12 results on '"Vittorio Vellani"'
Search Results
2. Depleted Calcium Stores and Increased Calcium Entry in Rod Photoreceptors of the Cacna2d4 Mouse Model of Cone-Rod Dystrophy RCD4
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Vittorio Vellani, Giovanna Mauro, and Gian Carlo Demontis
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Dietary ,Catalysis ,Inorganic Chemistry ,Mice ,voltage-gated calcium channels ,Retinal Rod Photoreceptor Cells ,cone-rod dystrophy ,α2δ-4 protein ,caffeine-sensitive calcium stores ,non-selective cationic channels ,calcium imaging ,patch-clamp ,Animals ,Physical and Theoretical Chemistry ,Molecular Biology ,Spectroscopy ,Animal ,Sodium ,Organic Chemistry ,General Medicine ,Computer Science Applications ,Calcium Channels ,Calcium, Dietary ,Disease Models, Animal ,Retinal Cone Photoreceptor Cells ,Calcium ,Cone-Rod Dystrophies ,Disease Models - Abstract
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.
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- 2022
3. TRPA1 Is Expressed in Central But Not in Peripheral Glia
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Chiara Giacomoni, Giorgia Pavesi, Massimiliano Prandini, Carolina Gomis-Perez, Marcello Pinti, Marco Caprini, Vittorio Vellani, Vellani, Vittorio, Gomis-Perez, Carolina, Pinti, Marcello, Prandini, Massimiliano, Pavesi, Giorgia, Giacomoni, Chiara, and Caprini, Marco
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0301 basic medicine ,Cell type ,Astrocytes, Intracellular TRPA1, DITNC-1, Ensheathing Schwann Cells ,Intracellular TRPA1 ,Ensheathing Schwann Cells ,chemistry.chemical_element ,food and beverages ,Calcium ,Biology ,Cell biology ,DITNC-1 ,03 medical and health sciences ,Electrophysiology ,030104 developmental biology ,0302 clinical medicine ,Calcium imaging ,chemistry ,Cell culture ,Astrocytes ,Extracellular ,Reversal potential ,030217 neurology & neurosurgery ,Intracellular ,psychological phenomena and processes - Abstract
TRPA1 are cation channels expressed in sensory neurons and in several other cell types. This channel is specifically activated by ally isothiocyanate (AITC), the pungent component of mustard oil, as well as by other electrophilic compounds. Although TRPA1 expression in central glia has been reported, its subcellular localization and its expression in peripheral glia have not been investigated before. In this paper we report the molecular and functional expression of TRPA1 in rat cortical astrocytes. Real-time RT-PCR identified low but significant amounts of TRPA1 mRNA in cortical astrocytes while no signal was seen in peripheral glia isolated from dorsal root ganglia (DRG) or in a glial cell line (DITNC-1). Calcium imaging showed AITC-induced signals in astro-cytes while no response in peripheral glia. AITC induced calcium signals in astrocytes in the presence and in the absence of extracellular calcium, suggesting an intracellular localization of TRPA1 channels. Whole cell electrophysiological recordings were performed in astrocytes, in peripheral glia and in DITNC-1 cells transfected with TRPA1 during AITC application. In TRPA1-transfected DITNC-1 cells typical TRPA1 currents were recorded with a reversal potential near 0 mV, consistent with the opening of a non-selective cation channel. No such currents were recorded in untransfected DITNC-1 cells, in astrocytes and in peripheral glial cells, where even high concentrations of AITC (up to 10 mM) induced no significant outward current. In astrocytes AITC transiently induced an outward rectifying current with the reversal potential near ?90 mV, consistent with K channel activation, likely activated by intracellular release of calcium. Our results suggest that TRPA1 channels are molecularly and functionally expressed in calcium-containing organelles of rat cortical astrocytes, with no expression in the plasma membrane.
