AMP-activated protein kinase (AMPK) is a metabolic sensor activated during metabolic stress and it regulates various enzymes and cellular processes to maintain metabolic homeostasis. We previously reported that activation of AMPK by glucose deprivation (GD) and leptin increases KATP currents by increasing the surface levels of KATP channel proteins in pancreatic beta-cells. Here, we show that the signaling mechanisms that mediate actin cytoskeleton remodeling are closely associated with AMPK-induced KATP channel trafficking. Using F-actin staining with Alexa 633-conjugated phalloidin, we observed that dense cortical actin filaments present in INS-1 cells cultured in 11 mM glucose were disrupted by GD or leptin treatment. These changes were blocked by inhibiting AMPK using compound C or siAMPK and mimicked by activating AMPK using AICAR, indicating that cytoskeletal remodeling induced by GD or leptin was mediated by AMPK signaling. AMPK activation led to the activation of Rac GTPase and the phosphorylation of myosin regulatory light chain (MRLC). AMPK-dependent actin remodeling induced by GD or leptin was abolished by the inhibition of Rac with a Rac inhibitor (NSC23766), siRac1 or siRac2, and by inhibition of myosin II with a myosin ATPase inhibitor (blebbistatin). Immunocytochemistry, surface biotinylation and electrophysiological analyses of KATP channel activity and membrane potentials revealed that AMPK-dependent KATP channel trafficking to the plasma membrane was also inhibited by NSC23766 or blebbistatin. Taken together, these results indicate that AMPK/Rac-dependent cytoskeletal remodeling associated with myosin II motor function promotes the translocation of KATP channels to the plasma membrane in pancreatic beta-cells.
AMP-Activated Protein Kinases/metabolism ; Actins/*metabolism ; Animals ; Cell Line ; Glucose/metabolism ; Insulin-Secreting Cells/*metabolism ; KATP Channels/*metabolism ; Leptin/metabolism ; Myosin Type II/*metabolism ; Phosphorylation ; Rats ; *Signal Transduction ; rac GTP-Binding Proteins/*metabolism

OBJECTIVETo investigate the effect of curcumin on oligomer formation and mitochondrial ATP-sensitive potassium channels (mitoKATP) induced by overexpression or mutation of α-synuclein.

METHODSRecombinant plasmids α-synuclein-pEGFP-A53T and α-synuclein-pEGFP-WT were transfected into PC12 cells by lipofectamin method, and intervened by application of curcumin (20 μmol/L) and 5-hydroxydecanoate (5-HD). Oligomer formation in the cultured cells was identified by Western blotting and Dot blotting. Cytotoxicity and apoptosis of the PC12 cells were measured by lactate dehydrogenase (LDH) and JC-1 assays. mitoKATP were identified by Western blotting and whole cell patch clamp.

RESULTSCurcumin has significantly reduced the oligomer formation induced by overexpression or mutation of α-synuclein in the cultured cells. LDH has decreased by 36.3% and 23.5%, and red/green fluorescence ratio of JC-1 was increased respectively by 48.46% and 50.33% after application of curcumin (P<0.05). Protein expression of Kir6.2 has decreased and mitoKATP channel current has significantly increased (P<0.05).

CONCLUSIONCurcumin can inhibit α-synuclein gene overexpression or mutation induced α-synuclein oligomers formation. It may block apoptosis induced by wild-type overexpression or mutation of α-synuclein. By stabilizing mitochondrial membrane potential. Opening of mitoKATP channel may have been the initiating protective mechanism of apoptosis induced by wild-type overexpression or mutation of α-synuclein. Curcumin may antagonize above cytotoxicity through further opening the mitoKATP channel.

