Inhibition Of Calcium Currents Research Articles

Inhibition of high-voltage-activated calcium currents by acute hypoxia in cultured retinal ganglion cells.

Hypoxia is a common factor of numerous ocular diseases that lead to dysfunctions and loss of retinal ganglion cells (RGCs) with subsequent vision loss. High-voltage-activated calcium channels are the main source of calcium entry into neurons. Their activity plays a central role in different signaling processes in health and diseases, such as enzyme activation, gene transcription, synaptic transmission, or the onset of cell death. This study aims to establish and evaluate the initial effect of the early stage of acute hypoxia on somatic HVA calcium currents in cultured RGCs. HVA calcium currents were recorded in RGCs using the whole-cell patch-clamp technique in the voltage-clamp mode. The fast local superfusion was used for a brief (up to 270 s) application of the hypoxic solution (pO2 < 5 mmHg). The switch from normoxic to hypoxic solutions and vice versa was less than 1 s. The HVA calcium channel activity was inhibited by acute hypoxia in 79% of RGCs (30 of 38 RGCs) in a strong voltage-dependent manner. The level of inhibition was independent of the duration of hypoxia or repeated applications. The hypoxia-induced inhibition of calcium currents had a strong correlation with the duration of hypoxia and showed the transition from reversible to irreversible at 75 s of hypoxia and longer. The results obtained are the first demonstration of the phenomena of HVA calcium current inhibition by acute hypoxia in RGCs and provide a conceptual framework for further research.

O1-conotoxin Tx6.7 cloned from the genomic DNA of Conus textile that inhibits calcium currents.

Conotoxins exhibit great potential as neuropharmacology tools and therapeutic candidates due to their high affinity and specificity for ion channels, neurotransmitter receptors or transporters. The traditional methods to discover new conotoxins are peptide purification from the crude venom or gene amplification from the venom duct. In this study, a novel O1 superfamily conotoxin Tx6.7 was directly cloned from the genomic DNA of Conus textile using primers corresponding to the conserved intronic sequence and 3' UTR elements. The mature peptide of Tx6.7 (DCHERWDWCPASLLGVIYCCEGLICFIAFCI) was synthesized by solid-phase chemical synthesis and confirmed by mass spectrometry. Patch clamp experiments on rat DRG neurons showed that Tx6.7 inhibited peak calcium currents by 59.29 ± 2.34% and peak potassium currents by 22.33 ± 7.81%. In addition, patch clamp on the ion channel subtypes showed that 10 μM Tx6.7 inhibited 56.61 ± 3.20% of the hCaV1.2 currents, 24.67 ± 0.91% of the hCaV2.2 currents and 7.30 ± 3.38% of the hNaV1.8 currents. Tx6.7 had no significant toxicity to ND7/23 cells and increased the pain threshold from 0.5 to 4 hours in the mouse hot plate assay. Our results suggested that direct cloning of conotoxin sequences from the genomic DNA of cone snails would be an alternative approach to obtaining novel conotoxins. Tx6.7 could be used as a probe tool for ion channel research or a therapeutic candidate for novel drug development.

Synthesis and evaluation of a novel analgesic conotoxin Lt7b that inhibits calcium currents and increases sodium currents

Conotoxins are promising neuropharmacological tools and drug candidates due to their high efficiency and specificity in targeting ion channels or neurotransmitter receptors. In this study, a novel O2‐superfamily conotoxin, Lt7b, was synthesized and its pharmacological functions were evaluated. Lt7b with three modified amino acids and three disulfide bonds was successfully synthesized. CD spectra showed that Lt7b had a typical α‐helix in the secondary structure. Patch clamp experiments on rat DRG neurons showed that Lt7b could significantly inhibit calcium currents with an IC50 value of 856 ± 95 nM. Meanwhile, 10 μM Lt7b could significantly increase the sodium currents by 77 ± 8%, but it had no obvious effects on the potassium currents in DRG neurons. In addition, patch clamp experiments on ion channel subtypes showed that 10 μM Lt7b could inhibit 7.0 ± 1.2%, 8.0 ± 1.5%, 4.6 ± 3.4%, and 9.5 ± 0.1% of the hCav1.2, hCav2.1, hCav2.2, and hCav3.2 currents, respectively, while it did not increase the rNav1.7, rNav1.8, hNav1.5, hNav1.7, and hNav1.8 currents. Lt7b had no obvious toxicity to HaCaT and ND7/23 cells up to 1 mM and significantly increased the pain threshold at the testing time of 0.5–4 h in a dose‐dependent manner in the mouse hotplate assay. This novel conotoxin Lt7b may be a useful tool for ion channel studies and analgesic drug development.

