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Vittorio VELLANI

Ricercatore Universitario
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Sc. Biomediche

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2022 - Depleted Calcium Stores and Increased Calcium Entry in Rod Photoreceptors of the Cacna2d4 Mouse Model of Cone-Rod Dystrophy RCD4 [Articolo su rivista]
Vellani, Vittorio; Mauro, Giovanna; Demontis, Gian Carlo

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.

2021 - The Calcium Channel Blocker Gabapentin, a Benchmark Drug in Pain Studies, Inhibits Translocation of the Epsilon Isoform of Protein Kinase C in Cultured Nociceptors: A Novel Mechanism of Action [Capitolo/Saggio]
Vellani, Vittorio; Giacomoni, Chiara

Gabapentin is a well-established anticonvulsant drug which is also effective for the treatment of neuropathic pain. Gabapentin, a structural analogue of the inhibitory neurotransmitter gamma-aminobutyric acid, was originally developed in the early nineties as a third-generation antiepileptic drug. 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 translocation of protein kinase C epsilon (PKC????) in cultured peripheral neurons isolated from rat dorsal root ganglia (DRGs). We found that gabapentin significantly reduced PKC???? translocation induced by the pronociceptive peptides bradykinin and prokineticin 2, which are involved in both inflammatory and chronic pain. We recently showed that paracetamol (acetaminophen), a very commonly used analgesic drug, also produces inhibition of PKCepsilon. We tested the effect of the combined use of paracetamol and gabapentin, and we found that the inhibition of translocation adds up in a non-cooperative fashion. Our study provides a novel mechanism of action for gabapentin in sensory neurons and suggests a rationale and 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.

2020 - Abnormal Pain Sensation in Mice Lacking the Prokineticin Receptor PKR2: Interaction of PKR2 with Transient Receptor Potential TRPV1 and TRPA1 [Articolo su rivista]
Maftei, D.; Vellani, V.; Artico, M.; Giacomoni, C.; Severini, C.; Lattanzi, R.

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.

2020 - Cone-Like Functional Properties of cGMP-Gated Channels in Rod-Like Retinal Photoreceptors of Nocturnal Geckoes [Capitolo/Saggio]
Vellani, Vittorio; Giacomoni, Chiara

Retinas of nocturnal geckoes only contain scotopic photoreceptors 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 ” originally derived from cones in cone-only retinas of ancestral diurnal geckoes as a consequence of evolutionary pressure that forced adaptation to 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 detached outer segment preparation, the voltage rectification properties of photoreceptor cGMP-gated channels in three species, Gekko gecko, Tarentola mauritanica, and Hemidactylus frenatus. We show that the currentvoltage 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. In conclusion, in this paper, we provide a novel, functional evidence of the presence of mechanisms in rod-shaped photoreceptors from nocturnal geckos that are reminiscent of their origin from ancestral cones. The molecular analysis of the retinal cGMP gated channel recently performed in Gekko gecko identified cone-like structural features, which agree with our observations.

2020 - CR4056, a powerful analgesic imidazoline-2 receptor ligand, inhibits the inflammation-induced PKCε phosphorylation and membrane translocation in sensory neurons [Articolo su rivista]
Vellani, V.; Sabatini, C.; Milia, C.; Caselli, G.; Lanza, M.; Letari, O.; Rovati, L. C.; Giacomoni, C.

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.

2017 - Effects of NSAIDs on the Release of Calcitonin Gene-Related Peptide and Prostaglandin E2 from Rat Trigeminal Ganglia [Articolo su rivista]
Vellani, Vittorio; Moschetti, Giorgia; Franchi, Silvia; Giacomoni, Chiara; Sacerdote, Paola; Amodeo, Giada

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 E 2 (PGE 2 ) 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 PGE 2 , although with some differences in timing and doses, and the action on CGRP does not seem to be related to PGE 2 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.

2017 - Gabapentin Inhibits Protein Kinase C Epsilon Translocation in Cultured Sensory Neurons with Additive Effects When Coapplied with Paracetamol (Acetaminophen) [Articolo su rivista]
Vellani, Vittorio; Giacomoni, Chiara

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.

2016 - TRPA1 Is Expressed in Central But Not in Peripheral Glia [Articolo su rivista]
Vellani, Vittorio; Gomis-Perez, Carolina; Pinti, Marcello; Prandini, Massimiliano; Pavesi, Giorgia; Giacomoni, Chiara; Caprini, Marco

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.

