TRPV1 Function Research Articles

Pharmacological Blockade of TRPA1 Inhibits Mechanical Firing in Nociceptors

BackgroundTRPA1 has been implicated in both chemo- and mechanosensation. Recent work demonstrates that inhibiting TRPA1 function reduces mechanical hypersensitivity produced by inflammation. Furthermore, a broad range of chemical irritants require functional TRPA1 to exert their effects. In this study we use the ex-vivo skin-nerve preparation to directly determine the contribution of TRPA1 to mechanical- and chemical-evoked responses at the level of the primary afferent terminal.ResultsAcute application of HC-030031, a selective TRPA1 antagonist, inhibited all formalin responses in rat C fibers but had no effect on TRPV1 function, assessed by capsaicin responsiveness. Genetic ablation experiments corroborated the pharmacological findings as C fibers from wild type mice responded to both formalin and capsaicin, but fibers from their TRPA1-deficient littermates responded only to capsaicin. HC-030031 markedly reduced the mechanically-evoked action potential firing in rat and wild type mouse C fibers, particularly at high-intensity forces, but had no effect on the mechanical responsiveness of Aδ fiber nociceptors. Furthermore, HC-030031 had no effect on mechanically-evoked firing in C fibers from TRPA1-deficient mice, indicating that HC-030031 inhibits mechanically-evoked firing via a TRPA1-dependent mechanism.ConclusionOur data show that acute pharmacological blockade of TRPA1 at the cutaneous receptive field inhibits formalin-evoked activation and markedly reduces mechanically-evoked action potential firing in C fibers. Thus, functional TRPA1 at sensory afferent terminals in skin is required for their responsiveness to both noxious chemical and mechanical stimuli.

Determinants of Molecular Specificity in Phosphoinositide Regulation: PHOSPHATIDYLINOSITOL (4,5)-BISPHOSPHATE (PI(4,5)P2) IS THE ENDOGENOUS LIPID REGULATING TRPV1

Once thought of as simply an oily barrier that maintains cellular integrity, lipids are now known to play an active role in a large variety of cellular processes. Phosphoinositides are of particular interest because of their remarkable ability to affect many signaling pathways. Ion channels and transporters are an important target of phosphoinositide signaling, but identification of the specific phosphoinositides involved has proven elusive. TRPV1 is a good example; although phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)) can potently regulate its activation, we show that phosphatidylinositol (4)-phosphate (PI(4)P) and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P(3)) can as well. To determine the identity of the endogenous phosphoinositide regulating TRPV1, we applied recombinant pleckstrin homology domains to inside-out excised patches. Although a PI(4,5)P(2)-specific pleckstrin homology domain inhibited TRPV1, a PI(3,4,5)P(3)-specific pleckstrin homology domain had no effect. Simultaneous confocal imaging and electrophysiological recording of whole cells expressing a rapamycin-inducible lipid phosphatase also demonstrates that depletion of PI(4,5)P(2) inhibits capsaicin-activated TRPV1 current; the PI(4)P generated by the phosphatases was not sufficient to support TRPV1 function. We conclude that PI(4,5)P(2), and not other phosphoinositides or other lipids, is the endogenous phosphoinositide regulating TRPV1 channels.

Behavioral and Electrophysiological Evidence for the Differential Functions of TRPV1 at Early and Late Stages of Chronic Inflammatory Nociception in Rats

We previously reported that vanilloid receptor type 1 (VR1, or TRPV1) was up-regulated in dorsal root ganglion (DRG) and the spinal dorsal horn after chronic inflammatory pain produced by complete Freund's adjuvant (CFA) injection into the plantar of rat hind paw. In the present study, we found that subcutaneous or intrathecal application of capsazepine (CPZ), a TRPV1 competitive antagonist, could inhibit thermal hyperalgesia on day 1 and on day 14 but not on day 28 after CFA injection. With extracellular electrophysiological recording, the effect of CPZ on noxious electrical or heat stimulation evoked responses of wide dynamic range (WDR) neurons in the deep layers of the spinal dorsal horn was evaluated. Under noxious electrical stimulation to sciatic nerve, CPZ applied to the spinal cord produced an inhibition on Adelta- and C-fiber evoked responses of WDR neurons on day 1 and 14, but not on day 28. Under radiant heat stimulation to the receptive field skin, subcutaneous application of CPZ significantly inhibited the background activity and extended the response latency of WDR neurons on day 14. These results provide new evidence for the functional significance of TRPV1 at the early stage, but not the late stage, in the rat model of CFA-induced inflammatory pain.

