Electroacupuncture stimulation (EAS) is known to change brain neurotransmitter release. In the present study, we investigated the effects of synchronous or asynchronous electroacupuncture stimulation with low versus high frequency on spinal opioid release and tail flick nociception. Rats were given b2/100HzQ EAS, which stands for an asynchronous mode of stimulation, in which 2Hz was alternated with 100Hz, each lasting for 3s, or b(2+100) HzQ EAS, a mode of stimulation in which 2Hz stimulation was applied to the left hind leg simultaneously with 100 Hz stimulation on the right hind leg. The rats were subjected to the same total number of electrical stimulations in these two modes. Results were as follows: (1) 2/100Hz EAS was 40% more potent than (2+100) Hz EAS (P b 0.01) in producing an antinociceptive effect. (2) Intrathecal (i.t.) injection of the A-opioid receptor antagonist d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr amide (CTOP) blocked in a dose-dependent manner the anti-nociceptive effect produced by 2/100Hz EAS but not by (2+100)Hz EAS, whereas i.t. injection of the n-opioid receptor antagonist norbinaltorphimide (Nor-BNI) blocked the anti-nociceptive effect induced by both modes of EAS. (3) I.t. injection of endomorphin-2 antiserum blocked in a dose-dependent manner the anti-nociceptive effect of 2/100Hz EAS but not that of (2+100)Hz EAS, whereas i.t. injection of dynorphin antiserum blocked the anti-nociceptive effect induced by both modes of stimulation. (4) 2/100Hz EAS increased the release of both endomorphin-2 and dynorphin, whereas (2+100)Hz EAS increased the release of dynorphin but not of endmorphin-2. We conclude that the more potent anti-nociceptive effect induced by 2/100Hz EAS, as compared with that of (2+100)Hz EAS, was due, at least partly, to the synergistic interaction of endomorphin-2 and dynorphin in rat spinal cord.

Peripheral electrical stimulation (PES) has been utilized to manage chronic pain associated with nerve injury. However, the data on clinical effectiveness are conflicting and the neurophysiological mechanism is not well known. This study was designed to assess whether PES relieved neuropathic pain and its possible mechanisms. The neuropathic pain model was made with lumbar 5th (L5) and 6th (L6) spinal nerve ligations in rats. Nociceptive responses of the rats were assessed by the cold plate test (the number and duration of paw lifts that occurred in 5 min on a 5 F 1jC cold plate). PES with a frequency of 2Hz and at increasing strengths was given for 30 min via stainless-steel needles inserted into standard acupoints on the leg and back, respectively. Immunochemistry was used to examine the immunoreactivity of the NMDA receptor 1 (NR1) subunit in the spinal cord dorsal horn. The results are as follows: (1) PES relieved neuropathic pain and the effect was blocked by 1.0mg/kg naloxone. (2) The effect of one session of PES lasted up to 12h. (3) Repetitive PES showed a cumulative effect and no tolerance was observed. (4) There was a significant increase of NR1 immunoreactivity in the superficial laminae of the spinal cord of neuropathic pain rats as compared with naive rats. This increase could be reversed by repetitive 2Hz PES. These results suggest that PES can relieve neuropathic pain, and that A-opioid receptors and NMDA receptors are involved in the effect of PES.

The capsaicin receptor VR1 is a polymodal nociceptor activated by multiple stimuli. It has been reported that protein kinase C plays a role in the sensitization of VR1. Protein kinase D/PKC is a member of the protein kinase D serine/threonine kinase family that exhibits structural, enzymological, and regulatory features distinct from those of the PKCs, with which they are related. As part of our effort to optimize conditions for evaluating VR1 pharmacology, we found that treatment of Chinese hamster ovary (CHO) cells heterologously expressing rat VR1 (CHO/rVR1) with butyrate enhanced rVR1 expression and activity. The expression of PKC and PKC1, but not of other PKC isoforms, was also enhanced by butyrate treatment, suggesting the possibility that these two isoforms might contribute to the enhanced activity of rVR1. In support of this hypothesis, we found the following. 1) Overexpression of PKC enhanced the response of rVR1 to capsaicin and low pH, and expression of a dominant negative variant of PKC reduced the response of rVR1. 2) Reduction of endogenous PKC using antisense oligonucleotides decreased the response of exogenous rVR1 expressed in CHO cells as well as of endogenous rVR1 in dorsal root ganglion neurons. 3) PKC localized to the plasma membrane when overexpressed in CHO/rVR1 cells. 4) PKC directly bound to rVR1 expressed in CHO cells as well as to endogenous rVR1 in dorsal root ganglia or to an N-terminal fragment of rVR1, indicating a direct interaction between PKC and rVR1.5) PKC directly phosphorylated rVR1 or a longer N-terminal fragment (amino acids 1-118) of rVR1 but not a shorter one (amino acids 1-99).6) Mutation of S116A in rVR1 blocked both the phosphorylation of rVR1 by PKC and the enhancement by PKC of the rVR1 response to capsaicin. We conclude that PKC functions as a direct modulator of rVR1.