Heat attenuates sensitivity of mammalian cells to capsaicin

The transient receptor potential (TRP) channels are thermo‐sensors, and transient receptor potential vanilloid (TRPV)1 and V4 are widely expressed in primary afferent neurons and nonneuronal cells. Although heat acclimation is considered as changes of thermoregulatory responses by thermo‐effectors to heat, functional changes of TRP channels in heat acclimation has not been fully elucidated. Here, we investigated whether heat acclimation induces capsaicin tolerance. NIH3T3 cells were incubated at 39.5°C. We determined the expression level of TRPV1 and TRPV4 messenger RNA (mRNA), performed cellular staining of TRPV1 and TRPV4, and investigated actin assembly and activation of the extracellular signal‐regulated kinase (ERK). Exposure to moderate heat decreased the levels of TRPV1 but not TRPV4 mRNA. It also induced stress fiber formation and the intensity of TRPV1 seemed to be decreased by chronic heat stimuli. In addition, heat acclimation attenuated the capsaicin‐induced activation of ERK. Heat acclimation may induce capsaicin tolerance via the downregulation of TRPV1.     

Monocyte chemoattractant protein-1 functions as a neuromodulator in dorsal root ganglia neurons

It has previously been observed that expression of chemokine monocyte chemoattractant protein-1 (MCP-1/CC chemokine ligand 2 (CCL2)) and its receptor CC chemokine receptor 2 (CCR2) is up-regulated by dorsal root ganglion (DRG) neurons in association with rodent models of neuropathic pain. MCP-1 increases the excitability of nociceptive neurons after a peripheral nerve injury, while disruption of MCP-1/CCR2 signaling blocks the development of neuropathic pain, suggesting MCP-1 signaling is responsible for heightened pain sensitivity. To define the mechanisms of MCP-1 signaling in DRG, we studied intracellular processing, release, and receptor-mediated signaling of MCP-1 in DRG neurons. We found that in a focal demyelination model of neuropathic pain both MCP-1 and CCR2 were up-regulated by the same neurons including transient receptor potential vanilloid receptor subtype 1 (TRPV1) expressing nociceptors. MCP-1 expressed by DRG neurons was packaged into large dense-core vesicles whose release could be induced from the soma by depolarization in a Ca²⁺-dependent manner. Activation of CCR2 by MCP-1 could sensitize nociceptors via transactivation of transient receptor potential channels. Our results suggest that MCP-1 and CCR2, up-regulated by sensory neurons following peripheral nerve injury, might participate in neural signal processing which contributes to sustained excitability of primary afferent neurons.     

TRPV1b overexpression negatively regulates TRPV1 responsiveness to capsaicin, heat and low pH in HEK293 cells

Transient receptor potential channel type V (TRPV) 1 is a non-selective cation channel that can be activated by capsaicin, endogenous vanilloids, heat and protons. The human TRPV1 splice variant, TRPV1b, lacking exon 7, was cloned from human dorsal root ganglia (DRG) RNA. The expression profile and relative abundance of TRPV1b and TRPV1 in 35 different human tissues were determined by quantitative RT-PCR using isoform-specific probes. TRPV1b was most abundant in fetal brain, adult cerebellum and DRG. Functional studies using electrophysiological techniques showed that recombinant TRPV1b was not activated by capsaicin (1 microM), protons (pH 5.0) or heat (50 degrees C). However, recombinant TRPV1b did form multimeric complexes and was detected on the plasma membrane of cells, demonstrating that the lack of channel function was not due to defects in complex formation or cell surface expression. These results demonstrate that exon 7, which encodes the third ankyrin domain and 44 amino acids thereafter, is required for normal channel function of human TRPV1. Moreover, when co-expressed with TRPV1, TRPV1b formed complexes with TRPV1, and inhibited TRPV1 channel function in response to capsaicin, acidic pH, heat and endogenous vanilloids, dose-dependently. Taken together, these data support the hypothesis that TRPV1b is a naturally existing inhibitory modulator of TRPV1.     

