Transient receptor potential vanilloid 4 deficiency suppresses unloading-induced bone loss

Mechanosensing is one of the crucial components of the biological events. In bone, as observed in unloading-induced osteoporosis in bed ridden patients, mechanical stress determines the levels of bone mass. Many molecules have been suggested to be involved in sensing mechanical stress in bone, while the full pathways for this event has not yet been identified. We examined the role of TRPV4 in unloading-induced bone loss. Hind limb unloading induced osteopenia in wild-type mice. In contrast, TRPV4 deficiency suppressed such unloading-induced bone loss. As underlying mechanism for such effects, TRPV4 deficiency suppressed unloading-induced reduction in the levels of mineral apposition rate and bone formation rate. In these mice, unloading-induced increase in the number of osteoclasts in the primary trabecular bone was suppressed by TRPV4 deficiency. Unloading-induced reduction in the longitudinal length of primary trabecular bone was also suppressed by TRPV4 deficiency. TRPV4 protein is expressed in both osteoblasts and osteoclasts. These results indicated that TRPV4 plays a critical role in unloading-induced bone loss.     

Warm temperature-sensitive transient receptor potential vanilloid 4 (TRPV4) plays an essential role in thermal hyperalgesia

Animals sense various ranges of temperatures by cutaneous thermal stimuli. Transient receptor potential vanilloid 4 (TRPV4) is a cation channel activated at a warm temperature (over 30 degrees C) in exogenously expressed cells. We found in the present study that TRPV4 is essential in thermal hyperalgesia at a warm temperature in vivo. TRPV4-/- and TRPV4+/+ mice exhibited the same latency of escape from 35-50 degrees C hotplates. Neuronal activity in the femoral nerve, however, revealed that the number and activity level of neurons decreased in response to a warm temperature in TRPV4-/- mice. TRPV4-/- mice displayed a significantly longer latency to escape from the plates at 35- 45 degrees C when hyperalgesia was induced by carrageenan without changes in foot volumes. TRPV4 therefore determines the sensitivity rather than the threshold of painful heat detection and plays an essential role in thermal hyperalgesia.     

瞬时受体电位香草酸亚型1(TRPV1)与炎性痛

瞬时受体电位香草酸亚型1(transient receptor potential vanilloid 1,TRPV1)是TRP超家族的成员之一,是一种非选择性的阳离子通道。TRPV1广泛分布于伤害性感受器上,并且在伤害性感受器中起重要作用。TRPV1能够感受伤害性刺激,将之转化为动作电位,传至中枢形成痛觉。炎症时释放的许多炎症介质都能够与TRPV1发生相互作用,产生疼痛或痛觉过敏,并且通过各种不同的信号通路来调制TRPV1的活性。深入研究TRPV1的作用机制,有助于理解痛觉生理和开发新型镇痛药物。     

Corticotropin-releasing factor receptor 1 mediates acute and delayed stress-induced visceral hyperalgesia in maternally separated Long-Evans rats

In rodents, maternal pup interactions play an important role in programming the stress responsiveness of the adult organism. The aims of this study were 1) to determine the effect of different neonatal rearing conditions on acute and delayed stress-induced visceral sensitivity as well as on other measures of stress sensitivity of the adult animal; and 2) to determine the role of corticotropin-releasing factor receptor (CRF-R) subtype 1 (CRF(1)R) in mediating visceral hypersensitivity. Three groups of male Long-Evans rat pups were used: separation from their dam for 180 min daily from postnatal days 2-14 (MS180), daily separation (handling) for 15 min (H), or no handling. The visceromotor responses (VMR) to colorectal distension, stress-induced colonic motility, and anxiety-like behavior were assessed in the adult rats. The VMR was assessed at baseline, immediately after a 1-h water avoidance (WA) stress, and 24 h poststress. Astressin B, a nonselective CRF-R antagonist, or CP-154,526, a selective CRF(1)R antagonist, was administered before the stressor and/or before the 24-h measurement. MS rats developed acute and delayed stress-induced visceral hyperalgesia. In contrast, H rats showed hypoalgesia immediately after WA and no change in VMR on day 2. MS rats with visceral hyperalgesia also exhibited enhanced stress-induced colonic motility and increased anxiety-like behavior. In MS rats, both CRF-R antagonists abolished acute and delayed increases in VMR. Rearing conditions have a significant effect on adult stress responsiveness including immediate and delayed visceral pain responses to an acute stressor. Both acute and delayed stress-induced visceral hypersensitivity in MS rats are mediated by the CRF/CRF(1)R system.     