New Phenylglycinamide Derivatives with Hybrid Structure as Candidates for New Broad-Spectrum Anticonvulsants.

In the present study, a focused combinatorial chemistry approach was applied to merge structural fragments of well-known TRPV1 antagonists with a potent anticonvulsant lead compound, KA-104, that was previously discovered by our group. Consequently, a series of 22 original compounds has been designed, synthesized, and characterized in the in vivo and in vitro assays. The obtained compounds showed robust in vivo antiseizure activity in the maximal electroshock (MES) test and in the 6 Hz seizure model (using both 32 and 44 mA current intensities). The most potent compounds 53 and 60 displayed the following pharmacological profile: ED50 = 89.7 mg/kg (MES), ED50 = 29.9 mg/kg (6 Hz, 32 mA), ED50 = 68.0 mg/kg (6 Hz, 44 mA), and ED50 = 73.6 mg/kg (MES), ED50 = 24.6 mg/kg (6 Hz, 32 mA), and ED50 = 56.3 mg/kg (6 Hz, 44 mA), respectively. Additionally, 53 and 60 were effective in the ivPTZ seizure threshold and had no influence on the grip strength and body temperature in mice. The in vitro binding and functional assays indicated a multimodal mechanism of action for 53 and 60. These molecules, beyond TRPV1 antagonism, inhibited calcium currents and fast sodium currents in patch-clamp assays. Further studies proved beneficial in vitro ADME-Tox properties for 53 and 60 (i.e., high metabolic stability, weak influence on CYPs, no neurotoxicity, etc.). Overall, 53 and 60 seem to be interesting candidates for future preclinical development in epilepsy and pain indications due to their interaction with the TRPV1 channel.

Characteristics of Bisphenol Cardiotoxicity: Impaired Excitability, Contractility, and Relaxation.

Bisphenol a (BPA) is a high production volume chemical that is frequently used to manufacture epoxy resins and polycarbonate plastics. BPA-containing products are now pervasive, and as a result, biomonitoring studies report widespread exposure in > 90% of adults, adolescents, and children. Both epidemiological and experimental studies have reported associations between BPA exposure and adverse cardiovascular health outcomes. With increasing concerns regarding BPA exposure, a few structurally similar bisphenol chemicals have been introduced as replacements, including bisphenol s (BPS) and bisphenol f (BPF). In accordance with the recently established "Key characteristics of cardiovascular toxicants", we reviewed the literature to highlight the immediate effects of bisphenol chemicals on (1) cardiac excitability, and (2) contractility and relaxation. BPA inhibits key cardiac ion channels, impairs cardiac excitability, and acts as a more potent inhibitor as compared to BPF and BPS. Through the inhibition of calcium current, some studies report that bisphenol chemicals can act as negative inotropic agents. Yet, others suggest that low dose exposures may increase contractility and precipitate triggered arrhythmias via the phosphorylation of key calcium handling proteins. Accordingly, we propose additional considerations for future work to comprehensively address the cardiac safety profile of BPA, as compared to replacement chemicals.

Presynaptic mechanisms underlying GABAB-receptor-mediated inhibition of spontaneous neurotransmitter release.

SUMMARYInhibition of neurotransmitter release by neurotransmitter substances constitutes a fundamental means of neuromodulation. In contrast to well-delineated mechanisms that underlie inhibition of evoked release via suppression of voltage-gated Ca2+ channels, processes that underlie neuromodulatory inhibition of spontaneous release remain unclear. Here, we interrogated inhibition of spontaneous glutamate and GABA release by presynaptic metabotropic GABAB receptors. Our findings show that this inhibition relies on Gβγ subunit action at the membrane, and it is largely independent of presynaptic Ca2+ signaling for both forms of release. In the case of spontaneous glutamate release, inhibition requires Gβγ interaction with the C terminus of the key fusion machinery component SNAP25, and it is modulated by synaptotagmin-1. Inhibition of spontaneous GABA release, on the other hand, is independent of these pathways and likely requires alternative Gβγ targets at the presynaptic terminal.

