N-glycosylation determines ionic permeability and desensitization of the TRPV1 capsaicin receptor

The balance of glycosylation and deglycosylation of ion channels can markedly influence their function and regulation. However, the functional importance of glycosylation of the TRPV1 receptor, a key sensor of pain-sensing nerves, is not well understood, and whether TRPV1 is glycosylated in neurons is unclear. We report that TRPV1 is N-glycosylated and that N-glycosylation is a major determinant of capsaicin-evoked desensitization and ionic permeability. Both N-glycosylated and unglycosylated TRPV1 was detected in extracts of peripheral sensory nerves by Western blotting. TRPV1 expressed in HEK-293 cells exhibited various degrees of glycosylation. A mutant of asparagine 604 (N604T) was not glycosylated but did not alter plasma membrane expression of TRPV1. Capsaicin-evoked increases in intracellular calcium ([Ca(2+)](i)) were sustained in wild-type TRPV1 HEK-293 cells but were rapidly desensitized in N604T TRPV1 cells. There was marked cell-to-cell variability in capsaicin responses and desensitization between individual cells expressing wild-type TRPV1 but highly uniform responses in cells expressing N604T TRPV1, consistent with variable levels of glycosylation of the wild-type channel. These differences were also apparent when wild-type or N604T TRPV1-GFP fusion proteins were expressed in neurons from trpv1(-/-) mice. Capsaicin evoked a marked, concentration-dependent increase in uptake of the large cationic dye YO-PRO-1 in cells expressing wild-type TRPV1, indicative of loss of ion selectivity, that was completely absent in cells expressing N604T TRPV1. Thus, TRPV1 is variably N-glycosylated and glycosylation is a key determinant of capsaicin regulation of TRPV1 desensitization and permeability. Our findings suggest that physiological or pathological alterations in TRPV1 glycosylation would affect TRPV1 function and pain transmission.     

Dendritic cells do not transduce inflammatory stimuli via the capsaicin receptor TRPV1

Inflammatory stimuli provide critical activation signals for dendritic cells (DC). Signaling through the capsaicin receptor TRPV1 is reported to initiate DC maturation and migration. We attempted to characterize TRPV1 channels in DC. Capsaicin or extracellular protons failed to elicit a change in intracellular [Ca(2+)] or membrane current in DC. In contrast, capsaicin evoked a sustained increase in [Ca(2+)] and large inwards currents in sensory neurons and TRPV1-expressing HEK293 cells. TRPV1 expression was confirmed by RT-PCR in sensory neurons, but was undetectable in DC. Interestingly, and in contrast to capsaicin, the inflammatory neuropeptide substance P evoked Ca(2+) transients in DC. Thus, our data do not support the hypothesis that DC express TRPV1 channels. Rather, signaling through TRPV1 in sensory nerves may modulate DC via neurogenic actions.     

TRPV1-mediated calcium signal couples with cannabinoid receptors and sodium-calcium exchangers in rat odontoblasts

Odontoblasts are involved in the transduction of stimuli applied to exposed dentin. Although expression of thermo/mechano/osmo-sensitive transient receptor potential (TRP) channels has been demonstrated, the properties of TRP vanilloid 1 (TRPV1)-mediated signaling remain to be clarified. We investigated physiological and pharmacological properties of TRPV1 and its functional coupling with cannabinoid (CB) receptors and Na(+)-Ca(2+) exchangers (NCXs) in odontoblasts. Anandamide (AEA), capsaicin (CAP), resiniferatoxin (RF) or low-pH evoked Ca(2+) influx. This influx was inhibited by capsazepine (CPZ). Delay in time-to-activation of TRPV1 channels was observed between application of AEA or CAP and increase in [Ca(2+)](i). In the absence of extracellular Ca(2+), however, an immediate increase in [Ca(2+)](i) was observed on administration of extracellular Ca(2+), followed by activation of TRPV1 channels. Intracellular application of CAP elicited inward current via opening of TRPV1 channels faster than extracellular application. With extracellular RF application, no time delay was observed in either increase in [Ca(2+)](i) or inward current, indicating that agonist binding sites are located on both extra- and intracellular domains. KB-R7943, an NCX inhibitor, yielded an increase in the decay time constant during TRPV1-mediated Ca(2+) entry. Increase in [Ca(2+)](i) by CB receptor agonist, 2-arachidonylglycerol, was inhibited by CB1 receptor antagonist or CPZ, as well as by adenylyl cyclase inhibitor. These results showed that TRPV1-mediated Ca(2+) entry functionally couples with CB1 receptor activation via cAMP signaling. Increased [Ca(2+)](i) by TRPV1 activation was extruded by NCXs. Taken together, this suggests that cAMP-mediated CB1-TRPV1 crosstalk and TRPV1-NCX coupling play an important role in driving cellular functions following transduction of external stimuli to odontoblasts.     

