Pharmacological evidence: a new therapeutic approach to the treatment of chronic heart failure through SUR2B/Kir6.1 channel in endothelial cells.

Both iptakalim (Ipt) and natakalim (Nat) activate the SUR2B/Kir6.1 channel, an ATP-sensitive potassium channel (KATP) subtype, with high selectivity. In this study we investigated the therapeutic effects of Ipt and Nat against isoproterenol-induced chronic heart failure (ISO-CHF) in rats, and demonstrated a new therapeutic approach to the treatment of CHF through activation of the SUR2B/Kir6.1 channel in endothelial cells. In ISO-CHF rats, oral administration of Nat (1, 3, 9 mg·kg-1·d-1) or Ipt (3 mg·kg-1·d-1) for 60 days significantly improved cardiac dysfunction, reversed cardiac remodeling, significantly attenuated the pathological increases in BNP levels, and improved endothelial dysfunction by adjusting the balance between endothelin and NO systems. The therapeutic effects of Nat were prevented by the selective KATP blocker glibenclamine (Gli, 50 mg·kg-1·d-1), confirming that these effects were mediated through activation of the SUR2B/Kir6.1 channel in endothelial cells. The molecular mechanisms underlying the therapeutic effects of Nat were further addressed using proteomic methods. We identified 724 proteins in the plasma of ISO-CHF rats; 55 proteins were related to Nat. These differentially expressed proteins were mainly involved in single-organism processes and the regulation of biological quality relative to CHF, including proteasome (Psm) and ATP protein clusters. We screened out PRKAR2ß, GAS6/eNOS/NO and NO/PKG/VASP pathways involved in the amelioration of CHF among the 24 enriched pathways. We further confirmed 6 protein candidates, including PRKAR2ß, GAS6 and VASP, which were involved in the endothelial mechanisms, and ATP, TIMP3 and AGT, which contributed to its cardiovascular actions. This study demonstrates a new pharmacological approach to the treatment of CHF through activation of the SUR2B/Kir6.1 channel in endothelial cells, and that the eNOS/VASP pathways are involved in its signaling mechanisms.

Natakalim Ameliorates Isoproterenol-Induced Chronic Heart Failure by Protecting against Endothelial Dysfunction.

The pharmacological effects and underlying mechanisms of natakalim, a novel SUR2B/Kir6.1-KATP channel opener, against chronic heart failure induced by isoproterenol in rats were investigated. Male Wistar rats were administered isoproterenol subcutaneously (85 mg/kg, 7 days) to induce chronic heart failure and were then treated with natakalim or saline for 6 weeks. Their blood pressure, heart rates and cardiac functions were measured using an 8-channel physiological recorder. Sophisticated technologies such as histological analysis, ELISA, radioimmunoassay, immunohistochemistry, real-time PCR and western blotting were employed for analysis. Natakalim (1, 3, 9 mg/kg/day, orally) or saline was administered for 6 weeks orally via a gastric tube to rats that had been injected with isoproterenol. Natakalim remarkably inhibited changes in left ventricular hemodynamic parameters and decreased the heart mass index, the left ventricular weight index, right ventricular weight index and lung weight index. Histological examination demonstrated no significant hypertrophy or fibrosis in the hearts of the natakalim-treated rats. Mechanistically, natakalim attenuates the elevation of plasma nitric oxide (NO) level and inducible NO synthase in cardiac tissue induced by isoproterenol. Additionally, natakalim inhibits the endothelin signaling system by decreasing both the content of endothelin-1 in the plasma and the protein levels of cardiac endothelin receptors A and B. Moreover, natakalim could augment the plasma prostacyclin concentration. In conclusion, our study provides evidence that natakalim effectively ameliorates isoproterenol-induced chronic heart failure in rats by protecting against endothelial dysfunction.

Brain protection against ischemic stroke using choline as a new molecular bypass treatment.

