Heat and AITC activate green anole TRPA1 in a membrane-delimited manner

Transient receptor potential ankyrin 1 (TRPA1) is a member of the large TRP super family of ion channels and functions as a Ca²⁺-permeable nonselective cation channel that is activated by various noxious stimuli. TRPA1 was initially identified as a potential mediator of noxious cold stimuli in mammalian nociceptive sensory neurons, while TRPA1s from nonmammalian vertebrates (snakes, green anole lizards, and frogs) were recently reported to be activated by heat, but not cold stimulus. In this study, we examined detailed properties of the green anole TRPA1 channel (gaTRPA1) related to thermal and chemical stimulation in whole-cell and single-channel recordings. Heat activates gaTRPA1 with a temperature threshold for activation of 35.8 °C, while heat together with allyl isothiocyanate (AITC), a chemical agonist, had synergistic effects on gaTRPA1 channel activation in that either the temperature threshold or activating AITC concentration was reduced in the presence of the other stimulus. Significant heat-evoked gaTRPA1 activation was observed in the presence but not absence of extracellular Ca²⁺. gaTRPA1 channels were also activated by heat and AITC in excised membrane patches with an inside-out configuration. By comparing the kinetics of heat- and AITC-evoked single-channel currents, we defined similarities and differences of gaTRPA1 channel responses to heat and AITC. We observed similar current-voltage relationship and unitary amplitudes for heat- and AITC-evoked currents and found that heat-activated currents showed shorter durations of both open and closed times. Our results suggest that the gaTRPA1 channel is directly activated by heat and chemical stimuli.     

Mustard oil enhances spinal neuronal responses to noxious heat but not cooling

The TRPA1 agonist mustard oil (allyl isothiocyanate = AITC) induces heat hyperalgesia and mechanical allodynia in human skin and sensitizes rat spinal wide dynamic range (WDR) neuronal responses to noxious skin heating. We presently used electrophysiological methods to investigate if AITC affects the responsiveness of individual spinal WDR neurons to intense skin cooling. Recordings were made from cold-sensitive WDR neurons in lamina I and deeper dorsal horn; 21/23 also responded to noxious skin heating. Topical application of AITC excited 8/18 units and significantly enhanced their responses to noxious heat while not significantly affecting responses to the cold stimulus. Vehicle (mineral oil) had no effect on thermal responses. The data confirm a role for the TRPA1 agonist AITC in enhancing heat nociception without significantly affecting cold sensitivity.     

Effects of TRPA1 agonists mustard oil and cinnamaldehyde on lumbar spinal wide-dynamic range neuronal responses to innocuous and noxious cutaneous stimuli in rats

Mustard oil [allyl isothiocyanate (AITC)] and cinnamaldehyde (CA), agonists of the ion channel TRPA1 expressed in sensory neurons, elicit a burning sensation and heat hyperalgesia. We tested whether these phenomena are reflected in the responses of lumbar spinal wide-dynamic range (WDR) neurons recorded in pentobarbital-anesthetized rats. Responses to electrical and graded mechanical and noxious thermal stimulation were tested before and after cutaneous application of AITC or CA. Repetitive application of AITC initially increased the firing rate of 52% of units followed by rapid desensitization that persisted when AITC was reapplied 30 min later. Responses to noxious thermal, but not mechanical, stimuli were significantly enhanced irrespective of whether the neuron was directly activated by AITC. Windup elicited by percutaneous or sciatic nerve electrical stimulation was significantly reduced post-AITC. These results indicate that AITC produced central inhibition and peripheral sensitization of heat nociceptors. CA did not directly excite WDR neurons, and significantly enhanced responses to noxious heat while not affecting windup or responses to skin cooling or mechanical stimulation, indicating a peripheral sensitization of heat nociceptors.     

