Internalization of FITC-albumin in Human Adipose-derived Stem Cells: Involvement of Clathrin and Caveolin.

Adipose tissue-derived stem cells (AdSCs) are one of the most promising cell types for cell-based therapies. In addition, AdSCs systematically injected into the body have been reported to localize to damaged tissues and certain types of tumor. As an important part of establishing a potent drug delivery system with AdSCs, the mechanism and efficiency of uptake into AdSCs has drawn much research attention. However, this remains to be fully clarified. The aim of this study was to examine the characteristics of endocytosis-mediated uptake in human AdSCs. We used fluorescein isothiocyanate-labeled albumin (FITC-albumin) as a potent marker of endocytosis. FITC-albumin uptake was time- and temperature-dependent. Confocal microscopy showed punctate localization of fluorescence in the cytoplasm. FITC-albumin uptake was inhibited by human serum albumin in a concentration-dependent manner. FITC-albumin uptake was inhibited by a metabolic inhibitor (2,4-dinitrophenol), a microtubule polymerization inhibitor (colchicine), an actin polymerization inhibitor (cytochalasin D), endosomal acidification inhibitors (chloroquine and bafilomycin A1), clathrin-dependent endocytosis inhibitors (chloropromazine, phenylarsine oxide, and Pitstop2), and caveolin-dependent endocytosis inhibitors (nystatin and methyl-ß-cyclodextrin). Furthermore, the knockdown of the clathrin heavy chain and caveolin-1 significantly reduced FITC-albumin uptake. These findings suggest that AdSCs take up albumin via endocytic pathways in which clathrin and caveolin are involved.

Pharmacokinetic-pharmacodynamic modelling of the hypoglycaemic effect of pulsatile administration of human insulin in rats.

The relationship between the plasma insulin (INS) concentration-time course and plasma glucose concentration-time course during and after pulsatile INS administration to rats was characterized using a pharmacokinetic-pharmacodynamic (PK-PD) model. A total INS dose of 0.5 IU/kg was intravenously injected in 2 to 20 pulses over a 2-h period. Compared with the single bolus administration, the area under the effect-time curve (AUE) increased depending on the number of pulses, and the AUEs for more than four pulses plateaued at a significantly larger value, which was similar to that after the infusion of a total of 0.5 IU/kg of INS over 2 h. No increase in plasma INS concentration occurred after pulsatile administration. Two indirect response models primarily reflecting the receptor-binding process (IR model) or glucose transporter 4 (GLUT4) translocation (GT model) were applied to describe the PK-PD relationship after single intravenous bolus administration of INS. These models could not explain the observed data after pulsatile administration. However, the IR-GT model, which was a combination of the IR and GT models, successfully explained the effects of pulsatile administration and intravenous infusion. These results indicate that the receptor-binding process and GLUT4 translocation are responsible for the change in AUE after pulsatile administration.

Effect of riboflavin deficiency on development of the cerebral cortex in Slc52a3 knockout mice.

Riboflavin transporter 3 (RFVT3), encoded by the SLC52A3 gene, is important for riboflavin homeostasis in the small intestine, kidney, and placenta. Our previous study demonstrated that Slc52a3 knockout (Slc52a3-/-) mice exhibited neonatal lethality and metabolic disorder due to riboflavin deficiency. Here, we investigated the influence of Slc52a3 gene disruption on brain development using Slc52a3-/- embryos. Slc52a3-/- mice at postnatal day 0 showed hypoplasia of the brain and reduced thickness of cortical layers. At embryonic day 13.5, the formation of Tuj1+ neurons and Tbr2+ intermediate neural progenitors was significantly decreased; no significant difference was observed in the total number and proliferative rate of Pax6+ radial glia. Importantly, the hypoplastic phenotype was rescued upon riboflavin supplementation. Thus, it can be concluded that RFVT3 contributes to riboflavin homeostasis in embryos and that riboflavin itself is required during embryonic development of the cerebral cortex in mice.

