Article

Antioxidants effects of Platinum Nanoparticles: A Potential Alternative Treatment to Lung Diseases

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

Imbalance oxidative status occurs when oxidative stress is higher in the body due to the production of reactive oxygen species. Thus, antioxidants are needed to counteract the production of free radicals. Reoccurrence of oxidative stress in the lung cells will eventually lead to inflammation and edema. This will result to a severe prognosis of lung diseases. Our interest is to populate certain mechanisms that can be activated during this process by reversing the oxidative stress status. Platinum nanoparticles (PtNPs) have been suggested as one of the powerful antioxidants that can quench free radicals. The mechanistic pathway may involve Protein Kinase C, which correlates well with the expression of the Epithelial Sodium Channel (ENaC). ENaC plays an important role in sodium uptake thus stimulate lung liquid clearance. The failure of lung clearance will interrupt gaseous exchange thus eventually lead to death. This review will discuss on the antioxidant properties of PtNPs as well as the underlying mechanism of PKC and ENaC in maintaining the oxidative status in the lung cells.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... [44][45][46][47][48][49][50][51][52] Actually, PtNPs have been already proposed as efficient and selective radical scavengers for therapies of oxidative stress diseases. [41][42][43][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69] However, their clinical potential has been slowed down by some toxicological concerns. Homogenous data about PtNP biocompatibility are by encapsulating PtNPs within dendrimers. ...
... 244,245 Fullerene, metal and metal oxide NPs have been suggested as antioxidant enzyme-mimetic nanomaterials, scavenging ROS in biological systems. [44][45][46][47]50,[247][248][249][250][251][252][253][254][255][256] Among these, PtNPs have attracted particular interest [41][42][43][53][54][55][56][57][58][59][60][61][63][64][65][66][67][68][69]126,[238][239][240][241]254,255,[257][258][259][260][261][262] owing to their high efficiency and selectivity as artificial CAT, HRP and SOD enzymes 41,128,129,[263][264][265] (Fig. 5). In particular, PtNPs are able to catalyze the reduction of H 2 O 2 to water and molecular oxygen acting as the biological enzyme CAT, or to promote the oxidation of a reduced substrate in order to decompose H 2 O 2 to water, as HRP mimics. ...
... 44,45,[247][248][249][250][251][252]255,[289][290][291][292] In this framework, PtNPs are showing safe applications to several human pathologies, as demonstrated in recently published works. [41][42][43][53][54][55][56][57][58][59][60][61]63,64,[66][67][68][69]126,[238][239][240][241]255,[257][258][259][260][261][262]293,294 Different from other metal nanoparticles, 193 PtNPs possess high stability in acidic cellular vesicle environments, forecasting cytocompatibility and tolerance in vivo. ...
Article
Oxidative stress-dependent inflammatory diseases represent a major concern for the population's health worldwide. Biocompatible nanomaterials with enzymatic properties could play a crucial role in the treatment of such pathologies. In this respect, platinum nanoparticles (PtNPs) are promising candidates, showing remarkable catalytic activity, able to reduce the intracellular reactive oxygen species (ROS) levels and impair the downstream pathways leading to inflammation. This review reports a critical overview of the growing evidence revealing the anti-inflammatory ability of PtNPs and their potential applications in nanomedicine. It provides a detailed description of the wide variety of synthetic methods recently developed, with particular attention to the aspects influencing biocompatibility. Special attention has been paid to the studies describing the toxicological profile of PtNPs with an attempt to draw critical conclusions. The emerging picture suggests that the material per se is not causing cytotoxicity, while other physicochemical features related to the synthesis and surface functionalization may play a crucial role in determining the observed impairment of cellular functions. The enzymatic activity of PtNPs is also summarized, analyzing their action against ROS produced by pathological conditions within the cells. In particular, we extensively discuss the potential of these properties in nanomedicine to down-regulate inflammatory pathways or to be employed as diagnostic tools with colorimetric readout. A brief overview of other biomedical applications of nanoplatinum is also presented.
... Advancement in nanotechnology has revealed several nanoparticles either from inorganic [19,20] or biological origins, such as melanin nanoparticles [21] as potent antioxidants by themselves. Novel metal nanoparticles (Au, Ag, Pt) and transition metal oxide (CuO, NiO) are the commonly used and tested for their antioxidant activity [19,20,[22][23][24]. ...
... Advancement in nanotechnology has revealed several nanoparticles either from inorganic [19,20] or biological origins, such as melanin nanoparticles [21] as potent antioxidants by themselves. Novel metal nanoparticles (Au, Ag, Pt) and transition metal oxide (CuO, NiO) are the commonly used and tested for their antioxidant activity [19,20,[22][23][24]. Moreover, nanocomposites either in single or bi-metallic combination, synthesized via chemical or green techniques using different phytochemicals (leaf extracts), were also evaluated for antioxidant activity [23,25,26]. ...
Article
Full-text available
Antioxidants interact with free radicals, terminating the adverse chain reactions and converting them to harmless products. Antioxidants thus minimize the oxidative stress and play a crucial role in the treatment of free radicals-induced diseases. However, the effectiveness of natural and/or synthetic antioxidants is limited due to their poor absorption, difficulties to cross the cell membranes, and degradation during delivery, hence contributing to their limited bioavailability. To address these issues, antioxidants covalently linked with nanoparticles, entrapped in nanogel, hollow particles, or encapsulated into nanoparticles of diverse origin have been used to provide better stability, gradual and sustained release, biocompatibility, and targeted delivery of the antioxidants with superior antioxidant profiles. This review aims to critically evaluate the recent scientific evaluations of nanoparticles as the antioxidant delivery vehicles, as well as their contribution in efficient and enhanced antioxidant activities.
... We found that the platinum-based nanoparticles actually reduced the level of superoxide formed upon treatment, similarly to what was seen in the Yusof., et al. paper that described the use of platinum nanoparticles as an antioxidant in treating lung disease [15,16]. They assessed the use of platinum-based nanoparticles as an alternative drug carrier due to their ability to produce less of an ROS-mediated effect to the system and suggested that platinum may reverse the oxidative imbalance by affecting the epithelial sodium channel, or ENaC. ...
... They assessed the use of platinum-based nanoparticles as an alternative drug carrier due to their ability to produce less of an ROS-mediated effect to the system and suggested that platinum may reverse the oxidative imbalance by affecting the epithelial sodium channel, or ENaC. ENaC works by downregulating the Protein Kinase C (PKC) pathway in the lungs and showed great promise in the treatment of lung disease [15]. In a paper by Shibuya., et al. it was seen that platinum nanoparticles share similar activity to that of catalase and superoxide dismutase, which are key endogenous antioxidant enzymes found in humans systems [17,18]. ...
... These nanoparticles display special features: the coexistence in both oxidation states (Ce 3+ and Ce 4+ ), reversible switching between these states, and reduction potential of 1.52 V [146]. Gold nanoparticles (Au NPs) have been extensively evaluated by the pharmacology and biomedical sectors due to their inert and non-toxic nature [147]. Silver nanoparticles (Ag NPs) also have a strong antioxidant capacity (reduction power and free-radical scavenging) [148]. ...
Article
Full-text available
M In the last few decades, the vast potential of nanomaterialsfor biomedical and healthcare applications has been extensively investigated. Several case studies demonstrated that nanomateri-alscan offer solutions to the current challenges ofraw materials in the biomedicalandhealthcare fields. This review describes the different nanoparticles and nanostructured materials synthesis ap-proaches and presents someemerging biomedical, healthcare, and agro-food applications. This re-view focuses on various nanomaterials types (e.g., spherical, nanorods, nanotubes, nanosheets, nan-ofibers, core-shell, andmesoporous)that can besynthesized from different raw materials and their emerging applications on bioimaging, biosensing, drug delivery, tissue engineering, antimicrobial, and agro-foods. Depending on their morphology (e.g., size, aspect ratio, geometry, porosity), nano-materials can be used as formulation modifiers, moisturizers, nanofillers, additives, membranes, and films. As toxicological assessment depends on sizes and morphologies, stringent regulation is needed from the testing of efficient nanomaterials dosages. The challenges and perspectives for an industrial breakthrough of nanomaterials are related to optimization of production and processing conditions.
... Another study also reveals that immobilized form of platinum films can be used for potential catalytic activity (Inbasekaran et al., 2014). PtNPs have a variety of medicinal applications, including anti-tumor therapy and contrast agents in medical imaging (Johnstone et al., 2016), antimicrobial activity , anti-bacterial activities (Deyhimihaghighi et al., 2014), biosensors and intracellular analysis (Yusof & Ismail 2015) and as biomarkers (Raghuwanshi et al., 2019;Manikandan et al., 2013). ...
Chapter
Full-text available
Nanomaterials have been attracting the attention of many researchers because of their size, high stability , affinity, and selectivity nature. Over the past decades, considerable intensive studies on many metal and metal-oxide nanomaterials have drawn consideration through their significant properties like size, shape, surface mass proportion, and their reactivity; all these properties are fundamental cornerstones for the turn of events and use of nanomaterials and nanoscale gadgets in biomedical areas. There is also a vast scope for a broad range of biofunctional applications such as antibacterial, antiviral, antifungal, antitumor, bioimaging, tissue engineering, biosensors, gene, and drug delivery. The authors review the nature, forms, and synthesis of nanomaterials here, with a thorough biological synthesis assessment. They also address the development of nanoparticles by microorganisms in depth, and this chapter also includes updates on different biological and biomedical applications of these bionanomaterials.
... Engineered nanostructured particles have recently been considered as an advanced strategy to provide novel antioxidants with enhanced characteristics. They are the commonly used and tested for their antioxidant activity [35][36][37][38][39][40]. Moreover, metal oxide nanoparticles either in single or bi-metallic combination, synthesized via physical, chemical or green techniques, were also studied for antioxidant activity. ...