- Published
- 2016
4. 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
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Laura Ravegnani, Pier Cosimo Magherini, Giorgia Pavesi, Chiara Giacomoni, Massimiliano Prandini, and Vittorio Vellani
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medicine.medical_specialty ,Sensory Receptor Cells ,Neurturin ,Protein Kinase C-epsilon ,sensitization ,Rats, Sprague-Dawley ,Versicans ,sensory neurons ,nociceptors ,pain ,hyperalgesia ,TRPV1 ,Ganglia, Spinal ,Lectins ,Internal medicine ,Nerve Growth Factor ,medicine ,Glial cell line-derived neurotrophic factor ,Animals ,Glial Cell Line-Derived Neurotrophic Factor ,Receptor ,Molecular Biology ,Cells, Cultured ,Glycoproteins ,Endothelin-1 ,biology ,Receptors, Endothelin ,General Neuroscience ,Immunohistochemistry ,Endothelin 1 ,Sensory neuron ,Rats ,Up-Regulation ,Cell biology ,Protein Transport ,Endocrinology ,Nerve growth factor ,medicine.anatomical_structure ,nervous system ,biology.protein ,Neuroglia ,Calcium ,Neurology (clinical) ,Endothelin receptor ,Developmental Biology - Abstract
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.
- Published
- 2011
5. Functional lipidomics. Calcium-independent activation of endocannabinoid/endovanilloid lipid signalling in sensory neurons by protein kinases C and A and thrombin
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Vittorio Vellani, Massimiliano Prandini, Pier Cosimo Magherini, Vincenzo Di Marzo, Luciano De Petrocellis, Marta Valenti, Peter A. McNaughton, and Stefania Petrosino
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Cannabinoid receptor ,medicine.medical_treatment ,2-Arachidonoylglycerol ,Anandamide ,Cannabinoid ,CB1 ,Channel ,Metabotropic ,Signalling ,TRPV1 ,Vanilloid ,Rats, Sprague-Dawley ,chemistry.chemical_compound ,Cannabinoid Vanilloid Signalling ,Ganglia, Spinal ,Egtazic Acid ,Cells, Cultured ,Protein Kinase C ,Forskolin ,Chemistry ,Thrombin ,Endocannabinoid system ,Tetradecanoylphorbol Acetate ,lipids (amino acids, peptides, and proteins) ,Signal Transduction ,medicine.medical_specialty ,Sensory Receptor Cells ,Receptors, Proteinase-Activated ,Arachidonic Acids ,Glycerides ,Cellular and Molecular Neuroscience ,Internal medicine ,Cannabinoid Receptor Modulators ,Thrombin receptor ,medicine ,Animals ,Humans ,Protein kinase C ,Pharmacology ,Colforsin ,Cyclic AMP-Dependent Protein Kinases ,Rats ,Endocrinology ,Animals, Newborn ,nervous system ,Calcium ,Endocannabinoids - Abstract
N-arachidonoylethanolamine (anandamide, AEA), is a full agonist at both cannabinoid CB1 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.
- Published
- 2008
6. Plant-Derived Cannabinoids Modulate the Activity of Transient Receptor Potential Channels of Ankyrin Type-1 and Melastatin Type-8
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Vittorio Vellani, Pietro Marini, Vincenzo Di Marzo, Pierangelo Orlando, Pier Cosimo Magherini, Luciano De Petrocellis, and Aniello Schiano-Moriello
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TRPM8 ,Ankyrins ,Cannabigerol ,TRPV1 ,TRPM Cation Channels ,ION-CHANNEL ,Pharmacology ,Transfection ,TRPA1 ,TRP CHANNELS ,Cell Line ,Rats, Sprague-Dawley ,Transient receptor potential channel ,chemistry.chemical_compound ,Cannabichromene ,endocannabinoids ,pain ,hyperalgesia ,Ganglia, Spinal ,medicine ,Animals ,Humans ,TRPA1 Cation Channel ,Cells, Cultured ,Cannabis ,TRPC Cation Channels ,Neurons ,Cannabinoids ,Icilin ,Endocannabinoid system ,PROSTATE-CANCER ,Rats ,chemistry ,SENSORY NEURONS ,Molecular Medicine ,Calcium ,Calcium Channels ,NOCICEPTIVE NEURONS ,Cannabidiol ,medicine.drug - Abstract
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.
