Trafficking and phosphorylation dynamics of AQP4 in histamine-treated human gastric cells.
BACKGROUND
AQP4 (aquaporin 4) internalization and a concomitant decrease in the osmotic water permeability coefficient (Pf) after histamine exposure has been reported in AQP4-transfected gastric HGT1 cells.


RESULTS
In the present study we report that AQP4 internalization is followed by an increase in AQP4 phosphorylation. Histamine treatment for 30 min resulted in an approx. 10-fold increase in AQP4 phosphorylation that was inhibited by 1 microM H89, a specific PKA (protein kinase A) inhibitor, but not by PKC (protein kinase C) and CK2 inhibitors. Moreover, measurement of PKA activity after 30 min of histamine treatment showed that PKA activity was approx. 3-fold higher compared with basal conditions. AQP4 phosphorylation was prevented in cells treated with histamine for 30 min after pre-incubation with PAO (phenylarsine oxide), an inhibitor of protein endocytosis. Using an endo-exocytosis assay we showed that, after histamine washed out, internalized AQP4 recycled back to the cell surface, even in cells in which de novo protein synthesis was inhibited by cycloheximide.


CONCLUSIONS
Phosphorylation experiments, combined with immunolocalization studies, indicated that AQP4 phosphorylation is mediated by PKA and occurs subsequently to its internalization in late endosomes. We suggest that phosphorylation might be a mechanism involved in retaining AQP4 in a vesicle-recycling compartment.
Carmosino
Monica
M
Department of General and Environmental Physiology, University of Bari, Via Amendola 165/A, 70126 Bari, Italy.
Procino
Giuseppe
G
Tamma
Grazia
G
Mannucci
Roberta
R
Svelto
Maria
M
Valenti
Giovanna
G
D000818:Animals; D051401:Aquaporin 4 / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000187:drug effects; D005089:Exocytosis / Q000187:drug effects; D006632:Histamine / Q000494:pharmacology*; D006801:Humans; D010295:Parietal Cells, Gastric / Q000166:cytology* / Q000187:drug effects*; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D014162:Transfection; D014721:Vesicular stomatitis Indiana virus
D051401:Aquaporin 4; D005576:Colforsin; D006632:Histamine; D017868:Cyclic AMP-Dependent Protein Kinases