Transient Receptor Potential Vanilloid Type 1 Function Research Articles

Neuronal TRPV1-CGRP axis regulates bone defect repair through Hippo signaling pathway

Transient receptor potential vanilloid type 1 (TRPV1) is highly expressed on sensory neurons where it serves as a polymodal receptor for detecting physical and chemical stimuli. However, the role of TRPV1 in bone metabolism remains largely unclear. This study aimed to investigate the underlying mechanism of neuronal TRPV1 in regulating bone defect repair. In vivo experiment verified that TRPV1 activation could trigger dorsal root ganglion (DRG) producing the neuropeptide calcitonin gene-related peptide (CGRP) in mice. The accelerated bone healing of femoral defect in this process was observed compared to the control group (p < 0.05). Conversely, Trpv1 knockdown led to reduced CGRP expression in DRG and nerves innervating femur bone tissue, following impaired bone formation and osteogenic capability in the defect region (p < 0.05), which could be rescued by local CGRP treatment. In vitro, results revealed that TRPV1 function in DRG neurons contributed essentially to the regulation of osteoblast physiology through affecting the production and secretion of CGRP. The capsaicin-activated neuronal TRPV1-CGRP axis could enhance the proliferation, migration and differentiation of osteoblasts (p < 0.05). Furthermore, we found that the promoting role of neuronal TRPV1 in osteogenesis was associated with Hippo signaling pathway, reflected by the phosphorylation protein level of large tumor suppressor 1 (LATS1), MOB kinase activator 1 (MOB1) and Yes-associated protein (YAP), as well as the subcellular location of YAP. Our study clarified the effects and intrinsic mechanisms of neuronal TRPV1 on bone defect repair, which might offer us a therapeutic implication for bone disorders.

Transient Receptor Potential Vanilloid Type 1 Protects Against Pressure Overload-Induced Cardiac Hypertrophy by Promoting Mitochondria-Associated Endoplasmic Reticulum Membranes.

:Transient receptor potential vanilloid type 1 (TRPV1) is a nonselective cation channel that mediates the relationship between mitochondrial function and pathological myocardial hypertrophy. However, its underlying mechanisms remain unclear. This study aimed to investigate whether TRPV1 activation improves the morphology and function of intracellular mitochondria to protect cardiomyocytes after pressure overload-induced myocardial hypertrophy. The myocardial hypertrophy model was established by performing transverse aortic constriction surgery in C57BL/6 J male mice. The data revealed that TRPV1 activation significantly reduced myocardial hypertrophy, promoted ejection fraction% and fractional shortening%, and decreased the left ventricular internal diameter in end-diastole and left ventricular internal diameter in end-systole after transverse aortic constriction. Moreover, in vitro experiments revealed that TRPV1 reduces cardiomyocyte area and improves mitochondrial function by promoting mitochondria-associated endoplasmic reticulum membranes (MAMs) formation in a phenylephrine-treated cardiomyocyte hypertrophy model. TRPV1 up-regulates the phosphorylation levels of AMP-activated protein kinase and expression of mitofusin2 (MFN2). TRPV1 function is blocked by single-stranded RNA interfering with silent interfering MFN2. Activation of TRPV1 reduced mitochondrial reactive oxygen species caused by phenylephrine, whereas disruption of MAMs by siMFN2 abolished TRPV1-mediated mitochondrial protection. Our findings suggest that TRPV1 effectively protects against pressure overload-induced cardiac hypertrophy by promoting MAM formation and conserved mitochondrial function via the AMP-activated protein kinase/MFN2 pathway in cardiomyocytes.

Knockout of TRPV1 Exacerbates Ischemia-reperfusion-induced Renal Inflammation and Injury in Obese Mice.

Transient receptor potential vanilloid type 1 (TRPV1) has anti-inflammatory properties. The present study aimed to investigate the role of TRPV1 in renal inflammatory responses and tissue injury following renal ischemia-reperfusion (I/R) in diet-induced obese mice. TRPV1 knockout and wild type mice were fed a normal or western diet (WD) for 23 weeks and were then subjected to renal I/R injury. TRPV1 knockout mice showed enhanced WD-induced renal macrophage infiltration and collagen deposition. Knocking out TRPV1 exacerbated renal I/R-induced increase of malondialdehyde, interleukin-6, monocyte chemoattractant protein-1, and NF-ĸB in obese mice. Similar results were observed in the expression of phosphorylated Smad1 and Smad2/3. Blockade of calcitonin gene-related peptide (CGRP) receptors with CGRP8-37 worsened the I/R-induced renal inflammation and injury. Our data indicate that preserving TRPV1 expression and function may prevent renal I/R injury in obesity likely through alleviating inflammatory responses.

