Different expression patterns of TRP genes in murine B and T lymphocytes.

A prolonged increase in the intracellular calcium concentration ([Ca2+]i) is essential for lymphocyte activation that includes cell proliferation and differentiation. This increase in [Ca2+]i results from Ca2+ release from the intracellular store and the subsequent Ca2+ influx from the extracellular environment via calcium channels located on the plasma membrane. Although transient receptor potential (TRP) channels have been reported to play important roles in the [Ca2+]i increase in lymphocytes, the function of these channels in lymphocyte activation remains unknown. Here, we report the comprehensive expression profile of TRP channel gene families including TRPC, TRPV, and TRPM in the murine immune system. RT-PCR analysis revealed different expression patterns of the TRP channel genes in B and T lymphocytes isolated from the spleen. Therefore, our results provide an appropriate reference of TRP gene expression in murine lymphocytes.

Lipophilicity of capsaicinoids and capsinoids influences the multiple activation process of rat TRPV1.

Analogs of capsaicin, such as capsaicinoids and capsinoids, activate a cation channel, transient receptor potential cation channel vanilloid subfamily 1 (TRPV1), and then increase the intracellular calcium concentration ([Ca2+]i). These compounds would be expected to activate TRPV1 via different mechanism(s), depending on their properties. We synthesized several capsaicinoids and capsinoids that have variable lengths of acyl moiety. The activities of these compounds towards TRPV1 heterologously expressed in HEK293 cells were determined by measuring [Ca2+]i. When an extracellular or intracellular Ca2+ source was removed, some agonists such as capsaicin could increase [Ca2+]i. However, a highly lipophilic capsaicinoid containing C18:0 and capsinoids containing C14:0, C18:0, or C18:1 (the latter was named olvanilate) could not elicit a large increase in [Ca2+]i in the absence of an extracellular or intracellular Ca2+ source. These results suggest that highly lipophilic compounds cause only a slight Ca2+ influx, via TRPV1 in the plasma membrane, and are not able to activate TRPV1 in the endoplasmic reticulum.

Enhanced thermal avoidance in mice lacking the ATP receptor P2X3.

P2X3 is an ATP-gated cation channel subtype expressed by a subpopulation of primary sensory neurons. In vivo spinal cord recordings in mice lacking P2X3 (P2X3-/-) have suggested that this protein may be important for the coding of peripheral warm stimuli. To explore this possibility more thoroughly, we examined behavioral and electrophysiological responses to thermal stimuli in P2X3-/- mice. As previously reported, recording from the spinal cord dorsal horn of anesthetized P2X3-/- mice revealed a blunted response of wide dynamic range neurons to hind paw heating. When placed in a thermal gradient, however, P2X3-/- mice exhibited an unexpectedly enhanced avoidance of both hot and cold temperatures, relative to controls. In the tail immersion test, mutant mice exhibited shorter withdrawal latencies at temperatures above and below thermoneutrality. Consistent with these changes, P2X3-/- mice exhibited enhanced induction of spinal cord c-FOS following hind paw heating to 45 degrees C. Thus, gain- and loss-of-function thermosensory phenotypes coexist in P2X3-/- mice. No changes in thermal preference were observed in wild-type mice injected subcutaneously with the P2X3 antagonist, A317491 or intrathecally with the P2X3 and P2X1 antagonist TNP-ATP. The reason for this apparent discrepancy is unclear, but we cannot exclude the possibility that compensatory events contribute, at least in part, to the P2X3-/- phenotype. Regardless, this study illustrates the utility of thermal preference assays as part of a comprehensive approach to the analysis of mouse thermosensation.

5-Iodoresiniferatoxin evokes hypothermia in mice and is a partial transient receptor potential vanilloid 1 agonist in vitro.

