VR1 Receptors Research Articles

The anandamide transport inhibitor AM404 reduces ethanol self‐administration

The endocannabinoid system mediates in the pharmacological actions of ethanol and genetic studies link endocannabinoid signaling to alcoholism. Drugs activating cannabinoid CB1 receptors have been found to promote alcohol consumption but their effects on self-administration of alcohol are less clear because of the interference with motor performance. To avoid this problem, a novel pharmacological approach to the study of the contribution of the cannabinoid system in alcoholism may be to use drugs that locally amplify the effects of alcohol on endogenous cannabinoids. In the present study we addressed this model by studying the effects of the anandamide transport inhibitor N-(4-hydroxyphenyl) arachidonoyl-ethanolamide (AM404) on both ethanol self-administration and reinstatement of alcohol-seeking behavior in rats. The results show that AM404 significantly reduced ethanol self-administration in a dose-dependent manner but failed to modify reinstatement for lever pressing induced by the stimulus associated with alcohol. This effect was not due to a motor depressant effect and was not related to a decrease in general motivational state, as it was not effective in other reward paradigms such as lever pressing for a saccharin solution. The mechanism of action of AM404 does not involve cannabinoid CB1 receptors as the CB1-selective antagonist SR141716A did not block the reduction of ethanol self-administration induced by the anandamide uptake blocker. Moreover, 3-(1,1-dimethylheptyl)-(-)-11-hydroxy-delta 8-tetrahydrocannabinol (HU-210), a classical cannabinoid receptor agonist, did not affect ethanol self-administration. The effects of AM404 are not mediated by either vanilloid VR1 receptors or cannabinoid CB2 receptors because it is not antagonized by either the VR1 receptor antagonist capsazepine or the CB2 antagonist AM630. These results indicate that AM404 may be considered as an innovative approach to reduce alcohol consumption.

The contribution of VR1 and CB1 receptors and the role of the afferent vagal pathway in modelling of cardio-respiratory effects of anandamide in rats

Anaesthetized and spontaneously breathing rats were used to study the cardio-respiratory effects of intravenous anandamide administration. To investigate the role of particular levels of the afferent pathway in this response rats were challenged with bolus injection of anandamide (1 mg kg − 1 ) into the femoral vein while intact, following bilateral superior laryngeal nerves (SLNs) section and after midcervical vagotomy. To test the hypothesis that the activation of the vanilloid receptors (VR1) as well as cannabinoid receptors (CB1) contributes to the anandamide-induced response administrations of anandamide were preceded by nonselective VR1 antagonist ruthenium red or selective CB1 antagonist AM281. Anandamide evoked apnoea of mean duration of 4.84 ± 0.75 s in all animals while intact which was shortened by subsequent neurotomies, after SLNs section to 3.3 ± 0.57 s ( P < 0.05) and after midcervical vagi section to 1.99 ± 0.24 s ( P < 0.01). In post-apnoeic breathing tidal volume ( V T) was reduced in all neural states. Anandamide evoked hypotension in the intact and SLNs neurotomized rats. Midcervical vagotomy reduced this fall in blood pressure. Both antagonists ruthenium red and AM281 eliminated post-anandamide apnoea and hypotension but had no effect on post-apnoeic depression of V T. Subsequent SLNs and cervical vagi sections did not eliminate but only reduced post-anandamide depression of breathing. Midcervical vagotomy lessened anandamide-induced hypotension. Apnoeic and hypotensive response to anandamide was mediated by both VR1 and CB1 receptors. Post-anandamide decline of V T might depend on different type of receptors.

Reduced Anxiety, Conditioned Fear, and Hippocampal Long-Term Potentiation in Transient Receptor Potential Vanilloid Type 1 Receptor-Deficient Mice

