Cardiac sarcolemmal ATP-sensitive K+ (K(ATP)) channels, composed of Kir6.2 and SUR2A subunits, are regulated by intracellular ATP and they couple the metabolic status of the cell with the membrane ...excitability. On the basis of previous studies, we have suggested that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) may be a part of the sarcolemmal K(ATP)-channel protein complex. A polypeptide of approximately 42 kDa was immunoprecipitated with an anti-SUR2A antibody from guinea-pig cardiac membrane fraction and identified as GAPDH. Immunoprecipitation/western blotting analysis with anti-Kir6.2, anti-SUR2A and anti-GAPDH antibodies showed that GAPDH is a part of the sarcolemmal K(ATP)-channel protein complex in vivo. Further studies with immunoprecipitation/western blotting and the membrane yeast two-hybrid system showed that GAPDH associates physically with the Kir6.2 but not the SUR2A subunit. Patch-clamp electrophysiology showed that GAPDH regulates K(ATP)-channel activity irrespective of high intracellular ATP, by producing 1,3-bisphosphoglycerate, a K(ATP)-channel opener. These results suggest that GAPDH is an integral part of the sarcolemmal K(ATP)-channel protein complex, where it couples glycolysis with the K(ATP)-channel activity.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Activation of PKCε confers protection against neuronal ischemia/reperfusion. Since activation of PKCε leads to its translocation to multiple intracellular sites, a mitochondrial-selective PKCε ...activator was used to test the importance of mitochondrial activation to the neuroprotective effect of PKCε. PKCε can regulate key cytoprotective mitochondrial functions including electron transport chain activity, reactive oxygen species (ROS) generation, mitochondrial permeability transition, and detoxification of reactive aldehydes. We tested the ability of mitochondrial selective activation of PKCε to protect primary brain cell cultures or mice subjected to ischemic stroke. Pre-treatment with either general PKCε activator peptide, ψεRACK, or mitochondrial-selective PKCε activator, ψεHSP90, reduced cell death induced by simulated ischemia/reperfusion in neurons, astrocytes, and mixed neuronal cultures. The protective effects of both ψεRACK and ψεHSP90 were blocked by the PKCε antagonist, εV
1–2
, indicating protection requires PKCε interaction with its anchoring protein, εRACK. Further supporting a mitochondrial mechanism for PKCε, neuroprotection by ψεHSP90 was associated with a marked delay in mitochondrial membrane depolarization and significantly attenuated ROS generation during ischemia. Importantly, ψεHSP90 reduced infarct size and reduced neurological deficit in C57/BL6 mice subjected to middle cerebral artery occlusion and 24 hours of reperfusion. Thus selective activation of mitochondrial PKCε preserves mitochondrial function
in vitro
and improves outcome
in vivo
, suggesting potential therapeutic value clinically when brain ischemia is anticipated, including neurosurgery and cardiac surgery.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Pulmonary arterial hypertension, group 1 of the pulmonary hypertension disease family, involves pulmonary vascular remodelling, right ventricular dysfunction and cardiac failure. Oxidative stress, ...through activation of mitogen-activated protein kinases is implicated in these changes. Inhibition of apoptosis signal-regulating kinase 1, an apical mitogen-activated protein kinase, prevented pulmonary arterial hypertension developing in rodent models. Here, we investigate apoptosis signal-regulating kinase 1 in pulmonary arterial hypertension by examining the impact that its inhibition has on the molecular and cellular signalling in established disease. Apoptosis signal-regulating kinase 1 inhibition was investigated in in vivo pulmonary arterial hypertension and in vitro pulmonary hypertension models. In the in vivo model, male Sprague Dawley rats received a single subcutaneous injection of Sugen SU5416 (20 mg/kg) prior to two weeks of hypobaric hypoxia (380 mmHg) followed by three weeks normoxia (Sugen/hypoxic), then animals were either maintained for three weeks on control chow or one containing apoptosis signal-regulating kinase 1 inhibitor (100 mg/kg/day). Cardiovascular measurements were carried out. In the in vitro model, primary cultures of rat pulmonary artery fibroblasts and rat pulmonary artery smooth muscle cells were maintained in hypoxia (5% O 2 ) and investigated for proliferation, migration and molecular signalling in the presence or absence of apoptosis signal-regulating kinase 1 inhibitor. Sugen/hypoxic animals displayed significant pulmonary arterial hypertension compared to normoxic controls at eight weeks. Apoptosis signal-regulating kinase 1 inhibitor decreased right ventricular systolic pressure to control levels and reduced muscularised vessels in lung tissue. Apoptosis signal-regulating kinase 1 inhibition was found to prevent hypoxia-induced proliferation, migration and cytokine release in rat pulmonary artery fibroblasts and also prevented rat pulmonary artery fibroblast-induced rat pulmonary artery smooth muscle cell migration and proliferation. Apoptosis signal-regulating kinase 1 inhibition reversed pulmonary arterial hypertension in the Sugen/hypoxic rat model. These effects may be a result of intrinsic changes in the signalling of adventitial fibroblast.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