瞬时受体电位香草酸亚型1激活通过抑制线粒体通透性转换孔开放保护急性心肌梗死小鼠的心肌细胞抗凋亡

瞬时受体电位香草酸亚型1（TRPV1）在心肌缺血激活后可传导心绞痛信号,释放神经肽,减轻心肌梗死后的心肌细胞凋亡。目前,TRPV1激活抑制心肌梗死后细胞凋亡的具体机制尚不清楚。线粒体通透性转换孔（MPTP）的开放与心肌细胞缺血再灌注损伤密切相关,抑制其开放可保护心肌缺血后的心肌细胞抗凋亡。本研究证明,TRPV1激活通过抑制MPTP开放而减少心肌细胞凋亡。首先,本研究利用左冠状动脉前降支结扎术建立了TRPV1基因敲除（TRPV1-/-）和野生型（WT）小鼠心肌梗死模型,辅以环孢素A（CSA）预处理抑制 MPTP开放,比较观察TRPV1、MPTP在心肌梗死中的作用。心肌组织切片氯化三苯基四氮唑（TTC）染色显示,心肌缺血24 h,TRPV1-/-小鼠的心肌梗死面积明显大于WT型小鼠。而经CSA预处理的TRPV1-/-小鼠比TRPV1-/-小鼠梗死面积明显减小。TUNEL检测心肌细胞凋亡指数（AI）揭示,WT型心肌梗死小鼠的AI明显低于TRPV1-/- 心肌梗死小鼠,而CSA预处理明显降低TRPV1-/-小鼠心肌细胞的AI。Western印迹检测胱天蛋白酶3、胱天蛋白酶9、Bcl-2、Bax、p53和细胞色素C（Cyt-C）水平。结果证明,TRPV1的激活可抑制MPTP的开放,减少线粒体Cyt-C的外溢,降低胱天蛋白酶9和胱天蛋白酶3的表达。GENMEN光度法检测MPTP开放实验显示,激活的TRPV1明显抑制了MPTP的开放。本研究证实,急性心肌梗死后的TRPV1激活可能通过抑制MPTP开放而抵抗心肌细胞凋亡,对心肌起保护作用。     

Capsaicinol: Synthesis by Allylic Oxidation and Its Effect on TRPV1-Expressing Cells and Adrenaline Secretion in Rats

Capsaicinol is an ingredient of hot red pepper. In this study, we developed a novel method for capsaicinol synthesis and examined capsaicinol’s physiological effects on capsaicin receptor (TRPV1)-related actions. Allylic oxidation of capsaicin by palladium acetate (Pd(OAc)₂) resulted in the formation of (±)-capsaicinol acetate at a 7.2% yield in a single step. The effectiveness of (±)-capsaicinol in TRPV1 activation (EC₅₀=1.1 μM) was found to be weaker than that of capsaicin (EC₅₀=0.017 μM), whereas the efficacy of (±)-capsaicinol reached 75% of that of capsaicin. Intravenous administration of (±)-capsaicinol in anesthetized rats dose-dependently enhanced adrenaline secretion from the adrenal gland. The response to a 5 mg/kg-dose of (±)-capsaicinol was comparable to that of a 0.05 mg/kg-dose of capsaicin. The relative pungency of capsaicinol to capsaicin was coincident with the relative effectiveness in inducing these TRPV1-related actions.     

Immunomodulatory effect of oleoylethanolamide in dendritic cells via TRPV1/AMPK activation

Oleoylethanolamide (OEA) is an endogenous lipid mediator involved in the control of feeding, body weight, and energy metabolism. However, whether OEA modulates maturation of dendritic cells (DCs) has never been addressed. Hence, we evaluated the effect of OEA on DCs maturation in bone marrow-derived DCs (BMDCs) in four aspects: (a) Cell surface markers were determined using flow cytometric analysis; (b) cell mobile ability was testified with the transwell assay; (c) stimulation of T cells proliferation was performed in a coculture system; and (d) cytokine production was measured using polymerase chain reaction (PCR). The result showed that, in mature BMDCs induced by lipopolysaccharides (LPS), the OEA treatment decreased expressions of cell surface markers, reduced cell migration, diminished the proliferation of cocultured T cells, and regulated cytokine production of BMDCs, indicating the modulatory effect of OEA on DCs maturation. Furthermore, to explore the underlying mechanism of the immunomodulatory effect of OEA, we used antagonists of transient receptor potential vanilloid-1 (TRPV1) and AMP-activated protein kinase (AMPK), determined the protein expressions of TRPV1/AMPK and Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) using western blot, and measured the intracellular calcium concentration using calcium imaging. The result illustrated that OEA downregulated TLR4/NF-κB, the classical pathway leading to DCs maturation induced by LPS, through the activation of TRPV1 and AMPK. Collectively, the present study suggests that OEA suppresses DCs maturation through the activation of TRPV1/AMPK. These findings increase our understanding of this endogenous lipid OEA.     