- Published
- 2008
7. Transport of K+ by Na(+)-Ca2+, K+ exchanger in isolated rods of lizard retina
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Vittorio Vellani, Giorgio Rispoli, and Anacleto Navangione
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Patch-Clamp Techniques ,Potassium Channels ,Biophysics ,chemistry.chemical_element ,Calcium ,In Vitro Techniques ,Michaelis–Menten kinetics ,Sodium-Calcium Exchanger ,Ion ,Diffusion ,Animals ,Homeostasis ,Patch clamp ,Na+/K+-ATPase ,Sodium-calcium exchanger ,Ion exchange ,Chemistry ,Lizards ,Models, Theoretical ,Rod Cell Outer Segment ,Potassium channel ,Crystallography ,Carrier Proteins ,Kinetics ,Biochemistry ,Potassium ,Thermodynamics ,Mathematics ,Research Article - Abstract
Transport of K+ by the photoreceptor Na(+)-Ca2+, K+ exchanger was investigated in isolated rod outer segments (OS) by recording membrane current under whole-cell voltage-clamp conditions. Known amounts of K+ were imported in the OS through the Ca(2+)-activated K+ channels while perfusing with high extracellular concentration of K+, [K+]o. These channels were detected in the recordings from the OS, which probably retained a small portion of the rest of the cell. The activation of forward exchange (Na+ imported per Ca2+ and K+ extruded) by intracellular K+, Ki+, was described by first-order kinetics with a Michaelis constant, Kapp(Ki+), of about 2 mM and a maximal current, Imax, of about -60 pA. [Na+]i larger than 100 mM had little effect on Kapp(Ki+) and Imax, indicating that Nai+ did not compete with Ki+ for exchange sites under physiological conditions, and that Na+ release at the exchanger intracellular side was not a rate-limiting step for the exchange process. Exchanger stoichiometry resulted in one K+ ion extruded per one positive charge imported. Exchange current was detected only if Ca2+ and K+ were present on the same membrane side, and Na+ was simultaneously present on the opposite side. Nonelectrogenic modes of ion exchange were tested taking advantage of the hindered diffusion found for Cai2+ and Ki+. Experiments were carried out so that the occurrence of a putative nonelectrogenic ion exchange, supposedly induced by the preapplication of certain extracellular ion(s), would have resulted in the transient presence of both Cai2+ and Ki+. The lack of electrogenic forward exchange in a subsequent switch to high Nao+, excluded the presence of previous nonelectrogenic transport.
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- 1995
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8. 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
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Frank Porreca, Elisa Giannini, Vittorio Vellani, Mariantonella Colucci, Pietro Melchiorri, Lucia Negri, Milena De Felice, Roberta Lattanzi, Hui Tian, and Federica Margheriti
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trpv1 ,pkr1 ,Body Temperature ,Receptors, G-Protein-Coupled ,Mice ,Dorsal root ganglion ,Ganglia, Spinal ,Drug Interactions ,nociception ,Cells, Cultured ,In Situ Hybridization ,Pain Measurement ,prokineticins ,Mice, Knockout ,Behavior, Animal ,Reverse Transcriptase Polymerase Chain Reaction ,General Neuroscience ,bv8 ,inflammation ,Nociceptors ,Articles ,Prokineticin ,PKR1 ,TRPV1 ,medicine.anatomical_structure ,Nociception ,Hyperalgesia ,Nociceptor ,medicine.symptom ,Pain Threshold ,medicine.medical_specialty ,Pain ,TRPV Cation Channels ,Gastrointestinal Hormones ,Internal medicine ,Physical Stimulation ,medicine ,Reaction Time ,Animals ,Neurons, Afferent ,Dose-Response Relationship, Drug ,business.industry ,Neuropeptides ,Prokineticin receptor 2 ,Prokineticin receptor 1 ,Mice, Inbred C57BL ,Endocrinology ,nervous system ,Gene Expression Regulation ,Calcium ,Capsaicin ,business - Abstract
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 [Ca(2+)](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 [Ca(2+)](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.
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- 2006
9. Sensitization of transient receptor potential vanilloid 1 by the prokineticin receptor agonist Bv8
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Pietro Melchiorri, Roberta Lattanzi, Vittorio Vellani, Lucia Negri, Peter A. McNaughton, Mariantonella Colucci, and Elisa Giannini
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Agonist ,medicine.medical_specialty ,medicine.drug_class ,trpv ,TRPV1 ,TRPV Cation Channels ,capsaicin ,sensitization ,Amphibian Proteins ,Membrane Potentials ,Gastrointestinal Hormones ,Rats, Sprague-Dawley ,heat ,hyperalgesia ,nociception ,nociceptor ,pain ,protein kinase ,sensory neurons ,substance p ,Mice ,Internal medicine ,Ganglia, Spinal ,medicine ,Glial cell line-derived neurotrophic factor ,Animals ,Receptor ,Cells, Cultured ,Neurons ,biology ,Chemistry ,General Neuroscience ,Neuropeptides ,Prokineticin receptor 2 ,Articles ,Prokineticin receptor 1 ,Prokineticin ,Cell biology ,Rats ,Endocrinology ,nervous system ,Animals, Newborn ,Hyperalgesia ,biology.protein ,Calcium ,Vascular Endothelial Growth Factor, Endocrine-Gland-Derived ,medicine.symptom ,substance P ,TRPV - Abstract
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 Bv8in vivocauses a potent and long-lasting hyperalgesia. Here, we investigate the cellular basis of hyperalgesia caused by activation of PKRs. Bv8 caused increases in [Ca]iin 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 Cε (PKCε), because Bv8 caused translocation of PKCε 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) inducedde novoexpression 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 PKCε. The results suggest a role for prokineticins in physiological inflammation and hyperalgesia.
