Vanilloid Receptor VR1 Research Articles

VR1- and VRL-1-like immunoreactivity in normal and injured trigeminal dental primary sensory neurons of the rat

The vanilloid receptor VR1 and the vanilloid receptor-like protein VRL-1 are associated with polymodal nociceptors, and may be important for pain processing in normal and injured teeth. Using immunohistochemistry, we have studied the distribution of these receptors in rat pulpal or gingival trigeminal ganglion neurons that were identified through retrograde labeling with fluoro-gold. Twenty-one percent to 34% of tooth pulp-innervating neurons were VR1-positive, while 32%-51% were VRL-1-immunoreactive. However, double-labeling experiments revealed that VR1 and VRL-1 rarely co-existed in the same cells, but rather seemed to be confined to separate subpopulations. Among the gingival neurons, about 25% were VR1-positive and about 41% were VRL-1-immunoreactive. A lesion of the inferior alveolar nerve, which supplies mandibular teeth and gingiva, resulted in a marked down-regulation of VR1 in the affected trigeminal ganglion cells. A down-regulation of VRL-1 was also indicated. The results suggest that both VR1 and VRL-1 could have significant roles in pulpal and gingival nociceptive transduction.

Anandamide and methanandamide induce both vanilloid VR1- and cannabinoid CB1 receptor-mediated changes in heart rate and blood pressure in anaesthetized rats.

In anaesthetized rats activation of vanilloid receptors on sensory vagal nerves elicits rapid bradycardia and hypotension (Bezold-Jarisch reflex). Recent in vitro experiments revealed that the endogenous cannabinoid ligand anandamide acts as an agonist at the vanilloid VRI receptors. The present study was aimed at examining whether vanilloid VR1 receptors are involved in the cardiovascular effects of anandamide in the anaesthetized rat. Intravenous injection of anandamide, its stable analogue methanandamide and the vanilloid receptor agonist capsaicin produced a dose-dependent immediate and short-lasting decrease in heart rate and blood pressure with the following rank order of potencies: capsaicin > methanandamide > anandamide. This bradycardia was dose-dependently diminished by the selective vanilloid receptor antagonist capsazepine (0.3-3 micromol/kg) and the nonselective inhibitor of these receptors, ruthenium red (1-10 micromol/kg). Both antagonists reduced or tended to reduce the hypotension stimulated by the agonists. Following this bradycardia and hypotension (presumably evoked by the Bezold-Jarisch reflex; phase I), capsaicin, anandamide and methanandamide led to a brief vasopressor effect (phase II). Subsequently both anandamides, but not capsaicin, induced a more prolonged decrease in blood pressure (phase III). Capsazepine and ruthenium red (at doses up to 3 tmol/kg and 10 micromol/kg, respectively) failed to affect these changes in blood pressure. The cannabinoid CB1 receptor antagonist SR 141716 at 3 micromol/kg abolished the prolonged decrease in blood pressure (phase III) induced by anandamide and methanandamide, but had no effect on the reflex bradycardia and hypotension (phase I) and on the subsequent vasopressor effect (phase II) evoked by capsaicin, anandamide and methanandamide. In conclusion, the endogenous cannabinoid receptor agonist anandamide and its stable analogue methanandamide induce reflex bradycardia and hypotension (phase I) by activating the vanilloid VRI receptor. Whereas the mechanism underlying the brief vasopressor effect (phase II) is unknown, the prolonged hypotension (phase III) results from stimulation of the cannabinoid CB1 receptor.

Biochemical characterization of the vanilloid receptor 1 expressed in a dorsal root ganglia derived cell line

The vanilloid receptor VR1 is an ion channel predominantly expressed by primary sensory neurons involved in nociception. Here we describe its biochemical properties and assess the subcellular localization, the glycosylation state and the quaternary structure of VR1 expressed in HEK293 cells and in the DRG-derived cell line F-11 (N18TG2 mouse neuroblastoma × rat dorsal root ganglia, hybridoma). VR1 was found to be glycosylated in both cell types. Of the five potential N-glycosylation sites, the predicted transient receptor potential channel-like transmembrane folding proposes N604 is localized extracellularly. We used site-directed mutagenesis to mutate the Asn at position 604 to Thr. This mutated VR1 was not glycosylated, confirming the extracellular location of N604 and its role as the exclusive site of glycosylation of the VR1 protein. VR1 occured in high molecular mass complexes as assessed by blue native PAGE. In the presence of limited amounts of SDS dimers, trimers and tetramers of VR1 were observed, consistent with the predicted tetrameric quaternary structure of the receptor. Cross-linking with dimethyladipimidate yielded almost exclusively dimers. Whereas VR1 localized both to the plasma membrane and to intracellular membranes in HEK293 cells, it localized predominantly to the plasma membrane in F-11 cells. Using confocal laserscanning microscopy, we observed an enrichment of anti-VR1 immunoreactivity in neurite-like structures of F-11 cells. In the light of conflicting literature data on biochemical characteristics of VR1, our data suggest that dorsal root ganglion-derived F-11 cells provide a powerful experimental system for the study of VR1 biochemistry.