The mechanism of μ-opioid receptor (MOR)-TRPV1 crosstalk in TRPV1 activation involves morphine anti-nociception,tolerance and dependence

Initiated by the activation of various nociceptors, pain is a reaction to specific stimulus modalities. The μ-opioid receptor (MOR) agonists, including morphine, remain the most potent analgesics to treat patients with moderate to severe pain. However, the utility of MOR agonists is limited by the adverse effects associated with the use of these drugs, including analgesic tolerance and physical dependence. A strong connection has been suggested between the expression of the transient receptor potential vanilloid type 1 (TRPV1) ion channel and the development of inflammatory hyperalgesia. TRPV1 is important for thermal nociception induction, and is mainly expressed on sensory neurons. Recent reports suggest that opioid or TRPV1 receptor agonist exposure has contrasting consequences for anti-nociception, tolerance and dependence. Chronic morphine exposure modulates TRPV1 activation and induces the anti-nociception effects of morphine. The regulation of many downstream targets of TRPV1 plays a critical role in this process, including calcitonin gene-related peptide (CGRP) and substance P (SP). Additional factors also include capsaicin treatment blocking the anti-nociception effects of morphine in rats, as well as opioid modulation of TRPV1 responses through the cAMP-dependent PKA pathway and MAPK signaling pathways. Here, we review new insights concerning the mechanism underlying MOR-TRPV1 crosstalk and signaling pathways and discuss the potential mechanisms of morphine-induced anti-nociception, tolerance and dependence associated with the TRPV1 signaling pathway and highlight how understanding these mechanisms might help find therapeutic targets for the treatment of morphine induced antinociception, tolerance and dependence.     

Melittin activates TRPV1 receptors in primary nociceptive sensory neurons via the phospholipase A2 cascade pathways

Previous studies demonstrated that melittin, the main peptide in bee venom, could cause persistent spontaneous pain, primary heat and mechanical hyperalgesia, and enhance the excitability of spinal nociceptive neurons. However, the underlying mechanism of melittin-induced cutaneous hypersensitivity is unknown. Effects of melittin applied topically to acutely dissociated rat dorsal root ganglion neurons were studied using whole-cell patch clamp and calcium imaging techniques. Melittin induced intracellular calcium increases in 60% of small (<25 μm) and medium (<40 μm) diameter sensory neurons. In current clamp, topical application of melittin evoked long-lasting firing in 55% of small and medium-sized neurons tested. In voltage clamp, melittin evoked inward currents in sensory neurons in a concentration-dependent manner. Repeated application of melittin caused increased amplitude of the inward currents. Most melittin-sensitive neurons were capsaicin-sensitive, and 65% were isolectin B4 positive. Capsazepine, the TRPV1 receptor inhibitor, completely abolished the melittin-induced inward currents and intracellular calcium transients. Inhibitions of signaling pathways showed that phospholipase A₂, but not phospholipase C, was involved in producing the melittin-induced inward currents. Inhibitors of cyclooxygenases (COX) and lipoxygenases (LOX), two key components of the arachidonic acid metabolism pathway, each partially suppressed the inward current evoked by melittin. Inhibitors of protein kinase A (PKA), but not of PKC, also abolished the melittin-induced inward currents. These results indicate that melittin can directly excite small and medium-sized sensory neurons at least in part by activating TRPV1 receptors via PLA2-COXs/LOXs cascade pathways.     