- Published
- 2006
10. Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide
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Andrea Moriondo, John B. Davis, Vittorio Vellani, Peter A. McNaughton, and Sarah Mapplebeck
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Hot Temperature ,Physiology ,Receptors, Drug ,ION-CHANNEL ,ROOT GANGLION NEURONS ,sensitization ,DESENSITIZATION ,chemistry.chemical_compound ,Ganglia, Spinal ,pain ,Cells, Cultured ,Protein Kinase C ,Neurons ,Anandamide ,Endocannabinoid system ,Cell biology ,Electrophysiology ,Hyperalgesia ,Nociceptor ,Tetradecanoylphorbol Acetate ,medicine.symptom ,Protons ,Capsazepine ,SUBPOPULATION ,Ion Channel Gating ,medicine.medical_specialty ,Polyunsaturated Alkamides ,TRPV1 ,Arachidonic Acids ,CALCIUM ,Cell Line ,Internal medicine ,medicine ,Animals ,Humans ,SENSORY NEURONS ,hyperalgesia ,RESPONSES ,Rats, Wistar ,Protein kinase C ,Original Articles ,Rats ,Enzyme Activation ,Endocrinology ,chemistry ,Capsaicin ,Calcium ,Endocannabinoids - Abstract
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. 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. 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. 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. Removing external Ca2+ 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 Ca2+ produced no further enhancement of the response to capsaicin in either DRG neurones or HEK293 cells stably transfected with VR1. 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. 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. Heat stimuli of above about 43 °C elicit a sensation of pain in humans, and initiate action potentials in nociceptive nerve terminals in humans and animals (Belmonte & Giraldez, 1981; Robinson et al. 1983). In experiments on isolated nociceptive neurones, heat was found to activate an inward current that has the properties expected from psychophysical and whole-animal experiments for the detector of painful levels of heat (Cesare & McNaughton, 1996; Reichling & Levine, 1997; Kirschstein et al. 1997; Nagy & Rang, 1999). Inflammation causes the threshold for the initiation both of a sensation of pain and of action potentials in nociceptive nerve fibres to fall to a lower temperature, a process known as sensitization (Treede et al. 1992). The heat-gated current also shows sensitization, in that the temperature threshold for activation of the current is lowered on exposure of the nociceptor to bradykinin, a nonapeptide released during inflammation (Cesare & McNaughton, 1996). Bradykinin sensitizes the heat-gated current by activating the ɛ isoform of protein kinase C (PKCɛ) (Cesare et al. 1999a). An important molecular sensor for heat is the receptor for capsaicin, which was recently cloned and named vanilloid receptor 1, or VR1 (Caterina et al. 1997). Capsaicin, the active ingredient of chilli peppers, excites nociceptive neurones (Baumann et al. 1991; LaMotte et al. 1992) resulting in a burning pain sensation (Buck & Burks, 1986). VR1 is a non-selective cation channel that is activated by a broad spectrum of stimuli, including capsaicin, heat, low pH and the endocannabinoid anandamide (Caterina et al. 1997; Tominaga et al. 1998; Zygmunt et al. 1999; Smart et al. 2000). Targeted disruption of the VR1 gene in mice produces animals that do not respond adversely to even high doses of capsaicin (Caterina et al. 2000), showing that VR1 is the only receptor for capsaicin linked to pain perception. VR1 knockout animals still respond to noxious heat, showing that other heat-sensitive nociceptor mechanisms exist in addition to VR1. A major difference between wild-type and VR1 knockout mice, however, was that the heat hyperalgesia observed in inflamed tissue was greatly reduced in the knockout, suggesting that VR1 is the principal mechanism responsible for heat hyperalgesia (Davis et al. 2000; Caterina et al. 2000). The experiments in the present study explore the role of PKC in the sensitization of VR1, using both nociceptive neurones and heterologously expressed VR1. In nociceptive neurones we found that the response to capsaicin is sensitized by PKC activation, in a similar manner to the response to heat, adding to the evidence that the same molecular mechanism is responsible for the response to the two stimuli. In HEK293 cells expressing VR1 (hVR1-HEK293 cells) the responses to heat, capsaicin, low pH and anandamide were found to be enhanced by PKC activation. The enhancement of current in response to capsaicin, in both nociceptive neurones and hVR1-HEK293 cells, was abolished in the absence of external Ca2+, consistent with the demonstration by other authors that desensitization of VR1 occurs by a calcium-sensitive process (Cholewinski et al. 1993; Docherty et al. 1996; Liu & Simon, 1996; Koplas et al. 1997). Finally, while heat, capsaicin, low pH and anandamide activate current through VR1, activation of PKC potentiates the effect of these stimuli but does not itself activate current, showing that PKC activation does not directly gate the VR1 channel. Preliminary reports of the data reported here have appeared elsewhere (Vellani et al. 1999).