Article
Full-text available
This work aims to investigate the effect of nickel nitrate concentration on the size and antioxidant activity of nickel oxide nanoparticles. For the first time Artemisia “herba-alba” leaves extract were employed to biosynthesize NiO NPs with four different nickel nitrate concentrations (0.01, 0.025, 0.05 and 0.075 M). Various analytical techniques were employed to study the properties of the obtained nanoparticles such as ultraviolet–visible (UV–Vis), X-ray diffraction (XRD), and Fourier transforms infrared spectroscopy (FTIR) analyses, SEM and EDX. Furthermore, the antioxidant activity of NiO NPs was investigated using total antioxidant capacity (TAC), DPPH· (2,2-diphenyl-1-picrylhydrazyl), and ferric ion reducing antioxidant power assay assays. The results have shown that the obtained NPs were assigned to a pure cubic NiO with an average size ranging between 7.49 and 10.7 nm. The results have also shown that both the size and antioxidant activity of NiO NPs are highly affected by nickel nitrate concentration. These findings can be used to control and optimize the antioxidant activity of NiO nanoparticles. Graphical Abstract
... PtNPs have emerged as a novel class of metal NPs with unique physicochemical properties [23]; they have demonstrated anti-inflammatory activity [24], radical scavenging and antioxidant activity in several previous studies [25][26][27][28][29]. Colloidal platinum was reported to protect intestinal epithelial cells from oxidative damage in a previous report [30]. ...
Article
Full-text available
Hypopigmentation disorders due to an underproduction of the pigment melanin by melanocytes cause uneven skin coloration, while in hair follicles they cause grey hair. There is a need for novel materials which can stimulate melanogenesis in the skin and hair for personal care use. While titanium dioxide, gold and silver nanoparticles have been extensively used for applications in cosmetic and personal-care products (PCP), the use of relatively inert platinum nanoparticles (PtNPs) has remained underappreciated. PtNPs have been reported to be a mimetic of the enzyme catechol oxidase with small size PtNPs reported to exhibit a higher catechol oxidase activity in a cell-free system, but no testing has been conducted in melanocytes to date. Herein, we have investigated if PtNPs of two sizes (SPtNP: 5 nm; LPtNP: 50 nm) might have an effect on melanogenesis. To this end, we have used MNT-1 human melanoma cells and primary human melanocytes from moderately-pigmented skin (HEMn-MP). Both SPtNP and LPtNP were nontoxic over a concentration range 6.25–25 μg/mL, hence these concentrations were used in further experiments. Both PtNPs stimulated higher extracellular melanin levels than control; SPtNP at concentrations 12.5 and 25 μg/mL significantly stimulated higher levels of extracellular melanin as compared to similar concentrations of LPtNP in MNT-1 cells, in the absence of ROS generation. The effects of PtNPs on melanin secretion were reversible upon removal of PtNPs from the culture medium. The results of primary particle size-specific augmentation of extracellular melanin by SPtNPs were also validated in HEMn-MP cells. Our results thus provide a proof-of-principle that SPtNP might hold potential as a candidate for the treatment of white skin patches, for sunless skin-tanning and for use in anti-greying hair products in cosmetics.
... Reoccurrence of oxidative stress eventually leads to inflammation. Studies have reported that PtNPs are suitable agents for reducing ROS levels under certain conditions [71]. Hence, we attempted to determine the effect of higher concentration and smaller size (larger surface area) of PtNPs on THP-1 cells. ...
Article
Full-text available
Generally, platinum nanoparticles (PtNPs) are considered non-toxic; however, toxicity depends on the size, dose, and physico-chemical properties of materials. Owing to unique physico-chemical properties, PtNPs have emerged as a material of interest for several biomedical applications, particularly therapeutics. The adverse effect of PtNPs on the human monocytic cell line (THP-1) is not well-established and remains elusive. Exposure to PtNPs may trigger oxidative stress and eventually lead to inflammation. To further understand the toxicological properties of PtNPs, we studied the effect of biologically synthesized ultra-small PtNPs on cytotoxicity, genotoxicity, and proinflammatory responses in the human monocytic cell line (THP-1). Our observations clearly indicated that PtNPs induce cytotoxicity in a dose-dependent manner by reducing cell viability and proliferation. The cytotoxicity of THP-1 cells correlated with an increase in the leakage of lactate dehydrogenase, generation of reactive oxygen species, and production of malondialdehyde, nitric oxide, and carbonylated proteins. The involvement of mitochondria in cytotoxicity and genotoxicity was confirmed by loss of mitochondrial membrane potential, lower ATP level, and upregulation of proapoptotic and downregulation of antiapoptotic genes. Decreases in the levels of antioxidants such as reduced glutathione (GSH), oxidized glutathione (GSH: GSSG), glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), and thioredoxin (TRX) were indicative of oxidative stress. Apoptosis was confirmed with the significant upregulation of key apoptosis-regulating genes. Oxidative DNA damage was confirmed by the increase in the levels of 8-oxodG and 8-oxoG and upregulation of DNA damage and repair genes. Finally, the proinflammatory responses to PtNPs was determined by assessing the levels of multiple cytokines such as interleukin-1β (IL-1β), IL-6, IL-8, tumor necrosis factor-α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF), and monocyte chemoattractant protein 1 (MCP-1). All the cytokines were significantly upregulated in a dose-dependent manner. Collectively, these observations suggest that THP-1 cells were vulnerable to biologically synthesized ultra-small PtNPs.
... The prominent biomedical applications of PtNPs are found to include, therapeutic effects 190 , anti-cancer therapy 191 , anti-tumor applications 192 , contrast agent in medical imaging 193 , antimicrobial activity 194 , anti-bacterial activities 195 , anti-oxidant effects 196 , cancer chemotherapy 197 , biosensors and intracellular analysis 198 , photothermal therapy 199 , as biocatalysts 200 and as biomarkers [201][202] . ...
Article
Full-text available
Nanobiomaterials are very effective components for several biomedical and pharmaceutical studies. Among the metallic, organic, ceramic and polymeric nanomaterials, metallic nanomaterials have shown certain prominent biomedical applications. Enormous works have been done to synthesize, analyse and administer the metallic nanoparticles for various kinds of medical and therapeutic applications, during the last forty years. In these analyses, the prominent biomedical applications of ten metallic nanobiomaterials have been reviewed from various sources and works. It has been found that almost nine of them are used in a very wide spectrum of medical and theranostic applications.
Article
Turbinaria decurrens Bory (T. decurrens), an Egyptian marine brown macroalga scattered along the coastline of the red sea, is used in some countries as food and medicine containing a diverse group of phytochemicals with unique bioactivities. Biosynthesis of gold nanoparticles (AuNPs) has also received a lot of attention as an alternative to physical and chemical methods. Thence, the primary goal of this work is to develop a green synthesis of AuNPs using hydromethanolic extract (HME) of T. decurrens. UV–visible spectra, Fourier transform infrared (FTIR) spectroscopy, XRD, and TEM analyses were used to investigate the size, shape, crystal structure, and agglomeration of biogenic AuNPs. AuNPs were virtually spherical and had a medium size ranges between 10.07 and 19.72nm. The presence of functional groups of algal extract compounds adsorbed on the surface of AuNPs was confirmed using FTIR spectroscopy. HME was assayed for polyphenols (28.86 ± 0.57mg gallic acid equivalent/g extract), flavonoids (11.63 ± 0.17mg quercetin equivalent/g extract), total protein (35.05 ± 0.45mg/g extract), and soluble sugars (15.45 ± 0.26 mg/g extract). Also, the HPLC analysis was used to analyze the phenolic profile of HME. Using the MTT assay against four cell lines of human cancer, the efficacy of both HME and biogenic AuNPs as cytotoxic agents was investigated and compared to both normal WI-38 cell lines and Doxorubicin as a standard drug. Results of the acridine orange/ethidium bromide (AO/EB) dual staining method, DNA fragmentation, and upgrade of the caspase activity confirmed the apoptotic mechanisms for cytotoxicity. In the case of HME, there was a discriminative inhibition of cancer cell growth of HEPG-2, whereas AuNPs had strong activity against three cancer cell lines and moderate activity against A549. Besides, HME and AuNPs were tested for antioxidant activity using DPPH and ABTS assays in comparison to Trolox, a standard drug, and the results revealed that both HME and AuNPs have effective antioxidant activity in a dose-based way. Our findings confirm the antioxidant and anticancer activities of HME and its AuNPs, as well as their potential as a therapeutic candidate in the treatment of oxidative stress diseases, particularly cancer.
Article
Full-text available
Pegylated colloidal platinum nanoparticles (PEG‐PtNPs) are widely used as a potential agent for diagnosis and therapy of various diseases including cancer. Prior to any preclinical applications, detailed investigations of toxicity, biodistribution, clearance, and pharmacokinetics (PKs) of new nanomaterials are essential. Extensive toxicological studies of PEG‐PtNPs are not reported in a systematic manner elsewhere. Herein, acute toxicity of PEG‐PtNPs is thoroughly investigated in mouse model. Prior to study in mice, a hemolytic analysis is performed with PtNPs that displays biocompatible nature. Administration of a single intraperitoneal dose of PEG‐PtNPs (10 and 50 mg kg−1 body weight) in mice does not induce any gross pathological changes. The data obtained from hematology, serum biochemistry, and histopathological analysis indicate no significant changes except for moderate nephrotoxicity at the higher dose. In addition, a PK analysis displays a maximum retention time and elimination half‐life at 10 mg kg−1 b.w. dose. Biodistribution studies demonstrate maximum accumulation of platinum in spleen tissue and tail of mice. Finally, detection of platinum in feces and urine confirms their excretion through a hepatobiliary system. Altogether, this study indicates that 10 mg kg−1 b.w. therapeutic dose of PEG‐PtNPs is safe for their potential future application in cancer theranostics. Acute toxicity of pegylated platinum nanoparticles (PEG‐PtNPs) is investigated in a mouse model with 10 and 50 mg kg−1 b.w. doses with no gross pathological changes. No toxicity is observed at low dose. Bioaccumulation in spleen and tail and sustained excretion is witnessed. This study indicates that 10 mg kg−1 b.w. dose of PtNPs is safe for clinical applications.
