Human breast cancer resistance protein (BCRP/ABCG2) is believed to act as an efflux pump to protect the body from drugs and toxins. BCRP is known to accept many kinds of endogenous and exogenous compounds as substrates and to be localized on the apical membrane of various tissues. Expression of BCRP is also reported on the side population cells, and a recent report suggested involvement of Akt in the modulation of the side population phenotype. In the present study, we have characterized the effect of Akt on the polarized expression of BCRP using LLC-PK1 cells. After treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors, internalization of stably transfected BCRP from the apical surface was observed after immunohistochemical staining, and the relative expression level of BCRP on the cell surface decreased to 49 +/- 14 and 51 +/- 8% of the control for LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride] and wortmannin treatment, respectively. This phenomenon was supported by the observation of internalized BCRP in presence of dominant negative-Akt. When the cells were treated with epidermal growth factor, the cell surface expression of BCRP was increased to 228 +/- 43% of the control accompanied by Akt stimulation. These results suggest that the relative expression of BCRP on the cell surface is regulated by the PI3K-Akt signaling pathway with a positive correlation in polarized cells. Alteration in Akt activities may influence the cellular extrusion of BCRP substrates by modifying epithelial BCRP localization.

Recently, we have provided evidence that the ABC-transporter MDR1 P-glycoprotein translocates analogs of various lipid classes across the apical plasma membrane of polarized LLC-PK1 cells transfected with MDR1 cDNA. Here, we show that expression of the basolateral ABC-transporter MRP1 (the multidrug resistance protein) induced lipid transport to the exoplasmic leaflet of the basolateral plasma membrane of LLC-PK1 cells at 15 degreesC. C6-NBD-glucosylceramide synthesized on the cytosolic side of the Golgi complex, but not C6-NBD-sphingomyelin synthesized in the Golgi lumen, became accessible to depletion by BSA in the basal culture medium. This suggests the absence of vesicular traffic and direct translocation of C6-NBD-glucosylceramide by MRP1 across the basolateral membrane. In line with this, transport of the lipid to the exoplasmic leaflet depended on the intracellular glutathione concentration and was inhibited by the MRP1-inhibitors sulfinpyrazone and indomethacin, but not by the MDR1 P-glycoprotein inhibitor PSC 833. In contrast to the broad substrate specificity of the MDR1 P-glycoprotein, MRP1 selectively transported C6-NBD-glucosylceramide and C6-NBD-sphingomyelin, the latter only when it was released from the Golgi lumen by brefeldin A. This shows the specific nature of the lipid translocation. We conclude that the transport activity of MDR1 P-glycoprotein and MRP1 must be taken into account in studies on the transport of lipids to the cell surface.

Recently, it was revealed that 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and 24R,25-dihydroxyvitamin D3 (24,25(OH)2D3) were metabolized to their respective epimers of the hydroxyl group at C-3 of the A-ring. We now report the isolation and structural assignment of 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3 as a major metabolite of 25-hydroxyvitamin D3 (25(OH)D3) and the further metabolism of C-3 epimers of vitamin D3 metabolites. When 25(OH)D3 was incubated with various cultured cells including osteosarcoma, colon adenocarcinoma, and hepatoblastoma cell lines, 3-epi-25(OH)D3 and 24,25 (OH)2D3 were commonly observed as a major and minor metabolite of 25(OH)D3, respectively. 25(OH)D3 was at least as sensitive to C-3 epimerization as 1alpha, 25(OH)2D3 which has been reported as a substrate for the C-3 epimerization reaction. Unlike these cultured cells, LLC-PK1 cells, a porcine kidney cell line, preferentially produced 24,25(OH)2D3 rather than 3-epi-25(OH)D3. We also confirmed the existence of 3-epi-25(OH)D3 in the serum of rats intravenously given pharmacological doses of 25(OH)D3. The cultured cells metabolized 3-epi-25OHD3 and 3-epi-1alpha,25(OH)2D3 to 3-epi-24,25(OH)2D3 and 3-epi-1alpha,24,25(OH)3D3, respectively. In addition, we demonstrated that 3-epi-25(OH)D3 was metabolized to 3-epi-1alpha,25(OH)2D3 by CYP27B1 and to 3-epi-24,25(OH)2D3 by CYP24 using recombinant Escherichia coli cell systems. 3-Epi-25(OH)D3, 3-epi-1alpha,25(OH)2D3, and 3-epi-24,25(OH)2D3 were biologically less active than 25(OH)D3, 1alpha,25(OH)2D3, and 24,25(OH)2D3, but 3-epi-1alpha,25(OH)2D3 showed to some extent transcriptional activity toward target genes and anti-proliferative/differentiation-inducing activity against human myeloid leukemia cells (HL-60). These results indicate that C-3 epimerization may be a common metabolic pathway for the major metabolites of vitamin D3.

