Effect of polycystin2 on differentiation and maturation of osteoblasts promoted by low-frequency pulsed electromagnetic fields / 生物工程学报

It is known that low-frequency pulsed electromagnetic fields (PEMFs) can promote the differentiation and maturation of rat calvarial osteoblasts (ROBs) cultured in vitro. However, the mechanism that how ROBs perceive the physical signals of PEMFs and initiate osteogenic differentiation remains unknown. In this study, we investigated the relationship between the promotion of osteogenic differentiation of ROBs by 0.6 mT 50 Hz PEMFs and the presence of polycystin2 (PC2) located on the primary cilia on the surface of ROBs. First, immunofluorescence staining was used to study whether PC2 is located in the primary cilia of ROBs, and then the changes of PC2 protein expression in ROBs upon treatment with PEMFs for different time were detected by Western blotting. Subsequently, we detected the expression of PC2 protein by Western blotting and the effect of PEMFs on the activity of alkaline phosphatase (ALP), as well as the expression of Runx-2, Bmp-2, Col-1 and Osx proteins and genes related to bone formation after pretreating ROBs with amiloride HCl (AMI), a PC2 blocker. Moreover, we detected the expression of genes related to bone formation after inhibiting the expression of PC2 in ROBs using RNA interference. The results showed that PC2 was localized on the primary cilia of ROBs, and PEMFs treatment increased the expression of PC2 protein. When PC2 was blocked by AMI, PEMFs could no longer increase PC2 protein expression and ALP activity, and the promotion effect of PEMFs on osteogenic related protein and gene expression was also offset. After inhibiting the expression of PC2 using RNA interference, PEMFs can no longer increase the expression of genes related to bone formation. The results showed that PC2, located on the surface of primary cilia of osteoblasts, plays an indispensable role in perceiving and transmitting the physical signals from PEMFs, and the promotion of osteogenic differentiation of ROBs by PEMFs depends on the existence of PC2. This study may help to elucidate the mechanism underlying the promotion of bone formation and osteoporosis treatment in low-frequency PEMFs.

Hipertensão resistente: análise dos estudos pathway 2 e 3 / Resistant hypertension: analysis of pathway 2 and 3 studies

A hipertensão arterial resistente (HAR) é definida quando, apesar do tratamento com pelo menos três medicações anti- -hipertensivas (incluindo um diurético) de diferentes classes a meta pressórica não é alcançada. Nesta sequência de fármacos, por muitos anos se utilizou empiricamente ou baseado em pequenos estudos, a espironolactona. Os estudos Pathway 2 e 3 vieram para corroborar a importância deste quarto fármaco, a espironolactona, como o mais eficaz em termos de potencia anti-hipertensiva, como também explicar os aspectos fisiopatológicos que levam o hipertenso a ficar resistente. Nesta revisão e análise crítica dos fármacos anti-hipertensivos na HAR destacamos os principais mecanismos envolvidos no não controle da pressão e as estratégias para um melhor controle pressórico

Resistant arterial hypertension (RAH) is defined when, despite treatment with at least three antihypertensive medications (including a diuretic) of different classes, the pressure target is not achieved. In this sequence of drugs, for many years it was used empirically or based on small studies, spironolactone. Pathway 2 and 3 studies have come to corroborate the importance of this fourth drug, spironolactone, as the most effective in terms of antihypertensive potency, as well explain the pathophysiological aspects that lead hypertensive patients to become resistant. In this review and critical analysis of antihypertensive drugs in hypertension, we highlight the main mechanisms involved in the lack of pressure control and the strategies for better pressure control

Expression and role of acid-sensitive ion channel-la in lung tissues of rats with acute lung injury / 中国药理学通报