Preprint
Mini Review Paper About INTRINSIC ANTIOXIDANT PROPERTIES, A DOUBLE-EDGED SWORD OF PLATINUM NANOPARTICLES (PtNPs)
Article
Drug repurpose or reposition has been recently recognized as a high‐performance strategy for developing therapeutic agents for cancer treatment. This approach can significantly reduce the risk of failure, shorten R&D time, and minimize cost and regulatory obstacles. Nanotechnology‐based delivery systems have been extensively investigated in cancer therapy due to their remarkable ability to overcome drug delivery challenges, enhance tumor specific targeting, and reduce toxic side effects. With increasing knowledge accumulated over the past decades, nanoparticle formulation and delivery have opened up a new avenue for repurposing drugs and demonstrated promising results in advanced cancer therapy. In this review, recent developments in nano‐delivery and formulation systems based on soft (i.e., DNA nanocages, nanogels, and dendrimers) and condensed (i.e., noble metal nanoparticles and metal–organic frameworks) nanomaterials, as well as their theranostic applications in drug repurpose against cancer are summarized. This review provides an account of soft and condensed nanoparticles and nanoformulations for cancer drug delivery and repurpose.
Article
Full-text available
Multi-drug resistance is a growing problem in the treatment of infectious diseases and the widespread use of broad-spectrum antibiotics has produced antibiotic resistance for many human bacterial pathogens. Advances in nanotechnology have opened new horizons in nanomedicine, allowing the synthesis of nanoparticles that can be assembled into complex architectures. Novel studies and technologies are devoted to understanding the mechanisms of disease for the design of new drugs, but unfortunately infectious diseases continue to be a major health burden worldwide. Since ancient times, silver was known for its anti-bacterial effects and for centuries it has been used for prevention and control of disparate infections. Currently nanotechnology and nanomaterials are fully integrated in common applications and objects that we use every day. In addition, the silver nanoparticles are attracting much interest because of their potent antibacterial activity. Many studies have also shown an important activity of silver nanoparticles against bacterial biofilms. This review aims to summarize the emerging efforts to address current challenges and solutions in the treatment of infectious diseases, particularly the use of nanosilver antimicrobials.
Article
Full-text available
Cancer ranks among the leading causes of human mortality. Cancer becomes intractable when it spreads from the primary tumor site to various organs (such as bone, lung, liver, and then brain). Unlike solid tumor cells, cancer stem cells and metastatic cancer cells grow in a non-attached (suspension) form when moving from their source to other locations in the body. Due to the non-attached growth nature, metastasis is often first detected in the circulatory systems, for instance in a lymph node near the primary tumor. Cancer research over the past several decades has primarily focused on treating solid tumors, but targeted therapy to treat cancer stem cells and cancer metastasis has yet to be developed. Because cancers undergo faster metabolism and consume more glucose than normal cells, glucose was chosen in this study as a reagent to target cancer cells. In particular, by covalently binding gold nanoparticles (GNPs) with thio-PEG (polyethylene glycol) and thio-glucose, the resulting functionalized GNPs (Glu-GNPs) were created for targeted treatment of cancer metastasis and cancer stem cells. Suspension cancer cell THP-1 (human monocytic cell line derived from acute monocytic leukemia patients) was selected because it has properties similar to cancer stem cells and has been used as a metastatic cancer cell model for in vitro studies. To take advantage of cancer cells' elevated glucose consumption over normal cells, different starvation periods were screened in order to achieve optimal treatment effects. Cancer cells were then fed using Glu-GNPs followed by X-ray irradiation treatment. For comparison, solid tumor MCF-7 cells (breast cancer cell line) were studied as well. Our irradiation experimental results show that Glu-GNPs are better irradiation sensitizers to treat THP-1 cells than MCF-7 cells, or Glu-GNPs enhance the cancer killing of THP-1 cells 20% more than X-ray irradiation alone and GNP treatment alone. This finding can help oncologists to design therapeutic strategies to target cancer stem cells and cancer metastasis.
Article
Full-text available
Lung cancer has been identified as one of the most deadly oncologies. The most influential causes for disease progression include smoking, genetic mutation and inflammatory lung diseases. Conventional therapies for lung cancer including chemo and radio-treatments often cause serious adverse effects. The advent of novel therapeutics that specifically target signalling pathways activated by genetic alterations has revolutionized the way patients with lung cancer are treated. These are comprised of various molecular targets on its carcinogen signalling pathways, among which the protein kinase C (PKC) family is a promising target. The 12 isotypes in the family demonstrate complex interactions. This inter-linked signalling loop has added complexity of developing effective therapies. An improved understanding of different molecules involved in these signalling pathways will provide several profound implications, ranging from preclinical work on the mechanisms to trial design. Therapies developed targeting individual/multiple PKCs combined with conventional strategies offer promising future combating cancer.
Article
Full-text available
Cu-Zn superoxide dismutase (Sod1) loss causes a redox imbalance as it leads to excess superoxide generation, which results in the appearance of various aging-related phenotypes, including skin atrophy. Noble metal nanoparticles, such as palladium (Pd) and platinum (Pt) nanoparticles, are considered to function as antioxidants due to their strong catalytic activity. In Japan, a mixture of Pd and Pt nanoparticles called PAPLAL has been used to treat chronic diseases over the past 60 years. In the present study, we investigated the protective effects of PAPLAL against aging-related skin pathologies in mice. Transdermal PAPLAL treatment reversed skin thinning associated with increased lipid peroxidation in Sod1-/- mice. Furthermore, PAPLAL normalized the gene expression levels of Col1a1, Mmp2, Has2, Tnf-α, Il-6, and p53 in the skin of the Sod1-/- mice. Pt nanoparticles exhibited marked SOD and catalase activity, while Pd nanoparticles only displayed weak SOD and catalase activity in vitro. Although the SOD and catalase activity of the Pt nanoparticles significantly declined after they had been oxidized in air, a mixture of Pd and Pt nanoparticles continued to exhibit SOD and catalase activity after oxidation. Importantly, a mixture of Pd and Pt nanoparticles with a molar ratio of 3 or 4 to 1 continued to exhibit SOD and catalase activity after oxidation, indicating that Pd nanoparticles prevent the oxidative deterioration of Pt nanoparticles. These findings indicate that PAPLAL stably suppresses intrinsic superoxide generation both in vivo and in vitro via SOD and catalase activity. PAPLAL is a potentially powerful tool for the treatment of aging-related skin diseases caused by oxidative damage.
Article
Full-text available
Hyperglycemia, hyperlipidemia and inflammation are key risk factors for atherosclerosis and can lead to overproduction of reactive oxygen species (ROS), which plays a critical role in vascular endothelial dysfunction and subsequent progress of atherosclerosis. However, there is currently a lack of effective drugs that deal with ROS. Platinum nanoparticles (Pt-NPs) have proven to be promising antioxidant drugs in vitro and in vivo. To optimize the efficacy of Pt-NP based drugs, we synthesized and characterized the ROS scavenging properties of three kinds of small molecules that capped Pt-NPs (Pt-AMP-NPs, Pt-ATT-NPs, Pt-MI-NPs) on a blood vessel-mimicking microfluidic chip. The Pt-NPs showed superior superoxide dismutase (SOD)-like functions and can scavenge ROS and recover compromised cell-cell junctions under hyperglycemic, hyperlipidemic and proinflammatory conditions. Amongst these NPs, Pt-AMP-NPs showed the most superior antioxidant properties, suggesting its potency to serve as a novel drug to treat vascular diseases such as atherosclerosis. Our microfluidic chip, providing physiological hemodynamic conditions for the experiments, is potentially a promising tool for a wide range of biological research on the vascular system.
Article
Full-text available
Rationale: Protein kinase C zeta (PKCζ) has been reported to act as a tumor suppressor. Deletion of PKCζ in experimental cancer models has been shown to increase tumor growth. However, the mechanisms of PKCζ down-regulation in cancerous cells have not been previously described. Objectives: To determine the molecular mechanisms that lead to decreased PKCζ expression and thus increased survival in cancer cells and tumor growth. Methods: The levels of expression of heme-oxidized IRP2 ubiquitin ligase 1L (HOIL-1L), HOIL-1-interacting protein (HOIP), Shank-associated RH domain-interacting protein (SHARPIN), and PKCζ were analyzed by Western blot and/or quantitative real-time polymerase chain reaction in different cell lines. Coimmunoprecipitation experiments were used to demonstrate the interaction between HOIL-1L and PKCζ. Ubiquitination was measured in an in vitro ubiquitination assay and by Western blot with specific antibodies. The role of hypoxia-inducible factor (HIF) was determined by gain/loss-of-function experiments. The effect of HOIL-1L expression on cell death was investigated using RNA interference approaches in vitro and on tumor growth in mice models. Increased HOIL-1L and decreased PKCζ expression was assessed in lung adenocarcinoma and glioblastoma multiforme and documented in several other cancer types by oncogenomic analysis. Measurements and main results: Hypoxia is a hallmark of rapidly growing solid tumors. We found that during hypoxia, PKCζ is ubiquitinated and degraded via the ubiquitin ligase HOIL-1L, a component of the linear ubiquitin chain assembly complex (LUBAC). In vitro ubiquitination assays indicate that HOIL-1L ubiquitinates PKCζ at Lys-48, targeting it for proteasomal degradation. In a xenograft tumor model and lung cancer model, we found that silencing of HOIL-1L increased the abundance of PKCζ and decreased the size of tumors, suggesting that lower levels of HOIL-1L promote survival. Indeed, mRNA transcript levels of HOIL-1L were elevated in tumor of patients with lung adenocarcinoma, and in a lung adenocarcinoma tissue microarray the levels of HOIL-1L were associated with high-grade tumors. Moreover, we found that HOIL-1L expression was regulated by HIFs. Interestingly, the actions of HOIL-1L were independent of LUBAC. Conclusions: These data provide first evidence of a mechanism of cancer cell adaptation to hypoxia where HIFs regulate HOIL-1L, which targets PKCζ for degradation to promote tumor survival. We provided a proof of concept that silencing of HOIL-1L impairs lung tumor growth and that HOIL-1L expression predicts survival rate in cancer patients suggesting that HOIL-1L is an attractive target for cancer therapy.
