Endothelium-derived relaxing factor has been recently identified as nitric oxide. The purpose of this study was to determine if vasodilator drugs that generate nitric oxide inhibit vascular smooth muscle mitogenesis and proliferation in culture. Three chemically dissimilar vasodilators, sodium nitroprusside, S-nitroso-N-acetylpenicillamine and isosorbide dinitrate, dose-dependently inhibited serum-induced thymidine incorporation by rat aortic smooth muscle cells. Moreover, 8-bromo-cGMP mimicked the antimitogenic effect of the nitric oxide-generating drugs. The antimitogenic effect of S-nitroso-N-acetylpenicillamine was inhibited by hemoglobin and potentiated by superoxide dismutase, supporting the view that nitric oxide was the ultimate effector. Sodium nitroprusside and S-nitroso-Nacetylpenicillamine significantly decreased the proliferation of vascular smooth muscle cells. Moreover, the inhibition of mitogenesis and proliferation was shown to be independent of cell damage, as documented by several criteria of cell viability. These results suggest that endogenous nitric oxide may function as a modulator of vascular smooth muscle cell mitogenesis and proliferation, by a cGMP-mediated mechanism.

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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.

Abstract-Vasoconstrictors that bind to phospholipase C-coupled receptors elevate inositol-1,4,5-trisphosphate (IP 3 The mechanism by which IP 3 regulates arterial contractility is generally well accepted. 1 Indeed, IP 3 -induced SR Ca 2ϩ release is considered to be the only mechanism by which this second messenger regulates arterial diameter. However, the physiological mechanisms by which IP 3 regulates intracellular Ca 2ϩ signaling and arterial diameter are poorly understood, and few studies have directly tested the accepted view. Arterial contractility regulation by IP 3 has primarily been studied by using vasoconstrictors that activate PLC. Because PLC activation elevates both DAG and IP 3 and reduces PIP 2 , mechanisms by which IP 3 specifically modulates arterial [Ca 2ϩ ] i signaling and diameter require additional study. Here, we investigated IP 3 regulation of ion channel activity, intracellular Ca 2ϩ signaling, and contractility in cerebral artery myocytes and pressurized arteries. We show that IP 3 activates a nonselective cation current (I Cat ) in myocytes and induces vasoconstriction via a mechanism that does not require the release of SR Ca 2ϩ but involves IP 3 R and TRPC3 (canonical transient receptor potential 3) channel activation. IP 3 -induced Na ϩ influx produces membrane depolarization, voltage-dependent Ca 2ϩ channel activation, an [Ca 2ϩ ] i elevation, and vasoconstriction. We also show that TRPC3 channel Original

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. INTRODUCTIONVillin, an epithelial cell-specific protein belongs to a family of actin-binding proteins that contain segments that display internal homology with each other (Arpin et al., 1988). The amino terminal core of villin retains the actin-capping and -severing functions of villin, whereas the carboxyl terminal headpiece enables villin to cross-link actin filaments. The actin-modifying properties of villin are regulated in vitro by calcium (Northrop et al., 1986), phosphoinositides (Janmey and Matsudaira, 1988; and tyrosine phosphorylation . It has been assumed for several years that villin's actin-bundling and not actin-severing functions are important because nonphysiologically high Ca 2ϩ concentrations (200 M) are required to activate villin's actin-severing activity. However, studies done with the villin knockout mice suggest that in the absence of villin, the actin-bundling properties associated with villin can be substituted by other proteins in the microvilli (Pinson et al., 1998); on the other hand, the actin-severing activity of the microvilli is lost (Ferrary et al., 1999). In recent years, we have demonstrated that villin's actin-modifying functions can be regulated in vitro by tyrosine phosphorylation and phosphatidylinositol bisphosphate (PIP 2 ), suggesting that villin has the potential to function as a link between receptor activation and actin cytoskeleton reorganization even in the absence of high calcium (Arora and McCulloch, 1996;Zhai et al., 2001;. In addition, we have recently demonstrated that the autoinhibited conformation of villin can be released by tyrosine phosp...

Recent studies indicate that endothelium-derived relaxing factor (EDRF) may be identical with nitric oxide (NO). The purpose of this study was to investigate the antimitogenic effect of NO-generating drugs in cultured mesangial cells. S-nitroso-N-acetylpenicillamine, sodium nitroprusside, and isosorbide dinitrate, which generate NO, dose dependently inhibited serum-stimulated DNA synthesis. All three drugs also inhibited the rate of cell proliferation, whereas sodium nitroprusside and S-nitroso-N-acetylpenicillamine decreased cell density at confluence. The antimitogenic activity of S-nitroso-N-acetylpenicillamine was labile in culture medium and could be inhibited by hemoglobin, supporting the view that NO, in free or bound form, was the ultimate effector. All three vasodilators increased cellular guanosine 3',5'-cyclic monophosphate (cGMP) levels dose dependently; moreover, 8-bromo-cGMP mimicked the effects of the NO-generating drugs, suggesting that cGMP may be an intracellular mediator of antimitogenesis. The growth-inhibitory effect of S-nitroso-N-acetylpenicillamine was reversible and was not due to cell toxicity as shown by several criteria of cell viability. The results raise the possibility that EDRF/NO may be a modulator of mesangial cell growth in vivo.

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Abstract-Migration of aortic smooth muscle cells is thought to be of essential importance in vascular restenosis, remodeling, and angiogenesis. Recent studies have shown that NO donors inhibit the migration of subcultured aortic smooth muscle cells. However, there is evidence that NO elicits opposite effects on cell proliferation in primary versus subcultured cells, indicating fundamental differences among different models of aortic smooth muscle cell cultures. The purpose of the current study was to investigate the effect of NO donors on migration of primary cultures of rat aortic smooth muscle cells and to compare and contrast their response with those in subcultured cells. A second purpose was to investigate some of the underlying mechanisms associated with NO-induced effects on cell migration. We report that 2 NO donors, S-nitroso-N-acetylpenicillamine (SNAP) and 2,2-(hydroxynitrosohydrazino)bis-ethanamine, stimulated the migration of primary cells in a wounded-culture model as well as in a transwell migration model. The effect of NO donors was mimicked by 2 cGMP analogues and C-type natriuretic peptide and blocked by a specific inhibitor of guanyl cyclase, 1H-(1,2,4)oxadiazolo[4,3,-a]quinoxalin-1-one, indicating the involvement of cGMP as second messenger. Moreover, neither NO donors nor cGMP analogues altered migration of primary cultures stimulated by either FBS or angiotensin II. In contrast to its effect in primary cultures, SNAP did not alter basal or stimulated migration of subcultured cells, except at a relatively high concentration of 1 mmol/L, at which migration was inhibited. The migration-stimulatory effect of NO donors and cGMP was associated with altered cell morphology and dissociation of actin filaments, consistent with recent studies indicating that cell morphology and cytoskeletal organization influence cell migration. The results suggest the possible involvement of NO-induced cell migration in vascular injury or remodeling, representing conditions in which vascular NO levels would be expected to be elevated. (Circ Res. 1999;84:655-667.)