Identification of species-specific determinants of the action of the antagonist capsazepine and the agonist PPAHV on TRPV1

The vanilloid receptor 1 (VR1 or TRPV1) ion channel is activated by noxious heat, low pH and by a variety of vanilloid-related compounds. The antagonist, capsazepine is more effective at inhibiting the human TRPV1 response to pH 5.5 than the rat TRPV1 response to this stimulus. Mutation of rat TRPV1 at three positions in the S3 to S4 region, to the corresponding human amino acid residues I514M, V518L, and M547L decreased the IC(50) values for capsazepine inhibition of the pH 5.5 response from >10,000 nm to 924 +/- 241 nm in [Ca(2+)](i) assays and increased capsazepine inhibition of the capsaicin response to levels seen for human TRPV1. We have previously noted that phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) is a strong agonist of rat TRPV1 but not human TRPV1 in [Ca(2+)](i) assays (1). Mutation of methionine 547 in S4 of rat TRPV1 to leucine, found in human TRPV1 (M547L), reduced the ability of PPAHV to activate TRPV1 by approximately 20-fold. The reciprocal mutation of human TRPV1 (L547M) enabled the human receptor to respond to PPAHV. These mutations did not significantly affect the agonist activity of capsaicin, resiniferatoxin (RTX) or olvanil in [Ca(2+)](i) assays. Introducing the equivalent mutation into guinea pig TRPV1 (L549M) increased the agonist potency of PPAHV by > 10-fold in the [Ca(2+)](i) assay and increased the amplitude of the evoked current. The rat M547L mutation reduced the affinity of RTX binding. Thus, amino acids within the S2-S4 region are important sites of agonist and antagonist interaction with TRPV1.     

Na(+)-dependent Ca(2+) transport modulates the secretory response to the Fcepsilon receptor stimulus of mast cells

Immunological stimulation of rat mucosal-type mast cells (RBL-2H3 line) by clustering of their Fcepsilon receptors (FcepsilonRI) causes a rapid and transient increase in free cytoplasmic Ca(2+) ion concentration ([Ca(2+)](i)) because of its release from intracellular stores. This is followed by a sustained elevated [Ca(2+)](i), which is attained by Ca(2+) influx. Because an FcepsilonRI-induced increase in the membrane permeability for Na(+) ions has also been observed, and secretion is at least partially inhibited by lowering of extracellular sodium ion concentrations ([Na(+)](o)), the operation of a Na(+)/Ca(2+) exchanger has been considered. We found significant coupling between the Ca(2+) and Na(+) ion gradients across plasma membranes of RBL-2H3 cells, which we investigated employing (23)Na-NMR, (45)Ca(2+), (85)Sr(2+), and the Ca(2+)-sensitive fluorescent probe indo-1. The reduction in extracellular Ca(2+) concentrations ([Ca(2+)](o)) provoked a [Na(+)](i) increase, and a decrease in [Na(+)](o) results in a Ca(2+) influx as well as an increase in [Ca(2+)](i). Mediator secretion assays, monitoring the released beta-hexosaminidase activity, showed in the presence of extracellular sodium a sigmoidal dependence on [Ca(2+)](o). However, the secretion was not affected by varying [Ca(2+)](o) as [Na(+)](o) was lowered to 0.4 mM, while it was almost completely inhibited at [Na(+)](o) = 136 mM and [Ca(2+)](o) < 0.05 mM. Increasing [Na(+)](o) caused the secretion to reach a minimum at [Na(+)](o) = 20 mM, followed by a steady increase to its maximum value at 136 mM. A parallel [Na(+)](o) dependence of the Ca(2+) fluxes was observed: Antigen stimulation at [Na(+)](o) = 136 mM caused a pronounced Ca(2+) influx. At [Na(+)](o) = 17 mM only a slight Ca(2+) efflux was detected, whereas at [Na(+)](o) = 0.4 mM no Ca(2+) transport across the cell membrane could be observed. Our results clearly indicate that the [Na(+)](o) dependence of the secretory response to FcepsilonRI stimulation is due to its influence on the [Ca(2+)](i), which is mediated by a Na(+)-dependent Ca(2+) transport.     