2014 - Cone-Like Rectification Properties of cGMP-Gated Channels in Transmutated Retinal Photoreceptors of Nocturnal Geckoes [Articolo su rivista]
Vellani, Vittorio; Giacomoni, Chiara

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.

2014 - Dispositivo termoregolatore per un sistema di microperfusione [Brevetto]
Vellani, Vittorio; Pini, Fabio; Leali, Francesco; Corradini, Matteo

Una forma di attuazione della presente invenzione riguarda un dispositivo termoregolatore (225) per un sistema di microperfusione (100), il quale comprende almeno un elemento Peltier (235) ed una piastra di supporto (255) in materiale termicamente conduttore, la quale è posta in relazione di scambio termico con l’elemento Peltier (235) e presenta un alloggiamento (270, 275, 280) per un tubo di convogliamento (265) di un fluido da erogare.

2014 - Testina di perfusione per un sistema di microapplicazione [Brevetto]
Vellani, Vittorio; Pini, Fabio; Leali, Francesco; Corradini, Matteo

Una forma di attuazione della presente invenzione rende disponibile una testina di perfusione per un sistema di microapplicazione, la quale comprende una pluralità di raccordi cilindrici ricavati in corpo monolitico e singolarmente atti ad accoppiarsi con un rispettivo tubo di convogliamento di un fluido da applicare, ed altrettanti condotti di erogazione ricavati all’interno di detto corpo monolitico e singolarmente atti a porre in comunicazione un 10 rispettivo raccordo cilindrico con una rispettiva luce di uscita. [fig. 3]

2013 - Effects of NSAIDs and paracetamol (acetaminophen) on protein kinase C epsilon translocation and on substance P synthesis and release in cultured sensory neurons. [Articolo su rivista]
Vellani, Vittorio; Franchi, S; Prandini, Massimiliano; Moretti, S; Castelli, M; Giacomoni, C; Sacerdote, P.

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.

2012 - Fisiologia [Monografia/Trattato scientifico]
Vellani, Vittorio

Libro di Testo di Fisiologia

2011 - 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. [Articolo su rivista]
Vellani, Vittorio; Prandini, Massimiliano; C., Giacomoni; Pavesi, Giorgia; Ravegnani, Laura; Magherini, Pier Cosimo

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.

2011 - Nimesulide inhibits protein kinase C epsilon and substance P in sensory neurons - comparison with paracetamol. [Articolo su rivista]
Vellani, Vittorio; Franchi, S; Prandini, Massimiliano; Moretti, S; Pavesi, Giorgia; Giacomoni, C; Sacerdote, P.

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.

2010 - Protease activated receptors 1 and 4 sensitize TRPV1 in nociceptive neurones [Articolo su rivista]
Vellani, Vittorio; A. M., Kinsey; Prandini, Massimiliano; S. C., Hechtfischer; P., Reeh; Magherini, Pier Cosimo; C., Giacomoni; P. A., Mcnaughton

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 PLCβ/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.

2008 - Functional lipidomics. Calcium-independent activation of endocannabinoid/endovanilloid lipid signalling in sensory neurons by protein kinases C and A and thrombin [Articolo su rivista]
Vellani, Vittorio; S., Petrosino; L., De Petrocellis; M., Valenti; Prandini, Massimiliano; Magherini, Pier Cosimo; P. A., Mcnaughton; V., Di Marzo

N-arachidonoylethanolamine (anandamide, AEA), is a full agonist at both cannabinoid CB, receptors and "transient receptor potential vanilloid" type I (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. (C) 2008 Elsevier Ltd. All rights reserved.

2008 - On the key role played by altered protein conformation in Parkinson’s disease [Articolo su rivista]
Agnati, Luigi Francesco; Baldelli, Enrica; Andreoli, Nicola; A. S., Woods; Vellani, Vittorio; D., Marcellino; D., Guidolin; K., Fuxe

