ABSTRACT
The carotid body is the main arterial chemoreceptor in mammals that mediates the cardiorespiratory reflexes activated by acute hypoxia. Here we describe the protocols followed in our laboratory to study responsiveness to hypoxia of single, enzymatically dispersed, glomus cells monitored by microfluorimetry and the patch-clamp technique.
Subject(s)
Carotid Body/cytology , Chemoreceptor Cells/metabolism , Patch-Clamp Techniques/methods , Single-Cell Analysis/methods , Animals , Carotid Body/physiology , Cell Hypoxia , Cells, Cultured , Cytophotometry , Mice , RatsABSTRACT
Overexpression of cell membrane aquaporins (AQPs) has recently been associated with tumor formation, particularly with angiogenesis, cell migration and proliferation. Additionally, the hypoxia inducible factor (HIF) family has been extensively implicated in tumor growth and recent studies evidence interplay between AQP expression and HIF stability. Therefore, we decided to explore the effect that AQP overexpression has on the long-term stability of HIF-2α in PC12 cells exposed to chronic hypoxia, characteristic of a growing tumor. HIF-2α levels were analyzed in five PC12 clones with stable overexpression of different proteins (AQP1, AQP3, AQP5, G6PD, and GDNF), in PC12 transiently expressing G6PD or Kv4.2, and in wild-type PC12 cells. Overexpression of AQP1, 3 or 5 in PC12 cells (o-AQP-c) prevented the HIF-2α down-expression otherwise observed, after 16 h at 1% O2, in wt-PC12 and in PC12 overexpressing non-AQP proteins. Longer HIF-2α stability was also observed in o-AQP-c exposed to cobalt chloride or dimethyloxallyl glycine. Normal proteasome activity was confirmed in all clones analyzed. Levels of HIF target genes (PHD2 and 3, VEGF, and PGK1) were 2-4 fold higher in hypoxic o-AQP-c than in wt-PC12 cells, and morphological changes in colony shape together with higher cell proliferation rates were observed in all o-AQP-c. Interestingly, analysis of PHD levels under normoxia revealed lower (50%) PHD3 expression in o-AQP-c than in controls. Our results indicate that AQP overexpression in PC12 cells prolongs HIF-2α stability during chronic hypoxia, leading to higher level of induction of its target genes and likely conferring to these cells a more tumor-like phenotype.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Hypoxia/physiology , Animals , Aquaporins/biosynthesis , Aquaporins/genetics , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Hypoxia/genetics , Cell Line, Tumor , PC12 Cells , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , RatsABSTRACT
Aquaporins (AQPS) are transmembrane water channels poorly investigated in birds. Using degenerated primers and RT-PCR, we identified in kidney and gastrointestinal tract of Hubbard chickens (Gallus gallus) three fragments, corresponding to ck-AQP2, ck-AQP4, and ck-AQP5 mRNAs. Comparison of nucleotide ck-AQPs sequences to their rat and human orthologues revealed an overall identity of 75-90%. Expression in the renal and gastrointestinal systems of the three ck-AQPs mRNA was analysed by Northern assays. Transcript of ck-AQP2 was only identified in kidney. ck-AQP4 mRNA was highly expressed in brain, and to a lesser extent in kidney and stomach. ck-AQP5 mRNA was found in jejunum and ileum, and to a lesser extent in colon and lung. In situ hybridisation showed ck-AQP5 mRNA in the crypt cells of jejunum, ileum and colon, whereas it was absent from the cells lining the villi. Levels of ck-AQP5 mRNA (analyzed by Northern and in situ hybridisation assays) and protein (analysed by immunohistochemistry) decreased from the jejunum to the colon. This work confirmed the presence of AQPs in chicken, and showed that chicken and mammalian AQPs share a high degree of similarity in nucleotide sequence and tissue distribution.