Article
Full-text available
The effect of gold nanoparticles on lung cancer cells is not yet clear. In this study, we investigated the cytotoxicity and cell invasion activity of lung cancer cells after treatment with gold nanoparticles and showed that small gold nanoparticles can be endocytosed by lung cancer cells and that they facilitate cell invasion. The growth of A549 cells was inhibited after treatment with 5-nm gold nanoparticles, but cell invasion increased. Endocytosed gold nanoparticles (size, 10 nm) notably promoted the invasion activity of 95D cells. All these effects of gold nanoparticles were not seen after treatment with larger particles (20 and 40 nm). The enhanced invasion activity may be associated with the increased expression of matrix metalloproteinase 9 and intercellular adhesion molecule-1. In this study, we obtained evidence for the effect of gold nanoparticles on lung cancer cell invasion activity in vitro. Moreover, matrix metalloproteinase 9 and intercellular adhesion molecule-1, key modulators of cell invasion, were found to be regulated by gold nanoparticles. These data also demonstrate that the responses of the A549 and 95D cells to gold nanoparticles have a remarkable relationship with their unique size-dependent physiochemical properties. Therefore, this study provides a new perspective for cell biology research in nanomedicine.
Article
Full-text available
Neural precursor cell expressed, developmentally down-regulated protein 4-2 (Nedd4-2) mediates the internalization / degradation of epithelial Na+ channel subunits (α-, β- and γ-ENaC). Serum / glucocorticoid inducible kinase 1 (SGK1) and protein kinase A (PKA) both appear to inhibit this process by phosphorylating Nedd4-2-Ser221, -Ser327 and -Thr246. This Nedd4-2 inactivation process is thought to be central to the hormonal control of Na+ absorption. The present study of H441 human airway epithelial cells therefore explores the effects of SGK1 and / or PKA upon the phosphorylation / abundance of endogenous Nedd4-2; the surface expression of ENaC subunits, and electrogenic Na+ transport. Effects on Nedd4-2 phosphorylation / abundance and the surface expression of ENaC were monitored by western analysis, whilst Na+ absorption was quantified electrometrically. Acutely (20 min) activating PKA in glucocorticoid-deprived (24 h) cells increased the abundance of Ser221-phosphorylated, Ser327-phosphorylated and total Nedd4-2 without altering the abundance of Thr246-phosphorylated Nedd4-2. Activating PKA under these conditions did not cause a co-ordinated increase in the surface abundance of α-, β- and γ-ENaC and had only a very small effect upon electrogenic Na+ absorption. Activating PKA (20 min) in glucocorticoid-treated (0.2 µM dexamethasone, 24 h) cells, on the other hand, increased the abundance of Ser221-, Ser327- and Thr246-phosphorylated and total Nedd4-2; increased the surface abundance of α-, β- and γ-ENaC and evoked a clear stimulation of Na+ transport. Chronic glucocorticoid stimulation therefore appears to allow cAMP-dependent control of Na+ absorption by facilitating the effects of PKA upon the Nedd4-2 and ENaC subunits.
Article
Full-text available
Silver nanoparticles (AgNPs) are currently one of the most manufactured nanomaterials. A wide range of toxicity studies have been performed on various AgNPs, but these studies report a high variation in toxicity and often lack proper particle characterization. The aim of this study was to investigate size- and coating-dependent toxicity of thoroughly characterized AgNPs following exposure of human lung cells and to explore the mechanisms of toxicity. BEAS-2B cells were exposed to citrate coated AgNPs of different primary particle sizes (10, 40 and 75 nm) as well as to 10 nm PVP coated and 50 nm uncoated AgNPs. The particle agglomeration in cell medium was investigated by photon cross correlation spectroscopy (PCCS); cell viability by LDH and Alamar Blue assay; ROS induction by DCFH-DA assay; genotoxicity by alkaline comet assay and gammaH2AX foci formation; uptake and intracellular localization by transmission electron microscopy (TEM); and cellular dose as well as Ag release by atomic absorption spectroscopy (AAS). The results showed cytotoxicity only of the 10 nm particles independent of surface coating. In contrast, all AgNPs tested caused an increase in overall DNA damage after 24 h assessed by the comet assay, suggesting independent mechanisms for cytotoxicity and DNA damage. However, there was no gammaH2AX foci formation and no increased production of intracellular reactive oxygen species (ROS). The reasons for the higher toxicity of the 10 nm particles were explored by investigating particle agglomeration in cell medium, cellular uptake, intracellular localization and Ag release. Despite different agglomeration patterns, there was no evident difference in the uptake or intracellular localization of the citrate and PVP coated AgNPs. However, the 10 nm particles released significantly more Ag compared with all other AgNPs (approx. 24 wt% vs. 4-7 wt%) following 24 h in cell medium. The released fraction in cell medium did not induce any cytotoxicity, thus implying that intracellular Ag release was responsible for the toxicity. This study shows that small AgNPs (10 nm) are cytotoxic for human lung cells and that the toxicity observed is associated with the rate of intracellular Ag release, a 'Trojan horse' effect.
Article
Full-text available
ENaC is negatively regulated by protein kinase C (PKC) as shown using PKC activators in a cell culture model. To determine whether PKCα influences ENaC activity in vivo, we examined the regulation of ENaC in renal tubules from PKCα(-/-) mice. Cortical collecting ducts were dissected and split open and the exposed principal cells were subjected to cell-attached patch clamp. In the absence of PKCα, open probability (Po) of ENaC was increased three-fold vs wild-type SV129 mice (0.52 ± 0.04 vs 0.17 ± 0.02). The number of channels per patch was also increased. Using confocal microscopy, we observed an increase in membrane localization of α, β, and γ subunits of ENaC in principal cells in the cortical collecting ducts of PKCα(-/-) mice compared to wild-type mice. To confirm this increase, one kidney from each animal was perfused with biotin and membrane protein was pulled down with streptavidin. The nonbiotinylated kidney was used to assess total protein. While total ENaC protein did not change in PKCα(-/-) mice, membrane localization of all the ENaC subunits was increased. The increase in membrane ENaC could be explained by the observation that ERK1/2 phosphorylation was decreased in the knockout mice. These results imply a reduction in ENaC membrane accumulation and Po by PKCα in vivo. The PKC-mediated increase in ENaC activity was associated with an increase in blood pressure in knockout mice fed a high-salt diet.
Article
Full-text available
The platinum (Pt)-group elements (PGEs) represent a new kind of environmental pollutant and a new hazard for human health. Since their introduction as vehicle-exhaust catalysts, their emissions into the environment have grown considerably compared with their low natural concentration in the earth crust. PGE emissions from vehicle catalysts can be also in the form of nanometer-sized particles (Pt nanoparticles [PtNPs]). These elements, both in their metallic form or as ions solubilized in biological media, are now recognized as potent allergens and sensitizers. Human skin is always exposed to toxic particles; therefore, in the present study we addressed the question of whether polyvinylpyrrolidone-coated PtNPs may have any negative effects on skin cells, including predominantly epidermal keratinocytes. In this study, PtNPs of two sizes were used: 5.8 nm and 57 nm, in concentrations of 6.25, 12.5, and 25 μg/mL. Both types of NPs were protected with polyvinylpyrrolidone. Primary keratinocytes were treated for 24 and 48 hours, then cytotoxicity, genotoxicity, morphology, metabolic activity, and changes in the activation of signaling pathways were investigated in PtNP-treated cells. We found that PtNPs trigger toxic effects on primary keratinocytes, decreasing cell metabolism, but these changes have no effects on cell viability or migration. Moreover, smaller NPs exhibited more deleterious effect on DNA stability than the big ones. Analyzing activation of caspases, we found changes in activity of caspase 9 and caspase 3/7 triggered mainly by smaller NPs. Changes were not so significant in the case of larger nanoparticles. Importantly, we found that PtNPs have antibacterial properties, as is the case with silver NPs (AgNPs). In comparison to our previous study regarding the effects of AgNPs on cell biology, we found that PtNPs do not exhibit such deleterious effects on primary keratinocytes as AgNPs and that they also can be used as potential antibacterial agents, especially in the treatment of Escherichia coli, representing a group of Gram-negative species.
Article
Full-text available
Platinum nanoparticles (PNPs) were synthesized by chemical reduction of potassium hexachloroplatinate (IV) with trisodium citrate under vigorous stirring and addition of sodium dodecyl sulfate as stabilizer reagent. Reducing agent was chosen depending on the oxidation reactions and potential values of the chemical materials used in the experiment. The aim of this study is to investigate the effects of PNPs on the different cancer cell lines and cytotoxicity study of this nanomaterial. The morphology of PNPs was investigated by scanning electron microscope (XL30, Philips Electronics, Amsterdam, The Netherlands) with the ability to perform elemental analysis by EDX. Malvern Zetasizer 3000 HSA (Malvern Instruments, Worcestershire, UK) was used to determine the distribution of particle size and zeta potential of PNPs. The cytotoxicity property of the nanoparticles was evaluated by MTT assay on MCF-7 and HepG-2 cell lines, and the cytotoxic concentration 50% values were determined for 24 h.
Article
Full-text available
Platinum nanoparticles are being utilized in various industrial applications, including in catalysis, cosmetics, and dietary supplements. Although reducing the size of the nanoparticles improves the physicochemical properties and provides useful performance characteristics, the safety of the material remains a major concern. The aim of the present study was to evaluate the biological effects of platinum particles less than 1 nm in size (snPt1). In mice administered with a single intravenous dose of snPt1, histological analysis revealed necrosis of tubular epithelial cells and urinary casts in the kidney, without obvious toxic effects in the lung, spleen, and heart. These mice exhibited dose-dependent elevation of blood urea nitrogen, an indicator of kidney damage. Direct application of snPt1 to in vitro cultures of renal cells induced significant cytotoxicity. In mice administered for 4 weeks with twice-weekly intraperitoneal snPt1, histological analysis of the kidney revealed urinary casts, tubular atrophy, and inflammatory cell accumulation. Notably, these toxic effects were not observed in mice injected with 8-nm platinum particles, either by single- or multiple-dose administration. Our findings suggest that exposure to platinum particles of less than 1 nm in size may induce nephrotoxicity and disrupt some kidney functions. However, this toxicity may be reduced by increasing the nanoparticle size.