The transient receptor potential vanilloid type 1 channel (TRPV1) (formerly called vanilloid receptor VR1) is known for its key role of functions in sensory nerves such as perception of inflammatory and thermal pain. Much less is known about the physiological significance of the TRPV1 expression in the brain. Here we demonstrate that TRPV1 knock-out mice (TRPV1-KO) show less anxiety-related behavior in the light-dark test and in the elevated plus maze than their wild-type littermates with no differences in locomotion. Furthermore, TRPV1-KO mice showed less freezing to a tone after auditory fear conditioning and stress sensitization. This reduction of conditioned and sensitized fear could not be explained by alterations in nociception. Also, tone perception per se was unaffected, as revealed by determination of auditory thresholds through auditory brainstem responses and distortion-product otoacoustic emissions. TRPV1-KO showed also less contextual fear if assessed 1 d or 1 month after strong conditioning protocols. These impairments in hippocampus-dependent learning were mirrored by a decrease in long-term potentiation in the Schaffer collateral-commissural pathway to CA1 hippocampal neurons. Our data provide first evidence for fear-promoting effects of TRPV1 with respect to both innate and conditioned fear and for a decisive role of this receptor in synaptic plasticity.

The Endogenous Cannabinoid Anandamide Produces δ-9-Tetrahydrocannabinol-Like Discriminative and Neurochemical Effects That Are Enhanced by Inhibition of Fatty Acid Amide Hydrolase but Not by Inhibition of Anandamide Transport

Anandamide is an endogenous ligand for brain cannabinoid CB(1) receptors, but its behavioral effects are difficult to measure due to rapid inactivation. Here we used a drug-discrimination procedure to test the hypothesis that anandamide, given i.v. or i.p., would produce discriminative effects like those of delta-9-tetrahydrocannabinol (THC) in rats when its metabolic inactivation was inhibited. We also used an in vivo microdialysis procedure to investigate the effects of anandamide, given i.v. or i.p., on dopamine levels in the nucleus accumbens shell in rats. When injected i.v., methanandamide (AM-356), a metabolically stable anandamide analog, produced clear dose-related THC-like discriminative effects, but anandamide produced THC-like discriminative effects only at a high 10-mg/kg dose that almost eliminated lever-press responding. Cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB-597), an inhibitor of fatty acid amide hydrolase (FAAH), the main enzyme responsible for metabolic inactivation of anandamide, produced no THC-like discriminative effects alone but dramatically potentiated discriminative effects of anandamide, with 3 mg/kg anandamide completely substituting for the THC training dose. URB-597 also potentiated the ability of anandamide to increase dopamine levels in the accumbens shell. The THC-like discriminative-stimulus effects of anandamide after URB-597 and methanandamide were blocked by the CB1 receptor antagonist rimonabant, but not the vanilloid VR1 receptor antagonist capsazepine. Surprisingly, the anandamide transport inhibitors N-(4-hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide (AM-404) and N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide (UCM-707) did not potentiate THC-like discriminative effects of anandamide or its dopamine-elevating effects. Thus, anandamide has THC-like discriminative and neurochemical effects that are enhanced after treatment with a FAAH inhibitor but not after treatment with transport inhibitors, suggesting brain area specificity for FAAH versus transport/FAAH inactivation of anandamide.

Evidences of submucosal neuronal plasticity in distal colon of chronic restraint stress rat

Chronic stress is involved in the pathogenesis of gastrointestinal disorders. The phenotype and function of neurons in submucosal reflex may be subjects for this evidence. We investigated whether enteric neuronal reflex was changed during chronic stress. Male Wistar rats were immobilized for 14 days (1-hr/day). Distal colonic epithelium was isolated to measure Isc and Rt by Ussing chamber techniques. Isc and Rt of stressed-rat colonic segments were lower than those in control rats. The secretory effects of vanilloid capsaicin (3 μM) mediated by VR-1 on sensory neurons was decreased by 70% in stressed-rat colon. Basolateral addition of DAMGO, DSLET or U-50,488 (1 μM), μ-, δ- or μ-opioid receptor (OR) agonists respectively, diminished the basal Isc of control rat colon by 20–30%, whereas only U-50,488 decreased the baseline Isc of stress rat colon by 20%. Substance P (SP, 1 μM) produced the same magnitude of Isc response in both control and stress rat colon. Pretreatment with DAMGO potentiated the SP response by 2-fold in stress rats. These findings suggested that the submucosal neuronal reflex mediated through VR-1 and opioid receptors was modified in distal colon of chronic stress rat. The changes in submucosal neuronal plasticity may be the cause of stress induced gastrointestinal disorders. This work was supported by the Thailand Research Fund (RSA 4780010) and Chulalongkorn University.

