Expression of transient receptor potential vanilloid 4 and effects of ruthenium red on detrusor overactivity associated with bladder outlet obstruction in rats

We previously demonstrated that endothelium-derived hyperpolarizing factor (EDHF)-mediated dilations in cerebral arteries are significantly reduced by inhibitors of PLA2. In this study we examined possible mechanisms by which PLA2 regulates endothelium-dependent dilation, specifically whether PLA2 is involved in endothelial Ca2+ 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 Ca2+ concentration ([Ca2+]i) and MCA dilation. Incubation with PACOCF3, a PLA2 inhibitor, significantly reduced both EC [Ca2+]i and dilation responses to UTP. EC [Ca2+]i was also partially reduced by a transient receptor potential vanilloid (TRPV) channel blocker, ruthenium red. Manganese quenching experiments demonstrated Ca2+ influx across the luminal and abluminal face of the endothelium in response to UTP. Interestingly, PLA2-sensitive Ca2+ influx occurred primarily across the abluminal face. Luminal application of arachidonic acid, the primary product of PLA2 and a demonstrated activator of certain TRPV channels, increased both EC [Ca2+]i and MCA diameter. TRPV4 mRNA and protein was demonstrated in the endothelium by RT-PCR and immunofluorescence, respectively. Finally, application of 4α-phorbol 12,13-didecanoate (4αPDD), a TRPV4 channel activator, produced an increase in EC [Ca2+]i that was significantly reduced in the presence of ruthenium red. We conclude that PLA2 is involved in EC Ca2+ regulation through its regulation of TRPV4 channels. Furthermore, the PLA2-sensitive component of Ca2+ influx may be polarized to the abluminal face of the endothelium.

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Thermal sensitivity of isolated vagal pulmonary sensory neurons: role of transient receptor potential vanilloid receptors

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00016.2006 ◽

2006 ◽

Vol 291 (3) ◽

pp. R541-R550 ◽

Cited By ~ 47

Author(s):

Dan Ni ◽

Qihai Gu ◽

Hong-Zhen Hu ◽

Na Gao ◽

Michael X. Zhu ◽

...

Keyword(s):

Sensory Neurons ◽

Transient Receptor Potential ◽

Thermal Sensitivity ◽

Receptor Potential ◽

Ruthenium Red ◽

Transient Receptor Potential Vanilloid ◽

Temperature Sensitive ◽

Inward Current ◽

Transient Receptor ◽

Increasing Temperature

A recent study has demonstrated that increasing the intrathoracic temperature from 36°C to 41°C induced a distinct stimulatory and sensitizing effect on vagal pulmonary C-fiber afferents in anesthetized rats ( J Physiol 565: 295–308, 2005). We postulated that these responses are mediated through a direct activation of the temperature-sensitive transient receptor potential vanilloid (TRPV) receptors by hyperthermia. To test this hypothesis, we studied the effect of increasing temperature on pulmonary sensory neurons that were isolated from adult rat nodose/jugular ganglion and identified by retrograde labeling, using the whole cell perforated patch-clamping technique. Our results showed that increasing temperature from 23°C (or 35°C) to 41°C in a ramp pattern evoked an inward current, which began to emerge after exceeding a threshold of ∼34.4°C and then increased sharply in amplitude as the temperature was further increased, reaching a peak current of 173 ± 27 pA ( n = 75) at 41°C. The temperature coefficient, Q10, was 29.5 ± 6.4 over the range of 35–41°C. The peak inward current was only partially blocked by pretreatment with capsazepine (Δ I = 48.1 ± 4.7%, n = 11) or AMG 9810 (Δ I = 59.2 ± 7.8%, n = 8), selective antagonists of the TRPV1 channel, but almost completely abolished (Δ I = 96.3 ± 2.3%) by ruthenium red, an effective blocker of TRPV1–4 channels. Furthermore, positive expressions of TRPV1–4 transcripts and proteins in these neurons were demonstrated by RT-PCR and immunohistochemistry experiments, respectively. On the basis of these results, we conclude that increasing temperature within the normal physiological range can exert a direct stimulatory effect on pulmonary sensory neurons, and this effect is mediated through the activation of TRPV1, as well as other subtypes of TRPV channels.

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MP4-10 PATHOPHYSIOLOGICAL ROLE OF TRANSIENT RECEPTOR POTENTIAL ANKYRIN 1 (TRPA1) CHANNEL IN BLADDER DYSFUNCTION IN MALE RATS WITH SUB-ACUTE BLADDER OUTLET OBSTRUCTION

The Journal of Urology ◽

10.1016/j.juro.2014.02.210 ◽

2014 ◽

Vol 191 (4S) ◽

Author(s):

Rino Sugiyama ◽

Naoki Aizawa ◽

Hiroki Ito ◽

Yukio Homma ◽

Yasuhiko Igawa

Keyword(s):

Bladder Outlet Obstruction ◽

Transient Receptor Potential ◽

Bladder Dysfunction ◽

Outlet Obstruction ◽

Receptor Potential ◽

Male Rats ◽

Pathophysiological Role ◽

Transient Receptor

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Abstract 326: TRPV2 Regulates Cardiomyocyte Function via Altering Ca 2+ Cycling

Circulation Research ◽

10.1161/res.111.suppl_1.a326 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Xiaoqian Gao ◽

Sheryl Koch ◽

Min Jiang ◽

Nathan Robbins ◽

Wenfeng Cai ◽

...