BACKGROUND

Espins are actin bundling proteins present in hair cell stereocilia. A recessive mutation in the espin gene (Espn) has been detected in the jerker mouse and causes deafness, vestibular dysfunction, and hair cell degeneration. More recently mutations in the human espin gene (ESPN) have been described in two families affected by autosomal recessive hearing loss and vestibular areflexia.

OBJECTIVE

To report the identification of four additional ESPN mutations (S719R, D744N, R774Q, and delK848) in patients affected by autosomal dominant hearing loss without vestibular involvement.

RESULTS

To determine whether the mutated ESPN alleles affected the biological activity of the corresponding espin proteins in vivo, their ability to target and elongate the parallel actin bundles of brush border microvilli was investigated in transfected LLC-PK1-CL4 epithelial cells. For three mutated alleles clear abnormalities in microvillar length or distribution were obtained.

CONCLUSIONS

The results further strengthen the causative role of the espin gene in non-syndromic hearing loss and add new insights into espin structure and function.

Crystals of calcium oxalate monohydrate (COM) in the renal tubule form the basis of most kidney stones. Tubular dysfunction resulting from COM-cell interactions occurs by mechanism(s) that are incompletely understood. We examined the production of reactive oxygen intermediates (ROI) by proximal (LLC-PK1) and distal (MDCK) tubular epithelial cells after treatment with COM (25-250 microg/ml) to determine whether ROI, specifically superoxide (O(2)(*-)), production was activated, and whether it was sufficient to induce oxidative stress. Employing inhibitors of cytosolic and mitochondrial systems, the source of ROI production was investigated. In addition, intracellular glutathione (total and oxidized), energy status (ATP), and NADH were measured. COM treatment for 1-24 h increased O(2)(*-) production 3-6-fold as measured by both lucigenin chemiluminescence in permeabilized cells and dihydrorhodamine fluorescence in intact cells. Using selective inhibitors we found no evidence of cytosolic production. The use of mitochondrial probes, substrates, and inhibitors indicated that increased O(2)(*-) production originated from mitochondria. Treatment with COM decreased glutathione (total and redox state), indicating a sustained oxidative insult. An increase in NADH in COM-treated cells suggested this cofactor could be responsible for elevating O(2)(*-) generation. In conclusion, COM increased mitochondrial O(2)(*-) production by epithelial cells, with a subsequent depletion of antioxidant status. These changes may contribute to the reported cellular transformations during the development of renal calculi.

The metabotropic glutamate receptors (mGluRs) share no sequence homology and show different structural features compared with most other G protein-coupled receptors (GPCRs). In particular, some isoforms of the phospholipase C (PLC)-coupled mGluRs (mGluR1a, mGluR5a, and mGluR5b) have a surprisingly long carboxyl-terminal intracellular domain of more than 350 residues, whereas the splice variants mGluR1b and mGluR1c have a much shorter carboxyl terminus. In the current study, the different splice variants of mGluR1 were expressed in porcine kidney epithelial (LLC-PK1) or the human embryonic kidney (HEK 293) cells, and their levels of expression were examined with the use of Western blot analysis. Expression of the short isoforms mGluR1b and mGluR1c did not modify the basal inositol phosphate production. In contrast, expression to similar levels of mGluR1a resulted in a 2-fold increase in the basal inositol phosphate formation. This increase in basal PLC activity was due to neither the presence of a low concentration of glutamate in the incubation medium nor a modification of the PLC pathway, resulting, for example, from the constant activation of mGluR1a++ by glutamate during the culture. Surprisingly none of the known competitive antagonists of mGluR1 inhibited the basal PLC activity, indicating that none of these molecules act as inverse agonists. Taken together, these results indicate that the long carboxyl-terminal domain confers a small agonist-independent activity to mGluR1. This indicates that, as already observed for other GPCRs, little constitutive activity of wild-type mGluRs can be detected. Our results also add to the splice variants and further suggest that the long carboxyl-terminal domain of mGluR1a confers better coupling efficiency to the G proteins.

