TRPV1 variants impair intracellular Ca2+ signaling and may confer susceptibility to malignant hyperthermia

PurposeMalignant hyperthermia (MH) is a pharmacogenetic disorder arising from uncontrolled muscle calcium release due to an abnormality in the sarcoplasmic reticulum (SR) calcium-release mechanism triggered by halogenated inhalational anesthetics. However, the molecular mechanisms involved are still incomplete.MethodsWe aimed to identify transient receptor potential vanilloid 1(TRPV1) variants within the entire coding sequence in patients who developed sensitivity to MH of unknown etiology. In vitro and in vivo functional studies were performed in heterologous expression system, trpv1^−/− mice, and a murine model of human MH.ResultsWe identified TRPV1 variants in two patients and their heterologous expression in muscles of trpv1^−/− mice strongly enhanced calcium release from SR upon halogenated anesthetic stimulation, suggesting they could be responsible for the MH phenotype. We confirmed the in vivo significance by using mice with a knock-in mutation (Y524S) in the type I ryanodine receptor (Ryr1), a mutation analogous to the Y522S mutation associated with MH in humans. We showed that the TRPV1 antagonist capsazepine slows the heat-induced hypermetabolic response in this model.ConclusionWe propose that TRPV1 contributes to MH and could represent an actionable therapeutic target for prevention of the pathology and also be responsible for MH sensitivity when mutated.     

TRPV4 as osmosensor: a transgenic approach

The transient receptor potential vanilloid 4 (TRPV4) ion channel was named initially vanilloid-receptor-related osmotically activated channel (VR-OAC). Preliminary answers to the question, “What is the function of the trpv4 gene in live animals ?” are highlighted briefly in this review. In trpv4 null mice, TRPV4 is necessary for the maintenance of osmotic equilibrium, and in Caenorhabditis elegans transgenic for mammalian TRPV4, TRPV4 directs the osmotic avoidance response in the context of the ASH “nociceptive” neuron. The molecular mechanisms of gating of TRPV4 in vivo need to be determined; in particular, whether TRPV4 in live animals is gated via phosphorylation of defined amino-acid residues or more directly through the osmotic stimulus itself.     

TRPV3 in keratinocytes transmits temperature information to sensory neurons via ATP

Transient receptor potential V3 (TRPV3) and TRPV4 are heat-activated cation channels expressed in keratinocytes. It has been proposed that heat-activation of TRPV3 and/or TRPV4 in the skin may release diffusible molecules which would then activate termini of neighboring dorsal root ganglion (DRG) neurons. Here we show that adenosine triphosphate (ATP) is such a candidate molecule released from keratinocytes upon heating in the co-culture systems. Using TRPV1-deficient DRG neurons, we found that increase in cytosolic Ca²⁺-concentration in DRG neurons upon heating was observed only when neurons were co-cultured with keratinocytes, and this increase was blocked by P2 purinoreceptor antagonists, PPADS and suramin. In a co-culture of keratinocytes with HEK293 cells (transfected with P2X₂ cDNA to serve as a bio-sensor), we observed that heat-activated keratinocytes secretes ATP, and that ATP release is compromised in keratinocytes from TRPV3-deficient mice. This study provides evidence that ATP is a messenger molecule for mainly TRPV3-mediated thermotransduction in skin.     

The TRPV1/2/3 activator 2-aminoethoxydiphenyl borate sensitizes native nociceptive neurons to heat in wildtype but not TRPV1 deficient mice

