The voltage-dependent K ϩ channel 4.3 (Kv4.3) is one of the major molecular correlates encoding a class of rapidly inactivating K ϩ currents, including the transient outward current in the heart (I to) and A currents (IA) in neuronal and smooth muscle preparations. Recent studies have shown that I to in human atrial myocytes and IA in murine colonic myocytes are modulated by Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII); however, the molecular target of CaMKII in these studies has not been elucidated. We performed experiments to investigate whether CaMKII could regulate K v4.3 currents directly. Inclusion of the autothiophosphorylated form of CaMKII in the patch pipette (10 nM) prolonged K v4.3 currents such that the time required to reach 50% inactivation from peak more than doubled, with positive shifts in voltage dependence of both activation and inactivation. In contrast, the rate of recovery from inactivation was accelerated under these conditions. CaMKII-inhibitory peptide or KN-93 produced effects opposite to that above; thus the rate of inactivation was increased, and recovery from inactivation decreased. A number of mutagenesis experiments were conducted on the three candidate CaMKII consensus sequence sites on the channel. Mutations at S550A, located at the COOH-terminal region of the channel, resulted in currents that inactivated more rapidly but recovered from inactivation at a slower rate than that of wild-type controls. In addition, these currents were unaffected by dialysis with either autothiophosphorylated CaMKII or the specific inhibitory peptide of CaMKII, suggesting that CaMKII slows the inactivation and accelerates the rate of recovery from inactivation of Kv4.3 currents by a direct effect at S550A, located at the COOH-terminal region of the channel. transient outward current; potassium channel; inactivation; phosphorylation

Vascular endothelial growth factor (VEGF) is an important regulator of endothelial cell function. VEGF stimulates NO production, proposed to be a result of phosphorylation and activation of endothelial NO synthase (eNOS) at Ser1177. Phosphorylation of eNOS at this site also occurs after activation of AMP-activated protein kinase (AMPK) in cultured endothelial cells. We therefore determined whether AMPK mediates VEGF-stimulated NO synthesis in endothelial cells. VEGF caused a rapid, dose-dependent stimulation of AMPK activity, with a concomitant increase in phosphorylation of eNOS at Ser1177. Infection of endothelial cells with an adenovirus expressing a dominant negative mutant AMPK partially inhibited both VEGF-stimulated eNOS Ser1177 phosphorylation and NO production. VEGF-stimulated AMPK activity was completely inhibited by the Ca2+/calmodulin-dependent protein kinase kinase inhibitor, STO-609. Stimulation of AMPK via Ca2+/calmodulin-dependent protein kinase kinase represents a novel signalling mechanism utilised by VEGF in endothelial cells that contributes to eNOS phosphorylation and NO production.

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QUB-TL1 corrects aberrant CAP activities, providing a mechanism to delay or prevent the development of CF lung disease in a manner independent of CF transmembrane conductance regulator mutation.

BackgroundVascular endothelial growth factors (VEGFs) are key regulators of endothelial cell function and angiogenesis. We and others have previously demonstrated that VEGF-A stimulates AMP-activated protein kinase (AMPK) in cultured endothelial cells. Furthermore, AMPK has been reported to regulate VEGF-mediated angiogenesis. The role of AMPK in the function of VEGF-B remains undetermined, as does the role of AMPK in VEGF-stimulated endothelial cell proliferation, a critical process in angiogenesis.MethodsHuman aortic endothelial cells (HAECs) were incubated with VEGF-A and VEGF-B prior to examination of HAEC AMPK activity, proliferation, migration, fatty acid oxidation and fatty acid transport. The role of AMPK in the functional effects of VEGF-A and/or VEGF-B was assessed after downregulation of AMPK activity with chemical inhibitors or infection with adenoviruses expressing a dominant negative mutant AMPK.ResultsIncubation of HAECs with VEGF-B rapidly stimulated AMPK activity in a manner sensitive to an inhibitor of Ca2+/calmodulin-dependent kinase kinase (CaMKK), without increasing phosphorylation of endothelial NO synthase (eNOS) phosphorylation at Ser1177. Downregulation of AMPK abrogated HAEC proliferation in response to VEGF-A or VEGF-B. However, activation of AMPK by agents other than VEGF inhibited proliferation. Downregulation of AMPK abrogated VEGF-A-stimulated HAEC migration, whereas infection with adenoviruses expressing constitutively active mutant AMPK stimulated chemokinesis. Neither VEGF-A nor VEGF-B had any significant effect on HAEC fatty acid oxidation, yet prolonged incubation with VEGF-A stimulated fatty acid uptake in an AMPK-dependent manner. Inhibition of eNOS abrogated VEGF-mediated proliferation and migration, but was without effect on VEGF-stimulated fatty acid transport, ERK or Akt phosphorylation.ConclusionsThese data suggest that VEGF-B stimulates AMPK by a CaMKK-dependent mechanism and stimulation of AMPK activity is required for proliferation in response to either VEGF-A or VEGF-B and migration in response to VEGF-A. AMPK activation alone was not sufficient, however, to stimulate proliferation in the absence of VEGF. VEGF-stimulated NO synthesis is required for the stimulation of proliferation by VEGF-A or VEGF-B, yet this may be independent of eNOS Ser1177 phosphorylation.