Inhibition of sensory neuronal TRPs contributes to anti-nociception by butamben

Butamben (n-butyl-p-aminobenzoic acid) is a pain-relieving local anesthetic for topical use. Blockade of voltage-gated channel expressed in the peripheral sensory neurons has been suggested as a mechanism of action. Its effects on another sensory neuronal channel family, transient receptor potential (TRP) have remained unclear. In this study we attempted to address this question using six sensory neuronal TRP channel-expressing heterologous systems, cultured sensory neurons and TRP-mediated acute animal pain tests. In Ca²⁺ imaging and whole cell electrophysiology, TRPA1 and TRPV4 were blocked by micromolar butamben. Butamben also activated TRPA1 at millimolar concentrations. The inhibitory effects on the two TRP channels were reproducible in sensory neurons. Moreover, butamben attenuated acute animal pain behaviors in a TRPA1- or TRPV4-dependent manner. Para-aminobenzoic acid (PABA), an analog of a simpler chemical structure, displayed similar in vitro and in vivo properties, suggestive that chemical structure is important for the two TRP-specificity. Our findings suggest that inhibition of TRPA1 and TRPV4 contribute to the peripheral analgesic mechanisms of butamben.     

Contribution of TRPV1 to the bradykinin-evoked nociceptive behavior and excitation of cutaneous sensory neurons

Bradykinin (BK), a major inflammatory mediator, excites and sensitizes nociceptor neurons/fibers, thus evoking pain and hyperalgesia. The cellular signaling mechanisms underlying these actions have remained unsolved, especially in regard to the identity of channels that mediate acute excitation. Here, to clarify the contribution of transient receptor potential vanilloid 1 (TRPV1), a heat-sensitive ion channel, to the BK-evoked nociceptor excitation and pain, we examined the behavioral and physiological BK-responses in TRPV1-deficient (KO) mice. A nocifencive behavior after BK injection (100 pmol/site) into mouse sole was reduced in TRPV1-KO mice compared with wild-type (WT). A higher dose of BK (1 nmol/site), however, induced the response in TRPV1-KO mice indistinguishable from that in the WT. BK-evoked excitation of cutaneous C-fibers in TRPV1-KO mice was comparable to that in WT. BK clearly increased intracellular calcium in cultured dorsal root ganglion (DRG) neurons of TRPV1-KO mice, although the incidence of BK-sensitive neurons was reduced. BK has been reported to activate TRPA1 indirectly, yet a considerable part of BK-sensitive DRG neurons did not respond to a TRPA1 agonist, mustard oil. These results suggest that BK-evoked nociception/nociceptor response would not be simply explained by activation of TRPV1 and A1, and that BK-evoked nociceptor excitation would be mediated by several ionic mechanisms.     

The transient receptor potential channel TRPA1: from gene to pathophysiology

The Transient Receptor Potential Ankyrin 1 channel (TRPA1), is a member of the large TRP family of ion channels, and functions as a Ca²⁺ permeable non-selective cation channel in many different cell processes, ranging from sensory to homeostatic tasks. TRPA1 is highly conserved across the animal kingdom. The only mammalian TRPA subfamily member, TRPA1, is widely expressed in neuronal (e.g. sensory dorsal root and trigeminal ganglia neurons)- and in non-neuronal cells (e.g. epithelial cells, hair cells). It exhibits 14–19 amino-(N-)terminal ankyrin repeats, an unusual structural feature. The TRPA1 channel is activated by noxious cold (<17?°C) as well as by a plethora of chemical compounds that includes not only electrophilic compounds and oxidants that can modify, in an alkylative or oxidative fashion, nucleophilic cysteine residues in the channel’s N-terminus, but also compounds that do not covalently bind to the channel proteins (e.g. menthol, nifedipin). Based on localization and functional properties, TRPA1 is considered a key player in acute and chronic (neuropathic) pain and inflammation. Moreover, its role in the (patho)physiology of nearly all organ systems is anticipated, and will be discussed along with the potential of TRPA1 as a drug target for the management of various pathological conditions.     