AIM: To determine whether administration of choline could attenuate brain injury in a rat model of ischemic stroke and the underlying mechanisms. METHODS: A rat model of ischemic stroke was established through permanent middle cerebral artery occlusion (pMCAO). After the surgery, the rats were treated with choline or choline plus the specific α7 nAChR antagonist methyllycaconitine (MLA), or with the control drug nimodipine for 10 days. The neurological deficits, brain-infarct volume, pial vessel density and the number of microvessels in the cortex were assessed. Rat brain microvascular endothelial cells (rBMECs) cultured under hypoxic conditions were used in in vitro experiments. RESULTS: Oral administration of choline (100 or 200 mg·kg(-1)·d(-1)) or nimodipine (20 mg·kg(-1)·d(-1)) significantly improved neurological deficits, and reduced infarct volume and nerve cell loss in the ischemic cerebral cortices in pMCAO rats. Furthermore, oral administration of choline, but not nimodipine, promoted the pial arteriogenesis and cerebral-cortical capillary angiogenesis in the ischemic regions. Moreover, oral administration of choline significantly augmented pMCAO-induced increases in the expression levels of α7 nAChR, HIF-1α and VEGF in the ischemic cerebral cortices as well as in the serum levels of VEGF. Choline-induced protective effects were prevented by co-treatment with MLA (1 mg·kg(-1)·d(-1), ip). Treatment of rBMECs cultured under hypoxic conditions in vitro with choline (1, 10 and 100 µmol/L) dose-dependently promoted the endothelial-cell proliferation, migration and tube formation, as well as VEGF secretion, which were prevented by co-treatment with MLA (1 µmol/L) or by transfection with HIF-1α siRNA. CONCLUSION: Choline effectively attenuates brain ischemic injury in pMCAO rats, possibly by facilitating pial arteriogenesis and cerebral-cortical capillary angiogenesis via upregulating α7 nAChR levels and inducing the expression of HIF-1α and VEGF.

Proteomics reveals potential non-neuronal cholinergic receptor-effectors in endothelial cells.

AIM: The non-neuronal acetylcholine system (NNAS) in endothelial cells participates in modulating endothelial function, vascular tone, angiogenesis and inflammation, thus plays a critical role in cardiovascular diseases. In this study, we used a proteomic approach to study potential downstream receptor-effectors of NNAS that were involved in regulating cellular function in endothelial cells. METHODS: Human umbilical vein endothelial cells were incubated in the presence of acetylcholine, oxotremorine, pilocarpine or nicotine at the concentration of 10 µmol/L for 12 h, and the expressed proteins in the cells were separated and identified with two-dimensional electrophoresis (2-DE) and LC-MS. The protein spots with the largest changes were identified by LC-MS. Biowork software was used for database search of the peptide mass fingerprints. RESULTS: Over 1200 polypeptides were reproducibly detected in 2-DE with a pH range of 3-10. Acetylcholine, oxotremorine, pilocarpine and nicotine treatment caused 16, 9, 8 and 9 protein spots, respectively, expressed differentially. Four protein spots were identified as destrin, FK506 binding protein 1A (FKBP1A), macrophage migration inhibitory factor (MIF) and profilin-1. Western blotting analyses showed that treatment of the cells with cholinergic agonists significantly decreased the expression of destrin, FKBP1A and MIF, and increased the expression of profilin-1. CONCLUSION: A set of proteins differentially expressed in endothelial cells in response to cholinergic agonists may have important implications for the downstream biological effects of NNAS.

[Mechanism of the inhibitory effects of thalidomide on expressions of VEGF and bFGF].

Objective: To investigate the inhibitory effects of thalidomide on the expressions of VEGF and bFGF in human lung adenocarcinoma A549 cells and human hepatocellular carcinomas HepG2 cells mediated by cereblon (CRBN). Methods: shRNA technology was used to construct the A549 cell line (A549CRBN) and HepG2 cell line (HepG2CRBN) with stable knockdown of CRBN, which was verified by real-time PCR and Western blot. A549 cells were divided into negative control group (A549luciferase) and CRBN down-regulation group (A549CRBN); HepG2 cells were divided into negative control group (HepG2luciferase) and CRBN down-regulation group (HepG2CRBN). The above cells were seeded into 6-well plates at 3×105 cells/well, and cultured in a 37℃, 5% CO2 incubator for 24 h. Then, 1 ml medium containing 100 µmol/L thalidomide (thalidomide group) and 1 ml medium containing 1‰ DMSO (control group) were added respectively, and the culture was continued for 24 hours before subsequent experiments. Each group was designed with three replicate wells. The effect of thalidomide on the activity of A549 cell line was detected by MTS assay. Real-time PCR was performed to detect mRNA expression levels of VEGF, bFGF and c-jun. ELISA assay was performed to detect protein expressions of VEGF and bFGF. Results: Compared with the control group, thalidomide at the concentrations of 1, 10, 50 and 100 µmol/L had no significant effects on the proliferation of A549 and HepG2 cells (P＞0.05). VEGF and bFGF levels in the A549CRBN or HepG2luciferase groups were significantly lower than those in the A549CRBN or HepG2CRBN groups (P＜0.05). Compared with the control group of the A549luciferase or HepG2luciferase, thalidomide inhibited the expressions of VEGF and bFGF in A549luciferase and HepG2luciferase cells (P＜ 0.05), but did not inhibit the expressions of VEGF and bFGF in A549CRBN and HepG2CRBN cells. Compared with the control group of the HepG2luciferase, thalidomide inhibited c-Jun expression in HepG2luciferase cells (P＜0.01), but did not significantly inhibit c-Jun expression in HepG2CRBN cells. Conclusion: The inhibitory effects of thalidomide on VEGF and bFGF expressions may be mediated by CRBN in A549 and HepG2 cells, and c-Jun may be one of the key transcription factors responsible for this inhibition.

