The movement of water across cell membranes is fundamental to life. Water constitutes roughly 70% of the mass of most living organisms, so the orderly distribution of water is required to maintain proper fluid balance within different anatomic compartments. Although water is known to diffuse through lipid bilayers, diffusion is not sufficiently rapid for many physiological processes. To accommodate these needs, a family of membrane channel proteins evolved for rapid transport of water across biological membranes. These proteins, termed “aquaporins,” are found in all life forms, including archaea, eubacteria, fungi, plants, and all phyla of animals.
Since the discovery of the aquaporins ten years ago (1), researchers around the world have sought to learn how these proteins work. Selectivity, rates of permeation, and gating mechanisms are three properties that characterize channel proteins. Aquaporins are exquisitely selective for the transport of water — even repelling hydronium ions (H3O+). The importance of the latter is emphasized by the normal function of mammalian renal tubules, which reabsorb 99% of the water from glomerular filtrate at the same time that acid is secreted by intercalated cells. Some members of the aquaporin superfamily are permeated by a variety of small neutral solutes such as glycerol or urea (termed “aquaglyceroporins”) (2). The transport of water or glycerol through aquaporins represents facilitated diffusion driven by osmotic or concentration gradients.
Mammalian cells are confronted with changes in extracellular osmolality at various sites, including the aqueous layer above the lung epithelium. Hypertonic shock induces the activation of mitogen-activated protein kinases and the expression of a defined set of genes, including aquaporins. We investigated upstream components of the response to hypertonicity in lung epithelial cells and found that before extracellular signal-regulated kinase activation and aquaporin synthesis, the membrane-bound prohormone neuregulin 1-β is cleaved and binds to human epidermal growth factor receptor 3 (HER3). The signaling is prevented by matrix metalloproteinase inhibition, inhibition of neuregulin 1-β binding to HER3, and inhibition of HER tyrosine kinase activity. Inhibition of HER activation interferes with the hypertonic induction of two different aquaporins in three distinct cell lines of mouse and human origin. We propose that ligand-dependent HER activation constitutes a generalized signaling principle in the mammalian hypertonic stress response relevant to aquaporin expression.
Introduction: Mechanisms leading to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) remain incompletely understood, yet the severity of illness and societal disease burden necessitate continued investigation. On-going lung inflammation and injury may occur due to inhibition of anti-inflammatory pathways in alveolar macrophages. With tissue injury, released adenosine preferentially activates the adenosine 2A receptor (A2aR), a pathway which has been implicated in mediating anti-inflammatory effects and tissue protection; however, modifiers of this pathway in macrophages are not well-described. In a murine model, we have observed that moderate levels of oxygen after intratracheal LPS increase and sustain macrophage inflammation. We hypothesize that moderate oxygen inhibits the A2aR pathway in macrophages to augment inflammation and lung injury. Methods: Murine model consists of intratracheal (IT) delivery of LPS, followed 12 hours later by 60% oxygen or 21% oxygen (RA) via sealed chamber. Results: Wild-type mice exposed to IT LPS (0.375 [[Unsupported Character - Symbol Font ]]g/g mouse) followed by 60% oxygen had significantly more lung injury at days 2-4 in comparison to mice exposed to LPS or 60% oxygen alone, with increased alveolar cell influx, histologic injury, and epithelial barrier permeability. 60% oxygen after LPS significantly reduced lung A2aR mRNA and alveolar cell intracellular cyclic AMP (IC cAMP), located directly downstream of A2aR. Reduction in IC cAMP also occurred in isolated macrophages exposed to both LPS and oxygen when compared to LPS alone; this reduction was reversed using a specific A2aR agonist, CGS 21680. Adding oxygen or ZM241385 (A2aR antagonist) to LPS-exposed macrophages increased secretion of pro-inflammatory cytokines TNF-[[Unsupported Character - Symbol Font ]] and MIP-2, and expression of co-stimulatory molecules CD86 and CD40. Murine delivery of CGS 21680 (125ug/dose/mouse) every 24 hours starting concurrently with oxygen exposure reduced lung injury in LPS and oxygen-exposed mice. Conclusion: After LPS exposure, 60% oxygen acts to decrease activation of the A2aR pathway in macrophages which may directly exacerbate lung injury.
Aquaporin-1 (AQP1) water channel protein expression is increased by hypertonic stress. The contribution of changes in protein stability to hypertonic induction of AQP1 have not been described. Incubation of BALB/c fibroblasts spontaneously expressing AQP1 with proteasome inhibitors increased AQP1 expression, suggesting basal proteasome-dependent degradation of the protein. Degradation by the proteasome is thought to be triggered by polyubiquitination of a target protein. To determine whether AQP1 is ubiquitinated, immunoprecipitation with anti-AQP1 antibodies was performed, and the resultant samples were probed by protein immunoblot for the presence of ubiquitin. Immunoblots demonstrated ubiquitination of AQP1 under control conditions that increased after treatment with proteasome inhibitors (MG132, lactacystin). Exposure of cells to hypertonic medium for as little as 4 h decreased ubiquitination of AQP1, an effect that persisted through 24 h in hypertonic medium. Using metabolic labeling with [ 35 S]methionine, the half-life of AQP1 protein under isotonic conditions was found to be <4 h. AQP1 protein half-life was markedly increased by exposure of cells to hypertonic medium. These observations provide evidence that aquaporins are a target for ubiquitination and proteasome-dependent degradation. Additionally, these studies demonstrate that reduced protein ubiquitination and increased protein stability lead to increased levels of AQP1 expression during hypertonic stress.
