Mechanisms Underlying NK-1 Activation-induced Potentiation Of TRPV1 And TTX-r Na Channel In Nociceptor

Chemical (capsaicin and acid) or physical (heat) variety can excite nociceptors, enabling the cell to generate action potentials which propagate to the dorsal horn of spinal cord after primary integration. The primary afferent nociceptive neurons as the first order neurons play a key role in nociception.Substance P (SP), which mainly exists in a subtype of small Dorsal root ganglion (DRG) neurons, is a kind of mammalian tachykinin. After synthesis, it is packed into the dense vesicles and transferred to the central and peripheral terminals. At the central terminals, the SP positive nerves terminate in the lamina I of spinal horn, and pass the nociceptive information to the spinal neurons. At the peripheral, SP is released as an inflammatory modulator, sensitizing the nociceptive perception. There are three kinds of tachykinin receptors, NK-1, NK-2 and NK-3, SP has the highest affinity to NK-1. When activated, it subsequently stimulates PLC-catalysed hydrolysis of PtdIns(4,5)P₂, consequently releasing Ca²⁺ from intracellular stores and activating protein kinase C (PKC).In peripheral, abundant NK-1 receptors existed in mast cells but whether the NK-1 autoreceptors in nociceptors existing remains controversy. Our previous work revealed that SP induced the thermal hyperalgesia without the activation of mast cells, which indicated a direct effect of SP on nociceptors.Using immunohistochemistry, western blot, whole cell patch clamp, calcium imaging and pain behavioral test, we for the first time characterized the NK-1 autoreceptors in primary sensory neurons.1. The existence of NK-1 in DRGEvidence of immunohistochemistry and western blot revealed a wide distribution of NK-1 receptors in primary sensory neurons and co-existent with SP and TRPV1, which significant up-regulated after CFA-induced inflammation. There are two key channels in thermal transduction and propagation, TRPV1 and the TTX-resistant sodium channels, which indicates that SP sensitizes nociceptors through the interaction of NK-1 with TRPV1 receptors and the TTX-resistant sodium channels.2. The "crosstalk" between NK-1 and TRPV1a) SP potentiates TRPV1 responsesSP potentiated TRPV1 responses through NK1 receptors. TRPV1 is a non-selective cation channel, which is activated by capsaicin or noxious heat stimuli. By the methods of whole cell patch clamp and calcium imaging, an inward non-selective cationic current and a transient increment of intracellular calcium concentration were elicited by capsaicin. 75.8% of recording neurons responded to capsaicin, in which, 49.6% responded to SP (1μM). The mean potentiation of TRPV1 responses were 409.12±44.54% of control. The effect of SP was mimicked by a selective NK-1 agonist [Sar⁹, Met(O₂)11]SP (Sar-SP, 1μM) and blocked by the specific NK-1 antagonists, WIN51,708 (5μM) and GR82,334 (1μM), while the NK-2 and NK-3 antagonists, L659, 187 (1μM) and SR-142, 801 (1μM) failed to block the effect of SP.b) Cellular mechanism of NK-1The effect of Sar-SP was also abolished by the antagonist of G protein, GDP-β-s (1 mM intracellular), the inhibitors of PKC, BIM (1μM) and Chel.C (5μM) and the PKCs specific inhibitor,εV1-2 (200μM, intracellular), but not the PKA inhibitor, H89 (1μM), pre-treatment with Phorbol 12-myristate 13-acetate (PMA, 0.6μM), the activator of PKC, prevented the further enhancement of TRPV1 response, all these results suggested a new signaling pathway from NK-1 to TRPV1 through PKCεphosphorylation.3. SP potentiates TTX-resistant sodium channels sensitivityThere are two TTX-resistant sodium channels subtypes, Nav1.8 and Nav1.9, specifically expressed in small DRG neurons, with distinct characters of activation and inactivation. By method of whole cell patch clamp, we successfully separated two kinds of TTX-resistant sodium currents, supposed to be induced by Nav1.8 and Nav1.9 seperatelly. Sar-SP potentiated both the Nav1.8 and Nav1.9 currents. a) Nav1.8Of 22 DRG neurons with Nav1.8 currents, 9 responded to Sar-SP (1μM), with a rapid increase in the peak of Nav1.8 currents (123.52±2.83%) and a hyperpolarizing shift of both the active and steady-state inactive curve.b) Nav1.9Of 20 DRG neurons with Nav1.9 currents, 7 responded to Sar-SP (1μM) with also an increase in Nav1.9 currents (110.27±2.33%), suggesting a role of Sar-SP on enhancement of cellular excitability.The present work not only supports the expression of NK-1 autoreceptors in nociceptors, but also strongly indicates a novel positive feedback signaling pathway through NK- 1 autoreceptor in processing peripheral nociceptive information.