Inhibition Of Cellular Uptake Research Articles

Effect of polyethyleneglycol (PEG) chain length on the bio–nano-interactions between PEGylated lipid nanoparticles and biological fluids: from nanostructure to uptake in cancer cells

When nanoparticles (NPs) enter a physiological environment, medium components compete for binding to the NP surface leading to formation of a rich protein shell known as the "protein corona". Unfortunately, opsonins are also adsorbed. These proteins are immediately recognized by the phagocyte system with rapid clearance of the NPs from the bloodstream. Polyethyleneglycol (PEG) coating of NPs (PEGylation) is the most efficient anti-opsonization strategy. Linear chains of PEG, grafted onto the NP surface, are able to create steric hindrance, resulting in a significant inhibition of protein adsorption and less recognition by macrophages. However, excessive PEGylation can lead to a strong inhibition of cellular uptake and less efficient binding with protein targets, reducing the potential of the delivery system. To reach a compromise in this regard we employed a multi-component (MC) lipid system with uncommon properties of cell uptake and endosomal escape and increasing length of PEG chains. Nano liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS) analysis allowed us to accurately determine the corona composition showing that apolipoproteins are the most abundant class in the corona and that increasing the PEG length reduced the protein adsorption and the liposomal surface affinity for apolipoproteins. Due to the abundance of apolipoproteins, we exploited the "protein corona effect" to deliver cationic liposome-human plasma complexes to human prostate cancer PC3 cells that express a high level of scavenger receptor class B type 1 in order to evaluate the cellular uptake efficiency of the systems used. Combining laser scanning confocal microscopy with flow cytometry analysis in PC3 cells we demonstrated that MC-PEG2k is the best compromise between an anti-opsonization strategy and active targeting and could be a promising candidate to treat prostate cancer in vivo.

Silica nanogelling of environment-responsive PEGylated polyplexes for enhanced stability and intracellular delivery of siRNA

In this study, poly(ethylene glycol) (PEG)-block-polycation/siRNA complexes (PEGylated polyplexes) were wrapped with a hydrated silica, termed "silica nanogelling", in order to enhance their stability and functionality. Silica nanogelling was achieved by polycondensation of soluble silicates onto the surface of PEGylated polyplexes comprising a disulfide cross-linked core. Formation of silica nanogel layer on the PEGylated cross-linked polyplexes was confirmed by particle size increase, surface charge reduction, and elemental analysis of transmission electron micrographs. Silica nanogelling substantially improved polyplex stability against counter polyanion-induced dissociation under non-reductive condition, without compromising the reductive environment-responsive siRNA release triggered by disulfide cleavage. Silica nanogelling significantly enhanced the sequence-specific gene silencing activity of the polyplexes in HeLa cells without associated cytotoxicity, probably due lower endosomal entrapment (or lysosomal degradation) of delivered siRNA. The lower endosomal entrapment of the silica nanogel system could be explained by an accelerated endosomal escape triggered by deprotonated silanol groups in the silica (the proton sponge hypothesis) and/or a modulated intracellular trafficking, possibly via macropinocytosis, as evidenced by the cellular uptake inhibition assay. Henceforth, silica nanogelling of PEGylated siRNA polyplexes is a promising strategy for preparation of stable and functional siRNA delivery vehicles.

Elevation of Serum Asymmetrical and Symmetrical Dimethylarginine in Patients with Advanced Glaucoma

Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are the dimethylated isomeric derivatives of the amino acid L-arginine. ADMA is an endogenous inhibitor of nitric oxide synthase (NOS), while SDMA is a competitive inhibitor of cellular uptake of L-arginine, the substrate for NOS. As such, these metabolites are associated with endothelial dysfunction. As the nitric oxide pathway and endothelial dysfunction have been implicated in glaucoma, the aim of this study was to investigate serum ADMA, SDMA, and L-arginine levels in individuals with advanced glaucoma compared with normal controls. In addition, we have investigated genetic variation in the DDAH1 and DDAH2 genes, encoding the enzymes responsible for degradation of ADMA, for association with ADMA level in glaucoma patients and controls. Two hundred eleven patients with advanced glaucoma and 295 normal controls were recruited. Liquid chromatography-tandem mass spectrometry was used to measure the serum ADMA, SDMA, and L-arginine levels of participants. Single nucleotide polymorphisms in the DDAH1 and DDAH2 genes reportedly associated with ADMA level were genotyped in all individuals. A significant increase in both serum ADMA and SDMA concentration was detected in advanced glaucoma cases compared with controls (P ≤ 0.0001). No significant change was detected in serum L-arginine concentration. No association of polymorphisms in DDAH1 and DDAH2 with either ADMA level or glaucoma was detected. The serum levels of two dimethylarginines, ADMA and SDMA, are associated with advanced glaucoma. These data further implicate the nitric-oxide pathway in glaucoma pathogenesis.