TRPV1 gene disruption results in a loss of capsaicin and proton responsiveness, but has minimal effects on heat-induced nocifensive behavior, suggesting that sensory transduction of heat is independent of TRPV1. TRPV3, another heat-activated ion channel but insensitive to capsaicin, was shown to be expressed in keratinocytes as well as in sensory neurons projecting to the skin. Recently, 2-aminoethoxydiphenyl borate was introduced as a TRPV3 agonist, but its selectivity was questioned by showing that it activated recombinant TRPV1 and TRPV2 as well. We used the isolated mouse skin-saphenous nerve preparation and whole-cell patch-clamping of cultured dorsal root ganglia neurons from TRPV1-/- and wildtype mice. We found no phenotypic differences between the heat responses of polymodal C-fibers, whereas cultured dorsal root ganglia neurons of TRPV1-/- hardly showed any heat-activated currents. Only C-fibers of wildtype but not TRPV1-/- mice were clearly sensitized to heat by 2-aminoethoxydiphenyl borate 10 and 100 microM; heat-activated current in wildtype neurons was only facilitated at 100 microM. Noxious heat-induced calcitonin gene-related peptide release showed clear deficits (<50%) in TRPV1 deficient skin, but the stimulated calcitonin gene-related peptide release from the isolated skull dura was unaffected. In both models, 2-aminoethoxydiphenyl borate was able to potentiate the heat response (46 degrees C, 5 min) in a concentration-dependent manner, again, only in wildtype but not TRPV1-/- mice, suggesting that TRPV2/3 are not involved in this sensitization to heat. The results further suggest that TRPV1 is not responsible for the normal heat response of native nociceptors but plays the essential role in thermal sensitization and a prominent one in controlling dermal calcitonin gene-related peptide release, i.e. neurogenic inflammation.     

TRPV4 plays an evolutionary conserved role in the transduction of osmotic and mechanical stimuli in live animals

The TRPV4 ion channel, previously named vanilloid receptor-related osmotically activated channel (VR-OAC), functions in vivo in the transduction of osmotic and mechanical stimuli. In trpv4 null mice, TRPV4 was found to be necessary for the maintenance of systemic osmotic equilibrium, and for normal thresholds in response to noxious mechanical stimuli. In a Caenorhabditis elegans TRPV mutant transgenic for mammalian TRPV4, the mammalian transgene was directing the osmotic and mechanical avoidance response in the context of the ASH 'nociceptive' neurone. Molecular mechanisms of gating of TRPV4 in vivo are not known at this point and have to be determined.     

Urothelium EP1 receptor facilitates the micturition reflex in mice

We investigated the presence of EP1 receptor in the urothelium and its role in micturition reflex by examining the effect of intravesical administration of prostaglandin E(2) (PGE2), an EP1 agonist (ONO-DI-004), acetic acid, and capsaicin. Age-matched EP1-KO mice and C57BL/6 wild-type (WT) mice were used. Western blots and standard immunohistochemical procedures were performed. Cystometrygram (CMG) was performed without anesthesia in a restraining cage. ATP release from the cultured urothelium cells was performed using luciferin-luciferase luminometry. The EP1 receptor was found to be present in the urothelium. In WT mice, PGE2 infusion shortened the intercontraction interval (ICI) in a dose-dependent fashion; however, it did not alter the ICI in EP1-KO mice. The EP1 agonist significantly shortened the ICI in WT mice, but not in EP1-KO mice. Acetic acid and capsaicin shortened the ICI in both WT mice and EP1-KO mice. EP1 agonist, PGE2 and capsaicin provoked ATP release from cultured urothelial cells. These results suggest that EP1 receptor was present in bladder urothelium, and could be activated by PGE2 to release ATP. EP1 receptor in urothelium might be important for reflex voiding in pathological conditions.     

The role of muscarinic receptor subtypes in acetylcholine release from urinary bladder obtained from muscarinic receptor knockout mouse

We investigated the subtype of prejunctional muscarinic receptors associated with inhibition of acetylcholine (ACh) released from the mouse bladder. We measured endogenous ACh release in the bladder obtained from the wild-type mice and muscarinic 1-5 (M1-M5) receptor knockout (KO) mice. Electrical field stimulation increased ACh release in all bladder preparations obtained from wild-type and M1-M5 receptor KO mice. The amount of ACh released from M1-M3 and M5 receptor KO mice was equal to that in the wild-type mice. In contrast, the amount of electrical field stimulation-induced ACh release in M4 receptor KO mice was significantly larger than that in the wild-type mice, but the extent of increase was small. Atropine increased electrical field stimulation-induced ACh release to levels found in wild-type mice in all M1-M5 receptor KO mice. In M2/M4 receptor double KO mice, the amount of electrical field stimulation-induced ACh release was equivalent to that in the M4 receptor KO mice. The cholinergic component of electrical field stimulation-induced contraction (in the presence of alpha,beta-methylene ATP) in the detrusor of M4 receptor KO mice was no different from that in the detrusor of wild-type mice. M4 receptor immunoreactivity was located between smooth muscle cells, colocalized with choline acetyltransferase immunoreactivity. These results indicate that the prejunctional inhibitory muscarinic receptors are of the M4 and non-M2 receptor subtypes. The nature of the non-M2 receptors remains unknown.     