Transient receptor potential vanilloid 1 (TRPV1) is a capsaicin- and heat-gated ion channel required for normal in vivo responses to these painful stimuli. However, growing evidence suggests that TRPV1 also participates in thermoregulation. Therefore, we examined the effects of a selective TRPV1 antagonist, 5-iodoresiniferatoxin (I-RTX), on mouse body temperature. Surprisingly, s.c. administration of I-RTX (0.1-1 micromol/kg) evoked a hypothermic response similar to that evoked by capsaicin (9.8 micromol/kg) in naive wild-type mice, but not in mice pretreated with resiniferatoxin, a potent TRPV1 agonist, or in naive TRPV1-null mice. In response to I-RTX in vitro, HEK293 cells expressing rat TRPV1 exhibited increases in intracellular Ca(2+) (biphasic, EC(50) = 56.7 nM and 9.9 microM) that depended on Ca(2+) influx and outwardly rectifying, capsazepine-sensitive currents that were smaller than those evoked by 1 microM capsaicin. Thus, I-RTX induces TRPV1-dependent hypothermia in vivo and is a partial TRPV1 agonist in vitro.

Sensitization of TRPV1 by EP1 and IP reveals peripheral nociceptive mechanism of prostaglandins.

Prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2) 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. PGE2 or PGI2increased or sensitized TRPV1 responses through EP1 or IP receptors, respectively predominantly in a PKC-dependent manner in both HEK293 cells expressing TRPV1 and mouse DRG neurons. In the presence of PGE2 or PGI2, the temperature threshold for TRPV1 activation was reduced below 35 degrees C, so that temperatures near body temperature are sufficient to activate TRPV1. A PKA-dependent pathway was also involved in the potentiation of TRPV1 through EP4 and IP receptors upon exposure to PGE2 and PGI2, respectively. Both PGE2-induced thermal hyperalgesia and inflammatory nociceptive responses were diminished in TRPV1-deficient mice and EP1-deficient mice. IP receptor involvement was also demonstrated using TRPV1-deficient mice and IP-deficient mice. Thus, the potentiation or sensitization of TRPV1 activity through EP1 or IP activation might be one important mechanism underlying the peripheral nociceptive actions of PGE2 or PGI2.

Attenuated fever response in mice lacking TRPV1.

TRPV1, the capsaicin receptor, is expressed not only in nociceptive neurons, but also in other locations, including the hypothalamus. Studies involving systemic or intrahypothalamic capsaicin administration have suggested a role for TRPV1 in body temperature control. To explore this possibility, we examined thermoregulatory responses in TRPV1-/- mice. These mutant animals exhibited no obvious changes in circadian body temperature fluctuation, tolerance to increased (35 degrees C) or decreased (4 degrees C) ambient temperature or ethanol-induced hypothermia. In contrast, fever production in response to the bacterial pyrogen, lipopolysaccharide (LPS) was significantly attenuated in TRPV1-/- mice. Despite this finding, we detected no significant differences between TRPV1-/- and control mice in the extent of LPS-induced c-Fos expression in numerous fever-related brain subregions. These results suggest that TRPV1 participates in the generation of polyphasic fever, perhaps at sites outside the brain.

Altered thermal selection behavior in mice lacking transient receptor potential vanilloid 4.

Transient receptor potential vanilloid 4 (TRPV4), a cation channel responsive to hypotonicity, can also be activated by warm temperatures. Moreover, TRPV4-/- mice reportedly exhibit deficits in inflammation-induced thermal hyperalgesia. However, it is unknown whether TRPV4 or related transient receptor potential channels account for warmth perception under injury-free conditions. We therefore investigated the contribution of TRPV4 to thermosensation and thermoregulation in vivo. On a thermal gradient, TRPV4-/- mice selected warmer floor temperatures than wild-type littermates. In addition, whereas wild-type mice failed to discriminate between floor temperatures of 30 and 34 degrees C, TRPV4-/- mice exhibited a strong preference for 34 degrees C. TRPV4-/- mice also exhibited prolonged withdrawal latencies during acute tail heating. TRPV4-/- and wild-type mice exhibited similar changes in behavior on a thermal gradient after paw inflammation. Circadian body temperature fluctuations and thermoregulation in a warm environment were also indistinguishable between genotypes. These results demonstrate that TRPV4 is required for normal thermal responsiveness in vivo.

Regulation mechanisms of vanilloid receptors.