Anandamide mediates hyperdynamic circulation in cirrhotic rats via CB1 and VR1 receptors

Hyperdynamic circulation and mesenteric hyperaemia are found in cirrhosis. To delineate the role of endocannabinoids in these changes, we examined the cardiovascular effects of anandamide, AM251 (CB(1) antagonist), AM630 (CB(2) antagonist) and capsazepine (VR1 antagonist), in a rat model of cirrhosis. Cirrhosis was induced by bile duct ligation. Controls underwent sham operation. Four weeks later, diameters of mesenteric arteriole and venule (intravital microscopy), arterial pressure, cardiac output, systemic vascular resistance and superior mesenteric artery (SMA) flow were measured after anandamide, AM251 (with or without anandamide), AM630 and capsazepine administration. CB(1), CB(2) and VR1 receptor expression in SMA was assessed by western blot and RT-PCR. Anandamide increased mesenteric vessel diameter and flow, and cardiac output in cirrhotic rats, but did not affect controls. Anandamide induced a triphasic arterial pressure response in controls, but this pattern differed markedly in cirrhotic rats. Pre-administration of AM251 blocked the effects of anandamide. AM251 (without anandamide) increased arterial pressure and systemic vascular resistance, constricted mesenteric arterioles, decreased SMA flow and changed cardiac output in a time-dependent fashion in cirrhotic rats. Capsazepine decreased cardiac output and mesenteric arteriolar diameter and flow, and increased systemic vascular resistance in cirrhotic rats, but lacked effect in controls. Expression of CB(1) and VR1 receptor proteins were increased in cirrhotic rats. AM630 did not affect any cardiovascular parameter in either group. These data suggest that endocannabinoids contribute to hyperdynamic circulation and mesenteric hyperaemia in cirrhosis, via CB(1)- and VR1-mediated mechanisms.

Transient Receptor Potential Vanilloid Receptor Subtype 1 in Painful Bladder Syndrome and its Correlation With Pain

Painful bladder syndrome is a chronic, debilitating bladder hypersensitivity disorder characterized by urinary frequency, urgency and bladder pain without an identifiable cause. Recent advances in understanding the molecular basis of hypersensitivity provide an opportunity to advance the understanding of and treatment for painful bladder syndrome. We studied the heat and capsaicin receptor transient receptor potential vanilloid receptor subtype 1 in the bladder in patients with painful bladder syndrome and their relationship to pain symptoms. Bladder biopsies were obtained from 20 characterized subjects with painful bladder syndrome and 25 with asymptomatic microscopic hematuria as controls. Specimens were immunostained using specific antibodies to transient receptor potential vanilloid receptor subtype 1 and neurofilaments as a structural maker. Nerve fiber and urothelial staining were quantified with computerized image analysis. The results of immunohistochemistry were correlated with the pain score. There was a marked increase in suburothelial nerve fibers expressing transient receptor potential vanilloid receptor subtype 1 in painful bladder syndrome in comparison with that in controls (p <0.0001). The ratio of transient receptor potential vanilloid receptor subtype 1 fibers to neurofilaments was also significantly increased in painful bladder syndrome, suggesting over expression of transient receptor potential vanilloid receptor subtype 1 (p <0.0001). When all specimens studied were included, the pain score correlated significantly with the relative nerve fiber density of transient receptor potential vanilloid receptor subtype 1 in the suburothelium (r = 0.6862, p = 0.0002) as well as the ratio of transient receptor potential vanilloid receptor subtype 1 fibers to neurofilaments (r = 0.5554, p = 0.004). Urothelial transient receptor potential vanilloid receptor subtype 1 showed a tendency toward an increase in the painful bladder syndrome group but it did not achieve statistical significance. No correlation was found between transient receptor potential vanilloid receptor subtype 1 immunoreactivity of urothelium or neurofilament fibers and the pain score. This study shows increased transient receptor potential vanilloid receptor subtype 1 in nerve fibers of the bladder in painful bladder syndrome and a correlation of the pain score with the relative density of transient receptor potential vanilloid receptor subtype 1 nerve fibers. Transient receptor potential vanilloid receptor subtype 1 may have a role in the pathophysiology of painful bladder syndrome and it is a potential target for novel therapeutic agents.

