Released nucleotides amplify the cilium-dependent, flow-induced [Ca2+]i response in MDCK cells.
OBJECTIVE
Changes in perfusate flow produce increases in [Ca(2+)](i) in renal epithelial cells. Cultured renal epithelia require primary cilia to sense subtle changes in flow. In perfused kidney tubules this flow response is caused by nucleotide signalling via P2Y(2) receptors. It is, however, not known whether nucleotides are released by mechanical stress applied to renal primary cilia. Here we investigate whether nucleotides are released during the cilium-dependent flow response and contribute to the flow-induced, cilium-dependent [Ca(2+)](i) signal.


METHODS
MDCK cells loaded with Fluo-4-AM were observed at 37 degrees C in semi-open single or closed-double perfusion chambers.


RESULTS
Our data suggest a purinergic component of the cilium-dependent flow-response: (1) ATP scavengers and P2 receptor antagonists reduced (55%) the cilium-dependent flow-response; (2) ATP added at subthreshold concentration sensitized the renal epithelia to flow changes; (3) increases in fluid flow transiently enhanced the ATP concentration in the superfusate (measured by biosensor-cells). To test if nucleotides were released in sufficient quantities to stimulate renal epithelia we used non-confluent MDCK cells without cilia as reporter cells. We confirmed that non-confluent cells do not respond to changes in fluid flow. Placing confluent, ciliated cells upstream in the in-flow path of the non-confluent cells made them responsive to fluid flow changes. This phenomenon was not observed if either non-confluent or de-ciliated confluent cells were placed upstream. The [Ca(2+)](i)-response in the non-confluent cells with ciliated cells upstream was abolished by apyrase and suramin.


CONCLUSIONS
This suggests that subtle flow changes sensed by the primary cilium induces nucleotide release, which amplifies the epithelial [Ca(2+)](i)-response.
Department of Physiology and Biophysics, Aarhus University, Aarhus, Denmark. hp@fi.au.dk
H A
HA
Praetorius
J
J
Leipziger
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001081:Apyrase / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D002923:Cilia / Q000187:drug effects / Q000378:metabolism*; D004285:Dogs; D009711:Nucleotides / Q000378:metabolism*; D011673:Pulsatile Flow / Q000502:physiology*; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D013498:Suramin / Q000494:pharmacology
D009711:Nucleotides; D018048:Receptors, Purinergic P2; D013498:Suramin; D000255:Adenosine Triphosphate; D001081:Apyrase