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

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

TRPV channels and modulation by hepatocyte growth factor/scatter factor in human hepatoblastoma (HepG2) cells

Using patch clamp and Ca(2+) imaging techniques, we have studied Ca(2+) entry pathways in human hepatoblastoma (HepG2) cells. These cells express the mRNA of TRPV1, TRPV2, TRPV3 and TRPV4 channels, but not those of TRPV5 and TRPV6. Functional assessment showed that capsaicin (10 microM), 4alpha-phorbol-12,13-didecanoate (4alphaPDD, 1 microM), arachidonic acid (10 microM), hypotonic stress, and heat all stimulated increases in [Ca(2+)](i) within minutes. The increase in [Ca(2+)](i) depended on extracellular Ca(2+) and on the transmembrane potential, which indicated that both driving forces affected Ca(2+) entry. Capsaicin also stimulated an increase in [Ca(2+)](i) in nominally Ca(2+)-free solutions, which was compatible with the receptor functioning as a Ca(2+) release channel. Hepatocyte growth factor/scatter factor (HGF/SF) modulated Ca(2+) entry. Ca(2+) influx was greater in HepG2 cells incubated with HGF/SF (20 ng/ml for 20 h) compared with non-stimulated cells, but this occurred only in those cells with a migrating phenotype as determined by presence of a lamellipodium and trailing footplate. The effect of capsaicin on [Ca(2+)](i) was greater in migrating HGF/SF-treated cells, and this was inhibited by capsazepine. The difference between control and HGF/SF-treated cells was not found in Ca(2+)-free solutions. 4alphaPDD also had no greater effect on HGF/SF-treated cells. We conclude that TRPV1 and TRPV4 channels provide Ca(2+) entry pathways in HepG2 cells. HGF/SF increases Ca(2+) entry via TRPV1, but not via TRPV4. This rise in [Ca(2+)](i) may constitute an early response of a signalling cascade that gives rise to cell locomotion and the migratory phenotype.     

Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives

We have studied activation by phorbol derivatives of TRPV4 channels, the human VRL-2, and murine TRP12 channels, which are highly homologous to the human VR-OAC, and the human and murine OTRPC4 channel. 4alpha-Phorbol 12,13-didecanoate (4alpha-PDD) induced an increase in intracellular Ca(2+) concentration, [Ca(2+)](i), in 1321N1 cells stably transfected with human VRL-2 (hVRL-2.1321N1) or HEK-293 cells transiently transfected with murine TRP12, but not in nontransfected or mock-transfected cells. Concomitantly with the increase in [Ca(2+)](i), 4alpha-PDD activated an outwardly rectifying cation channel with an Eisenman IV permeation sequence for monovalent cations that is Ca(2+)-permeable with P(Ca)/P(Na) = 5.8. Phorbol 12-myristate 13-acetate also induced an increase in [Ca(2+)](i) but was approximately 50 times less effective than 4alpha-PDD. EC(50) for Ca(2+) increase and current activation was nearly identical (pEC(50) approximately 6.7). Similar effects were observed in freshly isolated mouse aorta endothelial cells which express TRP12 endogenously. By using 4alpha-PDD as a tool to stimulate TRP12, we showed that activation of this channel is modulated by [Ca(2+)](i); an increase in [Ca(2+)](i) inhibits the channel with an IC(50) of 406 nm. Ruthenium Red at a concentration of 1 microm completely blocks inward currents at -80 mV but has a smaller effect on outward currents likely indicating a voltage dependent channel block. We concluded that the phorbol derivatives activate TRPV4 (VR-OAC, VRL-2, OTRPC4, TRP12) independently from protein kinase C, in a manner consistent with direct agonist gating of the channel.     

