Binding of dantrolene-Na to the ciliated membrane ofParamecium aurelia

Summary Dantrolene-Na is a muscular relaxant which binds to sarcoplasmic reticulum (SR) with high affinity and decreases the availability of Ca²⁺ channels. The binding of fluorescent compounds, dantrolene-Na, nifedipine and chlortetracycline to the ciliary membrane ofParamecium aurelia has been studied. Dantrolene at the concentrations of 1.9 · 10^–5, 3.8 · 10^–5 and 7.9 · 10^–5 M manifested a punctuated binding pattern to the cell membrane. Isolated cilia also bound dantrolene at their basal portion, whereas deciliated cell bodies lost their dotted binding pattern. Chlortetracycline showed a similar but weaker fluorescent staining. Nifedipine treated cells revealed no sign of fluorescent binding to the membrane and was only taken up in food vacuoles.Based on these observations we propose that dantrolene binding regular arrays ofParamecium cell membrane could be identical to granular plaques observed by electron microscope. The possible functioning of these structures as Ca²⁺ reservoirs is also discussed.     

Stimulation of Muscarinic Acetylcholine Receptors Increases Synaptosomal Free Calcium Concentration by Protein Kinase-Dependent Opening of L-Type Calcium Channels

In synaptosomes prepared from rat cerebral cortex, free cytosolic calcium concentration ([Ca2+]i) was measured using the fluorescent dye fura-2. Incubation of fura-2-loaded synaptosomes with carbachol increased [Ca2+]i in a dose-dependent manner (1-1,000 microM), with a maximum response of 22 +/- 2% at approximately 100 microM and an EC50 (calculated concentration producing 50% of the maximum response) of 30 microM. The effect of carbachol (100 microM) on [Ca2+]i was antagonised by atropine, but not by hexamethonium (10 microM). The calculated concentration of atropine needed for 50% inhibition (IC50) was 260 nM. The rise in [Ca2+]i produced by carbachol was reduced in the absence of extrasynaptosomal Ca2+ and effectively blocked by the L-type calcium channel blocker nifedipine (with an IC50 of 29 nM). The response to carbachol was reduced if the synaptosomes were preincubated with the protein kinase inhibitors H7 [1-(5-isoquinolinylsulfonyl)-2- methylpiperazine] (from 17% in the solvent control to 4%) and staurosporine (from 20% in the solvent control to 3%). These results show that stimulation of muscarinic acetylcholine receptors in synaptosomes increases [Ca2+]i by protein kinase-dependent activation of 1,4-dihydropyridine-sensitive calcium channels.     

Intracellular Calcium Dynamics and Cellular Energetics in Ischemic NG108-15 Cells Studied by Concurrent 31P/19F and 23Na Double-Quantum Filtered NMR Spectroscopy

Abstract: The role of voltage-sensitive Ca²⁺ channels in mediating Ca²⁺ influx during ischemia was investigated in NG108-15 cells, a neuronal cell line that does not express glutamate-sensitive receptor-mediated Ca²⁺ channels. Concurrent ³¹P/¹⁹F and ²³Na double-quantum filtered (DQF) NMR spectra were used to monitor cellular energy status, intracellular [Ca²⁺] ([Ca²⁺]_i), and intracellular Na⁺ content in cells loaded with the calcium indicator 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5FBAPTA) during ischemia and reperfusion. Cells loaded with 5FBAPTA were indistinguishable from unloaded cells except for small immediate decreases in levels of phosphocreatine (PCr) and ATP. Ischemia induced a steady decrease in intracellular pH and PCr and ATP levels, and a steady increase in intracellular Na⁺ content; however, a substantial increase in [Ca²⁺]_i (about threefold) was seen only following marked impairment of cellular energy status, when PCr was undetectable and ATP content was reduced to 55% of control levels. A depolarization-induced increase in [Ca²⁺]_i could be completely blocked by 1 µM nifedipine, whereas up to 20 µM nifedipine had no effect on the increase in [Ca²⁺]_i seen during ischemia. These data demonstrate that voltage-gated Ca²⁺ channels do not mediate significant Ca²⁺ flux during ischemia in this cell line and suggest an important role for Ca²⁺_i stores, the Na⁺/Ca²⁺ antiporter, or other processes linked to cellular energy status in the increase in cytosolic Ca²⁺ level during ischemia.     

