Characterization by Gene Expression Analysis of Two Groups of Dopaminergic Cells Isolated from the Mouse Olfactory Bulb.

The olfactory bulb (OB) is one of two regions of the mammalian brain which undergo continuous neuronal replacement during adulthood. A significant fraction of the cells added in adulthood to the bulbar circuitry is constituted by dopaminergic (DA) neurons. We took advantage of a peculiar property of dopaminergic neurons in transgenic mice expressing eGFP under the tyrosine hydroxylase (TH) promoter: while DA neurons located in the glomerular layer (GL) display full electrophysiological maturation, eGFP+ cells in the mitral layer (ML) show characteristics of immature cells. In addition, they also display a lower fluorescence intensity, possibly reflecting different degrees of maturation. To investigate whether this difference in maturation might be confirmed at the gene expression level, we used a fluorescence-activated cell sorting technique on enzymatically dissociated cells of the OB. The cells were divided into two groups based on their level of fluorescence, possibly corresponding to immature ML cells and fully mature DA neurons from the GL. Semiquantitative real-time PCR was performed to detect the level of expression of genes linked to the degree of maturation of DA neurons. We showed that indeed the cells expressing low eGFP fluorescence are immature neurons. Our method can be further used to explore the differences between these two groups of DA neurons.

Depleted Calcium Stores and Increased Calcium Entry in Rod Photoreceptors of the Cacna2d4 Mouse Model of Cone-Rod Dystrophy RCD4.

Unidentified pathogenetic mechanisms and genetic and clinical heterogeneity represent critical factors hindering the development of treatments for inherited retinal dystrophies. Frameshift mutations in Cacna2d4, which codes for an accessory subunit of voltage-gated calcium channels (VGCC), cause cone-rod dystrophy RCD4 in patients, but the underlying mechanisms remain unknown. To define its pathogenetic mechanisms, we investigated the impact of a Cacna2d4 frameshift mutation on the electrophysiological profile and calcium handling of mouse rod photoreceptors by patch-clamp recordings and calcium imaging, respectively. In mutant (MUT) rods, the dysregulation of calcium handling extends beyond the reduction in calcium entry through VGCC and surprisingly involves internal calcium stores' depletion and upregulation of calcium entry via non-selective cationic channels (CSC). The similar dependence of CSC on basal calcium levels in WT and MUT rods suggests that the primary defect in MUT rods lies in defective calcium stores. Calcium stores' depletion, leading to upregulated calcium and sodium influx via CSC, represents a novel and, so far, unsuspected consequence of the Cacna2d4 mutation. Blocking CSC may provide a novel strategy to counteract the well-known pathogenetic mechanisms involved in rod demise, such as the reticulum stress response and calcium and sodium overload due to store depletion.

Abnormal Pain Sensation in Mice Lacking the Prokineticin Receptor PKR2: Interaction of PKR2 with Transient Receptor Potential TRPV1 and TRPA1.

The amphibian Bv8 and the mammalian prokineticin 1 (PROK1) and 2 (PROK2) are new chemokine-like protein ligands acting on two G protein-coupled receptors, prokineticin receptor 1 (PKR1) and 2 (PKR2), participating to the mediation of diverse physiological and pathological processes. Prokineticins (PKs), specifically activating the prokineticin receptors (PKRs) located in several areas of the central and peripheral nervous system associated with pain, play a fundamental role in nociception. In this paper, to improve the understanding of the prokineticin system in the neurobiology of pain, we investigated the role of PKR2 in pain perception using pkr2 gene-deficient mice. We observed that, compared to wildtype, pkr2-null mice were more resistant to nociceptive sensitization to temperatures ranging from 46 to 48 °C, to capsaicin and to protons, highlighting a positive interaction between PKR2 and the non-selective cation channels TRPV1. Moreover, PKR2 knock-out mice showed reduced nociceptive response to cold temperature (4 °C) and to mustard oil-induced inflammatory hyperalgesia, suggesting a functional interaction between PKR2 and transient receptor potential ankyrin 1 ion (TRPA1) channels. This notion was supported by experiments in dorsal root ganglia (DRG) cultures from pkr1 and-pkr2-null mice, demonstrating that the percentage of Bv8-responsive DRG neurons which were also responsive to mustard oil was much higher in PKR1-/- than in PKR2-/- mice. Taken together, these findings suggest a functional interaction between PKR2 and TRP channels in the development of hyperalgesia. Drugs able to directly or indirectly block these targets and/or their interactions may represent potential analgesics.