Keyword(s):

Transient Receptor Potential ◽

Ventricular Myocytes ◽

Receptor Potential ◽

Ruthenium Red ◽

Transient Receptor Potential Vanilloid ◽

Dependent Manner ◽

Noxious Heat ◽

Cardiac Tissues ◽

Membrane Stretching ◽

Transient Receptor

TRPV2 is a member of transient receptor potential vanilloid (TRPV) family. As a Ca 2+ channel, it can detect various stimuli such as noxious heat (>52°C), membrane stretching, as well as a number of exogenous chemicals, including probenecid, 2-aminoethoxydiphenyl borate, and lysophospholipids. TRPV2 has been found in many tissue types, including neuron and kidney, but the function of TRPV2 in the heart is poorly understood. Here we show TRPV2 is involved in the Ca 2+ cycling process and then regulates the function of the cardiomyocyte. We identified the mRNA expression of TRPV2 in the cardiac tissues of mice using real-time PCR. By performing echocardiography we found that administration of probenecid, a selective TRPV2 agonist, increased cardiac ejection fraction in mice. This positive inotropic effect of probenecid was also shown in Langendorff perfused mice hearts as increased peak +dP/dt. In isolated ventricular myocytes, we found that probenecid significantly increased myocyte fractional shortening in a dose-dependent manner, which was fully blocked by ruthenium red, a non-selective TRPV2 blocker. We also performed fluorescent studies to examine myocyte Ca 2+ cycling. We found that probenecid significantly increased Ca 2+ transient and resting-state Ca 2+ sparks and this effect was eliminated by ruthenium red. When Ca 2+ storage in sarcoplasmic reticulum (SR) was depleted with caffeine, and SR Ca 2+ reuptake was blocked by thapsigargin at the same time, probenecid did not show any effects in either Ca 2+ transient or Ca 2+ sparks. Our patch clamp experiments indicate that probenecid treatment does not trigger any significant transmembrane Ca 2+ influx. These results point to the important role of TRPV2 in regulating SR Ca 2+ release. In conclusion, TRPV2 activation may contribute to increased SR Ca 2+ release, leading to the enhancement of myocyte contractility. Thus, TRPV2 plays a potentially important role in controlling the cellular function of heart.

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TRPV4 activation mediates flow-induced nitric oxide production in the rat thick ascending limb

AJP Renal Physiology ◽

10.1152/ajprenal.00619.2013 ◽

2014 ◽

Vol 307 (6) ◽

pp. F666-F672 ◽

Cited By ~ 18

Author(s):

Pablo D. Cabral ◽

Jeffrey L. Garvin

Keyword(s):

Nitric Oxide ◽

Transient Receptor Potential ◽

Receptor Potential ◽

Ruthenium Red ◽

Transient Receptor Potential Vanilloid ◽

No Production ◽

Thick Ascending Limb ◽

Different Types ◽

Luminal Flow ◽

Transient Receptor

Nitric oxide (NO) regulates renal function. Luminal flow stimulates NO production in the thick ascending limb (TAL). Transient receptor potential vanilloid 4 (TRPV4) is a mechano-sensitive channel activated by luminal flow in different types of cells. We hypothesized that TRPV4 mediates flow-induced NO production in the rat TAL. We measured NO production in isolated, perfused rat TALs using the fluorescent dye DAF FM. Increasing luminal flow from 0 to 20 nl/min stimulated NO from 8 ± 3 to 45 ± 12 arbitrary units (AU)/min ( n = 5; P < 0.05). The TRPV4 antagonists, ruthenium red (15 μmol/l) and RN 1734 (10 μmol/l), blocked flow-induced NO production. Also, luminal flow did not increase NO production in the absence of extracellular calcium. We also studied the effect of luminal flow on NO production in TALs transduced with a TRPV4shRNA. In nontransduced TALs luminal flow increased NO production by 47 ± 17 AU/min ( P < 0.05; n = 5). Similar to nontransduced TALs, luminal flow increased NO production by 39 ± 11 AU/min ( P < 0.03; n = 5) in TALs transduced with a control negative sequence-shRNA while in TRPV4shRNA-transduced TALs, luminal flow did not increase NO production (Δ10 ± 15 AU/min; n = 5). We then tested the effect of two different TRPV4 agonists on NO production in the absence of luminal flow. 4α-Phorbol 12,13-didecanoate (1 μmol/l) enhanced NO production by 60 ± 11 AU/min ( P < 0.002; n = 7) and GSK1016790A (10 ηmol/l) increased NO production by 52 ± 15 AU/min ( P < 0.03; n = 5). GSK1016790A (10 ηmol/l) did not stimulate NO production in TRPV4shRNA-transduced TALs. We conclude that activation of TRPV4 channels mediates flow-induced NO production in the rat TAL.