Biochemical characterization of the vanilloid receptor 1 expressed in a dorsal root ganglia derived cell line.

The vanilloid receptor VR1 is an ion channel predominantly expressed by primary sensory neurons involved in nociception. Here we describe its biochemical properties and assess the subcellular localization, the glycosylation state and the quaternary structure of VR1 expressed in HEK293 cells and in the DRG-derived cell line F-11 (N18TG2 mouse neuroblastoma x rat dorsal root ganglia, hybridoma). VR1 was found to be glycosylated in both cell types. Of the five potential N-glycosylation sites, the predicted transient receptor potential channel-like transmembrane folding proposes N604 is localized extracellularly. We used site-directed mutagenesis to mutate the Asn at position 604 to Thr. This mutated VR1 was not glycosylated, confirming the extracellular location of N604 and its role as the exclusive site of glycosylation of the VR1 protein. VR1 occured in high molecular mass complexes as assessed by blue native PAGE. In the presence of limited amounts of SDS dimers, trimers and tetramers of VR1 were observed, consistent with the predicted tetrameric quaternary structure of the receptor. Cross-linking with dimethyladipimidate yielded almost exclusively dimers. Whereas VR1 localized both to the plasma membrane and to intracellular membranes in HEK293 cells, it localized predominantly to the plasma membrane in F-11 cells. Using confocal laserscanning microscopy, we observed an enrichment of anti-VR1 immunoreactivity in neurite-like structures of F-11 cells. In the light of conflicting literature data on biochemical characteristics of VR1, our data suggest that dorsal root ganglion-derived F-11 cells provide a powerful experimental system for the study of VR1 biochemistry.

The distribution and regulation of vanilloid receptor VR1 and VR1 5′ splice variant RNA expression in rat

The vanilloid (capsaicin) receptor, VR1, is expressed in dorsal root ganglion and mediates the sensory response to vanilloids and other noxious stimuli. There is evidence for VR1 expression in CNS regions as well, but its function in these tissues is unknown. The identification of a rat VR1 5′ splice variant and the rat stretch inhibitable channel, which are also expressed in dorsal root ganglia and CNS, raises the possibility that these and/or other VR1 variants may regulate VR1 activity. We have used a quantitative ribonuclease protection assay to characterize the central and peripheral expression of VR1 and VR1 variant RNA in the rat. The data confirm that VR1 is widely expressed in CNS, with highest RNA levels found in cerebral cortex, hippocampus, and cerebellum. VR1 RNA expression in dorsal root ganglia is approximately 28 times greater than in any other tissue sample studied. VR1 5′ splice variant RNA is expressed at levels 12 times lower than VR1 in dorsal root ganglia, but at similar levels to VR1 in all other tissues examined. A VR1-related RNA expressed at high levels in kidney was detected, and was distinct from VR1 or stretch inhibitable channel. Our results also show that peripheral inflammation does not change VR1 RNA levels in rat dorsal root ganglia. Systemic resiniferatoxin administration, however, decreases VR1 expression in dorsal root ganglia by 65–80%, an effect that persists for at least 2 months. This study demonstrates that VR1 is expressed at high levels in dorsal root ganglia relative to other tissues and that VR1 5′ splice variant is expressed at low levels in dorsal root ganglia compared to VR1. VR1 gene expression in dorsal root ganglia is regulated in response to systemic resiniferatoxin but not peripheral inflammation.

