Selective activation of Ca²⁺ influx by extracellular ATP in a pancreatic β-cell line (HIT)

The action of exogenous ATP on cytoplasmic free Ca²⁺ ([Ca²⁺]_i) was studied in insulin secreting cells using fura-2. Stimulation of clonal pancreatic β-cells (HIT) with ATP (range 2-20 μM) evoked a sustained elevation in [Ca²⁺]_i. ATP selectively promoted Ca²⁺ influx and not Ca²⁺ mobilization since (1) the effect required external Ca²⁺ and (2) was observed in cells in which internal stores were depleted with ionomycin (3) the rate of Mn²⁺ influx, measured as the quenching of the fura-2 signal, was accelerated by ATP. The action of ATP was unaffected by the voltage-sensitive Ca²⁺ channel blockers nifedipine and verapamil as well as by a depolarizing concentration of K⁺. The effect on [Ca²⁺]_i was highly specific for ATP since AMP, ADP, adenosine 5′-[γ-thio]triphosphate, adenosine 5′-[β-γ-methylene]triphosphate, GTP and adenosine were ineffective. In normal pancreatic islet cells, both exogenous ATP (range 0.2-2 μM) and ADP induced a transient Ca²⁺ elevation that did not require external Ca²⁺. The nucleotide specificity of the effect on [Ca²⁺]_i suggests that ATP activates P_2y purinergic receptors in normal β-cells. Thus, ATP evokes a Ca²⁺ signal in clonal HIT cells and normal islet cells by different transducing systems involving distinct purinoreceptors. A novel mechanism for increasing [Ca²⁺]_i by extracellular ATP is reported in HIT cells, since the nucleotide specificity and the selective activation of Ca²⁺ influx without mobilization of internal Ca²⁺ stores cannot be explained by mechanism already described in other cell systems.