On the basis of the previously proposed hierarchic organisation of the central nervous system (CNS) and of its syntropic behaviour, a view of neurodegenerative diseases focusing on the assemblage of abnormal multimeric proteins (pathologic protein mosaics (PMs)) is proposed. Thus, the main focus of the present paper is on Parkinson’s disease (PD) as a neurodegenerative disease, which has as crucial feature protein conformational alterations and formation of pathological PMs. Two interconnected cellular dysfunctions are discussed as main pathogenic factors of PD syndromes, namely mitochondrial deficits (i.e. energy failure, especially critical for Substantia Nigra DA neurons) and conformational protein alterations (due to genetic or environmental causes). Conformational protein alterations can trigger pathological phenomena via the loss and/or the gain of new functions. In particular, altered proteins can lead to the formation of abnormal PMs, which can, inter alia, cause distortion of cellular structures, toxic functions and/or formation of improper membrane ion channels. In view of the fact that disordered proteins can easily acquire unwanted conformation, the “disorder index” (DI) for proteins involved in PD has been evaluated. It has been found that both α-synuclein and tau-protein have high DI. This datum is in agreement with the observation that these two proteins synergistically promote polymerisation of each other into amyloid fibrils, favouring the formation of Lewy bodies.

2008 - Plant-derived cannabinoids modulate the activity of transient receptor potential channels of ankyrin type-1 and melastatin type-8 [Articolo su rivista]
L., De Petrocellis; Vellani, Vittorio; A., Schiano Moriello; P., Marini; Magherini, Pier Cosimo; P., Orlando; V., Di Marzo

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 EC50 = 60 nM) and the least potent being CBG and CBD acid (EC50 = 3.4-12.0 mu M). CBC also activated MO-sensitive DRG neurons, although with lower potency (EC50 = 34.3 mu M). 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 (IC50 = 70-160 nM), whereas CBD acid was the least potent compound (IC50 = 0.9-1.6 mu M). CBG inhibited Ca2+ elevation also in icilin-sensitive DRG neurons with potency (IC50 = 4.5 mu M) similar to that of anandamide (IC50 = 10 mu M). 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.

2006 - Fisiologia: molecole, cellule e sistemi [Monografia/Trattato scientifico]
A. M., Angioy; Bardoni, Rita; Bigiani, Albertino; M., Brunelli; A., Contestabile; M., Crispino; E., D'Angelo; F., Franciolini; E., Fugassa; S., Fulle; A., Giuditta; R., Levi; M. G., Lionetto; V., Lombardi; E., Macchi; D., Negrini; P., Paggi; S., Palmero; A., Peres; M., Piccolino; C., Poggesi; G., Rispoli; P., Rossi; V. F., Sacchi; T., Schettino; R., Serio; G., Valenti; Vellani, Vittorio; A., Zaza

Questo libro nasce con l’intento di offrire agli studenti dei Corsi universitari di carattere biomedico uno strumento di riferimento per lo studio dei meccanismi alla base del funzionamento degli esseri viventi, in particolare nel regno animale: l’attenzione è principalmente rivolta ai processi fondamentali e unificanti dei fenomeni fisiologici, quelli che costituiscono le basi, di solito molecolari e cellulari, di funzioni fisiologiche anche apparentemente lontane tra loro. Poiché questo libro è destinato agli studenti di Corsi di Laurea differenti, dalle Scienze biologiche alle Biotecnologie, ma anche di Scienze naturali e Farmacia, si è ritenuto utile includere anche capitoli di fisiologia dei sistemi.La sequenza degli argomenti si articola dal livello molecolare e cellulare, per considerare successivamente le interazioni fra le cellule e concludersi con la trattazione dei più importanti organi e sistemi d’organo. Questa progressione dal microscopico al macroscopico segue una logica inversa rispetto alla cronologia della storia dell’indagine dei processi fisiologici, che è naturalmente partita molti secoli fa dallo studio del funzionamento degli apparati e degli organi più evidenti e considerati maggiormente importanti. La progressiva comprensione dei meccanismi di funzionamento e il parallelo sviluppo tecnologico degli strumenti d’indagine hanno facilitato nel corso del tempo dei processi a livelli via via più nascosti e microscopici per trovare una spiegazione soddisfacente dell’evento macroscopico. Laddove questo approccio è riuscito ad addentrarsi fino al livello molecolare si è constatato che molte funzioni macroscopiche, a prima vista significativamente diverse tra loro, si basano in realtà su processi microscopici molto simili. Per questo motivo ci è sembrato preferibile trattare inizialmente una serie di processi elementari che vengono successivamente ritrovati in azione per contribuire alla realizzazione di funzioni fisiologiche complesse.A loro volta i processi fisiologici elementari, a livello molecolare e cellulare, si basano sulle leggi della fisica, della chimica e della biologia molecolare e cellulare. Queste nozioni costituiscono un’indispensabile base propedeutica per lo studio della fisiologia e non sono normalmente contenute nei testi di fisiologia. Per comodità del lettore, tuttavia, sono stati inclusi alcuni capitoli che sintetizzano alcuni punti fondamentali delle discipline fisiche, chimiche e biologiche di particolare interesse in fisiologia.La grande variabilità dei programmi degli insegnamenti che fanno riferimento alla fisiologia – e la diversa estensione e importanza che nelle diverse Università viene a essi data – ha consigliato di impostare il libro in modo da facilitarne un utilizzo in una certa misura “personalizzato”. Così, accanto a un percorso base, contenente tutti gli argomenti più classici trattati in modo rigoroso, ma senza eccessivi approfondimenti, sono presenti parti differenziate, che potranno essere utilizzate secondo le necessità degli indirizzi di studio. Il testo in corpo ridotto riguarda approfondimenti non strettamente necessari alla preparazione di base. I riquadri trovano spazio nei diversi capitoli, per consentire approfondimenti specifici in aree diverse: biofisica molecolare (per le Classi di Biotecnologie e di Scienze Biologiche con indirizzi cellulari o di Neuroscienze), fisiologia comparata (per le Classi di Scienze naturali e di Scienze biologiche con indirizzi naturalistici), ricerca applicata (Biotecnologie e Farmacia), area storica (destinata a tutti coloro che sono interessati a conoscere la storia delle idee sul funzionamento degli esseri viventi e del corpo umano in particolare, una storia di grandissimo interesse scientifico, filosofico e umano, che può essere tracciata fin dai primordi delle civiltà conosciute).

