Tributyltin-binding protein type 1 has a distinctive lipocalin-like structure and is involved in the excretion of tributyltin in Japanese flounder,Paralichthys olivaceus

Tributyltin-binding protein type 1 (TBT-bp1) is a newly discovered protein that binds with TBT in the blood of the Japanese flounder, Paralichthys olivaceus. We determined the genomic sequence of TBT-bp1 and found that this protein has a conserved exon–intron structure that is common to the lipocalin protein family. The secondary and tertiary structures of TBT-bp1, predicted from amino acid sequence, included at least two α-helices and eight β-sheets that are conserved in all lipocalins and form a barrel structure that may bind with ligands. Analysis of the gene structure, secondary structure, and tertiary structure demonstrated that TBT-bp1 could be classified as a lipocalin. A homology search revealed the presence of TBT-bp1-like proteins in eight species of teleost. When flounder were injected intraperitoneally with TBT-d27 at 11.6 μg/fish, TBT-d27 was detected in the blood and in the skin mucus. The concentration of TBT-d27 in mucus was approximately 1/100 of that in the serum. Western blotting analysis revealed that TBT-bp1 was present in the skin mucus. These results suggest that TBT-bp1 in Japanese flounder binds with TBT and is excreted from the body via the mucus.     

Tributyltin-binding protein type 1, a lipocalin, prevents inhibition of osteoblastic activity by tributyltin in fish scales

Tributyltin-binding protein type 1 (TBT-bp1) is a member of the lipocalin family of proteins which bind to small hydrophobic molecules. In this study, we expressed a recombinant TBT-bp1 (rTBT-bp1, ca. 35kDa) in a baculovirus expression system and purified the protein from the hemolymph of silkworm larvae injected with recombinant baculovirus. After incubation of a mixture of rTBT-bp1 and TBT and its fractionation by means of gel filtration chromatography, TBT was detected in the elution peak of rTBT-bp1, confirming the binding potential of rTBT-bp1 for TBT. An assay of the ability of rTBT-bp1 or native TBT-bp1 (nTBT-bp1) to restore osteoblastic activity inhibited by TBT showed that co-treatment of the scales with rTBT-bp1 or nTBT-bp1 in combination with TBT restored osteoblastic activity in goldfish scales, whereas treatment with TBT alone significantly inhibited osteoblastic activity. These results suggest that TBT-bp1 as a lipocalin member might function to decrease the toxicity of TBT by binding to TBT.     

Characteristics of bioconcentration and depuration of endocrine disruption chemicals in the flounder,Paralichthys olivaceus, under flow-through system

This study investigated the bioaccumulation effects of endocrine disruption chemicals (EDCs) such as Tributyltin (TBT), Nonylphenol (NP) and Bisphenol-A (BPA) on flounder,Paralichthys olivaceus. The exposure experiment with the flow through system was performed to examine the effects of bioconcentration for single or multi-chemicals. In the muscle of flounder exposed to TBT, the concentrations of TBT were significantly increased compared with the control tank after two months. It is markedly more accumulated in the liver of the flounder than in the muscle. TBT in muscle of the low level of EDCs were highly accumulated in single exposures of TBT compared to multi-chemical exposure with NP and/or BPA. The concentrations of TBT in muscle were increased in the multi-chemical exposure system. The concentration of TBT in the liver of flounder showed a slight decrease in the multi-chemical exposure system. The metabolites of TBT, DBT and MBT were also concentrated in the muscle and liver. NP and BPA in the muscle of flounder were accumulated with high concentration of EDCs. NP and BPA in muscle were significantly decreased compared with TBT after the depuration period.     

Distribution and excretion of the mercury chelating agent sodium 2,3-dimercaptopropane-1-sulfonate in the rat

Summary The distribution and excretion of sodium 2,3-dimercapto-1,3 ¹⁴C-propane-1-sulfonate as dependent on time has been studied in the rat. The highest concentration is found in the kidneys, the lowest in the brain. The excretion is very rapid (T1/2=19 min) and follows a monoexponential curve during the first hour after administration. This holds for plasma and most of the organs too. The apparent distribution volume of the radioactivity is equivalent to the volume of the extracellular water. After oral administration, 30–40% is absorbed from the gut. The results lead to the conclusion that a fraction of the drug is weakly bound to plasma- and membrane-proteins. They are discussed with respect to the treatment of heavy metal poisoning.

