Mechanical stimuli are transduced into intracellular signals in lung alveolar epithelial cells (AEC). We studied whether mitogen-activated protein kinase (MAPK) pathways are activated during cyclic stretch of AEC. Cyclic stretch induced a rapid (within 5 min) increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in AEC. The inhibition of Na ϩ , L-type Ca 2ϩ and stretch-activated ion channels with amiloride, nifedipine, and gadolinium did not prevent the stretch-induced ERK1/2 activation. The inhibition of Grb2-SOS interaction with an SH3 binding sequence peptide, Ras with a farnesyl transferase inhibitor, and Raf-1 with forskolin did not affect the stretch-induced ERK1/2 phosphorylation. Moreover, cyclic stretch did not increase Ras activity, suggesting that stretch-induced ERK1/2 activation is independent of the classical receptor tyrosine kinase-MAPK pathway. Pertussis toxin and two specific epidermal growth factor receptor (EGFR) inhibitors (AG-1478 and PD-153035) prevented the stretch-induced ERK1/2 activation. Accordingly, in primary AEC, cyclic stretch activates ERK1/2 via G proteins and EGFR, in Na ϩ and Ca 2ϩ influxes and Grb2-SOS-, Ras-, and Raf-1-independent pathways. mechanotransduction; mechanical stress; mitogen-activated protein kinase; lung injury; epidermal growth factor receptor MECHANICAL VENTILATION with high lung volumes and pressures causes ventilator-induced lung injury characterized by increased permeability pulmonary edema and inflammation (26,35). During mechanical ventilation with high tidal volumes, alveolar epithelial cells (AEC) can be stretched. These cells are responsible for the normal function of the alveolar capillary barrier, alveolar fluid absorption, and the synthesis of pulmonary surfactant. It is known that AEC respond to mechanical stretch in different ways. A single stretch of AEC caused cytosolic Ca 2ϩ to increase, followed by a stimulation of surfactant secretion at a single cell (37) and at the whole organ level (1).Stretching AEC increased Na
Abstract-We set out to determine whether cellular hypoxia, via mitochondrial reactive oxygen species, promotes Na,K-ATPase degradation via the ubiquitin-conjugating system. Cells exposed to 1.5% O 2 had a decrease in Na,K-ATPase activity and oxygen consumption. The total cell pool of ␣1 Na,K-ATPase protein decreased on exposure to 1.5% O 2 for 30 hours, whereas the plasma membrane Na,K-ATPase was 50% degraded after 2 hours of hypoxia, which was prevented by lysosome and proteasome inhibitors. When Chinese hamster ovary cells that exhibit a temperature-sensitive defect in E1 ubiquitin conjugation enzyme were incubated at 40°C and 1.5% O 2 , the degradation of the ␣1 Na,K-ATPase was prevented. Exogenous reactive oxygen species increased the plasma membrane Na,K-ATPase degradation, whereas, in mitochondrial DNA deficient 0 cells and in cells transfected with small interfering RNA against Rieske iron sulfur protein, the hypoxia-mediated Na,K-ATPase degradation was prevented. The catalase/superoxide dismutase (SOD) mimetic (EUK-134) and glutathione peroxidase overexpression prevented the hypoxia-mediated Na,KATPase degradation and overexpression of SOD1, but not SOD2, partially inhibited the Na ϩ pump degradation. Accordingly, we provide evidence that during hypoxia, mitochondrial reactive oxygen species are necessary to degrade the plasma membrane Na,K-ATPase via the ubiquitin-conjugating system. Key Words: ATP Ⅲ oxygen Ⅲ proteasome Ⅲ antioxidants Ⅲ cell adaptation A daptation to hypoxia represents a well-defined means to improve ischemic tolerance. Unfortunately, there is no definitive understanding of the mechanisms associated with these phenomena. 1 As mammalian cells encounter lower oxygen levels, they develop mechanisms to prevent depletion of oxygen to anoxia that might result in cell death. 2 Cells respond to hypoxia through the stabilization of the transcription factor hypoxia-inducible factor (HIF)-1␣. In normoxic conditions, prolyl hydroxylases hydroxylate conserved proline residues in HIF-1␣. 3,4 This substrate modification is recognized by a ubiquitin ligase enzyme (Von-Hippel-Lindau protein [VHL]) that ubiquitinates and targets HIF-1␣ to the proteasome. During hypoxia, VHL-mediated degradation of HIF-1␣ is suppressed, allowing its transcriptional activation. 4,5 The intracellular mechanisms by which cells sense hypoxia to stabilize HIF-1␣ are not fully understood. The generation of mitochondrial reactive oxygen species (mROS) during hypoxia has been proposed as part of an oxygen sensing pathway for the hypoxic stabilization of HIF-1␣. 6 Another mechanism to prevent the depletion of oxygen during hypoxia is to decrease the cellular demand for oxygen by upregulating anaerobic ATP-producing pathways and downregulating ATP-consuming processes. 2 This regulation allows ATP levels to remain constant, even while ATP turnover rates greatly decline. The ATP requirements of ion pumping are downregulated by generalized "channel" arrest in hepatocytes and by the arrest of specific ion channels in neurons. 7 The Na,...
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