P-glycoprotein (P-gp) is an efflux transporter involved in limiting the oral bioavailability and tissue penetration of a variety of structurally divergent molecules. A better understanding of the structural requirements of modulators of P-gp function will aid in the design of therapeutic agents. Toward this goal, three-dimensional quantitative structure-activity relationship (3D-QSAR) models were generated using in vitro data associated with inhibition of P-gp function. Several approaches were undertaken with multiple iterations, yielding Catalyst 3D-QSAR models being able to qualitatively rank-order and predict IC(50) values for P-gp inhibitors excluded from the model in question. The success of these validations suggests that a P-gp pharmacophore for 27 inhibitors of digoxin transport in Caco-2 cells consisted of four hydrophobes and one hydrogen bond acceptor. A second pharmacophore generated with 21 inhibitors of vinblastine binding to plasma membrane vesicles derived from CEM/VLB(100) cells contained three ring aromatic features and one hydrophobic feature. A third pharmacophore generated with 17 inhibitors of vinblastine accumulation in P-gp expressing LLC-PK1 cells contained four hydrophobes and one hydrogen bond acceptor. A final pharmacophore was generated for inhibition of calcein accumulation in P-gp expressing LLC-PK1 cells and found to contain two hydrophobes, a ring aromatic feature, and a hydrogen bond donor. The similarity of features for the pharmacophores of P-gp inhibitors of digoxin transport and vinblastine binding suggest some commonality in their binding sites. Utilization of such models may prove to be of value for prediction of molecules that may modulate one or more P-gp binding sites.

Na+-dependent phosphate transport and its response to parathyroid hormone (PTH) has been investigated in three continuous cell lines of renal epithelial origin (LLC-PK1, JTC-12.P3, and OK). The apparent Km for phosphate was similar, but the maximal transport rate (Vmax) was markedly different in the three cell lines. PTH and forskolin produced an increase of cellular adenosine 3',5'-cyclic monophosphate (cAMP) in all cell lines, but Na+-dependent phosphate transport was inhibited exclusively in the OK cells (a threefold reduction of influx after 4 h of exposure to 10(-10) M PTH). The change in phosphate transport is accounted for by a lowered Vmax (30.8 +/- 5.3 vs. 10.2 +/- 1.1 pmol X mg-1 X 3 min-1). The reduction in phosphate transport was reversible, such that 5 h after removal of PTH the Vmax had increased threefold over the inhibited state. Addition of PTH did not alter Na+-dependent L-alanine influx in the OK cells. Experiments with apical membrane vesicles showed that the change in Vmax occurred at the membrane level. It is concluded that the regulatory event responsible for PTH-reduced phosphate transport is beyond cAMP. Of the cell lines studied, only OK cells have a complete regulatory cascade.

A novel translation, trans-translation, is facilitated by a highly structured RNA molecule, tmRNA. This molecule has two structural domains, a tRNA domain and an mRNA domain, the latter including four pseudoknot structures (PK1 to PK4). Here, we show that replacement of each of these pseudoknots, except PK1, in Escherichia coli tmRNA with a single stranded RNA did not seriously affect the functions as an alanine tRNA and as an mRNA. Furthermore, these three pseudoknots were interchangeable with only small losses of the two functions. These findings suggest that neither PK2, PK3 nor PK4 interacts in a functional manner with ribosome during the trans-translation process. Together with an earlier study showing the significance of PK1, it is concluded that among the four pseudoknots, PK1 is the most functional.

