Increase in intracellular Ca2+ and calcitonin gene-related peptide release through metabotropic P2Y receptors in rat dorsal root ganglion neurons

doi:10.1016/S0306-4522(02)00005-2

Neuroscience

Volume 111, Issue 2, 10 May 2002, Pages 413-422

https://doi.org/10.1016/S0306-4522(02)00005-2 Get rights and content

Abstract

We examined the effects of the activation of metabotropic P2Y receptors on the intracellular Ca²⁺ 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 Ca²⁺ concentration, while the removal of extracellular Ca²⁺ partly depressed the response to ATP, thus suggesting that the ATP-induced increase in intracellular Ca²⁺ concentration is due to both the release of Ca²⁺ from intracellular stores and the influx of extracellular Ca²⁺. Bath application of uridine 5′-triphosphate (UTP; 100 μM) also caused an increase in intracellular Ca²⁺ 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 Ca²⁺ elevation in response to UTP is mediated through metabotropic P2Y receptors. This intracellular Ca²⁺ increase was abolished by pretreating the neurons with thapsigargin (100 nM), suggesting that the UTP-induced increase in intracellular Ca²⁺ is primarily due to the release of Ca²⁺ from endoplasmic reticulum Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ concentration and subsequent release of neuropeptide CGRP in rat dorsal root ganglion neurons.

Section snippets

Preparation and culture of rat DRG neuron

DRG neurons were obtained from the L4-5 segments of male Sprague–Dawley rats (8–14 weeks, Japan SLC, Japan) using an enzymatic dissociation procedure similar to that described previously (Sango et al., 1991). Rats were anaesthetized by an intraperitoneal injection of sodium pentobarbital (50 mg/kg) and then were killed by decapitation. Four lumbar dorsal root ganglia were quickly dissected and transferred to a 35-mm plastic tissue culture dish containing Ham’s F12 medium (Gibco BRL, Grand

Size distribution of rat DRG neurons

We first characterized the size distribution of the rat DRG neurons prepared using the present enzymatic dissociation procedures. The diameter of dissociated DRG neurons, determined as the average lengths of the longest and the shortest axes of each cell body, ranged from 15 to 60 μm with a mean value of 33.1±0.7 μm (mean±S.E.M., n=206 neurons; Table 1). Based on the criteria proposed in previous studies (Caffrey et al., 1992), we classified these DRG neurons into three subpopulations; small-

Discussion

In the present study we characterized the [Ca²⁺]_i response to extracellular ATP and UTP in adult rat small-sized DRG neurons. Extracellular ATP evokes an increase in [Ca²⁺]_i in fura-2-loaded small-sized DRG neurons, consisting of both the release of Ca²⁺ from internal stores and the influx of Ca²⁺ (Fig. 1 and Table 2). The ionotropic P2X receptors elevate [Ca²⁺]_i by increasing membrane permeability for Ca²⁺ (Ralevic and Burnstock, 1998), thus extracellular Ca²⁺ is essential for their actions.

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Increase in intracellular Ca2+ and calcitonin gene-related peptide release through metabotropic P2Y receptors in rat dorsal root ganglion neurons

Abstract

Section snippets

Preparation and culture of rat DRG neuron

Size distribution of rat DRG neurons

Discussion

Neuroscience

Curr. Opin. Neurobiol.

Brain Res.

J. Biol. Chem.

Brain Res.

Neurosci. Lett.

J. Biol. Chem.

Neuroscience

Neuron

Neurosci. Lett.

Neuroscience

Curr. Opin. Neurobiol.

Pain

Neurosci. Lett.

Biochem. Biophys. Res. Commun.

Brain Res.

FEBS Lett.

Depolarization stimulates initial calcitonin gene-related peptide expression by embryonic sensory neurons in vitro

J. Neurosci.

Peripheral and spinal mechanisms of nociception

Pharmacol. Rev.

Molecular cloning and characterization of rat P2Y4 nucleotide receptor

Br. J. Pharmacol.

Calcium influx induced by stimulation of ATP receptors on neurons cultured from rat dorsal root ganglia

Eur. J. Neurosci.

New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor

Nature

An antagonist-insensitive P2X receptor expressed in epithelia and brain

EMBO J.

Increase in intracellular Ca²⁺ and calcitonin gene-related peptide release through metabotropic P2Y receptors in rat dorsal root ganglion neurons

Molecular cloning and characterization of rat P2Y₄ nucleotide receptor