Aim To explore the expression and possible effect of acid sensing ion channel-la( ASICla) in lung tissues of rats with acute lung injury ( ALI). Methods Thirty-two male Sprague-Dawley (SD) rats were randomly divided into four groups: Control group, model group, amiloride group and Dex group. Blood gas analysis of arterial blood, observation of lung histo-pathology were performed and wet/dry ratio was calculated after weighing of lung tissues, then the expression of TNF-a in serum was detected by ELISA, and the expression of ASICla in lung tissues was also detected by qPCR and Western blot. Results Compared with control group, the lung tissue damage was obvious in model group, the wet/dry ratio of lung tissues obviously increased, and the expression level of TNF-a markedly increased (P < 0. 01 ). Lung tissue injury was significantly alleviated in amiloride group and Dex group, the wet/dry ratio of lung tissues obviously decreased , and the expression level of TNF-a significantly decreased (P < 0.01). Immunohistochemistry, qPCR and Western blot results showed that, compared with control group, the expression of ASICla significantly increased in model group (P <0.01), and markedly decreased in amiloride group (P <0.01). Conclusions The expression of ASIC 1 a increases in acute lung injury induced by lipopolysaccharide, which may be involved in the occurrence and development of ALI.

Simultaneous quantification of amiloride and hydrochlorothiazide in human plasma by liquid chromatography–tandem mass spectrometry / 药物分析学报

A selective, sensitive and precise assay based on solid phase extraction and liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed for the simultaneous determination of amiloride (AMI) and hydrochlorothiazide (HCTZ) in human plasma. Sample clean-up with 250 μL of plasma was done on Phenomenex Strata?-X extraction cartridges using their labeled internal standards (AMI-15N3 and HCTZ- 13C,d2). Chromatography was performed on Hypersil Gold C18 (50 mm×3.0 mm, 5 μm) column using acetonitrile with 4.0 mM ammonium formate (pH 4.0, adjusted with 0.1% formic acid) (80:20, v/v) as the mobile phase. Detection was carried out on a triple quadrupole API 5500 mass spectrometer utilizing an electrospray ionization interface and operating in the positive ionization mode for AMI and negative ionization mode for HCTZ. Multiple reaction monitoring was used following the transitions at m/z 230.6/116.0, m/z 233.6/116.0, m/z 296.0/204.9 and m/z 299.0/205.9 for AMI, AMI-15N3, HCTZ and HCTZ-13C,d2, respectively. Calibration curves were linear (r2≥0.9997) over the concentration range of 0.050–50.0 and 0.50–500 ng/mL for AMI and HCTZ, respectively, with acceptable accuracy and precision. The signal-to-noise ratio at the limit of quantitation was ≥14 for both the analytes. The mean recovery of AMI and HCTZ from plasma was 89.0% and 98.7%, respectively. The IS-normalized matrix factors determined for matrix effect ranged from 0.971 to 1.024 for both the analytes. The validated LC–MS/MS method was successfully applied to a bioequivalence study using 5 mg AMI and 50 mg HCTZ fixed dose tablet formulation in 18 healthy Indian volunteers with good reproducibility.

Simultaneous quantification of amiloride and hydrochlorothiazide in human plasma by liquid chromatography–tandem mass spectrometry / 药物分析学报

A selective, sensitive and precise assay based on solid phase extraction and liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed for the simultaneous determination of amiloride (AMI) and hydrochlorothiazide (HCTZ) in human plasma. Sample clean-up with 250 μL of plasma was done on Phenomenex Strata?-X extraction cartridges using their labeled internal standards (AMI-15N3 and HCTZ- 13C,d2). Chromatography was performed on Hypersil Gold C18 (50 mm×3.0 mm, 5 μm) column using acetonitrile with 4.0 mM ammonium formate (pH 4.0, adjusted with 0.1% formic acid) (80:20, v/v) as the mobile phase. Detection was carried out on a triple quadrupole API 5500 mass spectrometer utilizing an electrospray ionization interface and operating in the positive ionization mode for AMI and negative ionization mode for HCTZ. Multiple reaction monitoring was used following the transitions at m/z 230.6/116.0, m/z 233.6/116.0, m/z 296.0/204.9 and m/z 299.0/205.9 for AMI, AMI-15N3, HCTZ and HCTZ-13C,d2, respectively. Calibration curves were linear (r2≥0.9997) over the concentration range of 0.050–50.0 and 0.50–500 ng/mL for AMI and HCTZ, respectively, with acceptable accuracy and precision. The signal-to-noise ratio at the limit of quantitation was ≥14 for both the analytes. The mean recovery of AMI and HCTZ from plasma was 89.0% and 98.7%, respectively. The IS-normalized matrix factors determined for matrix effect ranged from 0.971 to 1.024 for both the analytes. The validated LC–MS/MS method was successfully applied to a bioequivalence study using 5 mg AMI and 50 mg HCTZ fixed dose tablet formulation in 18 healthy Indian volunteers with good reproducibility.