Toxoplasma gondii genetic diversity varies in different geographical regions. In South America, T. gondii isolates are highly diverse, whereas in North America and Europe, the parasite is highly clonal, with 3 distinct lineage types (I, II, III). However, little is known of the T. gondii genotypes in the People's Republic of China. Because pork is considered the principal meat source for T. gondii infection in China, we conducted a survey to determine the prevalence and genetic diversity of this parasite in pigs from central China. In total, 434 DNA samples were extracted from the hilar lymph nodes of sick pigs in Hubei and Henan provinces in central China, and 34 were T. gondii B1 gene-positive. These T. gondii -positive DNA samples were typed at 10 genetic markers, including 9 nuclear loci, i.e., SAG1, SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico. Of these, 16 isolates could be genotyped with complete data for all loci. Two genotypes were present; one was the clonal type I lineage and the other was previously identified as a widespread lineage from many hosts in China. These results indicate that these 2 genotypes may be the major lineages in China. This is the first report of genetic typing of T. gondii isolates from pigs in central China. The results have implications for the prevention and control of T. gondii infections in humans and other animals.

Sodium- and chloride-coupled transport of dopamine from synapses into presynaptic terminals plays a key role in terminating dopaminergic neurotransmission. Regulation of the function of the dopamine transporter, the molecule responsible for this translocation, is thus of interest. The primary sequence of the dopamine transporter contains multiple potential phosphorylation sites, suggesting that the function of the transporter could be regulated by phosphorylation. Previous work from this laboratory has documented that phorbol ester activation of protein kinase C (PKC) decreases dopamine transport Vmax in transiently expressing COS cells. In the present report, we document in vivo phosphorylation of the rat dopamine transporter stably expressed in LLC-PK1, cells and show that phosphorylation is increased threefold by phorbol esters. Dopamine uptake is also regulated by phorbol esters in these cells; phorbol 12-myristate 13-acetate (PMA) reduces transport Vmax by 35%. Parallels between the time course, concentration dependency, and staurosporine sensitivity of alterations in transporter phosphorylation and transporter Vmax suggest that dopamine transporter phosphorylation involving PKC could contribute to this decreased transporter function. Phosphorylation of the dopamine transporter by PKC or by a PKC-activated kinase could be involved in rapid neuroadaptive processes in dopaminergic neurons.

Mutation of the Caenorhabditis elegans gene unc-89 results in disorganization of muscle A-bands. unc-89 encodes a giant polypeptide (900 kDa) containing two protein kinase domains, PK1 and PK2. Yeast two-hybrid screening using a portion of UNC-89 including PK2, yielded SCPL-1 (small CTD phosphatase-like-1), which contains a C terminal domain (CTD) phosphatase type domain. In addition to the PK2 domain, interaction with SCPL-1 required the putative autoinhibitory sequence, and immunoglobulin (Ig) and fibronectin type 3 (Fn3) domains lying N-terminal of the kinase domain. SCPL-1 also interacts with PK1, and it similarly requires the kinase domain and upstream Fn3 and Ig domains. Analogous regions from the two other giant kinases of C. elegans, twitchin and TTN-1, failed to interact with SCPL-1. The interaction between SCPL-1 and either Ig-Fn3-PK2 or Fn3-Ig-PK1 was confirmed by biochemical methods. The scpl-1b promoter is expressed in the same set of muscles as unc-89. Antibodies to SCPL-1 localize to the M-line and a portion of the I-band. Bacterially expressed SCPL-1 proteins have phosphatase activity in vitro with properties similar to previously characterized members of the CTD phosphatase family. RNA interference knockdown results in a defect in the function of egg-laying muscles. These studies suggest a new role for the CTD phosphatase family, that is, in muscle giant kinase signaling.