Site-specific contacts enable distinct modes of TRPV1 regulation by the potassium channel Kvβ1 subunit

Transient receptor potential vanilloid 1 (TRPV1) channel is a multimodal receptor that is responsible for nociceptive, thermal, and mechanical sensations. However, which biomolecular partners specifically interact with TRPV1 remains to be elucidated. Here, we used cDNA library screening of genes from mouse dorsal root ganglia combined with patch-clamp electrophysiology to identify the voltage-gated potassium channel auxiliary subunit Kvβ1 physically interacting with TRPV1 channel and regulating its function. The interaction was validated in situ using endogenous dorsal root ganglia neurons, as well as a recombinant expression model in HEK 293T cells. The presence of Kvβ1 enhanced the expression stability of TRPV1 channels on the plasma membrane and the nociceptive current density. Surprisingly, Kvβ1 interaction also shifted the temperature threshold for TRPV1 thermal activation. Using site-specific mapping, we further revealed that Kvβ1 interacted with the membrane-distal domain and membrane-proximal domain of TRPV1 to regulate its membrane expression and temperature-activation threshold, respectively. Our data therefore suggest that Kvβ1 is a key element in the TRPV1 signaling complex and exerts dual regulatory effects in a site-specific manner.     

Role of TRPM2 and TRPV1 cation channels in cellular responses to radiation-induced DNA damage

Background

Radiation exposure causes DNA damage, and DNA repair systems are essential to rescue damaged cells. Although DNA damage or oxidative stress activates transient receptor potential melastatin 2 (TRPM2) and vanilloid 1 (TRPV1) cation channels, it has not been established whether these TRP channels are involved in cellular responses to radiation-induced DNA damage. Here, we investigated the contribution of TRPM2 and TRPV1 channels to γ-irradiation- and UVB-induced DNA damage responses in human lung cancer A549 cells.

Methods

A549 cells were irradiated with γ-rays (2.0 Gy) or UVB (5–10 mJ/cm²). γH2AX foci, ATM activation, 53BP1 accumulation and EGFR expression were evaluated by immunofluorescence staining. Extracellular ATP concentration was measured by luciferin–luciferase assay. Knockdown of TRPM2 and TRPV1 expression was done by siRNA transfection.

Results

γ-Irradiation-induced γH2AX focus formation, ATM activation, 53BP1 accumulation and EGFR nuclear translocation, which are all associated with DNA repair, were suppressed by knockdown of TRPM2 and TRPV1 channels in A549 cells. Release of ATP, which mediates DNA damage response-associated activation of P2Y receptors, was suppressed by pre-treatment with catalase or knockdown of TRPM2 channel, but not TRPV1 channel. Similarly, UVB-induced γH2AX focus formation was suppressed in TRPM2- and TRPV1-knockdown cells, while UVB-induced ATP release was blocked in TRPM2- but not TRPV1-knockdown cells.

Conclusion

Our results suggest that the activation of TRPM2 channel, which mediates ATP release, and TRPV1 channel plays significant roles in the cellular responses to DNA damage induced by γ-irradiation and UVB irradiation.

General significance

Our results provide a new insight into the function of TRP channels from the viewpoint of radiation biology.     

Epac- and Rap- independent ERK1/2 phosphorylation induced by Gs-coupled receptor stimulation in HEK293 cells

Serotonin activates Ras and Ras-dependent ERK1/2 phosphorylation in HEK293 cells expressing G(s)-coupled 5-HT(4) or 5-HT(7) serotonin receptors through unknown mechanisms. Both Epac/Rap-dependent and -independent pathways for Ras-dependent ERK1/2 activation have been suggested. Epac overexpression or Epac-specific 8-CPT-2'-O-Me-cAMP did not cause ERK1/2 phosphorylation, despite Rap activation. The data did not support a role for PLCepsilon or DAG-dependent Ras GEFs of the Ras-GRP family in Ras-dependent ERK1/2 phosphorylation. However, serotonin stimulated phosphorylation of endogenous and recombinant Ras-GRF1, increased [Ca(2+)](i) and caused Ca(2+)- and calmodulin-dependent ERK1/2 phosphorylation. Different signalling pathways seem to be utilised by G(s)-coupled receptors in various isolates of HEK293 cells.     