- Published
- 2001
11. Turnover rate and number of Na+-Ca2+, K+ exchange sites in retinal photoreceptors
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Anacleto Navangione, Vittorio Vellani, and Giorgio Rispoli
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MECHANISM ,NA+/CA2+ EXCHANGER ,ROD OUTER SEGMENTS ,SODIUM-CALCIUM EXCHANGER ,NA-CA EXCHANGE ,MEMBRANE PATCHES ,ION-TRANSPORT ,PURIFICATION ,CELLS ,PHOTORECEPTORS ,phototransduction ,photoreceptor rod ,ion pumps ,electrophysiology ,Rod Outer Segments ,General Biochemistry, Genetics and Molecular Biology ,Sodium-Calcium Exchanger ,chemistry.chemical_compound ,History and Philosophy of Science ,Animals ,Photoreceptor Cells ,Ion transporter ,Sodium-calcium exchanger ,General Neuroscience ,Sodium ,Electric Conductivity ,Retinal ,Lizards ,Rod Cell Outer Segment ,chemistry ,Turnover ,Biophysics ,Potassium ,Calcium ,Carrier Proteins - Published
- 1996
12. Anandamide acts as an intracellular messenger amplifying Ca2+ influx via TRPV1 channels
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Aniello Schiano Moriello, P Geppetti, Mario van der Stelt, Vittorio Vellani, Luciano De Petrocellis, Peter A. McNaughton, Barbara Campi, Vincenzo Di Marzo, and Marcello Trevisani
- Subjects
Patch-Clamp Techniques ,Cannabinoid receptor ,Ca ,ROOT GANGLION NEURONS ,Inositol 1,4,5-Trisphosphate ,Pharmacology ,CALCIUM-ENTRY ,CAPSAICIN RECEPTOR ,Ion Channels ,Rats, Sprague-Dawley ,Metabotropic ,Store depletion ,chemistry.chemical_compound ,Adenosine Triphosphate ,Ganglia, Spinal ,homeostasis ,ENDOGENOUS CANNABINOID ANANDAMIDE ,MOLECULAR CHARACTERIZATION ,Vanilloid ,Anandamide ,2+ ,Cells, Cultured ,VANILLOID VR1 RECEPTORS ,General Neuroscience ,Purinergic receptor ,PRIMARY SENSORY NEURONS ,Endocannabinoid system ,Cell biology ,Ca2+ homeostasis ,store depletion ,PROTEIN-KINASE-C ,CELLULAR UPTAKE ,NERVOUS-SYSTEM ,Thapsigargin ,lipids (amino acids, peptides, and proteins) ,Erratum ,Signal Transduction ,Polyunsaturated Alkamides ,TRPV1 ,TRPV Cation Channels ,Arachidonic Acids ,Calcium-Transporting ATPases ,Muscarinic Agonists ,Biology ,Depolarization-induced suppression of inhibition ,Article ,General Biochemistry, Genetics and Molecular Biology ,Animals ,Humans ,Calcium Signaling ,Neurons, Afferent ,Molecular Biology ,General Immunology and Microbiology ,Cell Membrane ,Rats ,Metabotropic receptor ,chemistry ,nervous system ,Purinergic Agonists ,Type C Phospholipases ,GPR18 ,Calcium ,Carbachol ,Endocannabinoids - Abstract
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.
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