The epithelial canine and porcine kidney cell lines MDCK, MDCKII and LLC-PK1, respectively are employed to establish recombinant models of drug transport. Endogenous drug carriers in these cells may contribute to the activities of recombinant drug transporters, thus making it difficult to assess their properties. We analysed the expression of endogenous transporters in these cell lines by RT-PCR and by determining drug transporter activities. Concerning drug efflux, multidrug resistance protein 1 (MDR1) and MRP1 mRNAs were found in all lines. MRP2 mRNA was expressed in all cell lines except MDCK. Transepithelial transport of vinblastine and its modulation by a MDR1-specific inhibitor or by the MDR1- and MRP-inhibitor verapamil, indicated that MDCKII cells have, in comparisons to the other cell lines, relatively high levels of functional MDR1 while vinblastine transport in MDCK cells is likely to be mediated more by MRP1. Notably, LLC-PK1 cells displayed little activity attributable to either MDR1 and MRP1, thus making them suitable for the expression of these efflux pumps. Of the drug uptake carriers, OATP-A mRNA was only expressed in MDCK cells. OATP-C mRNA was barely detectable in MDCK cells and absent in MDCKII and LLC-PK1 cells. In agreement with transcriptional profiling, the OATP-mediated uptake of either estradiol-glucuronide or estrone-sulfate was either absent or barely detectable in all cell lines thus implying that they are suitable to establish recombinant models for human OATP's. Transcriptional profiling was also performed on porcine and canine tissues and revealed that MRP1 was expressed in canine but not in human or porcine liver, whereas surprisingly OATP-C was expressed in canine kidney but only in human and porcine liver. The findings presented are relevant to the use of porcine and canine models for drug disposition.

Aminoglycoside antibiotics are known to be internalized via endocytosis and have been associated with subcellular organelle dysfunction; however, the route of intracellular trafficking and their distribution remain largely unknown. To address these questions, a Texas Red conjugate of gentamicin (TRG) was synthesized for dual-labeling experiments with the endoplasmic reticulum, Golgi, and lysosomal markers DiOC6-3, C6-NBD-ceramide, and fluorescent dextrans, respectively. Confocal images were overlaid to determine areas of colocalization. Initial characterization studies of the fluorescent gentamicin analogue revealed that both internalization and accumulation were inhibited by excess unlabeled gentamicin. Furthermore, the fluorescent gentamicin label was colocalized with unlabeled gentamicin, using immunologic techniques. LLC-PK1 cells were exposed to the fluorescent gentamicin in media containing 1 mg/ml labeled gentamicin for 8 h and then either fixed or chased with gentamicin-free media for an additional 16 or 40 h (24 to 48 h total). Studies with fluorescent dextrans revealed rapid intracellular colocalization within the endosomal and lysosomal systems. Neither endoplasmic reticulum nor mitochondrial colocalization could be detected. However, Golgi colocalization was revealed using both confocal and electron microscopic techniques at 8 h of TRG incubation, and continued to be present for an additional 40 h. Protein synthetic rates were quantified and revealed decreased synthesis at the 24-h chase mark. These results suggest that TRG can serve as a fluorescent tracer for aminoglycoside trafficking within cells. The fluorescent marker remained associated with vesicular structures at all times and colocalized with the Golgi apparatus. It is postulated that this early association of gentamicin with the Golgi complex may be an avenue for delivery of aminoglycosides to other intracellular compartments.

Mutations in the gene encoding polycystin-2 (PC2) result in autosomal dominant polycystic kidney disease and defects in left-right asymmetry during embryogenesis. PC2 is a TRP-type Ca(2+)-permeable non-selective cation channel, which is expressed in kidney and other organs. PC2 is present and functional in microtubule-containing primary cilia of renal epithelial cells. However, no information is yet available as to whether PC2 interacts with microtubules. Here, we assessed the role of microtubular dynamics in regulating PC2 channel function in primary cilia. Isolated ciliary membranes from LLC-PK1 epithelial cells were reconstituted in a lipid bilayer system. The acute addition of the microtubular disrupter colchicine (15 mum) rapidly abolished, whereas the addition of the microtubular stabilizer paclitaxel (taxol, 15 mum) increased ciliary PC2 channel activity. The further addition of alpha-tubulin plus GTP also stimulated PC2 channel activity in ciliary membranes. However, alpha-tubulin and GTP had no effect on in vitro translated PC2. Using the yeast two-hybrid assay, we found that PC2 interacts with the microtubule-dependent motor kinesin-2 subunit KIF3A, a protein involved in polycystic kidney disease. The interaction occurred through the carboxyl termini domain of both proteins, which was further confirmed by in vitro glutathione S-transferase pull-down and dot blot overlay assays. Co-immunoprecipitation experiments showed that PC2 and KIF3A are in the same complex in native HEK293, Madin-Darby canine kidney cells (MDCK), and LLC-PK1 cells. Immunofluorescent staining also showed substantial PC2 and KIF3A co-localization in primary cilia of renal epithelial cells. The data indicate that microtubular organization regulates PC2 function, which may explain, among others, the regulatory role of PC2 in the sensory function of primary cilia.