Hematopoietic stem cell transplantation improves the high incidence of neutralizing allo-antibodies observed in Hurler's syndrome after pharmacological enzyme replacement therapy

Mucopolysaccharidosis type I is caused by deficiency of α-L-iduronidase. Currently available treatment options include an allogeneic hematopoietic stem cell transplant and enzyme replacement therapy. Exogenous enzyme therapy appears promising but the benefits may be attenuated, at least in some patients, by the development of an immune response to the delivered enzyme. The incidence and impact of alloimmune responses in these patients remain unknown. We developed an immunoglobulin G enzyme-linked immunosorbent assay as well as in vitro catalytic enzyme inhibition and cellular uptake inhibition assays and quantified enzyme inhibition by allo-antibodies. We determined the impact of these antibodies in eight patients who received enzyme therapy before and during hematopoietic stem cell transplantation. In addition, 20 patients who had previously received an allogeneic stem cell transplant were tested to evaluate this treatment as an immune tolerance induction mechanism. High titer immune responses were seen in 87.5% (7/8) patients following exposure to α-L-iduronidase. These patients exhibited catalytic enzyme inhibition (5/8), uptake inhibition of catalytically active enzyme (6/8) or both (4/8). High antibody titers generally preceded elevation of previously described biomarkers of disease progression. The median time to development of immune tolerance was 101 days (range, 26-137) after transplantation. All 20 patients, including those with mixed chimerism (22%), tested 1 year after transplantation were tolerized despite normal enzyme levels. We found a high incidence of neutralizing antibodies in patients with mucopolysaccharidosis type I treated with enzyme replacement therapy. We also found that allogeneic hematopoietic stem cell transplantation was an effective and rapid immune tolerance induction strategy.

Translocation mechanisms of chemically functionalised carbon nanotubes across plasma membranes

Understanding the mechanisms responsible for carbon nanotube (CNT) internalisation into live cells is considered critical both from a fundamental point of view and for further engineering of CNT-based delivery systems to intracellular targets. While several studies are focused on the development of such CNT-based delivery systems, attempts to systematically elucidate the cellular uptake mechanisms of CNTs are still rather limited. The aim of the present study is to evaluate the cellular internalisation of chemically functionalised multi-walled carbon nanotubes (f-MWCNTs) in the presence of different well-known cellular uptake inhibitors. Our data reveal how f-MWCNTs are able to translocate across cell membranes of both phagocytic and non-phagocytic cell lines. We have evidenced that at least 30–50% of f-MWCNTs are taken up by cells through an energy-independent mechanism. This characteristic makes nanotubes loaded with therapeutic or diagnostic cargos extremely interesting as the release of active molecules directly into the cytoplasm increase their biological activity and therapeutic efficacy.

Synthesis of novel folate conjugated fluorescent nanoparticles for tumor imaging

A novel folate fluorescent nanoconjugate was synthesized and used for detection of cancer cells overexpressing the folate receptor (FR). The folate conjugate (PCMS-NA-FA) was synthesized by conjugating folic acid (FA) and 4-ethylnyl-N-ethyl-1, 8-naphthalimide (NA) to the polychloromethylstyrene (PCMS) functionalized with azido group (PCMS-N₃) through click reaction. The obtained conjugate had clear structure and could form PCMS-NA-FA nanoparticles with particle size around 86 nm in aqueous solution. Ability of PCMS-NA-FA targeting to cancerous cells was investigated by comparing the uptake of the nanoparticles by human adenocarcinoma HeLa cells and by non-FR expressing human lung carcinoma A549 cells. Specificity of the PCMS-NA-FA nanoparticles targeting on FRs was verified with cellular uptake inhibition assay, in which HeLa cells were incubated with both nanoconjugate and free FA. In addition, the specificity was also confirmed by the collocation of the immunofluorescence staining of anti-FR and the cellular uptake of the PCMS-NA-FA nanoparticles. Furthermore, the organ distribution of this folate nanoconjugate was studied on HeLa cell-bearing mice via frozen slicing, and the results showed that the folate nanoparticles were preferentially accumulated in the tumor site rather than other tissues, indicating the desired specificity for tumor targeting and imaging. All these findings suggested that this practical synthetic strategy can potentially facilitate the preparation of multifunctional imaging probe in biology and diagnosis of disease.

