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Background and Purpose: Flupirtine is a non-opioid analgesic that has been in clinical use for more than 20 years. It is characterized as a selective neuronal potassium channel opener (SNEPCO). Nevertheless, its mechanisms of action remain controversial and are the purpose of this study.Experimental Approach: Effects of flupirtine on native and recombinant voltage- and ligand-gated ion channels were explored in patch-clamp experiments using the following experimental systems: recombinant K(IR)3 and K(V)7 channels and α3β4 nicotinic acetylcholine receptors expressed in tsA 201 cells; native voltage-gated Na(+), Ca(2+), inward rectifier K(+), K(V)7 K(+), and TRPV1 channels, as well as GABA(A), glycine, and ionotropic glutamate receptors expressed in rat dorsal root ganglion, dorsal horn and hippocampal neurons.Key Results: Therapeutic flupirtine concentrations (≤10 µM) did not affect voltage-gated Na(+) or Ca(2+) channels, inward rectifier K(+) channels, nicotinic acetylcholine receptors, glycine or ionotropic glutamate receptors. Flupirtine shifted the gating of K(V)7 K(+) channels to more negative potentials and the gating of GABA(A) receptors to lower GABA concentrations. These latter effects were more pronounced in dorsal root ganglion and dorsal horn neurons than in hippocampal neurons. In dorsal root ganglion and dorsal horn neurons, the facilitatory effect of therapeutic flupirtine concentrations on K(V)7 channels and GABA(A) receptors was comparable, whereas in hippocampal neurons the effects on K(V)7 channels were more pronounced.Conclusions and Implications: These results indicate that flupirtine exerts its analgesic action by acting on both GABA(A) receptors and K(V)7 channels. [ABSTRACT FROM AUTHOR]

Background and Purpose: The analgesic action of paracetamol involves K V 7 channels, and its metabolite N-acetyl-p-benzo quinone imine (NAPQI), a cysteine modifying reagent, was shown to increase currents through such channels in nociceptors. Modification of cysteine residues by N-ethylmaleimide, H 2 O 2 , or nitric oxide has been found to modulate currents through K V 7 channels. The study aims to identify whether, and if so which, cysteine residues in neuronal K V 7 channels might be responsible for the effects of NAPQI., Experimental Approach: To address this question, we used a combination of perforated patch-clamp recordings, site-directed mutagenesis, and mass spectrometry applied to recombinant K V 7.1 to K V 7.5 channels., Key Results: Currents through the cardiac subtype K V 7.1 were reduced by NAPQI. Currents through all other subtypes were increased, either by an isolated shift of the channel voltage dependence to more negative values (K V 7.3) or by such a shift combined with increased maximal current levels (K V 7.2, K V 7.4, K V 7.5). A stretch of three cysteine residues in the S2-S3 linker region of K V 7.2 was necessary and sufficient to mediate these effects., Conclusion and Implication: The paracetamol metabolite N-acetyl-p-benzo quinone imine (NAPQI) modifies cysteine residues of K V 7 subunits and reinforces channel gating in homomeric and heteromeric K V 7.2 to K V 7.5, but not in K V 7.1 channels. In K V 7.2, a triple cysteine motif located within the S2-S3 linker region mediates this reinforcement that can be expected to reduce the excitability of nociceptors and to mediate antinociceptive actions of paracetamol., (© 2024 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: The Kv 7 channel activator flupirtine is a clinical analgesic characterized as 'selective neuronal potassium channel opener'. Flupirtine was found to exert comparable actions at GABAA receptors and Kv 7 channels in neurons of pain pathways, but not in hippocampus., Experimental Approach: Expression patterns of GABAA receptors were explored in immunoblots of rat dorsal root ganglia, dorsal horns and hippocampi using antibodies for 10 different subunits. Effects of flupirtine on recombinant and native GABAA receptors were investigated in patch clamp experiments and compared with the actions on Kv 7 channels., Key Results: Immunoblots pointed towards α2, α3, β3 and γ2 subunits as targets, but in all γ2-containing receptors the effects of flupirtine were alike: leftward shift of GABA concentration-response curves and diminished maximal amplitudes. After replacement of γ2S by δ, flupirtine increased maximal amplitudes. Currents through α1β2δ receptors were more enhanced than those through Kv 7 channels. In hippocampal neurons, flupirtine prolonged inhibitory postsynaptic currents, left miniature inhibitory postsynaptic currents (mIPSCs) unaltered and increased bicuculline-sensitive tonic currents; penicillin abolished mIPSCs, but not tonic currents; concentration-response curves for GABA-induced currents were shifted to the left by flupirtine without changes in maximal amplitudes; in the presence of penicillin, maximal amplitudes were increased; GABA-induced currents in the presence of penicillin were more sensitive towards flupirtine than K(+) currents. In dorsal horn neurons, currents evoked by the δ-preferring agonist THIP (gaboxadol) were more sensitive towards flupirtine than K(+) currents., Conclusions and Implications: Flupirtine prefers δ-containing GABAA receptors over γ-containing ones and over Kv 7 channels., (© 2015 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: Amphetamines bind to the plasmalemmal transporters for the monoamines dopamine (DAT), noradrenaline (NET) and 5-HT (SERT); influx of amphetamine leads to efflux of substrates. Various models have been proposed to account for this amphetamine-induced reverse transport in mechanistic terms. A most notable example is the molecular stent hypothesis, which posits a special amphetamine-induced conformation that is not likely in alternative access models of transport. The current study was designed to evaluate the explanatory power of these models and the molecular stent hypothesis., Experimental Approach: Xenopus laevis oocytes and HEK293 cells expressing human (h) SERT were voltage-clamped and exposed to 5-HT, p-chloroamphetamine (pCA) or methylenedioxyamphetamine (MDMA)., Key Results: In contrast to the currents induced by 5-HT, pCA-triggered currents through SERT decayed slowly in Xenopus laevis oocytes once the agonist was removed (consistent with the molecular stent hypothesis). However, when SERT was expressed in HEK293 cells, currents induced by 3 or 100 μM pCA decayed 10 or 100 times faster, respectively, after pCA removal., Conclusions and Implications: This discrepancy in decay rates is inconsistent with the molecular stent hypothesis. In contrast, a multistate version of the alternative access model accounts for all the observations and reproduces the kinetic parameters extracted from the electrophysiological recordings. A crucial feature that explains the action of amphetamines is their lipophilic nature, which allows for rapid diffusion through the membrane., (© 2013 The Authors. British Journal of Pharmacology published by John Wiley &. Sons Ltd on behalf of The British Pharmacological Society.)

