Inhibitory Effects of Lidocaine and Mexiletine on Vasorelaxation Mediated by Adenosine Triphosphate-sensitive K ؉ Channels and the Role of Kinases in the Porcine Coronary Artery

Background: Effects of antiarrhythmic drugs on coronary vasodilation mediated by K ؉ channels have not been studied

RET PLCγ Phosphotyrosine Binding Domain Regulates Ca2+ Signaling and Neocortical Neuronal Migration

The receptor tyrosine kinase RET plays an essential role during embryogenesis in regulating cell proliferation, differentiation, and migration. Upon glial cell line-derived neurotrophic factor (GDNF) stimulation, RET can trigger multiple intracellular signaling pathways that in concert activate various downstream effectors. Here we report that the RET receptor induces calcium (Ca2+) signaling and regulates neocortical neuronal progenitor migration through the Phospholipase-C gamma (PLCγ) binding domain Tyr1015. This signaling cascade releases Ca2+ from the endoplasmic reticulum through the inositol 1,4,5-trisphosphate receptor and stimulates phosphorylation of ERK1/2 and CaMKII. A point mutation at Tyr1015 on RET or small interfering RNA gene silencing of PLCγ block the GDNF-induced signaling cascade. Delivery of the RET mutation to neuronal progenitors in the embryonic ventricular zone using in utero electroporation reveal that Tyr1015 is necessary for GDNF-stimulated migration of neurons to the cortical plate. These findings demonstrate a novel RET mediated signaling pathway that elevates cytosolic Ca2+ and modulates neuronal migration in the developing neocortex through the PLCγ binding domain Tyr1015

RET Tyr1015 mediates GDNF-stimulated migration <i>in vivo</i>.

<p>(<b>A</b>) Cartoon illustrating mouse embryo electroporation and GDNF-bead stimulated migration. (<b>B</b>–<b>D</b>) Migration of cortical progenitors in organotypic brain slices from embryos electroporated with RET^WT (<b>C</b>) or RET¹⁰¹⁵ (<b>D</b>) treated without beads (Control) or with beads (indicated with circles) soaked in PBS (Vehicle) or GDNF (500 ng/ml) placed in the cortical plate (CP). GFP positive RET^WT expressing progenitors (green) stimulated with GDNF beads (<b>B</b>, <b>C</b>) show significantly enhanced migration from the ventricular zone (VZ) towards the CP, as compared to Control, Vehicle, or inhibition of PLC with U73122 (5 µM). In RET¹⁰¹⁵ expressing progenitors GDNF beads failed to stimulate migration (<b>B</b>, <b>D</b>). Scale bars, 100 µm.</p

A RET/PLCγ/InsP₃R-cascade stimulates GDNF-induced Ca²⁺ release.

<p>(<b>A–H</b>) Representative single-cell Ca²⁺ recordings of GFP positive RET^WT expressing cells loaded with Fura-2/AM and preincubated with inhibitors as indicated, following treatment with GDNF (100 ng/ml). Inhibiting PLC with U73122 (5 µM) (<b>A</b>) or knocking down PLCγ with siRNA (<b>B</b>) blocked the cytosolic Ca²⁺ response induced by GDNF. Cells transfected with the Mock-siRNA retain the Ca²⁺ response (<b>C</b>). Inhibiting InsP₃R with 2-APB (5 µM) abolished the Ca²⁺ response induced by GDNF (<b>D</b>), while inhibiting RyR with ryanodine (a, 20 µM) or dantrolene (b, 10 µM) had no effect (<b>E</b>). Depleting intracellular Ca²⁺ stores with the SERCA Ca²⁺-ATPase inhibitor Thapsigargin (1 µM) blocked the Ca²⁺ response (<b>F</b>). Zero extracellular Ca²⁺ eliminated the GDNF-induced Ca²⁺ response (<b>G</b>), whereas a low extracellular concentration of Ca²⁺ (1 mM) produced a normal Ca²⁺ response (<b>H</b>).</p

Characteristics of Ca²⁺ responses triggered by GDNF in RET^WT cells treated with various inhibitors.

a<p>Non-responding cells have no Ca²⁺ increase exceeding 1.25 of the baseline.</p>b<p>Transient responding cells have one Ca²⁺ peak exceeding 1.25 of the baseline.</p>c<p>Oscillatory responding cells have at least three Ca²⁺ peaks exceeding 1.25 of the baseline.</p>d<p>[number of cells/number of experiments].</p>e<p>[concentration of extracellular Ca²⁺].</p

GDNF-induced Ca²⁺ signaling phosphorylates ERK1/2 and CaMKII.

<p>(<b>A</b>–<b>D</b>) Western blot of HeLa cells transfected with RET^WT or RET¹⁰¹⁵ treated with GDNF (100 ng/ml). GDNF triggers time dependent phosphorylation of ERK1/2 (pERK1/2) in RET^WT cells that is suppressed by BAPTA (10 µM) (<b>A</b>). Less pERK1/2 is observed in cells transfected with RET¹⁰¹⁵ than RET^WT (<b>B</b>). GDNF-induced phosphorylation of CaMKII (pCaMKII) or pERK1/2 is suppressed when blocking PLC with U73122 (5 µM) (<b>C</b>) or knocking down PLCγ with siRNA (PLCγ-siRNA) (<b>D</b>). Treating RET^WT cells with the U73122 analogue U73343 (5 µM) had no effect on GDNF-activated pCaMKII or pERK1/2 (<b>C</b>). Increased Caspase-3 cleavage was not detected in cells treated with the inhibitors BAPTA or U73122 (<b>C</b>).</p