Cinnamaldehyde inhibits L-type calcium channels in mouse ventricular cardiomyocytes and vascular smooth muscle cells

Cinnamaldehyde (CA), a major component of cinnamon, is known to have important actions in the cardiovascular system, including vasorelaxation and decrease in blood pressure. Although CA-induced activation of the chemosensory cation channel TRPA1 seems to be involved in these phenomena, it has been shown that genetic ablation of Trpa1 is insufficient to abolish CA effects. Here, we confirm that CA relaxes rat aortic rings and report that it has negative inotropic and chronotropic effects on isolated mouse hearts. Considering the major role of L-type Ca²⁺ channels in the control of the vascular tone and cardiac contraction, we used whole-cell patch-clamp to test whether CA affects L-type Ca²⁺ currents in mouse ventricular cardiomyocytes (VCM, with Ca²⁺ as charge carrier) and in mesenteric artery smooth muscle cells (VSMC, with Ba²⁺ as charge carrier). We found that CA inhibited L-type currents in both cell types in a concentration-dependent manner, with little voltage-dependent effects. However, CA was more potent in VCM than in VSMC and caused opposite effects on the rate of inactivation. We found these divergences to be at least in part due to the use of different charge carriers. We conclude that CA inhibits L-type Ca²⁺ channels and that this effect may contribute to its vasorelaxing action. Importantly, our results demonstrate that TRPA1 is not a specific target of CA and indicate that the inhibition of voltage-gated Ca²⁺ channels should be taken into account when using CA to probe the pathophysiological roles of TRPA1.     

Transient receptor potential ankyrin-1 participates in visceral hyperalgesia following experimental colitis

Transient receptor potential ankyrin-1 (TRPA1) is an important receptor that contributes to inflammatory pain. However, previous studies were mainly concerned with its function in somatic hyperalgesia while few referred to visceral, especially colonic inflammatory hyperalgesia. The present study was aimed to investigate the role of TRPA1 in visceral hyperalgesia after trinitrobenzene sulfonic acid (TNBS)-induced colitis. Results indicate that TNBS induced a significant increase in visceral sensitivity to colonic distension and chemical irritation accompanied by up-regulation of TRPA1 in colonic afferent dorsal root ganglia (DRG). Intrathecal administration of TRPA1 antisense (AS) oligodeoxynucleotide (ODN) reduced the TRPA1 expression in DRG as well as suppressed the colitis-induced hyperalgesia to nociceptive colonic distension and intracolonic allyl isothiocyanate (AITC). Meanwhile the TRPA1 antisense ODN had no effect on transient receptor potential vanilloid-1 (TRPV1) expression, which was proposed to highly co-express with TRPA1, and no effect on the response to TRPV1 agonist, capsaicin. These data suggest an apparent role of TRPA1 in visceral hyperalgesia following colitis that might provide a novel therapeutic target for the relief of pain.     

Histamine potentiates acid-induced responses mediating transient receptor potential V1 in mouse primary sensory neurons

In inflamed tissues, extracellular pH decreases and acidosis is an important source of pain. Histamine is released from mast cells under inflammatory conditions and evokes the pain sensation in vivo, but the cellular mechanism of histamine-induced pain has not been well understood. In the present study, we examined the effects of histamine on [Ca²⁺]_i and membrane potential responses to acid in isolated mouse dorsal root ganglion (DRG) neurons. In capsaicin-sensitive DRG neurons from wild-type mice, acid (>pH 5.0) evoked [Ca²⁺]_i increases, but not in DRG neurons from transient receptor potential V1 (TRPV1) (−/−) mice. Regardless of isolectin GS-IB4 (IB4)-staining, histamine potentiated [Ca²⁺]_i responses to acid (≥pH 6.0) that were mediated by TRPV1 activation. Histamine increased membrane depolarization induced by acid and evoked spike discharges. RT-PCR indicated the expression of all four histamine receptors (H1R, H2R, H3R, H4R) in mouse DRG. The potentiating effect of histamine was mimicked by an H1R agonist, but not H2R-H4R agonists and was inhibited only by an H1R antagonist. Histamine failed to potentiate the [Ca²⁺]_i response to acid in the presence of inhibitors for phospholipase C (PLC) and protein kinase C (PKC). A lipoxygenase inhibitor and protein kinase A inhibitor did not affect the potentiating effects of histamine. Carrageenan and complete Freund's adjuvant produced inflammatory hyperalgesia, but these inflammatory conditions did not change the potentiating effects of histamine in DRG neurons. The present results suggest that histamine sensitizes acid-induced responses through TRPV1 activation via H1R coupled with PLC/PKC pathways, the action of which may be involved in the generation of inflammatory pain.     