The CaSR/TRPV4 coupling mediates pro-inflammatory macrophage function

Aim

Although calcium-sensing receptor (CaSR) and transient receptor potential vanilloid 4 (TRPV4) channels are functionally expressed on macrophages, it is unclear if they work coordinately to mediate macrophage function. The present study investigates whether CaSR couples to TRPV4 channels and mediates macrophage polarization via Ca²⁺ signaling.

Methods

The role of CaSR/TRPV4/Ca²⁺ signaling was assessed in lipopolysaccharide (LPS)-treated peritoneal macrophages (PMs) from wild-type (WT) and TRPV4 knockout (TRPV4 KO) mice. The expression and function of CaSR and TRPV4 in PMs were analyzed by immunofluorescence and digital Ca²⁺ imaging. The correlation factors of M1 polarization, CCR7, IL-1β, and TNFα were detected using q-PCR, western blot, and ELISA.

Results

We found that PMs expressed CaSR and TRPV4, and CaSR activation-induced marked Ca²⁺ signaling predominately through extracellular Ca²⁺ entry, which was inhibited by selective pharmacological blockers of CaSR and TRPV4 channels. The CaSR activation-induced Ca²⁺ signaling was significantly attenuated in PMs from TRPV4 KO mice compared to those from WT mice. Moreover, the CaSR activation-induced Ca²⁺ entry via TRPV4 channels was inhibited by blocking phospholipases A2 (PLA2)/cytochromeP450 (CYP450) and phospholipase C (PLC)/Protein kinase C (PKC) pathways. Finally, CaSR activation promoted the expression and release of M1-associated cytokines IL-1β and TNFɑ, which were attenuated in PMs from TRPV4 KO mice.

Conclusion

We reveal a novel coupling of the CaSR and TRPV4 channels via PLA2/CYP450 and PLC/PKC pathways, promoting a Ca²⁺-dependent M1 macrophage polarization. Modulation of this coupling and downstream pathways may become a potential strategy for the prevention/treatment of immune-related disease.     

Expression and function of bradykinin B1 and B2 receptors in normal and inflamed rat urinary bladder urothelium

The bladder urothelium exhibits dynamic sensory properties that adapt to changes in the local environment. These studies investigated the localization and function of bradykinin receptor subtypes B1 and B2 in the normal and inflamed (cyclophosphamide (CYP)-induced cystitis) bladder urothelium and their contribution to lower urinary tract function in the rat. Our findings indicate that the bradykinin 2 receptor (B2R) but not the bradykinin 1 receptor (B1R) is expressed in control bladder urothelium. B2R immunoreactivity was localized throughout the bladder, including the urothelium and detrusor smooth muscle. Bradykinin-evoked activation of this receptor elevated intracellular calcium  (EC₅₀= 8.4 n m )  in a concentration-related manner and evoked ATP release from control cultured rat urothelial cells. In contrast, B1R mRNA was not detected in control rat urinary bladder; however, following acute (24 h) and chronic (8 day) CYP-induced cystitis in the rat, B1R mRNA was detected throughout the bladder. Functional B1Rs were demonstrated by evoking ATP release and increases in [Ca²⁺]_i in CYP (24 h)-treated cultured rat urothelial cells with a selective B1 receptor agonist (des-Arg⁹-bradykinin). Cystometry performed on control anaesthetized rats revealed that intravesical instillation of bradykinin activated the micturition pathway. Attenuation of this response by the P2 receptor antagonist PPADS suggests that bradykinin-induced micturition facilitation may be due in part to increased purinergic responsiveness. CYP (24 h)-treated rats demonstrated bladder hyperactivity that was significantly reduced by intravesical administration of either B1 (des-Arg¹⁰-Hoe-140) or B2 (Hoe-140) receptor antagonists. These studies demonstrate that urothelial expression of bradykinin receptors is plastic and is altered by pathology.     