Long-Term Diabetic Microenvironment Augments the Decay Rate of Capsaicin-Induced Currents in Mouse Dorsal Root Ganglion Neurons.

Individuals with end-stage diabetic peripheral neuropathy present with decreased pain sensation. Transient receptor potential vanilloid type 1 (TRPV1) is implicated in pain signaling and resides on sensory dorsal root ganglion (DRG) neurons. We investigated the expression and functional activity of TRPV1 in DRG neurons of the Ins2+/Akita mouse at 9 months of diabetes using immunohistochemistry, live single cell calcium imaging, and whole-cell patch-clamp electrophysiology. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence assay was used to determine the level of Reactive Oxygen Species (ROS) in DRGs. Although TRPV1 expressing neuron percentage was increased in Ins2+/Akita DRGs at 9 months of diabetes compared to control, capsaicin-induced Ca2+ influx was smaller in isolated Ins2+/Akita DRG neurons, indicating impaired TRPV1 function. Consistently, capsaicin-induced Ca2+ influx was decreased in control DRG neurons cultured in the presence of 25 mM glucose for seven days versus those cultured with 5.5 mM glucose. The high glucose environment increased cytoplasmic ROS accumulation in cultured DRG neurons. Patch-clamp recordings revealed that capsaicin-activated currents decayed faster in isolated Ins2+/Akita DRG neurons as compared to those in control neurons. We propose that in poorly controlled diabetes, the accelerated rate of capsaicin-sensitive TRPV1 current decay in DRG neurons decreases overall TRPV1 activity and contributes to peripheral neuropathy.

Transient Receptor Potential Vanilloid 1 Modulates Central Inflammation in Multiple Sclerosis.

Introduction: Disease course of multiple sclerosis (MS) is negatively influenced by proinflammatory molecules released by activated T and B lymphocytes and local immune cells. The endovanilloid system plays different physiological functions, and preclinical data suggest that transient receptor potential vanilloid type 1 (TRPV1) could modulate neuroinflammation in this disorder.Methods: The effect of TRPV1 activation on the release of two main proinflammatory cytokines, tumor necrosis factor (TNF) and interleukin (IL)-6, was explored in activated microglial cells. Furthermore, in a group of 132 MS patients, the association between the cerebrospinal fluid (CSF) levels of TNF and IL-6 and a single nucleotide polymorphisms (SNP) influencing TRPV1 protein expression and function (rs222747) was assessed.Results: In in vitro experiments, TRPV1 stimulation by capsaicin significantly reduced TNF and IL-6 release by activated microglial cells. Moreover, the anti-inflammatory effect of TRPV1 activation was confirmed by another TRPV1 agonist, the resiniferatoxin (RTX), whose effects were significantly inhibited by the TRPV1 antagonist, 5-iodoresiniferatoxin (5-IRTX). Vice versa, BV2 pre-treatment with 5-IRTX increased the inflammatory response induced by LPS. Moreover, in MS patients, a significant association emerged between TRPV1 SNP rs222747 and CSF TNF levels. In particular, the presence of a G allele, known to result in increased TRPV1 protein expression and function, was associated to lower CSF levels of TNF.Conclusions: Our results indicate that TRPV1 influences central inflammation in MS by regulating cytokine release by activated microglial cells. The modulation of the endovanilloid system may represent a useful approach to contrast neuroinflammation in MS.