Anandamide administration alone and after inhibition of fatty acid amide hydrolase (FAAH) increases dopamine levels in the nucleus accumbens shell in rats

Although endogenous cannabinoid systems have been implicated in the modulation of the rewarding effects of abused drugs and food, little is known about the direct effects of endogenous ligands for cannabinoid receptors on brain reward processes. Here we show for the first time that the intravenous administration of anandamide, an endogenous ligand for cannabinoid receptors, and its longer-lasting synthetic analog methanandamide, increase the extracellular dopamine levels in the nucleus accumbens shell of awake, freely moving rats, an effect characteristic of most drugs abused by humans. Anandamide produced two distinctly different effects on dopamine levels: (1) a rapid, transient increase that was blocked by the cannabinoid CB1 receptor antagonist rimonabant, but not by the vanilloid VR1 receptor antagonist capsazepine, and was magnified and prolonged by the fatty acid amide hydrolase (FAAH) enzyme inhibitor, URB597; (2) a smaller delayed and long-lasting increase, not sensitive to CB1, VR1 or FAAH blockade. Both effects were blocked by infusing either tetrodotoxin (TTX, 1 microm) or calcium-free Ringer's solution through the microdialysis probe, demonstrating that they were dependent on the physiologic activation of dopaminergic neurotransmission. Thus, these results indicate that anandamide, through the activation of the mesolimbic dopaminergic system, participates in the signaling of brain reward processes.

186 The involvement of CB1 and VR1 receptors in cardiovascular disturbances of bile-duct ligated rats

186 The involvement of CB1 and VR1 receptors in cardiovascular disturbances of bile-duct ligated rats

77 Vanilloid receptor expression in body and trigone of female urinary bladder: changes in urgency without detrusor overactivity

Introduction: Patients with sensory urgency (SU): the symptom complex of urgency associated with pain, burning or discomfort, often accompanied by frequency and nocturia, are difficult to treat. Vanilloid receptors on afferent neurons and on urothelium may participate in signalling from the bladder. Our aim was to examine whether changes in VR1 expression occur in bladder from SU patients compared with controls.Patients: Biopsies from body and trigone were obtained from patients with SU (female, aged 21–82) who had undergone videourodynamics to exclude detrusor overactivity and define first sensation. Asymptomatic age‐matched females with prior history of carcinoma acted as controls. Biopsies were collected into ‘RNALater’. Specimens were dissected into two layers, mucosa and detrusor muscle, before RNA extraction and quantitative competitive RT‐PCR (Mansfield et al 2005 Br J Pharmacol 144: 1089–99).Results: In control biopsies, VR1 mRNA was present in greater density in mucosa than in detrusor muscle (15.3 × 105cf. 4.6 × 105 copies/μg RNA, both n = 15, P &lt; 0.01). In SU, VR1 mRNA expression was significantly higher in trigonal mucosa than in body mucosa (19.1 × 105 cf. 7.4 × 105 copies/μg RNA, n = 21‐‐23, P &lt; 0.01). There was no significant change in VR1 expression in body mucosa from SU specimens compared with matched controls. In SU patients, the level of VR1 expression in trigonal mucosa (but not body mucosa) was significantly inversely correlated with the volume at first bladder sensation (r2 = 0.41, P &lt; 0.01).Conclusion: We hypothesise that excessive afferent signalling via VR1 receptors in the trigone region may be related to an early first bladder sensation and sensory urgency.

Pain Behaviors Produced by Capsaicin: Influence of Inflammatory Mediators and Nerve Injury

The present study was undertaken to characterize spontaneous (ie, nonevoked) pain behaviors (flinching, biting/licking) produced by local injections of capsaicin into the rat hindpaw as a model of chemogenic pain, and to determine effects of inflammatory mediators and nerve injury on such behaviors. Capsaicin antagonists are a potential class of novel topical analgesics, and this model may be of value for preclinical screening of novel compounds. Local injections of capsaicin (0.1-30 μg) into the hindpaw produced flinching and biting/licking behaviors over 5 min, and these were reduced by capsazepine, a competitive antagonist for capsaicin at the TRPV1 receptor. Coadministration of noradrenaline (NA), prostaglandin E 2 (PGE 2), and 5-hydroxytryptamine (5-HT) augmented capsaicin-evoked responses primarily by extending the duration of behaviors. Partial sciatic nerve ligation decreased flinching produced by capsaicin alone, by capsaicin in combination with each of NA, PGE 2, and 5-HT, and by formalin. Tibial nerve injury also reduced capsaicin-evoked flinching, and responses to formalin, but spinal nerve ligation did not affect either. These results indicate that (1) spontaneous pain behaviors occur as a result of TRPV1 receptor activation with a different time course than evoked responses, (2) inflammatory mediators augment capsaicin-evoked pain behaviors, and (3) various forms of nerve injury produce different effects on capsaicin-evoked pain behaviors. Perspective The VR1 receptor is a potential target for development of novel topical analgesics. This study characterized pain behaviors produced by local injections of capsaicin in the presence of inflammatory mediators and following various forms of nerve injury. Results are of interest for the preclinical screening of novel VR1 receptor antagonists.

