Fabio Arturo Iannotti

Nonpsychotropic Plant Cannabinoids, Cannabidivarin (CBDV) and Cannabidiol (CBD), Activate and Desensitize Transient Receptor Potential Vanilloid 1 (TRPV1) Channels in Vitro: Potential for the Treatment of Neuronal Hyperexcitability

Epilepsy is the most common neurological disorder, with over 50 million people worldwide affected. Recent evidence suggests that the transient receptor potential cation channel subfamily V member 1 (TRPV1) may contribute to the onset and progression of some forms of epilepsy. Since the two nonpsychotropic cannabinoids cannabidivarin (CBDV) and cannabidiol (CBD) exert anticonvulsant activity in vivo and produce TRPV1-mediated intracellular calcium elevation in vitro, we evaluated the effects of these two compounds on TRPV1 channel activation and desensitization and in an in vitro model of epileptiform activity. Patch clamp analysis in transfected HEK293 cells demonstrated that CBD and CBDV dose-dependently activate and rapidly desensitize TRPV1, as well as TRP channels of subfamily V type 2 (TRPV2) and subfamily A type 1 (TRPA1). TRPV1 and TRPV2 transcripts were shown to be expressed in rat hippocampal tissue. When tested on epileptiform neuronal spike activity in hippocampal brain slices exposed to a Mg(2+)-free solution using multielectrode arrays (MEAs), CBDV reduced both epileptiform burst amplitude and duration. The prototypical TRPV1 agonist, capsaicin, produced similar, although not identical effects. Capsaicin, but not CBDV, effects on burst amplitude were reversed by IRTX, a selective TRPV1 antagonist. These data suggest that CBDV antiepileptiform effects in the Mg(2+)-free model are not uniquely mediated via activation of TRPV1. However, TRPV1 was strongly phosphorylated (and hence likely sensitized) in Mg(2+)-free solution-treated hippocampal tissue, and both capsaicin and CBDV caused TRPV1 dephosphorylation, consistent with TRPV1 desensitization. We propose that CBDV effects on TRP channels should be studied further in different in vitro and in vivo models of epilepsy.

Endocannabinoids and endocannabinoid-related mediators: Targets, metabolism and role in neurological disorders.

The endocannabinoid system (ECS) is composed of two G protein-coupled receptors (GPCRs), the cannabinoid CB1 and CB2 receptors, and the two main endogenous lipid ligands of such receptors (also known as the endocannabinoids), anandamide and 2-arachidonoyl-glycerol. The ECS is a pleiotropic signalling system involved in all aspects of mammalian physiology and pathology, and for this reason it represents a potential target for the design and development of new therapeutic drugs. However, the endocannabinoids as well as some of their congeners also interact with a much wider range of receptors, including members of the Transient Receptor Potential (TRP) channels, Peroxisome Proliferator-Activated Receptors (PPARs), and other GPCRs. Indeed, following the discovery of the endocannabinoids, endocannabinoid-related lipid mediators, which often share the same metabolic pathways of the endocannabinoids, have also been identified or rediscovered. In this review article, we discuss the role of endocannabinoids and related lipids during physiological functions, as well as their involvement in some of the most common neurological disorders.

Involvement of KCNQ2 subunits in [3H]dopamine release triggered by depolarization and pre-synaptic muscarinic receptor activation from rat striatal synaptosomes

KCNQ2 and KCNQ3 subunits encode for the muscarinic‐regulated current (IKM), a sub‐threshold voltage‐dependent K+ current regulating neuronal excitability. In this study, we have investigated the involvement of IKM in dopamine (DA) release from rat striatal synaptosomes evoked by elevated extracellular K+ concentrations ([K+]e) and by muscarinic receptor activation. [3H]dopamine ([3H]DA) release triggered by 9u2003mmol/L [K+]e was inhibited by the IKM activator retigabine (0.01–30u2003μmol/L; Emaxu2003=u200354.80u2003±u20033.85%; IC50u2003=u20030.50u2003± 0.36u2003μmol/L). The IKM blockers tetraethylammonium (0.1–3u2003mmol/L) and XE‐991 (0.1–30u2003μmol/L) enhanced K+‐evoked [3H]DA release and prevented retigabine‐induced inhibition of depolarization‐evoked [3H]DA release. Retigabine‐induced inhibition of K+‐evoked [3H]DA release was also abolished by synaptosomal entrapment of blocking anti‐KCNQ2 polyclonal antibodies, an effect prevented by antibody pre‐absorption with the KCNQ2 immunizing peptide. Furthermore, the cholinergic agonist oxotremorine (OXO) (1–300u2003μmol/L) potentiated 9u2003mmol/L [K+]e‐evoked [3H]DA release (Emaxu2003=u2003155u2003± 9.50%; EC50u2003=u200325u2003±u20031.80u2003μmol/L). OXO (100u2003μmol/L)‐induced [3H]DA release enhancement was competitively inhibited by pirenzepine (1–10u2003nmol/L) and abolished by the M3‐preferring antagonist 4‐diphenylacetoxy N‐methylpiperidine methiodide (1u2003μmol/L), but was unaffected by the M1‐selective antagonist MT‐7 (10–100u2003nmol/L) or by Pertussis toxin (1.5–3u2003μg/mL), which uncouples M2‐ and M4‐mediated responses. Finally, OXO‐induced potentiation of depolarization‐induced [3H]DA release was not additive to that produced by XE‐991 (10u2003μmol/L), was unaffected by retigabine (10u2003μmol/L), and was abolished by synaptosomal entrapment of anti‐KCNQ2 antibodies. Collectively, these findings indicate that, in rat striatal nerve endings, IKM channels containing KCNQ2 subunits regulate depolarization‐induced DA release and that IKM suppression is involved in the reinforcement of depolarization‐induced DA release triggered by the activation of pre‐synaptic muscarinic heteroreceptors.