2006 - 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 [Articolo su rivista]
Negri, L; Lattanzi, R; Giannini, E; Colucci, M; Margheriti, F; Melchiorri, P; Vellani, Vittorio; Tian, H; De Felice, M; Porreca, F.

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.

2006 - Sensitization of transient receptor potential vanilloid 1 by the prokineticin receptor agonist Bv8 [Articolo su rivista]
Vellani, Vittorio; Colucci, M; Lattanzi, R; Giannini, E; Negri, L; Melchiorri, P; Mcnaughton, Pa

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 C epsilon ( PKC epsilon), because Bv8 caused translocation of PKC epsilon 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 PKC epsilon. The results suggest a role for prokineticins in physiological inflammation and hyperalgesia.

2005 - Anandamide acts as an intracellular messenger amplifying Ca2+ influx via TRPV1 channels [Articolo su rivista]
M., van der Stelt; M., Trevisani; Vellani, Vittorio; L., De Petrocellis; A. S., Moriello; B., Campi; P., Mcnaughton; P., Geppetti; V., Di Marzo

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 Ca2+-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.

2005 - Erratum: Anandamide acts as an intracellular messenger amplifying Ca 2+ influx via TRPV1 channels (EMBO Journal (2005) 24 (3026-3037) DOI: 10.1038/sj.emboj.7600784 [Articolo su rivista]
M., van der Stelt; M., Trevisani; Vellani, Vittorio; L., De Petrocellis; A. S., Moriello; B., Campi; P. A., Mcnaughton; P., Geppetti; V., Di Marzo

Correction to: The EMBO Journal (2005) 24, 3026–3037. doi:10.1038/sj.emboj.7600784

2004 - Effects of ketamine anesthesia on central nociceptive processing in the rat: a 2-deoxyglucose study. [Articolo su rivista]
Porro, Carlo Adolfo; Cavazzuti, Milena; Giuliani, Daniela; Vellani, Vittorio; Lui, Fausta; Baraldi, Patrizia

