Calcium sensing receptor modulates vacuolar H + ‐ATPase activity in a cell model of proximal tubule (OKP cells) (891.1)

The aim of the present study was to analyze the effects of CaSR stimulation on the activity of the vacuolar H + ‐ATPase in a cellular model of proximal tubule, OKP cells. H + ‐ATPase activity was accessed by a biochemical method. Intracellular pH (pHi) was monitored with BCECF and changes in [Ca 2+ ] i was determined using Fluo‐4 AM. A significant increase of H + ‐ATPase activity was observed when the CaSR was stimulated with agonists such as 300μM Gd 3+ and 200μM neomycin. This activity was also stimulated in a dose‐dependent fashion when Ca 2+ concentration was increased between 10 ‐4 mM and 2mM. Neomycin induced a significant alkalinization of pH i (∆pH i 0.57±0.04) that was significantly reduced in the presence of 10 ‐8 M concanamycin. Gd 3+ and neomycin produced a sustained rise of cytosolic ionized calcium (4.77±0.47 and 12.79±0.86%, respectively, p<0.0001 vs basal), an effect that disappeared when extracellular calcium was removed in the presence of 0.1μM thapsigargin. Inhibition of phospholipase C activity with U73122 (5x10 ‐8 M) reduced the increase in [Ca 2+ ] i induced by neomycin. These data allow us to conclude that CaSR stimulation of OKP cells induces an increase in vacuolar H + ‐ATPase activity. This effect involves an increase in intracellular calcium, resulting from the combination of Ca 2+ release from intracellular stores and the influx of Ca 2+ from the extracellular medium, an effect that requires PLC activity. Grant Funding Source : Supported by CAPES (Brazil)