Capsaicin, a pungent ingredient of hot peppers, causes excitation of small sensory neurons, and thereby produces severe pain. A nonselective cation channel activated by capsaicin has been identified in sensory neurons and a cDNA encoding the channel has been cloned recently. However, an endogenous activator of the receptor has not yet been found. In this study, we show that several products of lipoxygenases directly activate the capsaicinactivated channel in isolated membrane patches of sensory neurons. Among them, 12-and 15-(S)-hydroperoxyeicosatetraenoic acids, 5-and 15-(S)-hydroxyeicosatetraenoic acids, and leukotriene B4 possessed the highest potency. The eicosanoids also activated the cloned capsaicin receptor (VR1) expressed in HEK cells. Prostaglandins and unsaturated fatty acids failed to activate the channel. These results suggest a novel signaling mechanism underlying the pain sensory transduction.

The capsaicin-sensitive vanilloid receptor (VR1) was recently shown to play an important role in inflammatory pain (hyperalgesia), but the underlying mechanism is unknown. We hypothesized that pain-producing inflammatory mediators activate capsaicin receptors by inducing the production of fatty acid agonists of VR1. This study demonstrates that bradykinin, acting at B2 bradykinin receptors, excites sensory nerve endings by activating capsaicin receptors via production of 12-lipoxygenase metabolites of arachidonic acid. This finding identifies a mechanism that might be targeted in the development of new therapeutic strategies for the treatment of inflammatory pain. V R1, a cloned capsaicin receptor, is a nonspecific cation channel expressed preferentially in small sensory neurons and activated by the vanilloids, capsaicin and resiniferatoxin (1). Because VR1 is also activated by heat and acid (1, 2), it is now considered to be a molecular sensor that detects a variety of painful stimuli. Indeed, recent experiments performed in mice that lack VR1 demonstrated that the receptor is essential for inflammation-induced heat hyperalgesia (3, 4). Therefore, understanding the cellular mechanisms by which capsaicin receptors are activated by inflammatory mediators may be a key to identifying novel therapeutic targets for pain treatment. Because of the presence of VR1 in sensory neurons and an apparent role in inflammatory hyperalgesia, endogenous activators of VR1 have been suspected. We recently demonstrated that products of the lipoxygenase pathway of arachidonic acid (AA) metabolism can activate capsaicin receptors (5). Among the eicosanoids tested, the 12-lipoxygenase product, 12-hydroperoxyeicosatetraenoic acid (12-HPETE), structurally similar to capsaicin, was the most potent VR1 agonist. Thus, metabolic products of lipoxygenases become candidates for the endogenous capsaicinlike substances. However, the upstream signals that stimulate lipoxygenase and activate VR1 are elusive.Bradykinin (BK) is a potent inflammatory mediator that causes pain and hyperalgesia. BK is known to activate as well as sensitize sensory neurons to other stimuli. Various signaling pathways have been suggested to mediate the sensitizing effect of BK on sensory neurons (6, 7). However, activation mechanism by BK is not known. BK is now known to stimulate the production of AA in sensory neurons (8), a key substrate of lipoxygenases. Therefore, on the basis of previous observations that products of lipoxygenase activate VR1 (5), we hypothesized that BK excites sensory neurons by opening the capsaicin receptor via production of 12-lipoxygenase products of AA metabolism. Materials and MethodsCell Culture. Experiments were carried out according to the Ethical Guidelines of the International Association for the Study of Pain and approved by the research ethics committee for the use of animals of the Seoul National University and the University of California, San Francisco. Thoracic and lumbar dorsal root ganglia (DRGs) were dissected from 1-to 2 day-...

Vanilloid receptor 1 (VR1), a capsaicin receptor, is known to play a major role in mediating inflammatory thermal nociception. Although the physiological role and biophysical properties of VR1 are known, the mechanism of its activation by ligands is poorly understood. Here we show that VR1 must be phosphorylated by Ca 2؉ -calmodulin dependent kinase II (CaMKII) before its activation by capsaicin. In contrast, the dephosphorylation of VR1 by calcineurin leads to a desensitization of the receptor. Moreover, point mutations in VR1 at two putative consensus sites for CaMKII failed to elicit capsaicin-sensitive currents and caused a concomitant reduction in VR1 phosphorylation in vivo. Such mutants also lost their high affinity binding with [ 3 H]resiniferatoxin, a potent capsaicin receptor agonist. We conclude that the dynamic balance between the phosphorylation and dephosphorylation of the VR1 channel by CaMKII and calcineurin, respectively, controls the activation/ desensitization states by regulating VR1 binding. Furthermore, because sensitization by protein kinase A and C converge at these sites, phosphorylation stress in the cell appears to control a wide range of excitabilities in response to various adverse stimuli.

Capsaicin (CAP) excites small sensory neurons, causing pain, neurogenic inflammation, and other visceral reflexes. These effects have been proposed to be the result of CAP activation of a nonselective cation current. It is generally assumed that CAP binds to an extracellular domain of the membrane receptor. However, the exact binding site is not known because of the lipophilic nature of CAP. To determine whether the binding domain is extracellular or intracellular, we tested the effect of a synthetic watersoluble CAP analog, DA-5018⅐HCl, on current activation. CAP activated the 45 pS (at Ϫ60 mV) nonselective cation channel from either side of the membrane. However, DA-5018⅐HCl, which had a greater potency and efficacy than CAP, activated the channels only from the cytosolic side of the patch membrane in a capsazepine, a CAP receptor antagonist, reversible manner. When applied extracellularly, DA-5018⅐HCl did not, but CAP did, activate whole-cell currents in sensory neurons, as well as in oocytes expressing vanilloid receptor 1, a recently cloned CAP receptor. Hydrogen ions, reported as a possible endogenous activator of cation current, failed to elicit any current when acidic medium (pH 5.0-6.0) was applied intracellularly, indicating that H ϩ does not mediate the CAP effect. These results indicate that CAP and its analog bind to the cytosolic domain of the CAP receptor and suggest that an endogenous CAP-like substance other than H ϩ may be present in the cell.