Pre-synaptic BK channels selectively control glutamate versus GABA release from cortical and hippocampal nerve terminals

J. Neurochem. (2010) 115, 411–422.

EXPRESSION, LOCALIZATION, AND PHARMACOLOGICAL ROLE OF Kv7 POTASSIUM CHANNELS IN SKELETAL MUSCLE PROLIFERATION, DIFFERENTIATION AND SURVIVAL AFTER MYOTOXIC INSULTS

Changes in the expression of potassium channels regulate skeletal muscle development. The purpose of this study was to investigate the expression profile and pharmacological role of Kv7 voltage-gated potassium channels in skeletal muscle differentiation, proliferation, and survival after myotoxic insults. Transcripts for all Kv7 genes (Kv7.1–Kv7.5) were detected by polymerase chain reaction (PCR) and/or real-time PCR in murine C2C12 myoblasts; Kv7.1, Kv7.3, and Kv7.4 transcripts were up-regulated after myotube formation. Western blot experiments confirmed Kv7.2, Kv7.3, and Kv7.4 subunit expression, and the up-regulation of Kv7.3 and Kv7.4 subunits during in vitro differentiation. In adult skeletal muscles from mice and humans, Kv7.2 and Kv7.3 immunoreactivity was mainly localized at the level of intracellular striations positioned between ankyrinG-positive triads, whereas that of Kv7.4 subunits was largely restricted to the sarcolemmal membrane. In C2C12 cells, retigabine (10 μM), a specific activator of neuronally expressed Kv7.2 to Kv7.5 subunits, reduced proliferation, accelerated myogenin expression, and inhibited the myotoxic effect of mevastatin (IC50 ≈ 7 μM); all these effects of retigabine were prevented by the Kv7 channel blocker 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE-991) (10 μM). These data collectively highlight neural Kv7 channels as significant pharmacological targets to regulate skeletal muscle proliferation, differentiation, and myotoxic effects of drugs.

The dual blocker of FAAH/TRPV1 N-arachidonoylserotonin reverses the behavioral despair induced by stress in rats and modulates the HPA-axis

In recent years, several studies have explored the involvement of the deregulation of the hypothalamus-pituitary-adrenal (HPA) axis in the pathophysiology of stress-related disorders. HPA hyper-activation as a consequence of acute/chronic stress has been found to play a major role in the neurobiological changes that are responsible for the onset of such states. Currently available medications for depression, one of the most relevant stress-related disorders, present several limitations, including a time lag for treatment response and low rates of efficacy. N-Arachidonoylserotonin (AA-5-HT), a dual blocker at fatty acid amide hydrolase (FAAH, the enzyme responsible for the inactivation of the endocannabinoid anandamide) and transient receptor potential vanilloid type-1 channel (TRPV1), produces anxiolytic-like effects in mice. The present study was designed to assess the capability of AA-5-HT to reverse the behavioral despair following exposure to stress in rats and the role of the HPA-axis. Behavioral tasks were performed, and corticosterone and endocannabinoid (anandamide and 2-arachidonoylglycerol) levels were measured in selected brain areas critically involved in the pathophysiology of stress-related disorders (medial PFC and hippocampus) under basal and stress conditions, and in response to treatment with AA-5-HT. Our data show that AA-5-HT reverses the rat behavioral despair in the forced swim test under stress conditions, and this effect is associated with the normalization of the HPA-axis deregulation that follows stress application and only in part with elevation of anandamide levels. Blockade of FAAH and TRPV1 may thus represent a novel target to design novel therapeutic strategies for the treatment of stress-related disorders.