Ketamine is a dissociative anesthetic with complex actions on the CNS. We investigated here the effects of ketamine anesthesia on somatosensory processing in the rat spinal cord, thalamus, and cerebral cortex, using the quantitative 2-deoxyglucose mapping technique. Unanesthetized or ketamine-anesthetized male Sprague-Dawley rats received a s.c. injection of a dilute formaldehyde solution (5%, 0.08 ml) into a forepaw, inducing prolonged noxious afferent input, or an equal volume of isotonic saline as a control stimulus. The 2-deoxyglucose experiments started 30 min after the injection. In the cervical enlargement of the spinal cord, ketamine had no significant effect on glucose metabolic rates in saline-injected animals, whereas it prevented the metabolic increases elicited by prolonged noxious stimulation in unanesthetized animals. At the thalamic level, ketamine increased glucose uptake in both saline- and formalin-injected rats in the lateral posterior, lateral dorsal, medial dorsal, gelatinosus, antero-ventral and antero-medial thalamic nuclei, whereas it decreased metabolic activity in the ventro-basal complex. At the cortical level, the drug increased metabolic activity in both control and formalin groups in the lacunosus-molecularis layer of the dorsal hippocampus, posterior parietal, retrosplenial, cingulate and frontal cortex; significant metabolic decreases were found in the CA1 region of the dorsal hippocampus and in the parietal 1 and 2 cortical areas. In the investigated brain regions, ketamine did not abolish noxious-evoked increases in glucose uptake, which were in fact enhanced in the forelimb cortex and in the lacunosus-molecularis layer of the hippocampus. The dissociation between the spinal and supraspinal effects of ketamine suggests a specific antinociceptive action on spinal circuits, in parallel with complex changes of the activity of brain circuits involved in somatosensory processing. More generally, this study shows that functional imaging techniques are able to quantitatively assess the effects of anesthetic drugs on nociceptive processing at different levels of the neuraxis. (C) 2004 IBRO. Published by Elsevier Ltd. All rights reserved.

2004 - Functional bradykinin B1 receptors are expressed in nociceptive neurones and are upregulated by the neurotrophin GDNF [Articolo su rivista]
Vellani, Vittorio; O., Zachrisson; Pa, Mcnaughton

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 e 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 GDNE Neurones expressing functional B1 receptors were negative for the neuropeptides CGRP and substance P, but most expressed functional TRPVI receptors for capsaicin (60%) and bound the lectin IB4 (68%), both markers characteristic of nociceptors. B1 activation enhanced the heat-activated membrane current similar to3-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.

2004 - Involvement of PKC in the sensation of pain [Capitolo/Saggio]
Vellani, Vittorio; P. A., Mcnaughton

Pain is initiated by activation of specific pain-sensitive neurons, or nociceptors, whose sensory terminals express a range of specific membrane receptors responsible for detecting different noxious stimuli. Activation of protein kinase C (PKC) by inflammatorymediators causes an increase in the sensitivity of the nociceptor to a variety of noxious stimuli. The best-studied target of PKC is the heat-sensitive ion channel, VR1, which is phosphoryatedby PKC-ε at two intracellular serine residues, leading to an increase in sensitivity to heat and other pain-causing stimuli such as acid. PKC is also involved at the stage of transmission ofnociceptive information to second-order neurons in the spinal cord. PKC-γ in postsynaptic neurons is specifically activated in neuropathic pain, leading to enhanced transmission, probablybecause of phosphorylation of NMDA receptors in the membrane of the postsynaptic neuron. PKC is also involved in the development of opiate tolerance, though the isotypes involved and the targets of its action are less well defined. Isotype-specific antagonists may have therapeutic potential as analgesics, as PKC-ε antagonists are likely to be active in reducing inflammatory pain, while PKC-γ antagonists may reduce neuropathic pain.

2002 - Chapter 8 The cellular and molecular basis of the detection of pain [Capitolo/Saggio]
Bonnington, J. K.; Robinson, D. R.; Vellani, Vittorio; Mcnaughton, P. A.

In this volume of Cell and Molecular Responses to Stress articles provide up-to-date information on key areas of signal sensing (sensing of pain, heat, cold, light, infrared radiation), molecules involved in the intracellular transmission of these signals, metabolic responses to stress including changes in gene expression and production of specialized proteins that aid cell responses to factors including interrupted blood supply (ischemia), oxygen limitation (hypoxia/anoxia), freezing and dehydration, amino acid limitation, radiation and processing drugs. There are chapters which also provide insights into new technologies (such as cDNA arrays), analysis of metabolic control theory (a key method for analysing stress effects on cells), and examine how enzymes evolve in the face of stress.

2001 - Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide. [Articolo su rivista]
Vellani, Vittorio; S., Mapplebeck; A., Moriondo; J. B., Davis; P. A., Mcnaughton

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.