Background and Purpose: P2Y(1) , P2Y(2) , P2Y(4) , P2Y(12) and P2Y(13) receptors for nucleotides have been reported to mediate presynaptic inhibition, but unequivocal evidence for facilitatory presynaptic P2Y receptors is not available. The search for such receptors was the purpose of this study., Experimental Approach: In primary cultures of rat superior cervical ganglion neurons and in PC12 cell cultures, currents were recorded via the perforated patch clamp technique, and the release of [(3) H]-noradrenaline was determined., Key Results: ADP, 2-methylthio-ATP and ATP enhanced stimulation-evoked (3) H overflow from superior cervical ganglion neurons, treated with pertussis toxin to prevent the signalling of inhibitory G proteins. This effect was abolished by P2Y(1) antagonists and by inhibition of phospholipase C, but not by inhibition of protein kinase C or depletion of intracellular Ca(2+) stores. ADP and a specific P2Y(1) agonist caused inhibition of Kv7 channels, and this was prevented by a respective antagonist. In neurons not treated with pertussis toxin, (3) H overflow was also enhanced by a specific P2Y(1) agonist and by ADP, but only when the P2Y(12) receptors were blocked. ADP also enhanced K(+) -evoked (3) H overflow from PC12 cells treated with pertussis toxin, but only in a clone expressing recombinant P2Y(1) receptors., Conclusions and Implications: These results demonstrate that presynaptic P2Y(1) receptors mediate facilitation of transmitter release from sympathetic neurons most likely through inhibition of Kv7 channels., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

