Search

Background: Despite increasing evidence for the presence of voltage-gated Na+ channels (Nav) isoforms and measurements of Nav channel currents with the patch-clamp technique in arterial myocytes, no information is available to date as to whether or not Nav channels play a functional role in arteries. The aim of the present work was to look for a physiological role of Nav channels in the control of rat aortic contraction. Methodology/Principal Findings: Nav channels were detected in the aortic media by Western blot analysis and double immunofluorescence labeling for Nav channels and smooth muscle α-actin using specific antibodies. In parallel, using real time RT-PCR, we identified three Nav transcripts: Nav1.2, Nav1.3, and Nav1.5. Only the Nav1.2 isoform was found in the intact media and in freshly isolated myocytes excluding contamination by other cell types. Using the specific Nav channel agonist veratridine and antagonist tetrodotoxin (TTX), we unmasked a contribution of these channels in the response to the depolarizing agent KCl on rat aortic isometric tension recorded from endothelium-denuded aortic rings. Experimental conditions excluded a contribution of Nav channels from the perivascular sympathetic nerve terminals. Addition of low concentrations of KCl (2-10 mM), which induced moderate membrane depolarization (e.g., from -55.9±1.4 mV to -45.9±1.2 mV at 10 mmol/L as measured with microelectrodes), triggered a contraction potentiated by veratridine (100 μM) and blocked by TTX (1 μM). KB-R7943, an inhibitor of the reverse mode of the Na+/Ca2+ exchanger, mimicked the effect of TTX and had no additive effect in presence of TTX. Conclusions/Significance: These results define a new role for Nav channels in arterial physiology, and suggest that the TTXsensitive Nav1.2 isoform, together with the Na+/Ca2+ exchanger, contributes to the contractile response of aortic myocytes at physiological range of membrane depolarization. [ABSTRACT FROM AUTHOR]

Abstract: Changes in intracellular Ca2+ concentration ([Ca2+]i) control the setting up of the neuro-muscular synapse in vitro and probably in vivo. Dissociated cultures of purified embryonic (E15) rat motoneurons were used to explore the molecular mechanisms by which endoplasmic reticulum Ca2+ stores, via both ryanodine-sensitive and IP3-sensitive intracellular Ca2+ channels control [Ca2+]i homeostasis in these neurons during ontogenesis. Fura-2 microspectrofluorimetry monitorings in single neurons showed that caffeine-induced responses of [Ca2+]i increased progressively from days 1–7 in culture. These responses were blocked by ryanodine and nicardipine but not by ω-conotoxin-GVIA or ω-conotoxin-MVIIC suggesting a close functional relationship between ryanodine-sensitive and L-type Cav1 Ca2+ channels. Moreover, after 6 days in vitro, neurons exhibited spontaneous or caffeine-induced Ca2+ oscillations that were attenuated by nicardipine. In 1-day-old neurons, both thapsigargin or CPA, which deplete Ca2+ stores from the endoplasmic reticulum, induced an increase in [Ca2+]i in 75% of the neurons tested. The number of responding motoneurons declined to 25% at 5–6 days in vitro. Xestospongin-C, a membrane-permeable IP3 receptor inhibitor blocked the CPA-induced [Ca2+]i response in all stages. RT-PCR studies investigating the expression pattern of RYR and IP3 Ca2+ channels isoforms confirmed the presence of their different isoforms and provided evidence for a specific pattern of development for RYR channels during the first week in vitro. Taken together, present results show that the control of motoneuronal [Ca2+]i homeostasis is developmentally regulated and suggest the presence of an intracellular ryanodine-sensitive Ca2+ channel responsible for a Ca2+-induced Ca2+ release in embryonic motoneurons following voltage-dependent Ca2+ entry via L-type Ca2+ channels. [Copyright &y& Elsevier]

Ca2+ influx through voltage-gated calcium channels probably influences neuronal ontogenesis. Many developing neurones transiently express T-type/Cav3 calcium channels that contribute to their electrical activity and potentially to their morphological differentiation. Here we have characterized the electrophysiological properties and the functional role of a large T-type calcium current that is present in mouse developing primary vestibular neurones at embryonic day E17. This T-type current showed fast activation and inactivation, as well as slow deactivation kinetics. The overlap of activation and inactivation parameters produced a window current between −65 and −45 mV. Recovery from short-term inactivation was slow suggesting the presence of the Cav3.2 subunit. This T-type current was blocked by micromolar concentrations of Ni2+ and was inhibited by fast perfusion velocities in a similar fashion to recombinant Cav3.2 T-type channels expressed in HEK-293 cells. More importantly, current clamp experiments have revealed that the T-current could elicit afterdepolarization potentials during the repolarization phase of action potentials, and occasionally generate calcium spikes. Taken together, we demonstrate that the Cav3.2 subunit is likely to be the main T-type calcium channel subunit expressed in embryonic vestibular neurones and should play a key role in the excitability of these neurones during the ontogenesis of vestibular afferentation. [ABSTRACT FROM AUTHOR]

The mammalian utricular sensory receptors are commonly believed to be non-spiking cells with electrical activity limited to graded membrane potential changes. Here we provide evidence that during the first post-natal week, the sensory hair cells of the rat utricle express a tetrodotoxin (TTX)-sensitive voltage-gated Na[sup+] current that displays most of the biophysical and pharmacological characteristics of neuronal Na[sup+] current. Single-cell RT-PCR reveals that several α-subunit isoforms of the Na[sup+] channels are co-expressed within a single hair cell, with a major expression of Nav1.2 and Nav1.6 subunits. In neonatal hair cells, 30% of the Na[sup+] channels are available for activation at the resting potential. Depolarizing current injections in the range of the transduction currents are able to trigger TTX-sensitive action potentials. We also provide evidence of a TTX-sensitive activity-dependent brain-derived neurotrophic factor (BDNF) release by early post-natal utricle explants. Developmental analysis shows that Na[sup+] currents decrease dramatically from post-natal day 0 (PO) to P8 and become almost undetectable at P21. Concomitantly, depolarizing stimuli fail to induce both action potential and BDNF release at P20. The present findings reveal that vestibular hair cells express neuronal-like TTX-sensitive Na[sup+] channels able to generate Nat-driven action potentials only during the early post-natal period of development. During the same period an activity-dependent BDNF secretion by utricular explants has been demonstrated. This could be an important mechanism involved in vestibular sensory system differentiation and synaptogenesis. [ABSTRACT FROM AUTHOR]

Peripheral neuropathic pain (PNP) is a debilitating and intractable chronic disease, for which sensitization of somatosensory neurons present in dorsal root ganglia that project to the dorsal spinal cord is a key physiopathological process. Here, we show that hematopoietic cells present at the nerve injury site express the cytokine FL, the ligand of fms-like tyrosine kinase 3 receptor (FLT3). FLT3 activation by intra-sciatic nerve injection of FL is sufficient to produce pain hypersensitivity, activate PNP-associated gene expression and generate short-term and long-term sensitization of sensory neurons. Nerve injury-induced PNP symptoms and associated-molecular changes were strongly altered in Flt3-deficient mice or reversed after neuronal FLT3 downregulation in wild-type mice. A first-in-class FLT3 negative allosteric modulator, discovered by structure-based in silico screening, strongly reduced nerve injury-induced sensory hypersensitivity, but had no effect on nociception in non-injured animals. Collectively, our data suggest a new and specific therapeutic approach for PNP.