Congenital nephrogenic diabetes insipidus：2 cases report of brothers and review / 临床儿科杂志

Objective Congenital nephrogenic diabetes insipidus (CNDI) is a rare disease, the aim of this article is to help better understanding of this disease. Methods The clinical features, genetic analysis and treatments of two siblings with CNDI were retrospectively analyzed, and related literatures were reviewed. Results Both brothers had polydispia, polyuria and low concentrate urine continuously, and they both had a mutation in AQP 2 conifrmmed with Sanger sequencing. This novel frame shift mutation caused arginine of 254 to histidine, and prolonged AQP 2 protein. Conclusions Gene analysis can help diagnosis of CNDI. Amiloride is useful option for treatment.

Amiloride slows down calpain-mediated ABCA1 degradation through in-hibition of hypoxia-induced NHE1 expression / 中国病理生理杂志

AIM:To examine the effects of hypoxia on sodium-hydrogen exchange 1 (NHE1) expression, in-tracellular Ca2+concentration ( [ Ca2+] i ) and calpain activity, and to explore the effect of amiloride on adenosine triphos-phate-binding cassette transporter A1 (ABCA1) degradation and its calpain-related mechanism.METHODS:RAW264.7 cells were exposed to hypoxia for 0 h, 12 h, 24 h and 48 h.The cell viability was measured by MTT assay and the expres-sion of NHE1 at mRNA and protein levels was detected by real-time PCR and Western blot.[ Ca2+] i was analyzed by flow cytometry.Calpain activity was assessed by the method of Suc-LLVY-aminoluciferin.Furthermore, the protein levels of ABCA1 in the RAW264.7 cells exposed to hypoxia for 24 h were determined after 6 h or 12 h treatment with NHE1 inhibi-tor amiloride in the presence of cycloheximide.ABCA1 protein levels and calpain activity were detected after 12 h incuba-tion with calpain inhibitor ALLN or intracellular calcium-chelating agent BAPTA.RESULTS: Hypoxia inhibited the cell viability in a time-dependent manner.Hypoxia up-regulated the mRNA and protein expression of NHE1, and increased [ Ca2+] i and calpain activity.Hypoxia increased the degradation of ABCA1 and amiloride slowed down the ABCA1 degra-dation.ALLN or BAPTA increased ABCA1 protein level and decreased calpain activity.CONCLUSION:NHE1 inhibitor amiloride attenuates the calpain-mediated degradation of ABCA1, indicating that hypoxia-induced NHE1 might, at least in part, participate in the ABCA1 degradation.

The effect of Cratylia floribunda lectin on renal hemodynamics and ion transport

Lectins have been described as glycoproteins that reversibly and specifically bind to carbohydrates. Legume lectins isolated from the subtribe Diocleinae (Canavalia, Dioclea andCratylia) are structurally homologous with respect to their primary structures. The Diocleinae lectins of Canavalia brasiliensis, Dioclea guianensis andCanavalia ensiformis have been shown to distinctly alter physiological parameters in isolated rat kidneys. Thus, the aim of this study was to investigate the effect of Cratylia floribunda lectin (CFL) on renal hemodynamics and ion transport in rats. In isolated perfused kidneys, CFL (10 mg/mL, n=5) increased RPP, RVR and decreased %TK+, but did not change urinary flow, glomerular filtration rate, sodium or chloride tubular transport. In isolated perfused mesenteric bed, CFL (3 and 10 mg/mL/min; n=4) did not alter tissue basal tonus or tissue contraction by phenylephrine (1 mM/mL/min). In conclusion, the seed lectin of Cratylia floribunda increased renal hemodynamic parameters showing a kaliuretic effect. This effect could be of tubular origin, rather than a result from haemodynamic alterations.

