle cell motility by decreasing H2O2 levels: essential role of cGMP.

Insulin and insulin-like growth factor I (IGF-I) both play important roles in vascular remodeling. Moreover, nitric oxide (NO) is well established as a counterregulatory agent that opposes the actions of several vascular agonists, in part by decreasing smooth muscle motility. We tested the hypothesis that NO blocks insulin or IGF-I-induced rat aortic smooth muscle cell motility via a mechanism involving the attenuation of agonist-induced elevation of hydrogen peroxide levels and cGMP as mediator. Insulin or IGF-I induced an increase of hydrogen peroxide levels and cell motility. Both effects were blocked by catalase or diphenyleneiodonium, indicating that hydrogen peroxide elevation is necessary for induction of cell motility. Two NO donors mimicked the effects of catalase, indicating that NO decreases cell motility by suppressing agonist-induced elevation of hydrogen peroxide. A cGMP analogue mimicked the effect of NO, whereas a guanyl cyclase inhibitor blocked the effect of NO on hydrogen peroxide levels, indicating that elevation of cGMP is both necessary and sufficient to account for the reduction of hydrogen peroxide levels. A NO donor as well as a cGMP analogue attenuated insulin-stimulated NADPH activity, indicating that NO decreases hydrogen peroxide levels by inhibiting the generation of superoxide, via a cGMP-mediated mechanism. Finally, exogenous hydrogen peroxide increased cell motility and reversed the inhibitory effect of cGMP. These results support the view that NO plays an antioxidant role via reduction of hydrogen peroxide in cultured rat aortic smooth muscle cells and that this effect is both necessary and sufficient to account for its capacity to decrease cell motility.