The novel ATP-sensitive potassium channel opener iptakalim prevents insulin resistance associated with hypertension via restoring endothelial function.

AIM: To investigate the effects of iptakalim on endothelial dysfunction induced by insulin resistance (IR) and to determine whether iptakalim improved IR associated with hypertension in fructose-fed rats (FFRs) and spontaneously hypertensive rats (SHRs). METHODS: Human umbilical vein endothelial cells (HUVECs) were used for in vitro study. The levels of endothelial vasoactive mediators and eNOS protein expression were determined using radioimmunoassays, ELISAs, colorimetric assays or Western blotting. Sprague-Dawley rats were fed with a high-fructose diet. In both FFRs and SHRs, tail-cuff method was used to measure systolic blood pressure (SBP), and hyperinsulinemic- euglycemic clamp was used to evaluate IR states. RESULTS: (1) Cultured HUVECs incubated with the PI3-kinase inhibitor wortmannin (50 nmol/L) and insulin (100 nmol/L) induced endothelial dysfunction characterized by significantly reduced release of NO and expression of eNOS protein, and significantly increased production of ET-1. Pretreatment with iptakalim (0.1-10 µmol/L) could prevent the endothelial dysfunction. (2) In FFRs, the levels of SBP, fasting plasma glucose and insulin were significantly elevated, whereas the glucose infusion rate (GIR) and insulin sensitive index (ISI) were significantly decreased, and the endothelium-dependent vascular relaxation response to ACh was impaired. These changes could be prevented by oral administration of iptakalim (1, 3, or 9 mg·kg(-1)·d(-1), for 4 weeks). The imbalance between serum NO and ET-1 was also ameliorated by iptakalim. (3) In 2-4 month-old SHRs (IR was established at the age of 4 months), oral administration of iptakalim (1, 3, or 9 mg·kg(-1)·d(-1), for 8 weeks) significantly ameliorated hypertension and increased the GIR to the normal level. CONCLUSION: These results demonstrate that iptakalim could protect against IR-induced endothelial dysfunction, and ameliorate IR associated with hypertension, possibly via restoring the balance between NO and ET-1 signaling.

Targeting hypertension with a new adenosine triphosphate-sensitive potassium channel opener iptakalim.

Hypertension is the most common cardiovascular disease. The discovery of the antihypertensive action of adenosine triphosphate-sensitive potassium (K(ATP)) channel openers was a significant advance in the treatment of hypertension. Iptakalim is a novel K(ATP) channel opener with a unique chemical structure that differs from other K(ATP) openers. Among the 3 different subtypes of K(ATP) channels heterologously expressed in human embryonic kidney cells and Xenopus oocytes, iptakalim exhibits significant selectivity for SUR2B/Kir6.1 channels, mild effects on SUR2A/Kir6.2 channels, and fails to open SUR1/Kir6.2 channels. Iptakalim is a more potent activator of the SUR2B/Kir6.1 subtype of K(ATP) channels than diazoxide and pinacidil, the 2 most commonly studied K(ATP) channel openers. Iptakalim selectively produces arteriolar vasodilation with essentially no effect on the capacitance vessels. It can preferentially relax arterioles and small arteries, without affecting large arteries. Furthermore, iptakalim strongly lowers the blood pressure of hypertensive rodents and humans but has little effect on normotensive rodents and humans. Selective antihypertensive action is not observed with pinacidil or diazoxide and may be due to the high selectivity of iptakalim for the SUR2B/Kir6.1 subtype of K(ATP) channels, as well as its selective relaxation of resistance vessels. In pulmonary arterial smooth muscle cells, iptakalim inhibits the increase of cytoplasmic free Ca2+ concentration, as well as cell proliferation induced by endothelin-1. Furthermore, iptakalim has exerted protective effects against hypertensive damage to target organs in rats and improves endothelial dysfunction associated with cardiovascular diseases by selective activation of the SUR2B/Kir6.1 subtype of K(ATP) channels expressed in the endothelium. Clinical trials of iptakalim in the treatment of mild-moderate hypertension have been completed in China. In additional to strong antihypertensive efficacy, iptakalim seems to have a favorable safety and tolerability profile. Iptakalim is a promising new generation antihypertensive drug.