Fatty acids bound to albumin induce prostaglandin E2 production in human renal proximal tubular epithelial cell line HK-2.

Fatty acids bound to albumin have been reported to be involved in various responses in renal proximal tubular cells following albumin overload, leading to progression of tubulointerstitial damage in the kidneys. In addition, it has been reported that prostaglandin E2 (PGE2) plays an important role in nephrotoxicity. The aim of this study was to examine whether albumin-bound fatty acids induce PGE2 production in human renal proximal tubular epithelial cell line HK-2. Fatty acid-bearing human serum albumin increased PGE2 release in the culture medium in concentration-dependent and time-dependent manners, but fatty acid-depleted albumin had no effect on PGE2 production. Next, we investigated the effect of arachidonic acid, a precursor of eicosanoids, on PGE2 production. Arachidonic acid with fatty acid-free albumin significantly enhanced the release of PGE2 into the medium in a concentration-dependent manner. Furthermore, we examined the effect of arachidonic acid on mRNA expression of hypoxia inducible factor-1α (HIF-1α). Arachidonic acid increased HIF-1α mRNA expression in a concentration-dependent manner. These findings suggest that fatty acids, at least in part arachidonic acid, bound to albumin increase PGE2 production and expression of HIF-1α mRNA and protein, possibly resulting in various cell responses induced by albumin overload.

Pioglitazone induces hypoxia-inducible factor 1 activation in human renal proximal tubular epithelial cell line HK-2.

We previously reported that fatty acid-bearing albumin but not fatty acid-depleted albumin induces hypoxia-inducible factor-1 (HIF-1) activation in human renal proximal tubular epithelial cell line HK-2. Then, an increase in mRNA expression of peroxisome proliferator-activated receptor gamma (PPARγ) was observed on treatment with fatty acid-bearing albumin but not fatty acid-depleted albumin. The aim of this study was to determine whether a PPARγ agonist, pioglitazone, induces HIF-1 activation or not. Treatment with pioglitazone induced HIF-1α mRNA as well as PPARγ mRNA expression in a concentration dependent manner. In addition, pioglitazone increased HIF-1 target genes such as the mRNAs of glucose transporter 1 (GLUT1) and breast cancer resistance protein (BCRP/ABCG2), in a concentration-dependent manner. Consistent with the increases in GLUT1 and ABCG2 mRNAs, protein expression of GLUT1 and BCRP was increased by pioglitazone. In addition, GLUT inhibitor phloretin-sensitive D-[3H]glucose uptake activity and BCRP inhibitor Ko143-sensitive accumulation of Hoecsht33342, a BCRP substrate, were significantly enhanced by treatment with pioglitazone. These findings suggest that PPARγ activation by pioglitazone leads to HIF-1 protein expression induction followed by changes in HIF-1 target gene expression and protein product activity.

Cobalt Chloride Induces Expression and Function of Breast Cancer Resistance Protein (BCRP/ABCG2) in Human Renal Proximal Tubular Epithelial Cell Line HK-2.

The human breast cancer resistance protein (BCRP/ABCG2), a member of the ATP-binding cassette transporter family, is a drug transporter restricting absorption and enhancing excretion of many compounds including anticancer drugs. The cis-regulatory elements in the BCRP promoter include a hypoxia response element, i.e., the DNA binding site for hypoxia-inducible factor-1 (HIF-1). In this study, we investigated the effect of cobalt chloride, a chemical inducer of HIF-1α, on the expression and function of BCRP in human renal proximal tubular cell line HK-2. Cobalt chloride treatment significantly increased the mRNA expression of not only glucose transporter 1 (GLUT1), a typical HIF-1 target gene mRNA, but also ABCG2 mRNA in HK-2 cells. The BCRP inhibitor Ko143-sensitive accumulation of BCRP substrates such as Hoechst33342 and mitoxantrone was significantly enhanced by cobalt chloride treatment. In addition, treatment with cobalt chloride significantly increased the Ko143-sensitive accumulation of fluorescein isothiocyanate-labeled methotrexate in HK-2 cells. Furthermore, cobalt chloride treatment attenuated the cytotoxicity induced by mitoxantrone and methotrexate, which might be, at least in part, due to the increase in BCRP-mediated transport activity via HIF-1 activation. These findings indicate that HIF-1 activation protects renal proximal tubular cells against BCRP substrate-induced cytotoxicity by enhancing the expression and function of BCRP in renal proximal tubular cells.

Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins.

AIMS: Cisplatin (CDDP) is a platinum-based drug that is widely used in cancer chemotherapy, but the development of resistance in tumor cells is a major weakness of these treatments. Several mechanisms have been proposed to explain cisplatin resistance, and disruption of certain cellular pathways could modulate drug sensitivity to cisplatin. A lower level of cross-resistance to cisplatin leads to better outcomes in clinical use. MAIN METHODS: Cross-resistance was assessed using cisplatin resistant lung cancer cell line A549/CDDP. Cell cycle analysis was used to examine the effect of cisplatin on cell signaling pathways regulating G2/M transition in cisplatin resistant cells. KEY FINDINGS: A549/CDDP cells exhibited cross-resistance to carboplatin, but not oxaliplatin, which is often found in platinum analogues. Flow cytometry showed that nocodazole treatment caused a G2/M block in both A549/CDDP cells and cisplatin susceptible cells. However, A549/CDDP cells escaped the G2/M block following exposure to cisplatin. Activation of the Cdc2/CyclinB complex is required for transition from G2 to M phase, and the inactive form of phosphorylated Cdc2 is activated by Cdc25C dephosphorylation of Tyr15. In the cisplatin-treated susceptible cells, the levels of phosphorylated Cdc2 and Cdc25C were markedly decreased, leading to a loss of Cdc2 activity and G2/M arrest. In A549/CDDP cells, however, Cdc2 activity was supported by the expression of Cdc2 and Cdc25C after the addition of cisplatin, which resulted in G2/M progression. SIGNIFICANCE: The resistance phenotype of G2/M progression has been correlated with dysregulation of Cdc2 in a human lung cancer cell line selected for cisplatin.

Fatty acid-bearing albumin but not fatty acid-depleted albumin induces HIF-1 activation in human renal proximal tubular epithelial cell line HK-2.

Recently, we found that albumin overload induces expression of the transcription factor hypoxia-inducible factor-1α (HIF-1α) protein and several HIF-1 target genes in human renal proximal tubular epithelial cell line HK-2. In this study, the role of albumin-bound fatty acids in the albumin-induced HIF-1 activation was studied. The enhancing effect of fatty acid-bearing human serum albumin [FA(+)HSA] treatment on HIF-1α protein expression was much greater than that of fatty acid-depleted human serum albumin [FA(-)HSA] treatment. The FA(+)HSA treatment induced HIF-1 target gene mRNAs such as those of glucose transporter 1 (GLUT1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and breast cancer resistance protein (BCRP) in concentration-dependent manners, while FA(-)HSA caused no significant increases in these mRNAs. Consistent with increased GLUT1 mRNA, GLUT1 protein expression and GLUT inhibitor cytochalasin B-sensitive d-[(3)H]glucose uptake activity were significantly enhanced by treatment with FA(+)HSA, but not with FA(-)HSA. These findings indicate that fatty acids bound to albumin play a crucial role in albumin-induced HIF-1 activation followed by changes in HIF-1 target gene expression and protein product activity.

Entry of aminoglycosides into renal tubular epithelial cells via endocytosis-dependent and endocytosis-independent pathways.

