P2X3 purinoceptor cellular distribution was studied in rat sensory neurons in naive animals and following peripheral nerve injury using immunohistochemical methods. Specific antiserum was raised in rabbits and characterized by Western blot, absorption assays and labeling of recombinant receptors. In naive animals, P2X3 immunoreactivity was present predominantly in a subpopulation of small-diameter sensory neurons in dorsal root ganglia. In the spinal cord, immunoreactivity was observed in the superficial laminae of the dorsal horn. Following a chronic constriction injury to the sciatic nerve, the number of P2X3 positive small and medium diameter neurons increased in dorsal root ganglia when compared with sham-operated animals. In addition, the spinal cord immunoreactivity increased in magnitude on the side ipsilateral to the ligated nerve, consistent with up-regulation of receptors in presynaptic terminals of the primary sensory neurons.
P2 receptors have been identified in rat kidney by autoradiography, using the radioligand [3H]α,β-methylene ATP, and by immunohistochemistry, using a polyclonal antibody to the P2X1 purinoceptor. They have been localized to the vascular smooth muscle of intrarenal arteries, including arcuate and interlobular arteries, and afferent arterioles, but not glomeruli, postglomerular efferent arterioles, or renal tubules. We conclude that at least some of the P2 receptors present on vascular smooth muscle are of the P2X1 subtype. The functional significance of these findings in the vascular control of the kidney is discussed.
Background and purpose: Purinoceptors containing the P2X3 subunit (P2X3 homotrimeric and P2X2/3 heterotrimeric) are members of the P2X family of ion channels gated by ATP and may participate in primary afferent sensitization in a variety of pain-related diseases. The current work describes the in vitro pharmacological characteristics of AF-353, a novel, orally bioavailable, highly potent and selective P2X3/P2X2/3 receptor antagonist. Experimental approach: The antagonistic potencies (pIC50) of AF-353 for rat and human P2X3 and human P2X2/3 receptors were determined using methods of radioligand binding, intracellular calcium flux and whole cell voltage-clamp electrophysiology. Key results: The pIC50 estimates for these receptors ranged from 7.3 to 8.5, while concentrations 300-fold higher had little or no effect on other P2X channels or on an assortment of receptors, enzymes and transporter proteins. In contrast to A-317491 and TNP-ATP, competition binding and intracellular calcium flux experiments suggested that AF-353 inhibits activation by ATP in a non-competitive fashion. Favourable pharmacokinetic parameters were observed in rat, with good oral bioavailability (%F = 32.9), reasonable half-life (t1/2 = 1.63 h) and plasma-free fraction (98.2% protein bound). Conclusions and implications:The combination of a favourable pharmacokinetic profile with the antagonist potency and selectivity for P2X3 and P2X2/3 receptors suggests that AF-353 is an excellent in vivo tool compound for study of these channels in animal models and demonstrates the feasibility of identifying and optimizing molecules into potential clinical candidates, and, ultimately, into a novel class of therapeutics for the treatment of pain-related disorders.
P2X receptors have been suggested to play a role in the transduction of sensory signals such as pain and sound. In the present study, polyclonal antibodies against P2X1 to P2X6 receptors were used to localize P2X receptors in circumvallate and fungiform papillae of rats. Nerve fibres innervating the taste buds stained intensely with P2X3 receptor antibodies. P2X3 receptor-positive nerves were observed in the intra- and subgemmal regions. The nerve fibres were also stained with P2X2 receptor antibodies, but the intensity was much lower. The distribution of P2X2 receptor immunoreactivity overlaps with that of P2X3. These results suggest that ATP might be a neurotransmitter in taste reception cells in the taste buds, where it transducts the taste signals to the afferent taste nerves by activating P2X receptors at the synapses. This is the first experiment indicating such a role for ATP, although supplementary functional studies are required.
Polyclonal antisera generated to peptides corresponding to the carboxy termini of the seven cloned rat P2X receptors were compared using membranes from CHO-K1 or 1321N1 cells expressing recombinant rat or human P2X receptors (rat P2X 3 , P2X 4 , and P2X 5 and human P2X 1 , P2X 2 , P2X 6 , and P2X 7 ). Preimmune sera failed to recognize any bands in immunoblots against the membrane fractions. Antiserum to the P2X 1 receptor recognized a band of approximately 58 kDa while those to the rP2X 2 and the rP2X 3 receptors recognized doublets of approximately 60 and 64 kDa. The antiserum to the rP2X 4 receptor recognized a similarly sized doublet as well as a higher molecular weight species at the expected size for a receptor dimer. Antiserum to the rP2X 5 receptor revealed a single band at 64 kDa while antiserum to rP2X 6 and rP2X 7 gave single bands at 50 kDa and 95 kDa, respectively. In all cases, immunoreactivity was eliminated by preincubation of the antisera with the cognate peptide and there was no cross-reactivity of antisera with heterologous receptors. These antisera are useful reagents for the analysis of the P2X receptor subtypes in native tissues as well as for measuring receptor expression levels in transfected cell lines. Drug Dev.
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