CC chemokine ligand 2 upregulates the current density and expression of TRPV1 channels and Nav1.8 sodium channels in dorsal root ganglion neurons

ABSTRACT: BACKGROUND: Inflammation or nerve injury-induced upregulation and release of chemokine CC chemokine ligand 2 (CCL2) within the dorsal root ganglion (DRG) is believed to enhance the activity of DRG nociceptive neurons and cause hyperalgesia. Transient receptor potential vanilloid receptor 1 (TRPV1) and tetrodotoxin (TTX)-resistant Nav1.8 sodium channels play an essential role in regulating the excitability and pain transmission of DRG nociceptive neurons. We therefore tested the hypothesis that CCL2 causes peripheral sensitization of nociceptive DRG neurons by upregulating the function and expression of TRPV1 and Nav1.8 channels. METHODS: DRG neuronal culture was prepared from 3-week-old Sprague-Dawley rats and incubated with various concentrations of CCL2 for 24 to 36 hours. Whole-cell voltage-clamp recordings were performed to record TRPV1 agonist capsaicin-evoked inward currents or TTX-insensitive Na+ currents from control or CCL2-treated small DRG sensory neurons. The CCL2 effect on the mRNA expression of TRPV1 or Nav1.8 was measured by real-time quantitative RT-PCR assay. RESULTS: Pretreatment of CCL2 for 24 to 36 hours dose-dependently (EC50 value = 0.6 +/- 0.05 nM) increased the density of capsaicin-induced currents in small putative DRG nociceptive neurons. TRPV1 mRNA expression was greatly upregulated in DRG neurons preincubated with 5 nM CCL2. Pretreating small DRG sensory neurons with CCL2 also increased the density of TTX-resistant Na+ currents with a concentration-dependent manner (EC50 value = 0.7 +/- 0.06 nM). The Nav1.8 mRNA level was significantly increased in DRG neurons pretreated with CCL2. In contrast, CCL2 preincubation failed to affect the mRNA level of TTX-resistant Nav1.9. In the presence of the specific phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002 or Akt inhibitor IV, CCL2 pretreatment failed to increase the current density of capsaicin-evoked inward currents or TTX-insensitive Na+ currents and the mRNA level of TRPV1 or Nav1.8. CONCLUSIONS: Our results showed that CCL2 increased the function and mRNA level of TRPV1 channels and Nav1.8 sodium channels in small DRG sensory neurons via activating the PI3K/Akt signaling pathway. These findings suggest that following tissue inflammation or peripheral nerve injury, upregulation and release of CCL2 within the DRG could facilitate pain transmission mediated by nociceptive DRG neurons and could induce hyperalgesia by upregulating the expression and function of TRPV1 and Nav1.8 channels in DRG nociceptive neurons.     

GABA(B)-mediated inhibition of multiple modes of glutamate release in the nucleus of the solitary tract

In the caudal portions of the solitary tract (ST) nucleus, primary sensory afferents fall into two broad classes based on the expression of transient receptor potential vanilloid type 1 (TRPV1) receptors. Both afferent classes (TRPV1+/-) have indistinguishable glutamate release mechanisms for ST-evoked excitatory postsynaptic currents (EPSCs). However, TRPV1+ terminals release additional glutamate from a unique, TRPV1-operated vesicle pool that is temperature sensitive and facilitated by ST activity to generate asynchronous EPSCs. This study tested whether presynaptic γ-aminobutyric acid (GABA)(B) receptors inhibit both the evoked and TRPV1-operated release mechanisms on second-order ST nucleus neurons. In horizontal slices, shocks activated single ST axons and evoked the time-invariant (latency jitter <200 μs), glutamatergic EPSCs, which identified second-order neurons. Gabazine eliminated GABA(A) responses in all recordings. The GABA(B) agonist baclofen inhibited the amplitude of ST-EPSCs from both TRPV1+ and TRPV1- afferents with a similar EC(50) (～1.2 μM). In TTX, GABA(B) activation decreased miniature EPSC (mEPSC) rates but not amplitudes, suggesting presynaptic actions downstream from terminal excitability. With calcium entry through voltage-activated calcium channels blocked by cadmium, baclofen reduced mEPSC frequency, indicating that GABA(B) reduced vesicle release by TRPV1-dependent calcium entry. GABA(B) activation also reduced temperature-evoked increases in mEPSC frequency, which relies on TRPV1. Our studies indicate that GABA(B) G protein-coupled receptors are uniformly distributed across all ST primary afferent terminals and act at multiple stages of the excitation-release cascades to suppress both action potential-triggered and TRPV1-coupled glutamate transmission pathways. Moreover, the segregated release cascades within TRPV1+ ST primary afferents represent independent, potential targets for differential modulation.     