Spermine grafted galactosylated chitosan for improved nanoparticle mediated gene delivery

Despite multitude of beneficial features, chitosan has poor water solubility and transfection ability which affect its gene delivery efficacy. The two features are improved when certain chemical modifications are incorporated into the chitosan parent backbone. This strategy is adopted here, by coupling galactose and spermine into the chitosan backbone. The conjugation was determined with FTIR and 1H NMR and nanoparticle morphology was assessed by TEM and AFM techniques. Particle size, zeta potential, buffering capacity and DNA binding ability gave encouraging result of enhanced solubility and stability. In vitro studies of GCSM in HepG2 cell lines displayed low cytotoxicity and improved transfection. We also identified the preference of receptor mediated internalization for nanoparticles cellular uptake by treating with cellular uptake inhibitors. The results evidently led us to comprehend that galactosylated chitosan-g-spermine could be considered as a promising chitosan derivative for conducting nanoparticle mediated gene delivery.

Folate mediated l-arginine modified oligo (alkylaminosiloxane) graft poly (ethyleneimine) for tumor targeted gene delivery

Folate receptor mediated gene targeting provides several advantages such as delivery of high concentration of gene at specific tumor sites including brain, lung, ovary, uterus and kidney where folate receptors are over expressed. In the present study for both systemic stability and tumor targeting ability, poly (ethylene glycol)–folic acid (PEG–FA) conjugate was coupled with an arginine modified oligo (alkylaminosiloxane) graft poly (ethyleneimine) having enhanced transfection efficiency compared to poly (ethyleneimine). The resultant polymer P(SiDAAr)5FP2 complexed pDNA effectively and showed protection against nuclease degradation. The PEG group provided improved blood compatibility and cell viability. Uniformly oriented arginine moiety helped to enhance cellular and nuclear localisation, which led to improved transfection. The polymer was capable of releasing pDNA at the nucleus and being cleared from the cell after its purpose. Transfection in presence of cellular uptake inhibitors showed multiple pathways for cellular uptake of the targeted polymer, out of which folate receptor mediated uptake was more prominent. The folate mediated cellular uptake of P(SiDAAr)5FP2 was then confirmed by flow cytometric evaluation. The high accumulation of targeted polymers in the tumor tissues of tumor bearing mice from 2nd hour onwards proved the active targeting effect of the polymer. Besides tumor accumulation, the material showed capability to diffuse through the vascular endothelium. This property is expected to be beneficial for brain targeting experiments.

A Novel, Bio-Reducible Gene Vector Containing Arginine and Histidine Enhances Gene Transfection and Expression of Plasmid DNA

We have engineered a novel, non-viral, multifunctional gene vector (STR-CH(2)R(4)H(2)C) that contained stearoyl (STR) and a block peptide consisting of Cys (C), His (H), and Arg (R). STR-CH(2)R(4)H(2)C can form a stable nano-complex with plasmid DNA (pDNA) based on electronic interactions and disulfide cross linkages. In this study, we evaluated the efficacy of STR-CH(2)R(4)H(2)C as a gene vector. We first determined the optimal weight ratio for STR-CH(2)R(4)H(2)C/pDNA complexes. The complexes with a weight ratio of 50 showed the highest transfection efficacy. We also examined the transfection efficacy of STR-CH(2)R(4)H(2)C/pDNA complexes with or without serum and compared STR-CH(2)R(4)H(2)C/pDNA transfection efficacy with that of Lipofectamine. Even in the presence of serum, STR-CH(2)R(4)H(2)C showed higher transfection efficacy than did Lipofectamine. In addition, we determined the mechanism of transfection of the STR-CH(2)R(4)H(2)C/pDNA complexes using various cellular uptake inhibitors and evaluated its endosomal escape ability using chloroquine. Macropinocytosis was main cellular uptake pathway of STR-CH(2)R(4)H(2)C/pDNA complexes. Our results suggested that STR-CH(2)R(4)H(2)C is a promising gene delivery system.