CR4056, a powerful analgesic imidazoline-2 receptor ligand, inhibits the inflammation-induced PKCε phosphorylation and membrane translocation in sensory neurons.

BACKGROUND AND PURPOSE: CR4056 is a first-in-class imidazoline-2 (I2 ) receptor ligand characterized by potent analgesic activity in different experimental animal models of pain. In a recent phase II clinical trial, CR4056 effectively reduced pain in patients with knee osteoarthritis. In the present study, we investigated the effects of CR4056 on PKCε translocation in vitro and on PKCε activation in vivo in dorsal root ganglia (DRG) neurons. EXPERIMENTAL APPROACH: Effects of CR4056 on bradykinin-induced PKCε translocation were studied in rat sensory neurons by immunocytochemistry. PKCε activation was investigated by immunohistochemistry analysis of DRG from complete Freund's adjuvant-treated animals developing local hyperalgesia. The analgesic activity of CR4056 was tested on the same animals. KEY RESULTS: CR4056 inhibited PKCε translocation with very rapid and long-lasting activity. CR4056 decreased hyperalgesia and phospho-PKCε immunoreactivity in the DRG neurons innervating the inflamed paw. The effect of CR4056 on PKCε translocation was blocked by pertussis toxin, implying that the intracellular pathways involved Gi proteins. The inhibition of PKCε translocation by CR4056 was independent of the α2 -adrenoeceptor and, surprisingly, was also independent of idazoxan-sensitive I2 binding sites. The I2 agonist 2BFI had no effect alone but potentiated the activity of low concentrations of CR4056. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate that CR4056 shares the ability to inhibit PKCε translocation with other analgesics. Whether the inhibition of PKCε involves binding to specific subtype(s) of I2 receptors should be further investigated. If so, this would be a new mode of action of a highly specific I2 receptor ligand.

Effects of NSAIDs on the Release of Calcitonin Gene-Related Peptide and Prostaglandin E2 from Rat Trigeminal Ganglia.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently used to treat migraine, but the mechanisms of their effects in this pathology are not fully elucidated. The trigeminal ganglia and calcitonin gene-related peptide (CGRP) have been implicated in the pathophysiology of migraine. The release of CGRP and prostaglandin E2 (PGE2) from freshly isolated rat trigeminal ganglia was evaluated after oral administration of nimesulide, etoricoxib, and ketoprofen, NSAIDs with different pharmacological features. Thirty minutes after oral administration, nimesulide, 10 mg/Kg, decreased the GCRP release induced by an inflammatory soup, while the other NSAIDs were ineffective at this point in time. Two hours after oral nimesulide (5 and 10 mg/Kg) and ketoprofen (10 mg/Kg), but not of etoricoxib, a significant decrease in the CGRP release was observed. All drugs reduced PGE2, although with some differences in timing and doses, and the action on CGRP does not seem to be related to PGE2 inhibition. The reduction of CGRP release from rat trigeminal ganglia after nimesulide and ketoprofen may help to explain the mechanism of action of NSAIDs in migraine. Since at 30 minutes only nimesulide was effective in reducing CGRP release, these results suggest that this NSAID may exert a particularly rapid effect in patients with migraine.

Gabapentin Inhibits Protein Kinase C Epsilon Translocation in Cultured Sensory Neurons with Additive Effects When Coapplied with Paracetamol (Acetaminophen).