Analysis of Resibufogenin on Cardiac conduction reveals a species difference in the cardiac electrophysiology: Rats versus guinea pigs.

Resibufogenin (RBG) is a chemical ingredient of Chan Su. In our research, we found RBG affected cardiac rhythm in a negative chronotropic way in vivo. The cardiac Mapping system ex vivo and the patch clamp in vitro were used to explore how RBG influenced the cardiac electrophysiological properties. The negative chronotropic action of RBG at 100 μM might be attribute to prolongation in the atrioventricular conduction time and reduction in the ventricular conduction velocity. Using whole-cell patch clamp in ventricular myocytes of adult rats, we found that RBG prolonged the action potential duration (APD) in APD20, APD50, and APD90 at 100 μM and inhibited calcium currents (ICa), total outward potassium currents (IK), and transient outward potassium current (Ito) in a concentration-dependent manner, but not on the inward rectifying potassium current (IK1). Notably, RBG had a potent proarrhythmic action ex vivo in the isolated perfused guinea pig hearts at 10 μM, but not in rats. To avoid the potential cardiotoxicity derived from the distributional differences of ion channels among species, the effect of RGB on IKr in hERG-HEK293 cells was detected. The IC50 of RGB on IKr was more than 100 μM. In summary, all these results indicated that the negative chronotropic action of RBG relied on the blocking activities on multiple ion channels, and the species-difference of proarrhythmic effects might result from lack of the Ito on the myocardial membrane of guinea pigs. Anyhow, the cardiotoxicity observed in guinea pigs required further detailed studies to mitigate the potential risks in the clinical application of Chan Su.

Long-QT founder variant T309I-Kv7.1 with dominant negative pattern may predispose delayed afterdepolarizations under \u03b2-adrenergic stimulation

The variant c.926C > T (p.T309I) in KCNQ1 gene was identified in 10 putatively unrelated Czech families with long QT syndrome (LQTS). Mutation carriers (24 heterozygous individuals) were more symptomatic compared to their non-affected relatives (17 individuals). The carriers showed a mild LQTS phenotype including a longer QTc interval at rest (466 ± 24 ms vs. 418 ± 20 ms) and after exercise (508 ± 32 ms vs. 417 ± 24 ms), 4 syncopes and 2 aborted cardiac arrests. The same haplotype associated with the c.926C > T variant was identified in all probands. Using the whole cell patch clamp technique and confocal microscopy, a complete loss of channel function was revealed in the homozygous setting, caused by an impaired channel trafficking. Dominant negativity with preserved reactivity to β-adrenergic stimulation was apparent in the heterozygous setting. In simulations on a human ventricular cell model, the dysfunction resulted in delayed afterdepolarizations (DADs) and premature action potentials under β-adrenergic stimulation that could be prevented by a slight inhibition of calcium current. We conclude that the KCNQ1 variant c.926C > T is the first identified LQTS-related founder mutation in Central Europe. The dominant negative channel dysfunction may lead to DADs under β-adrenergic stimulation. Inhibition of calcium current could be possible therapeutic strategy in LQTS1 patients refractory to β-blocker therapy.

The Search for New Anticonvulsants in a Group of (2,5-Dioxopyrrolidin-1-yl)(phenyl)Acetamides with Hybrid Structure-Synthesis and In Vivo/In Vitro Studies.