Vanilloid receptor 1 (VR1), a ligand-gated ion channel activated by vanilloids, acid, and heat, is a molecular detector that integrates multiple modes of pain. Although the function and the biophysical properties of the channel are now known, the regions of VR1 that recognize ligands are largely unknown. By the stepwise deletion of VR1 and by chimera construction using its capsaicin-insensitive homologue, VRL1, we localized key amino acids, Arg-114 and Glu-761, in the N-and C-cytosolic tails, respectively, that determine ligand binding. Point mutations of the two key residues resulted in a loss of sensitivity to capsaicin and a concomitant loss of specific binding to [ 3 H]resiniferatoxin, a potent vanilloid. Furthermore, changes in the charges of the two amino acids blocked capsaicin-sensitive currents and ligand binding without affecting current responses to heat. Thus, these two regions in the cytoplasmic tails of VR1 provide structural elements for its hydrophilic interaction with vanilloids and might constitute a long-suspected binding pocket.Capsaicin, the principal pungent ingredient of hot peppers, excites sensory neurons by opening an ion channel, the vanilloid receptor 1 (VR1), 1 thereby causing pain. VR1 is a ligand-gated, cationic channel that is present mainly in small nociceptive sensory neurons (1-3). The presence of VR1 in sensory neurons leads to questions concerning the existence of endogenous capsaicin-like substances, and various lipid metabolic products of lipoxygenases or anandamide have been suggested as candidates, because they activate VR1 and are structurally similar to capsaicin (4, 5). Accordingly, a role for lipoxygenase products in the activation of VR1 during inflammation was suggested (5), and in fact, bradykinin, a potent pain-causing inflammatory mediator, is now known to activate VR1 via the lipoxygenase/VR1 pathway (6). In addition, bradykinin also has a potential to sensitize VR1 via a phospholipase C or protein kinase C pathway (7-9).VR1 is also activated by acid or heat at over 43°C, a threshold temperature for pain (3, 10 -12). Moreover, because ischemic or inflamed tissues become acidic, the acid activation of VR1 is a pathologically relevant event (13). More direct evidence for the pathophysiological role of VR1 in the production of inflammatory pain came from knock-out experiments. In mice lacking VR1, thermal hyperalgesia evoked by inflammation is reduced (14, 15). Furthermore, hyperalgesia induced by the key inflammatory mediators, bradykinin and nerve growth factor, is reduced in mice lacking VR1 (8). Thus, VR1 is now considered a primary molecular transducer that mediates inflammatory hyperalgesia (13).The putative topology of VR1 indicates that it belongs to a class of transient receptor potential channels possessing six transmembrane domains and two cytosolic domains at each Nand C terminus (3, 16). VR1 appears to form a homotetramer when expressed heterologously (17). However, VR1 may form a heteromultimer with another temperature-sensitive channel, transient rec...

The human cathelicidin antimicrobial protein hCAP18, which includes the C-terminal peptide LL-37, is a multifunctional protein. As a possible approach to enhancing the resistance to plant disease, a DNA fragment coding for hCAP18/LL-37 was fused at the C-terminal end of the leader sequence of endopolygalacturonase-inhibiting protein under the control of the cauliflower mosaic virus 35S promoter region. The construct was then introduced into Brassica rapa. LL-37 expression was confirmed in transgenic plants by reverse transcription-polymerase chain reaction and western blot analysis. Transgenic plants exhibited varying levels of resistance to bacterial and fungal pathogens. The average size of disease lesions in the transgenic plants was reduced to less than half of that in wild-type plants. Our results suggest that the antimicrobial LL-37 peptide is involved in wide-spectrum resistance to bacterial and fungal pathogen infection.

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The general stress response of Bacillus subtilis is triggered by a variety of environmental and metabolic stresses which activate the aB transcription factor. Among the more than 100 genes controlled b y aB.(the csb genes), the functions identified thus far include resistance to oxidative stress, resistance to protein denaturation and resistance to osmotic stress. T o understand the breadth of functions in which csb genes participate, the transcriptional organization and predicted products of two such genes previously identified in a screen for oB-dependent lacZ fusions were analysed. The csb-22::Tn917lacZ and csb-34::Tn917lacZ fusions are unusual among csb genes in that their expression appears to be completely dependent upon aB. By plasmidintegration experiments, fusion analyses and site-directed mutagenesis, stressinducible, aB-dependent promoters for both these fusions were identified. The csb-34 fusion marked an ORF (yxcC or csbC) which by sequence analysis lay in a monocistronic transcriptional unit. This ORF encoded a predicted 461-residue product which had high identity with Class I sugar transporters of the major facilitator superfamily. It was speculated that the csbC product could serve either a nutritional or an osmotic protection function. In contrast, the csb-22 fusion identified an ORF (ywmG or csbD) which appeared to be the second gene of a two-gene operon. This ORF encoded a predicted 62-residue product which resembled a small Escherichia coli protein of unknown function. The aBdependent promoter lay immediately upstream from csbD and appeared to be an internal promoter for the operon.