Polycystins: polymodal receptor/ion-channel cellular sensors

Transient receptor potential (TRP) channel proteins are divided into seven subgroups that are currently designated as TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPN (NOMP-C, from no mechanoreceptor potential-C), TRPA (ankyrin-like with transmembrane domains 1) and TRPP (polycystin). TRPC, TRPV and TRPM are related to canonical TRP proteins whereas TRPN, TRPA and TRPP (polycystin) are more divergent. Most TRP channels are linked to sensory stimuli, including phototransduction, thermosensation and mechanosensation. The TRPP subfamily was named after its founding member, polycystin kidney disease-2 (PKD2), a gene product mutated in many cases of autosomal dominant polycystic kidney disease (ADPKD). ADPKD is a major inherited nephropathy, affecting over 1:1,000 of the worldwide population, characterized by the progressive development of fluid-filled cysts from the tubules and collecting ducts of affected kidneys. Loss-of-function mutations in either polycystin-2, a non-selective cation channel, or polycystin-1 (PKD1), a large plasma membrane integral protein, give rise to ADPKD. PKD1 and PKD2 are thought to function together as part of a multiprotein receptor/ion-channel complex or independently and may be involved in transducing Ca²⁺-dependent mechanosensitive signals in response to cilia bending in renal epithelial cells and endodermally derived cells. Further information on the growing number and physiological properties of these TRP-polycystins is the basis of this review.     

Renoprotection: focus on TRPV1, TRPV4, TRPC6 and TRPM2

Members of the transient receptor potential (TRP) cation channel receptor family have unique sites of regulatory function in the kidney which enables them to promote regional vasodilatation and controlled Ca²⁺ influx into podocytes and tubular cells. Activated TRP vanilloid 1 receptor channels (TRPV1) have been found to elicit renoprotection in rodent models of acute kidney injury following ischaemia/reperfusion. Transient receptor potential cation channel, subfamily C, member 6 (TRPC6) in podocytes is involved in chronic proteinuric kidney disease, particularly in focal segmental glomerulosclerosis (FSGS). TRP vanilloid 4 receptor channels (TRPV4) are highly expressed in the kidney, where they induce Ca²⁺ influx into endothelial and tubular cells. TRP melastatin (TRPM2) non‐selective cation channels are expressed in the cytoplasm and intracellular organelles, where their inhibition ameliorates ischaemic renal pathology. Although some of their basic properties have been recently identified, the renovascular role of TRPV1, TRPV4, TRPC6 and TRPM2 channels in disease states such as obesity, hypertension and diabetes is largely unknown. In this review, we discuss recent evidence for TRPV1, TRPV4, TRPC6 and TRPM2 serving as potential targets for acute and chronic renoprotection in chronic vascular and metabolic disease.     

Activation of TRPA1 channels by fenamate nonsteroidal anti-inflammatory drugs

Transient receptor potential A1 (TRPA1) forms nonselective cation channels implicated in acute inflammatory pain and nociception. The mechanism of ligand activation of TRPA1 may involve either covalent modification of cysteine residues or conventional reversible ligand–receptor interactions. For certain electrophilic prostaglandins, covalent modification has been considered as the main mechanism involved in their stimulatory effect on TRPA1. Because some nonsteroidal anti-inflammatory drugs (NSAIDs) are structural analogs of prostaglandins, we examined several nonelectrophilic NSAIDs on TRPA1 activation using electrophysiological techniques and intracellular Ca²⁺ measurements and found that a selected group of NSAIDs can act as TRPA1 agonists. Extracellularly applied flufenamic, niflumic, and mefenamic acid, as well as flurbiprofen, ketoprofen, diclofenac, and indomethacin, rapidly activated rat TRPA1 expressed in Xenopus oocytes and human TRPA1 endogenously expressed in WI-38 fibroblasts. Similarly, the NSAID ligands activated human TRPA1 inducibly expressed in HEK293 cells, but the responses were absent in uninduced and parental HEK293 cells. The response to fenamate agonists was blocked by TRPA1 antagonists, AP-18, HC-030031, and ruthenium red. At subsaturating concentrations, the fenamate NSAIDs also potentiate the activation of TRPA1 by allyl isothiocyanate, cinnamaldehyde, and cold, demonstrating positive synergistic interactions with other well-characterized TRPA1 activators. Importantly, among several thermosensitive TRP channels, the stimulatory effect is specific to TRPA1 because flufenamic acid inhibited TRPV1, TRPV3, and TRPM8. We conclude that fenamate NSAIDs are a novel class of potent and reversible direct agonists of TRPA1. This selective group of TRPA1-stimulating NSAIDs should provide a structural basis for developing novel ligands that noncovalently interact with TRPA1 channels.     