Activation of SUR2B/Kir6.1 subtype of adenosine triphosphate-sensitive potassium channel improves pressure overload-induced cardiac remodeling via protecting endothelial function.

We sought to explore new strategies targeting SUR2B/Kir6.1, a subtype of adenosine triphosphate (ATP)-sensitive potassium channels (KATP), against pressure overload-induced heart failure. The effects of natakalim, a SUR2B/Kir6.1 selective channel opener, on progression of cardiac remodeling were investigated. Pressure overload-induced heart failure was induced in Wistar rats by abdominal aortic banding. The effects of natakalim (1, 3, and 9 mg·kg⁻¹·d⁻¹ for 10 weeks) on myocardial hypertrophy and heart failure, cardiac histology, vasoactive compounds, and gene expression were assessed. Ten weeks after the onset of pressure overload, natakalim treatment potently inhibited cardiac hypertrophy and prevented heart failure. Natakalim remarkably inhibited the changes of left ventricular hemodynamic parameters and reversed the increase of heart mass index, left ventricular weight index, and lung weight index. Histological examination demonstrated that there was no significant hypertrophy or fibrosis in pressure-overloaded hearts of natakalim-treated rats. Ultrastructural examination of hearts revealed well-organized myofibrils with mitochondria grouped along the periphery of longitudinally oriented fibers in rats from the natakalim group. The content of serum nitric oxide and plasma prostacyclin was increased, whereas that of plasma endothelin-1 and cardiac tissue hydroxyproline and atrial and B-type natriuretic peptide messenger RNA was downregulated in natakalim-treated rats. Natakalim at 0.01-100 µM had no effects on isolated working hearts derived from Wistar rats; however, natakalim had endothelium-dependent vasodilatory effects on the isolated tail artery helical strips precontracted with norepinephrine. These results indicate that natakalim reduces heart failure caused by pressure overloading by activating the SUR2B/Kir6.1 KATP channel subtype and protecting against endothelial dysfunction.

[Functional and morphological structure changes in the gut barrier during cholinesterase inhibitor intoxication and therapeutic effect of benthiactzine in rats].

OBJECTIVE: To investigate the functional and morphological structure changes in the gut barrier of rats induced by cholinesterase inhibitor VX poisoning, and the therapeutic effect of benthiactzine. METHODS: Forty male Wistar rats were randomly divided into five groups: normal saline control group, VX poisoning (model) group, benthiactzine 1, 3, 9 mg/kg treatment groups, with 8 rats in each group. In the benthiactzine treatment groups, different dosages of the drug were respectively given (intraperitoneal injection) 5 minutes after VX poisoning (13 microg/kg subcutaneous injection). The plasma concentration of D-lactate and the diamine oxidase (DAO) activity, which reflected the gut barrier function, were measured at 3 hours after VX poisoning. At the same time point, the specimens from jejunum and ileum were harvested. The morphological changes in the intestinal mucosa were determined with light microscope and electron microscope. RESULTS: After VX poisoning, the plasma D-lactate concentration and the DAO activity in model group were significantly increased compared with those of control group [D-lactate concentration in model group was (87.752 + or - 22.906) mg/L which was higher than that of control group (29.072 + or - 6.546) mg/L; DAO activity in model group was (6.72 + or - 0.93) U/L which was higher than that of control group (2.99 + or - 0.43) U/L, both P<0.01]. These values could be decreased dose-dependently after benthiactzine 1, 3, 9 mg/kg administration after the VX poisoning. Furthermore, the increase in D-lactate (45.290 + or - 11.141) mg/L and DAO activity (3.17 + or - 0.68) U/L could be totally reversed by 9 mg/kg of benthiactzine (both P<0.01). In model group, the intestinal mucosal epithelial injury was obvious at 3 hours after VX poisoning as shown under light microscope, including diminution of the mucosal thickness and the height of villi in jejunum and ileum, interstitial edema, angiotelectasis. Also electronic microscopic examination revealed damaged organelles and cell tight junction of mucosal epithelium. These pathological changes in intestine could be inhibited by benthiactzine in dose-dependent manner. CONCLUSION: The gut barrier function in rats was seriously damaged by the cholinesterase inhibitor agents poisoning, as a result of injury to intestinal mucosa and increase of intestinal permeability. Benthiactzine exerts protection against functional and morphological structure damages of the gut barrier during intoxication.

[Therapeutic effects of benthiactzine against respiratory failure induced by cholinesterase inhibitor in rats].

