Localization of functional calcitonin gene-related peptide binding sites in a subpopulation of cultured dorsal root ganglion neurons.

In this study we investigated whether cultured dorsal root ganglion (DRG) neurons from the adult rat express binding sites for calcitonin gene-related peptide (CGRP). These were identified on fixed cells by using CGRP labeled at the N-terminal site with 1.4-nm gold particles. After 1 day in culture, about 20% of small to medium-sized DRG neurons showed CGRP-gold binding. Binding of CGRP-gold was dose-dependently reduced by coadministration of CGRP. The calcium imaging technique in living cells revealed that the bath administration of CGRP evoked an increase of the intracellular calcium in up to 30% of the DRG neurons tested. Both depletion of intracellular calcium stores by thapsigargin or using a calcium-free medium blocked the CGRP-mediated increase of cytosolic calcium in most neurons. Thus intracellular and extracellular sources of calcium are relevant for the CGRP response. Using the whole-cell patch-clamp technique, about 30% of the neurons were found to exhibit an inward current and a depolarization upon administration of CGRP close to the neurons. Immunocytochemical double-labeling techniques showed that most of the CGRP-gold binding sites were expressed in unmyelinated (neurofilament 200-negative) DRG neurons. Most of the neurons with CGRP-gold binding sites also expressed the tyrosine kinase A receptor, and all of them showed CGRP-like immunoreactivity. This study shows, therefore, that a subpopulation of unmyelinated, peptidergic primary afferent neurons express CGRP binding sites that can be activated by CGRP in an excitatory direction. The binding sites may serve as autoreceptors because all of these neurons also synthesize CGRP. The activation of CGRP binding sites may sensitize primary afferent neurons and influence the release of mediators.