Involvement of vanilloid receptor VR1 and prostanoids in the acid-induced writhing responses of mice

We found that intraperitoneal injection of organic acids, such as propionic and lactic acid, are able to develop writhing responses in mice similarly as that of acetic acid. These acid-induced writhing reactions were significantly attenuated by capsazepine, a VR1 receptor-specific antagonist, but the phenylbenzoquinone-induced one was not, suggesting that the acids but not phenylbenzoquinone activate the VR1 receptor, which is involved in polymodal pain perception. Hoe 140, a bradykinin B 2 receptor antagonist, also suppressed the acid-induced writhing response. Furthermore, these writhing responses were significantly suppressed after neonatal treatment with capsaicin, which treatment is known to destroy peripheral sensory afferent C-fibers. Capsazepine and Hoe 140 did not further attenuate the already reduced writhing responses of capsaicin-treated mice, suggesting that the acids stimulate the VR1 and the bradykinin B 2 receptor in the pathway comprising sensory afferent C-fibers. On the other hand, indomethacin further significantly suppressed the writhing number of the capsaicin-treated animals, suggesting that the acid-induced pain perception requires prostanoid receptors not only in the pathway via capsaicin-sensitive C-fibers but also in other sensory pathways. These results provide the first evidence for the involvement of the vanilloid receptor in the acid-induced inflammatory pain perception via sensory C-fibers in addition to the known mediators bradykinin, neurokinins, and prostanoids.

Mechanisms of anandamide-induced vasorelaxation in rat isolated coronary arteries.

1. The cannabinoid arachidonyl ethanolamide (anandamide) caused concentration-dependent relaxation of 5-HT-precontracted, myograph-mounted, segments of rat left anterior descending coronary artery. 2. This relaxation was endothelium-independent, unaffected by the fatty acid amide hydrolase inhibitor, arachidonyl trifluoromethyl ketone (10 microM), and mimicked by the non-hydrolysable anandamide derivative, methanandamide. 3. Relaxations to anandamide were attenuated by the cannabinoid receptor antagonist, SR 141716A (3 microM), but unaffected by AM 251 (1 microM) and AM 630 (1 microM), more selective antagonists of cannabinoid CB(1) and CB(2) receptors respectively. Palmitoylethanolamide, a selective CB(2) receptor agonist, did not relax precontracted coronary arteries. 4. Anandamide relaxations were not affected by inhibition of sensory nerve transmission with capsaicin (10 microM) or blockade of vanilloid VR1 receptors with capsazepine (5 microM). Nevertheless capsaicin relaxed coronary arteries in a concentration-dependent and capsazepine-sensitive manner, confirming functional sensory nerves were present. In contrast, capsazepine and capsaicin did inhibit anandamide relaxations in methoxamine-precontracted rat small mesenteric arteries. 5. Relaxations to anandamide were inhibited by TEA (1 mM) or iberiotoxin (50 nM), blockers of large conductance, Ca(2+)-activated K(+) channels (BK(Ca)). Gap junction inhibition with 18alpha-glycyrrhetinic acid (100 microM) did not affect anandamide relaxations. 6. This study shows anandamide relaxes the rat coronary artery by a novel mechanism. Anandamide-induced relaxations do not involve the endothelium, degradation into active metabolites, or activation of cannabinoid CB(1) or CB(2) receptors, but may involve activation of BK(Ca). Vanilloid receptor activation also has no role in the effects of anandamide in coronary arteries, even though functional sensory nerves are present.

Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide.

1. The effects of activation of protein kinase C (PKC) on membrane currents gated by capsaicin, protons, heat and anandamide were investigated in primary sensory neurones from neonatal rat dorsal root ganglia (DRG) and in HEK293 cells (human embryonic kidney cell line) transiently or stably expressing the human vanilloid receptor hVR1. 2. Maximal activation of PKC by a brief application of phorbol 12-myristate 13-acetate (PMA) increased the mean membrane current activated by a low concentration of capsaicin by 1.65-fold in DRG neurones and 2.18-fold in stably transfected HEK293 cells. Bradykinin, which activates PKC, also enhanced the response to capsaicin in DRG neurones. The specific PKC inhibitor RO31-8220 prevented the enhancement caused by PMA. 3. Activation of PKC did not enhance the membrane current at high concentrations of capsaicin, showing that PKC activation increases the probability of channel opening rather than unmasking channels. 4. Application of PMA alone activated an inward current in HEK293 cells transiently transfected with VR1. The current was suppressed by the VR1 antagonist capsazepine. PMA did not, however, activate a current in the large majority of DRG neurones nor in HEK293 cells stably transfected with VR1. 5. Removing external Ca(2+) enhanced the response to a low concentration of capsaicin 2.40-fold in DRG neurones and 3.42-fold in HEK293 cells. Activation of PKC in zero Ca(2+) produced no further enhancement of the response to capsaicin in either DRG neurones or HEK293 cells stably transfected with VR1. 6. The effects of PKC activation on the membrane current gated by heat, anandamide and low pH were qualitatively similar to those on the capsaicin-gated current. 7. The absence of a current activated by PMA in most DRG neurones or in stably transfected HEK293 cells suggests that activation of PKC does not directly open VR1 channels, but instead increases the probability that they will be activated by capsaicin, heat, low pH or anandamide. Removal of calcium also potentiates activation, and PKC activation then has no further effect. The results are consistent with a model in which phosphorylation of VR1 by PKC increases the probability of channel gating by agonists, and in which dephosphorylation occurs by a calcium-dependent process.

