Extracellular ATP is via P2 receptors a significant modulator of the epithelial transport. Nucleotides are constitutively released by renal epithelial cells. Stimulation of the renal epithelium either mechanically or by various receptor-agonists provokes the cells to release additional ATP. Nucleotide release has been suggested to occur either via a conductive pore or by exocytosis. The aim of the present study is to define the nucleotide release pathway in renal epithelial cells.

Methods: 

We use various techniques to determine the ATP release pathway in renal epithelia using MDCK cells as a model system: (1) We have established that spontaneous [Ca²⁺]_i increments in renal epithelia reflect nucleotide release. The spontaneous [Ca²⁺]_i increments is monitored in Fluo-4 loaded MDCK cells. We used this method to screen for the most likely nucleotide release pathways. (2) Live cell imaging of quinacrine stained vesicles in MDCK cells (4) Luciferin-luciferase lumeometry.

Results: 

Inhibition of the H⁺-ATPase with Bafilomycin (1 mM) reduced the spontaneous nucleotide release with 78.8%. Bafilomycin also diminished the hypotonically (80%)- and the flow-induced [Ca²⁺]_i-response by 67.3% and 61.8% respectively. Interference with vesicular fusion by N-ethylamide (100 mM) dramatically lowered the spontaneous nucleotide release (88.5%) and the flow induced [Ca²⁺]_i-response (63.3%). The Golgi apparatus inhibitor brefeldin A (10 mg ml^-1) and the microfilament inhibitor cytochalasin B (50 mM) did not affect neither the spontaneous nucleotide release nor the flow-induced [Ca²⁺]_i-response. Neither did inhibition of gap-junction by 8ß-glucoronic acid, flufenamic acid, mefloquine and carbanoxolone.

MDCK cells loaded with quinacrine, a dye known to stain nucleotide-enriched granules, showed a distinct vesicular staining pattern. In time laps studies on living cells these vesicles moved vigorously around in the cytoplasm. After mechanical- (perfusate flow increase) or agonist- (ATP) stimulation the vesicle movements ceases and the overall fluorescence decreased with 5.8% and 7.6% respectively, from a very stable baseline fluorescence. This decrease likely reflects release of the content of quinacrine labelled vesicles. In summary, these data support spontaneous and mechanically induced ATP release occurs via exocytosis in renal epithelia.