Excitation of rat sympathetic neurons via M1 muscarinic receptors independently of Kv7 channels

Die langsame Komponente der synaptischen Übertragung in sympathischen Ganglien wird durch Aktivierung von muskarinischen M1 Rezeptoren und die dadurch vermittelte Hemmung von Kv7 Kanälen bestimmt. Allerdings liegt der Schwellenwert für eine Aktivierung von Kv7 Kanälen bei einem positiveren Potential als -60 mV und das tatsächliche Ruhemembranpotential eher zwis- chen -64 bis -67 mV. Außerdem war die ausgelöste Depolarisation durch den muskarinischen Agonisten Oxotremorin M (OxoM) in den untersuchten Zellen äußerst variabel im Gegensatz zur gleichförmigen OxoM- mediierten Hemmung von Kv7 Kanälen. Die Veränderung von intra- und extrazellulärem Chlorid und die daraus resultierenden Änderun- gen im Zellverhalten lieferten einen ersten Hinweis auf den Einfluss eines Chloridkanals. Die unspezifischen Chloridkanal-Blocker Niflumsäure und 2-[2-(4-Acetamido-2-Sulfophenyl)ethenyl]- 4-Isothiocyanatobenzen-1-Sulfonsäure (SITS) reduzierten sowohl OxoM-induzierte Depolarisa- tionen als auch Noradrenalinfreisetzung. Auch hier wurde kein Effekt auf Kv7 Kanäle und die durch OxoM induzierte Hemmung gefunden. Bei einem Haltepotential von -65 mV, löste die Applikation von OxoM einen langsam größer werdenden Einwärtsstrom aus. Dieser war durch selektive Inhibitoren von Anoctamin-1 (TMEM16A) und Anoctamin-2 (TMEM16B), welche ve- rantwortlich für einen Ca2+-aktivierten Cl--Strom sind, zu hemmen. Ebenso wurde die durch OxoM ausgelöste Noradrenalinfreisezung durch diese beiden Antagonisten reduziert. Aktivierung von M1 Rezeptoren führt zur Bildung von Diacylglycerol (DAG), welches wiederum Proteinkinase C (PKC) aktiviert. Im Gegensatz zur Hemmung der Kv7 Kanäle durch OxoM, waren sowohl die von OxoM ausgelösten Depolarisationen, als auch die Noradrenalinfreisetzung durch Hemmung der Proteinkinase C (PKC) reduziert. Zusammenfassend zeigt diese Studie, dass Ca2+-aktivierte Cl- Kanäle über einen PKC-abhängigen Mechanismus zur langsamen cholinergen Übertragung beitragen.Slow ganglionic transmission relies on activation of muscarinic M1 receptors and the subsequent inhibition of Kv7 channels. However, the activation threshold for Kv7 channels lies at more pos- itive potentials than the actually measured membrane voltage. Hence, inhibiting a non-active channel is unlikely to underlie M1 receptor mediated slow EPSPs. Additionally, the muscarinic agonist oxotremorine M (OxoM) triggered depolarizations in sympathetic neurons of the rat, but not in all investigated neurons. On the other hand, OxoM mediated Kv7 channel inhibition in all tested neurons. Changing intra- and extracellular chloride concentrations revealed an influence of chloride on both in OxoM-triggered depolarizations as well as noradrenaline release. Two non-selective chloride channel inhibitors niflumic acid and 2-[2-(4-acetamido-2-sulfophenyl)ethenyl]-4-isothiocyanato- benzene-1-sulfonic acid (SITS) reduced depolarizations and noradrenalin release evoked by OxoM, but left OxoM-mediated Kv7 channel inhibition unaltered. A slow rising inward current at -65 mV induced by OxoM was inhibited by selective antagonists of TMEM16A (anoctamin-1) and TMEM16B (anoctamin-2), who is responsible for Ca2+-activated Cl-currents. In addition, these selective inhibitors strongly reduced OxoM-evoked noradrenalin release from neuronal cultures of rat sympathetic ganglia. Activation of M1 receptors triggers formation of diacylglycerol, which activates protein kinase C (PKC). By using different kinds of PKC inhibitors, a dependence of OxoM-induced depolariza- tions and noradrenaline release from rat sympathetic neurons on classical PKCs was revealed. However, neither M-currents nor their inhibition via M1 receptors was influenced by PKC inhi- bition. Taken together, this study shows that slow cholinergic transmission is dependent on activation of Ca2+-activated Cl- channels in addition to being mediated by inhibition of Kv7 channels. Additionally, this pathway involves activity of PKC.submitted by Isabella SalzerAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheErschienen in Pflügers Archiv - European Journal of PhysiologyWien, Med. Univ., Diss., 2014OeBB(VLID)171388