Caged vanilloid ligands for activation of TRPV1 receptors by 1- and 2-photon excitation

Nociceptive neurons in the peripheral nervous system detect noxious stimuli and report the information to the central nervous system. Most nociceptive neurons express the vanilloid receptor, TRPV1, a nonselective cation channel gated by vanilloid ligands such as capsaicin, the pungent essence of chili peppers. Here, we report the synthesis and biological application of two caged vanilloids: biologically inert precursors that, when photolyzed, release bioactive vanilloid ligands. The two caged vanilloids, Nb-VNA and Nv-VNA, are photoreleased with quantum efficiency of 0.13 and 0.041, respectively. Under flash photolysis conditions, photorelease of Nb-VNA and Nv-VNA is 95% complete in approximately 40 micros and approximately 125 micros, respectively. Through 1-photon excitation with ultraviolet light (360 nm), or 2-photon excitation with red light (720 nm), the caged vanilloids can be photoreleased in situ to activate TRPV1 receptors on nociceptive neurons. The consequent increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) can be visualized by laser-scanning confocal imaging of neurons loaded with the fluorescent Ca(2+) indicator, fluo-3. Stimulation results from TRPV1 receptor activation, because the response is blocked by capsazepine, a selective TRPV1 antagonist. In Ca(2+)-free extracellular medium, photoreleased vanilloid can still elevate [Ca(2+)](i), which suggests that TRPV1 receptors also reside on endomembranes in neurons and can mediate Ca(2+) release from intracellular stores. Notably, whole-cell voltage clamp measurements showed that flash photorelease of vanilloid can activate TRPV1 channels in <4 ms at 22 degrees C. In combination with 1- or 2-photon excitation, caged vanilloids are a powerful tool for probing morphologically distinct structures of nociceptive sensory neurons with high spatial and temporal precision.     

Angiotensin II type I receptor modulates intracellular free Mg2+ in renally derived cells via Na+-dependent Ca2+-independent mechanisms

Treatment of Madin-Darby canine kidney (MDCK) cells with the peptide hormone angiotensin II (Ang II) results in an increase in the concentrations of cytosolic free calcium ([Ca(2+)](i)) and sodium ([Na(+)](i)) with a concomitant decrease in cytosolic free Mg(2+) concentration ([Mg(2+)](i)). In the present study we demonstrate that this hormone-induced decrease in [Mg(2+)](i) is independent of [Ca(2+)](i) but dependent on extracellular Na(+). [Mg(2+)](i), [Ca(2+)](i), and [Na(+)](i) were measured in Ang II-stimulated MDCK cells by fluorescence digital imaging using the selective fluoroprobes mag-fura-2AM, fura-2AM, and sodium-binding benzofuran isophthalate (acetoxymethyl ester), respectively. Ang II decreased [Mg(2+)](i) and increased [Na(+)](i) in a dose-dependent manner. These effects were inhibited by irbesartan (selective AT(1) receptor blocker) but not by PD123319 (selective AT(2) receptor blocker). Imipramine and quinidine (putative inhibitors of the Na(+)/Mg(2+) exchanger) and removal of extracellular Na(+) abrogated Ang II-mediated [Mg(2+)](i) effects. In cells pretreated with thapsigargin (reticular Ca(2+)-ATPase inhibitor), Ang II-stimulated [Ca(2+)](i) transients were attenuated (p < 0.01), whereas agonist-induced [Mg(2+)](i) responses were unchanged. Clamping the [Ca(2+)](i) near 50 nmol/liter with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) inhibited Ang II-induced [Ca(2+)](i) increases but failed to alter Ang II-induced [Mg(2+)](i) responses. Benzamil, a selective blocker of the Na(+)/Ca(2+) exchanger, inhibited [Na(+)](i) but not [Mg(2+)](i) responses. Our data demonstrate that in MDCK cells, AT(1) receptors modulate [Mg(2+)](i) via a Na(+)-dependent Mg(2+) transporter that is not directly related to [Ca(2+)](i). These data support the notion that rapid modulation of [Mg(2+)](i) is not simply a result of Mg(2+) redistribution from intracellular buffering sites by Ca(2+) and provide evidence for the existence of a Na(+)-dependent, hormonally regulated transporter for Mg(2+) in renally derived cells.     