Transient receptor potential vanilloid subtype 1 mediates microglial cell death in vivo and in vitro via Ca2+-mediated mitochondrial damage and cytochrome c release

The present study examined the expression of transient receptor potential vanilloid subtype 1 (TRPV1) in microglia, and its association with microglial cell death. In vitro cell cultures, RT-PCR, Western blot analysis, and immunocytochemical staining experiments revealed that rat microglia and a human microglia cell line (HMO6) showed TRPV1 expression. Furthermore, exposure of these cells to TRPV1 agonists, capsaicin (CAP) and resiniferatoxin (RTX), triggered cell death. This effect was ameliorated by the TRPV1 antagonists, capsazepine and iodo-resiniferatoxin (I-RTX), suggesting that TRPV1 is directly involved. Further examinations revealed that TRPV1-induced toxicity was accompanied by increases in intracellular Ca(2+), and mitochondrial damage; these effects were inhibited by capsazepine, I-RTX, and the intracellular Ca(2+) chelator BAPTA-AM. Treatment of cells with CAP or RTX led to increased mitochondrial cytochrome c release and enhanced immunoreactivity to cleaved caspase-3. In contrast, the caspase-3 inhibitor z-DEVD-fmk protected microglia from CAP- or RTX-induced toxicity. In vivo, we also found that intranigral injection of CAP or 12-hydroperoxyeicosatetraenoic acid, an endogenous agonist of TRPV1, into the rat brain produced microglial damage via TRPV1 in the substantia nigra, as visualized by immunocytochemistry. To our knowledge, this study is the first to demonstrate that microglia express TRPV1, and that activation of this receptor may contribute to microglial damage via Ca(2+) signaling and mitochondrial disruption.     

Transient receptor potential vanilloid 4 in the European sea bass Dicentrarchus labrax: a candidate protein for osmosensing

The Transient Receptor Potential Vanilloid 4 (TRPV4) protein is a member of the TRP ion channels superfamily that has been proposed as a potential fish osmosensor in previous studies. TRPV4 has been widely studied in mammals, particularly for its involvement in sensing the hypotonicity. The European sea bass, Dicentrarchus labrax, is a euryhaline teleost that is exposed to salinity changes due to its migrations between the sea and estuaries/lagoons. TRPV4 expression and localization in D. labrax was studied in seawater (SW)-adapted fish and in fish exposed to freshwater (FW) over different time-courses from 10 min to 30 days. TRPV4 mRNA expression was detected in gills, kidney and brain. In gills, the expression increased significantly in FW from 24 h to 30 d. In contrast, in the kidney, the TRPV4 expression decreased from 10 min to 7d of exposure to FW and then it increased at 30 d. In the brain, its expression was relatively low in SW compared to other organs and a significant decrease occurred in FW. The TRPV4 protein was localized in the basement membranes in branchial lamellae, the cartilage of gills, the posterior pituitary gland and in the collecting ducts. Possible roles of TRPV4 are discussed.     

Transient receptor potential vanilloid 1-mediated expression and secretion of endothelial cell-derived calcitonin gene-related peptide

Calcitonin gene-related peptide (CGRP), the principal transmitter in sensory nerves, could also be expressed in vascular endothelium. Transient receptor potential vanilloid 1(TRPV1), which modulates the synthesis and release of CGRP in sensory nerves, is also present in endothelial cells. The present study tested whether TRPV1 modulates the release and synthesis of CGRP in endothelial cells, and evaluated the protective effect of endothelial cell-derived CGRP. Human umbilical vein endothelial cells (HUVECs) were treated with capsaicin or hyperthermia. The level of CGRP mRNA was detected by RT-PCR, and protein level was measured by radioimmunoassay. Endothelial cell injury was induced by lysophosphatidylcholine, and evaluated by cell viability and lactate dehydrogenase activity. HUVECs expressed CGRP, both alpha- and beta-subtype. Capsaicin increased the level of CGRP in the culture medium, and up-regulated the expression of CGRP in endothelial cells. Hyperthermia also increased the level of CGRP mRNA. These effects were abolished by capsazepine, a competitive antagonist of TRPV1. Capsaicin significantly attenuated the endothelial cell damage induced by LPC, which was prevented and aggravated by capsazepine or CGRP(8-37,) antagonist of CGRP receptor. These results indicate that TRPV1 also regulates the expression and secretion of endothelial cell-derived CGRP, which affords protective effects on endothelial cells.     