Nociceptor Signalling through ion Channel Regulation via GPCRs

The prime task of nociceptors is the transformation of noxious stimuli into action potentials that are propagated along the neurites of nociceptive neurons from the periphery to the spinal cord. This function of nociceptors relies on the coordinated operation of a variety of ion channels. In this review, we summarize how members of nine different families of ion channels expressed in sensory neurons contribute to nociception. Furthermore, data on 35 different types of G protein coupled receptors are presented, activation of which controls the gating of the aforementioned ion channels. These receptors are not only targeted by more than 20 separate endogenous modulators, but can also be affected by pharmacotherapeutic agents. Thereby, this review provides information on how ion channel modulation via G protein coupled receptors in nociceptors can be exploited to provide improved analgesic therapy

Frontiers in Pharmacology / Electrophysiological Investigation of the Subcellular Fine Tuning of Sympathetic Neurons by Hydrogen Sulfide

HS is well-known as hypotensive agent, whether it is synthetized endogenously or administered systemically. Moreover, the HS donor NaHS has been shown to inhibit vasopressor responses triggered by stimulation of preganglionic sympathetic fibers. In contradiction with this latter result, NaHS has been reported to facilitate transmission within sympathetic ganglia. To resolve this inconsistency, HS and NaHS were applied to primary cultures of dissociated sympathetic ganglia to reveal how this gasotransmitter might act at different subcellular compartments of such neurons. At the somatodendritic region of ganglionic neurons, NaHS raised the frequency, but not the amplitudes, of cholinergic miniature postsynaptic currents via a presynaptic site of action. In addition, the HS donor as well as HS itself caused membrane hyperpolarization and decreased action potential firing in response to current injection. Submillimolar NaHS concentrations did not affect currents through K7 channels, but did evoke currents through KATP channels. Similarly to NaHS, the KATP channel activator diazoxide led to hyperpolarization and decreased membrane excitability; the effects of both, NaHS and diazoxide, were prevented by the KATP channel blocker tolbutamide. At postganglionic sympathetic nerve terminals, HS and NaHS enhanced noradrenaline release due to a direct action at the level of vesicle exocytosis. Taken together, HS may facilitate transmitter release within sympathetic ganglia and at sympatho-effector junctions, but causes hyperpolarization and reduced membrane excitability in ganglionic neurons. As this latter action was due to KATP channel gating, this channel family is hereby established as another previously unrecognized determinant in the function of sympathetic ganglia.(VLID)486035

Pain / The paracetamol metabolite N-acetylp-benzoquinone imine reduces excitability in first- and second-order neurons of the pain pathway through actions on KV7 channels