Potential role of transient receptor potential (TRP) channels in bladder cancer cells

Transient receptor potential (TRP) channels play important roles in thermal, chemical, and mechanical sensation in various tissues. In this study, we investigated the differences in urothelial TRP channels between normal urothelial cells and bladder cancer cells. TRPV2 and TRPM7 expression levels and TRPV2 activator-induced intracellular Ca²⁺ increases were significantly higher, whereas TRPV4 expression and TRPV4 activator-induced intracellular Ca²⁺ increases were significantly lower in mouse bladder cancer (MBT-2) cells compared to normal mouse urothelial cells. The proliferation rate of MBT-2 cells overexpressing dominant-negative TRPV2 was significantly increased. In contrast, treatment with TRPV2 activators significantly decreased the proliferation rate. TRPM7-overexpressing MBT-2 cells proliferated more slowly, as compared to mock-transfected cells. Moreover, expression of dominant-negative TRPV2 significantly decreased plasma membrane Ca²⁺ permeability of MBT-2 cells as compared to that in mock-transfected cells. Increases in the expression of TRPV2 mRNA, immunoreactivity, and TRPV2 activator-induced intracellular Ca²⁺ were also observed in T24 human bladder cancer cells. These results suggested that TRPV2 and TRPM7 were functionally expressed in bladder cancer cells and served as negative regulators of bladder cancer cell proliferation, most likely to prevent excess mechanical stresses.     

Toll-Like Receptor-Triggered Calcium Mobilization Protects Mice against Bacterial Infection through Extracellular ATP Release

Extracellular ATP (eATP), released as a “danger signal” by injured or stressed cells, plays an important role in the regulation of immune responses, but the relationship between ATP release and innate immune responses is still uncertain. In this study, we demonstrated that ATP was released through Toll-like receptor (TLR)-associated signaling in both Escherichia coli-infected mice and lipopolysaccharide (LPS)- or Pam3CSK4-treated macrophages. This ATP release could be blocked completely only by N-ethylmaleimide (NEM), not by carbenoxolone (CBX), flufenamic acid (FFA), or probenecid, suggesting the key role of exocytosis in this process. Furthermore, LPS-induced ATP release could also be reduced dramatically through suppressing calcium mobilization by use of {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, caffeine, and thapsigargin (TG). In addition, the secretion of interleukin-1β (IL-1β) and CCL-2 was enhanced significantly by ATP, in a time- and dose-dependent manner. Meanwhile, macrophage-mediated phagocytosis of bacteria was also promoted significantly by ATP stimulation. Furthermore, extracellular ATP reduced the number of invading bacteria and protected mice from peritonitis by activating purinergic receptors. Mechanistically, phosphorylation of AKT and ERK was overtly increased by ATP in antibacterial immune responses. Accordingly, if we blocked the P2X- and P2Y-associated signaling pathway by using suramin and pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid), tetrasodium salt (PPADS), the ATP-enhanced immune response was restrained significantly. Taken together, our findings reveal an internal relationship between danger signals and TLR signaling in innate immune responses, which suggests a potential therapeutic significance of calcium mobilization-mediated ATP release in infectious diseases.     