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Functional Expression of an Osmosensitive Cation Channel, Transient Receptor Potential Vanilloid 4, in Rat Vestibular Ganglia

Audiology and Neurotology ◽

10.1159/000449238 ◽

2016 ◽

Vol 21 (4) ◽

pp. 268-274 ◽

Cited By ~ 3

Author(s):

Takefumi Kamakura ◽

Makoto Kondo ◽

Yoshihisa Koyama ◽

Yukiko Hanada ◽

Yusuke Ishida ◽

...

Keyword(s):

Transient Receptor Potential ◽

Receptor Potential ◽

Functional Expression ◽

Ruthenium Red ◽

Cation Channel ◽

Trigeminal Ganglia ◽

Transient Receptor Potential Vanilloid ◽

Trpv Channels ◽

Polymerase Chain ◽

Transient Receptor

Transient receptor potential vanilloid (TRPV) 4 is a nonselective cation channel expressed in sensory neurons such as those in the dorsal root and trigeminal ganglia, kidney, and inner ear. TRPV4 is activated by mechanical stress, heat, low osmotic pressure, low pH, and phorbol derivatives such as 4α-phorbol 12,13-didecanoate (4α-PDD). We investigated the expression of TRPV4 in rat vestibular ganglion (VG) neurons. The TRPV4 gene was successfully amplified from VG neuron mRNA using reverse-transcription polymerase chain reaction. Furthermore, immunoblotting showed positive expression of TRPV4 protein in VG neurons. Immunohistochemistry indicated that TRPV4 was localized predominantly on the plasma membrane of VG neurons. Calcium (Ca2+) imaging of VG neurons showed that 4α-PDD and/or hypotonic stimuli caused an increase in intracellular Ca2+ concentration ([Ca2+]i) that was almost completely inhibited by ruthenium red, a selective antagonist of TRPV channels. Interestingly, a [Ca2+]i increase was evoked by both hypotonic stimuli and 4α-PDD in approximately 38% of VG neurons. These data indicate that TRPV4 is functionally expressed in VG neurons as an ion channel and that TRPV4 likely participates in VG neurons for vestibular neurotransmission as an osmoreceptor and/or mechanoreceptor.

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Transient Receptor Potential Vanilloid 5 Mediates Ca2+ Influx and Inhibits Chondrocyte Autophagy in a Rat Osteoarthritis Model

Cellular Physiology and Biochemistry ◽

10.1159/000477387 ◽

2017 ◽

Vol 42 (1) ◽

pp. 319-332 ◽

Cited By ~ 11

Author(s):

Yingliang Wei ◽

Yanfang Wang ◽

Yutong Wang ◽

Lunhao Bai

Keyword(s):

Articular Cartilage ◽

Transient Receptor Potential ◽

Joint Destruction ◽

Receptor Potential ◽

B Cell Lymphoma ◽

Transient Receptor Potential Channel ◽

Ruthenium Red ◽

Protective Mechanism ◽

Transient Receptor Potential Vanilloid ◽

Transient Receptor

Background: Autophagy, a self-protective mechanism of chondrocytes, has become a promising target to impede the progress of osteoarthritis (OA). Autophagy is regulated by cytosolic Ca2+ activity and may thus be modified by the Ca2+ permeable transient receptor potential channel vanilloid 5 (TRPV5). Therefore, we investigated the potential role of TRPV5 in mediating Ca2+ influx and in inhibiting chondrocyte autophagy in a rat OA model. Methods: The rat OA model was assessed by macroscopic and histological analyses. light chain 3B (LC3B) immunolocalization was detected by immunohistochemistry. TRPV5, LC3B and calmodulin in OA articular cartilage were assessed by real time polymerase chain reaction (RT-PCR) and western blotting. TRPV5 small interfering RNA (TRPV5 siRNA) were transfected into rat primary chondrocyte then the calmodulin and LC3B was detected by immunofluorescence. The functionality of the TRPV5 was assessed by Ca2+ influx. Western blot was used to measure autophagy-related proteins. Results: We constructed a monosodium iodoacetate (MIA) -induced rat OA model and found that ruthenium red (TRPV5 inhibitor) slowed the progression of joint destruction. We found that the TRPV5 and calmodulin were up-regulated but LC3B was down-regulated in articular cartilage following prolonged progression of OA. Furthermore, the up-regulated TRPV5 channel caused an increase in the Ca2+ influx in chondrocytes. The up-regulation of TRPV5 stimulated Ca2+ influx, which inhibited autophagy by increasing the production of calmodulin, phosphorylation of calmodulin dependent protein kinases II (p-CAMK II), phosphorylation of Beclin1 (p-Beclin1), and protein of B-cell lymphoma-2 (Bcl-2), and attenuating ratio of LC3-II/ LC3-. Conclusion: Up-regulated TRPV5 as an initiating factor inhibited chondrocyte autophagy via the mediation of Ca2+ influx.

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