Experimental colitis in mice and sensitization of converging visceral and somatic afferent pathways

Chronic pain syndromes affecting different organs often coexist. We hypothesized that sensitization of one afferent pathway may affect converging input from other areas of the body. We induced colitis in mice with 2,4,6-trinitrobenzenesulfonic acid (TNBS); control animals were treated with equal volumes of vehicle (50% ethanol) only. Visceromotor responses to graded colorectal distension, cystometrograms, and response thresholds to mechanical and thermal stimulation of both hind paws were determined on days 7 and 14. Inflammation of colon and bladder was assessed with validated histological markers and scores. TNBS caused significant colitis on day 7 that resolved by day 14; there was no evidence of bladder inflammation. There was a significant hypersensitivity to colorectal distension on day 7, which returned to normal on day 14. This was associated with bladder overactivity, as demonstrated by early onset of micturition and more frequent micturition on day 7 after TNBS administration. Colitis also significantly altered responses to mechanical and thermal stimulation of both hind paws on day 7 but not day 14. We conclude that cross talk between afferent visceral and somatic pathways may contribute to the coexistence of pain syndromes.     

Transient receptor potential vanilloid 1 receptors mediate acid-induced mucin secretion via Ca2+ influx in human airway epithelial cells

Mucin hypersecretion is a key pathological feature of inflammatory respiratory diseases. Previous studies have reported that acids (gastroesophageal reflux or environmental exposure) induce many respiratory symptoms and are implicated in the pathophysiology of obstructive airway diseases. To understand these mechanisms, we measured acid-induced mucin secretion in human bronchial epithelial cells. In the present study, acid induced inward currents of transient receptor potential vanilloid (TRPV)1 and mucin 5AC (MUC5AC) secretion dose dependently, which were inhibited by TRPV1 antagonist capsazepine in a concentration-dependent manner. TRPV1 agonist capsaicin mediated a concentration-dependent increase in TRPV1 inward currents and MUC5AC secretion. Furthermore, capsaicin enhanced acid-induced TRPV1 inward currents and MUC5AC secretion. Acid-induced Ca(2+) influx was prevented by capsazepine dose dependently and enhanced by capsaicin. Pretreatment only with capsaicin also increased the Ca(2+) concentration in a concentration-dependent manner. These data suggest that pharmacological inhibition of calcium-permeable TRPV1 receptors could be used to prevent acid-induced mucin secretion, thereby providing a potential mechanism to reduce their toxicity.     

Structures of peptide agonists for human protease activated receptor 2

Protease activated receptor 2 (PAR2) is an unusual G-protein coupled receptor in being self-activated, after pruning of the N-terminus by serine proteases like trypsin and tryptase. Short synthetic peptides corresponding to the newly exposed N-terminal hexapeptide sequence also activate PAR2 on immunoinflammatory, cancer and many normal cell types. (1)H nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy were used here to search for structural clues to activating mechanisms of the hexapeptide agonists SLIGRL (rat), SLIGKV (human) and the peptidomimetic analogue, 2-furoyl-LIGRLO. Either with a free or acetyl capped N-terminus, these agonist peptides display significant propensity in aprotic (DMSO) or lipidic (water-SDS) solvents for turn-like conformations, which are predicted to be receptor-binding conformations in the transmembrane or loops region of PAR2. These motifs may be valuable for the design of small molecule PAR2 agonists and antagonists as prospective new drugs for regulating inflammatory and proliferative diseases.     