Vanilloid receptor VR1 is both presynaptic and postsynaptic in the superficial laminae of the rat dorsal horn

Terminals in the rat spinal cord that express the vanilloid receptor VR1 are from small and medium dorsal root ganglion (DRG) neurons and appear prominent in lamina I and inner lamina II. Because primary afferents from these neurons can be myelinated or unmyelinated and their terminals in these laminae can be of various morphological and functional types, we undertook this study to identify the type(s) of VR1-positive afferent fibers and terminals. In the DRG, many small and medium-sized neurons are immunopositive. Under electron microscopy, dorsal root afferents that are immunopositive for VR1 are predominantly unmyelinated. Large numbers of VR1-positive terminals in lamina I are of the nonglomerular type and may contain dense core vesicles. VR1 immunoreactivity in terminals in lamina I is in good agreement with data on noxious, heat-sensitive neurons in the dorsal horn. Two types of glomerular afferent terminals in lamina II also are immunopositive for VR1. In both laminae, most VR1-positive terminals are distinct from substance P-positive terminals. However, the immunoreactivity in lamina II also is prominent in dendrites that are contacted by primary afferent endings. Because we also observed patchy immunostaining in cell bodies in lamina II, this unexpected result may reflect synthesis of VR1 by neurons in this lamina. However, because dorsal rhizotomy abolishes VR1 staining in both laminae I and II, it is suggested that the expression and intracellular dynamics of VR1 in lamina II neurons are controlled by presynaptic input. J. Comp. Neurol. 436:225–235, 2001. © 2001 Wiley-Liss, Inc.

Chemokines and Glycoprotein120 Produce Pain Hypersensitivity by Directly Exciting Primary Nociceptive Neurons

Human immunodeficiency virus-1 (HIV-1) infection is associated with numerous effects on the nervous system, including pain and peripheral neuropathies. We now demonstrate that cultured rat dorsal root ganglion (DRG) neurons express a wide variety of chemokine receptors, including those that are thought to act as receptors for the HIV-1 coat protein glycoprotein120 (gp120). Chemokines that activate all of the known chemokine receptors increased [Ca(2+)](i) in subsets of cultured DRG cells. Many neurons responded to multiple chemokines and also to bradykinin, ATP, and capsaicin. Immunohistochemical studies demonstrated the expression of the CXCR4 and CCR4 chemokine receptors on populations of DRG neurons that also expressed substance P and the VR1 vanilloid receptor. RT-PCR analysis confirmed the expression of CXCR4, CX3CR1, CCR4, and CCR5 mRNAs in DRG neurons. Chemokines and gp120 produced excitatory effects on DRG neurons and also stimulated the release of substance P. Chemokines and gp120 also produced allodynia after injection into the rat paw. Thus these results provide evidence that chemokines and gp120 may produce painful effects via direct actions on chemokine receptors expressed by nociceptive neurons. Chemokine receptor antagonists may be important therapeutic interventions in the pain that is associated with HIV-1 infection and inflammation.