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Zusammenfassung Die Zeitabhängigkeit der Verteilung und der Ausscheidung von Natrium-2,3-Dimercapto-1,3-¹⁴C-propan-1-Sulfonat wurde bei der Ratte untersucht. Die höchste Konzentration findet sich in den Nieren, die niedrigste im Gehirn. Die Ausscheidung ist sehr schnell (T 1/2 = 19 min) und folgt in der ersten Stunde nach der Verabreichung einer monoexponentiellen Kurve. Dies wird auch im Plasma und in den meisten Organen gefunden. Das Verteilungsvolumen der Radioaktivität ist gleich dem des extrazellulären Wassers. Nach oraler Verabreichung werden 30–40% aus dem Darm absorbiert. Die Ergebnisse führen zu der Schlußfolgerung, daß ein Teil des Chelatbildners lose an Plasma- und Membranproteine gebunden ist. Sie werden im Hinblick auf die Behandlung von Schwermetallvergiftungen diskutiert.

     

Dietary vitamin C reduced mercury contents in the tissues of juvenile olive flounder (Paralichthys olivaceus) exposed with and without mercury

A 2 × 3 factorial design was employed to evaluate the effects of dietary vitamin C (l-ascorblyl-2-monophosphate, C2MP) levels on growth and tissue mercury (Hg) accumulations in juvenile olive flounder, Paralichthys olivaceus. Six experimental diets with two levels of mercuric chloride (0 or 20 mg HgCl₂/kg diet) and three levels of vitamin C (0, 100, or 200 mg C2MP/kg diet) were added to the basal diet. At the end of 6 weeks feeding trial, in presence or absence of dietary Hg, fish body weight gain, specific growth rate, feed efficiency, protein efficiency ratio and whole body lipid content were increased in a dose-dependent manner as dietary vitamin C level increased in the diets. Interestingly, fish fed 100 or 200 mg C2MP/kg diets showed significant interactive effects on reducing Hg content in kidney tissue. These results revealed that dietary vitamin C as 100 or 200 mg C2MP/kg diet had protective effect against Hg accumulation in juvenile olive flounder.     

Effects of Guanosine on the Pharmacokinetics of Acriflavine in Rats Following the Administration of a 1:1 Mixture of Acriflavine and Guanosine,a Potential Antitumor Agent

Preclinical studies are currently underway to examine the potential antitumor effects of a 1:1 mixture of acriflavine (ACF; CAS 8063-24-9) and guanosine. Guanosine potentiates the anticancer activity of some compounds. However, the effects of guanosine on the pharmacokinetics of ACF in mammals are unknown. Therefore, this study investigated the effects of guanosine on the pharmacokinetics of ACF after administering a 1:1 mixture of ACF and guanosine in rats. The rats were given either 10 mg/kg of the mixture or 5 mg/kg ACF via an intravenous bolus injection; or 30 mg/kg of the mixture or 15 mg/kg ACF intramuscularly. An HPLC-based method, which was validated in this laboratory, was used to analyze the levels of trypaflavine (TRF) and proflavine (PRF) in the plasma, bile, urine, and tissue homogenates. It was found that TRF and PRF were rapidly cleared from the blood and transferred to the tissues after the i.v. bolus or i.m. injection of the combination mixture. Both TRF and PRF were found to be most highly concentrated in the kidneys after the i.v. bolus or i.m. injection, followed by slow excretion to the bile or urine. Guanosine had no effect on the plasma disappearance of TRF or PRF after the i.v. bolus injection. However, guanosine led to a prolongation of the plasma levels of PRF after the i.m. administration of the combination mixture, resulting in a 2 fold increase in the bioavailability (BA) of PRF The concentrations of TRF and PRF in all the tissues examined were similar in the groups given the mixture and ACF. However, guanosine led to a prolongation of the biliary and urinary excretions of both TRF and PRF after the i.v. bolus (1.25 fold) or i.m. (1.5-2.4 folds) injection. These prolonged effects of guanosine on the plasma disappearance or urinary excretion of TRF and PRF might be one reason for the enhanced antitumor effects of ACF. However, more study will be needed to further examine this potential mechanism.     