Vav-1 regulates NK T cell development and NK cell cytotoxicity

The hematopoietic-specific Rho-family GTP exchange factor Vav-1 is a regulator of lymphocyte antigen receptor signaling and mediates normal maturation and activation of B and T cells.Recent findings suggest that Vav-1 also forms part of signaling pathways required for natural and antibody dependent cellular cytotoxicity (ADCC) of human NK cells. In this study, we show that Vav-1 is also expressed in murine NK cells. Vav-1(-/-) mice had normal numbers of splenic NK cells, and these displayed a similar expression profile of NK cell receptors as wild-type mice. Unexpectedly, IL-2-activated Vav-1(-/-) NK cells retained normal ADCC. Fc-receptor mediated activation of ERK, JNK, and p38 was also normal. In contrast, Vav-1(-/-) NK cells exhibited reduced natural cytotoxicity against EL4, C4.4.25, RMA and RMA/S. Together, the results demonstrate that Vav-1 is dispensable for mainstream NK cell development, but is required for NK natural cytotoxicity. Unlike the findings for NK cells, NK T cells were dramatically diminished in Vav-1(-/-) mice and splenocytes from Vav-1 mutant mice failed to produce IL-4 in response to in vivo CD3 stimulation. These data highlight the important role of Vav-1 in NK T cell development and NK cell function.     

Direct evidence for activation and desensitization of the capsaicin receptor by N-oleoyldopamine on TRPV1-transfected cell, line in gene deleted mice and in the rat

Effects of the endogenous lipid N-oleoyldopamine (OLDA) were analyzed on the rTRPV1-expressing HT1080 human fibrosarcoma cell line (HT5-1), on cultured rat trigeminal neurons, on the noxious heat threshold of rats and on nocifensive behavior of TRPV1 knockout mice. The EC(50) of capsaicin and OLDA on (45)Ca accumulation of rTRPV1-expressing HT5-1 cells was 36 nM and 1.8 microM, respectively. The efficacy of OLDA was 60% as compared to the maximum response of capsaicin. OLDA (330 nM to 3.3 microM) caused a transient increase in fluorescence of fura-2 loaded cultured small trigeminal neurons of the rat and rTRPV1-transfected HT5-1 cells measured with a ratiometric technique. Repeated application of OLDA and capsaicin caused similar desensitization in the Ca(2+) transients both in cultured neurons and rTRPV1-transfected HT5-1 cells. In the rat intraplantar injection of OLDA (5 nmol) decreased the noxious heat threshold by 6-9 degrees C and this response was strongly inhibited by the TRPV1 antagonist iodoresiniferatoxin (0.05 nmol intraplantarly (i.pl.)). In wild-type mice OLDA (50 nmol i.pl.) evoked paw lifting/licking which was significantly less sustained in TRPV1 knockout mice. It is concluded that on TRPV1 capsaicin receptors OLDA is 50 times less potent than capsaicin and it might serve as an endogenous ligand for TRPV1 in the rat, but more likely in humans.     

The regulation of distention-induced ATP release from urothelium by the adenylyl cyclase-cyclic AMP pathway

Distention of the bladder during urine storage induces ATP release from urothelium, thereby facilitating transmission of visceral sensory signals to afferent nerve fibers. An excess of urothelial ATP release was found in interstitial cystitis, a condition accompanied by hyperesthesia of the urinary bladder; it remains unclear which signals are involved in this upregulation. The present study demonstrated that the adenylyl cyclase pathway enhances distention-induced ATP release in mouse bladder. In the absence of distention, adenylyl cyclase activation by forskolin or cyclic AMP increases by rolipram did not induce significant ATP release. However, forskolin or rolipram significantly enhanced ATP release from urothelium by a physiologically normal urine storage pressure (5 cmH(2)O). Blockade of adenylyl cyclases did not alter pressure-induced ATP release in normal condition. Thus, the adenylyl cyclase-cAMP pathway might be activated in pathological conditions and cause an excess of ATP release.     

TRPV channels and vascular function

Transient receptor potential channels, of the vanilloid subtype (TRPV), act as sensory mediators, being activated by endogenous ligands, heat, mechanical and osmotic stress. Within the vasculature, TRPV channels are expressed in smooth muscle cells, endothelial cells, as well as in peri-vascular nerves. Their varied distribution and polymodal activation properties make them ideally suited to a role in modulating vascular function, perceiving and responding to local environmental changes. In endothelial cells, TRPV1 is activated by endocannabinoids, TRPV3 by dietary agonists and TRPV4 by shear stress, epoxyeicosatrienoic acids (EETs) and downstream of Gq-coupled receptor activation. Upon activation, these channels contribute to vasodilation via nitric oxide, prostacyclin and intermediate/small conductance potassium channel-dependent pathways. In smooth muscle, TRPV4 is activated by endothelial-derived EETs, leading to large conductance potassium channel activation and smooth muscle hyperpolarization. Conversely, smooth muscle TRPV2 channels contribute to global calcium entry and may aid constriction. TRPV1 and TRPV4 are expressed in sensory nerves and can cause vasodilation through calcitonin gene-related peptide and substance P release as well as mediating vascular function via the baroreceptor reflex (TRPV1) or via increasing sympathetic outflow during osmotic stress (TRPV4). Thus, TRPV channels play important roles in the regulation of normal and pathological cellular function in the vasculature.     