Polyphenols and phenolic acids from strawberry and apple decrease glucose uptake and transport by human intestinal Caco‐2 cells

The effect of polyphenols, phenolic acids and tannins (PPTs) from strawberry and apple on uptake and apical to basolateral transport of glucose was investigated using Caco-2 intestinal cell monolayers. Substantial inhibition on both uptake and transport was observed by extracts from both strawberry and apple. Using sodium-containing (glucose transporters SGLT1 and GLUT2 both active) and sodium-free (only GLUT2 active) conditions, we show that the inhibition of GLUT2 was greater than that of SGLT1. The extracts were analyzed and some of the constituent PPTs were also tested. Quercetin-3-O-rhamnoside (IC₅₀ =31 μM), phloridzin (IC₅₀=146 μM), and 5-caffeoylquinic acid (IC₅₀=2570 μM) contributed 26, 52 and 12%, respectively, to the inhibitory activity of the apple extract, whereas pelargonidin-3-O-glucoside (IC₅₀=802 μM) contributed 26% to the total inhibition by the strawberry extract. For the strawberry extract, the inhibition of transport was non-competitive based on kinetic analysis, whereas the inhibition of cellular uptake was a mixed-type inhibition, with changes in both V(max) and apparent K(m) . The results in this assay show that some PPTs inhibit glucose transport from the intestinal lumen into cells and also the GLUT2-facilitated exit on the basolateral side.

Evaluation and mechanism studies of PEGylated dendrigraft poly-L-lysines as novel genedelivery vectors

Dendrimers have attracted great interest in the field of gene delivery due to their syntheticcontrollability and excellent gene transfection efficiency. In this work, dendrigraftpoly-L-lysines (DGLs) were evaluated as a novel gene vector for the first time. Derivativesof DGLs (generation 2 and 3) with different extents of PEGylation were successfullysynthesized and used to compact pDNA as complexes. The result of gel retardation assayshowed that pDNA could be effectively packed by all the vectors at a DGLs to pDNAweight ratio greater than 2. An increase in the PEGylation extent of vectors resulted ina decrease in the incorporation efficiency and cytotoxicity of complexes in 293cells, which also decreased the zeta potential a little but did not affect the meandiameter of complexes. Higher generation of DGLs could mediate higher genetransfection in vitro. Confocal microscopy and cellular uptake inhibition studiesdemonstrated that caveolae-mediated process and macropinocytosis were involvedin the cellular uptake of DGLs-based complexes. Also the results indicate thatproper PEGylated DGLs could mediate efficient gene transfection, showing theirpotential as an alternate biodegradable vector in the field of nonviral gene delivery.

Controllable inhibition of cellular uptake of oxidized low-density lipoprotein: Structure–function relationships for nanoscale amphiphilic polymers

A family of anionic nanoscale polymers based on amphiphilic macromolecules (AMs) was developed for controlled inhibition of highly oxidized low-density lipoprotein (hoxLDL) uptake by inflammatory macrophage cells, a process that triggers the escalation of a chronic arterial disease called atherosclerosis. The basic AM structure is composed of a hydrophobic portion formed from a mucic acid sugar backbone modified at the four hydroxyls with lauroyl groups conjugated to hydrophilic poly(ethylene glycol) (PEG). The AM structure-activity relationships were probed by synthesizing AMs with six key variables: length of the PEG chain, carboxylic acid location, type of anionic charge, number of anionic charges, rotational motion of the anionic group, and PEG architecture. All AM structures were confirmed by nuclear magnetic resonance spectroscopy and their ability to inhibit hoxLDL uptake in THP-1 human macrophage cells was compared in the absence and presence of serum. We report that AMs with one, rotationally restricted carboxylic acid within the hydrophobic portion of the polymer was sufficient to yield the most effective AM for inhibiting hoxLDL internalization by THP-1 human macrophage cells under serum-containing conditions. Further, increasing the number of charges and altering the PEG architecture in an effort to increase serum stabilization did not significantly impair the ability of AMs to inhibit hoxLDL internalization, suggesting that selected modifications to the AMs could potentially promote multifunctional characteristics of these nanoscale macromolecules.