Evaluation of the pharmacokinetics,tissue distribution and excretion studies of YMR-65, a tubulin polymerization inhibitor with potential anticancer activity,in rats using UPLC-MS/MS

1.YMR-65, 5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(3-methoxyphenyl)-4, 5-dihydro-1H-pyrazole-1-carboxamide, is a new tubulin polymerization inhibitor with encouraging anticancer activity.

2.The validated ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) method was successfully applied to the pharmacokinetics, tissue distribution and excretion study of YMR-65 after oral and intravenous administration. The area under concentration-time curve (AUC_0-∞) for YMR-65 were 151.67?±?54.48 and 459.45?±?49.23?ng/ml*h for oral and intravenous administration at the dosage of 1.5?mg/kg, respectively and the oral bioavailability was about 33.01%. Moreover, YMR-65 was extensively distributed in heart, liver, spleen, lung, kidney, stomach, intestine and testis and the highest were detected in heart, followed by stomach, intestine and liver. The majority of YMR-65 was excreted via feces and its accumulative excretion ratio during the period of 96?h was 19.83?±?3.01%, but only 1.54?±?0.37 and 0.215?±?0.026% for urine within 96?h and bile within 10?h after intravenous administration, respectively, though the fecal and urine excretion were incomplete within 96?h.

3.In summary, this study defined the pharmacokinetic characteristics of YMR-65 in vivo and the important data can be a useful resource for further research and development.     

Pharmacokinetics of guanosine in rats following intravenous or intramuscular administration of a 1:1 mixture of guanosine and acriflavine, a potential antitumor agent

A 1:1 mixture of acriflavine (ACF; CAS 8063-24-9) and guanosine is under evaluation in preclinical studies as a possible antitumor agent. Guanosine is known to potentiate the anti-cancer activity of ACF. We therefore investigated the pharmacokinetics of guanosine following administration of the ACF/guanosine mixture in rats. Rats were given guanosine (1 or 5 mg/kg) or ACF/guanosine (2 or 10 mg/kg) by i.v. bolus; or guanosine (3 or 15 mg/kg) or ACF/guanosine (6 or 30 mg/kg) by i.m. injection. We found that guanosine was rapidly cleared from the blood and transferred to tissues after i.m. administration of ACF/guanosine. The mean plasma half-lives (t_1/2) at the α and β phases were 0.091 and 6.86 h, or 0.09 and 7.51 h at a dose of 1 or 5 mg/kg guanosine, respectively. ACF had no effect on the plasma disappearance of guanosine following either i.v. bolus or i.m. administration of the combination mixture. Moreover, the ACF combination with guanosine did not significantly alter the values of MRT, V_dss, and CL_t of guanosine. Guanosine exhibited linear pharmacokinetics over the dose range from 1 to 5 mg/kg for i.v. doses and 3 to 15 mg/kg for i.m. doses. The bioavailability of guanosine after i.m. administration was 84% for 3 mg/kg dose and 88% for 15 mg/kg dose. ACF had no effects on biliary and urinary excretion of guanosine after i.m. administration. The cumulative amount of guanosine in urine after i.m. administration was about 5-fold larger than that in bile, indicating that guanosine is mostly excreted into the urine. Guanosine was widely distributed in all tissues examined in this study, but was most highly concentrated in the kidney after i.m. administration, followed by slow excretion to bile or urine. ACF had no effect on the tissue distribution of guanosine following i.m. administration. These characterizations of the pharmacokinetics of guanosine after administration of the ACF/guanosine combination will be useful in providing preclinical and clinical bases for the potential application of this combination to the treatment of cancer.     

Pharmacokinetics,metabolism, excretion and plasma protein binding of 14C-levofloxacin after a single oral administration in the Rhesus monkey