Protein Kinase A Anchoring via AKAP150 Is Essential for TRPV1 Modulation by Forskolin and Prostaglandin E2in Mouse Sensory Neurons

Phosphorylation-dependent modulation of the vanilloid receptor TRPV1 is one of the key mechanisms mediating the hyperalgesic effects of inflammatory mediators, such as prostaglandin E(2) (PGE(2)). However, little is known about the molecular organization of the TRPV1 phosphorylation complex and specifically about scaffolding proteins that position the protein kinase A (PKA) holoenzyme proximal to TRPV1 for effective and selective regulation of the receptor. Here, we demonstrate the critical role of the A-kinase anchoring protein AKAP150 in PKA-dependent modulation of TRPV1 function in adult mouse dorsal root ganglion (DRG) neurons. We found that AKAP150 is expressed in approximately 80% of TRPV1-positive DRG neurons and is coimmunoprecipitated with the capsaicin receptor. In functional studies, PKA stimulation with forskolin markedly reduced desensitization of TRPV1. This effect was blocked by the PKA selective inhibitors KT5720 [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylicacid hexyl ester] and H89 (N-[2-(p-bromo-cinnamylamino)-ethyl]-5-isoquinoline-sulfon-amide 2HCl), as well as by the AKAP inhibitory peptide Ht31. Similarly, PGE(2) decreased TRPV1 desensitization in a manner sensitive to the PKA inhibitor KT5720. Both the forskolin and PGE(2) effects were strongly impaired in DRG neurons from knock-in mice that express a mutant AKAP150 lacking the PKA-binding domain (Delta36 mice). Protein kinase C-dependent sensitization of TRPV1 remained intact in Delta36 mice. The PGE(2)/PKA signaling defect in DRG neurons from Delta36 mice was rescued by overexpressing the full-length human ortholog of AKAP150 in these cells. In behavioral testing, PGE(2)-induced thermal hyperalgesia was significantly diminished in Delta36 mice. Together, these data suggest that PKA anchoring by AKAP150 is essential for the enhancement of TRPV1 function by activation of the PGE(2)/PKA signaling pathway.

Impaired capsaicin-induced relaxation of coronary arteries in a porcine model of the metabolic syndrome

Recent studies implicate channels of the transient receptor potential vanilloid family (e.g., TRPV1) in regulating vascular tone; however, little is known about these channels in the coronary circulation. Furthermore, it is unclear whether metabolic syndrome alters the function and/or expression of TRPV1. We tested the hypothesis that TRPV1 mediates coronary vasodilation through endothelium-dependent mechanisms that are impaired by the metabolic syndrome. Studies were conducted on coronary arteries from lean and obese male Ossabaw miniature swine. In lean pigs, capsaicin, a TRPV1 agonist, relaxed arteries in a dose-dependent manner (EC50 = 116 +/- 41 nM). Capsaicin-induced relaxation was blocked by the TRPV1 antagonist capsazepine, endothelial denudation, inhibition of nitric oxide synthase, and K+ channel antagonists. Capsaicin-induced relaxation was impaired in rings from pigs with metabolic syndrome (91 +/- 4% vs. 51 +/- 10% relaxation at 100 microM). TRPV1 immunoreactivity was prominent in coronary endothelial cells. TRPV1 protein expression was decreased 40 +/- 11% in obese pigs. Capsaicin (100 microM) elicited divalent cation influx that was abolished in endothelial cells from obese pigs. These data indicate that TRPV1 channels are functionally expressed in the coronary circulation and mediate endothelium-dependent vasodilation through a mechanism involving nitric oxide and K+ channels. Impaired capsaicin-induced vasodilation in the metabolic syndrome is associated with decreased expression of TRPV1 and cation influx.

Thermal nociception and TRPV1 function are attenuated in mice lacking the nucleotide receptor P2Y 2