OBJECTIVE: To investigate the therapeutic effects of benthiactzine against respiratory failure induced by cholinesterase inhibitor dimethyl dichloro-vinyl phosphate (DDVP) in rats. METHODS: Forty-five male Wistar rats were divided into five groups randomly: control group, model group, and benthiactzine 0.5, 1.0, 2.0 mg/kg treatment groups (each n=9). Rats were treated with DDVP by intraperitoneal injection to reproduce respiratory failure model. The symptoms, respiratory rate (RR), blood gas analysis, electrolyte and plasma superoxide dismutase (SOD), malondialdehyde (MDA) and the pathological changes were observed before poisoning, during respiratory failure, and in different periods after the treatment. RESULTS: In rats with respiratory failure induced by DDVP, cyanosis and convulsion occurred in all groups. The success rates in three benthiactzine groups were 66.7% (6/9), 77.8% (7/9) and 88.9% (8/9). The rats of benthiactzine treatment groups recovered in 1-5 minutes after treatment and returned to normal state in 30 minutes. RR also returned to normal in 30 minutes. When respiratory failure occurred, arterial oxygen partial pressure (PaO2), arterial oxygen saturation (SaO2) and plasma SOD were decreased, plasma MDA was increased, and mixed acidosis was found. Thirty minutes after the treatment of benthiactzine, all above parameters in three groups returned to normal (all P<0.01). In respiratory failure rats, pathological examination of lung tissue revealed dilatation of pulmonary vessels with aggregation of erythrocytes, widening of alveolar space with presence of red blood cells in alveoli with heavy infiltration of inflammatory cells, and pulmonary edema and hemorrhage. The lungs of rats treated with benthiactzine showed less intense pathological changes. CONCLUSION: The new medicine against poisoning benthiactzine can be a favourable drug against respiratory failure induced by organophosphorus pesticides.

SUR2B/Kir6.1 channel openers correct endothelial dysfunction in chronic heart failure via the miR-1-3p/ET-1 pathway.

The SUR2B/Kir6.1 channel openers iptakalim and natakalim reverse cardiac remodeling and ameliorate endothelial dysfunction by re-establishing the balance between the nitric oxide and endothelin systems. In this study, we investigated the microRNAs (miRs) involved in the molecular mechanisms of SUR2B/Kir6.1 channel opening in chronic heart failure. Both iptakalim and natakalim significantly upregulated the expression of miR-1-3p, suggesting that this miR is closely associated with the therapeutic effects against chronic heart failure. Bioinformatic analysis showed that many of the 183 target genes of miR-1-3p are involved in cardiovascular diseases, suggesting that miR-1-3p plays a vital role in such diseases and vascular remodeling. Target gene prediction showed that miR-1-3p combines with the 3' untranslated region (UTR) of endothelin-1 (ET-1) mRNA. Iptakalim and natakalim upregulated miR-1-3p expression and downregulated ET-1 mRNA expression in vitro. The dual luciferase assay confirmed that there is a complementary binding sequence between miR-1-3p and the 3' UTR 158-165 sequence of ET-1 mRNA. To verify the effect of miR-1-3p on ET-1, lentiviral vectors overexpressing or inhibiting miR-1-3p were constructed for the transduction of rat primary cardiac microvascular endothelial cells. The results showed that natakalim enhanced the miR-1-3p level. miR-1-3p overexpression downregulated the expression of ET-1, whereas miR-1-3p inhibition had the opposite effect. Therefore, we verified that SUR2B/Kir6.1 channel openers could correct endothelial imbalance and ameliorate chronic heart failure through the miR-1-3p/ET-1 pathway in endothelial cells. Our study provides comprehensive insights into the molecular mechanisms behind the SUR2B/Kir6.1 channel's activity against chronic heart failure.

Activation of ATP-sensitive potassium channels protects vascular endothelial cells from hypertension and renal injury induced by hyperuricemia.