Inhibition of cellular uptake of folate by blocking synthesis of the membrane folate receptor.

The folate receptor, bound to the plasma membrane through a glycosylphosphatidylinositol anchor, requires both sphingolipids and cholesterol in the membrane for full activity. In recent studies, treatment of cells in culture with the mycotoxin fumonisin B1, which inhibits sphingolipid synthesis, virtually abolished uptake of 5-methyltetrahydrofolate.

Curcumin mediates time and concentration dependent regulation of redox homeostasis leading to cytotoxicity in macrophage cells

The present study was designed to test a hypothesis that curcumin may be modulating oxidative stress parameters including reactive oxygen species, non-protein thiols and expression of antioxidant genes in a concentration and time dependent manner in exhibiting cytotoxic effects in macrophage cell line RAW 264.7. The results have shown that curcumin elevated the reactive oxygen species levels accompanied by a decrease in levels of intracellular non-protein thiols at 2 h after its addition to cells. However, the levels of reactive oxygen species decreased and non-protein thiols content increased at 18 h after its addition. Whereas the expression of glutathione peroxidase ( GPx), catalase, Cu,Zn-superoxide dismutase ( Cu,Zn-SOD) and heme oxygenase -1 ( HO-1) increased with curcumin concentration and also with increase in time of incubation, the expression of Mn- superoxide dismutase ( Mn-SOD) showed concentration dependant repression upon treatment with curcumin. The cell viability was significantly reduced at high concentration (25 µM) of curcumin treatment but not at low concentration (5 µM). Curcumin at 5 µM scavenged γ-radiation induced reactive oxygen species and inhibited cell death. On the contrary, at 25 µM, curcumin increased radiation induced reactive oxygen species production and augmented cell death. Interestingly pretreatment with reducing agents glutathione (GSH) or N-acetyl-cysteine (NAC), modified the curcumin mediated redox changes and cell death differentially, due to the inhibition of cellular uptake of curcumin by GSH but not by NAC. The important finding of the study is that the concentration and time dependent dual effect of curcumin may be attributed to changes in oxidative stress and antioxidant gene expression levels leading to inhibition or promotion of cell death.

Uridine Protects Cortical Neurons from Glucose Deprivation-Induced Death: Possible Role of Uridine Phosphorylase

We previously reported that uridine blocked glucose deprivation-induced death of immunostimulated astrocytes by preserving ATP levels. Uridine phosphorylase (UPase), an enzyme catalyzing the reversible phosphorylation of uridine, was involved in this effect. Here, we tried to expand our previous findings by investigating the uridine effect on the brain and neurons using in vivo and in vitro ischemic injury models. Orally administrated uridine (50-200 mg/kg) reduced middle cerebral artery occlusion (1.5 h)/reperfusion (22 h)-induced infarct in mouse brain. Additionally, in the rat brain subjected to the same ischemic condition, UPase mRNA and protein levels were up-regulated. Next, we employed glucose deprivation-induced hypoglycemia in mixed cortical cultures of neurons and astrocytes as an in vitro model. Cells were deprived of glucose and, two hours later, supplemented with 20 mM glucose. Under this condition, a significant ATP loss followed by death was observed in neurons but not in astrocytes, which were blocked by treatment with uridine in a concentration-dependent manner. Inhibition of cellular uptake of uridine by S-(4-nitrobenzyl)-6-thioinosine blocked the uridine effect. Similar to our in vivo data, UPase expression was up-regulated by glucose deprivation in mRNA as well as protein levels. Additionally, 5-(phenylthio)acyclouridine, a specific inhibitor of UPase, prevented the uridine effect. Finally, the uridine effect was shown only in the presence of astrocytes. Taken together, the present study provides the first evidence that uridine protects neurons against ischemic insult-induced neuronal death, possibly through the action of UPase.