Recent studies indicate that ATP and UTP act at G protein-coupled (P2Y) nucleotide receptors to excite nociceptive sensory neurons; nucleotides also potentiate signaling through the pro-nociceptive capsaicin receptor, TRPV1. We demonstrate here that P2Y 2 is the principal UTP receptor in somatosensory neurons: P2Y 2 is highly expressed in dorsal root ganglia and P2Y 2(−/−) mice showed profound deficits in UTP-evoked calcium transients and potentiation of capsaicin responses. P2Y 2(−/−) mice were also deficient in the detection of painful heat: baseline thermal response latencies were increased and mutant mice failed to develop thermal hypersensitivity in response to inflammatory injury (injection of complete Freund’s adjuvant into the hindpaw). P2Y 2 was the only Gq-coupled P2Y receptor examined that showed an increase in DRG mRNA levels in response to inflammation. Surprisingly, TRPV1 function was also attenuated in P2Y 2(−/−) mice, as measured by the frequency and magnitude of capsaicin responses in vitro and behavioral responses to capsaicin administration in vivo. However, TRPV1 mRNA levels and immunoreactivity were not reduced, and behavioral sensitivity to capsaicin could be largely restored in P2Y 2(−/−) mice by pretreatment with bradykinin, suggesting that normal function of TRPV1 requires ongoing modulation by G protein-coupled receptors. These results indicate that nucleotide signaling through P2Y 2 plays a key role in thermal nociception.

Deficiency of TRPV4 abolishes shear stress‐induced vasodilation in mice.

In endothelial sensing of shear stress, the Ca2+‐permeable TRPV4 channel was proposed a candidate mechanosensor. Using TRPV4−/− mice, we investigated whether the absence of endothelial TRPV4 alters shear‐stress‐induced vasodilation. Loss of the TRPV4 protein and function was confirmed by IHC and electrophysiology in endothelium of TRPV4−/− mice. Agonist‐ and shear stress‐induced vasodilation was determined by pressure myography in carotid artery (CA) of TRPV4−/− and TRPV4+/+ mice (WT). In WT CAEC, TRPV4 was activated by the pharmacological TRPV4‐opener 4alphaPDD, arachidonic acid (AA), and by hypotonic cell swelling (HTS). In CAEC of TRPV4−/−, 4alphaPDD did not produce currents and currents in response to AA and HTS were greatly reduced. 4alphaPDD elicited a robust and strictly endothelium‐dependent vasodilation in WT mice, again strikingly absent in TRPV4−/− mice. Shear stress‐induced vasodilation was present in WT, but was eliminated in TRPV4−/− mice. Flow‐induced vasodilation was significantly reduced in TRPV4−/− vs. WT mice. In conclusion, loss of TRPV4 abolishes shear stress‐induced vasodilation. Thus, Ca2+‐influx through endothelial TRPV4 channels is a critical mechanism contributing to endothelial mechanotransduction.

Thermoregulation: Channels that Are Cool to the Core

TRP channels are molecular sensors that endow animals with the ability to sense external temperatures. Recent studies have provided strong evidence that at least one TRP channel, TRPV1, is also essential for the maintenance of internal body temperature.

4α‐PDD induces Ca2+ influx in human corneal epithelial cells by activating TRPV4 channels

Abstract Purpose: Transient receptor potential (TRP) isoform expression is evident in human corneal epithelial cells (HCEC‐SV40). However, their role in maintaining corneal epithelial homeostasis is not fully understood. We probed for gene and protein expression as well as functional activity of the vanilloid subtype, TRPV4, in immortalized HCEC‐SV40 since they elicit Ca2+ dependent regulatory volume decrease (RVD) responses during exposure to a hypotonic challenge. Methods: RT‐PCR and Western blotting analyses identified TRPV4 gene and protein expression. Functional activity was assessed based on determining whether the TRPV4 selective agonist, 4α‐PDD, induced transients increases in intracellular Ca2+ concentration. Results: Single cell fluorescence imaging results showed that 4α‐PDD (3 μM) increased intracellular Ca2+ concentration. The fura2 fluorescence ratio (f340/f380) was 0.39 ± 0.03578 in the resting state (n = 5). After application of 4α‐PDD it increased to 0.904 ± 0.14363 (n = 5; p = 5.72077×10‐5). This increase was abolished by the TRP channel blocker ruthenium red or by Ca2+‐free Ringer's medium. Conclusions: In conclusion, there is functional TRPV4 expression in HCEC‐SV40. TRPV4 expression may provide an osmosensor role to induce RVD behavior during exposure to a hypotonic challenge since this response is mediated through intracellular Ca2+ transients.Supported in part DFG Pl 150/14‐1 and NIH, EY04795. CR: none

Activation of Urothelial Transient Receptor Potential Vanilloid 4 by 4α-Phorbol 12,13-Didecanoate Contributes to Altered Bladder Reflexes in the Rat