Epilepsy belongs to the most common and debilitating neurological disorders with multifactorial pathophysiology and a high level of drug resistance. Therefore, with the aim of searching for new, more effective, and/or safer therapeutics, we discovered a focused series of original hybrid pyrrolidine-2,5-dione derivatives with potent anticonvulsant properties. We applied an optimized coupling reaction yielding several hybrid compounds that showed broad-spectrum activity in widely accepted animal seizure models, namely, the maximal electroshock (MES) test and the psychomotor 6 Hz (32 mA) seizure model in mice. The most potent anticonvulsant activity and favorable safety profile was demonstrated for compound 30 (median effective dose (ED50) MES = 45.6 mg/kg, ED50 6 Hz (32 mA) = 39.5 mg/kg, median toxic dose (TD50) (rotarod test) = 162.4 mg/kg). Anticonvulsant drugs often show activity in pain models, and compound 30 was also proven effective in the formalin test of tonic pain, the capsaicin-induced pain model, and the oxaliplatin (OXPT)-induced neuropathic pain model in mice. Our studies showed that the most plausible mechanism of action of 30 involves inhibition of calcium currents mediated by Cav1.2 (L-type) channels. Importantly, 30 revealed high metabolic stability on human liver microsomes, negligible hepatotoxicity, and relatively weak inhibition of CYP3A4, CYP2D6, and CYP2C9 isoforms of cytochrome P450, compared to reference compounds. The promising in vivo activity profile and drug-like properties of compound 30 make it an interesting candidate for further preclinical development.

Role of LVA Ca channels in norepinephrine-induced automatic activity in rat pulmonary vein myocardial sleeve

Ectopic foci in pulmonary veins (PV) are involved in the onset of atrial fibrillation. Norepinephrine (NE) induces in rat PV cardiomyocytes (CM) an automatic activity in saw-tooth pattern (catecholaminergic automatic activity or CAA), consisting of bursts of slow action potentials which depends on Ca (upstroke) and Na (inter burst) channels. We have previously shown the co-existence in rat PVCM, of a fast LVA (FLVA) non selective TTX-sensitive cationic channel and of T-type Ca channels. To evaluate in male Wistar rat PV myocardial sleeve the contribution of these channels to triggering NE-induced ectopic foci. Whole-cell currents were studied at room temperature in enzymatically isolated PVCM. Mechanical activity of PV rings was recorded in an organ bath at 37 °C and CAA was induced by application of 10 μM NE. The FLVA Ca current was increased by the Cav3.x blockers TTA-A2 (10-100 nM, n = 11) and NiCl2 (40 μM, n = 9) whereas a large LVA Ca current, present in 10 out of 80 PVCM, was partially reduced by 100 nM TTA-A2. Study of K currents revealed the presence of an inward rectifying current sensitive to TRAM-34 (1 μM, n = 6), a blocker of the Ca-activated K channel SK4. In PV rings, TTA-A2 (1-5 μM) decreased CAA incidence. Total arrhythmia duration, measured over the last 10 min of a 30 min long drug application, was reduced as well as burst duration and the number of spontaneous twitches within a burst. On the other hand, 40 μM NiCl2 or 1-2 μM TRAM-34 increased total arrhythmia duration by decreasing the interval between bursts (no significant effect on the burst characteristics). Only NiCl2 had a significant negative inotropic effect (∼80% inhibition of L-type calcium current). Our results suggest that Ca influx through Cav3.1 channels contribute to triggering bursts of automatic activity and that Cav3.2 Ca channels, through activation of SK4 channels, are involved in the regulation of the interval between bursts.

Nociceptin/orphanin FQ peptide receptor mediates inhibition of N-type calcium currents in vestibular afferent neurons of the rat.