Enhanced insulin secretion and sensitization in diabetic mice on chronic treatment with a transient receptor potential vanilloid 1 antagonist

AimsInhibition of transient receptor potential vanilloid 1 (TRPV1) suppresses calcitonin gene-related peptide (CGRP) secretion in pancreatic nerve fiber cells, thereby stimulating insulin secretion. We examined the effects of repeat administration of the TRPV1 antagonist N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamidte monohydrochloride (BCTC) to ob/ob mice, a model of type 2 diabetes with insulin resistance, on whole body glucose and lipid metabolism.Main methodsWe measured blood parameters, including levels of glucose, insulin, and triglycerides, and performed the oral glucose tolerance test (OGTT) after repeat administration of BCTC to ob/ob mice twice a day for four weeks.Key findingsWe found that BCTC treatment reduced fasting glucose, triglyceride, and insulin levels in the whole body. The effects were comparable to that of pioglitazone, a major insulin-sensitizing agent. Further, we found that administration of BCTC significantly increased plasma insulin secretion in the OGTT, which differed from the effect of pioglitazone treatment.SignificanceOur study is the first to show the anti-diabetic pharmacological effects of the TRPV1 signal inhibitor BCTC. These findings suggest that TRPV1 antagonists may represent a new class of drugs effective in treating type 2 diabetes mellitus because of their dual effects as insulin sensitizers and secretagogues.     

Transient receptor potential vanilloid 1, calcitonin gene-related peptide, and substance P mediate nociception in acute pancreatitis

The mechanism of pancreatitis-induced pain is unknown. In other tissues, inflammation activates transient receptor potential vanilloid 1 (TRPV1) on sensory nerves to liberate CGRP and substance P (SP) in peripheral tissues and the dorsal horn to cause neurogenic inflammation and pain, respectively. We evaluated the contribution of TRPV1, CGRP, and SP to pancreatic pain in rats. TRPV1, CGRP, and SP were coexpressed in nerve fibers of the pancreas. Injection of the TRPV1 agonist capsaicin into the pancreatic duct induced endocytosis of the neurokinin 1 receptor in spinal neurons in the dorsal horn (T10), indicative of SP release upon stimulation of pancreatic sensory nerves. Induction of necrotizing pancreatitis by treatment with L-arginine caused a 12-fold increase in the number of spinal neurons expressing the proto-oncogene c-fos in laminae I and II of L1, suggesting activation of nociceptive pathways. L-arginine also caused a threefold increase in spontaneous abdominal contractions detected by electromyography, suggestive of referred pain. Systemic administration of the TRPV1 antagonist capsazepine inhibited c-fos expression by 2.5-fold and abdominal contractions by 4-fold. Intrathecal, but not systemic, administration of antagonists of CGRP (CGRP(8-37)) and SP (SR140333) receptors attenuated c-fos expression in spinal neurons by twofold. Thus necrotizing pancreatitis activates TRPV1 on pancreatic sensory nerves to release SP and CGRP in the dorsal horn, resulting in nociception. Antagonism of TRPV1, SP, and CGRP receptors may suppress pancreatitis pain.     

Role of transient receptor potential vanilloid 2 in LPS-induced cytokine production in macrophages

There is considerable evidence indicating that intracellular Ca²⁺ participates as a second messenger in TLR4-dependent signaling. However, how intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) is increased in response to LPS and how they affect cytokine production are poorly understood. Here we examined the role of transient receptor potential (TRP), a major Ca²⁺ permeation pathway in non-excitable cells, in the LPS-induced cytokine production in macrophages. Pharmacologic experiments suggested that TRPV family members, but neither TRPC nor TRPM family members, are involved in the LPS-induced TNFα and IL-6 production in RAW264 macrophages. RT-PCR and immunoblot analyses showed that TRPV2 is the sole member of TRPV family expressed in macrophages. ShRNA against TRPV2 inhibited the LPS-induced TNFα and IL-6 production as well as IκBα degradation. Experiments using BAPTA/AM and EGTA, and Ca²⁺ imaging suggested that the LPS-induced increase in [Ca²⁺]_i involves both the TRPV2-mediated intracellular and extracellular Ca²⁺ mobilizations. BAPTA/AM abolished LPS-induced TNFα and IL-6 production, while EGTA only partially suppressed LPS-induced IL-6 production, but not TNFα production. These data indicate that TRPV2 is involved in the LPS-induced Ca²⁺ mobilization from intracellular Ca²⁺ store and extracellular Ca²⁺. In addition to Ca²⁺ mobilization through the IP₃-receptor, TRPV2-mediated intracellular Ca²⁺ mobilization is involved in NFκB-dependent TNFα and IL-6 expression, while extracellular Ca²⁺ entry is involved in NFκB-independent IL-6 production.     