Oxidant Injury in PC12 Cells—A Possible Model of Calcium "Dysregulation" in Aging: I. Selectivity of Protection Against Oxidative Stress

Abstract: Previous research has suggested that the initial effects of cellular free radical neurotoxic insult involve large increases in intracellular Ca²⁺. However, the exact role of oxidative stress on the various parameters involved in these increases has not been specified. The present experiments were performed to examine these parameters in PC12 cells exposed to 5, 25, or 300 µM H₂O₂ for 30 min in growth medium alone or containing either nifedipine (L-type Ca²⁺ antagonist), conotoxin (N-type antagonist), Trolox (vitamin E analogue), or α-phenyl-n-tert-butylnitrone (nitrone trapping agent; PBN). The concentrations of H₂O₂ were chosen by examining the degree of cell killing induced by exposure to graded concentrations of H₂O₂. The 5 and 25 µM concentrations of H₂O₂ produced no significant cell killing at either 30 min or 24 h after treatment, whereas the 300 µM concentration produced a moderate degree of cell killing that did not increase between the two times. Fluorescent imaging was used to visualize intracellular Ca²⁺ changes in fura-2-loaded cells. Baseline (pre-30 mM KCI) Ca²⁺ levels were increased significantly by H₂O₂ treatment (e.g., 300 µM, 200%), but the rise in the level of free intracellular Ca²⁺ after KCI stimulation (i.e., peak) was decreased (e.g., 300 µM, 50%) and the cell's ability to sequester or extrude the excess Ca²⁺ (i.e., Ca²⁺ recovery time) after depolarization was decreased significantly. All compounds prevented baseline Ca²⁺ increases and, with the exception of conotoxin, antagonized the peak decreases in Ca²⁺. It is interesting that after 300 µM H₂O₂ exposure, only Trolox was partially effective in preventing these deficits in recovery. Conotoxin increased the decrement recovery in the absence of H₂O₂. However, in cells exposed to 5 or 25 µM H₂O₂, conotoxin as well as the other agents were effective in preventing the deficits in recovery.     

Chronopharmacokinetics and Cardiovascular Effects of Nifedipine

Orcadian phase dependency in pharmacokinetics and hemodynamic effects on blood pressure and heart rate of different galenic formulations of nifedipine (immediate-release, sustained-release, and i.v. solution) were studied in healthy subjects or in hypertensive patients. Pharmacokinetics of immediate-release but not sustained-release and i.v. nifedipine were dependent on time of day: immediate-release nifedipine had higher C_max (peak concentration) and shorter t_max (time-to-peak concentration) after morning than evening application, and bioavailibility in the evening was reduced by about 40%. Orcadian rhythm in estimated hepatic blood flow as determined by indocyanine green kinetics may contribute to these chronokinetics. A circadian time dependency was also found in nifedipine-induced effects on blood pressure and heart rate as monitored by 24-h ambulatory blood pressure measurements. In conclusion, the dose response relationship of oral nifedipine is influenced by the circadian organization of the cardiovascular system as well as by the galenic drug formulation.     

硝苯地平联合硫酸镁对高龄孕妇妊娠高血压的临床疗效及机制研究

目的:探讨硝苯地平联合硫酸镁对高龄孕妇妊娠高血压的临床疗效及可能机制。方法:收集我院高龄孕妇妊娠高血压患者54例,随机分为A组和B组,各27例。A组给予100 m L 5%葡萄糖注射液加入20 m L 25%的硫酸镁注射液静脉滴注,30 min内滴完;再给予500 m L 5%葡萄糖注射液加入40 m L 25%硫酸镁注射液1~2 g/h,静脉滴注;B组在A组用药的基础上给予给予硝苯地平控释片30 mg/次口服,1次/日,2组患者均治疗7 d。治疗结束后,比较两组患者血压、C反应蛋白(CRP)、白细胞介素6(IL-6)、肿瘤坏死因子-α(TNF-α)及蛋白尿水平。结果:与治疗前相比,两组患者治疗后收缩压、舒张压、CRP、IL-6、TNF-α及蛋白尿水平均显著降低(P0.05);与A组比较,B组患者收缩压、舒张压、CRP、IL-6、TNF-α及蛋白尿水平较低(P0.05)。结论:硝苯地平联合硫酸镁对高龄孕妇妊娠高血压有较好的临床疗效,推测其机制与降低CRP、IL-6及TNF-α水平有关。     

Determination of nifedipine in human plasma by flow-injection tandem mass spectrometry

For use in clinical studies, a fast and sensitive assay method was developed for the determination of nifedipine in human plasma samples. The assay method is based on tandem mass spectrometry detection (HPLC–MS–MS). The effect of flow injection as well as HPLC separation on the results of the nifedipine determination were evaluated. The limit of quantification is 0.5 ng/ml and the accuracy (as determined by spiking recovery) was found to be good.     