BACKGROUND AND OBJECTIVES: It has been demonstrated that hyperuricemia induces reno-cardiovascular damage resulting in hypertension and renal injury because of vascular endothelial dysfunction. The pathogenesis of hyperuricemia, endothelial dysfunction, hypertension, and renal injury is progressive, and develops into a vicious cycle. It is reasonable to suggest that an antihypertensive drug with endothelial protection may block this vicious cycle. Iptakalim, a novel antihypertensive drug undergoing phase-three clinical trials, is a new ATP-sensitive potassium channel opener and can ameliorate endothelial dysfunction. We hypothesized that iptakalim could prevent hypertension and retard the pathogenesis of endothelial dysfunction and renal injury in hyperuricemic rats. METHODS AND RESULTS: In rats with hyperuricemia induced by 2% oxonic acid and 0.1 mmol/l uric acid, iptakalim prevented increases in systolic blood pressure, reduced the impairment of endothelial vasodilator function, and attenuated renal dysfunction and pathological changes in glomerular and renal interstitial tissue at 0.5, 1.5, and 4.5 mg/kg orally daily for 4 weeks. Serum levels of nitric oxide and prostacyclin, and gene expression of endothelial nitric oxide synthase in the aortic and intrarenal tissue, were increased, whereas the serum levels of endothelin-1 and gene expression of endothelin-1 in aortic and intrarenal tissue were decreased. However, serum levels of angiotensin II and renin remained unchanged in the hyperuricemic rats treated with iptakalim. In cultured rat aortic endothelial cells, amelioration of endothelial dysfunction by iptakalim was suggested by inhibition of the overexpression of intercellular adhesive molecule-1, vascular cell adhesive molecule-1, and monocyte chemoattractant protein-1 mRNA induced by uric acid, and reversal of the inhibitory effects of uric acid on nitric oxide release in a concentration-dependent manner, which could be abolished by pretreatment with glibenclamide, an ATP-sensitive potassium channel blocker. Iptakalim ameliorated hyperuricemia in this rat model by decreasing renal damage through its antihypertensive and endothelial protective properties, and it had no direct effects on anabolism, catabolism and excretion of uric acid. CONCLUSION: These findings suggest that the activation of ATP-sensitive potassium channels by iptakalim can protect endothelial function against hypertension and renal injury induced by hyperuricemia. Iptakalim is suitable for use in hypertensive individuals with hyperuricemia.

A new ATP-sensitive potassium channel opener protects endothelial function in cultured aortic endothelial cells.

OBJECTIVE: Endothelial dysfunction is an early risk factor for cardiovascular disease and hypertension. Mechanisms that participate in endothelial dysfunction include reduced nitric oxide (NO) generation and increased endothelin-1 (ET-1) generation. Endothelial ATP-sensitive potassium (K(ATP)) channels are responsible for maintaining the resting potential of endothelial cells and modulating the release of vasoactive compounds. We hypothesized that activation of endothelial K(ATP) channels might result in the protection against endothelial dysfunction. METHODS: Using cultured bovine or rat aortic endothelial cells, we examined the effects of a new K(ATP) channels opener, iptakalim, on the secretion of vasoactive substances. We also investigated its effects on the expression of adhesion molecules in metabolically disturbed cultured endothelial cells. RESULTS: In cultured aortic endothelial cells, iptakalim caused a concentration-dependent inhibition of ET-1 release and synthesis that correlated with reduced levels of mRNA for ET-1 and endothelin-converting enzyme. These effects of iptakalim were significantly inhibited by pretreatment with glibenclamide (a K(ATP) channel blocker) for 1 h. Similarly, iptakalim enhanced the release of NO in a concentration-dependent manner and increased basal levels of free intracellular calcium. Iptakalim at the concentrations of 100 and 1000 microM increased the activities of NO synthase (NOS) significantly. After the activity of NOS was blocked by L-N(omega)-nitro-arginine methyl ester (L-NAME), the inhibition of iptakalim on ET-1 release was abolished. In endothelial cell models of metabolic disturbance induced by low-density lipoprotein, homocysteine, or hyperglycemia, treatment with iptakalim could inhibit the overexpression of monocyte chemoattractant protein-1 (MCP-1), Intercellular adhesive molecule-1 (ICAM-1), and vascular cell adhesive molecule-1 (VCAM-1) mRNA. CONCLUSION: Iptakalim is a promising drug that could protect against endothelial dysfunction through activating K(ATP) channels in endothelial cells.

ATP-sensitive potassium channel openers and 2,3-dimethyl-2-butylamine derivatives.

ATP-sensitive potassium (K(ATP)) channels have important functions through their coupling of cellular energetic networks and their ability to decode metabolic signals, and they are implicated in diseases of many organs. K(ATP) channels are formed by the physical association between the inwardly rectifier potassium channels (Kir6.x) and the regulatory sulfonylurea receptor subunit (SUR), which form a hetero-octameric complex. Different subtypes of K(ATP) channels exist in various tissues. K(ATP) channel openers (KCOs) are classified into nine chemical families according to their molecular structures: (1) benzopyrans, (2) cyanoguanidines, (3) thioformamides, (4) pyrimidine derivatives, (5) pyridine derivatives, (6) benzothiadiazines, (7) dihydropyridines, (8) nicotinamide derivatives, and (9) aliphatic amines. Although the model also predicts that KCOs have four co-binding areas, it was hypothesized that the main contribution lies in the binding domain of hydrophobicity of the side chain. A series of compounds containing the skeleton of the aliphatic secondary amines as a side chain was designed. It was found that N-isopropyl 2,3-dimethyl-2-butylamine (iptakalim, 91) is a novel KCOs. Iptakalim regulates the pore selectively of the inwardly rectifier potassium channel and dilates smaller arteries, but has little effect on vasodilatation of the aorta. Iptakalim administered p.o. has selective and long-lasting antihypertensive effects in hypertensive animals and does not induce tolerance, but has little effect on blood pressure in normotensive animals. Meanwhile, it also reverses cardiovascular remodeling and protects the brain and kidney against damage caused by hypertension in animal models. Iptakalim is in phase II clinical trials now and has a promising future as a treatment for hypertension.

