Morphology of P2X3‐immunoreactive nerve endings in the rat tracheal mucosa

Nerve endings with immunoreactivity for the P2X3 purinoreceptor (P2X3) in the rat tracheal mucosa were examined by immunohistochemistry of whole‐mount preparations with confocal scanning laser microscopy. P2X3 immunoreactivity was observed in ramified endings distributed in the whole length of the trachea. The myelinated parent axons of P2X3‐immunoreactive nerve endings ramified into several branches that extended two‐dimensionally in every direction at the interface between the epithelial layer and lamina propria. The axonal branches of P2X3‐immunoreactive endings branched off many twigs located just beneath the epithelium, and continued to intraepithelial axon terminals. The axon terminals of P2X3‐immunoreactive endings were beaded, rounded, or club‐like in shape and terminated between tracheal epithelial cells. Flat axon terminals sometimes partly ensheathed neuroendocrine cells with immunoreactivity for SNAP25 or CGRP. Some axons and axon terminals with P2X3 immunoreactivity were immunoreactive for P2X2, while some terminals were immunoreactive for vGLUT2. Furthermore, a retrograde tracing method using fast blue (FB) revealed that 88.4% of FB‐labeled cells with P2X3 immunoreactivity originated from the nodose ganglion. In conclusion, P2X3‐immunoreactive nerve endings in the rat tracheal mucosa have unique morphological characteristics, and these endings may be rapidly adapting receptors and/or irritant receptors that are activated by mucosal irritant stimuli.     

The reduction of intraepidermal P2X3 nerve fiber density correlates with behavioral hyperalgesia in a rat model of nerve injury‐induced pain

Skin biopsies from patients with neuropathic pain often show changes in epidermal innervation, although it remains to be elucidated to what extent such changes can be linked to a particular subgroup of nerve fibers and how these changes are correlated with pain intensity. Here, we investigated to what extent behavioral signs of hyperalgesia are correlated with immunohistochemical changes of peptidergic and non‐peptidergic epidermal nerve fibers in a rat model of nerve injury‐induced pain. Rats subjected to unilateral partial ligation of the sciatic nerve developed significant mechanical and thermal hyperalgesia as tested by the withdrawal responses of the ipsilateral footpad to von Frey hairs and hotplate stimulation. At day 14, epidermal nerve fiber density and total epidermal nerve fiber length/mm² were significantly and consistently reduced compared to the contralateral side, following testing and re‐testing by two blinded observers. The expression of calcitonin gene‐related peptide, a marker for peptidergic nerve fibers, was not significantly changed on the ipsilateral side. In contrast, the expression of the P2X₃ receptor, a marker for non‐peptidergic nerve fibers, was not only significantly reduced but could also be correlated with behavioral hyperalgesia. When labeling both peptidergic and non‐peptidergic nerve fibers with the pan‐neuronal marker PGP9.5, the expression was significantly reduced, albeit without a significant correlation with behavioral hyperalgesia. In conjunction, our data suggest that the pathology of the P2X₃ epidermal nerve fibers can be selectively linked to neuropathy, highlighting the possibility that it is the degeneration of these fibers that drives hyperalgesia.     

Effects of doxapram on ionic currents recorded in isolated type I cells of the neonatal rat carotid body

Whole-cell patch-clamp recordings were used to investigate the effects of the respiratory stimulant doxapram on K+ and Ca2+ currents in isolated type I cells of the neonatal rat carotid body. Doxapram (1-100 microM) caused rapid, reversible and dose-dependent inhibitions of K+ currents recorded in type I cells (IC50 approximately 13 microM). Inhibition was voltage-dependent, in that the effects of doxapram were maximal at test potentials where a shoulder in the current-voltage relationship was maximal. These K+ currents were composed of both Ca(2+)-activated and Ca(2+)-independent components. Using high [Mg2+], low [Ca2+] solutions to inhibit Ca(2+)-activated K+ currents, doxapram was also seen to directly inhibit Ca(2+)-independent K+ currents. This effect was voltage-independent and was less potent (IC50 approximately 20 microM) than under control conditions, suggesting that doxapram was a more potent inhibitor of the Ca(2+)-activated K+ currents recorded under control conditions. Doxapram (10 microM) was without effect on L-type Ca2+ channel currents recorded under conditions where K+ channel activity was minimized and was also without significant effect on K+ currents recorded in the neuronal cell line NG-108 15, suggesting a selective effect on carotid body type I cells. The effects of doxapram on type I cells show similarities to those of the physiological stimuli of the carotid body, suggesting that doxapram may share a similar mechanism of action in stimulating the intact organ.     

Arachidonic acid inhibits both K and Ca currents in isolated type I cells of the rat carotid body

Whole-cell patch-clamp recordings were used to investigate the effects of arachidonic acid (AA) on K⁺ and Ca²⁺ channels in isolated rat type I carotid body cells. AA (2–20 μM) produced a concentration-dependent inhibition of both K⁺ currents and Ca²⁺ channel currents. The effects of AA on K⁺ currents were unaffected by indomethacin (5 μM), phenidone (5 μM) or 1-aminobenzotriazole (3 mM), suggesting that AA did not exert its effects via cyclo-oxygenase, lipoxygenase or cytochrome P-450 (cP-450) metabolism. Our results suggest that AA directly and non-selectively inhibits ionic currents in rat type I carotid body cells.