Simultaneous determination of nifedipine and dehydronifedipine in human plasma by liquid chromatography–tandem mass spectrometry

Quantitative analysis of therapeutic compounds and their metabolites in biological matrix (such as plasma, serum or urine) nowadays requires sensitive and selective methods to allow the determination of concentrations in the ng/ml range. A new on-line LC–MS–MS method using atmospheric pressure chemical ionisation (APCI) as interface for the simultaneous determination of nifedipine (NIF) and its metabolite in human plasma, dehydronifedipine (DNIF) has been developed. The compounds were extracted from plasma using solid-phase extraction (SPE) on disposable extraction cartridges (DECs). The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with phenyl modified silica was first conditioned with methanol and water. The washing step was performed with water. Finally, the analytes were successively eluted with methanol and water. The liquid chromatographic (LC) separation of NIF and DNIF was achieved on a RP-18 stationary phase (4 μm). The mobile phase consisted of methanol–50 mM ammonium acetate solution (50:50, v/v). The LC was then coupled to tandem mass spectrometry with an APCI interface in the positive ion mode.

The method developed was validated. The absolute recoveries evaluated over the whole concentration range were 95±2% and 95±4% for NIF and DNIF, respectively. The method was found to be linear in the 0.5–100 ng/ml concentration range for the two analytes (r²=0.999 for both NIF and DNIF). The mean R.S.D. values for repeatability and intermediate precision were 2.9 and 3.0% for NIF and 2.2–4.7% for the metabolite.The method developed was successfully used to investigate the plasma concentration of NIF and DNIF in the pharmacokinetic studies.     

Nifedipine, a calcium channel blocker, inhibits inflammatory and fibrogenic gene expressions in advanced glycation end product (AGE)-exposed fibroblasts via mineralocorticoid receptor antagonistic activity

Advanced glycation end products (AGE) are involved in tissue damage and remodeling. This study investigated whether AGE could elicit inflammatory and fibrogenic reactions in fibroblast cell line MRC-5 cells via autocrine production of aldosterone and if nifedipine could block the AGE actions through mineralocorticoid receptor (MR) antagonistic activity. AGE significantly up-regulated monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-β (TGF-β), type III collagen and receptor for AGE (RAGE) mRNA levels in MRC-5 cells, all of which were completely blocked by nifedipine or an MR antagonist spironolactone. Aldosterone also dose-dependently increased MCP-1, TGF-β and type III collagen mRNA levels in MRC-5 cells, which were suppressed by nifedipine, but not amlodipine, a control calcium channel blocker. Further, AGE significantly stimulated aldosterone generation in MRC-5 cells, which was partially blocked by nifedipine or spironolactone. In this study, we demonstrated for the first time that AGE could evoke inflammatory and fibrogenic reactions in MRC-5 cells via aldosterone production, which were blocked by the MR antagonistic activity of nifedipine. Our present study provides a unique beneficial aspect of nifedipine on tissue damage and remodeling; it could work as an anti-inflammatory and anti-fibrogenic agent against AGE via MR antagonistic activity.     

Nifedipine inhibits advanced glycation end products (AGEs) and their receptor (RAGE) interaction-mediated proximal tubular cell injury via peroxisome proliferator-activated receptor-gamma activation

There is a growing body of evidence that advanced glycation end products (AGEs) and their receptor (RAGE) interaction evokes oxidative stress generation and subsequently elicits inflammatory and fibrogenic reactions, thereby contributing to the development and progression of diabetic nephropathy. We have previously found that nifedipine, a calcium-channel blocker (CCB), inhibits the AGE-induced mesangial cell damage in vitro. However, effects of nifedipine on proximal tubular cell injury remain unknown. We examined here whether and how nifedipine blocked the AGE-induced tubular cell damage. Nifedipine, but not amlodipine, a control CCB, inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-γ (PPARγ). GW9662 treatment alone was found to increase RAGE mRNA levels in tubular cells. Further, nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-κB activation and increases in intercellular adhesion molecule-1 and transforming growth factor-beta gene expression in tubular cells, all of which were blocked by GW9662. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-oxidative and anti-inflammatory agent against AGEs in tubular cells by suppressing RAGE expression via PPARγ activation.