As lectinas são descritas como (glico)proteínas que se ligam, especificamente e reversivelmente, a carboidratos. Lectinas de leguminosas isoladas da subtribo Diocleinae (Canavalia, Dioclea eCratylia) são estruturalmente homólogas em relação às suas estruturas primárias. Demonstrou-se que as lectinas de DiocleinaeCanavalia brasiliensis, Dioclea guianensis eCanavalia ensiformis alteram diferentemente parâmetros fisiológicos em rins isolados de ratos. Dessa maneira, o objetivo deste estudo foi investigar o papel da lectina de Cratylia floribunda (CFL) na hemodinâmica renal e no transporte de íons em ratos. Em rins isolados perfundidos, CFL (10 mg/mL, n=5) aumentou a pressão de perfusão renal, a resistência vascular renal e reduziu o percentual do transporte tubular de K+, mas não alterou o fluxo urinário, a taxa de filtração glomerular e o percentual de transporte tubular dos íons sódio e cloreto. No leito mesentérico isolado perfundido, CFL (3 e 10 mg/mL/min, n=4) não alterou o tônus basal ou a contração do tecido induzida por fenilefrina (1 mM/mL/min). Em conclusão, a lectina de sementes de Cratylia floribunda altera parâmetros hemodinâmicos renais, provavelmente de origem tubular, e não por alterações hemodinâmicas.

The Influence of Epithelial Sodium Channels in Isolated Goat Ileum Segment; Interaction with Steroids.

The contractile effect of Acetylcholine (Ach) in the isolated longitudinal ileal muscle of adult goats was studied over a varying concentration range. Ach produced a concentration dependent-response curve indicative of an interaction with muscarinic receptors in the ileum, with a maximum contraction seen at 12 μM. On the other hand, pretreatment with the ENaC blocker, Amiloride (100 μM) substantially reduced the Ach induced contractions by 67.11 %. However, pretreatment with Prednisolone (2mM) restored this effect and the relaxation induced was only 14.26 %. This change was found to be statistically significant. This study emphasizes the importance of ENaC channels in the goat intestinal smooth muscle.

Acute Hypoxia Activates an ENaC-like Channel in Rat Pheochromocytoma (PC12) Cells

Cells can resist and even recover from stress induced by acute hypoxia, whereas chronic hypoxia often leads to irreversible damage and eventually death. Although little is known about the response(s) to acute hypoxia in neuronal cells, alterations in ion channel activity could be preferential. This study aimed to elucidate which channel type is involved in the response to acute hypoxia in rat pheochromocytomal (PC12) cells as a neuronal cell model. Using perfusing solution saturated with 95% N2 and 5% CO2, induction of cell hypoxia was confirmed based on increased intracellular Ca2+ with diminished oxygen content in the perfusate. During acute hypoxia, one channel type with a conductance of about 30 pS (2.5 pA at -80 mV) was activated within the first 2~3 min following onset of hypoxia and was long-lived for more than 300 ms with high open probability (Po, up to 0.8). This channel was permeable to Na+ ions, but not to K+, Ca+, and Cl- ions, and was sensitively blocked by amiloride (200 nM). These characteristics and behaviors were quite similar to those of epithelial sodium channel (ENaC). RT-PCR and Western blot analyses confirmed that ENaC channel was endogenously expressed in PC12 cells. Taken together, a 30-pS ENaC-like channel was activated in response to acute hypoxia in PC12 cells. This is the first evidence of an acute hypoxia-activated Na+ channel that can contribute to depolarization of the cell.