Endocannabinoid Degradation and Oxidative Defense Mechanisms Determine Anandamide-induced Cell Death in Liver Cell Populations

Background: We have shown that the endogenous cannabinoid anandamide (AEA) induces reactive oxygen species (ROS)-dependent cell death in primary hepatic stellate cells (HSCs), but not in primary hepatocytes. Aim: To determine cellular factors that control AEA-induced cell death in hepatic cell populations. Methods: Fatty acid amide hydrolase (FAAH) expression was analyzed by western blot and real time PCR in primary rat hepatocytes and primary rat HSCs. FAAH activity was determined colorimetrically. Cell death was analyzed by LDH release, propidium iodide uptake and western blot for caspase 3- and PARP cleavage. Glutathione (GSH) levels were measured by the DTNB method. mRNA expression of the endocannabinoid receptors CB1, CB2 and VR1 was determined by real time PCR. ROS production was monitored by DCFDA fluorescence. Results: FAAH mRNA, FAAH protein and FAAH activity were extremely high in primary rat hepatocytes, but virtually absent in primary rat HSCs. Hepatocytes from FAAH -/- mice, or wildtype hepatocytes which had been pretreated with the FAAH inhibitor URB597 were susceptible to AEA-mediated death (45% and 40% at 50 μM after 24h, respectively). Conversely, FAAH overexpression prevented AEA-induced death in HSCs. Anandamide induced an increase in ROS production in hepatocytes which had been pretreated with the FAAH inhibitor URB597 or depleted of GSH by BSO. GSH levels were 10-fold higher in hepatocytes than in HSCs. GSH depletion rendered hepatocytes susceptible to AEA-mediated death while GSH pretreatment prevented cell death in HSCs. The combination of FAAH inhibition and GSH depletion had additive effects on AEA-mediated hepatocyte cell death resulting in almost 70% death after 24h at 50 μM AEA and lowered the threshold for AEA-induced cell death to 500 nM. AEA-induced hepatocyte death after BSO and URB597 pretreatment was purely necrotic and occurred independently of the endocannabinoid receptors CB1, CB2 and VR1 which were virtually absent (CB1, CB2) or expressed at low levels (VR1) in hepatocytes. Conclusion: FAAH and GSH determine endocannabinoid-induced necrosis in hepatocytes and HSCs, independently of expression of cannabinoid receptors. High expression of FAAH and high intracellular GSH levels efficiently prevent AEA-induced cell death in hepatocytes. FAAH-mediated endocannabinoid degradation in hepatocytes leads to a new physiological concept allowing the specific targeting of HSCs in liver fibrosis.

No Pain, No Gain

Myocardial ischemia, the major cause of mortality and morbidity in the United States, accounts for nearly 20% of all deaths.1 The prognosis of patients with acute myocardial infarction is strongly related to the amount of tissue destroyed during ischemia/reperfusion; hence, limiting this injury is of paramount importance for reducing the morbidity and mortality associated with ischemic heart disease.2,3 Over the past 40 years, several hundred pharmacological or nonpharmacological therapies have been reported to alleviate ischemia/reperfusion injury in experimental animal models; unfortunately, few of these results have been reproducible, and, with the exception of early reperfusion, none have been translated into clinical therapies.4 These sobering facts provide a cogent rationale for a reassessment of the models and methodologies that have been used heretofore to test the efficacy of putative cardioprotective interventions. The obvious translational failure in this field calls for a new paradigm in which preclinical studies must be done as rigorously as their clinical counterparts, that is, using clinically relevant animal models, appropriate statistical methods, and a multicenter, randomized, blinded design using centralized core laboratories for data analysis.4 This paradigm would also improve our understanding of the mechanism of cardiomyocyte death during ischemia/reperfusion. Article p 3617 One facet of ischemic biology that has received relatively little attention is the role of cardiac sensory nerves. Myocardial ischemia/reperfusion is known to cause the release of bradykinin and protons, both of which activate the vanilloid receptor 1 (VR1, also known as transient receptor potential vanilloid type 1 [TRPV1]) on the capsaicin-sensitive cardiac sensory nerves (unmyelinated [type C] or thinly myelinated [A-δ] fibers).5 The VR1 is a nonselective cation channel that plays an important role in the polymodal detection of noxious stimuli such as low pH and heat.6 It can also be activated by chemicals such as capsaicin …