Article
Full-text available
Tight control of lung liquid (LL) regulation is vital for pulmonary function. The aim of this work was to determine whether PKC activation is involved in the physiological regulation of LL volume in a whole lung preparation. Rat lungs were perfused with a modified Ringer solution, and the lumen was filled with the same solution without glucose. LL volume was measured during a control period and after modulating drugs were administered, and net LL transepithelial movement (J(v)) was calculated. When the PKC activator PMA (10(-5) M) and the Ca(2+) ionophore ionomycin (10(-6) M) were instilled into the lung together, J(v) was significantly reduced (P = 0.03). This reduction was blocked by the PKC inhibitor chelerythrine chloride (10(-6) M; P = 0.56) and by a second PKC inhibitor GF109203X (10(-5) M; P = 0.98). When PMA and ionomycin were added with the β-adrenergic agonist terbutaline, the terbutaline-induced increase in J(v) was abolished. Addition of PMA and ionomycin with the epithelial Na(+) channel (ENaC) blocker amiloride had no additional inhibitory effect. Together, these results suggest that PKC is likely to be involved in LL absorption, and the ability of PMA/ionomycin to block the terbutaline-induced increase in J(v) suggests that the downstream target of PKC is ENaC.
Article
Full-text available
Aldosterone-induced increases in apical membrane epithelial sodium channel (ENaC) density and Na transport involve the induction of 14-3-3 protein expression and their association with Nedd4-2, a substrate of serum- and glucocorticoid-induced kinase (SGK1)-mediated phosphorylation. A search for other 14-3-3 binding proteins in aldosterone-treated cortical collecting duct (CCD) cells identified the Rab-GAP, AS160, an Akt/PKB substrate whose phosphorylation contributes to the recruitment of GLUT4 transporters to adipocyte plasma membranes in response to insulin. In CCD epithelia, aldosterone (10 nM, 24 h) increased AS160 protein expression threefold, with a time-course similar to increases in SGK1 expression. In the absence of aldosterone, AS160 overexpression increased total ENaC expression 2.5-fold but did not increase apical membrane ENaC or amiloride-sensitive Na current (I(sc)). In AS160 overexpressing epithelia, however, aldosterone increased apical ENaC and I(sc) 2.5-fold relative to aldosterone alone, thus recruiting the accumulated ENaC to the apical membrane. Conversely, AS160 knockdown increased apical membrane ENaC and I(sc) under basal conditions to approximately 80% of aldosterone-stimulated values, attenuating further steroid effects. Aldosterone induced AS160 phosphorylation at five sites, predominantly at the SGK1 sites T568 and S751, and evoked AS160 binding to the steroid-induced 14-3-3 isoforms, beta and epsilon. AS160 mutations at SGK1 phospho-sites blocked its selective interaction with 14-3-3beta and epsilon and suppressed the ability of expressed AS160 to augment aldosterone action. These findings indicate that the Rab protein regulator, AS160, stabilizes ENaC in a regulated intracellular compartment under basal conditions, and that aldosterone/SGK1-dependent AS160 phosphorylation permits ENaC forward trafficking to the apical membrane to augment Na absorption.
Article
Full-text available
Recently, the use of gold nanoparticles as potential tumor selective radiosensitizers has been proposed as a breakthrough in radiotherapy. Experiments in living cells and in vivo have demonstrated the efficiency of the metal nanoparticles when combined with low energy x-ray radiations (below conventional 1 MeV Linac radiation). Further studies on DNA have been performed in order to better understand the fundamental processes of sensitization and to further improve the method. In this work, we propose a new strategy based on the combination of platinum nanoparticles with irradiation by fast ions effectively used in hadron therapy. It is observed in particular that nanoparticles enhance strongly lethal damage in DNA, with an efficiency factor close to 2 for double strand breaks. In order to disentangle the effect of the nano-design architecture, a comparison with the effects of dispersed metal atoms at the same concentration has been performed. It is thus shown that the sensitization in nanoparticles is enhanced due to auto-amplified electronic cascades inside the nanoparticles, which reinforces the energy deposition in the close vicinity of the metal. Finally, the combination of fast ion radiation (hadron therapy) with platinum nanoparticles should strongly improve cancer therapy protocols.
Article
Full-text available
The epithelial Na+ channel (ENaC), composed of three subunits (alpha beta gamma), plays a critical role in salt and fluid homeostasis. Abnormalities in channel opening and numbers have been linked to several genetic disorders, including cystic fibrosis, pseudohypoaldosteronism type I and Liddle syndrome. We have recently identified the ubiquitin-protein ligase Nedd4 as an interacting protein of ENaC. Here we show that ENaC is a short-lived protein (t1/2 approximately 1 h) that is ubiquitinated in vivo on the alpha and gamma (but not beta) subunits. Mutation of a cluster of Lys residues (to Arg) at the N-terminus of gamma ENaC leads to both inhibition of ubiquitination and increased channel activity, an effect augmented by N-terminal Lys to Arg mutations in alpha ENaC, but not in beta ENaC. This elevated channel activity is caused by an increase in the number of channels present at the plasma membrane; it represents increases in both cell-surface retention or recycling of ENaC and incorporation of new channels at the plasma membrane, as determined by Brefeldin A treatment. In addition, we find that the rapid turnover of the total pool of cellular ENaC is attenuated by inhibitors of both the proteasome and the lysosomal/endosomal degradation systems, and propose that whereas the unassembled subunits are degraded by the proteasome, the assembled alpha beta gamma ENaC complex is targeted for lysosomal degradation. Our results suggest that ENaC function is regulated by ubiquitination, and propose a paradigm for ubiquitination-mediated regulation of ion channels.
Article
Full-text available
AlphaENaC-EGFP (enhanced green fluorescent protein-tagged alpha-subunit of the epithelial Na(+) channel) stably transfected clonal lines derived from the A6 parental cell line were used to study the physical mechanisms of insulin-stimulated Na(+) transport. Within 1 min of insulin stimulation, ENaC migrates from a diffuse cytoplasmic localization to the apical and lateral membranes. Concurrently, after insulin stimulation, phosphatidylinositol 3-kinase (PI 3-kinase) is colocalized with ENaC on the lateral but not apical membrane. An inhibitor of PI 3-kinase, LY-294002, does not inhibit ENaC/PI 3-kinase colocalization but does alter the intracellular site of the colocalization, preventing the translocation of ENaC to the lateral and apical membranes. These data show that insulin stimulation causes the migration of ENaC to the lateral and apical cell membranes and that this trafficking is dependent on PI 3-kinase activity.
Article
Full-text available
Epithelial sodium channels (ENaCs) are composed of three structurally related subunits that form a tetrameric channel. The Xenopus laevis oocyte expression system was used to identify regions within the ENaC alpha-subunit that confer a dominant negative phenotype on functional expression of alphabetagamma-ENaC to define domains that have a role in subunit-subunit interactions. Coexpression of full-length mouse alphabetagamma-ENaC with either 1) the alpha-subunit first membrane-spanning domain and short downstream hydrophobic domain (alpha-M1H1); 2) alpha-M1H1 and its downstream hydrophilic extracellular loop (alpha-M1H1-ECL); 3) the membrane-spanning domain of a control type 2 transmembrane protein (glutamyl transpeptidase; gamma-GT) fused to the alpha-ECL (gamma-GT-alpha-ECL); 4) the extracellular domain of a control type 1 transmembrane protein (Tac) fused to the alpha-subunit second membrane-spanning domain and short upstream hydrophobic domain (Tac-alpha-H2M2); or 5) the alpha-subunit cytoplasmic COOH terminus (alpha-Ct) significantly reduced amiloride-sensitive Na+ currents in X. laevis oocytes. Functional expression of Na+ channels was not inhibited when full-length alphabetagamma-ENaC was coexpressed with either 1) the alpha-ECL lacking a signal-anchor sequence, 2) alpha-M1H1 and alpha-Ct expressed as a fusion protein, 3) full-length gamma-GT, or 4) full-length Tac. Furthermore, the expression of ROMK channels was not inhibited when full-length ROMK was coexpressed with either alpha-M1H1-ECL or alpha-Ct. Full-length FLAG-tagged alpha-, beta-, or gamma-ENaC coimmunoprecipitated with myc-tagged alpha-M1H1-ECL, whereas wild-type gamma-GT did not. These data suggest that multiple sites within the alpha-subunit participate in subunit-subunit interactions that are required for proper assembly of the heterooligomeric ENaC complex.
Article
Full-text available
The epithelial Na+ channel (ENaC) functions as a pathway for epithelial Na+ transport, contributing to Na+ homeostasis and blood pressure control. Vasopressin increases ENaC expression at the cell surface through a pathway that includes cAMP and cAMP-dependent protein kinase (PKA), but the mechanisms that link PKA to ENaC are unknown. Here we found that cAMP regulates Na+ transport in part by inhibiting the function of Nedd4-2, an E3 ubiquitin-protein ligase that targets ENaC for degradation. Consistent with this model, we found that cAMP inhibited Nedd4-2 by decreasing its binding to ENaC. Moreover, decreased Nedd4-2 expression (RNA interference) or overexpression of a dominant negative Nedd4-2 construct disrupted ENaC regulation by cAMP. Nedd4-2 was a substrate for phosphorylation by PKA in vitro and in cells; three Nedd4-2 residues were phosphorylated by PKA and were required for cAMP to inhibit Nedd4-2 (relative functional importance Ser-327 > Ser-221 > Thr-246). Previous work found that these residues are also phosphorylated by serum and glucocorticoid-inducible kinase (SGK), a downstream mediator by which aldosterone regulates epithelial Na+ transport. Consistent with a functional interaction between these pathways, overexpression of SGK blunted ENaC stimulation by cAMP, whereas inhibition of SGK increased stimulation. Conversely, cAMP agonists decreased ENaC stimulation by SGK. The data suggest that cAMP regulates ENaC in part by phosphorylation and inhibition of Nedd4-2. Moreover, Nedd4-2 is a central convergence point for kinase regulation of Na+ transport.
