The capsaicin receptor, VR1, is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. It has been reported that ATP, one of the inflammatory mediators, potentiates the VR1 currents evoked by capsaicin or protons and reduces the temperature threshold for activation of VR1 through metabotropic P2Y 1 receptors in a protein Kinase C (PKC)-dependent pathway, suggesting the phosphorylation of VR1 by PKC. In this study, direct phosphorylation of VR1 upon application of phorbol 12-myristate 13-acetate (PMA) was proven biochemically in cells expressing VR1. An in vitro kinase assay using glutathione S-transferase fusion proteins with cytoplasmic segments of VR1 showed that both the first intracellular loop and carboxyl terminus of VR1 were phosphorylated by PKC⑀. Patch clamp analysis of the point mutants where Ser or Thr residues were replaced with Ala in the total 16 putative phosphorylation sites showed that two Ser residues, Ser 502 and Ser 800 were involved in the potentiation of the capsaicinevoked currents by either PMA or ATP. In the cells expressing S502A/S800A double mutant, the temperature threshold for activation was not reduced upon PMA treatment. The two sites would be promising targets for the development of substance modulating VR1 function, thereby reducing pain.The sensation of pain allows us to recognize injury and triggers appropriate protective responses. A specific population of primary afferent neurons called nociceptors are known to be involved in the detection of noxious thermal, mechanical, or chemical stimuli and can be distinguished by their sensitivity to capsaicin, the pungent ingredient in hot chili peppers (1-4). The capsaicin receptor VR1 1 is a nonspecific cation channel with six transmembrane domains expressed predominantly in unmyelinated C fibers and activated not only by capsaicin but also by noxious heat (with a thermal threshold Ͼ 43°C) or protons (acidification), both of which cause pain in vivo (5-9). This sensitivity of VR1 to multiple noxious stimuli might explain certain properties of so called polymodal nociceptors. Furthermore, analyses of mice lacking VR1 have shown that VR1 is essential for selective modalities of pain sensation and for tissue injury-induced thermal hyperalgesia, further suggesting a critical role for VR1 in the detection or modulation of pain (10, 11).Tissue damage associated with infection, inflammation, or ischemia produces an array of chemical mediators that activate or sensitize nociceptor terminals to elicit or exacerbate pain at the site of injury in addition to the release of the mediators from the niciceptor terminals themselves known as neurogenic inflammation. An important component of this pro-algesic response, adenosine 5Ј-triphosphate (ATP), has recently been found to potentiate the VR1 currents evoked by capsaicin or protons through metabotropic P2Y 1 receptor activation in a protein kinase C (PKC)-dependent pathway (12). In the presence of extracellular ATP, the temperature...

We have examined the role of the mouse Diaphanous-related formin (DRF) Rho GTPase binding proteins, mDia1 and mDia2, in cell regulation. The DRFs are required for cytokinesis, stress fiber formation, and transcriptional activation of the serum response factor (SRF). 'Activated' mDia1 and mDia2 variants, lacking their GTPase binding domains, cooperated with Rho-kinase or ROCK to form stress fibers but independently activated SRF. Src tyrosine kinase associated and co-localized with the DRFs in endosomes and in mid-bodies of dividing cells. Inhibition of Src also blocked cytokinesis, SRF induction by activated DRFs, and cooperative stress fiber formation with active ROCK. Our results show that the DRF proteins couple Rho and Src during signaling and the regulation of actin dynamics.

Prostaglandin E 2 (PGE 2 ) and prostaglandin I 2 (PGI 2 ) are major inflammatory mediators that play important roles in pain sensation and hyperalgesia. The role of their receptors (EP and IP, respectively) in inflammation has been well documented, although the EP receptor subtypes involved in this process and the underlying cellular mechanisms remain to be elucidated. The capsaicin receptor TRPV1 is a nonselective cation channel expressed in sensory neurons and activated by various noxious stimuli. TRPV1 has been reported to be critical for inflammatory pain mediated through PKA-and PKC-dependent pathways. PGE 2 or PGI 2 increased or sensitized TRPV1 responses through EP 1 or IP receptors, respectively predominantly in a PKC-dependent manner in both HEK293 cells expressing TRPV1 and mouse DRG neurons. In the presence of PGE 2 or PGI 2 , the temperature threshold for TRPV1 activation was reduced below 35°C, so that temperatures near body temperature are sufficient to activate TRPV1.