Uridine prevents the glucose deprivation-induced death of immunostimulated astrocytes via the action of uridine phosphorylase

We previously reported that in immunostimulated astrocytes, glucose deprivation induced cell death via the loss of ATP, reduced glutathione, and mitochondrial transmembrane potential. The cytotoxicity was due to reactive nitrogen and oxygen species and blocked by adenosine, a purine nucleoside, via the preservation of cellular ATP. Here, we investigated whether uridine, a pyrimidine nucleoside, could prevent the glucose deprivation-induced cytotoxicity in LPS + IFN-γ-treated (immunostimulated) astrocytes. Glucose deprivation induced the death of immunostimulated cells, which was significantly reduced by uridine. Glucose deprivation rapidly decreased cellular ATP levels in immunostimulated astrocytes, which was also reversed by uridine. The inhibition of cellular uptake of uridine by S-(4-nitrobenzyl)-6-thioinosine attenuated the protective effect of uridine. mRNA and protein expression for uridine phosphorylase, an enzyme catalyzing reversible phosphorolysis of uridine, were observed in rat brain as well as primary astrocytes. 5-(Phenylthio)acyclouridine (PTAU), a specific inhibitor of uridine phosphorylase, inhibited the protective effect of uridine. Additionally, the loss of mitochondrial transmembrane potential and reduced glutathione by glucose deprivation in immunostimulated cells was attenuated by uridine, which was also reversed by PTAU. These results provide the first evidence that uridine protects immunostimulated astrocytes against the glucose deprivation-induced death by preserving intracellular ATP through the action of uridine phosphorylase.

In vitroinvestigations on the mode of action of the hydroxypyridone antimycotics rilopirox and piroctone onCandida albicans

Rilopirox and piroctone belong to the class of hydroxypyridone antimycotics. This class is not related to other antimycotics. In contrast to azole antimycotics and polyene antimycotics the mode of action of hydroxypyridone antimycotics is not fully understood. Inhibition of cellular uptake of essential compounds as well as loss of other compounds seems to be only a secondary effect of a primary not known action of these drugs. The antifungal effect in vitro depends on the medium used. The hyphal induction of Candida albicans is inhibited by hydroxypyridone antimycotics, but this effect is compensated by iron ions. A damage of the cell membrane and a direct influence on adenosine triphosphate synthesis, respectively, do not seem to be part of the mode of action. But there are clear hints that reactive oxygen species (ROS) and available metabolic activity are important parts of the mode of action of the hydroxypyridone antimycotics rilopirox and piroctone.

The effect of block copolymer structure on the internalization of polymeric micelles by human breast cancer cells

The objective of this study was to assess the effect of hydrophilic/hydrophobic block chain lengths on the internalization of poly(ethylene oxide)- block-poly(ɛ-caprolactone) (PEO- b-PCL) micelles by cancer cells. PEO- b-PCL block copolymers with varied PEO and PCL chain lengths were synthesized, assembled to polymeric micelles and loaded with a hydrophobic fluorescent probe (DiI) through a co-solvent evaporation method of physical encapsulation. The slow release of the fluorescent probe from the micellar structure was evidenced following DiI transfer to lipid vesicles. The extent of micellar uptake by cancer cells was investigated through their incubation with MCF-7 cells followed by measurement of the fluorescent emission intensity of DiI ( λ = 550 nm) in separated lysed cells. Cellular internalization of polymeric micelles was confirmed by laser scanning microscopy. The mechanism of micellar uptake was investigated by pretreatment of MCF-7 cells with chlorpromazine and cytochalasin B. Encapsulation of DiI in PEO- b-PCL micelles lowered the extent and rate of hydrophobic probe internalization by cancer cells. For polymeric micelles with 5000 g mol −1 of PCL and varied PEO molecular weights of 2000, 5000 and 13,000 g mol −1, maximum uptake was observed at a PEO molecular weight of 5000 g mol −1. For polymeric micelles with 5000 g mol −1 of PEO and varied PCL molecular weights of 5000, 13,000 and 24,000 g mol −1, maximum uptake was observed at 13,000 g mol −1 of PCL. Chlorpromazine reduced the cellular uptake of PEO- b-PCL micelles independent from the block copolymer structure, pointing to the involvement of clathrin mediated endocytosis mechanisms in the uptake of polymeric micelles by cancer cells. Inhibition of cellular uptake of PEO- b-PCL micelles by cytochalasin B, on the other hand, was found to be dependent on the chemical structure of the core/shell forming blocks.