Paracetamol (acetaminophen, APAP) is one of the most frequently used analgesic agents worldwide. It is generally preferred over nonsteroidal anti-inflammatory drugs because it does not cause typical adverse effects resulting from the inhibition of cyclooxygenases, such as gastric ulcers. Nevertheless, inhibitory impact on these enzymes is claimed to contribute to paracetamols mechanisms of action which, therefore, remained controversial. Recently, the APAP metabolites N-arachidonoylaminophenol (AM404) and N-acetyl-p-benzoquinone imine (NAPQI) have been detected in the central nervous system after systemic APAP administration and were reported to mediate paracetamol effects. In contrast to nonsteroidal anti-inflammatory drugs that rather support seizure activity, paracetamol provides anticonvulsant actions, and this dampening of neuronal activity may also form the basis for analgesic effects. Here, we reveal that the APAP metabolite NAPQI, but neither the parent compound nor the metabolite AM404, reduces membrane excitability in rat dorsal root ganglion (DRG) and spinal dorsal horn (SDH) neurons. The observed reduction of spike frequencies is accompanied by hyperpolarization in both sets of neurons. In parallel, NAPQI, but neither APAP nor AM404, increases currents through KV7 channels in DRG and SDH neurons, and the impact on neuronal excitability is absent if KV7 channels are blocked. Furthermore, NAPQI can revert the inhibitory action of the inflammatory mediator bradykinin on KV7 channels but does not affect synaptic transmission between DRG and SDH neurons. These results show that the paracetamol metabolite NAPQI dampens excitability of first- and second-order neurons of the pain pathway through an action on KV7 channels.(VLID)491090

Analgesic Action of Acetaminophen via Kv7 Channels

The mechanism of acetaminophen (APAP) analgesia is at least partially unknown. Previously, we showed that the APAP metabolite N-acetyl-p-benzoquinone imine (NAPQI) activated Kv7 channels in neurons in vitro, and this activation of Kv7 channels dampened neuronal firing. Here, the effect of the Kv7 channel blocker XE991 on APAP-induced analgesia was investigated in vivo. APAP had no effect on naive animals. Induction of inflammation with λ-carrageenan lowered mechanical and thermal thresholds. Systemic treatment with APAP reduced mechanical hyperalgesia, and co-application of XE991 reduced APAP’s analgesic effect on mechanical pain. In a second experiment, the analgesic effect of systemic APAP was not antagonized by intrathecal XE991 application. Analysis of liver samples revealed APAP and glutathione-coupled APAP indicative of metabolization. However, there were no relevant levels of these metabolites in cerebrospinal fluid, suggesting no relevant APAP metabolite formation in the CNS. In summary, the results support an analgesic action of APAP by activating Kv7 channels at a peripheral site through formation of the metabolite NAPQI

Membrane coordination of receptors and channels mediating the inhibition of neuronal ion currents by ADP

ADP and other nucleotides control ion currents in the nervous system via various P2Y receptors. In this respect, Cav2 and Kv7 channels have been investigated most frequently. The fine tuning of neuronal ion channel gating via G protein coupled receptors frequently relies on the formation of higher order protein complexes that are organized by scaffolding proteins and harbor receptors and channels together with interposed signaling components. However, ion channel complexes containing P2Y receptors have not been described. Therefore, the regulation of Cav2.2 and Kv7.2/7.3 channels via P2Y1 and P2Y12 receptors and the coordination of these ion channels and receptors in the plasma membranes of tsA 201 cells have been investigated here. ADP inhibited currents through Cav2.2 channels via both P2Y1 and P2Y12 receptors with phospholipase C and pertussis toxin-sensitive G proteins being involved, respectively. The nucleotide controlled the gating of Kv7 channels only via P2Y1 and phospholipase C. In fluorescence energy transfer assays using conventional as well as total internal reflection (TIRF) microscopy, both P2Y1 and P2Y12 receptors were found juxtaposed to Cav2.2 channels, but only P2Y1, and not P2Y12, was in close proximity to Kv7 channels. Using fluorescence recovery after photobleaching in TIRF microscopy, evidence for a physical interaction was obtained for the pair P2Y12/Cav2.2, but not for any other receptor/channel combination. These results reveal a membrane juxtaposition of P2Y receptors and ion channels in parallel with the control of neuronal ion currents by ADP. This juxtaposition may even result in apparent physical interactions between receptors and channels.W 1205-B09(VLID)309167