Animals ; Apoptosis ; drug effects ; Cell Line ; Curcumin ; pharmacology ; Humans ; KATP Channels ; chemistry ; genetics ; metabolism ; Mitochondria ; drug effects ; genetics ; metabolism ; Mutation ; drug effects ; PC12 Cells ; Parkinson Disease ; drug therapy ; genetics ; metabolism ; physiopathology ; Rats ; alpha-Synuclein ; genetics

The aim of the present study is to investigate the expressions of ATP-sensitive K(+) channels (KATP) in pulmonary artery smooth muscle cells (PASMCs) and the relationship with p38 MAPK signal pathway in rats. Male SD rat PASMCs were cultured in vitro, and a model of hypoxia and hypercapnia was reconstructed. PASMCs were divided to normal (N), hypoxia-hypercapnia (H), hypoxia-hypercapnia+DMSO incubation (HD), hypoxia-hypercapnia+SB203580 (inhibitor of p38 MAPK pathway) incubation (HS) and hypoxia-hypercapnia+Anisomycin (agonist of p38 MAPK pathway) incubation (HA) groups. Western blot was used to detect the protein expression of SUR2B and Kir6.1; semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of SUR2B and Kir6.1. The results demonstrated that: (1) Compared with N, H, HD and HS groups, the expressions of Kir6.1 mRNA and protein in PASMCs of HA group were decreased significantly (P < 0.01), but there were no differences among N, H, HD and HS groups (P > 0.05); (2) Compared with N group, the expressions of SUR2B mRNA and protein in H, HD, HS and HA groups were increased significantly (P < 0.05), but there were no differences among H, HD, HS and HA groups (P > 0.05). The results imply that: (1) Hypoxia-hypercapnia, SB203580 didn't change the expressions of Kir6.1 mRNA and protein in PASMCs, but Anisomycin decreased the expressions of Kir6.1 mRNA and protein, so Kir6.1 may be regulated by the other subfamily of MAPK pathway; (2) Hypoxia-hypercapnia raised SUR2B mRNA and protein expressions in PASMCs, but SB203580 and Anisomycin did not affect the changes, so the increasing of SUR2B mRNA and protein induced by hypoxia-hypercapnia may be not depend on p38 MAPK pathway.
Animals ; Anisomycin ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Hypercapnia ; Imidazoles ; pharmacology ; KATP Channels ; metabolism ; MAP Kinase Signaling System ; Male ; Myocytes, Smooth Muscle ; metabolism ; Pulmonary Artery ; cytology ; Pyridines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Sulfonylurea Receptors ; metabolism ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors

The present study was designed to investigate the role of opioid receptors in the vasorelaxation effect of chronic intermittent hypobaric hypoxia (CIHH) in thoracic aorta rings and the underlying mechanism in rats. Adult male Sprague-Dawley (SD) rats were randomly divided into 2 groups: CIHH treatment group and control group. The rats in CIHH group were exposed to hypoxia in a hypobaric chamber (simulated 5 000 m altitude) for 28 days, 6 h per day. The rats in control group were kept in the same environment as CIHH rats except no hypoxia exposure. The relaxation of thoracic aorta rings was recorded by organ bath perfusion technique, and expression of opioid receptors was measured by Western blot. Results are shown as follows. (1) The acetylcholine (ACh)-induced endothelium-dependent relaxation of thoracic aorta in CIHH rats was increased obviously in a concentration-dependent manner compared with that in control rats (P < 0.05). (2) This enhancement of ACh-induced relaxation in CIHH rats was abolished by naloxone, a non-specific opioid receptor blocker (P < 0.05). (3) The expressions of δ, μ and κ opioid receptors in thoracic aorta of CIHH rats were up-regulated compared with those in control rats (P < 0.05). (4) The enhancement of CIHH on relaxation of thoracic aorta was reversed by glibenclamide, an ATP-sensitive potassium channel (KATP) blocker (P < 0.05). The results suggest that opioid receptors are involved in CIHH-enhanced ACh-induced vasorelaxation of thoracic aorta through KATP channel pathways.
Acetylcholine ; pharmacology ; Altitude ; Animals ; Aorta, Thoracic ; drug effects ; Glyburide ; pharmacology ; Hypoxia ; physiopathology ; KATP Channels ; antagonists & inhibitors ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid ; metabolism ; Vasodilation

OBJECTIVETo study the effects of iptakalim (Ipt), an ATP-sensitive potassium channel opener, on cardiac remodeling induced by isoproterenol (ISO) in Wistar rats.