Gabapentin is a well-established anticonvulsant drug which is also effective for the treatment of neuropathic pain. Although the exact mechanism leading to relief of allodynia and hyperalgesia caused by neuropathy is not known, the blocking effect of gabapentin on voltage-dependent calcium channels has been proposed to be involved. In order to further evaluate its analgesic mechanisms, we tested the efficacy of gabapentin on protein kinase C epsilon (PKCε) translocation in cultured peripheral neurons isolated from rat dorsal root ganglia (DRGs). We found that gabapentin significantly reduced PKCε translocation induced by the pronociceptive peptides bradykinin and prokineticin 2, involved in both inflammatory and chronic pain. We recently showed that paracetamol (acetaminophen), a very commonly used analgesic drug, also produces inhibition of PKCε. We tested the effect of the combined use of paracetamol and gabapentin, and we found that the inhibition of translocation adds up. Our study provides a novel mechanism of action for gabapentin in sensory neurons and suggests a mechanism of action for the combined use of paracetamol and gabapentin, which has recently been shown to be effective, with a cumulative behavior, in the control of postoperative pain in human patients.

Cone-like rectification properties of cGMP-gated channels in transmutated retinal photoreceptors of nocturnal geckoes.

Photoreceptors of nocturnal geckoes are scotopic, with rod-shaped outer segments, and sensitivities to light similar to the one of retinal rods from other species of lower vertebrates. However, these cells are not rods, but they originated from cones of ancestral diurnal geckoes with pure-cone retinas, after being forced to adapt to a nocturnal behavior. Several interesting adaptations of these rod-like cones have been studied to date; molecular biology and functional studies confirmed that several proteins of the phototransductive cascade display structural and functional properties that indicate their origin from cones rather than from rods. In this paper, we investigate, with whole cell voltage clamp in the photoreceptor detached outer segment preparation, the voltage rectification properties of cGMP-gated channels in three species, Gekko gecko, Tarentola mauritanica, and Hemidactylus frenatus. We show that the current-voltage properties in the physiological voltage range are reminiscent of the ones of cGMP-gated channels from cones rather than from rods of other cold-blooded vertebrates. The origin and the relevance of the mechanisms investigated are discussed.

Effects of NSAIDs and paracetamol (acetaminophen) on protein kinase C epsilon translocation and on substance P synthesis and release in cultured sensory neurons.

Celecoxib, diclofenac, ibuprofen, and nimesulide are nonsteroidal anti-inflammatory drugs (NSAIDs) very commonly used for the treatment of moderate to mild pain, together with paracetamol (acetaminophen), a very widely used analgesic with a lesser anti-inflammatory effect. In the study reported here, we tested the efficacy of celecoxib, diclofenac, and ibuprofen on preprotachykinin mRNA synthesis, substance P (SP) release, prostaglandin E(2) (PGE(2)) release, and protein kinase C epsilon (PKCɛ) translocation in rat cultured sensory neurons from dorsal root ganglia (DRGs). The efficacy of these NSAIDs was compared with the efficacy of paracetamol and nimesulide in in vitro models of hyperalgesia (investigated previously). While nimesulide and paracetamol, as in previous experiments, decreased the percentage of cultured DRG neurons showing translocation of PKCɛ caused by 100 nM thrombin or 1 µM bradykinin in a dose-dependent manner, the other NSAIDs tested did not have a significant effect. The amount of SP released by peptidergic neurons and the expression level of preprotachykinin mRNA were assessed in basal conditions and after 70 minutes or 36 hours of stimulation with an inflammatory soup (IS) containing potassium chloride, thrombin, bradykinin, and endothelin-1. The release of SP at 70 minutes was inhibited only by nimesulide, while celecoxib and diclofenac were effective at 36 hours. The mRNA basal level of the SP precursor preprotachykinin expressed in DRG neurons was reduced only by nimesulide, while the increased levels expressed during treatment with the IS were significantly reduced by all drugs tested, with the exception of ibuprofen. All drugs were able to decrease basal and IS-stimulated PGE(2) release. Our study demonstrates novel mechanisms of action of commonly used NSAIDS.