[Molecular of pulmonary arterioles relaxation through SUR2B/Kir6.1 channel opening].

OBJECTIVE: To study the dilatation characteristics of ATP-sensitive potassium channel (KATP) SUR2B/Kir6.1 subtype opener iptakalim (Ipt) in pulmonary arterioles, and to explore its possible mechanism. METHODS: Vessels pressure-diameter monitoring perfusion technique was used to observe the dilatation effects of Ipt in rats fourth pulmonary arterioles (n=6~8). After the pulmonary arterioles were pre-treated with removing endothelium or pre-incubated with KATP channel blocker glibenclamide (Gli), cyclo-oxygenase (COX) inhibitor indomethacin (Indo) and nitric oxide synthase (NOS) inhibitor L-Nω-Nitro-arginine methyl ester(L-NAME), the dilatation effects of Ipt were observed. RESULTS: Pulmonary arterioles could be relaxed by Ipt, the maximal relaxation rate was (60.53±2.08)%. Compaired with control group, the effects of Ipt in endothelium denuded arterioles were significantly decreased, the maximal relaxation rate was (9.47±1.56)% (P<0.01). The maximal relaxation rate were decreased to(17.49±1.47)%,(37.00±3.88)% and(24.91±2.30)% respectively after Gli,Indo,L-NAME were pre-incubated (P<0.01). CONCLUSIONS: Pulmonary arterioles can be relaxed by Ipt. The selective activation of KATP SUR2B/Kir6.1 subtype by Ipt was involved in its mechanisms. The endothelium-dependently dilatation of Ipt was related to nitric oxide (NO) and prostacyclin (PGI2) released by endothelial cells.

[Construction of T vectors based on Xcm I recognition site and optimization of PCR fragments for ligation].

OBJECTIVE: To construct T vectors based on Xcm I recognition site and optimize the PCR fragments for its ligation. METHODS: We firstly cloned the human histone H4 cDNA containing one Xcm I recognition site at both its 5' and 3' end into pCDNA 3.0 vector and then generated T vector with pCDNA 3.0 backbone by cutting the recombinant plasmid with Xcm I. To increase the ligation efficiency, the primers were firstly phosphorylated before DNA fragments amplification and then the PCR products were treated with Taq DNA polymerase and dATP after PCR amplification. Two DNA fragments with the length of 312 bp and 1 329 bp were ligated to it and the ligation mixture was transformed into E. coli DH5α competent cells and the positive rates of the transformants were evaluated by PCR and DNA agarose gel electrophoresis. RESULTS: Our results showed that the T vector produced by our method could ligate to the target DNA fragments with high efficiency. Besides, the phosphorylation state of the primers used for PCR amplification is also an important factor determining the cloning efficiency. What's more, as for longer DNA fragments, retreatment with Taq DNA polymerase and dATP after PCR amplification and purification could improve the ligation efficiency significantly. CONCLUSION: Our protocol may overcome the dependence on blue/white screening to get positive clones and provide a potent way to generate T vectors and ligate them to the target PCR fragment.

[The effect of pioglitazone on apoptotic cardiomyocytes for ischemia reperfusion].