Excitation and sensitization of nociceptors by bradykinin: what do we know?

Bradykinin is an endogenous nonapeptide known to induce pain and hyperalgesia to heat and mechanical stimulation. Correspondingly, it excites nociceptors in various tissues and sensitizes them to heat, whereas sensitizing effect on the mechanical response of nociceptors is not well established. Protein kinase C and TRPV1 contribute to the sensitizing mechanism of bradykinin to heat. In addition, TRPA1 and other ion channels appear to contribute to excitation caused by bradykinin. Finally, prostaglandins sensitize bradykinin-induced excitation in normal tissues by restoring desensitized responses due to the inhibition of protein kinase A.     

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.     

Calcium-dependent decrease in the single-channel conductance of TRPV1

TRPV1 is a Ca²⁺ permeable cation channel gated by multiple stimuli including noxious heat, capsaicin, protons, and extracellular cations. In this paper, we show that Ca²⁺ causes a concentration and voltage-dependent decrease in the capsaicin-gated TRPV1 single-channel conductance. This Ca²⁺-dependent effect on conductance was strongest at membrane potentials between −60 and +20 mV, but was diminished at more hyperpolarised potentials. Using simultaneous recordings of membrane current and fura-2 fluorescence to measure the fractional Ca²⁺ current of whole-cell currents evoked through wild-type and mutant TRPV1, we investigated a possible link between the mechanisms underlying Ca²⁺ permeation and the Ca²⁺-dependent effect on conductance. Surprisingly, we found no evidence of a structural correlation, and observed that the substitution of amino acids known to regulate Ca²⁺ permeability had little effect on the ability for Ca²⁺ to decrease TRPV1 conductance. However, we did observe that the Ca²⁺-dependent effect on conductance was not diminished by negative hyperpolarisation for a mutant receptor with severely impaired Ca²⁺ permeability, TRPV1-D646N/E648Q/E651Q. This would be consistent with the idea that Ca²⁺ reduces conductance by interacting with an intra-pore binding site, and that negative hyperpolarization reduces occupancy of this site by speeding the exit of Ca²⁺ into the cell. Taken together, our data show that in addition to directly and indirectly regulating channel gating, Ca²⁺ also directly reduces the conductance of TRPV1. Surprisingly, the mechanism underlying this Ca²⁺-dependent effect on conductance is largely independent of mechanisms governing Ca²⁺ permeability.     

Role of transient receptor potential ankyrin subfamily member 1 in pruritus induced by endothelin-1

Noxious cold reduces pruritus and transient receptor potential ankyrin subfamily member 1 (TRPA1), a non-selective cation channel, is known as a noxious cold-activated ion channel. Recent findings implicated the involvement of TRPA1 in pain induced by endothelin-1 (ET-1). Therefore, we evaluated its potential role in pruritus induced by ET-1. We found that ruthenium red (RR; a nonselective TRP inhibitor) and AP18 (a TRPA1 antagonist) significantly increased scratching bouts caused by ET-1, while capsazepine (a TRPV1 antagonist) and morphine showed no effects in the ET-1-induced scratching response. However, RR and capsazepine significantly reduced scratching bouts caused by histamine. Our results suggested that activation of TRPA1 could suppress itch induced by ET-1 and this is not related to pain induced by ET-1.     