Nimesulide inhibits protein kinase C epsilon and substance P in sensory neurons - comparison with paracetamol.

In this paper we describe new actions of nimesulide and paracetamol in cultured peripheral neurons isolated from rat dorsal root ganglia (DRG). Both drugs were able to decrease in a dose-dependent fashion the number of cultured DRG neurons showing translocation of protein kinase C epsilon (PKCɛ) caused by exposure to 1 µM bradykinin or 100 nM thrombin. In addition, the level of substance P (SP) released by DRG neurons and the level of preprotachykinin mRNA expression were measured in basal conditions and after 70 minutes or 36 hours of stimulation with nerve growth factor (NGF) or with an inflammatory soup containing bradykinin, thrombin, endothelin-1, and KCl. Nimesulide (10 µM) significantly decreased the mRNA levels of the SP precursor preprotachykinin in basal and in stimulated conditions, and decreased the amount of SP released in the medium during stimulation of neurons with NGF or with the inflammatory soup. The effects of paracetamol (10 µM) on such response was lower. Nimesulide completely inhibited the release of prostaglandin E2 (PGE2) from DRG neurons, either basal or induced by NGF and by inflammatory soup, while paracetamol decreased PGE2 release only partially. Our data demonstrate, for the first time, a direct effect of two drugs largely used as analgesics on DRG neurons. The present results suggest that PKCɛ might be a target for the effect of nimesulide and paracetamol, while inhibition of SP synthesis and release is clearly more relevant for nimesulide than for paracetamol mechanism of action.

Functional endothelin receptors are selectively expressed in isolectin B4-negative sensory neurons and are upregulated in isolectin B4-positive neurons by neurturin and glia-derived neurotropic factor.

Activation of endothelin receptors expressed in DRG neurons is functionally coupled to translocation of PKCε from cytoplasm to the plasma membrane. Using immunocytochemistry we show that in DRG cultured neurons PKCε translocation induced by endothelin-1 was prominently seen in a peptidergic subpopulation of cultured DRG neurons largely negative for isolectin B4 staining, indicating that in basal conditions functional expression of endothelin receptors does not occur in non-peptidergic, RET-expressing nociceptors. Translocation was blocked by the specific ETA-R antagonist BQ-123 while it was unaffected by the ETB-R antagonist BQ-788. No calcium response in response to endothelin-1 was observed in sensory neurons, while large and long-lasting responses were observed in the majority of non-neuronal cells present in DRG cultures, which are ensheathing Schwann cells and satellite cells, identified with the glial marker S-100. Calcium responses in non-neuronal cells were abolished by BQ-788. The fraction of peptidergic PKCε-translocated neurons was significantly increased by nerve growth factor, while in the presence of neurturin or glia-derived neurotropic factor (GDNF), an IB4-positive subpopulation of small- and medium-sized neurons showed PKCε translocation induced by endothelin-1 which could be blocked by BQ-123 but not by BQ-788. Our in vitro results show that the level of expression of functional endothelin receptors coupled to PKCε is different in peptidergic and non-peptidergic nociceptors and is modulated with different mechanisms in distinct neuronal subpopulations.

Protease activated receptors 1 and 4 sensitize TRPV1 in nociceptive neurones.

