Sensory neurons can be activated by a variety of endogenous chemical mediators. Many of these mediators including ATP, serotonin (5-HT) and histamine are released from mast cells following an inflammatory stimulus. ATP and 5-HT are known pain-producing substances while histamine elicits either pain or itch, depending on the site of stimulation. While histamine has been shown to act via the H1 histamine receptor (Nicolson et al. 2002), the receptor subtypes responding to ATP and 5-HT remain unclear. Here we describe some experiments in which we have used calcium imaging to investigate the receptor subtypes involved in these responses.
Adult Wistar rats (200-250 g) were killed humanely by a rising CO2 atmosphere. Sensory neurones were subsequently isolated from the dorsal root ganglion and maintained in Ham’s F14 medium supplemented with nerve growth factor for 2-3 days as described previously (Bevan & Winter, 1995). The ratiometric dye fura-2 was used to estimate intracellular free calcium and the emitted fluorescence was collected with a cooled CCD system (see Nicolson et al. 2002). All experiments were performed at room temperature (21-25 °C).
In agreement with the data of Nicolson et al. (2002), the proportion of neurons responding to histamine with a rise in intracellular calcium was concentration dependent, with 32 % (n = 74/233) of neurons responding to 10 mM histamine. More than half of the sensory neurons (71 %, n = 66/93) responded to ATP (30 µM). The P2Y agonist 2-methyl thio ATP also elicited responses in a significant proportion of the sensory neuron population (72 %, n = 91/126). The similarity of the time course and percentage of cells responding to ATP in 0 mM Ca2+ together with the activation of neurons by 2-methyl thio ATP suggests the involvement of a P2Y receptor. Approximately a fifth (17 %, n = 56/333) of sensory neurons responded to 5-HT (100 µM). The percentage of cells that responded to 100 µM 5-HT in 0 mM Ca2+ (18 %, n = 17/93) is unchanged, implying that the 5-HT response is mediated via 5-HT2 receptors.
In conclusion, it appears that the calcium responses elicited by 5-HT and ATP in rat sensory neurons are primarily due to activation of G protein-coupled receptors rather than ligand-activated ion channels.
A.F.F. is in receipt of a MRC collaborative studentship.