Activation of endogenous TRPV1 fails to induce overstimulation-based cytotoxicity in breast and prostate cancer cells but not in pain-sensing neurons

Vanilloids including capsaicin and resiniferatoxin are potent transient receptor potential vanilloid type 1 (TRPV1) agonists. TRPV1 overstimulation selectively ablates capsaicin-sensitive sensory neurons in animal models in vivo. The cytotoxic mechanisms are based on strong Na+ and Ca2+ influx via TRPV1 channels, which leads to mitochondrial Ca2+ accumulation and necrotic cell swelling. Increased TRPV1 expression levels are also observed in breast and prostate cancer and derived cell lines. Here, we examined whether potent agonist-induced overstimulation mediated by TRPV1 might represent a means for the eradication of prostate carcinoma (PC-3, Du 145, LNCaP) and breast cancer (MCF7, MDA-MB-231, BT-474) cells in vitro. While rat sensory neurons were highly vanilloid-sensitive, normal rat prostate epithelial cells were resistant in vivo. We found TRPV1 to be expressed in all cancer cell lines at mRNA and protein levels, yet protein expression levels were significantly lower compared to sensory neurons. Treatment of all human carcinoma cell lines with capsaicin didn't lead to overstimulation cytotoxicity in vitro. We assume that the low vanilloid-sensitivity of prostate and breast cancer cells is associated with low expression levels of TRPV1, since ectopic TRPV1 expression rendered them susceptible to the cytotoxic effect of vanilloids evidenced by plateau-type Ca2+ signals, mitochondrial Ca2+ accumulation and Na+- and Ca2+-dependent membrane disorganization. Moreover, long-term monitoring revealed that merely the ectopic expression of TRPV1 stopped cell proliferation and often induced apoptotic processes via strong activation of caspase-3 activity. Our results indicate that specific targeting of TRPV1 function remains a putative strategy for cancer treatment.

Abstract 453: Ablation of TRPV1 Impairs Exendin-4 Induced Suppression in Renal Cytokine Release

Impairment of function and expression of the transient receptor potential vanilloid type 1 (TRPV1) occurs in obesity and diabetes, which may aggravate inflammation and tissue injury given that the TRPV1 mediated pathway has been shown to be anti-inflammatory. Glucagon-like peptide-1 (GLP-1), an incretin hormone released in response to food intake, is capable of protecting tissues from injury, but its mechanism largely unknown. We tested the hypothesis that exendin-4 (Ex4), a GLP-1 receptor agonist, suppresses hypoxia-induced cytokine release via a TRPV1 mediated pathway. Using gene-targeted TRPV1-null mutant (TRPV1 -/- ) or wild-type (WT, C57BL/6) mice, plasma creatinine or urine lactate dehydrogenase (LDH) levels were measured 40- min after ischemia plus 3-hr reperfusion (I/R). Renal I/R increased plasma creatinine and urine LDH levels with a greater magnitude in TRPV1-/- than WT mice (creatinine, uM: WT: 99.4 ± 4.4 to TRPV1: 129.7 ± 11.9, p &lt; 0.05; LDH, mU: WT: 132.3± 39.8 to TRPV1-/-: 178.5 ± 44.4, p &lt; 0.05). In vitro, cytokine or chemokine secretion from renal tissues incubated either with 95% O 2 -5% CO 2 or 99% nitrogen (hypoxia) was measured. TNFα, IL-6, or MCP-1 release in response to nitrogen (1 hr) was increased in both strains with a bigger magnitude in TRPV1-/- than WT mice (p&lt;0.05). Blockade of receptors for calcitonin gene-related peptide (CGRP) with CGRP8-38 (10 -7 M) further increased hypoxia-induced MCP-1 release in WT mice (p&lt;0.05). Ex4 suppressed nitrogen (0.5hr)-induced cytokine/chemokine release from the kidneys in WT but not TRPV1-/-mice (WT: IL-6: 0.74 ± 0.08 to 0.66 ± 0.03; TNF-a: 0.73 ± 0.07 to 0.66 ± 0.03; MCP-1: 16.1± 3.5 to 12.7 ± 1.3 pg/g/min, p &lt; 0.05. TRPV1-/-: p&gt;0.05 for all). Moreover, Ex4 increased CGRP release from the renal pelvis in WT ( p &lt; 0.05) but not TRPV1 -/- mice, which can be inhibited by Ex9-39, an Ex4 antagonist. Thus, ablation of TRPV1 or blockade of CGRP receptors enhances hypoxia-induced inflammation and renal injury following I/R. Ex4 suppresses hypoxia-induced cytokine/chemokine release and enhances CGRP release from the kidney only when TRPV1 function and expression are intact, suggesting that TRPV1/CGRP may play a key role mediating Ex4-induced anti-inflammatory effects.