Protease-activated receptors (PAR1-4) are activated by proteases released by cell damage or blood clotting, and are known to be involved in promoting pain and hyperalgesia. Previous studies have shown that PAR2 receptors enhance activation of TRPV1 but the role of other PARs is less clear. In this paper we investigate the expression and function of the PAR1, 3 and 4 thrombin-activated receptors in sensory neurones. Immunocytochemistry and in situ hybridization show that PAR1 and PAR4 are expressed in 10 - 15% of neurons, distributed across all size classes. Thrombin or a specific PAR1 or PAR4 activating peptide (PAR1/4-AP) caused functional effects characteristic of activation of the PLCß/PKC pathway: intracellular calcium release, sensitisation of TRPV1, and translocation of the epsilon isoform of PKC (PKCε) to the neuronal cell membrane. Sensitisation of TRPV1 was significantly reduced by PKC inhibitors. Neurons responding to thrombin or PAR1-AP were either small nociceptive neurones of the peptidergic subclass, or larger neurones which expressed markers for myelinated fibres. Sequential application of PAR1-AP and PAR4-AP showed that PAR4 is expressed in a subset of the PAR1-expressing neurons. Calcium responses to PAR2-AP were by contrast seen in a distinct population of small IB4+ nociceptive neurones. PAR3 appears to be non-functional in sensory neurones. In a skin-nerve preparation the release of the neuropeptide CGRP by heat was potentiated by PAR1-AP. Culture with nerve growth factor (NGF) increased the proportion of thrombin-responsive neurons in the IB4- population, while glial-derived neurotropic factor (GDNF) and neurturin upregulated the proportion of thrombin-responsive neurons in the IB4+ population. We conclude that PAR1 and PAR4 are functionally expressed in large myelinated fibre neurons, and are also expressed in small nociceptors of the peptidergic subclass, where they are able to potentiate TRPV1 activity.

Plant-derived cannabinoids modulate the activity of transient receptor potential channels of ankyrin type-1 and melastatin type-8.

The plant cannabinoids (phytocannabinoids), cannabidiol (CBD), and Delta(9)-tetrahydrocannabinol (THC) were previously shown to activate transient receptor potential channels of both vanilloid type 1 (TRPV1) and ankyrin type 1 (TRPA1), respectively. Furthermore, the endocannabinoid anandamide is known to activate TRPV1 and was recently found to antagonize the menthol- and icilin-sensitive transient receptor potential channels of melastatin type 8 (TRPM8). In this study, we investigated the effects of six phytocannabinoids [i.e., CBD, THC, CBD acid, THC acid, cannabichromene (CBC), and cannabigerol (CBG)] on TRPA1- and TRPM8-mediated increase in intracellular Ca2+ in either HEK-293 cells overexpressing the two channels or rat dorsal root ganglia (DRG) sensory neurons. All of the compounds tested induced TRPA1-mediated Ca2+ elevation in HEK-293 cells with efficacy comparable with that of mustard oil isothiocyanates (MO), the most potent being CBC (EC(50) = 60 nM) and the least potent being CBG and CBD acid (EC(50) = 3.4-12.0 microM). CBC also activated MO-sensitive DRG neurons, although with lower potency (EC(50) = 34.3 microM). Furthermore, although none of the compounds tested activated TRPM8-mediated Ca2+ elevation in HEK-293 cells, they all, with the exception of CBC, antagonized this response when it was induced by either menthol or icilin. CBD, CBG, THC, and THC acid were equipotent (IC(50) = 70-160 nM), whereas CBD acid was the least potent compound (IC(50) = 0.9-1.6 microM). CBG inhibited Ca2+ elevation also in icilin-sensitive DRG neurons with potency (IC(50) = 4.5 microM) similar to that of anandamide (IC(50) = 10 microM). Our findings suggest that phytocannabinoids and cannabis extracts exert some of their pharmacological actions also by interacting with TRPA1 and TRPM8 channels, with potential implications for the treatment of pain and cancer.

Functional lipidomics. Calcium-independent activation of endocannabinoid/endovanilloid lipid signalling in sensory neurons by protein kinases C and A and thrombin.