Cannabinoid activation of recombinant and endogenous vanilloid receptors

The effects of three structurally related cannabinoids on human and rat recombinant vanilloid VR1 receptors expressed in human embryonic kidney (HEK293) cells and at endogenous vanilloid receptors in the rat isolated mesenteric arterial bed were studied. In the recombinant cells, all three were full agonists, causing concentration-dependent increases in [Ca 2+] i (FLIPR™), with a rank order of potency relative to the vanilloids capsaicin and olvanil, of olvanil≥capsaicin>AM404 ((allZ)- N-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide)>anandamide>methanandamide. These responses were inhibited by the vanilloid VR1 receptor antagonist, capsazepine. In the mesenteric arterial bed, vasorelaxation was evoked by these ligands with a similar order of potency. The AM404-induced vasorelaxation was virtually abolished by capsaicin pretreatment. AM404 inhibition of capsaicin-sensitive sensory neurotransmission was blocked by ruthenium red, but not by cannabinoid CB 1 and CB 2 receptor antagonists. AM404 had no effect on relaxations to calcitonin gene-related peptide. These data demonstrate that the vasorelaxant and sensory neuromodulator properties of AM404 in the rat isolated mesenteric arterial bed are mediated by vanilloid VR1 receptors.

Temperature-dependent activation of recombinant rat vanilloid VR1 receptors expressed in HEK293 cells by capsaicin and anandamide

Capsaicin activates vanilloid (VR1) receptors found on sensory neurons. These ligand-gated ion channels are also sensitive to low pH, elevated temperature and the endocannabinoid, anandamide. In this study, we have measured capsaicin- and anandamide-induced elevations in intracellular calcium concentrations ([Ca 2+] i) in fura-2 loaded HEK293 cells stably expressing the rat VR1 receptor at 22, 37 and 50 °C. Both capsaicin and anandamide produced a concentration-dependent elevation in [Ca 2+] i at all temperatures. pEC 50 values were 7.74 and 5.69 at 22 °C and 6.90 and 5.15 at 37 °C for capsaicin and anandamide, respectively. At 50 °C, the pEC 50 value for capsaicin was 6.36 but the response to anandamide did not saturate. Responses to both agonists were sensitive to ruthenium red and capsazepine at all temperatures. This temperature-dependent reduction in potency may result from desensitization.

Vanilloid receptor VR1 is both presynaptic and postsynaptic in the superficial laminae of the rat dorsal horn

Terminals in the rat spinal cord that express the vanilloid receptor VR1 are from small and medium dorsal root ganglion (DRG) neurons and appear prominent in lamina I and inner lamina II. Because primary afferents from these neurons can be myelinated or unmyelinated and their terminals in these laminae can be of various morphological and functional types, we undertook this study to identify the type(s) of VR1-positive afferent fibers and terminals. In the DRG, many small and medium-sized neurons are immunopositive. Under electron microscopy, dorsal root afferents that are immunopositive for VR1 are predominantly unmyelinated. Large numbers of VR1-positive terminals in lamina I are of the nonglomerular type and may contain dense core vesicles. VR1 immunoreactivity in terminals in lamina I is in good agreement with data on noxious, heat-sensitive neurons in the dorsal horn. Two types of glomerular afferent terminals in lamina II also are immunopositive for VR1. In both laminae, most VR1-positive terminals are distinct from substance P-positive terminals. However, the immunoreactivity in lamina II also is prominent in dendrites that are contacted by primary afferent endings. Because we also observed patchy immunostaining in cell bodies in lamina II, this unexpected result may reflect synthesis of VR1 by neurons in this lamina. However, because dorsal rhizotomy abolishes VR1 staining in both laminae I and II, it is suggested that the expression and intracellular dynamics of VR1 in lamina II neurons are controlled by presynaptic input.

Capsaicin receptor VR1 and ATP-gated ion channel P2X3 in human urinary bladder.

To determine the presence, distribution and molecular forms of the vanilloid receptor VR1, and confirm the presence and distribution of the ATP-gated ion channel P2X3 in the human urinary bladder. Materials and methods Normal urinary bladder tissues were obtained at postmortem from four subjects. Eight urinary bladder biopsies were also taken from patients with detrusor hyper-reflexia treated with intravesical resiniferatoxin. The specimens were studied using affinity-purified specific antibodies to VR1 and P2X3 by Western blotting and immunocytochemistry, and compared with immunostaining using antibodies to the pan-neuronal marker PGP 9.5 and Schwann cell marker S-100. VR1- and P2X3-immunoreactive fine nerve fibres were scattered throughout the suburothelium of the normal bladder and cystoscopic biopsies, and traversed the muscle layer. They had a similar distribution to PGP 9.5-immunoreactive fibres, but there were fewer, suggesting localization in subsets of axons. Western blot studies showed an expected 100-kDa VR1 protein and a P2X3-immunoreactive 66-kDa protein. Conclusion VR1 and P2X3 are present in the human urinary bladder and may contribute to distinct pathophysiological states of bladder overactivity, in accord with their differential expression in sensory neurones. Intravesical vanilloids act via VR1 and are effective in the treatment of detrusor hyper-reflexia. P2X3 may represent a selective therapeutic target for other causes of overactive bladder.