The vestibular system is modulated by various neuromodulators including opioid peptides. The current study was conducted to determine whether activation of nociceptin/orphanin FQ peptide (NOP) receptors modulates voltage-gated calcium currents and action potential discharge of rat vestibular afferent neurons. We performed whole cell patch-clamp recordings on cultured vestibular afferent neurons from P7-P10 Long-Evans rats. Application of nociceptin/orphanin FQ (N/OFQ), a 17-amino acid neuropeptide that is the endogenous ligand for NOP receptor, inhibits the high-voltage activated (HVA) component of the calcium current in a concentration-dependent manner with a half inhibitory concentration of 26 nM. Said inhibitory action on the calcium current is voltage-dependent, which was made clear by the fact that it was reverted in 80% by a depolarizing prepulse. Furthermore, the effect of N/OFQ was blocked by application of the specific NOP-antagonist UFP101, by preincubation with G-protein blocker pertussis toxin, and by coapplication of the specific N-type calcium-current blocker ω-conotoxin-MVIIA. N/OFQ application causes an increase in the duration and maximum rate of repolarization of action potentials. It also decreases repetitive discharge and discharge elicited by sinusoidal stimulation. These results show that in vestibular afferents, NOP receptor activation inhibits N-type calcium current by activating G proteins, mostly through the Gβγ subunit. This suggests that NOP activation produces a presynaptic modulation of primary vestibular afferent neurons' output into the vestibular nuclei, thus taking part in the integration and gain setting of vestibular information in second-order vestibular nucleus neurons.NEW & NOTEWORTHY Our results show that in primary vestibular afferent neurons, activation of the nociceptin/orphanin FQ peptide receptor inhibits the N-type calcium current by a mechanism mediated by G proteins. We propose that calcium current inhibition modulates neurotransmitter release from vestibular afferents, producing a presynaptic modulation of vestibular input to vestibular nuclei, thus contributing to gain control in the vestibular afferent input.

Metabolic insights from a GHSR-A203E mutant mouse model

ObjectiveBinding of ghrelin to its receptor, growth hormone secretagogue receptor (GHSR), stimulates GH release, induces eating, and increases blood glucose. These processes may also be influenced by constitutive (ghrelin-independent) GHSR activity, as suggested by findings in short people with naturally occurring GHSR-A204E mutations and reduced food intake and blood glucose in rodents administered GHSR inverse agonists, both of which impair constitutive GHSR activity. In this study, we aimed to more fully determine the physiologic relevance of constitutive GHSR activity. MethodsWe generated mice with a GHSR mutation that replaces alanine at position 203 with glutamate (GHSR-A203E), which corresponds to the previously described human GHSR-A204E mutation, and used them to conduct ex vivo neuronal electrophysiology and in vivo metabolic assessments. We also measured signaling within COS-7 and HEK293T cells transfected with wild-type GHSR (GHSR-WT) or GHSR-A203E constructs. ResultsIn COS-7 cells, GHSR-A203E resulted in lower baseline IP3 accumulation than GHSR-WT; ghrelin-induced IP3 accumulation was observed in both constructs. In HEK293T cells co-transfected with voltage-gated CaV2.2 calcium channel complex, GHSR-A203E had no effect on basal CaV2.2 current density while GHSR-WT did; both GHSR-A203E and GHSR-WT inhibited CaV2.2 current in the presence of ghrelin. In cultured hypothalamic neurons from GHSR-A203E and GHSR-deficient mice, native calcium currents were greater than those in neurons from wild-type mice; ghrelin inhibited calcium currents in cultured hypothalamic neurons from both GHSR-A203E and wild-type mice. In brain slices, resting membrane potentials of arcuate NPY neurons from GHSR-A203E mice were hyperpolarized compared to those from wild-type mice; the same percentage of arcuate NPY neurons from GHSR-A203E and wild-type mice depolarized upon ghrelin exposure. The GHSR-A203E mutation did not significantly affect body weight, body length, or femur length in the first ∼6 months of life, yet these parameters were lower in GHSR-A203E mice after 1 year of age. During a 7-d 60% caloric restriction regimen, GHSR-A203E mice lacked the usual marked rise in plasma GH and demonstrated an exaggerated drop in blood glucose. Administered ghrelin also exhibited reduced orexigenic and GH secretagogue efficacies in GHSR-A203E mice. ConclusionsOur data suggest that the A203E mutation ablates constitutive GHSR activity and that constitutive GHSR activity contributes to the native depolarizing conductance of GHSR-expressing arcuate NPY neurons. Although the A203E mutation does not block ghrelin-evoked signaling as assessed using in vitro and ex vivo models, GHSR-A203E mice lack the usual acute food intake response to administered ghrelin in vivo. The GHSR-A203E mutation also blunts GH release, and in aged mice leads to reduced body length and femur length, which are consistent with the short stature of human carriers of the GHSR-A204E mutation.