N-arachidonoylethanolamine (anandamide, AEA), is a full agonist at both cannabinoid CB(1) receptors and "transient receptor potential vanilloid" type 1 (TRPV1) channels, and N-palmitoylethanolamine (PEA) potentiates these effects. In neurons of the rat dorsal root ganglia (DRG), TRPV1 is activated and/or sensitised by AEA as well as upon activation of protein kinases C (PKC) and A (PKA). We investigated here the effect on AEA levels of PKC and PKA activators in DRG neurons. AEA levels were significantly enhanced by both phorbol-miristoyl-acetate (PMA), a typical PKC activator, and forskolin (FSK), an adenylate cyclase stimulant, as well as by thrombin, which also activates PKC by stimulating protease-activated receptors (PARs). The levels of the other endocannabinoid and TRPV1-inactive compound, 2-arachidonoylglycerol (2-AG), were enhanced only by thrombin and to a lesser extent than AEA, whereas PEA was not affected by any of the treatments. Importantly, FSK- and PMA-induced elevation of AEA levels was not sensitive to intracellular Ca2+ chelation with BAPTA-acetoxymethyl (AM) ester. In human embryonic kidney (HEK-293) cells, which constitutively express PARs, thrombin, PMA and FSK elevated AEA levels, and the effects of the two former compounds were counteracted by the PKC inhibitor, RO318220, whereas the effect of FSK was reduced by the PKA inhibitor RpcAMPs. In conclusion, we report that AEA levels are stimulated by both PKC, either directly or after thrombin receptor activation, and PKA, possibly in a way independent from intracellular calcium. Since AEA activates TRPV1, these findings may suggest the existence of an amplificatory cascades on this receptor in sensory neurons.

Impaired nociception and inflammatory pain sensation in mice lacking the prokineticin receptor PKR1: focus on interaction between PKR1 and the capsaicin receptor TRPV1 in pain behavior.

Bv8, prokineticin-1 or EG-VEGF (endocrine gland-derived vascular endothelial growth factor), and prokineticin-2, are naturally occurring peptide agonists of two G-protein-coupled receptors (GPCRs), prokineticin receptor 1 (PKR1) and PKR2. PKRs are expressed in neurons in the CNS and peripheral nervous system and many dorsal root ganglion (DRG) cells expressing PKRs also express transient receptor potential vanilloid receptor-1 (TRPV1). Mice lacking the pkr1 gene were generated to explore the role of the PKR1 receptor in nociceptive signaling and in nociceptor sensitization. When compared with wild-type littermates, mice lacking the pkr1 gene showed impaired responsiveness to noxious heat, mechanical stimuli, capsaicin, and protons. In wild-type mice, activation of PKRs by the PKR agonist Bv8 caused hyperalgesia and sensitized to the actions of capsaicin. pkr1-null mice exhibited impaired responses to Bv8 but showed normal hyperalgesic responses to bradykinin and PGE2 (prostaglandin E2). Conversely, trpv1-null mice showed a reduced pronociceptive response to Bv8. Additionally, pkr1-null mice showed diminished thermal hyperalgesia after acute inflammation elicited by mustard oil and reduced pain behavior after chronic inflammation produced by complete Freund's adjuvant. The number of neurons that responded with a [Ca2+]i increase to Bv8 exposure was five times lower in pkr1-null DRG cultures than in wild-type cultures. Furthermore, Bv8-responsive neurons from pkr1-null mice showed a significant reduction in the [Ca2+]i response to capsaicin. These findings indicate a modulatory role of PKR1 in acute nociception and inflammatory pain and disclose a pharmacological interaction between PKR1 and TRPV1 in nociceptor activation and sensitization.

Sensitization of transient receptor potential vanilloid 1 by the prokineticin receptor agonist Bv8.

