Abstract
O(2) is essential for aerobic life, and the classic view is that it diffuses freely across the plasma membrane. However, measurements of O(2) permeability of lipid bilayers have indicated that it is much lower than previously thought, and therefore, the existence of membrane O(2) channels has been suggested. We hypothesized that, besides its role as a water channel, aquaporin-1 (AQP-1) could also work as an O(2) transporter, because this transmembrane protein appears to be CO(2)-permeable and is highly expressed in cells with rapid O(2) turnover (erythrocytes and microvessel endothelium). Here we show that in mammalian cells overexpressing AQP-1 and exposed to hypoxia, the loss of cytosolic O(2), as well as stabilization of the O(2)-dependent hypoxia-inducible transcription factor and expression of its target genes, is accelerated. In normoxic endothelial cells, knocking down AQP-1 produces induction of hypoxia-inducible genes. Moreover, lung AQP-1 is markedly up-regulated in animals exposed to hypoxia. These data suggest that AQP-1 has O(2) permeability and thus could facilitate O(2) diffusion across the cell membrane.
Highlights
(O2/CO2) turnover such as erythrocytes [5] and microvessel endothelium [6, 7], and experiments performed with recombinant AQP-1 expressed in Xenopus oocytes have suggested that it confers upon the cells increased membrane CO2 permeability (8 –10)
We showed that HAQP cells responded more rapidly to an hyposmotic challenge than LAQP cells (Fig. 2B), indicating that recombinant AQP-1 expressed in the PC12 cell membrane was functional as a water-permeable channel
The results in this study provide evidence that AQP-1 can accelerate the establishment of cytosolic hypoxia possibly through facilitation of O2 transport across the plasma membrane
Summary
Cell Culture, Transfections, siRNA, and in Vitro Hypoxic Treatments—Rat cDNAs for AQP-1 and AQP-3 were cloned into pcDNA3 (Invitrogen). From 40 clones analyzed, 20 were positive for either AQP-1 or AQP-3 with variable levels of expression. Hypoxia experiments performed to analyze mRNA levels of tyrosine hydroxylase (TH), phosphoglycerate kinase 1 (PGK1), or vascular endothelial growth factor (VEGF) were done on a cell incubator with 1–10% O2 (CO2 10%) [17]. MRNA expressed in the various PC12 cell clones permanently transfected with either AQP-1 or AQP-3 gene. The black symbol indicates the value in wild type PC-12 cells, which do not express either AQP-1 or AQP-3. D, hypoxic (6% O2 for 12 h) TH mRNA expression as a function of the amount of AQP-1 protein detected by Western blot in the different cell clones. Statistical Analysis—Data were presented as mean Ϯ S.E. and were analyzed with either paired Student’s t test or the one-way analysis of variance followed by Tukey’s test
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