OBJECTIVE: This study was to investigate the effect of pioglitazone on apoptotic cardiomyocytes with the model of ischemia-reperfusion at rat heart in vivo. METHODS: Sprague-Dawley rats were randomly divided into two groups. One was 30 min reperfusion group, which was subdivided into sham (n = 5), model (vehicle, n = 6) and pioglitazone 3 mg/kg (n = 7) with 30 min ischemia followed by 30 min reperfusion to detect the area of myocardial infarction (MI). Another was 2 h reperfusion group, which was further subdivided into sham (n = 5), model (vehicle, n = 6), and pioglitazone 0.3 mg/kg (n = 6), 1 mg/kg (n = 7) and 3 mg/kg (n = 6). Apart from the sham, pioglitazone and vehicle were administered intravenously 30 min before occlusion. Then hearts were excised, paraffined and cut into 4 microm thick. Immunohistochemistry, in situ hybridization, TUNEL and DNA agarose gel electrophoresis were performed to detect the expression of Bax, Bcl-2, Caspase-3 and PPARgamma protein and PPARgamma mRNA. RESULTS: (1) Compared with model, nec/aar of pioglitazone decreased by 28% (P < 0.01). The nec/lv ratio reduced by 32% (P < 0.01). (2) In a dose-dependent manner, the expressions of Bax and Caspase-3 were depressed, while the expression of Bcl-2, PPARgamma protein and PPARgamma mRNA were enhanced by pioglitazone. (3) The apoptotic index of subgroups injected pioglitazone reduced significantly by TUNEL compared with model (P < 0.05). Agarose gel electrophoresis demonstrated that DNA ladder existed in model, pioglitazone 0.3 mg/kg and pioglitazone 1 mg/kg, but not pioglitazone 3 mg/kg. CONCLUSIONS: Pioglitazone could protect the heart from I/R injury evidenced by the improvement in the expression of PPARgamma at the levels of protein and mRNA after pioglitazone administrated, and by the decrease in the apoptotic cardiomyocytes.

[Effects of hypoxic acclimatization on myocardial sarcoplasmic reticulum ATPase and 45Ca2+ uptake in rats].

OBJECTIVE: To study the effect of acute hypoxia and hypoxic acclimatization on myocardial function of rats. METHOD: Eighteen male Wistar rats were randomly divided into three groups: normoxic control, acute hypoxia and intermittent hypoxic acclimatization group (n=6). After being exposed to hypoxia (8000 m) for 4 h before and after intermittent hypoxic acclimatization (3000 m and 5000 m, 14 d respectively, 4 h/d), the rats were decapitated and then myocardial sarcoplasmic reticulum (SR) were derived from cardiac muscles. Activities of Na+, K(+)-ATPase, Ca2+, Mg2(+)-ATPase in SR, phosphorylation of phospholamban (PLB) and the ability of 45Ca2+ uptake in SR were observed in all these three groups. RESULT: 1) Hypoxia had no effects on the activity of Na+, K(+)-ATPase in rats myocardial SR of rats. 2) Compared with normoxic control rats, the activity of Ca2+, Mg2(+)-ATPase in myocardial SR of rats after acute hypoxia was reduced significantly (P<0.01). After intermittent hypoxic acclimatization, its activity increased significantly as compared with that of acute hypoxic rats (P<0.01). 3) The phosphorylation of PLB in acute hypoxic rats was reduced significantly compared with normoxic control rats. After intermittent hypoxic acclimatization, its phosphorylation was increased significantly compared with that of acute hypoxic rats. It suggests that hypoxic acclimatization could alleviate the inhibition of calcium pump. 4) The ability of 45Ca2+ uptake of SR in acute hypoxic rats was decreased significantly. After hypoxic acclimatization, its ability was strengthened significantly. CONCLUSION: These results suggest that the increased function of myocardial SR calcium pump, the strengthened phosphorylation of PLB to alleviate the inhibition of calcium pump and the increased function of Ca2+ transport in SR are the mechanisms of hypoxic acclimatization protecting cardiac functions from injury induced by severe hypoxia.

The molecular pathway of ATP-sensitive potassium channel in endothelial cells for mediating arteriole relaxation.

AIMS: The endothelial molecular pathway of a new ATP-sensitive potassium channel (KATP) opener natakalim was investigated in mesenteric arterioles of rats. MAIN METHODS: A DMT wire myograph was used to evaluate the vasorelaxation effects of natakalim. Ca(2+) responses of endothelial cells induced by natakalim were measured by laser confocal fluorescence microscopy. NO assay kits and Western blotting were used. KEY FINDINGS: The new KATP opener natakalim significantly produced endothelium-dependent arteriolar dilation and increased endothelial cell intracellular calcium concentration ([Ca(2+)]i) as well as NO release, which could be inhibited by SB366791 and capsazepine, the specific TRPV1 blockers. Additionally, down-regulation of endothelial TRPV1 by RNA interference inhibited the Ca(2+) influx induced by natakalim. SIGNIFICANCE: These results suggest that endothelial KATP mediated natakalim-induced vasorelaxation through increasing [Ca(2+)]i and NO production. Activation of endothelial TRPV1 channels and subsequent Ca(2+) entry, and NO release at least partly contribute to endothelium-dependent vasorelaxation induced by natakalim.