Ana Gomis

Hypoosmotic- and pressure-induced membrane stretch activate TRPC5 channels

Transient receptor potential (TRP) channels mediate a wide array of sensory functions. We investigated the role of TRPC5, a poorly characterized channel widely expressed in the central and peripheral nervous system, as a potential osmosensory protein. Here we show that hypoosmotic stimulation activates TRPC5 channels resulting in a large calcium influx. The response to osmotically induced membrane stretch is blocked by GsMTx‐4, an inhibitor of stretch activated ion channels. Direct hypoosmotic activation of TRPC5 is independent of phospholipase C function. However, the osmotic response is inhibited in a cell line in which PIP2 levels are reduced by regulated overexpression of a lipid phosphatase. The response was restored by increasing intracellular PIP2 levels through the patch pipette. The mechano‐sensitivity of the channel was probed in the whole‐cell configuration by application of steps of positive pressure through the patch pipette. Pressure‐induced membrane stretch also activated TRPC5 channels, suggesting its role as a transducer of osmo‐mechanical stimuli. We also demonstrated the expression of TRPC5 in sensory neurones which together with the osmo‐mechanical characteristics of TRPC5 channels suggest its putative role in mechanosensory transduction events.

Identification of molecular determinants of channel gating in the transient receptor potential box of vanilloid receptor I

Transient receptor potential vanilloid receptor subtype I (TRPV1) is an ion channel gated by physical and chemical stimuli that belongs to the TRPV protein family. TRPV receptors contain a highly conserved, 6‐mer segment near the channel gate, known as the TRP box, whose function remains unknown. Here, we performed an alanine scanning mutagenesis of the TRP box of TRPV1 (IWKLQR) and found that mutation of this motif affected channel gating by raising the free energy of channel activation. Functional characterization of TRPV1 mutants showed that substitution of I696, W697, and R701 by alanine severely affected voltage‐ and heat‐dependent activation and notably reduced the capsaicin responsiveness and tachyphylaxia, while mutation of K698, L699, and Q700 had minor effects. In addition, mutation of I696 to alanine promoted a strong outward rectification at negative membrane potentials, and slowed the kinetics of channel activation. Taken together, our findings suggest that modification of I696, W697, and R701 to alanine altered channel function by affecting events downstream of the initial stimuli‐sensing step and imply that inter‐subunit interactions within the TRP box play an important role in TRPV1 gating.—Valente, P., García‐Sanz, N., Gomis, A., Fernández‐Carvajal, A., Fernández‐Ballester, G., Viana, F., Belmonte, C., and Ferrer‐Montiel, A. Identification of molecular determinants of channel gating in the transient receptor potential box of vanilloid receptor I. FASEB J. 22, 3298–3309 (2008)

Monoacylglycerols Activate TRPV1 – A Link between Phospholipase C and TRPV1

Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H1 receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H1 receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous “entourage” compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.

Intra-articular injections of hyaluronan solutions of different elastoviscosity reduce nociceptive nerve activity in a model of osteoarthritic knee joint of the guinea pig

OBJECTIVE To study in guinea pigs knee joints the effects of intra-articular injection of HYADD 4-G (Fidia-Farmaceutici), a novel hyaluronan (HA)-derived elastoviscous material and of Hyalgan (Fidia-Farmaceutici), a HA product with very low viscoelasticity, on movement-evoked nociceptor impulse activity from normal and inflamed knee joints. DESIGN Nociceptor impulse activity was recorded from single Adelta and C fibers of the medial articular nerve either under control conditions or after induction of an experimental knee joint osteoarthritis (OA) by partial medial menisectomy and transection of the anterior cruciate ligament (PMM-TACL). The stimuli consisted of standardized innocuous and noxious inward and outward rotations of the tibia against the femur of 50s duration, repeated every 5min for 1.5h. RESULTS The number of movement-evoked impulses was significantly augmented 1 day and 1 week after PMM-TACL compared with intact knee joint. The enhanced impulse response to joint movements 1 week following surgery was attenuated by repeated intra-articular injection of HYADD 4-G and even more prominently by Hyalgan. CONCLUSIONS HA products have a reducing action on joint nociceptor discharges that appears to depend predominantly on their role as an elastoviscous filter associated with their rheological properties, but also on a chemical effect on sensitized nociceptive terminals of inflamed joint tissues, possibly linked to the HA concentration.

