BACKGROUND AND PURPOSEFlupirtine 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 APPROACHEffects of flupirtine on native and recombinant voltage-and ligand-gated ion channels were explored in patch-clamp experiments using the following experimental systems: recombinant KIR3 and KV7 channels and a3b4 nicotinic acetylcholine receptors expressed in tsA 201 cells; native voltage-gated Na, and TRPV1 channels, as well as GABAA, glycine, and ionotropic glutamate receptors expressed in rat dorsal root ganglion, dorsal horn and hippocampal neurons. KEY RESULTSTherapeutic flupirtine concentrations (Յ10 mM) 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 KV7 K + channels to more negative potentials and the gating of GABAA 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 KV7 channels and GABAA receptors was comparable, whereas in hippocampal neurons the effects on KV7 channels were more pronounced. CONCLUSIONS AND IMPLICATIONSThese results indicate that flupirtine exerts its analgesic action by acting on both GABAA receptors and KV7 channels. AbbreviationsBMI, bicuculline methiodide; CNQX, cyano-2,3-dihydroxy-7-nitroquinoxaline; DRG, dorsal root ganglion; NBQX, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide; SCG, superior cervical ganglion; SNEPCO, selective neuronal potassium channel opener

Phosphorylation of cortactin on S405 and S418 residues is required for its interaction with WAVE2 in monocyte chemotactic protein 1–induced cytoskeleton remodeling, facilitating human aortic smooth muscle cell migration.

Oxytocin (OT)- and vasopressin (VP)-secreting magnocellular neurons of the supraoptic nucleus (SON) display calcium-dependent afterhyperpolarizations (AHPs) following a train of action potentials that are critical to shaping the firing patterns of these cells. Previous work demonstrated that the lipid phosphatidylinositol 4,5-bisphosphate (PIP ) enabled the slow AHP component (sAHP) in cortical pyramidal neurons. We investigated whether this phenomenon occurred in OT and VP neurons of the SON. Using whole cell recordings in coronal hypothalamic slices from adult female rats, we demonstrated that inhibition of PIP synthesis with wortmannin robustly blocked both the medium and slow AHP currents (I and I ) of OT, but not VP neurons with high affinity. We further tested this by introducing a water-soluble PIP analogue (diC -PIP ) into neurons, which in OT neurons not only prevented wortmannin's inhibitory effect, but slowed rundown of the I and I . Inhibition of phospholipase C (PLC) with U73122 did not inhibit either I or I in OT neurons, consistent with wortmannin's effects not being due to reducing diacylglycerol (DAG) or IP availability, i.e. PIP modulation of AHPs is not likely to involve downstream Ca release from inositol 1,4,5-trisphosphate (IP )-triggered Ca -store release, or channel modulation via DAG and protein kinase C (PKC). We found that wortmannin reduced [Ca ] increase induced by spike trains in OT neurons, but had no effect on AHPs evoked by uncaging intracellular Ca . Finally, wortmannin selectively reduced whole cell Ca currents in OT neurons while leaving VP neurons unaffected. The results indicate that PIP modulates both the I and I in OT neurons, most likely by controlling Ca entry through voltage-gated Ca channels opened during spike trains.

Most cells express more than one receptor plus degrading enzymes for adenine nucleotides or nucleosides, and cellular responses to purines are rarely compatible with the actions of single receptors. Therefore, these receptors are viewed as components of a combinatorial receptor web rather than self-dependent entities, but it remained unclear to what extent they can associate with each other to form signalling units. P2Y 1 , P2Y 2 , P2Y 12 , P2Y 13 , P2X 2 , A 1 , A 2A receptors and NTPDase1 and -2 were expressed as fluorescent fusion proteins which were targeted to membranes and signalled like the unlabelled counterparts. When tested by FRET microscopy, all the G protein-coupled receptors proved able to form heterooligomers with each other, and P2Y 1 , P2Y 13 , A 1 , A 2A , and P2X 2 receptors also formed homooligomers. P2Y receptors did not associate with P2X, but G protein-coupled receptors formed heterooligomers with NTPDase1, but not with NTPDase2. The specificity of prototypic interactions (P2Y 1 /P2Y 1 , A 2A / P2Y 1 , A 2A /P2Y 12 ) was corroborated by FRET competition or co-immunoprecipitation. These results demonstrate that G protein-coupled purine receptors associate with each other and with NTPDase1 in a highly promiscuous manner. Thus, purinergic signalling is not only determined by the expression of receptors and enzymes but also by their direct interaction within a previously unrecognized multifarious membrane network.