Roles of reactive oxygen and nitrogen species in pain

Peroxynitrite (PN; ONOO⁻) and its reactive oxygen precursor superoxide (SO; O₂^•−) are critically important in the development of pain of several etiologies including pain associated with chronic use of opiates such as morphine (also known as opiate-induced hyperalgesia and antinociceptive tolerance). This is now an emerging field in which considerable progress has been made in terms of understanding the relative contributions of SO, PN, and nitroxidative stress in pain signaling at the molecular and biochemical levels. Aggressive research in this area is poised to provide the pharmacological basis for development of novel nonnarcotic analgesics that are based upon the unique ability to selectively eliminate SO and/or PN. As we have a better understanding of the roles of SO and PN in pathophysiological settings, targeting PN may be a better therapeutic strategy than targeting SO. This is because, unlike PN, which has no currently known beneficial role, SO may play a significant role in learning and memory [1]. Thus, the best approach may be to spare SO while directly targeting its downstream product, PN. Over the past 15 years, our team has spearheaded research concerning the roles of SO and PN in pain and these results are currently leading to the development of solid therapeutic strategies in this important area.     

Molecular mechanism of 2-APB-induced Ca influx in external acidification in PC12

2-Aminoethoxydiphenyl borate (2-APB) is used as a pharmacological tool because it antagonizes inositol 1,4,5-trisphosphate receptors and store-operated Ca²⁺ (SOC) channels, and activates some TRP channels. Recently, we reported that 2-APB enhanced the increase in cytotoxic [Ca²⁺]_i, resulting in cell death under external acidic conditions in rat pheochromocytoma cell line PC12. However, the molecular mechanism and functional role of the 2-APB-induced Ca²⁺ influx in PC12 have not been clarified. In this study, to identify the possible target for the action of 2-APB we examined the pharmacological and molecular properties of [Ca²⁺]_i and secretory responses to 2-APB under extracellular low pH conditions. 2-APB dose-dependently induced a [Ca²⁺]_i increase and dopamine release, which were greatly enhanced by the external acidification (pH 6.5). [Ca²⁺]_i and secretory responses to 2-APB at pH 6.5 were inhibited by the removal of extracellular Ca²⁺ and SOC channel blockers such as SK&F96365, La³⁺ and Gd³⁺. PC12 expressed all SOC channel molecules, Orai 1, Orai 2 and Orai 3. When we used an siRNA system, downregulation of Orai 3, but not Orai 1 and Orai 2, attenuated both [Ca²⁺]_i and secretory responses to 2-APB. These results suggest that 2-APB evokes external acid-dependent increases of [Ca²⁺]_i and dopamine release in PC12 through the activation of Orai 3. The present results indicate that 2-APB may be a useful pharmacological tool for Orai channel-related signaling.     

TRPC3-mediated Ca influx contributes to Rac1-mediated production of reactive oxygen species in MLP-deficient mouse hearts

Dilated cardiomyopathy (DCM) is a myocardial disorder that is characterized by dilation and dysfunction of the left ventricle (LV). Accumulating evidence has implicated aberrant Ca²⁺ signaling and oxidative stress in the progression of DCM, but the molecular details are unknown. In the present study, we report that inhibition of the transient receptor potential canonical 3 (TRPC3) channels partially prevents LV dilation and dysfunction in muscle LIM protein-deficient (MLP (−/−)) mice, a murine model of DCM. The expression level of TRPC3 and the activity of Ca²⁺/calmodulin-dependent kinase II (CaMKII) were increased in MLP (−/−) mouse hearts. Acitivity of Rac1, a small GTP-binding protein that participates in NADPH oxidase (Nox) activation, and the production of reactive oxygen species (ROS) were also increased in MLP (−/−) mouse hearts. Treatment with pyrazole-3, a TRPC3 selective inhibitor, strongly suppressed the increased activities of CaMKII and Rac1, as well as ROS production. In contrast, activation of TRPC3 by 1-oleoyl-2-acetyl-sn-glycerol (OAG), or by mechanical stretch, induced ROS production in rat neonatal cardiomyocytes. These results suggest that up-regulation of TRPC3 is responsible for the increase in CaMKII activity and the Nox-mediated ROS production in MLP (−/−) mouse cardiomyocytes, and that inhibition of TRPC3 is an effective therapeutic strategy to prevent the progression of DCM.     

Functional expression of 5-HT2A receptor in osteoblastic MC3T3-E1 cells

In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT₂ receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT_2A and 5-HT_2C receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT_2A receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT_2A receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells.