Novel insight into the mechanisms involved in the regulation of the m1 muscarinic receptor,iNOS and nNOS mRNA levels

In this paper we have determined the different signaling pathways involved in M(1) muscarinic acetylcholine receptor (mAChR)-dependent stimulation of m1 mAChRs, neural and inducible isoforms of nitric oxide synthase (nNOS and iNOS)-mRNA gene expression of rat frontal cortex. Carbachol-stimulation of M(1) mAChRs exerts an increase in m1 mAChR-mRNA, activation of phosphoinositide (PI) turnover, translocation of protein kinase C (PKC) and stimulation of NOS activity. Inhibitors of phospholipase C (PLC), calcium/calmodulin and NOS, but not guanylate cyclase, prevent the carbachol-dependent increase of m1 mAChR-mRNA levels. These inhibitors also attenuate the muscarinic receptor-dependent increase in nNOS and iNOS mRNA levels. These results suggest that carbachol-activation of M(1) mAChRs increases m1 mAChR, nNOS and iNOS mRNA levels associated with increased production of nitric oxide (NO). The mechanism appears to occur secondarily to stimulation of PI turnover via PLC activation. This in turn, triggers a cascade reaction involving calcium/calmodulin and PKC, leading to activation of NOS. On the basis of our results, the activation of M(1) mAChRs appears to induce nNOS and iNOS expression and, reciprocally, the activator of NOS up-regulates m1 mAChR gene expression. These results may contribute to a better understanding of the effects and side effects of cholinomimetic treatment in patients with neurodegenerative diseases.     

Elevated neuronal nitric oxide synthase expression in chronic haloperidol-treated rats

Long-term administration of neuroleptic drugs, such as haloperidol, in the management of psychiatric disorders may adversely cause an irreversible neurological syndrome of tardive dyskinesia, which is associated with dopamine (DA(2)) receptor supersensitivity in the basal ganglia. Recent studies also indirectly suggest an involvement of nitric oxide synthase (NOS) in dopaminergic supersensitivity; however, chronic neuroleptic effects on neuronal NOS (nNOS) expression in the basal ganglia have not been reported. In this investigation, we treated rats with saline or haloperidol (1 mg/kg, s.c.) daily for 21 days. Five days later, we detected a significant increase of NOS activity in the striatum of haloperidol-treated rats when compared to saline controls. This effect was associated with elevated levels of nNOS mRNA and protein expression in the striatum, but not in the nucleus accumbens, as evidenced by the use of in situ hybridization, Western blot and immunohistochemical techniques. The involvement of the nNOS system after chronic haloperidol treatment coincides with increases of striatal DA(2) receptor sites, calmodulin kinase II activity, animal locomotor and stereotypy behaviors. This study suggests an integral role between nNOS, DA(2) receptor and calmodulin system in the development of dopaminergic behavioral supersensitivity resulting from chronic neuroleptic drug treatment. Furthermore, the toxic effect of chronic haloperidol on NOS system selectivity takes place in the neostriatum.     

Evidence for a calmodulin inhibitory substance(s) isolated from human meningiomas

Summary Calmodulin (CaM), a calcium-binding protein, is present in human tumor tissues and in meningioma. Following a purification procedure using DEAE-cellulose and the polymeric resin 3520, the CaM content of tumor extracts was assayed using CaM-deficient phosphodiesterase (PDE). In the presence of low amounts of the extracts, a concentration dependent stimulation of PDE was observed. However, further addition of higher concentrations of the extract produced a marked inhibition of the CaM stimulation of PDE in 13 of 15 specimens. A wide range (2.44–51.31 units/1 mg tumor [wet weight]) of inhibitor concentration was noted. However, no detectable inhibitory activity of this magnitude was observed in normal human meningeal extracts. The final extracts showed no calcineurin-phosphatase activity in the presence of Ni⁺⁺, a known activator of this phosphatase. SDS-polyacrylamide gel (10%) electrophoresis of the extracts revealed the typical calmodulin band at 17 kDa plus two additional bands with apparent molecular masses of 21 and 36 kDa respectively. These bands were not seen using normal meningeal extracts.     

Calmodulin antagonists promote TRA-8 therapy of resistant pancreatic cancer

Pancreatic cancer is highly malignant with limited therapy and a poor prognosis. TRAIL-activating therapy has been promising, however, clinical trials have shown resistance and limited responses of pancreatic cancers. We investigated the effects of calmodulin(CaM) antagonists, trifluoperazine(TFP) and tamoxifen(TMX), on TRA-8-induced apoptosis and tumorigenesis of TRA-8-resistant pancreatic cancer cells, and underlying mechanisms. TFP or TMX alone did not induce apoptosis of resistant PANC-1 cells, while they dose-dependently enhanced TRA-8-induced apoptosis. TMX treatment enhanced efficacy of TRA-8 therapy on tumorigenesis in vivo. Analysis of TRA-8-induced death-inducing-signaling-complex (DISC) identified recruitment of survival signals, CaM/Src, into DR5-associated DISC, which was inhibited by TMX/TFP. In contrast, TMX/TFP increased TRA-8-induced DISC recruitment/activation of caspase-8. Consistently, caspase-8 inhibition blocked the effects of TFP/TMX on TRA-8-induced apoptosis. Moreover, TFP/TMX induced DR5 expression. With a series of deletion/point mutants, we identified CaM antagonist-responsive region in the putative Sp1-binding domain between −295 to −300 base pairs of DR5 gene. Altogether, we have demonstrated that CaM antagonists enhance TRA-8-induced apoptosis of TRA-8-resistant pancreatic cancer cells by increasing DR5 expression and enhancing recruitment of apoptotic signal while decreasing survival signals in DR5-associated DISC. Our studies support the use of these readily available CaM antagonists combined with TRAIL-activating agents for pancreatic cancer therapy.     

Central nicotinic acetylcholine receptor involved in Ca(2+) -calmodulin-endothelial nitric oxide synthase pathway modulated hypotensive effects

BACKGROUND AND PURPOSE

Recent evidence has suggested that nicotine decreases blood pressure (BP) and heart rate (HR) in the nucleus tractus solitarii (NTS), indicating that nicotinic acetylcholine receptors (nAChRs) play an important role in BP control in the NTS. However, the signalling mechanisms involved in nAChR-mediated depressor effects in the NTS are unclear. Hence, the aim of this study was to investigate these signalling mechanisms.

EXPERIMENTAL APPROACH

Depressor responses to nicotine microinjected into the NTS of Wistar-Kyoto rats were elicited in the absence and presence of an antagonist of α7 nAChR, the calcium chelator ethylene glycol tetraacetic acid, a calmodulin-specific inhibitor, nitric oxide (NO) synthase (NOS) inhibitor, endothelial NOS (eNOS)-selective inhibitor or neuronal NOS (nNOS)-specific inhibitor.

KEY RESULTS

Microinjection of nicotine into the NTS produced a dose-dependent decrease in BP and HR, and increased nitrate levels. This depressor effect of nicotine was attenuated after pretreatment with a nAChR antagonist or blockers of the calmodulin-eNOS pathway. In contrast, N5-(1-Imino-3-butenyl)-L-ornithine (vinyl-L-NIO), nNOS-specific inhibitor, did not diminish these nicotine-mediated effects. Calmodulin was found to bind eNOS after nicotine injection into NTS. However, nicotine did not affect the eNOS phosphorylation level or eNOS upstream extracellular signal-regulated kinases (ERK)1/2 and Akt phosphorylation levels. Furthermore, pretreatment with an ERK1/2 or Akt inhibitor did not attenuate nicotine-induced depressor effects in the NTS.

CONCLUSIONS AND IMPLICATIONS

These results suggest that the nAChR-Ca²⁺-calmodulin-eNOS-NO signalling pathway, but not nNOS, plays a significant role in central BP regulation, and neither the ERK1/2 nor Akt signalling pathway are significantly involved in the activation of eNOS by nAChRs in the NTS.     

钙调素拮抗剂O-(4-乙氧基-丁基)-小檗胺对人白血病细胞K562和K562/A02生长的抑制作用

目的研究钙调素拮抗剂O-(4-乙氧基-丁基)-小檗胺(EBB)体外抗白血病作用及机制。方法四唑盐(MTT)比色法评价EBB的细胞毒作用;流式细胞术检测细胞凋亡;荧光分光光度计检测细胞ROS水平;激光共聚焦显微镜检测[Ca2+]i含量。结果EBB明显抑制敏感K562和耐药K562/A02细胞增殖,IC50分别为(8.22±1.67)mol.L-1和(8.97±1.48)mol.L-1。EBB作用早期诱导凋亡,晚期引起坏死。K562/A02细胞的ROS静息值是K562细胞的2倍,EBB诱导两种细胞ROS生成呈浓度和时间依赖性,加入SOD明显降低坏死率。K562/A02细胞的[Ca2+]i低于K562细胞。EBB能明显增加两种细胞的[Ca2+]i。结论EBB明显抑制K562和K562/A02细胞增殖。信号分子Ca2+、ROS参与了EBB的细胞毒作用。     

糖皮质激素对下丘脑-垂体-肾上腺轴钙调素基因表达的影响

目的　探讨糖皮质激素醋酸可的松对大鼠下丘脑 垂体 肾上腺轴 (hypothalamus pituitay adrenal,HPAA)钙调素基因表达的影响。方法　以逆转录多聚酶链反应 (RT PCR)半定量法测定大鼠注射醋酸可的松后钙调素 (calmodulin ,CaM)mRNA的表达水平。结果　醋酸可的松诱导大鼠下丘脑、肾上腺CaMmRNA水平升高 ,对垂体组织CaMmRNA水平没有显著影响。结论　CaMmRNA在糖皮质激素对HPAA的功能调控中起着重要作用     

Bepridil up-regulates cardiac Na+ channels as a long-term effect by blunting proteasome signals through inhibition of calmodulin activity

Background and purpose:

Bepridil is an anti-arrhythmic agent with anti-electrical remodelling effects that target many cardiac ion channels, including the voltage-gated Na⁺ channel. However, long-term effects of bepridil on the Na⁺ channel remain unclear. We explored the long-term effect of bepridil on the Na⁺ channel in isolated neonatal rat cardiomyocytes and in a heterologous expression system of human Na_v1.5 channel.

Experimental approach:

Na⁺ currents were recorded by whole-cell voltage-clamp technique. Na⁺ channel message and protein were evaluated by real-time RT-PCR and Western blot analysis.

Key results:

Treatment of cardiomyocytes with 10 µmol·L⁻¹ bepridil for 24 h augmented Na⁺ channel current (I_Na) in a dose- and time-dependent manner. This long-term effect of bepridil was mimicked or masked by application of W-7, a calmodulin inhibitor, but not KN93 [2-[N-(2-hydroxyethyl)-N-(4-methoxy benzenesulphonyl)]-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine], a Ca²⁺/calmodulin-dependent kinase inhibitor. During inhibition of protein synthesis by cycloheximide, the I_Na increase due to bepridil was larger than the increase without cycloheximide. Bepridil and W-7 significantly slowed the time course of Na_v1.5 protein degradation in neonatal cardiomyocytes, although the mRNA levels of Na_v1.5 were not modified. Bepridil and W-7 did not increase I_Na any further in the presence of the proteasome inhibitor MG132 [N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide]. Bepridil, W-7 and MG132 but not KN93 significantly decreased 20S proteasome activity in a concentration-dependent manner.

Conclusions and implications:

We conclude that long-term exposure of cardiomyocytes to bepridil at therapeutic concentrations inhibits calmodulin action, which decreased degradation of the Na_v1.5 α-subunit, which in turn increased Na⁺ current.     

白芍总甙对大鼠腹腔巨噬细胞产生肿瘤坏死因子的调节机制

目的：研究不同浓度白芍总甙（ＴＧＰ）调节大鼠腹腔巨噬细胞（ＭΦ）产生肿瘤坏死因子（ＴＮＦ）作用。方法：在ＭΦ培养系统中有或无环氧酶抑制剂和钙调蛋白抑制剂等工具药，测定４５Ｃａ内流、ＰＧＥ２和ＴＮＦ含量。结果：ＴＧＰ（０．５～１０ｍｇ·Ｌ－１）明显促进ＬＰＳ诱导ＭΦ的４５Ｃａ内流和ＴＮＦ产生。线性回归分析表明，４５Ｃａ内流和ＴＮＦ产生呈明显正相关。三氟拉嗪（４０μｍｏｌ·Ｌ－１）可阻断ＴＧＰ促进ＬＰＳ诱导ＭΦ产生ＴＮＦ。ＴＧＰ－ＬＰＳ的ＴＮＦ释放曲线呈钟罩形，而ＴＧＰ－ＬＰＳ的ＰＧＥ２产生曲线呈浓度依赖性升高。当ＴＧＰ在低浓度（０．５～１２．５ｍｇ·Ｌ－１）时，ＴＮＦ与ＰＧＥ２产生明显正相关，而高浓度（１２．５～２５０ｍｇ·Ｌ－１）两者呈明显负相关。吲哚美辛（１０μｍｏｌ·Ｌ－１）可使ＴＮＦ量效曲线下降支消失，而Ｎω亚硝基－Ｌ－精氨酸（１５μｍｏｌ·Ｌ－１）对此无明显影响。结论：低浓度ＴＧＰ对ＴＮＦ产生上调作用可能与促进４５Ｃａ内流，提高钙调蛋白活性从而促进ＰＧＥ２分泌等有关，而高浓度下调作用是可能与ＭΦ自身产生大量ＰＧＥ２介导有关。