The capsaicin receptor TRPV1 (also known as the vanilloid receptor VR1) is a non-selective cation channel and is activated not only by capsaicin but also by noxious heat or protons. Tissue damage associated with infection, inflammation or ischaemia, produces an array of chemical mediators that activate or sensitize nociceptor terminals. An important component of this pro-algeic response is ATP. In cells expressing TRPV1, ATP increased the currents evoked by capsaicin or protons through activation of P2Y metabotropic receptors in a PKC-dependent manner. In the presence of ATP, the temperature threshold for TRPV1 activation was reduced from 42 degrees C to 35 degrees C, such that normal body temperature could activate TRPV1. Functional interaction between P2Y receptors and TRPV1 was confirmed in a behavioural analysis using TRPV1-deficient mice. Direct phosphorylation of TRPV1 by PKC was confirmed biochemically and the two serine residues involved were identified. Extracellular Ca2+ -dependent desensitization of TRPV1 is thought to be one mechanism underlying the paradoxical effectiveness of capsaicin as an analgesic therapy. The Ca2+ -binding protein calmodulin binds to the C-terminus of TRPV1. We found that disruption of the calmodulin binding segment prevented TRPV1 desensitization even in the presence of extracellular Ca2+.

[Molecular mechanisms of nociception].

Capsaicin, the main ingredient in 'hot' chili peppers, elicits burning pain by activating nociceptors. The cloned capsaicin receptor (TRPV1) is a nonselective cation channel with six transmembrane domains, and is activated not only by capsaicin but also by noxious heat (> 43 degrees C) or protons (acidification), both of which cause pain in vivo. Furthermore, analyses of mice lacking VR1 showed that VR1 is essential for selective modalities of pain sensation and for tissue injury-induced thermal hyperalgesia. Tissue damage produces an array of chemical mediators that activate or sensitize nociceptor terminals to elicit pain. Important components of this pro-algesic response are ATP and bradykinin. In cells expressing TRPV1, ATP or bradykinin increased the currents evoked by capsaicin or protons through activation of metabotropic P2Y or B2 bradykinin receptors. In the presence of ATP or bradykinin, the temperature threshold for VR1 activation was reduced from 42 degrees C to 30-35 degrees C, such that normally non-painful normal body temperatures were capable of activating TRPV1, thereby leading to the sensation of pain. Direct phosphorylation of TRPV1 by PKC epsilon was confirmed and the involved two serine residues were determined. This represents a novel mechanism through which ATP or bradykinin in response to tissue trauma might trigger the sensation of pain.

Possible involvement of P2Y2 metabotropic receptors in ATP-induced transient receptor potential vanilloid receptor 1-mediated thermal hypersensitivity.

The capsaicin receptor transient receptor potential V1 (TRPV1; also known as vanilloid receptor 1) 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 extracellular ATP potentiates the TRPV1 currents evoked by capsaicin or protons and reduces the temperature threshold for its activation through metabotropic P2Y receptors in a PKC-dependent pathway, suggesting that TRPV1 activation could trigger the sensation of pain at normal body temperature in the presence of ATP. Here, we show that ATP-induced thermal hyperalgesia was abolished in mice lacking TRPV1, suggesting the functional interaction between ATP and TRPV1 at a behavioral level. However, thermal hyperalgesia was preserved in P2Y1 receptor-deficient mice. Patch-clamp analyses using mouse dorsal root ganglion neurons indicated the involvement of P2Y2 rather than P2Y1 receptors. Coexpression of TRPV1 mRNA with P2Y2 mRNA, but not P2Y1 mRNA, was determined in the rat lumbar DRG using in situ hybridization histochemistry. These data indicate the importance of metabotropic P2Y2 receptors in nociception through TRPV1.

Comparative histopathological studies in the early stages of acute pathogenic and nonpathogenic SHIV-infected lymphoid organs.

To clarify the early pathological events in simian and human immunodeficiency chimeric virus (SHIV)-infected lymphoid organs, we examined rhesus macaques infected with an acute pathogenic SHIV (SHIV89.6P) or a nonpathogenic SHIV (NM-3rN) by sequential biopsies and serial necropsies. In the SHIV89.6P-infected monkeys, acute thymic involution as shown by increased cortical tingible-body macrophages and by neutrophilic infiltrates without follicular aggregation in the medulla began within 14 days postinoculation (dpi). Cells that were strongly positive for the virus were identified in the thymic medulla. SHIV89.6P-infected lymph nodes showed severe paracortical lymphadenitis with scattered virus-positive cells at 14 dpi and they developed paracortical depletion without the obvious follicular involution. In contrast, NM-3rN-infected monkeys showed no signs of thymic dysinvolution and the lymph nodes exhibited only follicular hyperplasia. NM-3rN-infected monkeys showed much fewer virus-positive cells in these lymphoid tissues than did SHIV89.6P-infected monkeys during the same period. These differences clearly reflect the difference in the virulence of these SHIVs.