METHODSISO was given subcutaneously (85 mg/(kg x d), sc, 7 days) to induce cardiac remodeling in rats. The rats in Ipt treated group were administrated with Ipt 3 mg/kg (po) after ISO injection. After treated with Ipt for 6 weeks, the hemodynamic parameters were tested by an eight channel physiological recorder (RM-6000). Then the heart weight was weighed and the cardiac remodeling index was calculated. HE stain and Masson's stain were employed to perform histological analysis, the hydroxyproline(Hyp) content in cardiac tissue was detected by colorimetric method, radioimmunoassay was used to measure the plasma levels of endothelin-1 (ET-1) and prostacyclin (PGI2).

RESULTSSix weeks after ISO injection, the cardiac functions of model group were damaged markedly compared with those of normal group. The characteristics of ventricular remodeling in model group included that the heart weight index, myocyte cross-sectional area, myocardial fibrosis, and the hydroxyproline content in cardiac tissue were all increased significantly. The plasma level of ET-1 was increased, while the plasma level of PGI2 was decreased significantly. These changes could be reversed by Ipt treatment (3 mg/(kg x d) for 6 weeks).

CONCLUSIONIpt can reverse cardiac remodeling induced by isoproterenol in rats. The endothelial protective effect regulating effects of Ipt on the balance between the ET-1 and PGI2 system may be involved in its mechanisms.

Animals ; Endothelin-1 ; blood ; Hemodynamics ; Hydroxyproline ; metabolism ; Isoproterenol ; pharmacology ; KATP Channels ; drug effects ; Male ; Myocardium ; metabolism ; Propylamines ; pharmacology ; Prostaglandins I ; blood ; Rats ; Rats, Wistar ; Ventricular Remodeling ; drug effects

The present study aimed to investigate the protective effect and mechanism of hydrogen sulfide donor NaHS administration against gastric mucosal injury induced by gastric ischemia-reperfusion (GI-R) in rats. GI-R injury was induced by clamping the celiac artery of adult male SD rats for 30 min and followed by reperfusion for 1 h. The rats were randomly divided into sham group, GI-R group, NaHS group, glibenclamide group and pinacidil group. Gastric mucosal damage was analyzed with macroscopic injured area, deep damage was assessed with histopathology scores, and the hydrogen sulfide concentration in plasma was determined by colorimetric method. The results showed that pretreatment of NaHS significantly reduced the injured area and deep damage of the gastric mucosa induced by GI-R. However, NaHS did not significantly alter the levels of hydrogen sulfide in plasma 14 d after NaHS administration. The gastric protective effect of NaHS during reperfusion could be attenuated by glibenclamide, an ATP-sensitive potassium channel (K(ATP)) blocker. However, K(ATP) opener pinacidil inhibited the GI-R-induced injury. These results suggest that exogenous hydrogen sulfide plays a protective role against GI-R injury in rats possibly through modulation of K(ATP) channel opening.
Animals ; Gastric Mucosa ; pathology ; Hydrogen Sulfide ; metabolism ; Ischemic Preconditioning ; methods ; KATP Channels ; metabolism ; physiology ; Male ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; prevention & control ; Stomach ; blood supply ; Sulfides ; pharmacology

