Characteristics of P2X7-like receptor activated by adenosine triphosphate in HIT-T15 cells.

OBJECTIVES

The study examined the presence of a P2X7 receptor subtype and its functional roles in pancreatic beta cells.

METHODS

In a hamster beta-cell line, HIT-T15 cells, purinergic stimulation was investigated using fluorometry, electrophysiology, flow cytometry, and electrophoresis.

RESULTS

Adenosine triphosphate (ATP) and 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) increased in the intracellular free Ca2+ concentration, with an EC50 of 398.0 and 136.6 microM, respectively. Preincubation with oxidized ATP, a P2X7 receptor antagonist, inhibited the ATP- and BzATP-induced increase in the intracellular Ca2+ level. The BzATP-induced increase in the intracellular Ca2+ level was dependent on the extracellular Ca2+ concentration. The extracellular Mg2+ had a significant effect on the ATP-induced increase in the intracellular Ca2+ level. The ATP also induced depolarization like high potassium chloride. In the voltage-clamp experiments, ATP evoked inward currents, which were reversed at almost 0 mV. The ATP stimulated the slow influx of ethidium bromide, indicating permeability to larger molecules. Flow cytometry showed that the number of hypodiploid cells (A0), which are indicative of apoptosis, increased when the cells were exposed to ATP for 24 hours. The ATP also induced DNA fragmentation.

CONCLUSIONS

These results suggest that the HIT-T15 cells have endogenous P2X7-like receptors and that purinergic stimulation increased the level of intracellular Ca2+, depolarization, inward current, permeability, and apoptosis.