Abstract
Activation of vanilloid receptor (VR1) by protein kinase C (PKC) was investigated in cells ectopically expressing VR1 and primary cultures of dorsal root ganglion neurons. Submicromolar phorbol 12,13-dibutyrate (PDBu), which stimulates PKC, acutely activated Ca(2+) uptake in VR1-expressing cells at pH 5.5, but not at mildly acidic or neutral pH. PDBu was antagonized by bisindolylmaleimide, a PKC inhibitor, and ruthenium red, a VR1 ionophore blocker, but not capsazepine, a vanilloid antagonist indicating that catalytic activity of PKC is required for PDBu activation of VR1 ion conductance, and is independent of the vanilloid site. Chronic PDBu dramatically down-regulated PKC(alpha) in dorsal root ganglion neurons or the VR1 cell lines, whereas only partially influencing PKCbeta, -delta, -epsilon, and -zeta. Loss of PKC(alpha) correlated with loss of response to acute re-challenge with PDBu. Anandamide, a VR1 agonist in acidic conditions, acts additively with PDBu and remains effective after chronic PKC down-regulation. Thus, two independent VR1 activation pathways can be discriminated: (i) direct ligand binding (anandamide, vanilloids) or (ii) extracellular ligands coupled to PKC by intracellular signaling. Experiments in cell lines co-expressing VR1 with different sets of PKC isozymes showed that acute PDBu-induced activation requires PKC(alpha), but not PKC(epsilon). These studies suggest that PKC(alpha) in sensory neurons may elicit or enhance pain during inflammation or ischemia.
Highlights
From the Neuronal Gene Expression Unit, Pain and Neurosensory Mechanisms Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892
The vanilloid receptor type 1 (VR1)1 is highly expressed in the small size neurons, which are involved in transmitting noxious heat and chemical stimuli from the periphery
In contrast to the isozyme pattern detected in sensory neurons from newborns, protein kinase C (PKC)␣, an additional isozyme was positively identified in dorsal root ganglia (DRG) cultures prepared from 3-month-old rats, which may regulate VR1 in adults (25)
Summary
Experiments in cell lines co-expressing VR1 with different sets of PKC isozymes showed that acute PDBu-induced activation requires PKC␣, but not PKC⑀. These studies suggest that PKC␣ in sensory neurons may elicit or enhance pain during inflammation or ischemia. Knockout of the PKC␥ isozyme in mice suggested that this enzyme, which is normally highly expressed in specific second order neurons in spinal inner lamina II, was involved in the proper processing of incoming afferent nociceptive signals (20 –23) Another approach to identify specific isozyme participation is selective down-regulation of PKC isotypes by exposure to active phorbol esters. Our observations emphasize the conditional requirements for the actions of endovanilloids and PKC and their capacity to work in a concerted, additive fashion on the nociceptive nerve terminal
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