Aminoglycoside antibiotics such as gentamicin and amikacin are well recognized as a clinically important antibiotic class because of their reliable efficacy and low cost. However, the clinical use of aminoglycosides is limited by their nephrotoxicity and ototoxicity. Nephrotoxicity is induced mainly due to high accumulation of the antibiotics in renal proximal tubular cells. Therefore, a lot of studies on characterization of the renal transport system for aminoglycosides so far reported involved various in-vivo and in-vitro techniques. Early studies revealed that aminoglycosides are taken up through adsorptive endocytosis in renal epithelial cells. Subsequently, it was found that megalin, a multiligand endocytic receptor abundantly expressed on the apical side of renal proximal tubular cells, can bind aminoglycosides and that megalin-mediated endocytosis plays a crucial role in renal accumulation of aminoglycosides. Therefore, megalin has been suggested to be a promising molecular target for the prevention of aminoglycoside-induced nephrotoxicity. On the other hand, recently, some reports have indicated that aminoglycosides are transported via a pathway that does not require endocytosis, such as non-selective cation channel-mediated entry, in cultured renal tubular cells as well as cochlear outer hair cells. In this commentary article, we review the cellular transport of aminoglycosides in renal epithelial cells, focusing on endocytosis-dependent and -independent pathways.

Characterization of protamine uptake by opossum kidney epithelial cells.

Protamine, a mixture of polypeptides that is rich in arginine, has been used clinically as an antidote to heparin overdoses and a complexing agent in a long-acting insulin preparation. When protamine is administered intravenously, its abundant accumulation in the kidneys has been reported. However, the renal uptake mechanism for protamine is not clear. In this study, we examined the transport mechanism for protamine in opossum kidney (OK) cells, a suitable in vitro model for renal proximal tubular epithelial cells. Flow cytometric analysis revealed that the association of fluorescein isothiocyanate (FITC)-labeled protamine from salmon (FITC-protamine) by OK cells was inhibited by unlabeled protamine in a concentration-dependent manner. The association of FITC-protamine was temperature- and energy-dependent. Confocal microscopy analysis showed that the fluorescence was localized in the cytoplasm and nucleus of OK cells. In addition, FITC-protamine association was inhibited by cationic drugs such as polycationic gentamicin and polymixin B, but it was increased by a basic amino acid, arginine. Inhibitors for clathrin- and caveolin-dependent endocytosis showed inhibitory effects on FITC-protamine association. Pretreatment with heparinase III partially but significantly decreased the association of FITC-protamine. These results suggest that protamine may be taken up by OK cells via receptor-mediated endocytosis, which may result in its localization in the cytoplasm and nucleus of the cells.

Expression and function of PEPT2 during transdifferentiation of alveolar epithelial cells.

AIMS: The purpose of this study was to clarify the expression and function of peptide transporter 2 (PEPT2) in primary cultured alveolar type II epithelial cells and in transdifferentiated type I-like cells. MAIN METHODS: Real-time PCR analysis, uptake study of [(3)H]Gly-Sar, and immunostaining were performed in alveolar epithelial cells. KEY FINDINGS: The expression of PEPT2 mRNA in type II cells isolated from rat lungs was highest at day 0, and decreased rapidly during culture of the cells. In accordance with this change, PEPT2 activity estimated as cefadroxil-sensitive [(3)H]Gly-Sar uptake also decreased along with transdifferentiation. The expression of PEPT2 protein in type II cells was confirmed by immunostaining and Western blot analysis. The uptake of [(3)H]Gly-Sar in type II cells was time- and pH-dependent. In contrast, minimal time-dependence and no pH-dependence of [(3)H]Gly-Sar uptake were observed in type I-like cells. The maximal [(3)H]Gly-Sar uptake was observed at pH6.0, and the uptake decreased at higher pHs in type II cells. The uptake of [(3)H]Gly-Sar in type II cells was inhibited by cefadroxil in a concentration-dependent manner, the IC50 value being 4.3 µM. On the other hand, no significant inhibition by cefadroxil was observed in type I-like cells. In addition, [(3)H]Gly-Sar uptake in type II cells was saturable, the Km value being 72.0 µM. SIGNIFICANCE: PEPT2 is functionally expressed in alveolar type II epithelial cells, but the expression decreases along with transdifferentiation, and PEPT2 would be almost completely lost in type I cells.

Enhancing effect of poly(amino acid)s on albumin uptake in human lung epithelial A549 cells.