TRPV1 involvement in inflammatory tissue fibrosis in mice

We examined whether absence or blocking of transient receptor potential vanilloid subtype 1 (TRPV1) affects the level of inflammation and fibrosis/scarring during healing of injured tissue using an alkali burn model of cornea in mice. A cornea burn was produced with 1 N NaOH instilled into one eye of TRPV1-/- (KO) (n = 88) or TRPV1+/+ (n = 94) mice. Examinations of the corneal surface and eye globe size suggested that the loss of TRPV1 suppressed inflammation and fibrosis/scarring after alkali burn, and this was confirmed by histology, IHC, and gene expression analysis. The loss of TRPV1 inhibited inflammatory cell invasion and myofibroblast generation in association with reduction of expression of proinflammatory and profibrogenic components. Experiments of bone marrow transplantation between either genotype of mice showed that KO corneal tissue resident cells, but not KO bone marrow-derived cells, are responsible for KO-type wound healing with reduced inflammation and fibrosis. The absence of TRPV1 attenuated expression of transforming growth factor β 1 (TGFβ1) and other proinflammatory gene expression in cultured ocular fibroblasts, but did not affect TGFβ1 expression in macrophages. Loss of TRPV1 inhibited myofibroblast transdifferentiation in cultured fibroblasts. Systemic TRPV1 antagonists reproduced the KO type of healing. In conclusion, absence or blocking of TRPV1 suppressed inflammation and fibrosis/scarring during healing of alkali-burned mouse cornea. TRPV1 is a potential drug target for improving the outcome of inflammatory/fibrogenic wound healing.     

Mustard oil induces a transient receptor potential vanilloid 1 receptor-independent neurogenic inflammation and a non-neurogenic cellular inflammatory component in mice

A neurogenic component has been suggested to play a pivotal role in a range of inflammatory/immune diseases. Mustard oil (allyl-isothiocyanate) has been used in studies of inflammation to mediate neurogenic vasodilatation and oedema in rodent skin. The aim of the present study was to analyse mustard oil-induced oedema and neutrophil accumulation in the mouse ear focussing on the roles of neurokinin 1 (NK(1)) and vanilloid (TRPV1) receptors using normal (BALB/c, C57BL/6) as well as NK(1) and TRPV1 receptor knockout mice. A single or double treatment of 1% mustard oil on the BALB/c mouse ear induced ear oedema with responses diminished by 6 h. However a 25-30% increase in ear thickness was maintained by the hourly reapplication of mustard oil. Desensitisation of sensory nerves with capsaicin, or the NK(1) receptor antagonist SR140333, inhibited oedema but only in the first 3 h. Neutrophil accumulation in response to mustard oil was inhibited neither by SR140333 nor capsaicin pre-treatment. An activating dose of capsaicin (2.5%) induced a large oedema in C57BL/6 wild-type mice that was minimal in TRPV1 receptor knockout mice. By comparison, mustard oil generated ear swelling was inhibited by SR140333 in wild-type and TRPV1 knockout mice. Repeated administration of mustard oil maintained 35% oedema in TRPV1 knockout animals and the lack of TRPV1 receptors did not alter the leukocyte accumulation. In contrast repeated treatment caused about 20% ear oedema in Sv129+C57BL/6 wild-type mice but the absence of NK(1) receptors significantly decreased the response. Neutrophil accumulation showed similar values in both groups. This study has revealed that mustard oil can act via both neurogenic and non-neurogenic mechanisms to mediate inflammation in the mouse ear. Importantly, the activation of the sensory nerves was still observed in TRPV1 knockout mice indicating that the neurogenic inflammatory component occurs via a TRPV1 receptor independent process.