1. In PC12 cells, adenine nucleotides inhibit voltage-activated Ca(2+) currents and adenylyl cyclase activity, and the latter effect was reported to involve P2Y(12) receptors. To investigate whether these two effects are mediated by one P2Y receptor subtype, we used the antithrombotic agents 2-methylthio-AMP (2-MeSAMP) and N(6)-(2-methyl-thioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene-ATP (AR-C69931MX). 2. ADP reduced A(2A) receptor-dependent cyclic AMP synthesis with half maximal effects at 0.1-0.17 micro M. In the presence of 30 micro M 2-MeSAMP or 100 nM AR-C69931MX, concentration response curves were shifted to the right by factors of 39 and 30, indicative of pA(2) values of 6.1 and 8.5, respectively. 3. The inhibition of Ca(2+) currents by ADP was attenuated by 10-1000 nM AR-C69931MX and by 3-300 micro M 2-MeSAMP. ADP reinhibited Ca(2+) currents after removal of 2-MeSAMP within less than 15 s, but required 2 min to do so after removal of AR-C69931MX. 4. ADP inhibited Ca(2+) currents with half maximal effects at 5-20 micro M. AR-C69931MX (10-100 nM) displaced concentration response curves to the right, and the resulting Schild plot showed a slope of 1.09 and an estimated pK(B) value of 8.7. Similarly, 10-100 micro M 2-MeSAMP also caused rightward shifts resulting in a Schild plot with a slope of 0.95 and an estimated pK(B) of 5.4. 5. The inhibition of Ca(2+) currents by 2-methylthio-ADP and ADPbetaS was also antagonized by AR-C69931MX, which (at 30 nM) caused a rightward shift of the concentration response curve for ADPbetaS by a factor of 3.8, indicative of a pA(2) value of 8.1. 6. These results show that antithrombotic drugs antagonize the inhibition of neuronal Ca(2+) channels by adenine nucleotides, which suggests that this effect is mediated by P2Y(12) receptors.

P2Y receptors inhibiting adenylyl cyclase have been found in blood platelets, glioma cells, and endothelial cells. In platelets and glioma cells, these receptors were identified as P2Y(12). Here, we have used PC12 cells to search for adenylyl cyclase inhibiting P2Y receptors in a neuronal cellular environment. ADP and ATP (0.1 - 100 microM) left basal cyclic AMP accumulation unaltered, but reduced cyclic AMP synthesis stimulated by activation of endogenous A(2A) or recombinant beta(2) receptors. Forskolin-dependent cyclic AMP production was reduced by ADPbetaS (71 nM)>ATP (164 nM)=ADP (244 nM). The inhibition by ADP was not antagonized by suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid, or adenosine-3'-phosphate-5'-phosphate, but attenuated by reactive blue 2, ATP(alpha)S, and 2-methylthio-AMP. RT - PCR demonstrated the expression of P2Y(2), P2Y(4), P2Y(6), and P2Y(12), but not P2Y(1), receptors in PC12 cells. In Northern blots, only P2Y(2) and P2Y(12) were detectable. Differentiation with NGF did not alter these hybridization signals and left the nucleotide inhibition of adenylyl cyclase unchanged. We conclude that P2Y(12) receptors are expressed in neuronal cells and inhibit adenylyl cyclase activity.

The effect of acetyl - tyrosyl-valyl-alanyl-aspartyl - chloromethylketone (ac-YVAD-cmk), an irreversible caspase-1 (IL-1beta converting enzyme, ICE) inhibitor on mortality, leukocyte and platelet counts and cytokine levels was investigated in a double-blind rat model of endotoxaemia. Intravenous (i.v.) bolus administration of lipopolysaccharide (LPS) (25-75 mg kg(-1), n=12 per group) to anaesthetized rats induced a dose dependent increase in mortality over 8 h (LD(50)=48 mg kg(-1)). During this period, animals became leukopenic and thrombocytopenic. Serum levels of IL-beta, IL-6, and TNF-alpha were highly elevated. Pretreatment of rats with ac-YVAD-cmk at a dose of 12.5 micromol kg(-1) significantly reduced mortality from 83 to 33% using Log Rank analysis. However, ac-YVAD-cmk did not modify blood cell counts or cytokine profiles as compared with the LPS-drug vehicle group. These data lay credence to the potential importance of caspase-1-inhibition in modifying the inflammatory response to endotoxin. Further investigations are warranted in understanding the relationship between caspase-1 inhibition, cytokine production and animal survival in different experimental paradigms of sepsis.