Thymol provokes burst of action potentials in neurons of snail Caucasotachea atrolabiata

Thymol, a phenolic monoterpene, is well known for its antimicrobial, antifungal and antioxidant properties. In spite of wide use in oral care products, pharmaceutical and cosmetic preparation and in food industry, the effects of thymol on the neuronal activity and intrinsic properties have not been well studied. We studied the effects of thymol on the spontaneous activity and action potential properties of central neurons of snail Caucasotachea atrolabiata. Thymol (1 mM) altered action potentials characteristics and provoked epileptiform burst firing in snail neurons, which were partially reversible after washout. Before burst firing, action potentials had lower amplitude and maximum rising slope, while the threshold voltage was raised. These results suggest the inhibition of ion channels underlying action potential initiation and upstroke. The maximum falling slope and afterhyperpolarization were also considerably reduced, suggesting the inhibition of potassium channels. Thymol (0.5 mM) that was not able to induce burst firing in snail neurons, synergistically acted with potassium channel blocker, tetraethyl ammonium, to induce burst firing, which also supports the importance of potassium channel inhibition, especially delayed rectifier potassium channels, to the thymol-induced alteration of firing pattern. The thymol-induced burst firing seems to be dependent on both sodium and calcium currents. Our findings provide evidences for the ability of thymol in altering the firing mode of central neurons of snail, which apparently involves the inhibition of calcium and potassium currents. These results further support the interaction of thymol with ion channels and emphasize on the vulnerability of nervous system to this compound.

Excitation of murine cardiac myocytes by nanosecond pulsed electric field.

Opening of voltage-gated sodium channels takes tens to hundreds of microseconds, and mechanisms of their opening by nanosecond pulsed electric field (nsPEF) stimuli remain elusive. This study was aimed at uncovering the mechanisms of how nsPEF elicits action potentials (APs) in cardiomyocytes. Fluorescent imaging of optical APs (FluoVolt) and Ca2+ -transients (Fluo-4) was performed in enzymatically isolated murine ventricular cardiomyocytes stimulated by 200-nanosecond trapezoidal pulses. nsPEF stimulation evoked tetrodotoxin-sensitive APs accompanied or preceded by slow sustained depolarization (SSD) and, in most cells, by transient afterdepolarization waves. SSD threshold was lower than the AP threshold (1.26 ± 0.03 vs 1.34 ± 0.03 kV/cm, respectively, P < 0.001). Inhibition of l-type calcium and sodium-calcium exchanger currents reduced the SSD amplitude and increased the AP threshold ( P < 0.05). The threshold for Ca 2+ -transients (1.40 ± 0.04 kV/cm) was not significantly affected by a tetrodotoxin-verapamil cocktail, suggesting the activation of a Ca 2+ entry pathway independent from the opening of Na + or Ca 2+ voltage-gated channels. Removal of external Ca 2+ decreased the SSD amplitude ( P = 0.004) and blocked Ca 2+ -transients but not APs. The incidence of transient afterdepolarization waves was decreased by verapamil and by removal of external Ca 2+ ( P = 0.002). The study established that nsPEF stimulation caused calcium entry into cardiac myocytes (including routes other than voltage-gated calcium channels) and SSD. Tetrodotoxin-sensitive APs were mediated by SSD, whose amplitude depended on the calcium entry. Plasma membrane electroporation was the most likely primary mechanism of SSD with additional contribution from l-type calcium and sodium-calcium exchanger currents.

Effect of heptanol and ethanol on excitation wave propagation in a neonatal rat ventricular myocyte monolayer

In this work, the action of heptanol and ethanol was investigated in a two-dimensional (2D) model of cardiac tissue: the neonatal rat ventricular myocyte monolayer. Heptanol is known in electrophysiology as a gap junction uncoupler but may also inhibit voltage-gated ionic channels. Ethanol is often associated with the occurrence of arrhythmias. These substances influence sodium, calcium, and potassium channels, but the complete mechanism of action of heptanol and ethanol remains unknown. The optical mapping method was used to measure conduction velocities (CVs) in concentrations of 0.05–1.8 mM heptanol and 17–1342 mM ethanol. Heptanol was shown to slow the excitation wave significantly, and a mechanism that involves a simultaneous action on cell coupling and activation threshold was suggested. Whole-cell patch-clamp experiments showed inhibition of sodium and calcium currents at a concentration of 0.5 mM heptanol. Computer modeling was used to estimate the relative contribution of the cell uncoupling and activation threshold increase caused by heptanol. Unlike heptanol, ethanol slightly influenced the CV at clinically relevant concentrations. Additionally, the critical concentrations for re-entry formation in ethanol were determined.