Antinociceptive Effects of Amiloride and Benzamil in Neuropathic Pain Model Rats

Amiloride and benzamil showed antinocicepitve effects in several pain models through the inhibition of acid sensing ion channels (ASICs). However, their role in neuropathic pain has not been investigated. In this study, we investigated the effect of the intrathecal amiloride and benzamil in neuropathic pain model, and also examined the role of ASICs on modulation of neuropathic pain. Neuropathic pain was induced by L4-5 spinal nerve ligation in male Sprague-Dawley rats weighing 100-120 g, and intrathecal catheterization was performed for drug administration. The effects of amiloride and benzamil were measured by the paw-withdrawal threshold to a mechanical stimulus using the up and down method. The expression of ASICs in the spinal cord dorsal horn was also analyzed by RT-PCR. Intrathecal amiloride and benzamil significantly increased the paw withdrawal threshold in spinal nerve-ligated rats (87%+/-12% and 76%+/-14%, P=0.007 and 0.012 vs vehicle, respectively). Spinal nerve ligation increased the expression of ASIC3 in the spinal cord dorsal horn (P=0.01), and this increase was inhibited by both amiloride and benzamil (P<0.001 in both). In conclusion, intrathecal amiloride and benzamil display antinociceptive effects in the rat spinal nerve ligation model suggesting they may present an alternative pharmacological tool in the management of neuropathic pain at the spinal level.

Effects of acid sensing ion channels inhibitor amiloride on pain behavior in a rat model of post-incision pain / 中华行为医学与脑科学杂志

ObjectiveTo explore the effects and underlying mechanisms of acid sensing ion channels (ASICs) on pain behavior in a rat model of post-incision pain.MethodsFifty-eight adult male Sprague Dawley rats were used in this study,four rats were used for immunofluorescence test,thirty rats were employed for pain behavior test,and twenty-four rats were used for Western blot.Rats used for pain behavior test and Western blot were randomly divided into 3 groups:control group ( C group),incision pain model group ( I group) and amiloride group (A group).Plantar skin of rats in A group were infiltrated with 20 μl(200 μg)amiloride solution.Paw withdrawal mechanical threshold(PWMT) and paw withdrawal thermal latency(PWTL) of all rats in pain behavior test was tested at 24 h preoperative,2 h,4 h,8 h,12 h,24 h postoperative.Western blot was tested at 4 h postoperative.ResultsImmunofluorescence test displayed ASIC3 was expressed in plantar skin of all rats.The basal level of PWMT and PWTL of all rats in three groups was C group( (23.15 ± 5.10) g,( 11.32 ± 1.21 ) s),I group ( (23.26 ± 5.69) g,( 11.75 ± 2.01 ) s),A group ( (23.63 ± 4.96 ) g,( 11.47 ± 1.96) s) respectively,which was no significantly difference (P ＞ 0.05 ).PWMT and PWTL of I group and A group was significantly lower than that of C group at all time points postoperative (P ＜ 0.05) ; PWMT and PWTL of A group was at 2 h( ( 13.75 ±3.25)g,(9.96±1.32)s),4h((14.05±3.75)g,(9.17±2.11)s),8 h((9.75 ±2.74)g,(8.11 ±1.22)s)postoperative,which was significantly higher than that of I group (P ＜ 0.05 ).Compared with that of C group,the level of pERK1/2 expression was significantly increased in I group at 4 h postoperative (P ＜ 0.05 ),which could be inhibited by amiloride local infiltration (P ＜ 0.05 ).ConclusionASIC3 can mediate incision pain in a rat model of post-incision pain,through pERK1/2 signaling pathway,which can be inhibited by amiloride.