The endocannabinoid 2-AG controls skeletal muscle cell differentiation via CB1 receptor-dependent inhibition of Kv7 channels

Significance Although CB1 cannabinoid receptors control skeletal muscle insulin signaling, little is known of their role in muscle formation during differentiation from myoblasts to myotubes. The voltage-dependent Kv7 K+ channels, which are tonically activated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), instead activate myotube formation. We found that the levels of the endogenous CB1 agonist 2-arachidonoylglycerol are decreased during murine myoblast differentiation into myotubes, whereas CB1 expression is up-regulated. CB1 activation inhibits myotube formation. This effect is exerted by reducing PIP2 binding to Kv7.4, which represents the Kv7 subunit responsible for the pro-myogenic effects. Accordingly, CB1 activation inhibits Kv7.4-mediated currents in transfected CHO cells. The endocannabinoid system might thus play a role in skeletal muscle dystrophies. Little is known of the involvement of endocannabinoids and cannabinoid receptors in skeletal muscle cell differentiation. We report that, due to changes in the expression of genes involved in its metabolism, the levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) are decreased both during myotube formation in vitro from murine C2C12 myoblasts and during mouse muscle growth in vivo. The endocannabinoid, as well as the CB1 agonist arachidonoyl-2-chloroethylamide, prevent myotube formation in a manner antagonized by CB1 knockdown and by CB1 antagonists, which, per se, instead stimulate differentiation. Importantly, 2-AG also inhibits differentiation of primary human satellite cells. Muscle fascicles from CB1 knockout embryos contain more muscle fibers, and postnatal mice show muscle fibers of an increased diameter relative to wild-type littermates. Inhibition of Kv7.4 channel activity, which plays a permissive role in myogenesis and depends on phosphatidylinositol 4,5-bisphosphate (PIP2), underlies the effects of 2-AG. We find that CB1 stimulation reduces both total and Kv7.4-bound PIP2 levels in C2C12 cells and inhibits Kv7.4 currents in transfected CHO cells. We suggest that 2-AG is an endogenous repressor of myoblast differentiation via CB1-mediated inhibition of Kv7.4 channels.

Specification of skeletal muscle differentiation by repressor element-1 silencing transcription factor (REST)-regulated Kv7.4 potassium channels

Kv7.4-potassium channel expression plays a permissive role in skeletal muscle differentiation. The transcriptional repressor REST controls the changes in Kv7.4 levels during myogenesis by binding to regulatory regions in the Kv7.4 gene. This mechanism may be a target for intervention against abnormal repair and differentiation of skeletal muscle.

Neuronal potassium channel openers in the management of epilepsy: role and potential of retigabine

Despite the availability of over 20 antiepileptic drugs, about 30% of epileptic patients do not achieve seizure control. Thus, identification of additional molecules targeting novel molecular mechanisms is a primary effort in today’s antiepileptic drug research. This paper reviews the pharmacological development of retigabine, an antiepileptic drug with a novel mechanism of action, namely the activation of voltage-gated potassium channels of the Kv7 subfamily. These channels, which act as widespread regulators of intrinsic neuronal excitability and of neurotransmitter-induced network excitability changes, are currently viewed among the most promising targets for anticonvulsant pharmacotherapy. In particular, the present work reviews the pathophysiological role of Kv7 channels in neuronal function, the molecular mechanisms involved in the Kv7 channel-opening action of retigabine, the activity of retigabine in preclinical in vitro and in vivo studies predictive of anticonvulsant activities, and the clinical status of development for this drug as an add-on treatment for pharmacoresistant epilepsy. Particular efforts are devoted to highlighting the potential advantages and disadvantages of retigabine when compared with currently available compounds, in order to provide a comprehensive assessment of its role in therapy for treatment-resistant epilepsies.

Activation of pre-synaptic M-type K+ channels inhibits [ 3H]d-aspartate release by reducing Ca2+ entry through P/Q-type voltage-gated Ca2+channels

In this study, the functional consequences of the pharmacological modulation of the M‐current (IKM) on cytoplasmic Ca2+ intracellular Ca2+concentration ([Ca2+]i) changes and excitatory neurotransmitter release triggered by various stimuli from isolated rat cortical synaptosomes have been investigated. Kv7.2 immunoreactivity was identified in pre‐synaptic elements in cortical slices and isolated glutamatergic cortical synaptosomes. In cerebrocortical synaptosomes exposed to 20u2003mM [K+]e, the IKM activator retigabine (RT, 10u2003μM) inhibited [3H]d‐aspartate ([3H]d‐Asp) release and caused membrane hyperpolarization; both these effects were prevented by the IKM blocker XE‐991 (20u2003μM). The IKM activators RT (0.1–30u2003μM), flupirtine (10u2003μM) and BMS‐204352 (10u2003μM) inhibited 20u2003mM [K+]e‐induced synaptosomal [Ca2+]i increases; XE‐991 (20u2003μM) abolished RT‐induced inhibition of depolarization‐triggered [Ca2+]i transients. The P/Q‐type voltage‐sensitive Ca2+channel (VSCC) blocker ω‐agatoxin IVA prevented RT‐induced inhibition of depolarization‐induced [Ca2+]i increase and [3H]d‐Asp release, whereas the N‐type blocker ω‐conotoxin GVIA failed to do so. Finally, 10u2003μM RT did not modify the increase of [Ca2+]i and the resulting enhancement of [3H]d‐Asp release induced by [Ca2+]i mobilization from intracellular stores, or by store‐operated Ca2+channel activation. Collectively, the present data reveal that the pharmacological activation of IKM regulates depolarization‐induced [3H]d‐Asp release from cerebrocortical synaptosomes by selectively controlling the changes of [Ca2+]i occurring through P/Q‐type VSCCs.