(206) Modulation of multiple modalities of somatosensory information by peripheral kappa opioid receptors

Peripherally-restricted kappa opioid receptor (KOR) agonists are emerging as a novel treatment for pain and itch conditions and have shown efficacy in several recent clinical trials. One primary site of action is thought to be at KORs on primary afferent nerves that innervate the body. Yet, the subtypes of primary afferent somatosensory neurons that express the kappa opioid receptor remain undefined. Using a newly developed KOR-cre knockin allele, viral tracing, and single-cell RT-PCR we discovered that that KOR is expressed in a specific subset of peptidergic afferents. This subset targeted multiple tissue types and expressed higher levels of transcripts known to be involved in neurogenic inflammation. Consistent with this, peripherally-restricted KOR agonists inhibited behavioral responses to chemical pain and itch stimuli, but not acute heat stimuli, and also decreased mechanical hypersensitivity following an incision injury. Unexpectedly, we also found that KOR is expressed in subsets of primary afferents that form lanceolate or circumferential endings around hair follicles, suggesting an unappreciated role for KOR signaling in the modulation of low-threshold mechanosensation. At a functional level, genetically-labeled afferents showed inhibition of voltage-gated calcium current in response to kappa agonists, and optogenetic experiments revealed that dynorphin inhibited evoked-EPSCs from the central terminals of KOR-expressing afferents. These experiments provide key insight for the rationale use of peripherally-restricted KOR agonists for the modulation of inflammatory pain, itch, and potentially mechanical allodynia.

Isoflurane, but Not the Nonimmobilizers F6 and F8, Inhibits Rat Spinal Cord Motor Neuron CaV1 Calcium Currents.

Volatile anesthetics decrease Ca²⁺ entry through voltage-dependent Ca²⁺ channels. Ca influences neurotransmitter release and neuronal excitability. Because volatile anesthetics act specifically on the spinal cord to produce immobility, we examined the effect of isoflurane and the nonimmobilizers F6 (1, 2-dichlorohexafluorocyclobutane) and F8 (2, 3-dichlorooctafluorobutane) on CaV1 and CaV2 Ca²⁺ channels in spinal cord motor neurons and dorsal root ganglion neurons. Using patch clamping, we compared the effects of isoflurane with those of F6 and F8 on CaV1 and CaV2 channels in isolated, cultured adult rat spinal cord motor neurons and on CaV1 and CaV2 channels in adult rat dorsal root ganglion sensory neurons. In spinal cord motor neurons, isoflurane, but not F6 or F8, inhibited currents through CaV1 channels. Isoflurane and at least one of the nonimmobilizers inhibited currents through CaV1 and CaV2 channels in dorsal root ganglion neurons and CaV2 in spinal cord motor neurons. The findings that isoflurane, but not nonimmobilizers, inhibited CaV1 Ca²⁺ channels in spinal cord motor neurons are consistent with the notion that spinal cord motor neurons might mediate isoflurane-induced immobility. Additional studies are required to examine whether inhibition of CaV1 calcium currents in spinal cord motor neurons is sufficient or whether actions on other channels/proteins contribute to isoflurane-induced immobility.

The antiallodynic action of pregabalin in neuropathic pain is independent from the opioid system.