Effect of Amiloride to Retinal Toxicity Induced by Tissue Plasminogen Activator

PURPOSE: The effects of amiloride on cellular toxicity caused by tissue plasminogen activator (tPA) in mouse primary retinal cells were investigated. METHODS: Primary retinal cell cultures were maintained using glial conditioned medium. Commercial tPA and L-arginine were added, and the level of cyclic guanosine monophosphate (cyclic-GMP) in the culture supernatant was assessed using an ELISA assay. We measured the cell viability of cultured retinal cells pretreated with three different concentrations of amiloride (1, 10, and 100 microm) in addition to commercial tPA or L-arginine treatment. RESULTS: After exposing the cultured mouse retinal cells to tPA plus L-arginine or L-arginine alone, cyclic-GMP concentrations were 61.9 +/- 5.1 pmole/mL and 63.1 +/- 6.1 pmole/mL, respectively. However, the control group had a significantly lower concentration of cyclic-GMP (37.2 +/- 3.4 pmole/mL, p < 0.01). The cyclic GMP-dissolved solution did not cause retinal cell death. In the control group and the group treated with 1 microm amiloride and tPA containing L-arginine, the cell viability was 43.7% and 44.5%, respectively. However, cell viability increased to 70.6% with 10 microm amiloride and 78.4% with 100 microm amiloride (p = 0.015). CONCLUSIONS: L-arginine increases intracellular cyclic-GMP and may give rise to retinal cells through this mechanism. In addition, amiloride in concentrations greater than 10 microm protects against L-arginine-induced retinal cell death.

Effect of amiloride on the invasion capacity of esophageal carcinoma EC9706 cell line and its possible mechanisms / 肿瘤研究与临床

Objective To investigate the effect of amiloride on the invasion capacity of esophageal carcinoma EC9706 cell line in vitro and to elucidate its possible mechanism.Methods The invasion capacities of EC9706 cells pretreated with amiloride were measured by transwell chamber assay. The urokinase-type plasminogen activator (uPA) transcription were determined by RT-PCR.The protein expression of uPA were assessed by Western blot.Results After the EC9706 cells were pretreated with amiloride at different concentrations,the number of invaded cells was obviously less than those of control group with obvious dosage dependent pattern (96±7,78±6,57±6,33±4,15±3,F =43.46,P ＜ 0.01).The transcription levels of uPA mRNA and the protein expression levels of uPA in EC9706 cells decreased significantly compared with the control (mRNA:0.623±0.065,0.526±0.054,0.389±0.041,0.312±0.038,0.247±0.025,F =6.71,P ＜0.01; protein:0.732±0.064,0.644±0.057,0.533±0.058,0.391±0.036,0.267±0.043,F =6.71,P ＜0.01).Conclusion Amiloride inhibits the invasion capacity of esophageal carcinoma EC9706 cells.The mechanism might be associated with down-regulation of the expression of uPA.

Renal Expression of Ammonia Transporters in Rats with Amiloride-Induced Renal Tubular Acidosis / 대한내과학회지

BACKGROUND/AIMS: Renal tubular acidosis (RTA) decreases the net acid excretion, predominantly due to a decrease in urinary ammonia excretion. This study examined whether this decrement is associated with changes in the renal expression of the ammonia transporter family members, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg), in rats with amiloride-induced RTA. METHODS: Male Sprague-Dawley rats were treated intraperitoneally with amiloride (3 mg/kg/day) for 6 days. Rhbg and Rhcg expression was evaluated by immunoblotting and immunohistochemistry. Cell height, total cellular expression, expression in the apical 25% of the cell, and apical expression as a percentage of total expression were quantified using immunohistochemistry with quantitative morphometric analysis. RESULTS: After amiloride treatment for 6 days, the serum bicarbonate level was decreased, and serum potassium was increased. The total urinary ammonia excretion and potassium excretion were decreased. The total Rhbg and Rhcg protein expression levels were not changed in the cortex or outer medulla of the kidney. Light microscopy and immunohistochemistry with quantitative morphometric analysis demonstrated that total Rhcg expression was decreased in the cortical collecting duct (CCD) and outer medullary collecting duct (OMCD) in amiloride-induced RTA, whereas Rhbg immunoreactivity was unchanged. CONCLUSIONS: Rats with amiloride-induced RTA have decreased urinary ammonia excretion associated with decreased Rhcg expression in the CCD and OMCD, suggesting that the ammonia transporter Rhcg plays an important role in the pathogenesis of amiloride-induced RTA.