Berberine via suppression of transient receptor potential vanilloid 4 channel improves vascular stiffness in mice

Berberine, as an alkaloid found in many Chinese herbs, improves vascular functions in patients with cardiovascular diseases. We determined the effects of berberine in hypertension and vascular ageing, and elucidated the underlying mechanisms. In isolated aortas, berberine dose‐dependently elicited aortic relaxation. In cultured cells, berberine induced the relaxation of vascular smooth muscle cells (VSMCs). Overexpression of transient receptor potential vanilloid 4 (TRPV4) channel by genetic approaches abolished the berberine‐induced reduction in intracellular Ca²⁺ concentration in VSMCs and attenuated berberine‐elicited vessel dilation in mice aortas. In deoxycorticosterone acetate (DOCA)‐induced hypertensive model, treatment of mice with berberine or RN‐1734, a pharmacological inhibitor of TRPV4, significantly decreased systemic blood pressure (BP) in control mice or mice infected with an adenovirus vector. However, berberine‐induced effects of lowering BP were reversed by overexpressing TRPV4 in mice by infecting with adenovirus. Furthermore, long‐term administration of berberine decreased mean BP and pulse BP, increased artery response to vasodilator and reduced vascular collagen content in aged mice deficient in apolipoprotein E (Apoe‐KO), but not in Apoe‐KO old mice with lentivirus‐mediated overexpression of TRPV4 channel. In conclusion, berberine induces direct vasorelaxation to lower BP and reduces vascular stiffness in aged mice through suppression of TRPV4.     

Rap1 mediates galanin receptor 2-induced proliferation and survival in squamous cell carcinoma

Previously we showed that galanin, a neuropeptide, is secreted by human squamous cell carcinoma of the head and neck (SCCHN) in which it exhibits an autocrine mitogenic effect. We also showed that rap1, a ras-like signaling protein, is a critical mediator of SCCHN progression. Given the emerging importance of the galanin cascade in regulating proliferation and survival, we investigated the effect of GAL on SCCHN progression via induction of galanin receptor 2 (GALR2)-mediated rap1 activation. Studies were performed in multiple SCCHN cell lines by inducing endogenous GALR2, by stably overexpressing GALR2 and by downregulating endogenous GALR2 with siGALR2. Cell proliferation and survival, mediated by the ERK and AKT signaling cascades, respectively, were evaluated by functional and immunoblot analysis. The role of rap1 in GALR2-mediated proliferation and survival was evaluated by modulating expression. Finally, the effect of GALR2 on tumor growth was determined. GALR2 stimulated proliferation and survival via ERK and AKT activation, respectively. Knockdown or inactivation of rap1 inhibited GALR2-induced, AKT and ERK-mediated survival and proliferation. Overexpression of GALR2 promoted tumor growth in vivo. GALR2 promotes proliferation and survival in vitro, and promotes tumor growth in vivo, consistent with an oncogenic role for GALR2 in SCCHN.     

Silencing of vanilloid receptor TRPV1 by RNAi reduces neuropathic and visceral pain in vivo

RNA interference (RNAi) has proven to be a powerful technique to study the function of genes by producing knock-down phenotypes. Here, we report that intrathecal injection of an siRNA against the transient receptor potential vanilloid receptor 1 (TRPV1) reduced cold allodynia of mononeuropathic rats by more than 50% over a time period of approximately 5 days. A second siRNA targeted to a different region of the TRPV1 gene was employed and confirmed the analgesic action of a TRPV1 knock-down. Furthermore, siRNA treatment diminished spontaneous visceral pain behavior induced by capsaicin application to the rectum of mice. The analgesic effect of siRNA-mediated knockdown of TRPV1 in the visceral pain model was comparable to that of the low-molecular weight receptor antagonist BCTC. Our data demonstrate that TRPV1 antagonists, including TRPV1 siRNAs, have potential in the treatment of both, neuropathic and visceral pain.     

Essential role of platelet activation via protease activated receptor 4 in tissue factor-initiated inflammation

Introduction  

Tissue factor (TF) activation of the coagulation proteases enhances inflammation in animal models of arthritis and endotoxemia, but the mechanism of this effect is not yet fully understood – in particular, whether this is primarily due to fibrin formation or through activation of protease activated receptors (PARs).