ATP-sensitive potassium (K(ATP)) channels are widely distributed in vasculatures, and play an important role in the vascular tone regulation. The K(ATP) channels consist of 4 pore-forming inward rectifier K(+) channel (Kir) subunits and 4 regulatory sulfonylurea receptors (SUR). The major vascular isoform of K(ATP) channels is composed of Kir6.1/SUR2B, although low levels of other subunits are also present in vascular beds. The observation from transgenic mice and humans carrying Kir6.1/SUR2B channel mutations strongly supports that normal activity of the Kir6.1/SUR2B channel is critical for cardiovascular function. The Kir6.1/SUR2B channel is regulated by intracellular ATP and ADP. The channel is a common target of several vasodilators and vasoconstrictors. Endogenous vasopressors such as arginine vasopressin and α-adrenoceptor agonists stimulate protein kinase C (PKC) and inhibit the K(ATP) channels, while vasodilators such as β-adrenoceptor agonists and vasoactive intestinal polypeptide increase K(ATP) channel activity by activating the adenylate cyclase-cAMP-protein kinase A (PKA) pathway. PKC phosphorylates a cluster of 4 serine residues at C-terminus of Kir6.1, whereas PKA acts on Ser1387 in the nucleotide binding domain 2 of SUR2B. The Kir6.1/SUR2B channel is also inhibited by oxidants including reactive oxygen species allowing vascular regulation in oxidative stress. The molecular basis underlying such a channel inhibition is likely to be mediated by S-glutathionylation at a few cysteine residues, especially Cys176, in Kir6.1. Furthermore, the channel activity is augmented in endotoxemia or septic shock, as a result of the upregulation of Kir6.1/SUR2B expression. Activation of the nuclear factor-κB dependent transcriptional mechanism contributes to the Kir6.1/SUR2B channel upregulation by lipopolysaccharides and perhaps other toll-like receptor ligands as well. In this review, we summarize the vascular K(ATP) channel regulation under physiological and pathophysiological conditions, and discuss the importance of K(ATP) channel as a potentially useful target in the treatment and prevention of cardiovascular diseases.
ATP-Binding Cassette Transporters ; genetics ; physiology ; Animals ; Endotoxemia ; metabolism ; physiopathology ; Humans ; KATP Channels ; genetics ; physiology ; Mice ; Mice, Transgenic ; Muscle, Smooth, Vascular ; metabolism ; physiology ; Potassium Channels, Inwardly Rectifying ; genetics ; physiology ; Receptors, Drug ; genetics ; physiology ; Shock, Septic ; metabolism ; physiopathology ; Sulfonylurea Receptors ; Vasoconstriction ; physiology ; Vasodilation ; physiology ; Vasomotor System ; physiology

Cumulative evidence suggests that renal vascular endothelial injury play an important role in initiating and extending tubular epithelial injury and contribute to the development of ischemic acute renal failure. Our previous studies have demonstrated that iptakalim's endothelium protection is related to activation of SUR2B/Kir6.1 subtype of ATP sensitive potassium channel (K(ATP)) in the endothelium. It has been reported that SUR2B/Kir6.1 channels are widely distributed in the tubular epithelium, glomerular mesangium, and the endothelium and the smooth muscle of blood vessels. Herein, we hypothesized that activating renal K(ATP) channels with iptakalim might have directly neroprotective effects. In this study, glomerular endothelial, mesangial and tubular epithelial cells which are the main cell types to form nephron were exposed to oleic acid (OA) at various concentrations for 24 h. 0.25 microl/ml OA could cause cellular damage of glomerular endothelium and mesangium, while 1.25 microl/ml OA could lead to the injury of three types of renal cells. It was observed that pretreatment with iptakalim at concentrations of 0.1, 1, 10 or 100 micromol/L prevented cellular damage of glomerular endothelium and tubular epithelium, whereas iptakalim from 1 to 100 micromol/L prevented the injury of mesangial cells. Our data showed iptakalim significantly increased survived cell rates in a concentration-dependent manner, significantly antagonized by glibenclamide, a K(ATP) blocker. Iptakalim played a protective role in the main cell types of kidney, which was consistent with natakalim, a highly selective SUR2B/Kir6.1 channel opener. Iptakalim exerted protective effects through activating SUR2B/Kir6.1 channels, suggesting a new strategy for renal injury by its endothelial and renal cell protection.
Cells, Cultured ; Epithelial Cells ; metabolism ; Glyburide ; adverse effects ; Humans ; KATP Channels ; metabolism ; Kidney ; cytology ; metabolism ; physiopathology ; Kidney Diseases ; drug therapy ; metabolism ; Oleic Acid ; adverse effects ; Propylamines ; pharmacology ; Protective Agents ; pharmacology