Article
Full-text available
The epithelial Na+ channel (ENaC) is assembled in the endoplasmic reticulum from three structurally related subunits (α, β, and γ). Channel maturation within the biosynthetic pathway involves cleavage of the α and γ subunits by furin and processing of N-linked glycans on α, β, and γ to complex type. Both mature and immature subunits have been observed at the surface of stably transfected Madin-Darby canine kidney cells. We have examined whether channel maturation is an all-or-none event or whether heterogeneous processing of channel subunits occurs within an individual channel complex. Using an immobilized lectin to isolate proteins with complex type N-glycans, we found that individual channel complexes with mature subunits lack immature subunits. Furthermore, terminal processing of N-glycans on ENaC subunits was not dependent on cleavage of ENaC subunits, and proteolysis of channel subunits was not dependent on prior processing of N-glycans. Our results suggest that processing of subunits within an individual channel complex is an all-or-none event such that channels present on the cell surface contain either all mature or all immature subunits. The presence of immature channel complexes at the plasma membrane provides epithelial cells with a reserve of poorly functional channels that can be activated by proteases in post-Golgi compartments.
Article
Full-text available
The intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the alpha, beta and gamma subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na+-K+-ATPase. Pseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains. The mRNA expression of ENaC and Na+-K+-ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection. The infection of the different mouse strains evoked regulation of alpha and beta ENaC mRNA. Following Pseudomonas instillation, the expression of alphaENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p < 0.05). The relative expression of betaENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p < 0.05). No significant modulation of gammaENaC mRNA was detected although the general pattern of expression of the subunit was similar to alpha and beta subunits. No modulation of alpha1Na+-K+-ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains. These results show that Pseudomonas infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs.
Article
In order to investigate the pulmonary toxicity of titanium dioxide (TiO2) nanoparticles, we performed an intratracheal instillation study with rats of well-dispersed TiO2 nanoparticles and examined the pulmonary inflammation and histopathological changes in the lung. Wistar Hannover rats were intratracheally administered 0.2 mg (0.66 mg/kg) and 1.0 mg (3.3 mg/kg) of well-dispersed TiO2 nanoparticles (P90; diameter of agglomerates: 25 nm), then the pulmonary inflammation responses were examined from 3 days to 6 months after the instillation, and the pathological features were examined up to 24 months. Transient inflammation and the upregulation of chemokines in the broncho-alveolar lavage fluid were observed for 1 month. No respiratory tumors or severe fibrosis were observed during the recovery time. These data suggest that transient inflammation induced by TiO2 may not lead to chronic, irreversible legions in the lung, and that TiO2 nanoparticles may not have a high potential for lung disorder.
Article
The antimicrobial properties of silver nanoparticles (Ag-NPs) have resulted in their extensive application in consumer and health care products. Although Ag-NPs have great potential benefits, their side effects are unknown and seem inevitable due to their ability to reach the nucleus and damage genetic material. This study aimed to determine genotoxic potential of Ag-NPs using mitotic index (MI), DNA damage (comet assay), structural chromosome aberrations (SCA), micronuclei (MN) formation as genetic endpoints and induction of reactive oxygen species (ROS) as oxidative stress endpoint in bone marrow of Sprague-Dawley rats. Four groups of five male rats were orally administered Ag-NPs, once a day for five days with doses of 5, 25, 50, 100, mg/Kg. A control group was also made of five rats. Bone marrow samples were collected 24 h after the last treatment following standard protocols. Ag-NPs exposure significantly increased (p<0.05) the induction of ROS, number of SCA, the frequency of micro-nucleated cells, damaged the DNA and decreased the mitotic index compared to negative control. The results suggest that Ag-NPs may have the potential to induce oxidative stress mediated genotoxicity in rats. Further characterization of their genotoxicity and also their potential health implications should be monitored regularly. Copyright © 2015. Published by Elsevier Ltd.
Article
The aim of this project was to synthesize and characterize gold nanoparticles (GNPs) to trace the sequence of the hnRNPB1as a lung cancer biomarker. In the synthesis of GNPs with characteristics appropriate for conjugation, the size, morphology, and shape of the synthesized GNPs were determined by using spectrophotometry and transmission electron microscopy (TEM), followed by designing a probe for hnRNPB1biomarker with characteristics suitable for conjugation. Next, the GNPs were functionalized with a single-stranded DNA probe that was specific for the biomarker, for the characterization and confirmation of the conjugation process. Finally, for determination of minimum level of detection in solution including DNA target and probe aggregation, the changes in the absorption spectra of the samples in the range of 250-750 nm were determined using the NanoDrop ND 1000 spectrophotometer. The surface of GNPs can be modified by utilizing ligands to selectively attach biomarkers. Thiol-bonding of DNA and chemical functionalization of GNPs are the most common approaches. Colloidal gold was synthesized with the citrate reduction method, as described by Turkevich et al. in 1951. In this study, the probe for hnRNPB1 was designed with a thiol crosslinker. Every set of conjugated GNPs was complementary to one end of the hnRNPB1 biomarker, and the probes were aligned in a tail to tail fashion onto the target. Uniform GNPs were synthesized by the citrate reduction technique, and the outcomes of trials with variation in factors (shape and size of the nanoparticles, gold concentration, and conjugation between GNP and probes) were investigated. The gold nanoprobe-based technique is better than the PCR-based techniques, because there are no requirements of enzymatic amplification and gel electrophoresis, and the evaluation can be done using small amounts of sample.
Article
Concerns about the risk of titanium dioxide nanoparticles (TiO2 NPs) to human health and environment are gradually increasing due to their wide range of applications. In this study, cytotoxicity, DNA damage, and apoptosis induced by TiO2 NPs (5 nm) in A549 cells were investigated. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays revealed the time- and concentration-dependent cytotoxic effects of TiO2 NPs in a concentration range of 50 to 200 μg/mL. A statistically significant (p < 0.05) induction in DNA damage was observed by the comet assay in cells exposed to 50 to 200 μg/mL TiO2 NPs for 48 h. A significant (p < 0.05) induction in micronucleus formation determined by 4,6-diamino-2-phenylindole (DAPI) staining was also observed at the above concentrations. Typical apoptotic morphological feature and apoptotic bodies in A549 cells induced by TiO2 NPs at the above concentrations were observed by scanning electron micrographs. Flow cytometric analysis demonstrated that the cells treated with TiO2 NPs at concentrations of 100 and 200 μg/mL showed a significant G2/M phase arrest and a significant increased proportion of apoptotic cells. TiO2 NPs also disrupted the mitochondrial membrane potential evaluated by rhodamine 123 staining. Further analysis by quantitative real-time PCR (qRT-PCR) indicated that the expression of caspase-3 and caspase-9 messenger RNA (mRNA) was increased significantly at the concentrations of 100 and 200 μg/mL TiO2 NPs for 48 h. Taken together, these findings suggest that TiO2 NPs can inhibit A549 cell proliferation, cause DNA damage, and induce apoptosis via a mechanism primarily involving the activation of the intrinsic mitochondrial pathway. The assay data provide strong evidence that TiO2 NPs can induce cytotoxicity, significant DNA damage, and apoptosis of A549 cells, suggesting that exposure to TiO2 NPs could cause cell injury and be hazardous to health.
Article
Secondhand smoke (SHS) is a confirmed lung carcinogen that introduces thousands of toxic chemicals into the lungs. SHS contains chemicals that have been implicated in causing oxidative DNA damage in the airway epithelium. Although DNA repair is considered a key defensive mechanism against various environmental attacks, such as cigarette smoking, the associations of individual repair enzymes with susceptibility to lung cancer are largely unknown. This study investigated the role of NEIL2, a DNA glycosylase excising oxidative base lesions, in human lung cells treated with sidestream smoke (SSS), the main component of SHS. To do so, we generated NEIL2 knockdown cells using siRNA-technology and exposed them to SSS-laden medium. Representative SSS chemical compounds in the medium were analyzed by mass spectrometry. An increased production of reactive oxygen species (ROS) in SSS-exposed cells was detected through the fluorescent detection and the induction of HIF-1α. The long amplicon-quantitative PCR (LA-QPCR) assay detected significant dose-dependent increases of oxidative DNA damage in the HPRT gene of cultured human pulmonary fibroblasts (hPF) and BEAS-2B epithelial cells exposed to SSS for 24 h. These data suggest that SSS exposure increased oxidative stress, which could contribute to SSS-mediated toxicity. siRNA knockdown of NEIL2 in hPF and HEK 293 cells exposed to SSS for 24 h resulted in significantly more oxidative DNA damage in HPRT and POLB than in cells with control siRNA. Taken together, our data strongly suggest that decreased repair of oxidative DNA base lesions due to an impaired NEIL2 expression in non-smokers exposed to SSS would lead to accumulation of mutations in genomic DNA of lung cells over time, thus contributing to the onset of SSS-induced lung cancer.
Article
We have successfully demonstrated the potential of surface enhanced Raman spectroscopy (SERS) in monitoring the real time damage to genomic DNA. To reveal the capabilities of this technique, we exposed DNA to reactive oxygen species (ROS), an agent that has been implicated in causing DNA double-strand breaks, and the various stages of free radical-induced DNA damage has been monitored by using SERS. Besides this, we showed that prompt DNA aggregation followed by DNA double-strand scission and residual damage to the DNA bases caused by the ROS could be substantially reduced by the protective effect of Pt nanocages and nearly cubical Pt nanopartcles. The antioxidant activity of Pt nanoparticles was further confirmed by the cell viability studies. Based on SERS results, we identified various stages involved in the mechanism of action of ROS towards DNA damage, which involves the DNA double-strand scission and its aggregation followed by the oxidation of DNA bases. We found that Pt nanoparticles inhibits the DNA double-strand scission to a significant extent by the degradation of ROS. Our method illustrates the capability of SERS technique in giving vital information about the DNA degradation reactions at molecular level, which may provide insight into the effectiveness and mechanism of action of many drugs in cancer therapy.
