P2X purinoceptors in cultured myenteric neurons of guinea-pig small intestine

1. Fast excitatory postsynaptic currents (fEPSCs) and responses to exogenously applied purinoceptor agonists were studied in primary cultures of myenteric neurons from guinea-pig small intestine. Whole-cell and outside-out configurations of the patch clamp technique were used. Hexamethonium (100 mu M) partly inhibited fEPSCs in 28% of neurons. Hexamethonium-resistant fEPSCs were inhibited by 97 +/- 2% by the P2X receptor antagonist, pyridoxal-phosphate-6-azophenyl-2'4'-disulphonic acid (PPADS, 10 mu M). 2. ATP caused two types of inward currents. In 92% of neurons (n = 123), ATP caused a slowly desensitizing current that declined with a double exponential time course (tau(1) = 7.1 +/- 2.0 s; tau(2) = 57 +/- 7.4 s, n = 4). The rank order potency for purinoceptor agonists in these neurons was ATP > 2-methylthio-ATP (2-MeSATP) >> alpha,beta-methylene ATP (alpha,beta-meATP) > beta,gamma-meATP > ADP. The EC(50) values for ATP and 2-MeSATP were 40 and 65 mu M, respectively. alpha,beta-MeATP acted as a partial agonist at these receptors. In 8% of neurons (n = 11), ATP-induced currents desensitized rapidly with a double exponential time course (tau(1) = 0.13 +/- 0.015 s; tau(2) = 2.2 +/- 1.3 s, n = 4); alpha,beta-meATP caused similar responses in these cells. Both types of ATP-induced current were associated with an increased conductance and an inwardly rectifying I-V relationship (E(rev) = 10 mV). Halving [Na+](o) shifted the reversal potential of ATP currents by -22 +/- 6 mV. 3. ATP activated single channel currents in outside-out patches. The single channel I-V relationship was linear between -120 and 60 mV (E(rev) approximate to 0 mV). Single channel conductance between -100 and -60 mV ws 25 +/- 2 pS. Single channel open probability was voltage dependent and decreased from 0.05 +/- 0.01 at -100 mV to 0.007 +/- 0.002 at +40 mV. 4. These data show that P2X purinoceptors mediate some fEPSCs in cultured myenteric neurons. Myenteric neurons express the fast-desensitizing alpha,beta-meATP-sensitive subtype of P2X receptor that has the properties of cloned P2X(1) receptors and is similar to native receptors in smooth muscle cells. Myenteric neurons also express a P2X receptor that desensitized slowly as was alpha,beta-meATP-insensitive. This receptor has the properties of cloned P2X(2) or P2X(5) receptors and is similar to native receptors found in PC-12 cells and superior cervical ganglion neurons. The known distribution of P2X(2) and P2X(5) receptors suggests that myenteric neurons are likely to express predominately P2X(2) receptors.