Increase in intracellular Ca 2+ and calcitonin gene-related peptide release through metabotropic P2Y receptors in rat dorsal root ganglion neurons

Abstract

We examined the effects of the activation of metabotropic P2Y receptors on the intracellular Ca 2+ concentration and the release of neuropeptide calcitonin gene-related peptide (CGRP) in isolated adult rat dorsal root ganglion neurons. In small-sized dorsal root ganglion neurons (soma diameter<30 μm) loaded with fura-2, a bath application of ATP (100 μM) evoked an increase in intracellular Ca 2+ concentration, while the removal of extracellular Ca 2+ partly depressed the response to ATP, thus suggesting that the ATP-induced increase in intracellular Ca 2+ concentration is due to both the release of Ca 2+ from intracellular stores and the influx of extracellular Ca 2+. Bath application of uridine 5′-triphosphate (UTP; 100 μM) also caused an increase in intracellular Ca 2+ concentration in small-sized dorsal root ganglion neurons and the P2 receptor antagonists suramin (100 μM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS; 10 μM) virtually abolished the response, indicating that the intracellular Ca 2+ elevation in response to UTP is mediated through metabotropic P2Y receptors. This intracellular Ca 2+ increase was abolished by pretreating the neurons with thapsigargin (100 nM), suggesting that the UTP-induced increase in intracellular Ca 2+ is primarily due to the release of Ca 2+ from endoplasmic reticulum Ca 2+ stores. An enzyme-linked immunosorbent assay showed that an application of UTP (100 μM) significantly stimulated the release of CGRP and that suramin (100 μM) totally abolished the response, suggesting that P2Y receptor-mediated increase in intracellular Ca 2+ is accompanied by CGRP release from dorsal root ganglion neurons. These results suggest that metabotropic P2Y receptors contribute to extracellular ATP-induced increase in intracellular Ca 2+ concentration and subsequent release of neuropeptide CGRP in rat dorsal root ganglion neurons.