Role of IFN-gamma and tumor necrosis factor-alpha in herpes simplex virus type 1 infection.

One of the characteristics of herpes simplex virus type 1 (HSV-1) is that recurrent diseases often develop from latent infection established after acute infection. Cytokines have been proposed to play an important role in each stage of HSV-1 infection, but the exact role of cytokines remains unclear. In the present study, we investigated the role of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in acute infection and reactivation using IFN-gamma gene knockout (IFN-gamma(-/-)) mice and TNF-alpha gene knockout (TNF-alpha(-/-)) mice. We first examined the survival rate after corneal infection with HSV-1. The survival rates of wild-type C57BL/6 (B6) mice, IFN-gamma(-/-) mice, and TNF-alpha(-/-) mice were 97% (73 of 75), 57% (24 of 42), and 83% (60 of 72), respectively. These results suggest that TNF-alpha and IFN-gamma play a protective role in acute infection with HSV-1. We also examined the rate of reactivation induced by ultraviolet (UV) light in latently infected mice over 60 days postinoculation. The reactivation was confirmed by detecting viral DNA extracted from eyeballs by the polymerase chain reaction (PCR) method at day 2 after the UV light stimulation. The rates of reactivation in IFN-gamma(-/-) mice and TNF-alpha(-/-) mice were significantly higher than that in B6 mice; 16% (4 of 25) showed reactivation in B6 mice, 47% (9 of 19) in IFN-gamma(-/-) mice, and 48% (10 of 21) in TNF-alpha(-/-) mice. These results suggest that IFN-gamma and TNF-alpha play an important role in acute infection and reactivation from latency.

Heat-evoked activation of the ion channel, TRPV4.

The mammalian nervous system constantly evaluates internal and environmental temperatures to maintain homeostasis and to avoid thermal extremes. Several members of the transient receptor potential (TRP) family of ion channels have been implicated as transducers of thermal stimuli, including TRPV1 and TRPV2, which are activated by heat, and TRPM8, which is activated by cold. Here we demonstrate that another member of the TRP family, TRPV4, previously described as a hypo-osmolarity-activated ion channel, also can be activated by heat. In response to warm temperatures, TRPV4 mediates large inward currents in Xenopus oocytes and both inward currents and calcium influx into human embryonic kidney 293 cells. In both cases these responses are observed at temperatures lower than those required to activate TRPV1 and can be inhibited reversibly by ruthenium red. Heat-evoked TRPV4-mediated responses are greater in hypo-osmotic solutions and reduced in hyperosmotic solutions. Consistent with these functional properties, we observed TRPV4 immunoreactivity in anterior hypothalamic structures involved in temperature sensation and the integration of thermal and osmotic information. Together, these data implicate TRPV4 as a possible transducer of warm stimuli within the hypothalamus.

Co-expression of interleukin-5 influences replication of simian/human immunodeficiency viruses in vivo.

The positive effect of the co-expression of T helper (Th) cell type 2 cytokine interleukin-5 (IL-5) on nef-deleted simian/human immunodeficiency virus (SHIV) replication in vitro has been observed previously. To analyse whether the growth advantage of IL-5-containing SHIV (NI-IL5) in vitro would be relevant in vivo, the virus was inoculated into monkeys. Three rhesus macaques were inoculated intravenously with 10(4) TCID(50) of NI-IL5. Results were compared with those obtained previously from SHIV NM-3rN (intact) and SHIV-dn (nef-deleted)-infected monkeys. Cytokine production, analysed by IL-5 ELISA, showed a twofold increase in IL-5 concentration in the plasma soon after the peak of virus replication. Virus replication and antibody production were greater in monkeys inoculated with IL-5-expressing SHIV than in monkeys inoculated with nef-deleted SHIV without IL-5. These findings show a stimulation of SHIV replication by co-expression of IL-5 and suggest the important role of Th2-type cytokines in human immunodeficiency virus type 1 infection.