Evidence for a role of protein kinase C in the sustained activation of rat dorsal horn neurons evoked by cutaneous mustard oil application.

The intracellular mechanisms involved in the sensitisation of spinal dorsal horn neurons brought about by sustained or repeated nociceptive inputs are unknown. The present experiments addressed any role of protein kinase (PKC) in sustained nociceptive responses of rat dorsal horn neurons by: (i) ionophoretic administration of PKC inhibitors whilst recording activity evoked by repeated cutaneous application of mustard oil; and (ii) assessing subcellular translocation of PKC evoked in spinal cord by cutaneous application of mustard oil. Both marked attenuation of mustard oil-induced neuronal activity by PKC inhibitors and selective translocation of PKC in spinal cord tissue ipsilateral to mustard oil application strongly supported a critical role of PKC in sustained nociceptive responses to mustard oil.

Evidence for a role of metabotropic glutamate receptors in sustained nociceptive inputs to rat dorsal horn neurons.

Several antagonists at metabotropic glutamate (mGlu) receptors, when applied ionophoretically, inhibited the excitation of single dorsal horn neurons elicited by cutaneous administration of the C fibre-selective algogen, mustard oil. The selectivity and stereospecificity of AP3 isomers at mGlu, compared to NMDA receptors was confirmed on responses to agonists and matched by their effects on mustard oil-evoked activity.

The effects of neurokinin receptor antagonists on mustard oil-evoked activation of rat dorsal horn neurons.

Previous evidence indicated that brief nociceptive responses of neurons in laminae IV/V of both rat and cat dorsal horn are more readily inhibited by antagonists at NK2 rather than at NK1 neurokinin receptors. Further support for a role of spinal NK2 receptors in nociception has been provided from experiments assessing modulation of the nociceptive flexor reflex by tachykinins and activation of dorsal horn neurons by brief application of capsaicin to afferents. The present experiments were designed to compare the contribution of NK1 and NK2 receptors in dorsal horn to the sustained neuronal activity induced by peripheral application of the chemical algogen mustard oil (reported to be a selective activator of C afferents). In nearly all of the multireceptive laminae IV/V neurons tested, a selective NK2 receptor antagonist L 659,874 inhibited previously established mustard oil-induced activity. In contrast, two selective NK1 receptor antagonists L 668,169 and GR 82334 were only rarely effective. These results further underline the apparent importance of NK2 receptors in spinal nociceptive processing. NK1 receptors do not appear to play a major role in the present experimental protocol, but they may of course do so under different circumstances.

Inhibition by NK2 but not NK1 antagonists of carrageenan-induced preprodynorphin mRNA expression in rat dorsal horn lamina I neurons.

Previous evidence indicated that NK2 rather than NK1 receptors play a central role in mediating the electrophysiological responses of dorsal horn neurons to brief cutaneous stimuli such as noxious heat (but not noxious pinch) and moderately sustained stimuli such as mustard oil, topically applied over 10-20 min. The present experiments were designed to investigate, by in situ hybridisation histochemistry, a delayed genomic response in dorsal horn neurons (the expression of preprodynorphin mRNA induced by intraplantar carrageenan injection) and explore the role of NK1 and NK2 receptors in mediating this response. In anaesthetised rats with bilateral intraplantar injections of carrageenan, neurokinin receptor antagonists were administered unilaterally by prolonged ionophoresis into the superficial dorsal horn. The marked increase in preprodynorphin mRNA expression elicited by carrageenan was inhibited (both in terms of number of expressing cells and their level of expression) by NK2 but not NK1 antagonists.

Evidence for a role of tachykinin NK2 receptors in mediating brief nociceptive inputs to rat dorsal horn (laminae III-V) neurons.

Since the NK2 receptor-selective tachykinin, neurokinin A is present in fine primary afferent neurons in addition to the NK1 receptor-selective tachykinin, substance P, we have addressed the relative role of NK1 and NK2 receptors in somatosensory processing in spinal dorsal horn. Recording extracellularly from rat laminae III-V neurons whilst ionophoresing drugs nearby, the selective NK1 receptor antagonists L 688,169, GR 82334 and [D-Pro4,D-Trp7,910Phe11]substance P-(4-11) failed to influence neuronal responses to cutaneous pinch or noxious heat but often enhanced responses to innocuous brush. In contrast, the highly selective NK2 receptor antagonist L 659,874 profoundly inhibited responses to noxious heat but not pinch or brush. Highly selective synthetic agonists for both NK1 and NK2 receptors ([N-acetyl-Arg6,Sar9,Met(O2)11]substance P-(6-11) and GR 64349, respectively) and also NKA showed the inverse effects on sensory responses to those brought about by their antagonists. At higher ionophoretic currents, both NK1 and NK2 receptor agonists increased spontaneous activity. This increased basal firing induced by GR 64349 and neurokinin A (but not that due to [N-acetyl-Arg6,Sar9,Met(O2)11]substance P-(6-11) appeared to partially pre-empt further excitatory responses to noxious heat. It is concluded that although both NK1 and NK2 receptors can clearly mediate excitation of dorsal horn neurons, it is not NK1, but rather NK2 receptors that are important as the physiological transducer of brief thermal nociceptive inputs in this model.