With countless research papers using preclinical models and showing the superiority of nanoparticle design over current drug therapies used to treat cancers, it is surprising how deficient the translation of these nano-sized drug carriers into the clinical setting is. This review article seeks to compare the preclinical and clinical results for Doxil®, PK1, Abraxane®, Genexol-PM®, Xyotax™, NC-6004, Mylotarg®, PK2, and CALAA-01. While not comprehensive, it covers nano-sized drug carriers designed to improve the efficacy of common drugs used in chemotherapy. While not always available or comparable, effort was made to compare the pharmacokinetics, toxicity, and efficacy between the animal and human studies. Discussion is provided to suggest what might be causing the gap. Finally, suggestions and encouragement are dispensed for the potential that nano-sized drug carriers hold.

We established stable human canalicular multispecific organic anion transporter (cMOAT/MRP2) cDNA transfectants, CHO/cMOAT from non-polarized Chinese hamster ovary (CHO)-K1 and LLC/cMOAT from polarized pig kidney epithelial LLC-PK1. Human cMOAT was mainly localized in the plasma membrane of CHO/cMOAT and in the apical membrane of LLC/cMOAT. The ATP-dependent uptake of leukotriene C4 (LTC4) into CHO/cMOAT membrane vesicles was enhanced compared with empty vector transfectants. Km values in CHO/cMOAT membrane vesicles were 0.24 microM for LTC4 and 175 microM for ATP. Drug sensitivity to vincristine and cisplatin in human cMOAT cDNA transfectants decreased, but not to etoposide. Cellular accumulation of vincristine and cisplatin in human cMOAT cDNA transfectants decreased, but not of etoposide. The uptake of LTC4 into CHO/cMOAT membrane vesicles was inhibited by exogenous administration of vincristine or cisplatin, but not that of etoposide. Moreover, this inhibition was more enhanced in the presence of glutathione. These consequences indicate that drug resistance to vincristine or cisplatin appears to be modulated by human cMOAT through transport of the agents, possibly in direct or indirect association with glutathione.

Fourteen isolates of Toxoplasma gondii were isolated from cats from 4 different geographic provinces (Anhui, Hubei, Shanxi and Guangdong) in China and their genetic diversity with 8 nuclear loci SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico, was analysed by restriction fragment length polymorphisms (RFLPs). Two genotypes from these 14 isolates were identified but none of them belongs to the typical genetic types (types I, II and III). It is unexpected that such high similarity was observed in these 14 isolates although their original regions are significantly distant. Our results strongly indicate that the three traditional clonal lineages of types I, II and III of this parasite may not be preponderant in China. In addition, our results show that the genotypes of T. gondii in China may be highly clonal with atypical genotypes and higher virulence.

