Molecular mechanisms of pain signal transmission and evaluation of analgesias inn vitro

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1)Serotonin (5-HT) potentiated Ca^<2+> response to capsaicin in cultured rat dorsal root ganglion (DRG) cells expressing 5-HT_<2A> and 5-HT_7 receptors. The increased response was inhibited by the blocker of protein kinase C or protein kinaseA. 2)In isolated neonatal rat spinal cord, electrical stimulation of the L3 ventral root elicited a fast monosynaptic reflex potential (MSR) followed by very slow potential change (sVRP) in the ipsilateral L3 ventral root. The sVRP was inhibited by α2 adrenoceptor agonists, dexmedetomidine (DEX), xylazine (XY) and clonidine (CLO) or opiate receptor agonists, morphine (MOR) and DPDPE. The order of potency was DEX>DPDPE=MOR>CLO>XY. 3)MSR and sVRP were inhibited by perfusion of the isolated spinal cord with bicarbonate-buffeted physiological solution bubbled with 20% CO_2 (pH6.7). The inhibitory effect of hypercapnia was attenuated by adenosine A1 receptor antagonists. An adenosine kinase inhibitor evoked a similar inhibitory effect on MSR and sVRP. 4)Zinc selectively potentiated sVRP, which inhibited by glutamate NMDA receptor antagonists, ketamine and AP-5. 5)DEC, XY and MOR inhibited struggling body movement induced by formalin. DEC and MOR inhibited capsaicin-induced body movement but not XY, indicative of the difference in the site of action of XY. 6)We cloned cDNA coding porcine vanilloid receptor protein (2484 bases, porcine TRPV1), which was 84% homology to rat or human TRPV1. 7)TRPV1 were expressed in HEK293 cells. In these cells, capsaicin caused increases in intracellular Ca^<2+> concentration and non-selective cation currents. Porcine TRPV1 was activated by reducing extracellular pH or increasing temperature of the perfusing solution (>42℃), indicative of fiunctionin Ca2+g polymodal receptors. 8)Although responsiveness to vanilloid agonists in porcine TRPV1 was quite similar to that in rat TRPV1, the reduction of extracellular pH was more effective in porcine than rat TRPV1.