Fluorescent leukotriene B4: potential applications

Leukotriene B4 (LTB4) is a potent lipid mediator of inflammation that acts primarily via a seven-transmembrane-spanning, G-protein-coupled receptor denoted BLT1. Here, we describe the synthesis and characterization of fluorescent analogs of LTB4 that are easy to produce, inexpensive, and without the disadvantages of a radioligand. Fluorescent LTB4 is useful for labeling LTB4 receptors for which no antibodies are available and for performing one-step fluorescence polarization assays conducive to high-throughput screening. We found that orange and green fluorescent LTB4 were full agonists that activated the LTB4 receptor BLT1 with EC50 values of 68 and 40 nM, respectively (4.5 nM for unmodified LTB4). Flow cytometric measurements and confocal imaging showed that fluorescent LTB4 colocalized with BLT1. Fluorescence polarization measurements showed that orange fluorescent LTB4 bound to BLT1 with a Kd of 66 nM and that this binding could be displaced by unlabeled LTB4 and other BLT1-specific ligands. Fluorescent LTB4 analogs were also able to displace tritiated LTB4. Orange fluorescent LTB4 binding to enhanced green fluorescent protein-tagged BLT1 could be observed using fluorescence resonance energy transfer. In addition to being a useful alternative to radiolabeled LTB4, the unique properties of fluorescently labeled LTB4 allow a variety of detection technologies to be used.

Anandamide induces necrosis in primary hepatic stellate cells

The endogenous cannabinoid anandamide (AEA) is a lipid mediator that blocks proliferation and induces apoptosis in many cell types. Although AEA levels are elevated in liver fibrosis, its role in fibrogenesis remains unclear. This study investigated effects of AEA in primary hepatic stellate cells (HSCs). Anandamide blocked HSC proliferation at concentrations of 1 to 10 micromol/L but did not affect HSC proliferation or activation at nanomolar concentrations. At higher concentrations (25-100 micromol/L), AEA rapidly and dose-dependently induced cell death in primary culture-activated and in vivo-activated HSCs, with over 70% cell death after 4 hours at 25 micromol/L. In contrast to treatment with Fas ligand or gliotoxin, AEA-mediated death was caspase independent and showed typical features of necrosis such as rapid adenosine triphosphate depletion and propidium iodide uptake. Anandamide-induced reactive oxygen species (ROS) formation, and an increase in intracellular Ca(2+). Pretreatment with the antioxidant glutathione or Ca(2+)-chelation attenuated AEA-induced cell death. Although the putative endocannabinoid receptors CB1, CB2, and VR1 were expressed in HSCs, specific receptor blockade failed to block cell death. Depletion of membrane cholesterol by methyl-beta-cyclodextrin inhibited AEA binding, blocked ROS formation and intracellular Ca(2+)-increase, and prevented cell death. In primary hepatocytes, AEA showed significantly lower binding and failed to induce cell death even after prolonged treatment. In conclusion, AEA efficiently induces necrosis in activated HSCs, an effect that depends on membrane cholesterol and a subsequent increase in intracellular Ca(2+) and ROS. The anti-proliferative effects and the selective killing of HSCs, but not hepatocytes, indicate that AEA may be used as a potential anti-fibrogenic tool.