Role of vanilloid VR1 receptor in thermal allodynia and hyperalgesia in diabetic mice

We examined the role of the vanilloid VR1 receptor in the thermal hyperalgesia and allodynia seen in diabetic mice. Tail-flick latencies at source voltages of 35 and 50 V for a 50-W projection bulb in diabetic mice were significantly shorter than those in non-diabetic mice. Tail-flick latencies at 35 and 50 V in diabetic mice were increased by pretreatment with anti-vanilloid VR1 receptor serum. Intrathecal (i.t.) injection of anti-VR1 serum resulted in a significant increase in the tail-flick latency at 50 V in non-diabetic mice. However, i.t. pretreatment with anti-vanilloid VR1 receptor serum did not affect the tail-flick latency at a heat intensity of 35 V in non-diabetic mice. Thus, it seems likely that thermal allodynia and hyperalgesia in diabetic mice may be due to the sensitization of vanilloid VR1 receptors in primary sensory neurons in the spinal cord.

The Activity of Anandamide at Vanilloid VR1 Receptors Requires Facilitated Transport across the Cell Membrane and Is Limited by Intracellular Metabolism

The endogenous ligand of CB(1) cannabinoid receptors, anandamide, is also a full agonist at vanilloid VR1 receptors for capsaicin and resiniferatoxin, thereby causing an increase in cytosolic Ca(2+) concentration in human VR1-overexpressing (hVR1-HEK) cells. Two selective inhibitors of anandamide facilitated transport into cells, VDM11 and VDM13, and two inhibitors of anandamide enzymatic hydrolysis, phenylmethylsulfonyl fluoride and methylarachidonoyl fluorophosphonate, inhibited and enhanced, respectively, the VR1-mediated effect of anandamide, but not of resiniferatoxin or capsaicin. The nitric oxide donor, sodium nitroprusside, known to stimulate anandamide transport, enhanced anandamide effect on the cytosolic Ca(2+) concentration. Accordingly, hVR1-HEK cells contain an anandamide membrane transporter inhibited by VDM11 and VDM13 and activated by sodium nitroprusside, and an anandamide hydrolase activity sensitive to phenylmethylsulfonyl fluoride and methylarachidonoyl fluorophosphonate, and a fatty acid amide hydrolase transcript. These findings suggest the following. (i) Anandamide activates VR1 receptors by acting at an intracellular site. (ii) Degradation by fatty acid amide hydrolase limits anandamide activity on VR1; and (iii) the anandamide membrane transporter inhibitors can be used to distinguish between CB(1) or VR1 receptor-mediated actions of anandamide. By contrast, the CB(1) receptor antagonist SR141716A inhibited also the VR1-mediated effect of anandamide and capsaicin on cytosolic Ca(2+) concentration, although at concentrations higher than those required for CB(1) antagonism.

Characterisation using FLIPR of human vanilloid VR1 receptor pharmacology

A full pharmacological characterisation of the recently cloned human vanilloid VR1 receptor was undertaken. In whole-cell patch clamp studies, capsaicin (10 μM) elicited a slowly activating/deactivating inward current in human embryonic kidney (HEK293) cells stably expressing human vanilloid VR1 receptor, which exhibited pronounced outward rectification (reversal potential −2.1±0.2 mV) and was abolished by capsazepine (10 μM). In FLIPR™-based Ca 2+ imaging studies the rank order of potency was resiniferatoxin>olvanil>capsaicin>anandamide, and all were full agonists. Isovelleral and scutigeral were inactive (1 nM–30 μM). The potencies of capsaicin, olvanil and resiniferatoxin, but not anandamide, were enhanced 2- to 7-fold at pH 6.4. Capsazepine, isovelleral and ruthenium red inhibited the capsaicin (100 nM)-induced Ca 2+ response (p K B=6.58±0.02, 5.33±0.03 and 7.64±0.03, respectively). In conclusion, the recombinant human vanilloid VR1 receptor stably expressed in HEK293 cells acted as a ligand-gated, Ca 2+-permeable channel with similar agonist and antagonist pharmacology to rat vanilloid VR1 receptor, although there were some subtle differences.