Nociceptive nerve activity in an experimental model of knee joint osteoarthritis of the guinea pig: Effect of intra-articular hyaluronan application

Abstract Nociceptive impulse activity was recorded extracellularly from single Aδ and C primary afferents of the guinea pig’s medial articular nerve after induction of an experimental osteoarthritis in the knee joint by partial medial menisectomy and transection of the anterior cruciate ligament (PMM + TACL). Also, the analgesic effects of intra‐articular hyaluronan solutions were evaluated. Healthy, PMM + TACL operated, sham‐operated (opening of the joint capsule without PMM and TACL surgery) and acutely inflamed (intra‐articular kaolin–carrageenan, K–C) animals were used. The stimulus protocol consisted of torque meter‐controlled, standardized innocuous and noxious inward and outward rotations of the joint. This stimulus protocol of 50 s duration was repeated every 5 min for 70 min. One day, one week and three weeks after PMM + TACL, the movement‐evoked discharges of Aδ articular afferents were increased significantly over values found in sham‐operated animals. The discharges of C fibers were significantly augmented only one week after PMM + TACL surgery. Filling of the joint cavity with a high viscosity hyaluronan solution (hylan G‐F 20, Synvisc®) immediately and three days after surgery reduced significantly the enhanced nerve activity observed in joint afferent fibers one day and one week after surgery. Augmentation of movement‐evoked discharges in K–C acutely inflamed knee joints was similar to that observed one week after PMM + TACL. Our results indicate that in the PMM + TACL model of osteoarthritis in guinea pigs, enhancement of nociceptive responses to joint movement was primarily associated to post‐surgical inflammation. Intra‐articular injection of an elastoviscous hyaluronan solution reduced the augmented nerve activity.

GAP43 stimulates inositol trisphosphate-mediated calcium release in response to hypotonicity

The identification of osmo/mechanosensory proteins in mammalian sensory neurons is still elusive. We have used an expression cloning approach to screen a human dorsal root ganglion cDNA library to look for proteins that respond to hypotonicity by raising the intracellular Ca2+ concentration ([Ca2+]i). We report the unexpected identification of GAP43 (also known as neuromodulin or B50), a membrane‐anchored neuronal protein implicated in axonal growth and synaptic plasticity, as an osmosensory protein that augments [Ca2+]i in response to hypotonicity. Palmitoylation of GAP43 plays an important role in the protein osmosensitivity. Depletion of intracellular stores or inhibition of phospholipase C (PLC) activity abrogates hypotonicity‐evoked, GAP43‐mediated [Ca2+]i elevations. Notably, hypotonicity promoted the selective association of GAP43 with the PLC‐δ1 isoform, and a concomitant increase in inositol‐1,4,5‐trisphosphate (IP3) formation. Collectively, these findings indicate that hypo‐osmotic activation of GAP43 induces Ca2+ release from IP3‐sensitive intracellular stores. The osmosensitivity of GAP43 furnishes a mechanistic framework that links axon elongation with phospho inositide metabolism, spontaneous triggering of cytosolic Ca2+ transients and the regulation of actin dynamics and motility at the growth cone in response to temporal and local mechanical forces.

Oscillatory patterns of electrical activity in mouse pancreatic islets of Langerhans recorded in vivo

Pancreatic β-cells secrete insulin as a function of blood glucose concentration. One of the key steps in stimulus-secretion coupling is the depolarisation of the membrane and the appearance of bursts of calcium action potentials. Recently, the characteristics and glucose dependence of the oscillations in electrical activity in vivo have been described. The experiments described here were designed to determine the temporal evolution of such electrical activity when no experimental changes in the glycaemia are imposed. The absolute duration of the active and silent phases has been analysed and compared with the values obtained in vitro. We have found that in vivo, at glycaemia ranging from 6.0 to 7.5 mM, the electrical activity of the islets of Langerhans is permanently oscillatory, the mean duration of the depolarisation phase being 28 s. In general, the oscillatory pattern remains very constant for relatively long (up to 60 min) periods of time. In some experiments, slow or transitory changes in the degree of β-cell activation could be observed, as well as the existence, in a very few cases, of oscillatory non-periodic patterns.