The transient receptor potential vanilloid-1 is localized at excitatory synapses in the mouse dentate gyrus

The transient receptor potential vanilloid type 1 (TRPV1) is a non-selective cation channel that plays an important role in pain perception and modulates neurotransmitter release and synaptic plasticity in the brain. TRPV1 function must lay on its anatomical distribution in the peripheral and central nervous system regions involved in the physiological roles of the channel. However, the anatomical localization of TRPV1 is well established in the periphery, but in the brain it is a matter of debate. While some studies support the presence of TRPV1 in several brain regions, recent evidences suggest a restricted distribution of the channel in the central nervous system. To investigate to what extent central TRPV1 function stands on a precise brain distribution of the channel, we examined the mouse hippocampal dentate molecular layer (ML) where TRPV1 mediates long-term synaptic plasticity. Using pre-embedding immunocytochemistry for high resolution electron microscopy, we show that TRPV1 immunoparticles are highly concentrated in postsynaptic dendritic spines to asymmetric perforant path synapses in the outer 2/3 of the ML. However, TRPV1 is poorly expressed at the excitatory hilar mossy cell synapses in the inner 1/3 of this layer. Importantly, the TRPV1 pattern distribution disappeared in the ML of TRPV1-knockout mice. Taken together, these findings support the notion of the presence of TRPV1 in a brain region where the channel has been shown to have a functional role, such as the perforant path synapses in the hippocampal dentate ML.

Abstract 94: High Sucrose Intake-induced Endoplasmic Reticulum Stress, Inflammation and Fibrosis in hte Pancreas: Role of Trpv1

Excessive consumption of sugar may serve as a primary and independent factor for increased morbidity of diabetes. We test the hypothesis that the transient receptor potential vanilloid type 1 (TRPV1) channels play a protective role against sugar-induced pancreatic endoplasmic reticulum (ER) stress, inflammatory cell infiltration, and tissue fibrosis. Wild-type (WT, C57BL/6) or gene-targeted TRPV1-null mutant (TRPV1 -/- ) mice were fed a normal (CON) or high sucrose diet for 22-23 weeks. TRPV1 ablation impaired glucose tolerance and suppressed glucose-induced insulin release when fed a CON diet (p&lt;0.05). Sucrose intake further impaired glucose tolerance and insulin release in TRPV1 -/- compared to WT mice (p&lt;0.05). Sucrose intake increased pancreatic ER stress, showing elevated levels of chop protein content in both strains and GRP78, XBP-1, and CREB-2 in TRPV1 -/- mice only (p&lt;0.05). TRPV1 ablation increased levels of phosphorylated JNK (WT, 0.12±0.14; TRPV1-/-, 0.31±0.13) and decreased glut2 contents (WT, 1.38±0.24; TRPV1-/-, 0.83±0.09) in the pancreas when fed a CON diet (p&lt;0.05). Sucrose intake further increased levels of pancreatic phosphorylated JNK (WT, 0.89±0.19; TRPV1-/-, 1.05±0.19) and decreased glut2 contents (WT, 0.39±0.04; TRPV1-/-, 0.26±0.16) in both strains (p&lt;0.05). Sucrose intake increased pancreatic TGF-β levels in both strains with exacerbation in TRPV1-/- mice (p&lt;0.05), and increased pancreatic phosphorylated Smad2/3 and collagen levels in TRPV1 -/- mice only (p&lt;0.05). Quantitative immunostaining revealed that sucrose intake enhanced pancreatic infiltration of CD3 positive cells in both strains and macrophage in TRPV1 -/- mice only (p&lt;0.05). Thus, TRPV1 ablation impairs insulin release and glucose tolerance at baseline possibly via increased pancreatic phosphorylated JNK signaling and decreased glut2 expression. Sucrose intake aggravates impairment in insulin release and glucose tolerance when TRPV1 is ablation, possibly via enhancing ER stress, macrophage infiltration, and activation of the TGF-β/Smad pathway leading to pancreatic fibrosis, suggesting preserving TRPV1 expression and function may attenuate pancreatic injury via alleviating inflammatory responses.