Article
Please cite this paper as: Protective effects of platinum nanoparticles against UV-light-induced epidermal inflammation. Experimental Dermatology 2010; 19: 1000–1006. Abstract: Intracellular reactive oxygen species (ROS) and apoptosis play important roles in the ultraviolet (UV)-induced inflammatory responses in the skin. Metal nanoparticles have been developed to increase the catalytic activity of metals, which is because of the large surface area of smaller particles. Platinum nanoparticles (nano-Pt) protected by poly acrylic acid were manufactured by reduction with ethanol. A marked increase in ROS production was observed in UV-treated HaCaT keratinocytes cell lines, while a decrease in ROS production was observed in nano-Pt-treated cells. Pretreatment of the cells with nano-Pt also caused a significant inhibition of UVB- and UVC-induced apoptosis. Furthermore, we found that mice treated with nano-Pt gel prior to UV irradiation showed significant inhibition of UVB-induced inflammation and UVA-induced photoallergy compared to UV-irradiated control mice. These results suggest that nano-Pt effectively protects against UV-induced inflammation by decreasing ROS production and inhibiting apoptosis in keratinocytes.
Article
Pt nanoparticle is a strong reductant and has been used as an antioxidant in cosmetics and medicine. It was reported to have catalase-like activity, which converts hydrogen peroxide to water and oxygen. However, in this study, freshly prepared Pt nanoparticle was almost inert towards decomposing hydrogen peroxide. The catalase-like activity of Pt nanoparticle increased with increasing weeks of storage at room temperature and became more significant when the Pt nanoparticle was exposed to air. No hydroxyl radical formation was confirmed by several methods such as ESR spin-trapping, dimethyl sulphoxide oxidation, salicylic acid hydroxylation and hydroxytoluene oxidation, indicating that the decomposition of hydrogen peroxide proceeds by the two-electron oxidation/reduction reaction. The oxidatively deteriorated Pt nanoparticle catalytically decomposed ascorbic acid, which is one of the most important biological antioxidants. We found that such oxidation was effectively prevented by the addition of Pd nanoparticle. We also discussed the reaction mechanisms and application of Pt nanoparticle.
Article
Platinum nanoparticles (nano-Pt) have been reported to possess anti-oxidant and anti-tumor activities. However, the biological activity and mechanism of action of nano-Pt in inflammation are still unknown. The present study was designed to determine the in-vitro anti-inflammatory effects of nano-Pt on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. RAW 264.7 macrophages were used for the study. The LPS-induced production of reactive oxygen species (ROS) was determined by flow cytometry. The prostaglandin E(2) (PGE(2)) concentration was measured using a PGE(2) assay kit. The protein levels and mRNA expression of the pro-inflammatory cytokines [tumor necrosis factor-α, interleukin (IL)-1β and IL-6], along with cyclooxygenase (COX-2) and inducible nitric oxide synthase, were analyzed by Western blotting and reverse transcription-polymerase chain reaction analysis. The phosphorylation of extracellular signal regulated kinase (ERK1/2) and Akt, and the phosphorylation and degradation of inhibitory kappa B-alpha (IκB-α) was determined by Western blot analysis. Nano-Pt significantly reduced the LPS-induced production of intracellular ROS and inflammatory mediators. In addition, nano-Pt suppressed the phosphorylation of ERK1/2 and Akt, and significantly inhibited the phosphorylation/degradation of IκB-α as well as nuclear factor kappa-B (NFκB) transcriptional activity. These results suggest that the anti-inflammatory properties of nano-Pt may be attributed to their downregulation of the NFκB signaling pathway in macrophages, thus supporting the use of nano-Pt as an anti-inflammatory agent.
Article
Glucocorticoids appear to control Na⁺ absorption in pulmonary epithelial cells via a mechanism dependent upon serum and glucocorticoid-inducible kinase 1 (SGK1), a kinase that allows control over the surface abundance of epithelial Na⁺ channel subunits (α-, β- and γ-ENaC). However, not all data support this model and the present study re-evaluates this hypothesis in order to clarify the mechanism that allows glucocorticoids to control ENaC activity. Electrophysiological studies explored the effects of agents that suppress SGK1 activity upon glucocorticoid-induced ENaC activity in H441 human airway epithelial cells, whilst analyses of extracted proteins explored the associated changes to the activities of endogenous protein kinase substrates and the overall/surface expression of ENaC subunits. Although dexamethasone-induced (24 h) ENaC activity was dependent upon SGK1, prolonged exposure to this glucocorticoid did not cause sustained activation of this kinase and neither did it induce a coordinated increase in the surface abundance of α-, β- and γ-ENaC. Brief (3 h) exposure to dexamethasone, on the other hand, did not evoke Na⁺ current but did activate SGK1 and cause SGK1-dependent increases in the surface abundance of α-, β- and γ-ENaC. Although glucocorticoids activated SGK1 and increased the surface abundance of α-, β- and γ-ENaC, these responses were transient and could not account for the sustained activation of ENaC. The maintenance of ENaC activity did, however, depend upon SGK1 and this protein kinase must therefore play an important but permissive role in glucocorticoid-induced ENaC activation.
Article
Ischemic stroke is a major, urgent neurologic disorder in which reactive oxygen species (ROS) are deeply involved in the detrimental effects. Platinum nanoparticle (nPt) species are a novel and strong scavenger of such ROS, so we examined the clinical and neuroprotective effects of nPts in mouse ischemic brain. Mice were subjected to transient middle cerebral artery occlusion (tMCAO) for 60 min. Upon reperfusion, nPt or vehicle was administered intravenously. At 48 hr after the tMCAO, motor function, infarct volume, immunohistochemistry of neurovascular components (endothelial NAGO, tight junctional occludin, and basal laminal collagen IV), and zymography for MMP-9 activity were examined. Superoxide anion generation at 2 hr after tMCAO was determined with oxidized hydroethidine. Compared with vehicle, treatment with nPts significantly improved the motor function and greatly reduced the infarct volume, especially in the cerebral cortex. Immunohistochemical analyses revealed that tMCAO resulted in a minimal decrease of NAGO and occludin but a great decrease of collagen IV and a remarkable increase of MMP-9. Treatment with nPts greatly reduced this decrease of collagen IV and activation of MMP-9 and, with large reductions of MMP-9 activation on zymography and superoxide production. The present study demonstrates that treatment with nPts ameliorates the neurological scores with a large reduction in infarct size as well as the preservation of outer components of the neurovascular unit (collagen IV) and inactivation of MMP-9. A strong reduction of superoxide anion production by nPts could account for such remarkable neurobehavioral and neuroprotective effects on ischemic stroke.
Article
Platinum is recognized as a harmless metal and is widely used in many industrial products. Recent studies have proposed that platinum in the form of nanoparticles has antioxidant properties, suggesting potential uses for platinum nanoparticles as additives in foods and cosmetics, with direct exposure consequences for humans. However, the influence of platinum nanoparticles on humans has not been sufficiently evaluated, thus far. Therefore, to investigate the influence of platinum nanoparticles on a living body, we comprehensively examined the expression profiles of genes obtained from 25 organs and tissues of rats after oral administration of platinum nanoparticles by gavage. Comparative analysis revealed that the expression levels of 18 genes were altered in 12 organs and tissues after the administration (approximately 0.17% of all the genes examined). Of the tissues examined, those of the glandular stomach, which were most directly exposed to the orally administered platinum nanoparticles, showed altered expression levels of genes associated with inflammation. In subcutaneous adipose tissue, the expression levels of genes whose products exhibited ATPase activity were altered. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) analysis confirmed the alteration in the expression levels of these genes in these 2 different tissues. Our findings indicate that orally administered platinum nanoparticles do not have a marked effect on systemic gene expression levels, except on a small number of genes expressed in rat tissues, including peripheral tissues indirectly exposed to the orally administered nanoparticles.
Article
Insulin-induced Na(+) retention in the distal nephron may contribute to the development of oedema/hypertension in patients with type 2 diabetes. This response to insulin is usually attributed to phosphatidylinositol-3-kinase (PI3K)/serum and glucocorticoid-inducible kinase 1 (SGK1) but a role for protein kinase B (PKB) has been proposed. The present study therefore aimed to clarify the way in which insulin can evoke Na(+) retention. We examined the effects of nominally selective inhibitors of PI3K (wortmannin, PI103, GDC-0941), SGK1 (GSK650394A) and PKB (Akti-1/2) on Na(+) transport in hormone-deprived and insulin-stimulated cortical collecting duct (mpkCCD) cells, while PI3K, SGK1 and PKB activities were assayed by monitoring the phosphorylation of endogenous proteins. Wortmannin substantially inhibited basal Na(+) transport whereas PI103 and GDC-0941 had only very small effects. However, these PI3K inhibitors all abolished insulin-induced Na(+) absorption and inactivated PI3K, SGK1 and PKB fully. GSK650394A and Akti-1/2 also inhibited insulin-evoked Na(+) absorption and while GSK650394A inhibited SGK1 without affecting PKB, Akti-1/2 inactivated both kinases. While studies undertaken using PI103 and GDC-0941 show that hormone-deprived cells can absorb Na(+) independently of PI3K, PI3K seems to be essential for insulin induced Na(+) transport. Akti-1/2 does not act as a selective inhibitor of PKB and data obtained using this compound must therefore be treated with caution. GSK650394A, on the other hand, selectively inhibits SGK1 and the finding that GSK650394A suppressed insulin-induced Na(+) absorption suggests that this response is dependent upon signalling via PI3K/SGK1.
Article
The great potential for medical applications of inorganic nanoparticles in living organisms is severely restricted by the concern that nanoparticles can harmfully interact with biological systems, such as lipid membranes or cell proteins. To enable an uptake of such nanoparticles by cells without harming their membranes, platinum nanoparticles were synthesized within cavities of hollow protein nanospheres (apoferritin). In vitro, the protein-platinum nanoparticles show good catalytic efficiency and long-term stability. Subsequently the particles were tested after ferritin-receptor-mediated incorporation in human intestinal Caco-2 cells. Upon externally induced stress, for example, with hydrogen peroxide, the oxygen species in the cells decreased and the viability of the cells increased.