Ruthenium red inhibits selectively chromaffin cell calcium channels

The effect of Ruthenium red (RR) on ionic currents and catecholamine secretion was studied in chromaffin cells. This polycation inhibited 59 mM potassium-stimulated 45Ca2+ uptake in a concentration-dependent manner (IC50 = 5 +/- 0.2 microM). This effect was more evident at extracellular calcium concentrations over 1 mM and was not abolished by neuraminidase pretreatment. RR also inhibited potassium-stimulated catecholamine secretion (IC50 = 6 +/- 0.9 microM). These results were corroborated by patch-clamp in whole-cell recordings. RR inhibited chromaffin cell calcium currents (IC50 = 7 microM) without affecting significantly either sodium or potassium currents. Radioligand binding studies in adrenomedullary plasma membranes showed that RR inhibited [125I]omega-conotoxin GVIA binding but it had no effect on specific binding of [3H]nitrendipine. The effect of the RR on calcium currents was additive with the inhibitory effect observed with 10 microM nitrendipine. The residual dihydropyridine-resistant calcium current was inhibited with a potency similar to that determined under control conditions in the absence of nitrendipine. These results demonstrate that RR selectively inhibits calcium channels; however, this polycation was not selective for a particular calcium channel subtype.

Membrane-tethered peptides patterned after the TRP domain (TRPducins) selectively inhibit TRPV1 channel activity

The transient receptor potential vanilloid 1 (TRPV1) channel is a thermosensory receptor implicated in diverse physiological and pathological processes. The TRP domain, a highly conserved region in the C terminus adjacent to the internal channel gate, is critical for subunit tetramerization and channel gating. Here, we show that cell‐penetrating, membrane‐anchored peptides patterned after this protein domain are moderate and selective TRPV1 antagonists both in vitro and in vivo, blocking receptor activity in intact rat primary sensory neurons and their peripheral axons with mean decline time of 30 min. The most potent lipopeptide, TRP‐p5, blocked all modes of TRPV1 gating with micromolar efficacy (IC50<10 µM), without significantly affecting other thermoTRP channels. In contrast, its retrosequence or the corresponding sequences of other TRPV channels did not alter TRPV1 channel activity (IC50»100 µM). TRP‐p5 did not affect the capsaicin sensitivity of the vanilloid receptor. Our data suggest that TRP‐p5 interferes with protein‐protein interactions at the level of the TRP domain that are essential for the “conformational” change that leads to gate opening. Therefore, these palmitoylated peptides, which we termed TRPducins, are noncompetitive, voltage‐independent, sequence‐specific TRPV1 blockers. Our findings indicate that TRPducin‐like peptides may embody a novel molecular strategy that can be exploited to generate a selective pharmacological arsenal for the TRP superfamily of ion channels.—Valente, P., Fernández‐Carvajal, A., Camprubí‐Robles, M., Gomis, A., Quirce, S., Viana, F., Fernández‐Ballester, G., González‐Ros, J. M., Belmonte, C., Planells‐Cases, R., Ferrer‐Montiel, A. Membrane‐tethered peptides patterned after the TRP domain (TRPducins) selectively inhibit TRPV1 channel activity. FASEB J. 25, 1628–1640 (2011). www.fasebj.org

MECHANOPROTECTIVE ACTIONS OF ELASTOVISCOUS HYLANS ON ARTICULAR PAIN RECEPTORS

ABSTRACT The sensitivity of articular nociceptors can be influenced directly by drugs either applied systemically or locally, and this is a very promising way to exert analgesic actions. For instance, Pozo et al. reported in 19971 that an elastoviscous solution of hylan, a hyaluronan derivative, significantly reduced both the ongoing activity and the movement evoked responses of primary afferent units from inflamed knee joints of the cat. These results support the assumption that hylans injected into the knee joint cavity may act as an elastoviscous filter, reducing the transmission of stretch to the nociceptive terminals where the mechano-electrical transduction takes place. More recently we have extended these observation to the knee joint of the rat. So far, with no appreciable exception, the results from nociceptors with fine afferent fibers (A δ- and C-fibers) of the medial articular nerve of the rat correspond to those obtained in the cat. The mechanoprotective effects found in the knee joint of rat are in the same order of magnitude as those obtained by systemic application of peripherally acting analgesic drugs. This duplication allows a further generalization of the results obtained in the cat. In addition it has opened the possibility to test in a much more controlled and systematic manner the effects of hylans and other substances with different elastic and viscous properties (alone or in connection with various analgesic drugs) on the discharge characteristics of nociceptors innervating normal and acutely inflamed knee joints