ATP-sensitive K(+) (K(ATP)) channels in the heart are normally closed by high intracellular ATP, but are activated during ischemia to promote cellular survival. These channels are heteromultimers ...composed of Kir6.2 subunit, an inwardly rectifying K(+) channel core, and SUR2A, a regulatory subunit implicated in ligand-dependent regulation of channel gating. Here, we have shown that the muscle form (M-LDH), but not heart form (H-LDH), of lactate dehydrogenase is directly physically associated with the sarcolemmal K(ATP) channel by interacting with the Kir6.2 subunit via its N-terminus and with the SUR2A subunit via its C-terminus. The species of LDH bound to the channel regulated the channel activity despite millimolar concentration of intracellular ATP. The presence of M-LDH in the channel protein complex was required for opening of K(ATP) channels during ischemia and ischemia-resistant cellular phenotype. We conclude that M-LDH is an integral part of the sarcolemmal K(ATP) channel protein complex in vivo, where, by virtue of its catalytic activity, it couples the metabolic status of the cell with the K(ATP) channels activity that is essential for cell protection against ischemia.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Radiation-induced dermatitis is a debilitating clinical problem in cancer patients undergoing cancer radiation therapy. It is also a possible outcome of exposure to high levels of radiation due to ...accident or hostile activity. We report that activation of aldehyde dehydrogenase 2 (ALDH2) enzymatic activity using the allosteric agonist, Alda-1, significantly reduced 4-hydroxynonenal adducts accumulation, delayed the onset of radiation dermatitis and substantially reduced symptoms in a clinically-relevant model of radiation-induced dermatitis. Importantly, Alda-1 did not radioprotect tumors in mice. Rather, it increased the sensitivity of the tumors to radiation therapy. This is the first report of reactive aldehydes playing a role in the intrinsic radiosensitivity of normal and tumor tissues. Our findings suggest that ALDH2 represents a novel target for the treatment of radiation dermatitis without reducing the benefit of radiotherapy.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Late Na(+) current (I(NaL)) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are both increased in the diseased heart. Recently, CaMKII was found to phosphorylate the Na(+) channel 1.5 ...(Na(v)1.5), resulting in enhanced I(NaL). Conversely, an increase of I(NaL) would be expected to cause elevation of intracellular Ca(2+) and activation of CaMKII. However, a relationship between enhancement of I(NaL) and activation of CaMKII has yet to be demonstrated. We investigated whether Na(+) influx via Na(v)1.5 leads to CaMKII activation and explored the functional significance of this pathway. In neonatal rat ventricular myocytes (NRVM), treatment with the I(NaL) activators anemone toxin II (ATX-II) or veratridine increased CaMKII autophosphorylation and increased phosphorylation of CaMKII substrates phospholamban and ryanodine receptor 2. Knockdown of Na(v)1.5 (but not Na(v)1.1 or Na(v)1.2) prevented ATX-II-induced CaMKII phosphorylation, providing evidence for a specific role of Na(v)1.5 in CaMKII activation. In support of this view, CaMKII activity was also increased in hearts of transgenic mice overexpressing a gain-of-function Na(v)1.5 mutant (N(1325)S). The effects of both ATX-II and the N(1325)S mutation were reversed by either I(NaL) inhibition (with ranolazine or tetrodotoxin) or CaMKII inhibition (with KN93 or autocamtide 2-related inhibitory peptide). Furthermore, ATX-II treatment also induced CaMKII-Na(v)1.5 coimmunoprecipitation. The same association between CaMKII and Na(v)1.5 was also found in N(1325)S mice, suggesting a direct protein-protein interaction. Pharmacological inhibitions of either CaMKII or I(NaL) also prevented ATX-II-induced cell death in NRVM and reduced the incidence of polymorphic ventricular tachycardia induced by ATX-II in rat perfused hearts. Taken together, these results suggest that a Na(v)1.5-dependent increase in Na(+) influx leads to activation of CaMKII, which in turn phosphorylates Na(v)1.5, further promoting Na(+) influx. Pharmacological inhibition of either CaMKII or Na(v)1.5 can ameliorate cardiac dysfunction caused by excessive Na(+) influx.