Agonist-Dependent Potentiation of Vanilloid Receptor Transient Receptor Potential Vanilloid Type 1 Function by Stilbene Derivatives

Transient receptor potential vanilloid type 1 (TRPV1) is a nonselective cation channel activated by capsaicin, low pH, and noxious heat and plays a key role in nociception. Understanding mechanisms for functional modulation of TRPV1 has important implications. One characteristic of TRPV1 is that channel activity induced by either capsaicin or other activators can be sensitized or modulated by factors involving different cell signaling mechanisms. In this study, we describe a novel mechanism for the modulation of TRPV1 function: TRPV1 function is modulated by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and its analogs. We found that, in rat dorsal root ganglion neurons, although DIDS did not induce the activation of TRPV1 per se but drastically increased the TRPV1 currents induced by either capsaicin or low pH. DIDS also blocked the tachyphylaxis of the low pH-induced TRPV1 currents. 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), a DIDS analog, failed to enhance the capsaicin-evoked TRPV1 current but increased the low pH-evoked TRPV1 currents, with an effect comparable with that of DIDS. SITS also blocked the low pH-induced tachyphylaxis. DIDS also potentiated the currents of TRPV1 channels expressed in human embryonic kidney 293 cells, with an effect of left-shifting the concentration-response curve of the capsaicin-induced TRPV1 currents. This study demonstrates that DIDS and SITS, traditionally used chloride channel blockers, can modify TRPV1 channel function in an agonist-dependent manner. The results provide new input for understanding TRPV1 modulation and developing new modulators of TRPV1 function.

Increased transient receptor potential vanilloid type 1 (TRPV1) signaling in idiopathic overactive bladder urothelial cells

To measure transient receptor potential vanilloid type 1 (TRPV1) signaling in human bladder urothelial cells (BUC) from non-neurogenic overactive bladder (OAB) patients and control subjects (NB) BUC. Primary BUC cell cultures were derived from cystoscopic biopsies from two OAB and two NB subjects. TRPV1 expression was detected by immunofluorescence, PCR and Western blot staining. TRPV1 function was assessed by capsaicin (CAP, 6 µM)-evoked intracellular calcium ([Ca(2+)](i)) changes measured by microfluorimetry imaging. CAP evoked changes in inward and outward currents were recorded electrophysiologically using excised outside-out patches and whole cell configurations using various protocols. OAB BUC had significantly increased expression of TRPV1 compared to NB BUC on Western blot. CAP evoked significantly higher maximal [Ca(2+)](i) change over baseline in OAB (84.71 ± 8.96%) compared to NB BUC (60.32 ± 7.93%) (P < 0.05). CAP induced significantly greater percent change in single channel open probability (205.94 ± 20.53% OAB vs. 141.26 ± 16.53% NB, P < 0.05) and normalized inward currents (13.54 ± 1.6 4 pA/pF OAB vs. 8.28 ± 0.89 pA/pF NB, P < 0.05). CAP caused significantly higher percent increase from baseline of whole cell outward currents in OAB (177.12 ± 44.46%) compared to NB BUC (135.98 ± 44.28%) (P < 0.05). Similarly thermal stimulus (45°C solution) evoked significantly higher percent increase in whole cell outward currents in OAB (183.93 ± 14.07%) compared to NB (145.61 ± 10.12%) BUC (P < 0.05). These responses were blocked by 10 µM capsazepine (CPZ), a TRPV1 antagonist. Because only a few subjects were studied, augmented TRPV1 signaling cannot be generalized to all OAB subjects. However, the findings are consistent with the hypothesis that BUC are involved in sensory signaling.

Corticosterone Mediates Reciprocal Changes in CB 1 and TRPV1 Receptors in Primary Sensory Neurons in the Chronically Stressed Rat

Chronic stress is associated with visceral hyperalgesia in functional gastrointestinal disorders. We investigated whether corticosterone plays a role in chronic psychological stress-induced visceral hyperalgesia. Male rats were subjected to 1-hour water avoidance (WA) stress or subcutaneous corticosterone injection daily for 10 consecutive days in the presence or absence of corticoid-receptor antagonist RU-486 and cannabinoid-receptor agonist WIN55,212-2. The visceromotor response to colorectal distension was measured. Receptor protein levels were measured and whole-cell patch-clamp recordings were used to assess transient receptor potential vanilloid type 1 (TRPV1) currents in L6-S2 dorsal root ganglion (DRG) neurons. Mass spectrometry was used to measure endocannabinoid anandamide content. Chronic WA stress was associated with visceral hyperalgesia in response to colorectal distension, increased stool output and reciprocal changes in cannabinoid receptor 1 (CB1) (decreased) and TRPV1 (increased) receptor expression and function. Treatment of WA stressed rats with RU-486 prevented these changes. Control rats treated with serial injections of corticosterone in situ showed a significant increase in serum corticosterone associated with visceral hyperalgesia, enhanced anandamide content, increased TRPV1, and decreased CB1 receptor protein levels, which were prevented by co-treatment with RU-486. Exposure of isolated control L6-S2 DRGs in vitro to corticosterone reproduced the changes in CB1 and TRPV1 receptors observed in situ, which was prevented by co-treatment with RU-486 or WIN55,212-2. These results support a novel role for corticosterone to modulate CB1 and TRPV1-receptor pathways in L6-S2 DRGs in the chronic WA stressed rat, which contributes to visceral hyperalgesia observed in this model.