1. UTP and UDP depolarize rat superior cervical ganglion neurons and trigger noradrenaline release from these cells. The present study investigated the mechanisms underlying this excitatory action of uridine nucleotides by measuring whole-cell voltage-dependent K+ and Ca2+ currents. 2. Steady-state outward (holding) currents measured in the amphotericin B perforated-patch configuration at a potential of -30 mV were reduced by 10 microM UTP in a reversible manner, but steady-state inward (holding) currents at -70 mV were not affected. This action of UTP was shared by the muscarinic agonist oxotremorine-M. In current-voltage curves between -20 and -100 mV, UTP diminished primarily the outwardly rectifying current components arising at potentials positive to -60 mV. 3. Slow relaxations of muscarinic K+ currents (IM) evoked by hyperpolarizations from -30 to -55 mV were also reduced by 10 microM UTP (37% inhibition) and oxotremorine-M (81% inhibition). In contrast, transient K+-currents, delayed rectifier currents, fast and slow Ca2+-dependent K+ currents, as well as voltage-dependent Ca2+ currents were not altered by UTP. 4. In conventional (open-tip) whole-cell recordings, replacement of GTP in the pipette by GDPbetaS abolished the UTP-induced inhibition of IM, whereas replacement by GTPgammaS rendered it irreversible. 5. The UTP-induced reduction of IM was half maximal at 1.5 microM with a maximum of 37% inhibition; UDP was equipotent and equieffective, while ADP was less potent (half maximal inhibition at 29 microM). ATP had no effect at < or = 30 microM. 6. The inhibition of IM induced by 10 microM UTP was antagonized by pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) at > or = 30 microM and by reactive blue 2 at > or = 10 microM, but not by suramin at concentrations up to 30 microM. 7. These results show that rat superior cervical ganglion neurons possess uridine nucleotide preferring P2Y receptors which inhibit KM channels. This effect presumably forms the basis of the excitatory action of uridine nucleotides in rat sympathetic neurons.

1. The role of bradykinin receptors in the regulation of sympathetic transmitter release was investigated in primary cultures of neurones dissociated from superior cervical ganglia of neonatal rats. These cultures were loaded with [3H]-noradrenaline and the outflow of radioactivity was determined under continuous superfusion. 2. Bradykinin (100 nmol l[-1] applied for 10 min) caused a transient increase in tritium outflow that reached a peak within four minutes after the beginning of the application and then declined towards the baseline, despite the continuing presence of the peptide. ATP (100 micromol l[-1]) and nicotine (10 micromol l[-1]) caused elevations in 3H outflow with similar kinetics, whereas outflow remained elevated during a 10 min period of electrical field stimulation (0.5 ms, 50 mA, 50 V cm[-1], 1.0 Hz). 3. When bradykinin was applied for periods of 2 min, the evoked 3H overflow was half-maximal at 12 nmol l(-1) and reached a maximum of 2.3% of cellular radioactivity. The preferential B1 receptor agonist des-Arg9-bradykinin failed to alter 3H outflow. The B2 receptor antagonists, [D-Phe7]-bradykinin (1 micromol l[-1]) and Hoe 140 (10 nmol l[-1]), per se did not alter 3H outflow, but shifted the concentration-response curve for bradykinin-evoked 3H overflow to the right by a factor of 7.9 and 4.3, respectively. 4. Bradykinin-induced overflow was abolished in the absence of extracellular Ca2+ and in the presence of either 1 micromol l(-1) tetrodotoxin or 300 micromol l(-1) Cd2+, as was electrically-induced overflow. Activation of alpha2-adrenoceptors by 1 micromol l(-1) UK 14,304 reduced both bradykinin- and electrically-triggered overflow. The Ca2+-ATPase inhibitor thapsigargin (0.3 micromol l[-1]) failed to alter either type of stimulated overflow. Caffeine (10 mmol l[-1]) enhanced bradykinin-induced overflow, but reduced overflow triggered by electrical field stimulation. 5. Inclusion of Ba2+ (0.1 to 1 mmol l[-1]) in the superfusion medium enhanced electrically induced overflow by approximately 100% and potentiated bradykinin-triggered overflow by almost 400%. Application of 1 mmol l(-1) Ba2+ for periods of 2 min triggered 3H overflow, and this overflow was abolished by 1 micromol l(-1) tetrodotoxin and enhanced by 10 mmol l(-1) caffeine. In contrast, inclusion of tetraethylammonium (0.1 to 1 mmol l[-1]) in the superfusion buffer caused similar increases of bradykinin- and electrically evoked 3H overflow (by about 100%), and tetraethylammonium, when applied for 2 min, failed to alter 3H outflow. 6. Treatment of cultures with 100 ng ml(-1) pertussis toxin caused a significant increase in bradykinin-, but not in electrically-, evoked tritium overflow. Treatment with 100 ng ml(-1) cholera toxin reduced both types of stimulated 3H overflow. 7. These data reveal bradykinin as a potent stimulant of action potential-mediated and Ca2+-dependent transmitter release from rat sympathetic neurones in primary cell culture. This neurosecretory effect of bradykinin involves activation of B2-receptors, presumably linked to pertussis- and cholera toxin-insensitive G proteins, most likely members of the Gq family. Results obtained with inhibitors of muscarinic K+ (KM) channels, like caffeine and Ba2+, indicate that the secretagogue action of bradykinin probably involves inhibition of these K+ channels.

