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.

Identification of T-type alpha1H Ca2+ channels (Ca(v)3.2) in major pelvic ganglion neurons.

Among autonomic neurons, sympathetic neurons of the major pelvic ganglia (MPG) are unique by expressing low-voltage-activated T-type Ca2+ channels. To date, the T-type Ca2+ channels have been poorly characterized, although they are believed to be potentially important for functions of the MPG neurons. In the present study, thus we investigated characteristics and molecular identity of the T-type Ca2+ channels using patch-clamp and RT-PCR techniques. When the external solution contained 10 mM Ca2+ as a charge carrier, T-type Ca2+ currents were first activated at -50 mV and peaked around -20 mV. Besides the low-voltage activation, T-type Ca2+ currents displayed typical characteristics including transient activation/inactivation and voltage-dependent slow deactivation. Overlap of the activation and inactivation curves generated a prominent window current around resting membrane potentials. Replacement of the external Ca2+ with 10 mM Ba2+ did not affect the amplitudes of T-type Ca2+ currents. Mibefradil, a known T-type Ca2+ channel antagonist, depressed T-type Ca2+ currents in a concentration-dependent manner (IC50 = 3 microM). Application of Ni2+ also produced a concentration-dependent blockade of T-type Ca2+ currents with an IC50 of 10 microM. The high sensitivity to Ni2+ implicates alpha1H in generating the T-type Ca2+ currents in MPG neurons. RT-PCR experiments showed that MPG neurons predominantly express mRNAs encoding splicing variants of alpha1H (called pelvic Ta and Tb, short and long forms of alpha1H, respectively). Finally, we tested whether the low-threshold spikes could be generated in sympathetic MPG neurons expressing T-type Ca2+ channels. When hyperpolarizing currents were injected under a current-clamp mode, sympathetic neurons produced postanodal rebound spikes, while parasympathetic neurons were silent. The number of the rebound spikes was reduced by 10 microM Ni2+ that blocked 50% of T-type Ca2+ currents and had a little effect on HVA Ca2+ currents in sympathetic MPG neurons. Furthermore, generation of the rebound spikes was completely prevented by 100 microM Ni2+ that blocked most of the T-type Ca2+ currents. In conclusions, T-type Ca2+ currents in MPG neurons mainly arise from alpha1H among the three isoforms (alpha1G, alpha1H, and alpha1I) and may contribute to generation of low-threshold spikes in sympathetic MPG neurons.