P2Y2receptors mediate ATP-induced resensitization of TRPV1 expressed by kidney projecting sensory neurons

The transient receptor potential vanilloid type 1 (TRPV1) channel is a ligand-gated cation channel expressed by sensory nerves. P2Y receptors are G protein-coupled receptors that are also expressed by TRPV1-positive sensory neurons. Therefore, we studied interactions between P2Y receptors and TRPV1 function on kidney projecting sensory neurons. Application of Fast Blue (FB) to nerves surrounding the renal artery retrogradely labeled neurons in dorsal root ganglia of rats. Whole cell recording was performed on FB-labeled neurons maintained in primary culture. Capsaicin was used to activate TRPV1. Four types of kidney projecting neurons were identified based on capsaicin responses: 1) desensitizing (35%), 2) nondesensitizing (29%), 3) silent (3%), and 4) insensitive (30%). Silent neurons responded to capsaicin only after ATP (100 microM) pretreatment. ATP reversed desensitization in desensitizing neurons. Insensitive neurons never responded to capsaicin. UTP, a P2Y purinoceptor 2 (P2Y(2))/P2Y(4) receptor agonist, reversed capsaicin-induced TRPV1 desensitization. 2-methyl-thio-ATP (2-Me-S-ATP), a P2Y(1) receptor agonist, did not change desensitization. MRS 2179 and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), drugs that block P2Y(1) receptors, did not block ATP-induced resensitization of TRPV1. Suramin, a P2Y(2) receptor antagonist, blocked resensitization caused by UTP. Immunocytochemical studies showed that FB-labeled neurons coexpressed P2Y(2) receptors and TRPV1. We conclude that P2Y(2) receptor activation can maintain TRPV1 function perhaps during sustained episodes of activity of kidney projecting sensory neurons.

Role of TRPV1 channels in renal haemodynamics and function in Dahl salt-sensitive hypertensive rats.

This study tests the hypothesis that dysfunction of transient receptor potential vanilloid type 1 (TRPV1) channels occurs and contributes to the decrease in the glomerular filtration rate (GFR) and sodium/water excretion in Dahl salt-sensitive hypertensive rats. Recirculating Krebs-Henseleit buffer added with inulin was perfused at a constant flow in the isolated kidneys of Dahl salt-sensitive (DS) or Dahl salt-resistant (DR) rats fed a high-salt (HS) or low-salt (LS) diet for 3 weeks. Perfusion pressures (PP) were pre-adjusted to three levels ( approximately 100, approximately 150 or approximately 190 mmHg) with or without phenylephrine. Capsaicin, a selective TRPV1 agonist, in the presence or absence of capsazepine, a selective TRPV1 antagonist, was perfused. Basal GFR, urine flow rate (UFR) and Na(+) excretion (U(Na)V) were significantly lower in DS-HS than in DR-HS, DS-LS and DR-LS rats. Capsaicin caused pressure-dependent decreases in PP and increases in GFR, UFR and U(Na)V in all groups, with less magnitude of decreases in PP and increases in GFR, UFR and U(Na)V in DS-HS than in DR-HS, DS-LS and DR-LS rats. Capsazepine completely blocked the effect of capsaicin on PP, GFR, UFR and U(Na)V in all groups. Thus, these results show that TRPV1 function is impaired in the kidney of DS rats fed a high-salt diet, which may contribute to the decrease in GFR and renal excretory function in DS rats in the face of salt challenge.