Late Na+ current (...) and .../calmodulin-dependent protein kinase II (CaMKII) are both increased in the diseased heart. Recently, CaMKII was found to phosphorylate the Na+ channel 1.5 (Na...1.5), ...resulting in enhanced ... Conversely, an increase of ... would be expected to cause elevation of intracellular ... and activation of CaMKII. However, a relationship between enhancement of ... and activation of CaMKII has yet to be demonstrated. We investigated whether Na+ influx via Na...1.5 leads to CaMKII activation and explored the functional significance of this pathway. In neonatal rat ventricular myocytes (NRVM), treatment with the ... activators anemone toxin II (ATX-II) or veratridine increased CaMKII autophosphorylation and increased phosphorylation of CaMKII substrates phospholamban and ryanodine receptor 2. Knockdown of Na...1.5 (but not Na...1.1 or Na...1.2) prevented ATX-II-induced CaMKII phosphorylation, providing evidence for a specific role of Na...1.5 in CaMKII activation. In support of this view, CaMKII activity was also increased in hearts of transgenic mice overexpressing a gain-of-function Na...1.5 mutant (...). The effects of both ATX-II and the ... mutation were reversed by either ... inhibition (with ranolazine or tetrodotoxin) or CaMKII inhibition (with KN93 or autocamtide 2-related inhibitory peptide). Furthermore, ATX-II treatment also induced CaMKII-Na...1.5 coimmunoprecipitation. The same association between CaMKII and Na...1.5 was also found in ... mice, suggesting a direct protein-protein interaction. Pharmacological inhibitions of either CaMKII or ... also prevented ATX-II-induced cell death in NRVM and reduced the incidence of polymorphic ventricular tachycardia induced by ATX-II in rat perfused hearts. Taken together, these results suggest that a Na...1.5-dependent increase in Na+ influx leads to activation of CaMKII, which in turn phosphorylates Na...1.5, further promoting Na+ influx. Pharmacological inhibition of either CaMKII or Na...1.5 can ameliorate cardiac dysfunction caused by excessive Na+ influx. (ProQuest: ... denotes formulae/symbols omitted.)
Late Na + current ( I NaL ) and Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) are both increased in the diseased heart. Recently, CaMKII was found to phosphorylate the Na + channel 1.5 (Na v ...1.5), resulting in enhanced I NaL . Conversely, an increase of I NaL would be expected to cause elevation of intracellular Ca 2+ and activation of CaMKII. However, a relationship between enhancement of I NaL and activation of CaMKII has yet to be demonstrated. We investigated whether Na + influx via Na v 1.5 leads to CaMKII activation and explored the functional significance of this pathway. In neonatal rat ventricular myocytes (NRVM), treatment with the I NaL activators anemone toxin II (ATX-II) or veratridine increased CaMKII autophosphorylation and increased phosphorylation of CaMKII substrates phospholamban and ryanodine receptor 2. Knockdown of Na v 1.5 (but not Na v 1.1 or Na v 1.2) prevented ATX-II-induced CaMKII phosphorylation, providing evidence for a specific role of Na v 1.5 in CaMKII activation. In support of this view, CaMKII activity was also increased in hearts of transgenic mice overexpressing a gain-of-function Na v 1.5 mutant (N 1325 S). The effects of both ATX-II and the N 1325 S mutation were reversed by either I NaL inhibition (with ranolazine or tetrodotoxin) or CaMKII inhibition (with KN93 or autocamtide 2-related inhibitory peptide). Furthermore, ATX-II treatment also induced CaMKII-Na v 1.5 coimmunoprecipitation. The same association between CaMKII and Na v 1.5 was also found in N 1325 S mice, suggesting a direct protein-protein interaction. Pharmacological inhibitions of either CaMKII or I NaL also prevented ATX-II-induced cell death in NRVM and reduced the incidence of polymorphic ventricular tachycardia induced by ATX-II in rat perfused hearts. Taken together, these results suggest that a Na v 1.5-dependent increase in Na + influx leads to activation of CaMKII, which in turn phosphorylates Na v 1.5, further promoting Na + influx. Pharmacological inhibition of either CaMKII or Na v 1.5 can ameliorate cardiac dysfunction caused by excessive Na + influx.
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