Many clinically important drug interactions occur due to inhibition of human liver cytochrome P450 3A (CYP3A) metabolism. The drug efflux pump P-glycoprotein (Pgp) can be an additional locus contributing to these drug interactions because there is overlap in drugs that are substrates for both proteins. We screened a number of CYP3A inhibitors (macrolide antibiotics, azole antifungals, and ergotpeptides) for their ability to interact with Pgp, compared with prototypical Pgp inhibitors. We used cell lines expressing human, mouse, and rat mdr1 genes. Pgp antagonism was defined by interactions of the drugs with four cell lines (LLC-PK1, L-MDR1, L-mdr1a, and L-mdr1b) using a microfluorometric calcein-AM assay and characterized for their inhibitor constant (K(i)) toward calcein-AM. The compounds were further defined for their ability to inhibit MDR1 by their effect on vinblastine accumulation into L-MDR1 cells. Representative compounds from each class of drugs were further tested as Pgp substrates, defined by the ability of human Pgp or mouse mdr1a/Pgp to transport them across a polarized kidney epithelial cell in vitro. These same compounds were administered radiolabeled in vivo to mdr1a (+/+) and (-/-) mice and the distribution of radioactivity compared. The results are summarized as follows: 1) Some drug interactions with Pgp were substrate- and/or assay-dependent. 2) Ergot alkaloids were identified as a class of MDR1/Pgp chemosensitizers. 3) The Ergot alkaloids revealed species differences in the structure-activity relationships for inhibition of Pgp. Simultaneous inhibition of Pgp by many CYP3A inhibitors contributes to human variation in the extent of drug-drug interactions.

In renal medullas during antidiuresis, the extracellular fluid is hyperosmotic because of high concentrations of NaCl and urea. Under those conditions, the cells contain high concentrations of organic osmolytes, namely sorbitol, myo-inositol, glycerophosphorylcholine (GPC), and betaine to balance the extracellular hyperosmolality. These organic osmolytes increase cell osmolality without perturbing the intracellular milieu in ways that would degrade the function of cellular macromolecules. The present study surveyed a number of cell lines for the ability to survive in media with high concentrations of NaCl and/or urea and for the accumulation of organic osmolytes. Of the renal cell lines tested, MDCK, GRB-MAL1, and A6 cells proliferated in hyperosmotic media, but medullary interstitial cells LLC-PK1 and LLC-PK3 did not proliferate, nor did nonrenal HTC-BH cells, MDCK, LLC-PK1, and LLC-PK3 cells contained higher concentrations of myo-inositol, GPC, and betaine when cultured in media containing high NaCl (with or without high urea) and much lower or undetectable levels of these osmolytes when grown in isosmotic media. Sorbitol, and to a lesser extent myo-inositol, were elevated in GRB-MAL1 cells in media hyperosmotic with NaCl but not in isosmotic media. There was less accumulation of organic osmolytes when only urea was added to increase osmolality. Thus the same osmolytes were accumulated by one or another cell line in vitro as were previously found in renal medullas. These cell lines provide models for studying osmolyte accumulation.

Fimbrins/plastins are a family of highly conserved actin-bundling proteins. They are present in all eukaryotic cells including yeast, but each isoform displays a remarkable tissue specificity. T-plastin is normally found in epithelial and mesenchymal cells while L-plastin is present in hematopoietic cells. However, L-plastin has been also found in tumor cells of non-hematopoietic origin (Lin, C.-S., R. H. Aebersold, S. B. Kent, M. Varma, and J. Leavitt. 1988. Mol. Cell. Biol. 8:4659-4668; Lin, C.-S., R. H. Aebersold, and J. Leavitt. 1990. Mol. Cell. Biol. 10: 1818-1821). To learn more about the biological significance of their tissue specificity, we have overproduced the T- and L-plastin isoforms in a fibroblast-like cell line, CV-1, and in a polarized epithelial cell line, LLC-PK1. In CV-1 cells, overproduction of T- and L-plastins induces cell rounding and a concomitant reorganization of actin stress fibers into geodesic structures. L-plastin remains associated with microfilaments while T-plastin is almost completely extracted after treatment of the cells with non-ionic detergent. In LLC-PK1 cells, T-plastin induces shape changes in microvilli and remains associated with microvillar actin filaments after detergent extraction while L-plastin has no effect on these structures and is completely extracted. The effect of T-plastin on the organization of microvilli differs from that of villin, another actin-bundling protein. Our experiments indicate that these two isoforms play differing roles in actin filament organization, and do so in a cell type-specific fashion. Thus it is likely that these plastin isoforms play fundamentally different roles in cell function.