Interdependent Regulation of Afferent Renal Nerve Activity and Renal Function: Role of Transient Receptor Potential Vanilloid Type 1, Neurokinin 1, and Calcitonin Gene-Related Peptide Receptors

Our previous studies have shown that the activation of the transient receptor potential vanilloid type 1 (TRPV1) expressed in the renal pelvis leads to an increase in ipsilateral afferent renal nerve activity (ARNA) and contralateral renal excretory function, but the molecular mechanisms of TRPV1 action are largely unknown. This study tests the hypothesis that activation of receptors of neurokinin 1 (NK1) or calcitonin gene-related peptide (CGRP) by endogenously released substance P (SP) or CGRP following TRPV1 activation, respectively, governs TRPV1-induced increases in ARNA and renal excretory function. Capsaicin (CAP; 0.04, 0.4, and 4 nM), a selective TRPV1 agonist, administered into the renal pelvis dose-dependently increased ARNA. CAP (4 nM)-induced increases in ipsilateral ARNA or contralateral urine flow rate (Uflow) and urinary sodium excretion (UNa) were abolished by capsazepine (CAPZ), a selective TRPV1 antagonist, or 2-[1-imino-2-(2-methoxyphenyl)ethyl]-7,7-diphenyl-4-perhydroisoindolone (3aR,7aR) (RP67580) or cis-2-(diphenylmethyl)-N-[(2-iodophenyl)-methyl]-1 azabicyclo[2.2.2]octan-3-amine (L703,606), selective NK1 antagonists, but not by CGRP8-37, a selective CGRP receptor antagonist. Both SP (7.4 nM) and CGRP (0.13 muM) increased ARNA, Uflow, or UNa, and increases in these parameters induced by CGRP but not SP were abolished by CAPZ. CAP at 4 nM perfused into the renal pelvis caused the release of SP and CGRP, which was blocked by CAPZ but not by RP67580, L703,606, or CGRP8-37. Immunofluorescence results showed that NK1 receptors were expressed in sensory neurons in dorsal root ganglion and sensory nerve fibers innervating the renal pelvis. Taken together, our data indicate that NK1 activation induced by SP release upon TRPV1 activation governs TRPV1 function and that a TRPV1-dependent mechanism is operant in CGRP action.

TRPV1 gene knockout impairs preconditioning protection against myocardial injury in isolated perfused hearts in mice

Although the transient receptor potential vanilloid type 1 (TRPV1)-containing afferent nerve fibers are widely distributed in the heart, the relationship between TRPV1 function and cardiac ischemic preconditioning (PC) has not been well defined. Using TRPV1 knockout mice (TRPV1(-/-)), we studied the role of TRPV1 in PC-induced myocardial protection. Hearts of gene-targeted TRPV1-null mutant (TRPV1(-/-)) or wild-type (WT) mice were Langendorffly perfused in the presence or absence of CGRP(8-37), a selective calcitonin gene-related peptide (CGRP) receptor antagonist; or RP-67580, a selective neurokinin-1 receptor antagonist when hearts were subjected to three 5-min periods of ischemia PC followed by 30 min of global ischemia and 40 min of reperfusion (I/R). PC before I/R decreased left ventricular (LV) end-diastolic pressure and increased LV developed pressure, coronary flow (CF), peak-positive maximum rate of rise of LV pressure in WT mice (PC-WT) compared with PC-TRPV1(-/-), TRPV1(-/-), or WT hearts (P < 0.05), and PC also decreased LV end-diastolic pressure in PC-TRPV1(-/-) compared with TRPV1(-/-). CGRP(8-37) or RP-67580 abolished PC-induced protection in WT but not TRPV1(-/-) hearts (P < 0.05). Moreover, PC decreased lactate dehydrogenase release and infarct size in PC-WT compared with PC-TRPV1(-/-), TRPV1(-/-), or WT hearts, and it also lowered these parameters in PC-TRPV1(-/-) compared with TRPV1(-/-) hearts (P < 0.05). Radioimmunoassay showed that the release of substance P and CGRP after PC was higher in WT hearts than in TRPV1(-/-) hearts (P < 0.05), which was attenuated by capsazepine in WT but not TRPV1(-/-) hearts. Thus PC-induced protection of the heart was impaired in TRPV1(-/-) hearts, indicating that TRPV1 contributes to the beneficial effects of preconditioning against I/R injury through release substance P and CGRP.