Dopamine-induced graded intracellular Ca2+ elevation via the Na+Ca2+ exchanger operating in the Ca2+-entry mode in cockroach salivary ducts

Stimulation with the neurotransmitter dopamine causes an amplitude-modulated increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in epithelial cells of the ducts of cockroach salivary glands. This is completely attributable to a Ca(2+) influx from the extracellular space. Additionally, dopamine induces a massive [Na(+)](i) elevation via the Na(+)K(+)2Cl(-) cotransporter (NKCC). We have reasoned that Ca(2+)-entry is mediated by the Na(+)Ca(2+) exchanger (NCE) operating in the Ca(2+)-entry mode. To test this hypothesis, [Ca(2+)](i) and [Na(+)](i) were measured by using the fluorescent dyes Fura-2, Fluo-3, and SBFI. Inhibition of Na(+)-entry from the extracellular space by removal of extracellular Na(+) or inhibition of the NKCC by 10 microM bumetanide did not influence resting [Ca(2+)](i) but completely abolished the dopamine-induced [Ca(2+)](i) elevation. Simultaneous recordings of [Ca(2+)](i) and [Na(+)](i) revealed that the dopamine-induced [Na(+)](i) elevation preceded the [Ca(2+)](i) elevation. During dopamine stimulation, the generation of an outward Na(+) concentration gradient by removal of extracellular Na(+) boosted the [Ca(2+)](i) elevation. Furthermore, prolonging the dopamine-induced [Na(+)](i) rise by blocking the Na(+)/K(+)-ATPase reduced the recovery from [Ca(2+)](i) elevation. These results indicate that dopamine induces a massive NKCC-mediated elevation in [Na(+)](i), which reverses the NCE activity into the reverse mode causing a graded [Ca(2+)](i) elevation in the duct cells.     

Feedback inhibition of sodium/calcium exchange by mitochondrial calcium accumulation

Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger were subjected to two periods of 5 and 3 min, respectively, during which the extracellular Na(+) concentration ([Na(+)](o)) was reduced to 20 mm; these intervals were separated by a 5-min recovery period at 140 mm Na(+)(o). The cytosolic Ca(2+) concentration ([Ca(2+)](i)) increased during both intervals due to Na(+)-dependent Ca(2+) influx by the exchanger. However, the peak rise in [Ca(2+)](i) during the second interval was only 26% of the first. The reduced rise in [Ca(2+)](i) was due to an inhibition of Na(+)/Ca(2+) exchange activity rather than increased Ca(2+) sequestration since the influx of Ba(2+), which is not sequestered by internal organelles, was also inhibited by a prior interval of Ca(2+) influx. Mitochondria accumulated Ca(2+) during the first interval of reduced [Na(+)](o), as determined by an increase in fluorescence of the Ca(2+)-indicating dye rhod-2, which preferentially labels mitochondria. Agents that blocked mitochondrial Ca(2+) accumulation (uncouplers, nocodazole) eliminated the observed inhibition of exchange activity during the second period of low [Na(+)](o). Conversely, diltiazem, an inhibitor of the mitochondrial Na(+)/Ca(2+) exchanger, increased mitochondrial Ca(2+) accumulation and also increased the inhibition of exchange activity. We conclude that Na(+)/Ca(2+) exchange activity is regulated by a feedback inhibition process linked to mitochondrial Ca(2+) accumulation.     

Activation of recombinant human TRPV1 receptors expressed in SH-SY5Y human neuroblastoma cells increases [Ca(2+)](i), initiates neurotransmitter release and promotes delayed cell death

The transient receptor potential (TRP) vanilloid receptor subtype 1 (TRPV1) is a ligand-gated, Ca(2+)-permeable ion channel in the TRP superfamily of channels. We report the establishment of the first neuronal model expressing recombinant human TRPV1 (SH-SY5Y(hTRPV1)). SH-SY5Y human neuroblastoma cells were stably transfected with hTRPV1 using the Amaxa Biosystem (hTRPV1 in pIREShyg2 with hygromycin selection). Capsaicin, olvanil, resiniferatoxin and the endocannabinoid anandamide increased [Ca(2+)](i) with potency (EC(50)) values of 2.9 nmol/L, 34.7 nmol/L, 0.9 nmol/L and 4.6 micromol/L, respectively. The putative endovanilloid N-arachidonoyl-dopamine increased [Ca(2+)](i) but this response did not reach a maximum. Capsaicin, anandamide, resiniferatoxin and olvanil mediated increases in [Ca(2+)](i) were inhibited by the TRPV1 antagonists capsazepine and iodo-resiniferatoxin with potencies (K(B)) of approximately 70 nmol/L and 2 nmol/L, respectively. Capsaicin stimulated the release of pre-labelled [(3)H]noradrenaline from monolayers of SH-SY5Y(hTRPV1) cells with an EC(50) of 0.6 nmol/L indicating amplification between [Ca(2+)](i) and release. In a perfusion system, we simultaneously measured [(3)H]noradrenaline release and [Ca(2+)](i) and observed that increased [Ca(2+)](i) preceded transmitter release. Capsaicin treatment also produced a cytotoxic response (EC(50) 155 nmol/L) that was antagonist-sensitive and mirrored the [Ca(2+)](I) response. This model displays pharmacology consistent with TRPV1 heterologously expressed in standard non-neuronal cells and native neuronal cultures. The advantage of SH-SY5Y(hTRPV1) is the ability of hTRPV1 to couple to neuronal biochemical machinery and produce large quantities of cells.     