Heart Failure Modulates the Muscle Reflex

Sympathetic nervous activity (SNA) increases with exercise in normal subjects and this response is accentuated in heart failure (HF). The muscle metaboreceptor contribution to regulation of muscle SNA is blunted in HF whereas the mechanoreceptor contribution is augmented. The underlying causes of these changes in the muscle afferent function are poorly understood. This review is based on recently published data from our laboratory demonstrating that: 1) muscle interstitial ATP rises with twitch contraction and stimulates P2X receptors on thin fibers muscle afferents; 2) ATP accentuates muscle mechanoreceptor responses; 3) activation of VR1 receptor induces an increase in arterial blood pressure; and 4) cardiovascular responses to VR1 receptor stimulation are blunted and P2X mediated responses are augmented in rats with HF as compared with a control group of animals. These results suggest that alternations in VR1 and P2X receptors on muscle afferent nerves influence the processing of sensory information in HF and in turn may alter the magnitude and distribution of SNA during exercise in HF. Keywords: muscle afferent, muscle metabolite, p2x, vr1, sympathetic nerve, muscle pressor reflex

Lipopolysaccharide fever is initiated via a capsaicin‐sensitive mechanism independent of the subtype‐1 vanilloid receptor

As pretreatment with intraperitoneal capsaicin (8-methyl-N-vanillyl-6-nonenamide, CAP), an agonist of the vanilloid receptor known as VR1 or transient receptor potential channel-vanilloid receptor subtype 1 (TRPV-1), has been shown to block the first phase of lipopolysaccharide (LPS) fever in rats, this phase is thought to depend on the TRPV-1-bearing sensory nerve fibers originating in the abdominal cavity. However, our recent studies suggest that CAP blocks the first phase via a non-neural mechanism. In the present work, we studied whether this mechanism involves the TRPV-1. Adult Long-Evans rats implanted with chronic jugular catheters were used. Pretreatment with CAP (5 mg kg(-1), i.p.) 10 days before administration of LPS (10 microg kg(-1), i.v.) resulted in the loss of the entire first phase and a part of the second phase of LPS fever. Pretreatment with the ultrapotent TRPV-1 agonist resiniferatoxin (RTX; 2, 20, or 200 microg kg(-1), i.p.) 10 days before administration of LPS had no effect on the first and second phases of LPS fever, but it exaggerated the third phase at the highest dose. The latter effect was presumably due to the known ability of high doses of TRPV-1 agonists to cause a loss of warm sensitivity, thus leading to uncontrolled, hyperpyretic responses. Pretreatment with the selective competitive TRPV-1 antagonist capsazepine (N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamidem, CPZ; 40 mg kg(-1), i.p.) 90 min before administration of LPS (10 microg kg(-1), i.v.) or CAP (1 mg kg(-1), i.p.) did not affect LPS fever, but blocked the immediate hypothermic response to acute administration of CAP. It is concluded that LPS fever is initiated via a non-neural mechanism, which is CAP-sensitive but RTX- and CPZ-insensitive. The action of CAP on this mechanism is likely TRPV-1-independent. It is speculated that this mechanism may be the production of prostaglandin E(2) by macrophages in LPS-processing organs.

Interaction between Protein Kinase Cμ and the Vanilloid Receptor Type 1

The capsaicin receptor VR1 is a polymodal nociceptor activated by multiple stimuli. It has been reported that protein kinase C plays a role in the sensitization of VR1. Protein kinase D/PKCmu is a member of the protein kinase D serine/threonine kinase family that exhibits structural, enzymological, and regulatory features distinct from those of the PKCs, with which they are related. As part of our effort to optimize conditions for evaluating VR1 pharmacology, we found that treatment of Chinese hamster ovary (CHO) cells heterologously expressing rat VR1 (CHO/rVR1) with butyrate enhanced rVR1 expression and activity. The expression of PKCmu and PKCbeta1, but not of other PKC isoforms, was also enhanced by butyrate treatment, suggesting the possibility that these two isoforms might contribute to the enhanced activity of rVR1. In support of this hypothesis, we found the following. 1) Overexpression of PKCmu enhanced the response of rVR1 to capsaicin and low pH, and expression of a dominant negative variant of PKCmu reduced the response of rVR1. 2) Reduction of endogenous PKCmu using antisense oligonucleotides decreased the response of exogenous rVR1 expressed in CHO cells as well as of endogenous rVR1 in dorsal root ganglion neurons. 3) PKCmu localized to the plasma membrane when overexpressed in CHO/rVR1 cells. 4) PKCmu directly bound to rVR1 expressed in CHO cells as well as to endogenous rVR1 in dorsal root ganglia or to an N-terminal fragment of rVR1, indicating a direct interaction between PKCmu and rVR1. 5) PKCmu directly phosphorylated rVR1 or a longer N-terminal fragment (amino acids 1-118) of rVR1 but not a shorter one (amino acids 1-99). 6) Mutation of S116A in rVR1 blocked both the phosphorylation of rVR1 by PKCmu and the enhancement by PKCmu of the rVR1 response to capsaicin. We conclude that PKCmu functions as a direct modulator of rVR1.