In rat cultured sympathetic neurones, UDP, UTP and ATP at micromolar concentrations triggered Ca(2+)-dependent and tetrodotoxin-sensitive [3H]-noradrenaline release. The overflow evoked by UTP or ATP was similar at 100 mumol l-1, the concentration used in all subsequent experiments. Pre-exposure of the neurones to 100 mumol l-1 UTP significantly reduced ensuing secretory effects of UTP but not of ATP. Conversely, pre-exposure to ATP diminished the overflow due to ATP but not that due to UTP. In the presence of 10 mumol l-1 pyridoxal-5'-phosphate or 30 mumol l-1 suramin, the secretory response to ATP was reduced, but the effect of UTP was unaltered. Zn2+ (10 mumol l-1) reduced the overflow triggered by UTP, but increased the overflow due to ATP. These results indicate the presence of separate receptors for pyrimidine nucleotides and for purine nucleotides which both trigger transmitter release.

1. The release of previously incorporated [3H]-noradrenaline was investigated in cultures of dissociated chick or rat sympathetic neurones and in cerebrocortical slices from neonatal or adult rats. Noradrenaline, in the presence of 10 mumol l-1 of the uptake inhibitor, cocaine, or the selective alpha 2-adrenoceptor agonist, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK 4,304), was applied for different periods of time in order to detect a possible time-dependence of the alpha 2-adrenoceptor-mediated inhibition of electrically evoked tritium outflow. 2. In chick sympathetic neurones, stimulation-evoked overflow was reduced to 30%, 42%, or 56% of control when noradrenaline (1 mumol l-1) was present for 2, 8, or 16 min, respectively. Likewise, UK 14,304 (1 mumol l-1) present for these periods of time reduced 3H overflow to 35%, 51%, and 53% of control, respectively. Addition of 1 nmol l-1 to 10 mumol l-1 UK 14,304 for either 2 or 16 min did not produce significantly different IC50 values, but the inhibitory effects were smaller with 16 min as compared to 2 min exposure at concentrations > or = 10 nmol l-1. 3. In rat sympathetic neurones, noradrenaline (100 nmol l-1) reduced stimulation-evoked overflow to 33%, 56%, or 57% of control, when present for 2, 8, or 16 min, respectively. Addition of UK 14,304 (1 mumol l-1) for these periods of time caused inhibition to 11%, 41%, and 46% of control. Applying UK14,304 for either 2 or 16 min did not result in significantly different IC5o values, but the inhibition induced by 16 min as compared to 2 min exposure was smaller at concentrations > 10 nmol 1-1.4. In cerebrocortical slices from either neonatal or adult rats, exposure to 0.1 to 1.0 micromol 1-1 UK14,304 for 16 min never caused a smaller inhibition than a corresponding 3 min exposure, although various experimental conditions were investigated.5 The results demonstrate that alpha 2-adrenoceptors which regulate noradrenaline release from sympathetic neurones undergo agonist-induced desensitization within minutes. Such rapid desensitization of alpha 2-autoreceptors was not detected in brain slice preparations.