二甲基氨氯吡咪对心肌肥厚大鼠离体心功能的影响及机制探讨

目的研究二甲基氨氯吡咪(d im ethylam iloride,DMA)对心肌肥厚大鼠离体心功能的影响,并探讨其可能的作用机制。方法应用Langendorff心脏灌流方法观察DMA对心肌肥厚大鼠离体心脏功能的影响,并使用L-型钙通道阻滞剂和Na+/Ca+交换(sod ium calc ium exchanger,NCX)抑制剂探讨其可能机制。结果在Langendorff灌流的大鼠心脏,DMA(0.5~2)μmol.L-1能增强心肌肥厚大鼠离体心脏功能,即增强左室主动收缩压(LVSP-LVDP)、左室压最大上升速率(+dp/dtm ax)、和左室压最大下降速率(-dp/dtm ax),与用药前比较,差异均具有显著性(P<0.05或P<0.01)。DMA对心肌肥厚大鼠离体心功能的增强作用不能被L-型钙通道阻滞剂尼卡地平(n icard ip ine)阻断,但能被NCX抑制剂氯化镍(N iC l2)阻断。结论DMA(0.5~2μmol.L-1)能增强心肌肥厚大鼠心脏的收缩和舒张功能。DMA增强心肌肥厚大鼠心脏功能的作用可能是通过激动NCX实现的,与L-型钙通道无关。     

Adenovirally delivered shRNA strongly inhibits Na+-Ca2+ exchanger expression but does not prevent contraction of neonatal cardiomyocytes

The cardiac Na(+)-Ca(2+) exchanger (NCX1) is the main mechanism for Ca(2+) efflux in the heart and is thought to serve an essential role in cardiac excitation-contraction (E-C) coupling. The demonstration that an NCX1 gene knock-out is embryonic lethal provides further support for this essential role. However, a recent report employing the Cre/loxP technique for cardiac specific knock-out of NCX1 has revealed that cardiac function is remarkably preserved in these mice, which survived to adulthood. This controversy highlights the necessity for further investigation of NCX1 function in the heart. In this study, we report on a novel approach for depletion of NCX1 in postnatal rat myocytes that utilizes RNA interference (RNAi), administered with high efficiency via adenoviral transfection. Depletion of NCX1 was confirmed by immunocytochemical detection, Western blots and radioisotopic assays of Na(+)-Ca(2+) exchange activity. Exchanger expression was inhibited by up to approximately 94%. Surprisingly, spontaneous beating of these cardiomyocytes was still maintained, although at a lower frequency. Electrical stimulation could elicit a normal beating rhythm, although NCX depleted cells exhibited a depressed Ca(2+) transient amplitude, a depressed rate of Ca(2+) rise and decline, elevated diastolic [Ca(2+)], and shorter action potentials. We also observed a compensatory increase in sarcolemmal Ca(2+) pump expression. Our data support an important, though non-essential, role for the NCX1 in E-C coupling in these neonatal heart cells. Furthermore, this approach provides a valuable means for assessing the role of NCX1 and could be utilized to examine other cardiac proteins in physiological and pathological studies.     

Two sides of the same coin: Sodium homeostasis and signaling in astrocytes under physiological and pathophysiological conditions

The intracellular sodium concentration of astrocytes is classically viewed as being kept under tight homeostatic control and at a relatively stable level under physiological conditions. Indeed, the steep inwardly directed electrochemical gradient for sodium, generated by the Na⁺/K⁺‐ATPase, contributes to maintain the electrochemical gradient of K⁺ and the highly K⁺‐based negative membrane potential, and is a central element in energizing membrane transport. As such it is tightly coupled to the homeostasis of extra‐ and intracellular potassium, calcium or pH and to the reuptake of transmitters such as glutamate. Recent studies, however, have demonstrated that this picture is far too simplistic. It is now firmly established that transmitters, most notably glutamate, and excitatory neuronal activity evoke long‐lasting sodium transients in astrocytes, the properties of which are distinctly different from those of activity‐related glial calcium signals. From these studies, it emerges that sodium homeostasis and signaling are two sides of the same coin: sodium‐dependent transporters, primarily known for their role in ion regulation and homeostasis, also generate relevant ion signals during neuronal activity. The functional consequences of activity‐related sodium transients are manifold and are just coming into view, enabling surprising and important new insights into astrocyte function and neuron‐glia interaction in the brain. The present review will highlight current knowledge about the mechanisms that contribute to sodium homeostasis in astrocytes, present recent data on the spatial and temporal properties of activity‐related glial sodium signals and discuss their functional consequences with a special emphasis on pathophysiological conditions. GLIA 2013;61:1191–1205     

