Mamenko M. Opioid modulation of P2X receptors in rat sensory neurones

Modulation of P2X receptors by opioids was studied in rat primary sensory neurones. Electrophysiological recordings were performed using conventional whole-cell patch clamp method in combination with concentration clamp technique. P2X-mediated currents were recorded in neuronal cell bodies isolated from dorsal root and nodose ganglia. It was shown that opioid receptors are functionally coupled with P2X receptors in rat sensory neurones. In the majority (80%) of tested nodose neurones opioids modulated P2X-mediated currents in a biphasic manner: the initial transient phase of potentiation (lasting for 2-3 min) was followed by long-lasting inhibition of the response, which reached its steady-state level within the next 8-10 min. At saturating concentrations opioids augmented ATP-activated currents by 51-60% of their initial amplitude during the first 2-3 min of application. Then the currents were inhibited by 39-52% of their control amplitude within the next 10 mins. In the rest 20% of tested neurones we observed monophasic inhibition of ATP-activated currents by 46-52% of their initial amplitude. In this case opioid-induced inhibition reached its steady-state level within 10 min. Desensitization kinetics of ATP-activated currents varied from cell to cell. Opioid modulation of P2X currents did not depend on their desensitization kinetics. The observed effects were similar for a variety of used opioids: Leu-enkephalin, DAMGO, endomorphin-1. Endomorphin-1 was more effective at lower concentrations indicating that the action of opioids could be mediated by -opioid receptors. Opioids could not alter ATP-activated currents when applied on the background of competitive antagonist of opioid receptors - naloxone. Naloxone itself did not affect ATP-induced responses in nodose neurones. Addition of GTP S (2 M) to the intracellular solution resulted in partial inhibition of P2X2/3-mediated currents and opioids were not effective against the residual response. When GDP (200 M) was added to the intracellular solution instead of GTP, opioids could not alter P2X2/3-mediated currents. Preincubation with pertussis toxin (200 ng/ml) abolished only the inhibition phase. Therefore, P2X2/3 receptors in rat nodose ganglion neurones were affected by opioids via several G-protein dependent pathways. Opioid receptor agonists partially inhibited P2X3-mediated currents in dorsal root ganglion neurones. Application of morphine decreased the amplitudes of ATP-activated currents in a wide concentration range with IC50 = 8.5 nМ, indicating that the effect of morphine on P2X3 receptors is mediated by -opioid receptors. At saturating concentrations (10 M) morphine inhibited ATP-induced current by 55% at the average. Morphine also suppressed the currents activated by , -Ме-АТP by 45% at the average, indicating that P2Y receptors are not involved in the observed inhibitory effects. Leu-enkephalin and its non-hydrolysable analogue D-Leu-enkephalin inhibited ATP-activated P2X3-mediated currents by 36% at the average. The inhibitory action of Leu-enkephalin was blocked by naloxone. When 10 M Leu-enkephalin were applied on the background of 50 M naloxone, the former opioid did not inhibit ATP-evoked current. Naloxone itself did not affect P2X3-mediated responses in dorsal root ganglion neurones. Application of 40 nM phorbol ester (phorbol-12-myristate-13-acetate) mimicked the effects induced by opioids, suggesting that their action is mediated via G-protein dependent pathway with protein kinase C involved. In order to elucidate possible mechanisms, by which cancer cells can interact with nociceptors and alter their excitability, we used a recently developed in vitro model, in which mice fibrosarcoma cells (NCTC 2472 cell line) were co-cultured with rat nodose neurones (on separate glass cover slips). We have tested the possibility that cancer cells affect the sensitivity of P2X receptors in nodose neurones to endomorphin-1. It was found that in co-cultured neurones inhibition of ATP-activated currents by endomorphin-1 was inversely dependent on their desensitization kinetics. The currents with slower desensitization kinetics were less sensitive to the inhibitory effect of the opioid. Sensitivity of "ultra-slow" responses to endomorphin-1 was completely abolished. The number of neurones with slowly desensitizing P2X-mediated currents increased with the duration of co-culturing. The obtained data indicate that diffusible tumour-derived factors decrease sensitivity of ATP responses to endomorphin-1 in nodose neurons co-cultured with fibrosarcoma cells. Key words: sensory neurones, P2X receptors, opioid receptors, modulation, G-proteins, co-culture model, fibrosarcoma.