The ion channel transient receptor potential vanilloid (TRPV) 4 can be activated by hypo-osmolarity, heat, or certain lipid compounds. Here, we demonstrate expression of functional TRPV4 protein in the urothelium lining the renal pelvis, ureters, urinary bladder, and urethra. Exposure of cultured rat urothelial cells from the urinary bladder to the TRPV4-selective agonist 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) promoted Ca2+ influx, evoked ATP release, and augmented the ATP release evoked by hypo-osmolarity. In awake rats during continuous infusion cystometrograms, intravesical administration of 4alpha-PDD (10-100 microM) increased maximal micturition pressure by 51%, specifically by augmenting the portion of each intravesical pressure wave that follows high-frequency urethral oscillations and voiding. This unusual pharmacological effect was prevented by intravesical pretreatment with the nonselective ATP receptor antagonist, pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (100 microM), systemic treatment with the selective P2X3 purinergic antagonist 5-([(3-phenoxybenzyl)[1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl)-1,2,4-benzenetricarboxylic acid (A317491) (250 micromol/kg), or urethane anesthesia, but was unaffected by capsaicin pretreatment (100 mg/kg s.c.) or denervation of the urethral sphincter. 4Alpha-PDD (1-100 microM) did not alter the contractility to electrical stimulation of excised bladder strips. We conclude that activation of urothelial TRPV4 by 4alpha-PDD and release of mediators such as ATP trigger a novel neural mechanism that regulates the late phase of detrusor muscle contraction after micturition. These data raise the possibility that TRPV4 channels in the urothelium could contribute to abnormal bladder activity.

Estrogen Amplifies Pain Responses to Uterine Cervical Distension in Rats by Altering Transient Receptor Potential-1 Function

Estrogen sensitizes responses to painful stimuli, but its contribution to acute and chronic pain from the uterine cervix is unknown. Previous studies link the excitatory transient receptor potiential-1 channel (TRPV-1) to sensitization in viscera, and show that estrogen increases TRPV-1 expression in afferents from the uterine cervix. Here, we tested whether estrogen enhanced responses to uterine cervical distension in rats, and whether this involved TRPV-1 channels. Ovariectomized rats, with or without estrogen replacement, were anesthetized and hypogastric nerve and abdominal muscle contraction reflex responses to graded uterine cervical distension were recorded. Single unit hypogastric nerve fiber firing was measured before and after acute treatment with the TRPV-1 antagonist, capsaizepine, or vehicle. Abdominal muscle contraction reflex responses to uterine cervical distension were enhanced in estrogen-treated rats. Hypogastric afferent responses to cervical distension were reduced by capsaizepine in estrogen-treated animals, but were unaffected in ovariectomized animals without estrogen replacement. These data suggest that the TRPV-1 channel is unimportant for normal mechanosensation in the cervix in the absence of estrogen, since capsaizepine failed to reduce responses to uterine cervical distension in rats without estrogen replacement. In contrast, TRPV-1 function is important for estrogen-induced sensitization. These data raise the possibility that acute and chronic pain coming from the cervix, such as labor or cancer, may be enhanced by estrogen and might be reduced by antagonists of TRPV-1.

TRPV1 + Sensory Neurons Control β Cell Stress and Islet Inflammation in Autoimmune Diabetes

In type 1 diabetes, T cell-mediated death of pancreatic beta cells produces insulin deficiency. However, what attracts or restricts broadly autoreactive lymphocyte pools to the pancreas remains unclear. We report that TRPV1(+) pancreatic sensory neurons control islet inflammation and insulin resistance. Eliminating these neurons in diabetes-prone NOD mice prevents insulitis and diabetes, despite systemic persistence of pathogenic T cell pools. Insulin resistance and beta cell stress of prediabetic NOD mice are prevented when TRPV1(+) neurons are eliminated. TRPV1(NOD), localized to the Idd4.1 diabetes-risk locus, is a hypofunctional mutant, mediating depressed neurogenic inflammation. Delivering the neuropeptide substance P by intra-arterial injection into the NOD pancreas reverses abnormal insulin resistance, insulitis, and diabetes for weeks. Concordantly, insulin sensitivity is enhanced in trpv1(-/-) mice, whereas insulitis/diabetes-resistant NODxB6Idd4-congenic mice, carrying wild-type TRPV1, show restored TRPV1 function and insulin sensitivity. Our data uncover a fundamental role for insulin-responsive TRPV1(+) sensory neurons in beta cell function and diabetes pathoetiology.

Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane

Sensitization of the pain-transducing ion channel TRPV1 underlies thermal hyperalgesia by proalgesic agents such as nerve growth factor (NGF). The currently accepted model is that the NGF-mediated increase in TRPV1 function during hyperalgesia utilizes activation of phospholipase C (PLC) to cleave PIP2, proposed to tonically inhibit TRPV1. In this study, we tested the PLC model and found two lines of evidence that directly challenge its validity: (1) polylysine, a cationic phosphoinositide sequestering agent, inhibited TRPV1 instead of potentiating it, and (2) direct application of PIP2 to inside-out excised patches dramatically potentiated TRPV1. Furthermore, we show four types of experiments indicating that PI3K is physically and functionally coupled to TRPV1: (1) the p85β subunit of PI3K interacted with the N-terminal region of TRPV1 in yeast 2-hybrid experiments, (2) PI3K-p85β coimmunoprecipitated with TRPV1 from both HEK293 cells and dorsal root ganglia (DRG) neurons, (3) TRPV1 interacted with recombinant PI3K-p85 in vitro, and (4) wortmannin, a specific inhibitor of PI3K, completely abolished NGF-mediated sensitization in acutely dissociated DRG neurons. Finally, simultaneous electrophysiological and total internal reflection fluorescence (TIRF) microscopy recordings demonstrate that NGF increased the number of channels in the plasma membrane. We propose a new model for NGF-mediated hyperalgesia in which physical coupling of TRPV1 and PI3K in a signal transduction complex facilitates trafficking of TRPV1 to the plasma membrane.

G02 Identification of functional TRPV1 vanilloid receptor in MCF‐7 cells

G02 Identification of functional TRPV1 vanilloid receptor in MCF‐7 cells

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

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

TRPV1, but not P2X3, requires cholesterol for its function and membrane expression in rat nociceptors

We examined the importance of membrane cholesterol for the function and expression of TRPV1 (vanilloid receptor subtype 1) and P2X(3) in adult rat dorsal root ganglion (DRG) neurons. Cholesterol, an essential component of lipid rafts, was depleted using methyl beta-cyclodextrin (MCD). We found that MCD significantly reduced TRPV1-mediated capsaicin- and proton-activated currents. By contrast, inward currents activated by alpha,beta-methylene ATP (alpha,beta-meATP), a non-hydrolysable ATP analogue, were not altered. Immunoreactivity for TRPV1, but not P2X(3), in the plasma membrane was markedly reduced by MCD. A reduction of TRPV1 protein in membrane fractions was found following cholesterol depletion. Our data show that the level of cholesterol determines the activity and the amount of membrane TRPV1, suggesting that TRPV1 might be localized in cholesterol-rich microdomains in nociceptors. The differential dependence on the membrane cholesterol of TRPV1 and P2X(3) may have physiological significance in nociception during inflammation.

Der Capsaicinrezeptor

The capsaicin receptor TRPV1, once discovered as a receptor for pungent spices, is a polymodal sensor molecule for painful chemical and thermal stimuli. However, TRPV1 plays an important role not only for the integration of acute painful stimuli but also in the genesis of inflammatory processes. The persistent functional sensitization of TRPV1 as well as an up-regulation of its expression may contribute to the development and maintenance of chronic pain states. Thus, TRPV1 is an excellent target for a rational pharmacological treatment of pain. Several additional physiological and pathophysiological functions of TRPV1 are assumed beyond nociception and pain. Activation of TRPV1 seems to contribute to the etiology and pathogenesis of inflammatory diseases concerning, e.g., the gastrointestinal tract, the bladder, and the respiratory system. Therefore, the therapeutic potential of a pharmacological manipulation of TRPV1 may not be restricted to a symptomatic therapy of pain.

Capsaicin-resistant arterial baroreceptors.