Effect of Amiloride on Invasive Activity and uPA System of Lung Carcinoma Cell Line / 中国肿瘤临床

Objective: To investigate the effect of amiloride on in vitro invasive activity and uPA (urokinase-type plasminogen activator)system of human highly metastatic lung carcinoma cell line PGCL3. Methods: At 6 hours after treatment with amiloride at the concentrations of 25μmol/L，50μmol/L and 100μmol/L for PGCL3 cells，Transwell Chamber assay was performed to detect the effect of amiloride on the invasive and migratory capacity of PGCL3 cells.Effect of amiloride on the activity of uPA and PAI-1(plasminogen activator inhibitor-1)secreted by PGCL3 cells were measured by chromogenic substrate assay after PGCL3 cells were incubated with amiloride for 24 hours.RT-PCR was used to analyze the effect of amilorede on mRNA levels of uPA，uPAR(urokinase-type plasminogen activator receptor)and PAI-1.The expression levels of uPA，ERK2(extracellular regulated protein kinases 2)and ras protein were assessed by Western blot. Results: The number of cells through membrane was significantly decreased in invasion and migration test in vitro.The inhibitory rates of invasion and migration after treatment with amiloride of 100μmol/L were 37.7%±4.1%and 64.9%±4.9%.respectively,with a significant difference from those in the control group(P<0.01).At 24 hours after amiloride treatment，the chromogenic substrate assay showed direct inhibition of the activity of uPA and PAI-1 secreted by PGCL3 cells.No effect on the expression of uPAR in mRNA level was observed,but the expression of PAI-1 in mRNA level was significantly inhibited.Amiloride of 100μmol/L dramatically inhibited the expression of uPA mRNA.The expression level of uPA protein was decreased with the increase of the concentration of amiloride,but no effect was observed on the expression of ERK2 and ras in protein level.Conclusion: Amiloride can inhibit the invasion and migration of PGCL3 cells,through inhibiting the expression and activity of uPA and PAI-1.Amiloride is a potential agent to inhibit cancer invasion and metastasis.

Effect of amiloride pretreatment on lipopolysaccharide-induced acute lung injury in rats / 中华麻醉学杂志

Objective To investigate the effect of amiloride pretreatment on the acute lung injury (ALI)induced by lipopolysaccharide (LPS) in rats. Methods Thirty-two pathogen-free male SD rats weighing 200-250 g were randomly divided into 4 groups (n = 8 each); group Ⅰ received iv normal saline (group C); group Ⅱ ALI received iv LPS 6 mg/kg (group ALI); group Ⅲ received iv amiloride 10 mg/kg (group A) and group Ⅳ received amiloride 10 mg/kg iv 30 min before iv LPS ( group AL). The animals were killed by exsanguination at 6 h after iv LPS infusion. The lungs were immediately removed. Microscopic examination of lung tissue was performed. The left lung was lavaged. The total protein (TP), TNF-α and macrophage inflammatory protein-2 (MIP-2)concentrations in broncho-alveolar lavage fluid (BALF) were measured. The W/D weight ratio and the myeloperoxidase (MPO) activity and expression of Na-H exchanger-1 ( NHE1 ), p38MAPK and extracellular signal-regulated kinase (ERK) in lung tissue were determined. Results LPS significantly increased ALI score (0 = slightest, 4 = severest), W/D lung weight ratio, TP, TNF-α and MIP-2 concentrations in BALF and MPO activity and the expression of NHE1, p38MAPK and ERK in the lung as compared with. control group. Amiloride pretreatment significantly attenuated LPS-induced changes except p38MAPK expression. Conclusion Pretreatment with amiloride can attenuate LPS-induced ALI by inhibition of ERK activation.