Role of adenosine A(1) receptor in angiotensin II- and norepinephrine-induced renal vasoconstriction.

We investigated the contributions of adenosine A(1) receptors to angiotensin II- and norepinephrine-induced renal vasoconstriction. Intrarenal administrations of angiotensin II (3, 10, and 30 ng) or norepinephrine (100 and 500 ng) produced dose-dependent renal vasoconstriction in anesthetized dogs. Under resting conditions, angiotensin II (30 ng) and norepinephrine (500 ng) significantly decreased renal blood flow by -43 +/- 3 and -19 +/- 2%, respectively (n = 21). Intra-arterial infusion of adenosine (5 microg/kg/min) significantly augmented renal blood flow responses to both angiotensin II and norepinephrine (-64 +/- 4 and -45 +/- 14%, n = 7). Renal blood flow responses to angiotensin II and norepinephrine were also augmented by inhibition of cellular uptake of adenosine with dipyridamole (10 microg/kg/min, n = 6). Blockade of adenosine A(1) receptors with 8-(noradamantan-3-yl)-1,3-dipropylxanthine (KW-3902; 10 microg/kg/min) did not alter basal renal blood flow but significantly attenuated angiotensin II- and norepinephrine-induced renal vasoconstriction (-34 +/- 6 and -9 +/- 3%, n = 7). Furthermore, KW-3902 completely prevented augmentation of renal blood flow responses to angiotensin II and norepinephrine produced by adenosine or dipyridamole (n = 7 and 6, respectively). Administrations of angiotensin II (30 ng) or norepinephrine (500 ng) into the common carotid artery significantly decreased carotid blood flow by -20 +/- 5 and -41 +/- 10%, respectively; however, neither adenosine (5 microg/kg/min) nor KW-3902 (10 microg/kg/min) affected the carotid blood flow responses to angiotensin II and norepinephrine (n = 5, respectively). Adenosine concentrations in dialysates were not significantly changed by administrations of angiotensin II (from 19 +/- 3 to 24 +/- 4 nM, n = 6) or norepinephrine (from 16 +/- 3 to 19 +/- 3 nM, n = 6). These results suggest that basal interstitial adenosine levels influence both angiotensin II and norepinephrine-induced vasoconstriction via A(1) receptors in the kidney but not in the area drained by the common carotid artery. The responses of adenosine to angiotensin II- and norepinephrine-induced renal vasoconstriction may not be mediated through de novo intrarenal adenosine accumulation due to angiotensin II- and norepinephrine-induced renal vasoconstriction.

Evaluation of GL331 in combination with paclitaxel: GL331's interference with paclitaxel-induced cell cycle perturbation and apoptosis.

Combination of selecting agents that act on different cellular mechanisms is a common strategy in cancer chemotherapy. GL331 is a new potent topoisomerase II (Topo II) poison; distinctly, paclitaxel is a microtubule-interfering cancer chemotherapeutic agent. In this study, we intended to evaluate the efficacy of combining GL331 with paclitaxel in cell killing and apoptotic induction in nasopharyngeal carcinoma NPC-TW01 cells. By MTT and internucleosomal DNA cleavage assays, we found that pretreatment or simultaneous treatment of NPC-TW01 cells with GL331 could significantly interfere with paclitaxel's cell killing and apoptosis-inducing activity. When the administration schedule was reversed, the cytotoxicity of GL331 was attenuated by paclitaxel pretreatment. The anti-cancer activity produced by combining GL331 with paclitaxel was obviously lower than the addition of the activities of two individual agents. NPC-TW01 cells were treated with GL331 and 3H-labeled paclitaxel simultaneously or with GL331 before 3H-labeled paclitaxel. In both conditions, GL331 did not reduce the [3H]paclitaxel level in the cells, suggesting that GL331's interference with paclitaxel's cell-killing and apoptosis-inducing efficacy did not result from any inhibition of cellular uptake or retention of paclitaxel. In addition, we found that GL331-induced perturbation of cell cycle progression dramatically over-rode the patterns of mitotic arrest induced by paclitaxel, and the mechanism could be the inhibition of cyclin B1/CDC2 kinase and MAD2 checkprotein activities.