Discovery and characterization of ORM‐11372, a novel inhibitor of the sodium‐calcium exchanger with positive inotropic activity

Background and PurposeThe lack of selective sodium–calcium exchanger (NCX) inhibitors has hampered the exploration of physiological and pathophysiological roles of cardiac NCX 1.1. We aimed to discover more potent and selective drug like NCX 1.1 inhibitor.Experimental ApproachA flavan series‐based pharmacophore model was constructed. Virtual screening helped us identify a novel scaffold for NCX inhibition. A distinctively different NCX 1.1 inhibitor, ORM‐11372, was discovered after lead optimization. Its potency against human and rat NCX 1.1 and selectivity against other ion channels was assessed. The cardiovascular effects of ORM‐11372 were studied in normal and infarcted rats and rabbits. Human cardiac safety was studied ex vivo using human ventricular trabeculae.Key ResultsORM‐11372 inhibited human NCX 1.1 reverse and forward currents; IC₅₀ values were 5 and 6 nM respectively. ORM‐11372 inhibited human cardiac sodium 1.5 (I _Na) and hERG K_V11.1 currents (I _hERG) in a concentration‐dependent manner; IC₅₀ values were 23.2 and 10.0 μM. ORM‐11372 caused no changes in action potential duration; short‐term variability and triangulation were observed for concentrations of up to 10 μM. ORM‐11372 induced positive inotropic effects of 18 ± 6% and 35 ± 8% in anaesthetized rats with myocardial infarctions and in healthy rabbits respectively; no other haemodynamic effects were observed, except improved relaxation at the lowest dose.Conclusion and ImplicationsORM‐11372, a unique, novel, and potent inhibitor of human and rat NCX 1.1, is a positive inotropic compound. NCX inhibition can induce clinically relevant improvements in left ventricular contractions without affecting relaxation, heart rate, or BP, without pro‐arrhythmic risk.     

Biphasic effects of nitric oxide on calcium influx in human platelets

In this study the effects of nitric oxide (NO) donors on intracellular free calcium ([Ca²⁺]_i) in human platelets was examined. Inhibition of guanylyl cyclase (GC) with either methylene blue or ODQ slightly inhibited the ability of submaximal concentrations of thrombin to increase [Ca²⁺]_i which suggests that a small portion of the thrombin mediated increase in [Ca²⁺]_i was due to an increase in NO and subsequent increase in cGMP and activation of cGMP dependent protein kinase (cGPK). Thrombin predominantly increases [Ca²⁺]_i by stimulating store-operated Ca²⁺ entry (SOCE). The NO donor GEA3162 was previously shown to stimulate SOCE in some cells. In platelets GEA3162 had no effect to increase [Ca²⁺]_i however it inhibited the ability of thrombin to increase [Ca²⁺]_i and this effect was reversed by ODQ. The addition of low concentrations (2.0 - 20 nM) of the NO donor sodium nitroprusside (SNP) slightly potentiated the ability of thrombin to increase [Ca²⁺]_i whereas higher concentrations (> 200 nM) of SNP inhibited thrombin induced increases in [Ca²⁺]_i. Both of these effects of SNP were reversed by ODQ which implies that they were both mediated by cGPK. Ba²⁺ influx was stimulated by low concentrations (2.0 nM) of SNP and inhibited by high concentrations (> 200 nM) of SNP and both effects were inhibited by ODQ. Previous studies showed that Ba²⁺ influx was blocked by the SOCE inhibitors 2-aminoethoxydipheny borate and diethylstilbestrol. It was concluded that low levels of SNP can stimulate SOCE in platelets and this effect may account for the increased aggregation and secretion previously observed with low concentrations of NO donors. Of the proteins known to be involved in SOCE (e.g. stromal interaction molecule 1 (Stim1), Stim2 and Orai1) only Stim2 has cGPK phosphorylation sites. The possibility that Stim2 phosphorylation regulates SOCE in platelets is discussed.     

Emerging therapeutic targets in chronic heart failure: Part I

Chronic heart failure (CHF) is a life threatening disease with an enormous medical requirement. Approximately 15 million people worldwide suffer from CHF. The prevalence will inevitably increase due to the ageing population. Nevertheless, current treatment options based on angiotensin-converting enzyme inhibitors and β-adrenergic receptor antagonists merely slow progression of the disease. Novel treatment concepts based on new therapeutic targets must have the capability to reverse the severity of this disease. This review, focusing on the emerging targets in the most promising therapeutic areas for the treatment of CHF, will be divided into two parts. In Part I, disease concepts such as altered calcium handling and ion channel activity, pathophysiological hypertrophy and inefficient cardiac metabolism are discussed. Validation status and potential therapeutic value for new targets in each research field is given by summarising the results of in vitro and in vivo studies.     