Primary alcohols activate human TRPA1 channel in a carbon chain length-dependent manner

Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable non-selective cation channel that is mainly expressed in primary nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent compounds such as mustard oil and cinnamaldehyde, and intracellular alkalization. Here, we show that primary alcohols, which have been reported to cause skin, eye or nasal irritation, activate human TRPA1 (hTRPA1). We measured intracellular Ca²⁺ changes in HEK293 cells expressing hTRPA1 induced by 1 mM primary alcohols. Higher alcohols (1-butanol to 1-octanol) showed Ca²⁺ increases proportional to the carbon chain length. In whole-cell patch-clamp recordings, higher alcohols (1-hexanol to 1-octanol) activated hTRPA1 and the potency increased with the carbon chain length. Higher alcohols evoked single-channel opening of hTRPA1 in an inside-out configuration. In addition, cysteine at 665 in the N terminus and histidine at 983 in the C terminus were important for hTRPA1 activation by primary alcohols. Furthermore, straight-chain secondary alcohols increased intracellular Ca²⁺ concentrations in HEK293 cells expressing hTRPA1, and both primary and secondary alcohols showed hTRPA1 activation activities that correlated highly with their octanol/water partition coefficients. On the other hand, mouse TRPA1 did not show a strong response to 1-hexanol or 1-octanol, nor did these alcohols evoke significant pain in mice. We conclude that primary and secondary alcohols activate hTRPA1 in a carbon chain length-dependent manner. TRPA1 could be a sensor of alcohols inducing skin, eye and nasal irritation in human.     

Behavioral evidence of thermal hyperalgesia and mechanical allodynia induced by intradermal cinnamaldehyde in rats

TRPA1 agonists cinnamaldehyde (CA) and mustard oil (allyl isothiocyanate = AITC) induce heat hyperalgesia and mechanical allodynia in human skin, and sensitize responses of spinal and trigeminal dorsal horn neurons to noxious skin heating in rats. TRPA1 is also implicated in cold nociception. We presently used behavioral methods to investigate if CA affects sensitivity to thermal and mechanical stimuli in rats. Unilateral intraplantar injection of CA (5–20%) induced a significant, concentration-dependent reduction in latency for ipsilateral paw withdrawal from a noxious heat stimulus, peaking (61.7% of pre-injection baseline) by 30 min with partial recovery at 120 min. The highest dose of CA also significantly reduced the contralateral paw withdrawal latency. CA significantly reduced mechanical withdrawal thresholds of the injected paw that peaked sooner (3 min) and was more profound (44.4% of baseline), with no effect contralaterally. Bilateral intraplantar injections of CA resulted in a significant cold hyperalgesia (cold plate test) and a weak enhancement of innocuous cold avoidance (thermal preference test). The data are consistent with roles for TRPA1 in thermal (hot and cold) hyperalgesia and mechanical allodynia.     

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.     

Emerging roles of calcium‐activated K channels and TRPV4 channels in lung oedema and pulmonary circulatory collapse

It has been suggested that the transient receptor potential cation (TRP) channel subfamily V (vanilloid) type 4 (TRPV4) and intermediate conductance calcium‐activated potassium (KCa3.1) channels contribute to endothelium‐dependent vasodilation. Here, we summarize very recent evidence for a synergistic interplay of TRPV4 and KCa3.1 channels in lung disease. Among the endothelial Ca²⁺‐permeable TRPs, TRPV4 is best characterized and produces arterial dilation by stimulating Ca²⁺‐dependent nitric oxide synthesis and endothelium‐dependent hyperpolarization. Besides these roles, some TRP channels control endothelial/epithelial barrier functions and vascular integrity, while KCa3.1 channels provide the driving force required for Cl^? and water transport in some cells and most secretory epithelia. The three conditions, increased pulmonary venous pressure caused by left heart disease, high inflation pressure and chemically induced lung injury, may lead to activation of TRPV4 channels followed by Ca²⁺ influx leading to activation of KCa3.1 channels in endothelial cells ultimately leading to acute lung injury. We find that a deficiency in KCa3.1 channels protects against TRPV4‐induced pulmonary arterial relaxation, fluid extravasation, haemorrhage, pulmonary circulatory collapse and cardiac arrest in vivo. These data identify KCa3.1 channels as crucial molecular components in downstream TRPV4 signal transduction and as a potential target for the prevention of undesired fluid extravasation, vasodilatation and pulmonary circulatory collapse.