Renal secretion of organic cations involves at least two distinct transporters, located in the basolateral and apical membranes of proximal tubule cells. Whereas the basolateral transporter has recently been cloned, sequence information about the apical type was not yet available. An organic cation transporter, OCT2p, was cloned from LLC-PK1 cells, a porcine cell line with properties of proximal tubular epithelial cells. OCT2p was heterologously expressed and characterized in human embryonic kidney 293 cells. OCT2p-mediated uptake of the prototypical organic cation [14C]tetraethylammonium ([14C]TEA) into 293 cells was saturable. There was a highly significant correlation between the Ki values for the inhibition of apical [14C]TEA uptake into LLC-PK1 cells and 293 cells transfected with OCT2p (r = 0.995; p < 0.001; n = 6). Although OCT2p is structurally related to OCT1r, the basolateral organic cation transporter from rat kidney, the transporters could be clearly discriminated pharmacologically with corticosterone, decynium22, and O-methylisoprenaline. The findings at hand suggest that OCT2 corresponds to the apical type of organic cation transporter. Reverse transcriptase-polymerase chain reaction indicates that mRNA of OCT1r is limited to non-neuronal tissue, whereas OCT2r, the OCT2p homologue from rat, was found in both the kidney and central nervous regions known to be rich in the monoamine transmitter dopamine.

Tyrosine-dependent sequence motifs are implicated in sorting membrane proteins to the basolateral domain of Madin-Darby canine kidney (MDCK) cells. We find that these motifs are interpreted differentially in various polarized epithelial cell types. The H, K-ATPase beta subunit, which contains a tyrosine-based motif in its cytoplasmic tail, was expressed in MDCK and LLC-PK1 cells. This protein was restricted to the basolateral membrane in MDCK cells, but was localized to the apical membrane in LLC-PK1 cells. Similarly, HA-Y543, a construct in which a tyrosine-based motif was introduced into the cytoplasmic tail of influenza hemagglutinin, was sorted to the basolateral membrane of MDCK cells and retained at the apical membrane of LLC-PK1 cells. A chimera in which the cytoplasmic tail of the H,K-ATPase beta subunit protein was replaced with the analogous region of the Na,K-ATPase beta subunit polypeptide was localized to both surface domains of MDCK cells. Mutation of tyrosine-20 of the H,K-ATPase beta subunit cytoplasmic sequence to an alanine was sufficient to disrupt basolateral localization of this polypeptide. In contrast, these constructs all remain localized to the apical membrane in LLC-PK1 cells. The FcRII-B2 protein bears a di-leucine motif and is found at the basolateral membrane of both MDCK and LLC-PK1 cells. These results demonstrate that polarized epithelia are able to discriminate between different classes of specifically defined membrane protein sorting signals.

The long-term effects of ouabain on transepithelial Na(+) transport involve transcriptional downregulation of apical Na(+)/H(+) exchanger isoform 3 (NHE3). The aim of this study was to determine whether ouabain could acutely regulate NHE3 via a posttranscriptional mechanism in LLC-PK1 cells. We observed that the basolateral, but not apical, application of ouabain for 1 h significantly reduced transepithelial Na(+) transport. This effect was not due to changes in the integrity of tight junctions or increases in the intracellular Na(+) concentration. Ouabain regulated the trafficking of NHE3 and subsequently inhibited its activity, a process independent of intracellular Na(+) concentration. Ouabain-induced NHE3 trafficking was abolished by either cholesterol depletion or Src inhibition. Moreover, ouabain increased the intracellular Ca(2+) concentration. Pretreatment of cells with the intracellular Ca(2+) chelator BAPTA-AM blocked ouabain-induced trafficking of NHE3. Also, blockade of Na(+)-K(+)-ATPase endocytosis by a phosphatidylinositol 3-kinase inhibitor was equally effective in attenuating ouabain-induced NHE3 trafficking. These data indicate that ouabain acutely stimulates NHE3 trafficking by activating the basolateral Na(+)-K(+)-ATPase signaling complex. Taken together with our previous observations, we propose that ouabain can simultaneously regulate basolateral Na(+)-K(+)-ATPase and apical NHE3, leading to inhibition of transepithelial Na(+) transport. This mechanism may be relevant to proximal tubular Na(+) handling during conditions associated with increases in circulating endogenous cardiotonic steroids.