Hypoxic pulmonary hypertension (HPH) is a syndrome characterized by the increase of pulmonary vascular tone and the structural remodeling of peripheral pulmonary arteries. The aim of specific therapies for hypoxic pulmonary hypertension is to reduce pulmonary vascular resistance, reverse pulmonary vascular remodeling, and thereby improving right ventricular function. Iptakalim, a lipophilic para-amino compound with a low molecular weight, has been demonstrated to be a new selective ATP-sensitive potassium (K(ATP)) channel opener via pharmacological, electrophysiological, biochemical studies, and receptor binding tests. In hypoxia-induced animal models, iptakalim decreases the elevated mean pressure in pulmonary arteries, and attenuates remodeling in the right ventricle, pulmonary arteries and airways. Furthermore, iptakalim has selective antihypertensive effects, selective vasorelaxation effects on smaller arteries, and protective effects on endothelial cells, but no effects on the central nervous, respiratory, digestive or endocrine systems at therapeutic dose. Our previous studies demonstrated that iptakalim inhibited the effects of endothelin-1, reduced the intracellular calcium concentration and inhibited the proliferation of pulmonary artery smooth muscle cells. Since iptakalim has been shown safe and effective in both experimental animal models and phase I clinical trials, it can be a potential candidate of HPH in the future.
Animals ; Antihypertensive Agents ; therapeutic use ; Calcium ; metabolism ; Disease Models, Animal ; Endothelin-1 ; metabolism ; Hypertension, Pulmonary ; drug therapy ; Hypoxia ; drug therapy ; KATP Channels ; drug effects ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Propylamines ; therapeutic use ; Pulmonary Artery ; drug effects

The present study is aimed to investigate the effect of chronic intermittent hypobaric hypoxia (CIHH) on contractile activities in isolated thoracic aorta and pulmonary artery rings and the underlying mechanism in rats. Sprague-Dawley (SD) rats were randomly divided into 4 groups: control group (CON), 14 days CIHH treatment group (CIHH14), 28 days CIHH treatment group (CIHH28) and 42 days CIHH treatment group (CIHH42). CIHH rats were exposed to hypoxia in a hypobaric chamber simulating 5 000 m altitude, 6 h daily for 14, 28 and 42 d, respectively. After artery rings were prepared from pulmonary artery and thoracic aorta, the contractile activity of the artery rings was recorded using organ bath technique. Results are shown as follows. (1) There were no significant differences of noradrenaline (NA)- and KCl-induced contractions in thoracic aorta and pulmonary artery rings among CIHH and CON rats. (2) Angiotensin Ⅱ (ANGⅡ)-induced contraction in thoracic aorta rings, not in pulmonary artery rings, of CIHH rats was decreased compared with that in CON rats. There was no significant difference of ANGⅡ-induced contraction in thoracic aorta rings among CIHH rats. (3) Inhibitory effect of CIHH on ANGⅡ-induced contraction in thoracic aorta rings was endothelium-independent, and was reversed by glibenclamide (Gli), an ATP-sensitive potassium channels (K(ATP)) blocker, and L-NAME, a NO synthase inhibitor, but not by indomethacin (Indo), a cyclooxygenase inhibitor. These results suggest that CIHH attenuates the contraction induced by ANGⅡ in thoracic aorta rings of rat, which is related to the opening of K(ATP) channel and the increased production of NO.
Angiotensin II ; pharmacology ; Animals ; Aorta, Thoracic ; physiopathology ; Hypoxia ; physiopathology ; KATP Channels ; metabolism ; Male ; Muscle Contraction ; physiology ; Muscle, Smooth, Vascular ; physiopathology ; Nitric Oxide ; biosynthesis ; Pulmonary Artery ; physiopathology ; Rats ; Rats, Sprague-Dawley ; Vasoconstriction ; physiology