Small mammalian proteins called the prokineticins [prokineticin 1 (PK1) and PK2] and two corresponding G-protein-coupled receptors [prokineticin receptor 1 (PKR1) and PKR2] have been identified recently, but the physiological role of the PK/PKR system remains mostly unexplored. Bv8, a protein extracted from frog skin, is a convenient and potent agonist for both PKR1 and PKR2, and injection of Bv8 in vivo causes a potent and long-lasting hyperalgesia. Here, we investigate the cellular basis of hyperalgesia caused by activation of PKRs. Bv8 caused increases in [Ca]i in a population of isolated dorsal root ganglion (DRG) neurons, which we identified as nociceptors, or sensors for painful stimuli, from their responses to capsaicin, bradykinin, mustard oil, or proteases. Bv8 enhanced the inward current carried by the heat and capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1) via a pathway involving activation of protein kinase Cepsilon (PKCepsilon), because Bv8 caused translocation of PKCepsilon to the neuronal membrane and because PKC antagonists reduced both the enhancement of current carried by TRPV1 and behavioral hyperalgesia in rodents. The neuronal population expressing PKRs consisted partly of small peptidergic neurons and partly of neurons expressing the N52 marker for myelinated fibers. Using single-cell reverse transcriptase-PCR, we found that mRNA for PKR1 was mainly expressed in small DRG neurons. Exposure to GDNF (glial cell line-derived neurotrophic factor) induced de novo expression of functional receptors for Bv8 in a nonpeptidergic population of neurons. These results show that prokineticin receptors are expressed in nociceptors and cause heat hyperalgesia by sensitizing TRPV1 through activation of PKCepsilon. The results suggest a role for prokineticins in physiological inflammation and hyperalgesia.

Anandamide acts as an intracellular messenger amplifying Ca2+ influx via TRPV1 channels.

The endocannabinoid anandamide is able to interact with the transient receptor potential vanilloid 1 (TRPV1) channels at a molecular level. As yet, endogenously produced anandamide has not been shown to activate TRPV1, but this is of importance to understand the physiological function of this interaction. Here, we show that intracellular Ca2+ mobilization via the purinergic receptor agonist ATP, the muscarinic receptor agonist carbachol or the Ca(2+)-ATPase inhibitor thapsigargin leads to formation of anandamide, and subsequent TRPV1-dependent Ca2+ influx in transfected cells and sensory neurons of rat dorsal root ganglia (DRG). Anandamide metabolism and efflux from the cell tonically limit TRPV1-mediated Ca2+ entry. In DRG neurons, this mechanism was found to lead to TRPV1-mediated currents that were enhanced by selective blockade of anandamide cellular efflux. Thus, endogenous anandamide is formed on stimulation of metabotropic receptors coupled to the phospholipase C/inositol 1,4,5-triphosphate pathway and then signals to TRPV1 channels. This novel intracellular function of anandamide may precede its action at cannabinoid receptors, and might be relevant to its control over neurotransmitter release.

Functional bradykinin B1 receptors are expressed in nociceptive neurones and are upregulated by the neurotrophin GDNF.

Bradykinin (BK) has long been recognized as an important mediator of pain and inflammation. In normal tissue bradykinin causes an acute sensation of pain by an action at B2 receptors, but in inflamed tissue the pharmacology of the response changes to that of B1 receptors. Attempts to demonstrate the presence of functional B1 receptors in sensory neurones have failed, however, and the actions of B1 agonists have therefore been presumed to be indirect. Here we show that specific B1 receptor activation causes translocation of the epsilon isoform of protein kinase C (PKC-epsilon) to the membrane of a small fraction of freshly isolated sensory neurones from rats and mice. The proportion of neurones in which PKC-epsilon translocation was observed increased to around 20% of neurones after 3 days in culture with the neurotrophins glial cell line derived neurotrophic factor (GDNF) and neurturin, but not with nerve growth factor (NGF). Using in situ hybridization we found that the proportion of neurones expressing B1 mRNA increased from close to zero to 20.4% after 8 h culture in GDNF. Neurones expressing functional B1 receptors were negative for the neuropeptides CGRP and substance P, but most expressed functional TRPV1 receptors for capsaicin (60%) and bound the lectin IB4 (68%), both markers characteristic of nociceptors. B1 activation enhanced the heat-activated membrane current approximately 3-fold, and the enhancement was much more prolonged than was the case with B2 activation, consistent with a role for B1 receptors in sustained pain. We conclude that GDNF and neurturin potently upregulate functional B1 receptor expression in small non-peptidergic nociceptive neurones.