Vanilloid receptor ligands: hopes and realities for the future.

Neurons possessing C-fibers transmit nociceptive information into the central nervous system and participate in various reflex responses. These neurons carry receptors that bind capsaicin, recently identified as the vanilloid VR1 receptor. Excitation of these cells by capsaicin is followed by a lasting refractory state, termed desensitisation, in which the neurons fail to respond to a variety of noxious stimuli. Desensitisation to capsaicin has a clear therapeutic potential in relieving neuropathic pain and ameliorating urinary bladder overactivity, just to cite 2 important examples. Vanilloids may also be beneficial in the treatment of benign prostate hyperplasia (BPH). Since the majority of elderly patients have neuropathic pain co-existent with urinary incontinence and/or BPH, a drug that ameliorates pain and improves urinary symptoms at the same time promises to be of great clinical value in geriatric medicine. In fact, capsaicin has already been shown to have a role in the treatment of conditions that can arise in the elderly, including herpes zoster-related neuropathic pain, diabetic neuropathy, postmastectomy pain, uraemic itching associated with renal failure, and urinary incontinence. The potent VR1 agonist resiniferatoxin, now in phase II clinical trials, appears to be superior to capsaicin in terms of its tolerability profile. Recent discoveries enhance the therapeutic potential of vanilloids. The recognition that VR1 also functions as a principal receptor for protons and eicosanoids implies that VR1 antagonists may be of value in the treatment of inflammatory hyperalgesia and pain. Animal experimentation has already lent support to this assumption. The discovery of VR1-expressing cells in the brain as well as in non-neural tissues such as the kidney and urothelium places VR1 in a much broader perspective than peripheral pain perception, and is hoped to identify further, yet unsuspected, indications for vanilloid therapy. The realisation that VR1 and cannabinoid CB1 receptors have overlapping ligand recognition properties may also have far-reaching implications for vanilloid therapy. In fact, arvanil, a combined agonist of VR1 and CB1 receptors, has already proved to be a powerful analgesic drug in the mouse. From academic molecular biology laboratories to industrial drug discovery centres to the clinics, there is a steady flow of new data, forcing us to constantly revise the ways we are thinking about vanilloid receptor ligands and their hopes and realities for the future. This review covers the most promising current trends in vanilloid research with special emphasis on geriatric medicine.

Overlap between the ligand recognition properties of the anandamide transporter and the VR1 vanilloid receptor: inhibitors of anandamide uptake with negligible capsaicin-like activity

Some synthetic agonists of the VR1 vanilloid (capsaicin) receptor also inhibit the facilitated transport into cells of the endogenous cannabinoid anandamide (arachidonoylethanolamide, AEA). Here we tested several AEA derivatives containing various derivatized phenyl groups or different alkyl chains as either inhibitors of the AEA membrane transporter (AMT) in intact cells or functional agonists of the VR1 vanilloid receptor in HEK cells transfected with the human VR1. We found that four known AMT inhibitors, AM404, arvanil, olvanil and linvanil, activate VR1 receptors at concentrations 400–10 000-fold lower than those necessary to inhibit the AMT. However, we also found three novel AEA derivatives, named VDM11, VDM12 and VDM13, which inhibit the AMT as potently as AM404 but exhibit little or no agonist activity at hVR1. These compounds are weak inhibitors of AEA enzymatic hydrolysis and poor CB 1/CB 2 receptor ligands. We show for the first time that, despite the overlap between the chemical moieties of AMT inhibitors and VR1 agonists, selective inhibitors of AEA uptake that do not activate VR1 (e.g. VDM11) can be developed.

Impaired nociception and pain sensation in mice lacking the capsaicin receptor.

The capsaicin (vanilloid) receptor VR1 is a cation channel expressed by primary sensory neurons of the "pain" pathway. Heterologously expressed VR1 can be activated by vanilloid compounds, protons, or heat (>43 degrees C), but whether this channel contributes to chemical or thermal sensitivity in vivo is not known. Here, we demonstrate that sensory neurons from mice lacking VR1 are severely deficient in their responses to each of these noxious stimuli. VR1-/- mice showed normal responses to noxious mechanical stimuli but exhibited no vanilloid-evoked pain behavior, were impaired in the detection of painful heat, and showed little thermal hypersensitivity in the setting of inflammation. Thus, VR1 is essential for selective modalities of pain sensation and for tissue injury-induced thermal hyperalgesia.