BackgroundClinical management of neuropathic pain, which is pain arising as a consequence of a lesion or a disease affecting the somatosensory system, partly relies on the use of anticonvulsant drugs such as gabapentinoids. Therapeutic action of gabapentinoids such as gabapentin and pregabalin, which act by the inhibition of calcium currents through interaction with the α2δ-1 subunit of voltage-dependent calcium channels, is well documented. However, some aspects of the downstream mechanisms are still to be uncovered. Using behavioral, genetic, and pharmacological approaches, we tested whether opioid receptors are necessary for the antiallodynic action of acute and/or long-term pregabalin treatment in the specific context of neuropathic pain.ResultsUsing the cuff model of neuropathic pain in mice, we show that acute pregabalin administration at high dose has a transitory antiallodynic action, while prolonged oral pregabalin treatment leads to sustained antiallodynic action, consistent with clinical observations. We show that pregabalin remains fully effective in μ-opioid receptor, in δ-opioid receptor and in κ-opioid receptor deficient mice, either female or male, and its antiallodynic action is not affected by acute naloxone. Our work also shows that long-term pregabalin treatment suppresses tumor necrosis factor-α overproduction induced by sciatic nerve constriction in the lumbar dorsal root ganglia.ConclusionsWe demonstrate that neither acute nor long-term antiallodynic effect of pregabalin in a context of neuropathic pain is mediated by the endogenous opioid system, which differs from opioid treatment of pain and antidepressant treatment of neuropathic pain. Our data are also supportive of an impact of gabapentinoid treatment on the neuroimmune aspect of neuropathic pain.

Epinephrine reversed high-concentration bupivacaine-induced inhibition of calcium channels and transient outward potassium current channels, but not on sodium channel in ventricular myocytes of rats.

BackgroundEpinephrine is a first-line drug for cardiopulmonary resuscitation, but its efficacy in the treatment of bupivacaine-induced cardiac toxicity is still in question. We hypothesized that epinephrine can reverse cardiac inhibition of bupivacaine by modulating ion flows through the ventricular myocyte membrane channels of rats. The aim of this study was to observe and report the effects of epinephrine on high-concentration bupivacaine-induced inhibition of sodium (INa), L-type calcium (ICa-L), and transient outward potassium (Ito) currents in the ventricular myocytes of rats.MethodsThe ventricular myocytes were isolated from Sprague-Dawley rats (250-300 g) by acute enzymatic dissociation. The whole-cell patch clamp technique was used to record the ion channel currents in single ventricular myocytes both before and after administration of medications.ResultAdministration of bupivacaine 100 μmol/L significantly reduced INa, (P < 0.05). However, administration of bupivacaine 100 μmol/L in conjunction with epinephrine 0.15 μg/ml had no effect in restoring INa to its previous state. Similarly, a sharp decline of ICa-L and Ito was observed after administration of bupivacaine 100 μmol/L (P < 0.05). In contrast to INa, ICa-L and Ito were significantly improved after the administration of the aforementioned combination of bupivacaine and epinephrine (P < 0.05).ConclusionEpinephrine can reverse high-concentration bupivacaine induced inhibition of ICa-L and Ito, but not INa. Thus, epinephrine’s effectiveness in reversal of bupivacaine-induced cardiac toxicity secondary to sodium channel inhibition may be limited.

Biphasic modulation by mGlu5 receptors of TRPV1-mediated intracellular calcium elevation in sensory neurons contributes to heat sensitivity.

Elevation of glutamate, an excitatory amino acid, during inflammation and injury plays a crucial role in the reception and transmission of sensory information via ionotropic and metabotropic receptors. This study aimed to investigate the mechanisms underlying the biphasic effects of metabotropic glutamate mGlu5 receptor activation on responses to noxious heat. We assessed the effects of intraplantar quisqualate, a non-selective glutamate receptor agonist, on heat and mechanical pain behaviours in mice. In addition, the effects of quisqualate on the intracellular calcium response and on membrane currents mediated by TRPV1 channels, were examined in cultured dorsal root ganglion neurons from mice. Activation of mGlu5 receptors in hind paw transiently increased, then decreased, the response to noxious heat. In sensory neurons, activation of mGlu5 receptors potentiated TRPV1-mediated intracellular calcium elevation, while terminating activation of mGlu5 receptors depressed it. TRPV1-induced currents were potentiated by activation of mGlu5 receptors under voltage clamp conditions and these disappeared after washout. However, voltage-gated calcium currents were inhibited by the mGlu5 receptor agonist, even after washout. These results suggest that, in sensory neurons, mGlu5 receptors biphasically modulate TRPV1-mediated intracellular calcium response via transient potentiation of TRPV1 channel-induced currents and persistent inhibition of voltage-gated calcium currents, contributing to heat hyper- and hypoalgesia.