2000 - Modulation of the synaptic Ca2+ current in salamander photoreceptors by polyunsaturated fatty acids and retinoids [Articolo su rivista]
Vellani, Vittorio; A. M., Reynolds; P. A., Mcnaughton

Synaptic transmission between retinal photoreceptors and second-order neurones is controlled by an L-type Ca2+ conductance (gCa) in the photoreceptor inner segment. Modulation of this conductance therefore influences the flow of visual information to higher centres. Possible modulation of gCa by retinal factors was investigated using patch clamp and Ca2+ imaging. No significant modulation of gCa by retinal neurotransmitters nor by intracellular signalling pathways was found. gCa was inhibited by retinoids (all-trans retinal) and by polyunsaturated fatty acids (PUFAs) such as arachidonic acid and docosahexaenoic acid, which are known to be released in the retina by exposure to light. Some PUFAs tested are physiological substrates for the cyclo-oxygenase, lipoxygenase and epoxygenase pathways, but specific inhibitors of these pathways had no effect on the inhibition of gCa. Treatments designed to activate or inhibit G-protein-coupled pathways or protein kinases A and C similarly had no effect on the inhibition by PUFAs nor on gCa itself. Inhibitors of phosphatases 1 and 2A were also largely ineffective. The inhibition by PUFAs is, however, dependent on membrane potential, suggesting that it arises from a direct interaction of fatty acids with the Ca2+ channel. The effect was not use or frequency dependent, suggesting that the effect does not depend on channel gating state. Control by retinoids and by PUFAs may be an important mechanism by which the Ca2+ conductance, and consequently the transmission of the visual signal, is modulated at the first retinal synapse.

1999 - Ion channels gated by heat [Articolo su rivista]
Cesare, P.; Moriondo, A.; Vellani, V.; Mcnaughton, P. A.

All animals need to sense temperature to avoid hostile environments and to regulate their internal homeostasis. A particularly obvious example is that animals need to avoid damagingly hot stimuli. The mechanisms by which temperature is sensed have until recently been mysterious, but in the last couple of years, we have begun to understand how noxious thermal stimuli are detected by sensory neurons. Heat has been found to open a nonselective cation channel in primary sensory neurons, probably by a direct action. In a separate study, an ion channel gated by capsaicin, the active ingredient of chili peppers, was cloned from sensory neurons. This channel (vanilloid receptor subtype 1, VR1) is gated by heat in a manner similar to the native heat-activated channel, and our current best guess is that this channel is the molecular substrate for the detection of painful heat. Both the heat channel and VR1 are modulated in interesting ways. The response of the heat channel is potentiated by phosphorylation by protein kinase C, whereas VR1 is potentiated by externally applied protons. Protein kinase C is known to be activated by a variety of inflammatory mediators, including bradykinin, whereas extracellular acidification is characteristically produced by anoxia and inflammation. Both modulatory pathways are likely, therefore, to have important physiological correlates in terms of the enhanced pain (hyperalgesia) produced by tissue damage and inflammation. Future work should focus on establishing, in molecular terms, how a single ion channel can detect heat and how the detection threshold can be modulated by hyperalgesic stimuli.

1999 - Manipulation of synaptic sign and strength with divalent cations in the vertebrate retina: pushing the limits of tonic, chemical neurotransmission. [Articolo su rivista]
Piccolino, M.; Vellani, Vittorio; Rakotobe, La; Pignatelli, A.; Barnes, S.; Mcnaughton, P.

At the first synaptic level of the vertebrate retina, photoreceptor light responses are transmitted to second order neurones through a chemical synapse based on a tonic release of neurotransmitter modulated by graded changes of presynaptic potential. The possibility that such synapses could work through a Ca2+-independent process had been proposed by previous authors, based on the persistence of transmission process in low Ca2+ media containing Co2+ or Ni2+ ions. Recently, we were able to explain these results within the framework of the classical calcium-hypothesis of synaptic transmission by taking into account the modifications of presynaptic surface potential brought about by changes of divalent cation concentrations. Here we report data showing how a surface-charge hypothesis could account for several apparently paradoxical effects of divalent cation manipulations such as: the enhancement of neurotransmitter release induced by low Ca2+ media; the transmission "unblocking" effect of Zn2+, Co2+ and Ni2+; and the reversal of transmission polarity induced by application of low Ca2+ media containing Cd2+ or Mg2+ ions.

1996 - Turnover rate and number of Na+-Ca2+, K+ exchange sites in retinal photoreceptors [Articolo su rivista]
G., Rispoli; A., Navangione; Vellani, Vittorio

Study of Na:Ca,K exchange in outer segnments of vertebrate photoreceptors

1995 - Transport of K+ by Na(+)-Ca2+, K+ exchanger in isolated rods of lizard retina. [Articolo su rivista]
Rispoli, G.; Navangione, A.; Vellani, Vittorio

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.