The clearance of albumin from the alveolar space is a critical process in the recovery from edema. In this study, we investigated the effect of poly(amino acid)s such as poly-l-ornithine (PLO) on albumin uptake in the cultured lung epithelial cell line A549. FITC-albumin uptake as well as cell surface binding was markedly stimulated by co-incubation with PLO, and there was a good correlation between them. After being taken up by A549 cells, FITC-albumin was predominantly targeted to lysosomes. Interestingly, pretreatment of A549 cells with PLO further stimulated FITC-albumin uptake, even in the absence of PLO in the uptake buffer. FITC-albumin uptake in the presence of PLO was inhibited by a metabolic inhibitor, clathrin-mediated endocytosis inhibitors, and a macropinocytosis inhibitor, indicating the involvement of clathrin-mediated endocytosis and/or macropinocytosis. The effect of PLO on FITC-albumin clearance was also examined in an in vivo pulmonary administration method in rats, and co-administration of PLO enhanced fluorescence elimination from the lungs. These findings suggest that pulmonary administration of poly(amino acid)s such as PLO is a possible strategy for enhancing albumin clearance from the alveolar space, and thereby facilitating the recovery from pulmonary edema.

Albumin overload induces expression of hypoxia-inducible factor 1α and its target genes in HK-2 human renal proximal tubular cell line.

The aim of this study was to investigate the effect of human serum albumin (HSA) overload on the expression of the transcription factor hypoxia-inducible factor-1α (HIF-1α) in human renal proximal tubular cell line HK-2. First, the cell viability and cytotoxic activity were examined to assess the cellular conditions in HK-2 cells with HSA treatment employed in this study. HSA treatment for 48h decreased the cell viability and increased the leakage of lactate dehydrogenase (LDH) into the medium in a concentration-dependent manner, but the toxicity was relatively mild. Western Blot analysis revealed that HSA treatment induced the expression of HIF-1α protein in a concentration-dependent manner without a change in ß-actin protein expression. Confocal microscopy analysis revealed that HIF-1α protein was predominantly localized in the nucleus but was also observed in the cytoplasm. The HIF-1 target gene mRNAs, glucose transporter 1 and glyceraldehyde 3-phosphate dehydrogenase, were up-regulated by HSA treatment, leading to the increases in the protein expression levels. In addition, the mRNA of HIF-1α was increased by HSA treatment. In conclusion, albumin loading induces HIF-1α in HK-2 cells, resulting in the increases in the expression of proteins of its target genes.

Effect of protamine on the accumulation of gentamicin in opossum kidney epithelial cells.

OBJECTIVES: The purpose of this study was to examine whether or not protamine, an arginine-rich basic protein mixture, inhibits the accumulation of gentamicin, a nephrotoxic drug, in cultured opossum kidney (OK) epithelial cells. METHODS: The effect of protamine from salmon on accumulation and binding of [(3) H]gentamicin was investigated in OK cells. KEY FINDINGS: Protamine inhibited the binding and accumulation of [(3) H]gentamicin in a concentration-dependent manner. The accumulation of [(14) C]inulin, a marker of fluid-phase endocytosis, was not affected by protamine at concentrations up to 1 mm. l-Arginine at concentrations up to 10 mm had no significant effect on the accumulation of [(3) H]gentamicin. On the other hand, preincubation with 100 µm protamine for 5 min decreased the accumulation of [(3) H]gentamicin to almost the same extent as coincubation with 100 µm protamine for 60 min. CONCLUSIONS: Our results indicate that protamine decreases the accumulation of gentamicin in OK cells. These findings suggest that protamine or its derivatives might be useful in preventing the nephrotoxicity of aminoglycoside antibiotics including gentamicin.

Flow cytometry analysis of cancer cell death induced by the extract of Thai plant Ellipeiopsis cherrevensis.