Modulation of TRPV1 by nonreceptor tyrosine kinase, c-Src kinase

The capsaicin receptor TRPV1 is a nonselective cation channel that is expressed in sensory neurons. In this study, we examined the role of the nonreceptor cellular tyrosine kinase c-Src kinase in the modulation of the rat TRPV1. Capsaicin-induced currents in identified colonic dorsal root ganglion neurons were blocked by the c-Src kinase inhibitor PP2 and enhanced by the tyrosine phosphatase inhibitor sodium orthovandate. PP2 also abolished currents in human embryonic kidney-293 cells transfected with rat TRPV1, whereas cotransfection of TRPV1 with v-Src resulted in fivefold increase in capsaicin-induced currents. In cells transfected with dominant-negative c-Src and TRPV1, capsaicin-induced currents were decreased by approximately fourfold. TRPV1 co-immunoprecipitated with Src kinase and was tyrosine phosphorylated. These studies demonstrate that TRPV1 is a potential target for cellular tyrosine kinase-dependent phosphorylation.     

PLA2 and TRPV4 channels regulate endothelial calcium in cerebral arteries

We previously demonstrated that endothelium-derived hyperpolarizing factor (EDHF)-mediated dilations in cerebral arteries are significantly reduced by inhibitors of PLA(2). In this study we examined possible mechanisms by which PLA(2) regulates endothelium-dependent dilation, specifically whether PLA(2) is involved in endothelial Ca(2+) regulation through stimulation of TRPV4 channels. Studies were carried out with middle cerebral arteries (MCA) or freshly isolated MCA endothelial cells (EC) of male Long-Evans rats. Nitro-l-arginine methyl ester (l-NAME) and indomethacin were present throughout. In pressurized MCA, luminally delivered UTP produced increased EC intracellular Ca(2+) concentration ([Ca(2+)](i)) and MCA dilation. Incubation with PACOCF(3), a PLA(2) inhibitor, significantly reduced both EC [Ca(2+)](i) and dilation responses to UTP. EC [Ca(2+)](i) was also partially reduced by a transient receptor potential vanilloid (TRPV) channel blocker, ruthenium red. Manganese quenching experiments demonstrated Ca(2+) influx across the luminal and abluminal face of the endothelium in response to UTP. Interestingly, PLA(2)-sensitive Ca(2+) influx occurred primarily across the abluminal face. Luminal application of arachidonic acid, the primary product of PLA(2) and a demonstrated activator of certain TRPV channels, increased both EC [Ca(2+)](i) and MCA diameter. TRPV4 mRNA and protein was demonstrated in the endothelium by RT-PCR and immunofluorescence, respectively. Finally, application of 4alpha-phorbol 12,13-didecanoate (4alphaPDD), a TRPV4 channel activator, produced an increase in EC [Ca(2+)](i) that was significantly reduced in the presence of ruthenium red. We conclude that PLA(2) is involved in EC Ca(2+) regulation through its regulation of TRPV4 channels. Furthermore, the PLA(2)-sensitive component of Ca(2+) influx may be polarized to the abluminal face of the endothelium.     