BackgroundAortic baroreceptors (BRs) comprise a class of cranial afferents arising from major arteries closest to the heart whose axons form the aortic depressor nerve. BRs are mechanoreceptors that are largely devoted to cardiovascular autonomic reflexes. Such cranial afferents have either lightly myelinated (A-type) or non-myelinated (C-type) axons and share remarkable cellular similarities to spinal primary afferent neurons. Our goal was to test whether vanilloid receptor (TRPV1) agonists, capsaicin (CAP) and resiniferatoxin (RTX), altered the pressure-discharge properties of peripheral aortic BRs.ResultsPeriaxonal application of 1 μM CAP decreased the amplitude of the C-wave in the compound action potential conducting at <1 m/sec along the aortic depressor nerve. 10 μM CAP eliminated the C-wave while leaving intact the A-wave conducting in the A-δ range (<12 m/sec). These whole nerve results suggest that TRPV1 receptors are expressed along the axons of C- but not A-conducting BR axons. In an aortic arch – aortic nerve preparation, intralumenal perfusion with 1 μM CAP had no effect on the pressure-discharge relations of regularly discharging, single fiber BRs (A-type) – including the pressure threshold, sensitivity, frequency at threshold, or maximum discharge frequency (n = 8, p > 0.50) but completely inhibited discharge of an irregularly discharging BR (C-type). CAP at high concentrations (10–100 μM) depressed BR sensitivity in regularly discharging BRs, an effect attributed to non-specific actions. RTX (≤ 10 μM) did not affect the discharge properties of regularly discharging BRs (n = 7, p > 0.18). A CAP-sensitive BR had significantly lower discharge regularity expressed as the coefficient of variation than the CAP-resistant fibers (p < 0.002).ConclusionWe conclude that functional TRPV1 channels are present in C-type but not A-type (A-δ) myelinated aortic arch BRs. CAP has nonspecific inhibitory actions that are unlikely to be related to TRV1 binding since such effects were absent with the highly specific TRPV1 agonist RTX. Thus, CAP must be used with caution at very high concentrations.

Evidence for a Functional Role of Endothelial Transient Receptor Potential V4 in Shear Stress–Induced Vasodilatation

Ca2+-influx through transient receptor potential (TRP) channels was proposed to be important in endothelial function, although the precise role of specific TRP channels is unknown. Here, we investigated the role of the putatively mechanosensitive TRPV4 channel in the mechanisms of endothelium-dependent vasodilatation. Expression and function of TRPV4 was investigated in rat carotid artery endothelial cells (RCAECs) by using in situ patch-clamp techniques, single-cell RT-PCR, Ca2+ measurements, and pressure myography in carotid artery (CA) and Arteria gracilis. In RCAECs in situ, TRPV4 currents were activated by the selective TRPV4 opener 4alpha-phorbol-12,13-didecanoate (4alphaPDD), arachidonic acid, moderate warmth, and mechanically by hypotonic cell swelling. Single-cell RT-PCR in endothelial cells demonstrated mRNA expression of TRPV4. In FURA-2 Ca2+ measurements, 4alphaPDD increased [Ca2+]i by &140 nmol/L above basal levels. In pressure myograph experiments in CAs and A gracilis, 4alphaPDD caused robust endothelium-dependent and strictly endothelium-dependent vasodilatations by &80% (K(D) 0.3 microL), which were suppressed by the TRPV4 blocker ruthenium red (RuR). Shear stress-induced vasodilatation was similarly blocked by RuR and also by the phospholipase A2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF3). 4alphaPDD produced endothelium-derived hyperpolarizing factor (EDHF)-type responses in A gracilis but not in rat carotid artery. Shear stress did not produce EDHF-type vasodilatation in either vessel type. Ca2+ entry through endothelial TRPV4 channels triggers NO- and EDHF-dependent vasodilatation. Moreover, TRPV4 appears to be mechanistically important in endothelial mechanosensing of shear stress.

TRPV4 channel expression and function in blood‐brain barrier (BBB) microvessel endothelial cells

RT-PCR analysis revealed TRPV4 mRNA expression in microvessels isolated from mouse brain, in rat brain microvessel endothelial cell cultures, and in an immortalized mouse BBB endothelial cell line, bEnd3. The analysis was extended with bEnd3 cells grown on coverslips. Both immunoblotting and immunohistochemistry verified expression of TRPV4 protein in the bEnd3 cells and immunofluorescent imaging showed TRPV4 within the cytoplasm and at the cell membrane. To evaluate function, intracellular calcium levels were measured in bEnd3 cells on coverslips using fura 2 fluorescence imaging (37 °C). Cells were activated by application of: 1) the PKC-inactive phorbol ester, 4α-phorbol 12,13-decanoate (4α-PDD, 100 nM), a selective activator of TRPV4; 2) the PKC-active phorbol ester, phorbol 12-myristate 13-acetate (PMA, 100 nM); and 3) mechanical stimulation via hypotonic swelling (225 mOsm/kg). All stimuli activated Ca influx and were partially inhibited by the general TRPV channel blocker, ruthenium red (RR, 1 μM). Similar responses were demonstrated in TRPV4-transfected HEK 293 cells, but not in non-transfected cells. It is concluded that TRPV4 is expressed in BBB endothelial cells and that it may function as a mechanosensitive channel similar to that previously demonstrated for TRPV4 function in TRPV4-transfected HEK 293 cells.