Effect of dimethyl amiloride on invasive activity of highly-metastatic lung carcinoma cell line and its possible mechanisms / 肿瘤

Objective:To investigate the effect of dimethyl amiloride (DMA) on invasive activity of PGCL3 cells from a human highly-metastatic lung carcinoma cell line in vitro and elucidate its possible mechanism. Methods:The invasion and migration capacities of PGCL3 cells were measured by using Transwell chamber assay after pretreatment with DMA. The effects of DMA on the activity of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) secreted by PGCL3 cells were measured by chromogenic substrate assay. The effects of DMA on uPA, urokinase-type plasminogen activator receptor (uPAR), and PAI-1 mRNAs transcription were determined by RT-PCR. The expression levels of extracellular regulated protein kinases 2 (ERK2) and ras protein were assessed by Western blot. Results:DMA inhibited invasion and migration capabilities of PGCL3 cells in vitro, down-regulated the mRNA transcription of uPA, uPAR and PAI-1, as well as up-regulated the expression of ras protein. After 24-hour treatment, DMA reduced the activity of uPA at higher concentration, but DMA had no effects on the activity of secreted PAI-1 protein and expression of ERK2 protein. Conclusion:DMA inhibits the invasion and migration of highly-metastatic lung cancer PGCL3 cells. The mechanism might be associated with down-regulation of the expression of uPA system.

Neuroprotective Effects of Amiloride / 国际脑血管病杂志

Amiloride is an epithelial Na+ channel(EnaC)blocker.As a potassium-sparing diuretic,it has been used in clinical practice for decades of years.Studies have shown that many ion channels were semitive to amiloride in the central nervous system,such as acid-sensitive ion channel(ASIC)and Na+/H+ exchanger.These channels have important physiological functions,and paticipate in pathological processes such as cerebral ischemia and tissue acidosis.It has demonstrated that amailoride reduces the effects of ischemia-and acid-mediated neuronal injury by blocking these channels,which may become a novel neuroprotective agent for the treatment of cerebral ischemia.

Repetition of Apoptosis Induced by Amiloride Derivatives in Human Umbilical Vein Endothelial Cells

PURPOSE: Human umbilical vein endothelial cells(HUVECs) play an important role in regulating blood flow by releasing vasoactive substances. It has been reported that endothelial impairment and dysfunction might be a primary cause of placental vascular disease, which is manifested clinically as preeclampsia in mother and intrauterine growth restriction in fetus. Furthermore, the frequency of apoptotic changes is increased in umbilical and placental tissues from growth-restricted pregnancies. However, the various mechanisms of umbilical endothelial cell apoptosis have not been broadly proposed. We investigate the effects of amiloride derivatives on apoptotic death of HUVECs and identify their ionic mechanism. METHODS: HUVECs were purchased from Clonetics, and cultured on endothelial cell growth medium. MTT assay and flow cytometry were used for assessing cytotoxic effect and confirming the apoptosis. Changes in intracellular ion concentrations were measured with specific fluorescent dyes and fluorescence imaging analysis system. RESULTS: Amiloride derivatives elicited cytotoxic effects on HUVECs with dose-dependent manners and the rank order of potency is HMA(IC50 11.2 micrometer), MIA>EIPA>>amiloride. HMA-induced cytotoxicity is dependent on extra- and intracellular pH, that is, increase extra- and intracellular pH augmented the cytotoxic effects of HMA. HMA dose-dependently reduced intracellular major ions, such as K+ and Cl-. Interestingly, the depletion of intracellular ions induced by HMA was also significantly enhanced at alkaline extracellular pH. CONCLUSION: Amiloride derivatives induce apoptosis of HUVECs with dose and pH dependent manners. They reduce intracellular K+ and Cl- concentration, which is also extracellular pH dependent.