Voltage-dependent changes of TRPV6-mediated Ca2+ currents

The physiological role and activation mechanism for most proteins of the transient receptor potential (TRP) family are unknown. This is also the case for the highly Ca(2+) selective transient receptor potential vanilloid type 6 (TRPV6) channel. Patch clamp experiments were performed on transiently transfected human embryonic kidney (HEK) cells to address this issue. Currents were recorded under various conditions of intracellular Ca(2+) buffering and monitored at the same voltage throughout. No TRPV6-mediated Ca(2+) entry was detected under in vivo Ca(2+) buffering conditions at a slightly negative holding potential; however, moderate depolarization resulted in current activation. Very similar results were obtained with different Ca(2+) chelators, either EGTA or BAPTA dialyzing the cell. TRPV6 channel activity showed a negative correlation with the intracellular free Ca(2+) concentration ([Ca(2+)](i)) and was modulated by the membrane potential: Hyperpolarization decreases and depolarization increases TRPV6-mediated currents. Monovalent ions permeated TRPV6 channels in the absence of extracellular divalent cations. These currents were resistant to changes in the holding potential while the negative correlation to the [Ca(2+)](i) was conserved, indicating that the voltage-dependent current changes depend on blocking and unblocking the charge carrier Ca(2+) within the pore. In summary, these results suggest that the voltage dependence of TRPV6-mediated Ca(2+) influx is of physiological importance since it occurs at cytosolic Ca(2+) buffering and takes place within a physiologically relevant membrane potential range.     

Sarcolemmal cation channels and exchangers modify the increase in intracellular calcium in cardiomyocytes on inhibiting Na+-K+-ATPase

Although inhibition of the sarcolemmal (SL) Na(+)-K(+)-ATPase is known to cause an increase in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) by stimulating the SL Na(+)/Ca(2+) exchanger (NCX), the involvement of other SL sites in inducing this increase in [Ca(2+)](i) is not fully understood. Isolated rat cardiomyocytes were treated with or without different agents that modify Ca(2+) movements by affecting various SL sites and were then exposed to ouabain. Ouabain was observed to increase the basal levels of both [Ca(2+)](i) and intracellular Na(+) concentration ([Na(+)](i)) as well as to augment the KCl-induced increases in both [Ca(2+)](i) and [Na(+)](i) in a concentration-dependent manner. The ouabain-induced changes in [Na(+)](i) and [Ca(2+)](i) were attenuated by treatment with inhibitors of SL Na(+)/H(+) exchanger and SL Na(+) channels. Both the ouabain-induced increase in basal [Ca(2+)](i) and augmentation of the KCl response were markedly decreased when cardiomyocytes were exposed to 0-10 mM Na(+). Inhibitors of SL NCX depressed but decreasing extracellular Na(+) from 105-35 mM augmented the ouabain-induced increase in basal [Ca(2+)](i) and the KCl response. Not only was the increase in [Ca(2+)](i) by ouabain dependent on the extracellular Ca(2+) concentration, but it was also attenuated by inhibitors of SL L-type Ca(2+) channels and store-operated Ca(2+) channels (SOC). Unlike the SL L-type Ca(2+)-channel blocker, the blockers of SL Na(+) channel and SL SOC, when used in combination with SL NCX inhibitor, showed additive effects in reducing the ouabain-induced increase in basal [Ca(2+)](i). These results support the view that in addition to SL NCX, SL L-type Ca(2+) channels and SL SOC may be involved in raising [Ca(2+)](i) on inhibition of the SL Na(+)-K(+)-ATPase by ouabain. Furthermore, both SL Na(+)/H(+) exchanger and Na(+) channels play a critical role in the ouabain-induced Ca(2+) increase in cardiomyocytes.     

Osmosensitivity of transient receptor potential vanilloid 1 is synergistically enhanced by distinct activating stimuli such as temperature and protons