Levofloxacin's metabolism, excretion, and in vitro plasma protein binding, together with its pharmacokinetics, were studied in the Rhesus monkey in support of an anthrax efficacy study in this species. Three males and three female Rhesus monkeys were dosed with a single oral dose of ¹⁴C-levofloxacin at 15?mg?kg^?1 (2?MBq?kg^?1). Following dose administration, blood samples were collected up to 48?h post-dose, and urine and faeces were quantitatively collected up to 168?h post-dose. Blood, plasma, urine, and faeces were analysed for total radioactivity. Metabolite profiling and identification was performed using radio-high-performance liquid chromatography (HPLC) and liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS). Additionally, the plasma protein binding of levofloxacin was determined in vitro by means of equilibrium dialysis. Peak plasma levels of total radioactivity and levofloxacin were rapidly reached after oral administration with a total radioactivity blood: plasma ratio close to unity. The elimination half-life of levofloxacin was estimated at about 2?h. Total radioactivity was mainly excreted in urine (about 57–86% of the dose) with faecal excretion accounting for only a minor fraction of the total amount of excreted radioactivity (about 7.4–14.7%). In the plasma, the majority of total radioactivity was accounted for by levofloxacin. In addition, two minor metabolites, i.e. levofloxacin n-oxide and presumably a glucuronide conjugate of levofloxacin, were detected. In the urine, five components were found, with levofloxacin being the major component. Minor metabolites included desmethyl levofloxacin, levofloxacin n-oxide, and a glucuronide conjugate of levofloxacin. In the faeces, the major analyte was a polar metabolite, tentatively identified as a levofloxacin glucuronide. The in vitro plasma protein binding was low (on average 11.2%) and independent of concentration (1.0–10.0?µg?ml^?1). No sex differences were noted in any of the investigations. The present data indicated that the metabolism and excretion pattern, and also the in vitro plasma protein binding of levofloxacin in the Rhesus monkey, were comparable with those previously reported in man, hereby supporting the use of this animal species in the efficacy evaluation of levofloxacin against inhalation anthrax. The shorter half-life of levofloxacin in the Rhesus monkey relative to man (2 versus 7?h) prompted the development of an alternative dosing strategy for use in the efficacy study.     

Purification and characterization of Cop, a protein involved in the copy number control of plasmid pE194

Cop protein has been overexpressed inEscherichia coli using a T7 RNA polymerase system. Purification to apparent homogeneity was achieved by the sequential chromatography on ion exchange, affinity chromatography, and reverse phase high performance liquid chromatography system. The molecular weight of the purified Cop was estimated as 6.1 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). But the molecular mass of the native state Cop was shown to be 19 kDa by an analytical high performance size exclusion chromatography, suggesting a trimer-like structure in 50 mM Tris-HCI buffer (pH 7.5) containing 100 mM NaCl. Cop protein was calculated to contain 39.1% α-helix, 16.8% β-sheet, 17.4% turn, and 26.8% random structure. The DNA binding property of Cop protein expressed inE. coli was preserved during the expression and purification process. The isoelectric point of Cop was determined to be 9.0. The results of amino acid composition analysis and N-terminal amino acid sequencing of Cop showed that it has the same amino acid composition and N-terminal amino acid sequence as those deduced from its DNA sequence analysis, except for the partial removal of N-terminal methionine residue by methionyl-aminopeptidase inE. coli.     

周期素D1与p16蛋白及Rb基因在大肠癌中的表达及意义

目的研究cyclinD1（周期素D1），p16及Rb基因表达的蛋白产物在大肠癌组织中的变化及其意义。方法应用免疫组化S-P法，在55例原发性大肠癌中检测cyclinD1，p16及Rb基因蛋白产物的表达。结果cyclinD1蛋白表达阳性率为43．64％（24／55），p16蛋白阳性表达率为47．27％，Rb蛋白阳性表达率为76．36％；cyclinD1，p16及Rb阳性表达与正常结肠直肠黏膜组织之间有非常显著性差异（P〈0．01）；Rb失活与p16表达之间呈负相关关系（P〈0．01）；Rb蛋白表达与肿瘤分化呈反比（P〈0．01）；p16蛋白表达与大肠癌淋巴结转移有关（P〈0．01）；cyclinD1，p16及Rb蛋白阳性表达率在死亡组与生存组之间有显著性差异（P〈0．05）。结论大肠癌发生过程中，存在cyclinD1，p16及Rb蛋白表达的异常，p16、Rb及cyclinD1过度表达与患者的预后密切相关，对肿瘤预后评估有重要意义。     

The multifunctional protein PEA-15 is involved in the control of apoptosis and cell cycle in astrocytes