  The mechanism of cancer cell death induced by KP018, an ethanol extract of the Thai plant Ellipeiopsis cherrevensis, was examined in paclitaxel-resistant HepG2 (PR-HepG2) and colon-26 cells using flow cytometry. In PR-HepG2 cells, KP018 induced necrosis in a concentration-dependent manner. Necrosis of PR-HepG2 cells induced by KP018 as well as by hydrogen peroxide was suppressed by co-treatment of the cells with N-acetylcysteine. KP018 decreased the viability of colon-26 cells with an IC50 value of 15.1 µg/mL, which was estimated by XTT assay. As observed in PR-HepG2 cells, KP018 induced necrosis and the necrosis was suppressed by N-acetylcysteine in colon-26 cells. In addition, using colon-26 solid tumor-bearing mice, KP018 was found to suppress tumor growth without apparent toxicities under in vivo conditions. These results indicate that KP018 induces necrosis rather than apoptosis in these cancer cells, and reactive oxygen species such as hydrogen peroxide would be involved in KP018-induced necrosis. KP018 may be a useful source to search for a new anticancer drug that can be used for the chemotherapy of multidrug-resistant tumors.

Effect of cigarette smoke extract on insulin transport in alveolar epithelial cell line A549.

BACKGROUND: The main purpose of this study was to evaluate the effect of cigarette smoke extract (CSE) on insulin transport in alveolar epithelial cells. METHODS: We first examined the effect of CSE pretreatment on cell viability, mRNA expression, and lamellar body structures in A549 cells. Then the effect of CSE pretreatment on FITC-insulin transport was examined. RESULTS: When A549 cells were treated with 30 µg/ml of CSE for 48 h, the expression of some mRNAs abundantly expressed in type II alveolar epithelial cells such as surfactant protein B was significantly increased. Lamellar bodylike structures became more evident with CSE treatment. FITC-insulin uptake from the apical side and subsequent efflux to the basal side was enhanced by CSE treatment in A549 cells. The enhancing effect of CSE on FITC-insulin uptake was concentration-dependent and reversible. A concentration-dependent enhancing effect of CSE on FITC-insulin uptake was also observed in normal, primary cultured alveolar type II epithelial cells isolated from rats. CONCLUSIONS: Treatment of A549 cells by CSE may direct the cells to a more type II-like phenotype. In accordance with this observation, FITC-insulin uptake was enhanced by CSE treatment. These results may partly explain the higher insulin absorption from the lung in smokers than in nonsmokers.

Gadolinium modulates gentamicin uptake via an endocytosis-independent pathway in HK-2 human renal proximal tubular cell line.

The aim of this study was to characterize the uptake mechanism of gentamicin, an aminoglycoside antibiotic, in human renal proximal tubular cell line HK-2. Sodium-dependent uptake of D-[(3)H]glucose and L-[(3)H]alanine was observed in HK-2 cells, indicating that the cells employed in this study retain functional characteristics of the renal proximal tubular cells. On the other hand, mRNA and protein expression of megalin, an endocytic receptor which is responsible for the internalization of gentamicin into the renal proximal tubular cells, was very faint in HK-2 cells. Various aminoglycoside antibiotics including amikacin and kanamycin inhibited the uptake of [(3)H]gentamicin. Colchicine and cytochalasin D, general endocytosis inhibitors, had no significant effect on [(3)H]gentamicin uptake in HK-2 cells, which was in contrast to the results observed in OK cells, a renal proximal tubular cell line expressing megalin. Furthermore, unlike OK cells, [(3)H]gentamicin uptake in HK-2 cells was not inhibited by N-WASP181-200, a cationic 20-amino acid peptide. Ruthenium red, a nonspecific cation channel blocker, decreased the uptake of [(3)H]gentamicin in HK-2 cells. In contrast, the trivalent cation gadolinium biphasically modulated [(3)H]gentamicin uptake with a maximum increase at 0.3 mM gadolinium. The enhanced effect of gadolinium on [(3)H]gentamicin uptake was independent of gadolinium-induced increase in intracellular calcium concentration. These findings indicate that gentamicin is primarily taken up via an endocytosis-independent pathway in HK-2 cells with very low expression of megalin, and that the uptake of gentamicin is modulated by gadolinium.