Article
The mechanisms by which replicating influenza viruses decrease the expression and function of amiloride-sensitive epithelial sodium channels (ENaCs) have not been elucidated. We show that expression of M2, a transmembrane influenza protein, decreases ENaC membrane levels and amiloride-sensitive currents in both Xenopus oocytes, injected with human alpha-, beta-, and gamma-ENaCs, and human airway cells (H441 and A549), which express native ENaCs. Deletion of a 10-aa region within the M2 C terminus prevented 70% of this effect. The M2 ENaC down-regulation occurred at normal pH and was prevented by MG-132, a proteasome and lysosome inhibitor. M2 had no effect on Liddle ENaCs, which have decreased affinity for Nedd4-2. H441 and A549 cells transfected with M2 showed higher levels of reactive oxygen species, as shown by the activation of redox-sensitive dyes. Pretreatment with glutathione ester, which increases intracellular reduced thiol concentrations, or protein kinase C (PKC) inhibitors prevented the deleterious effects of M2 on ENaCs. The data suggest that M2 protein increases steady-state concentrations of reactive oxygen intermediates that simulate PKC and decrease ENaCs by enhancing endocytosis and its subsequent destruction by the proteasome. These novel findings suggest a mechanism for the influenza-induced rhinorrhea and life-threatening alveolar edema in humans.
Article
In the present work, platinum nanoparticles were prepared by in situ reduction with polyethylene glycols (PEGs). The catalytic performance of Pt nanoparticles immobilized in PEGs (Pt-PEGs) is discussed for the hydrogenation of o-chloronitrobenzene (o-CNB). A high selectivity to o-chloroaniline (o-CAN) of about 99.7% was obtained with the Pt-PEGs catalysts at the complete conversion of o-CNB, which is much higher than that (83.4%) obtained over the conventional catalyst of Pt/C. The Pt nanoparticles could be immobilized in PEGs stably and recycled for four times with the same activity and selectivity. It presents a promising performance in the hydrogenation and its wide application in catalytic reactions is expected.
Article
Recent evidence implicates increased oxidative stress as an important mechanism of the pulmonary inflammation that occurs in cigarette smokers. Since cigarette smoke (CS) contains and generates a large amount of reactive oxygen species (ROS) that elicit pulmonary inflammation, antioxidants may become effective therapeutic agents for CS-related inflammatory lung diseases, such as chronic obstructive pulmonary disease. Platinum nanoparticles stabilized with polyacrylate to form a stable colloid solution (PAA-Pt) are a new class of antioxidants that has been shown to efficiently quench ROS. In the present study we investigated the therapeutic effects of PAA-Pt on pulmonary inflammation in smoking mice. PAA-Pt or saline was administered intranasally to DBA/2 mice, which were then exposed to CS or control air daily for 3 days. Mice were sacrificed 4h after their final exposure to CS or control air. CS exposure caused depletion of antioxidant capacity, NFkappaB activation, and neutrophilic inflammation in the lungs of mice, and intranasal administration of PAA-Pt prior to CS exposure was found to inhibit these changes. Intranasal administration of PAA-Pt alone did not elicit pulmonary inflammation or toxicity. In in vitro experiments, treatment of alveolar-type-II-like A549 cells with PAA-Pt inhibited cell death after exposure to a CS extract. These results suggest that platinum nanoparticles act as antioxidants that inhibit pulmonary inflammation induced by acute cigarette smoking.
Article
The epithelial Na+ channel (ENaC) transports Na+ across tight epithelia, including the distal nephron. Different paradigms of ENaC regulation include extrinsic and intrinsic factors that affect the expression, single-channel properties, and intracellular trafficking of the channel. In particular, recent discoveries highlight new findings regarding proteolytic processing, ubiquitination, and recycling of the channel. Understanding the regulation of this channel is critical to the understanding of various clinical phenomena, including normal physiology and several diseases of kidney and lung epithelia, such as blood pressure (BP) control, edema, and airway fluid clearance. Significant progress has been achieved in this active field of research. Although ENaC is classically thought to be a mediator of BP and volume status through Na+ reabsorption in the distal nephron, several studies in animal models highlight important roles for ENaC in lung pathophysiology, including in cystic fibrosis. The purpose of this review is to highlight the various modes and mechanisms of ENaC regulation, with a focus on more recent studies and their clinical implications.
Article
The molecular composition of a core conduction element formed by the alpha-subunit of cloned epithelial Na+ channels (ENaC) was studied in planar lipid bilayers. Two pairs of in vitro translated proteins were employed in combinatorial experiments: 1) wild-type (WT) and an N-terminally truncated alphaDeltaN-rENaC that displays accelerated kinetics (tauo = 32 +/- 13 ms, tauc = 42 +/- 11 ms), as compared with the WT channel (tauc1 = 18 +/- 8 ms, tauc2 = 252 +/- 31 ms, and tauo = 157 +/- 43 ms); and 2) WT and an amiloride binding mutant, alphaDelta278-283-rENaC. The channels that formed in a alphaWT:alphaDeltaN mixture fell into two groups: one with tauo and tauc that corresponded to those exhibited by the alphaDeltaN-rENaC alone, and another with a double-exponentially distributed closed time and a single-exponentially distributed open time that corresponded to the alphaWT-rENaC alone. Five channel subtypes with distinct sensitivities to amiloride were found in a 1alphaWT:1alphaDelta278-283 protein mixture. Statistical analyses of the distributions of channel phenotypes observed for either set of the WT:mutant combinations suggest a tetrameric organization of alpha-subunits as a minimal model for the core conduction element in ENaCs.
Article
The epithelial Na(+) channel (ENaC) plays a key role in the regulation of Na(+) and water absorption in several epithelia, including those of the distal nephron, distal colon, and lung. Accordingly, mutations in ENaC leading to reduced or increased channel activity cause human diseases such as pseudohypoaldosteronism type I or Liddle's syndrome, respectively. The gain of ENaC function in Liddle's syndrome is associated with increased activity and stability of the channel at the plasma membrane. Thus understanding the regulation of channel processing and trafficking to and stability at the cell surface is of fundamental importance. This review describes some of the recent advances in our understanding of ENaC trafficking, including the role of glycosylation, ENaC solubility in nonionic detergent, targeting signal(s) and hormones. It also describes the regulation of ENaC stability at the cell surface and the roles of the ubiquitin ligase Nedd4 (and ubiquitination) and clathrin-mediated endocytosis in that regulation.
Article
Renal A6 epithelial cells were used to determine the mechanism by which protein kinase C (PKC) decreases epithelial Na(+) channel (ENaC) activity. Activation of PKC reduced relative Na(+) reabsorption to <20% within 60 min. This decrease was sustained over the next 24-48 h. In response to PKC signaling, alpha-, beta-, and gamma-ENaC levels were 0.97, 0.36, and 0.39, respectively, after 24 h, with the levels of the latter two subunits being significantly decreased. The PKC-mediated decreases in beta- and gamma-ENaC were significantly reversed by simultaneous addition of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase-1/2 inhibitors U-0126 and PD-98059. These inhibitors, in addition, protected Na(+) reabsorption from PKC, demonstrating that the MAPK1/2 cascade, in some instances, plays a central role in downregulation of ENaC activity. The effects of PKC on beta- and gamma-ENaC levels were additive with those of inhibitors of transcription (actinomycin D) and translation (emetine and cycloheximide), suggesting that PKC promotes subunit degradation and does not affect subunit synthesis. The bulk of whole cell gamma-ENaC was degraded within 1 h after treatment with inhibitors of synthesis; however, a significant pool was "protected" from inhibitors for up to 12 h. PKC affected this protected pool of gamma-ENaC. Moreover, proteosome inhibitors (MG-132 and lactacystin) reversed PKC effects on this protected pool of gamma-ENaC. Thus PKC signaling via MAPK1/2 cascade activation in A6 cells promotes degradation of gamma-ENaC.
Article
11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which requires oxidized nicotinamide adenine dinucleotide as a cofactor, metabolizes endogenous glucocorticoids. Since 11beta-HSD2 has been detected in lung epithelial cells, we examined whether carbenoxolone, a potent inhibitor of 11beta-HSD, would enhance endogenous glucocorticoid action on lung fluid balance and inflammation. Controlled laboratory study. University research laboratory. Adult Sprague-Dawley rats (n = 66). Rats were intraperitoneally injected with carbenoxolone (2 x 10 mg.kg(-1).day(-1) for 3 days) and allowed free access to water and food. Rats were further challenged with endotoxin instillation (1 mg/kg). We discovered that carbenoxolone significantly increased messenger RNA expression of all three epithelial sodium channel subunits in distal lung tissues (two-fold increase of alpha-subunit, four-fold increase of beta-subunit, and two-fold increase of gamma-subunit) as well as in trachea. Carbenoxolone increased the amiloride-sensitive alveolar fluid clearance significantly. When rats were further challenged by endotoxin instillation (1 mg/kg), pretreatment with carbenoxolone significantly inhibited endotoxin-induced increase in lung neutrophils as well as tumor necrosis factor-alpha and cytokine-induced neutrophil chemoattractant-1 concentrations in serum and bronchoalveolar lavage fluid. These beneficial effects of carbenoxolone on lung fluid balance and inflammation are very similar to those expected when glucocorticoids are introduced exogenously. We conclude that carbenoxolone increased the actions of endogenous bioactive glucocorticoids on lung cells by reducing local steroid breakdown.
Article
The platinum group elements (PGE) Pt, Pd and Rh are increasingly emitted into the environment by automobile catalytic converters. Whereas the biological availability of PGE to plants and animals has been demonstrated, only few studies concentrate on the influence of PGE on a cellular level. The effects of Pt, Pd and Rh compared with Cd, Ni and Cr on cell viability and oxidative stress response using soluble metal salts were studied in the human bronchial epithelial cell line BEAS-2B. Whilst Rh(III) showed little influence, both Pt(II) and Pt(IV) as well as Pd(II) had significant effects on cell viability at levels comparable to Cd(II) and Cr(VI). Arranging metal species in order of increasing toxicity as determined by LC50 yields: Rh(III)=1.2 mmol/L<Ni(II)=0.8 mmol/L<Pt(II)=Pd(II)=0.4 mmol/L<Pt(IV)=0.05 mmol/L<Cr(VI)=0.02 mmol/L<Cd(II)=0.005 mmol/L. ROS induction can be used as a biomarker for oxidative cell stress. The maximum relative increase in ROS production for Pt(IV) (1134%) was more than twice as high as for Cr(VI) (560%), with Pt(II) still resulting in an increase of 238%. These findings underline the strong effects of PGE on cell metabolism and emphasize the need for further studies of their toxic properties.