SEA0400, a specific Na+/Ca2+ exchange inhibitor, prevents dopaminergic neurotoxicity in an MPTP mouse model of Parkinson's disease

We have recently shown that the Na⁺/Ca²⁺ exchanger (NCX) is involved in nitric oxide (NO)-induced cytotoxicity in cultured astrocytes and neurons. However, there is no in vivo evidence suggesting the role of NCX in neurodegenerative disorders associated with NO. NO is implicated in the pathogenesis of neurodegenerative disorders such as Parkinson’s disease. This study examined the effect of SEA0400, the specific NCX inhibitor, on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity, a model of Parkinson’s disease, in C57BL/6J mice. MPTP treatment (10 mg/kg, four times at 2-h intervals) decreased dopamine levels in the midbrain and impaired motor coordination, and these effects were counteracted by S-methylthiocitrulline, a selective neuronal NO synthase inhibitor. SEA0400 protected against the dopaminergic neurotoxicity (determined by dopamine levels in the midbrain and striatum, tyrosine hydroxylase immunoreactivity in the substantia nigra and striatum, striatal dopamine release, and motor deficits) in MPTP-treated mice. SEA0400 had no radical-scavenging activity. SEA0400 did not affect MPTP metabolism and MPTP-induced NO production and microglial activation, while it attenuated MPTP-induced increases in extracellular signal-regulated kinase (ERK) phosphorylation and lipid peroxidation product, thiobarbituric acid reactive substance. These findings suggest that SEA0400 protects against MPTP-induced neurotoxicity probably by blocking ERK phosphorylation and lipid peroxidation which are downstream of NCX-mediated Ca²⁺ influx.     

Subeffective doses of nitroparacetamol (NCX-701) enhance the antinociceptive activity of the α₂-adrenoceptor agonist medetomidine

The α₂-adrenergic system is involved in pain processing and inflammation-induced sensitization. α₂-adrenoceptor agonists induce analgesia, and this effect is greater when administered in combination with other analgesics. In the present study, we assessed a possible enhancement of antinociception combining the α₂-adrenoceptor agonist medetomidine with subeffective doses of NCX701 (nitroparacetamol). The effects of the drugs were studied in spinal cord neuronal responses from adult male Wistar rats with carrageenan-induced inflammation, using the recording of single motor unit technique. The experiments showed that the i.v. administration of medetomidine and NCX701 induced a more potent and effective antinociceptive effect than medetomidine when given alone (ID₅₀: 0.47 ± 0.1 vs. 1.1 ± 0.1 μg/kg) or in the presence of paracetamol, in naturally-evoked nociceptive responses. In addition, the duration of antinociception was significantly longer (P < 0.001, 100 min after administration). The use of low doses of NCX701 and α₂-adrenoceptor agonists might open new perspectives in the treatment of inflammatory pain.     

SNL大鼠鞘内注射KB-R7943的镇痛作用研究

目的 评价SNL大鼠鞘内注射KB-R7943的镇痛作用,并探讨其可能的机制。方法 选取成年雄性SD大鼠（180~220 g）,建立SNL模型,通过测定机械痛阈选取SNL模型建立成功的大鼠,采用免疫荧光检测其手术侧L4~6脊髓背角钠钙交换蛋白（sodium calcium exchanger,NCX）定位情况;成年雄性SD大鼠进行鞘内置管,置管成功后1周,制作SNL模型,于SNL模型建立的第7天给予鞘内注射不同剂量（5 μg、10 μg、20 μg）KB-R7943以及1% DMSO对照溶剂,通过行为学检测其鞘内注射后引起的机械痛阈和热痛阈变化;记录成年雄性SD大鼠脊髓背角C纤维诱发电位,给予坐骨神经强直电刺激诱发C纤维电位长时程增强（long-term potentiation,LTP）,局部脊髓表面给予KB-R7943以及1% DMSO对照溶剂,记录C纤维电位ＬＴＰ的幅度变化。结果 NCX表达在大鼠L4~6脊髓背角浅层的C纤维投射区内。SNL大鼠在术后第7天给予鞘内注射NCX反向转运抑制剂KB-R7943,KB-R7943注射组大鼠手术侧足底的机械痛阈及热痛阈均高于溶剂组（P＜0.05）,其抑制作用与剂量呈正相关。大鼠诱发脊髓C纤维电位LTP后,给予局部孵育KB-R7943后可降低C纤维电位幅度,单纯给予溶剂不会影响C纤维电位幅度。结论 SNL大鼠鞘内注射KB-R7943具有剂量依赖性的抗神经病理性疼痛作用,其机制与抑制大鼠脊髓背角内的NCX反向转运功能,并抑制脊髓背角C纤维诱发电位的长时程增强有关。