In animals, body-fluid osmolality is continuously monitored to keep it within a narrow range around a set point (～300 mOsm/kg). Transient receptor potential vanilloid 1 (TRPV1), a cation channel, has been implicated in body-fluid homeostasis in vivo based on studies with the TRPV1-knockout mouse. However, the response of TRPV1 to hypertonic stimuli has not been demonstrated with heterologous expression systems so far, despite intense efforts by several groups. Thus, the molecular entity of the hypertonic sensor in vivo still remains controversial. Here we found that the full-length form of TRPV1 is sensitive to an osmotic increase exclusively at around body temperature using HEK293 cells stably expressing rat TRPV1. At an ambient temperature of 24°C, a slight increase in the intracellular calcium concentration ([Ca(2+)](i)) was rarely observed in response to hypertonic stimuli. However, the magnitude of the osmosensitive response markedly increased with temperature, peaking at around 36°C. Importantly, the response at 36°C showed a robust increase over a hypertonic range, but a small decrease over a hypotonic range. A TRPV1 antagonist, capsazepine, and a nonspecific TRP channel inhibitor, ruthenium red, completely blocked the increase in [Ca(2+)](i). These results endorse the view that the full-length form of TRPV1 is able to function as a sensor of hypertonic stimuli in vivo. Furthermore, we found that protons and capsaicin likewise synergistically potentiated the response of TRPV1 to hypertonic stimuli. Of note, HgCl(2), which blocks aquaporins and inhibits cell-volume changes, significantly reduced the osmosensitive response. Our findings thus indicate that TRPV1 integrates multiple different types of activating stimuli, and that TRPV1 is sensitive to hypertonic stimuli under physiologically relevant conditions.     

Coexpression and activation of TRPV1 suppress the activity of the KCNQ2/3 channel

Transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated nonselective cation channel expressed predominantly in peripheral nociceptors. By detecting and integrating diverse noxious thermal and chemical stimuli, and as a result of its sensitization by inflammatory mediators, the TRPV1 receptor plays a key role in inflammation-induced pain. Activation of TRPV1 leads to a cascade of pro-nociceptive mechanisms, many of which still remain to be identified. Here, we report a novel effect of TRPV1 on the activity of the potassium channel KCNQ2/3, a negative regulator of neuronal excitability. Using ion influx assays, we revealed that TRPV1 activation can abolish KCNQ2/3 activity, but not vice versa, in human embryonic kidney (HEK)293 cells. Electrophysiological studies showed that coexpression of TRPV1 caused a 7.5-mV depolarizing shift in the voltage dependence of KCNQ2/3 activation compared with control expressing KCNQ2/3 alone. Furthermore, activation of TRPV1 by capsaicin led to a 54% reduction of KCNQ2/3-mediated current amplitude and attenuation of KCNQ2/3 activation. The inhibitory effect of TRPV1 appears to depend on Ca(2+) influx through the activated channel followed by Ca(2+)-sensitive depletion of phosphatidylinositol 4,5-bisphosphate and activation of protein phosphatase calcineurin. We also identified physical interactions between TRPV1 and KCNQ2/3 coexpressed in HEK293 cells and in rat dorsal root ganglia neurons. Mutation studies established that this interaction is mediated predominantly by the membrane-spanning regions of the respective proteins and correlates with the shift of KCNQ2/3 activation. Collectively, these data reveal that TRPV1 activation may deprive neurons from inhibitory control mediated by KCNQ2/3. Such neurons may thus have a lower threshold for activation, which may indirectly facilitate TRPV1 in integrating multiple noxious signals and/or in the establishment or maintenance of chronic pain.     

Xenon reduces activation of transient receptor potential vanilloid type 1 (TRPV1) in rat dorsal root ganglion cells and in human TRPV1-expressing HEK293 cells

AimsXenon provides effective analgesia in several pain states at sub-anaesthetic doses. Our aim was to examine whether xenon may mediate its analgesic effect, in part, through reducing the activity of transient receptor potential vanilloid type 1 (TRPV1), a receptor known to be involved in certain inflammatory pain conditions.Main methodsWe studied the effect of xenon on capsaicin-evoked cobalt uptake in rat cultured primary sensory neurons and in human TRPV1 (hTRPV1)-expressing human embryonic kidney 293 (HEK293) cells. We also examined xenon's effect on the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the rat spinal dorsal horn evoked by hind-paw injection of capsaicin.Key findingsXenon (75%) reduced the number of primary sensory neurons responding to the TRPV1 agonist, capsaicin (100 nM–1 μM) by ~ 25% to ~ 50%. Xenon reduced the number of heterologously-expressed hTRPV1 activated by 300 nM capsaicin by ~ 50%. Xenon (80%) reduced by ~ 40% the number of phosporylated ERK1/2-expressing neurons in rat spinal dorsal horn resulting from hind-paw capsaicin injection.SignificanceXenon substantially reduces the activity of TRPV1 in response to noxious stimulation by the specific TRPV1 agonist, capsaicin, suggesting a possible role for xenon as an adjunct analgesic where hTRPV1 is an active contributor to the excitation of primary afferents which initiates the pain sensation.