PEA-15 is a small protein (15 kDa) that was first identified as an abundant phosphoprotein in brain astrocytes [Araujo et al., J Biol Chem 1993;268(8):5911-20], and subsequently shown to be widely expressed in different tissues and highly conserved among mammals [Estelles et al., J Biol Chem 1996;271(25):14800-6; Danziger et al., J Neurochem 1995;64(3):1016-25]. It is composed of a N-terminal death effector domain and a C-terminal tail of irregular structure. PEA-15 is regulated by multiple calcium-dependent phosphorylation pathways that account for its different forms: a non-phosphorylated form in equilibrium with a mono and a biphosphorylated variety. This already suggested that PEA-15 may play a major role in signal integration. Accordingly, it has been demonstrated to modulate signaling pathways that control apoptosis and cell proliferation. In particular, PEA-15 diverts astrocytes from TNFalpha-triggered apoptosis and regulates the actions of the ERK MAP kinase cascade by binding to ERK and altering its subcellular localization. The three-dimensional structure of PEA-15 has been modelized and recently determined using NMR spectroscopy, and may help to understand the various functions played by the protein through its molecular interactions.     

P-gp is involved in the intestinal absorption and biliary excretion of afatinib in vitro and in rats

Background

Afatinib is an irreversible multi-targeted TKI, used in the treatment with EGFR mutated non-small cell lung cancer (NSCLC). The purpose of this study is to explore the molecular pharmacokinetic mechanism underlying the effect of P-gp inhibitors on the intestinal absorption and biliary excretion and to understand how P-gp inhibitors affect afatinib pharmacokinetics.

In vivo and in vitro renal metabolism and excretion of benzoic acid by a marine teleost, the southern flounder

In mammals, benzoic acid is primarily metabolized to its glycine conjugate, hippuric acid, which is readily excreted via the renal organic anion transport system. Teleost fish have not been shown to make glycine conjugates of carboxylic acids. Therefore, metabolism, excretion, and renal transport of benzoic acid were examined in the southern flounder, Paralichthys lethostigma. Excretion of injected [14C]benzoic acid was slow (10% of the administrated dose/day) and followed zero order kinetics. More than 95% of the excreted label was a single metabolite which was shown by hydrolysis and TLC to be benzoyltaurine. Isolated renal tubules and both hepatic and renal mitochondria produced benzoyltaurine in vitro. Excretion of benzoic acid and benzoyltaurine were not limited by plasma binding (less than 5% bound). To examine whether the slow excretion reflected renal transport, the transport of 100 microM benzoic acid, benzoyltaurine, and hippuric acid were determined in isolated renal tubules. All three compounds were accumulated via a probenecid- and cyanide-sensitive mechanism. Probenecid-sensitive uptake at 60 min of hippuric acid (436 nmol/mg) was greater than that of benzoyltaurine (164 nmol/mg) and both exceeded uptake of benzoic acid (63 nmol/mg). The same pattern was seen at 10 microM, except that tissue:medium ratios were even greater, indicating at least partial saturation of transport at 100 microM. Thus, the slow excretion of benzoic acid by these flounder was due to a combination of factors, including slow metabolism of benzoic acid to benzoyltaurine, saturation of the transport of benzoyltaurine, and low affinity of secretory transport for benzoic acid and benzoyltaurine, relative to hippuric acid, the mammalian metabolite.     

Mrp2 is involved in the efflux and disposition of fosinopril

The multidrug‐resistance‐associated proteins 1 and 2 (MRP1/MRP2) are transporters responsible for the efflux of drugs and endogenous compounds. Madin Darby canine kidney (MDCK) cells transfected with the human MRP1 or MRP2 genes were used to assess whether several widely used pharmaceuticals are potential substrates by examining their differential toxicity, accumulation and efflux. Loratadine, an antihistamine, was 1.4‐fold less toxic to MRP1 cells and its retention was 1.3‐fold lower than that from MDCK control cells. Fosinopril, an angiotensin converting enzyme inhibitor, was 2.4‐fold less toxic and its retention was 4.5‐fold lower in MRP2‐transfected cells compared with control cells. To determine whether fosinopril contributed to a drug–drug interaction, fosinopril efflux was examined in vitro in combination with other known or suspected MRP2 substrates over a period of 20 min. When fosinopril was coincubated with desloratadine, loratadine or methotrexate, its retention was increased by 2‐, 4.7‐ and 2‐fold, respectively, which likely indicates that a drug–drug interaction is occurring. In vivo studies were conducted, in which FVB wild‐type and FVB/Mrp2^?/? mice were dosed with fosinopril and the known MRP2 substrate methotrexate, and tissues collected after 1 h. In mice lacking Mrp2, drug levels were reduced in the intestine by 1.5‐fold, but increased in the liver, serum and kidneys, by 2.1‐, 2.9‐ and 3‐fold, respectively. These data suggest that, in the absence of Mrp2, fosinopril alters the retention of a second drug. These findings will help increase our understanding of the role that MRP2 plays in altering the retention and disposition of coadministered pharmaceuticals. Copyright © 2011 John Wiley & Sons, Ltd.     