Most cells express more than one receptor plus degrading enzymes for adenine nucleotides or nucleosides, and cellular responses to purines are rarely compatible with the actions of single receptors. Therefore, these receptors are viewed as components of a combinatorial receptor web rather than self-dependent entities, but it remained unclear to what extent they can associate with each other to form signalling units. P2Y(1), P2Y(2), P2Y(12), P2Y(13), P2X(2), A(1), A(2A) receptors and NTPDase1 and -2 were expressed as fluorescent fusion proteins which were targeted to membranes and signalled like the unlabelled counterparts. When tested by FRET microscopy, all the G protein-coupled receptors proved able to form heterooligomers with each other, and P2Y(1), P2Y(12), P2Y(13), A(1), A(2A), and P2X(2) receptors also formed homooligomers. P2Y receptors did not associate with P2X, but G protein-coupled receptors formed heterooligomers with NTPDase1, but not NTPDase2. The specificity of prototypic interactions (P2Y(1)/P2Y(1), A(2A)/P2Y(1), A(2A)/P2Y(12)) was corroborated by FRET competition or co-immunoprecipitation. These results demonstrate that G protein-coupled purine receptors associate with each other and with NTPDase1 in a highly promiscuous manner. Thus, purinergic signalling is not only determined by the expression of receptors and enzymes but also by their direct interaction within a previously unrecognized multifarious membrane network.

Molecularly defined P2Y receptor subtypes are known to regulate the functions of neurons through an inhibition of K V 7 K + and Ca V 2 Ca 2+ channels and via an activation or inhibition of Kir3 channels. Here, we searched for additional neuronal ion channels as targets for P2Y receptors. Rat P2Y 1 receptors were expressed in PC12 cells via an inducible expression system, and the effects of nucleotides on membrane currents and intracellular Ca 2+ were investigated. At a membrane potential of −30 mV, ADP induced transient outward currents in a concentration-dependent manner with half-maximal effects at 4 μm. These currents had reversal potentials close to the K + equilibrium potential and changed direction when extracellular Na + was largely replaced by K + , but remained unaltered when extracellular Cl − was changed. Currents were abolished by P2Y 1 antagonists and by blockade of phospholipase C. ADP also caused rises in intracellular Ca 2+ , and ADP-evoked currents were abolished when inositol trisphosphate-sensitive Ca 2+ stores were depleted. Blockers of K Ca 2, but not those of K Ca 1.1 or K Ca 3.1, channels largely reduced ADP-evoked currents. In hippocampal neurons, ADP also triggered outward currents at −30 mV which were attenuated by P2Y 1 antagonists, depletion of Ca 2+ stores, or a blocker of K Ca 2 channels. These results demonstrate that activation of neuronal P2Y 1 receptors may gate Ca 2+ -dependent K + (K Ca 2) channels via phospholipase C-dependent increases in intracellular Ca 2+ and thereby define an additional class of neuronal ion channels as novel effectors for P2Y receptors. This mechanism may form the basis for the control of synaptic plasticity via P2Y 1 receptors.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.The physiological demands of parturition and lactation lead to the increased pulsatile release of oxytocin (OT) into the circulation from the neurohypophysial axons of OT neurones in the supraoptic (SON) and paraventricular (PVN) nuclei. These states of increased OT release are accompanied by a significant plasticity in magnocellular OT neurones and their synaptic connections, and many of these changes require activation of a central OT receptor. The mitogen-activated protein kinase/extracellular signal-regulated kinase pathway (MAPK/ERK) is assumed to be up-regulated in the PVN during lactation, and many of the effects of OT in peripheral and brain tissue are mediated through a MAPK/ERK pathway. The present study investigated whether this pathway is altered in the SON and PVN during late pregnancy [embryonic day (E)20-21], which is a critical period for OT plasticity induction, and for lactation, when plastic changes are sustained. Based on immunoreactivity for phosphorylated ERK1/2 (pERK1/2), the results suggest an enhanced activation of MAPK/ERK pathway in OT neurones specifically during late pregnancy in both the SON and PVN. Although immunoblots from the SON confirm this pregnancy-associated up-regulation in late pregnancy, they also suggest enhancement into lactation as well. Together, the results suggest an important role for the MAPK/ERK pathway during reproductive changes in the SON and PVN.

BACKGROUND AND PURPOSEP2Y1, P2Y2, P2Y4, P2Y12 and P2Y13 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 APPROACHIn 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 [3H]-noradrenaline was determined.KEY RESULTSADP, 2-methylthio-ATP and ATP enhanced stimulation-evoked 3H overflow from superior cervical ganglion neurons, treated with pertussis toxin to prevent the signalling of inhibitory G proteins. This effect was abolished by P2Y1 antagonists and by inhibition of phospholipase C, but not by inhibition of protein kinase C or depletion of intracellular Ca2+ stores. ADP and a specific P2Y1 agonist caused inhibition of Kv7 channels, and this was prevented by a respective antagonist. In neurons not treated with pertussis toxin, 3H overflow was also enhanced by a specific P2Y1 agonist and by ADP, but only when the P2Y12 receptors were blocked. ADP also enhanced K+-evoked 3H overflow from PC12 cells treated with pertussis toxin, but only in a clone expressing recombinant P2Y1 receptors.CONCLUSIONS AND IMPLICATIONSThese results demonstrate that presynaptic P2Y1 receptors mediate facilitation of transmitter release from sympathetic neurons most likely through inhibition of Kv7 channels.