Enhancement effect of poly(amino acid)s on insulin uptake in alveolar epithelial cells.

In this study, we elucidated the effect of poly(amino acid)s such as poly-L-ornithine (PLO) on FITC-insulin uptake in cultured alveolar type II epithelial cells, RLE-6TN. FITC-insulin uptake by RLE-6TN cells as well as its cell surface binding was markedly increased by PLO without cytotoxicity. The uptake of FITC-insulin in the presence of PLO was shown to be mediated by endocytosis, but in contrast to the uptake in the absence of PLO, the contribution of macropinocytosis emerged. Colocalization of FITC-insulin and LysoTracker Red was observed by confocal laser scanning microscopy both in the absence and presence of PLO, indicating that FITC-insulin was partly targeted to lysosomes in the cells and degraded. The half-life of the intracellular degradation of FITC-insulin was, however, prolonged by the presence of PLO. PLO also stimulated the uptake of other FITC-labeled compounds. Among them, the enhancement effects of PLO on FITC-albumin and FITC-insulin uptake were prominent. The effect of PLO on insulin absorption was also examined in in-vivo pulmonary administration in rats, and co-administration of PLO enhanced the hypoglycemic action of insulin. These findings suggest that co-administration of poly(amino acid)s such as PLO is a useful strategy for enhancing insulin uptake by alveolar epithelial cells and subsequent absorption from the lung.

Endocytic uptake of FITC-albumin by human alveolar epithelial cell line A549.

The uptake mechanism of FITC-labeled albumin (FITC-albumin) was examined in human alveolar epithelial cell line A549. FITC-albumin uptake by A549 cells was time- and temperature-dependent, and was markedly suppressed at 4°C compared with that at 37°C. The uptake was saturable, and was mediated by a high-affinity, low-capacity system and by a low-affinity, high-capacity system. In the following experiments, we focused on the low-affinity system. FITC-albumin uptake was markedly inhibited by metabolic inhibitors and by a vacuolar H⁺-ATPase, bafilomycin A1. The uptake was inhibited by clathrin-mediated endocytosis inhibitors (phenylarsine oxide and chlorpromazine). Potassium depletion and hypertonicity that inhibit clathrin-mediated endocytosis also decreased FITC-albumin uptake. On the other hand, caveolae-mediated endocytosis inhibitors (indomethacin and nystatin) did not affect FITC-albumin uptake. In addition, FITC-albumin uptake was inhibited by macropinocytosis inhibitors such as 5-(N-ethyl-N-isopropyl) amiloride. These results suggest that the low-affinity system of FITC-albumin uptake is mediated by endocytosis in A549 cells, predominantly via a clathrin-mediated pathway. Macropinocytosis, but not caveolae-mediated endocytosis, may also be involved. Considering our previous findings, albumin may be transported by a similar mechanism and/or pathway in rat and human alveolar epithelial cells.

Nanoparticulation of probucol, a poorly water-soluble drug, using a novel wet-milling process to improve in vitro dissolution and in vivo oral absorption.

To improve the dissolution and oral absorption properties of probucol, a novel wet-milling process using the ULTRA APEX MILL was investigated. The particle size of bulk probucol powder was 17.1 µm. However, after wet-milling with dispersing agents such as Gelucire 44/14, Gelucire 50/13, vitamin E-TPGS, and Pluronic F-108, the probucol particle sizes decreased to about 77-176 nm. Scanning electron microscopy (SEM) analysis also suggested that the probucol particles were successfully milled into the nanometer range. An in vitro dissolution study showed that the dissolution rates of all nanopowders were several folds higher than those of the corresponding mixed powders. When orally administered to rats, the AUC values of probucol nanopowders treated with Gelucire 44/14 and 50/13, and vitamin E-TPGS were about 3.06-3.54-folds greater than that of the bulk powder. Therefore, through this study, we have developed a new pharmaceutical technique to improve the dissolution rate and oral absorption of probucol using the ULTRA APEX MILL by wet-milling with various dispersing agents.