PTP-1B及其抑制剂与2型糖尿病的研究现状

蛋白酪氨酸磷酸酶-1B (PTP-1B)是蛋白酪氨酸磷酸酶(PTP)家族中的一员,在胰岛素信号转导途径中发挥重要作用.经研究发现,PTP-1B与2型糖尿病的发生、发展有密切关系. 本文按PTP-1B 小分子抑制剂的结构类别对近年来文献报道的代表性小分子PTP-1B 抑制剂进行综述.表明深入研究PTP-1B及其有效的抑制剂对于2型糖尿病治疗具有良好的发展前景.     

A mitogen-activated protein kinase is involved in the inotropic but not chronotropic actions of adrenoceptor agonists and endothelin-1

The activation of mitogen-activated protein kinase (MAPK) pathways in the heart, for instance by alpha(1)-adrenoceptor agonists and endothelin-1, has primarily been associated with cellular growth regulation. Here we have investigated a possible role of MAPK pathways in the inotropic and chronotropic effects of adrenoceptor agonists and endothelin-1 in isolated rat left and right atria. Inotropic and chronotropic responses of the isolated atria to methoxamine, isoprenaline and endothelin-1 were measured in the absence and presence of inhibitors of MAPK pathways. The MAPK kinase (MKK(mek)) inhibitors PD98059 (100 microM) and U0126 (10 microM) significantly inhibited the inotropic responses to the alpha(1)-adrenoceptor agonist methoxamine (300 microM) and endothelin-1 (50 nM), but not the chronotropic responses to these agonists. U0126 but not PD98059 inhibited the inotropic response to 3 microM isoprenaline. None of the aforementioned inotropic and chronotropic effects were inhibited by the MAPKP(p38) inhibitor SB203580 (2 microM). We conclude that activation of the PD98059/U0126-sensitive MAPK pathway is essential for the inotropic but not chronotropic actions of adrenoceptor agonists and endothelin-1.     

A nitric oxide/Ca(2+)/calmodulin/ERK1/2 mitogen-activated protein kinase pathway is involved in the mitogenic effect of IL-1beta in human astrocytoma cells

Background and purpose:

Evidence is accumulating to support a role for interleukin-1β (IL-1β) in astrocyte proliferation. However, the mechanism by which this cytokine modulates this process is not fully elucidated.

Experimental approach:

In this study we used human astrocytoma U-373MG cells to investigate the role of nitric oxide (NO), intracellular Ca²⁺ concentration ([Ca²⁺]_i), and extracellular signal-regulated protein kinase (ERK) in the signalling pathway mediating IL-1β-induced astrocyte proliferation.

Key results:

Low IL-1β concentrations induced dose-dependent ERK activation which paralleled upregulation of cell division, whereas higher concentrations gradually reversed both these responses by promoting apoptosis. Pretreatment with the nonspecific NOS inhibitor, N-ω-nitro-l-arginine methyl ester (L-NAME) or the selective iNOS inhibitor, N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride (1400W), antagonized ERK activation and cell proliferation induced by IL-1β. Inhibition of cGMP formation by the guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), partially inhibited ERK activation and cell division. Functionally blocking Ca²⁺ release from endoplasmic reticulum with ryanodine or 2-aminoethoxydiphenylborane (2-APB), inhibiting calmodulin (CaM) activity with N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide hydrochloride (W7) or MAPK kinase activity with 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthiol]butadiene (U0126) downregulated IL-1β-induced ERK activation as well as cell proliferation. The cytokine induced a transient and time-dependent increase in intracellular NO levels which preceded elevation in [Ca²⁺]_i.

Conclusions and implications:

These data identified the NO/Ca²⁺/CaM/ERK signalling pathway as a novel mechanism mediating the mitogenic effect of IL-1β in human astrocytes. As astrocyte proliferation is a hallmark of reactive astrogliosis, our results reveal a new potential target for therapeutic intervention in neuroinflammatory disorders.