VR-1 receptor blockade attenuates the pressor response to capsaicin but has no effect on the pressor response to contraction in cats

Vanilloid type 1 (VR-1) receptors are stimulated by capsaicin and hydrogen ions, the latter being a by-product of muscular contraction. We tested the hypothesis that activation of VR-1 receptors during static contraction contributes to the exercise pressor reflex. We established a dose of iodoresinaferatoxin (IRTX), a VR-1 receptor antagonist, that blocked the pressor response to capsaicin injected into the arterial supply of muscle. Specifically, in eight decerebrated cats, we compared pressor responses to capsaicin (10 mug) injected into the right popliteal artery, which was subsequently injected with IRTX (100 mug), with those to capsaicin injected into the left popliteal artery, which was not injected with IRTX. The pressor response to capsaicin injected into the right popliteal artery averaged 49 +/- 9 mmHg before IRTX and 9 +/- 2 mmHg after IRTX (P < 0.05). In contrast, the pressor response to capsaicin injected into the left popliteal artery averaged 46 +/- 10 mmHg "before" and 43 +/- 6 mmHg "after" (P > 0.05). We next determined whether VR-1 receptors mediated the pressor response to contraction of the triceps surae. During contraction without circulatory occlusion, the pressor response before IRTX (100 mug) averaged 26 +/- 3 mmHg, whereas it averaged 22 +/- 3 mmHg (P > 0.05) after IRTX (n = 8). In addition, during contraction with occlusion, the pressor responses averaged 35 +/- 3 mmHg before IRTX injection and 49 +/- 7 mmHg after IRTX injection (n = 7). We conclude that VR-1 receptors play little role in evoking the exercise pressor reflex.

Functional Vanilloid Receptor VR1 Is Expressed on Megakaryocytes and Platelets, and Acts as a Sensor for Hypertheimia Generating Endocannabinoids.

Recent investigations have been revealing that platelet can sense shear stress and cold stimulation. These mechanical and physical stimuli alter the platelet functions, including induction of activation and irreversible dysfunction. These data may suggest that platelets, most small-sized cells, can efficiently circulate in the microcirculatory vasculature, and can sense the peripheral physical conditions including temperature.Here we show that human megakaryocytic cell line, MEG-01 and platelets express vanilloid receptor VR1, a receptor for capsaicin. It has been identified that VR1 is linked to thermal sensor, and this can sense hyperthermia around 43 °. Therefore we examined the physiological significance of VR1 expressed on MEG-01 and platelets.First we identified the expression of VR1 on human platelets and MEG-01 by immunoblotting, flowcytometer and RT-PCR, and identified the expression of VR1 on these cells. Hyperthermia stimulation of MEG-01 at 40–43 ° generated a endocannabinoids, 2-arachidonylglycerol(2-AG) and anandamide. Since this effect of hyperthermia stimulation was abolished by the incubation with capsazepine, an antagonistic analogue of capsaicin, it was suggested that the effect of hyperthermia might through VR1. It is well known that 2-AG and anandamide act on their specific receptors, CB1, CB2 and VR1 expressed on various cell-types, and act as an endocannabinoids, and accomplish diverse physiological functions, including vessel dilatation. Hyperthermia stimulation also affected the cell-cycle and induced G1-phase arrest in MEG-01 cells, partially through autocrine manner of generated endocannabinoids by acting of CB1 and CB2 receptors. Moreover prolonged hyperthermia stimulation up to 6 hours induced cellular apoptosis in MEG-01 by releasing cytocrome C and resulting caspase cascade activation.Based on these results, we hypothesize that VR1 on platelets may act as a hyperthermal sensor and generate endocannabinoid, and this may regulate the diverse systems including vessel functions.