Hypoxia-inducible factor-1 (HIF-1) is a regulator of metabolic adaptation to hypoxia. It is now appreciated that HIF-1alpha accumulation is achieved under normoxic conditions by various factors, such as TNF-alpha. Here, it was our intention to gain insight into the signaling mechanisms used by TNF-alpha to stimulate HIF-1alpha. In tubular LLC-PK1 or human embryonic kidney cells, TNF-alpha induced accumulation of HIF-1alpha protein but not HIF-1alpha mRNA. Blocking nuclear factor (NF)-kappaB with sulfasalazine or expression of an IkappaB superrepressor attenuated HIF-1alpha accumulation, whereas transfection of active p50/p65-NF-kappaB subunits mimicked a TNF-alpha response. Experiments with actinomycin D and cycloheximide also pointed to a transcriptional and translational process in facilitating the TNF-alpha response. Interestingly, and in contrast to established hypoxic signaling concepts, TNF-alpha elicited HIF-1alpha accumulation in a ubiquitinated form that still bound the von Hippel-Lindau (pVHL) protein. These data indicate that HIF-1alpha accumulation by TNF-alpha demands the NF-kappaB pathway, preserves ubiquitination of HIF-1alpha, and allows the HIF-1alpha-pVHL interaction.

CD147, a type I integral membrane protein of the immunoglobulin superfamily, exhibits reversed polarity in retinal pigment epithelium (RPE). CD147 is apical in RPE in contrast to its basolateral localization in extraocular epithelia. This elicited our interest in understanding the basolateral sorting signals of CD147 in prototypic Madin-Darby canine kidney (MDCK) cells. The cytoplasmic domain of CD147 has basolateral sorting information but is devoid of well-characterized basolateral signals, such as tyrosine and di-leucine motifs. Hence, we carried out systematic site-directed mutagenesis to delineate basolateral targeting information in CD147. Our detailed analysis identified a single leucine (252) as the basolateral targeting motif in the cytoplasmic tail of CD147. Four amino acids (243-246) N-terminal to leucine 252 are also critical basolateral determinants of CD147, because deletion of these amino acids leads to mistargeting of CD147 to the apical membranes. We ruled out the involvement of adaptor complex 1B (AP1B) in the basolateral trafficking of CD147, because LLC-PK1 cells lacking AP1B, target CD147 basolaterally. At variance with MDCK cells, the human RPE cell line ARPE-19 does not distinguish between CD147 (WT) and CD147 with leucine 252 mutated to alanine and targets both proteins apically. Thus, our study identifies an atypical basolateral motif of CD147, which comprises a single leucine and is not recognized by RPE cells. This unusual basolateral sorting signal will be useful in unraveling the specialized sorting machinery of RPE cells.

Genomic clones including the 5' flanking regions of the AQP2 (aquaporin 2) gene were isolated, and the promoter region was examined by transiently transfecting a promoter-luciferase reporter fusion gene into renal cultured epithelial cells. An orientation specific promoter for the AQP2 gene was found within the proximal 3 kb of 5'-flanking region. Minimal basal promoter activity of the AQP2 gene was found within 198 bp upstream from the transcription start site by deletion analysis. Sequencing the transcriptionally active region revealed a typical TATA box, adenosine 3',5'-cyclic monophosphate (cAMP) responsive element (CRE) and three putative CCAAT boxes in the proximal 1.2-kb region. Significantly, a GATA motif, AP1, AP2, and SP1 transcriptional factor consensus sites were also found in this region. Exposure to cAMP-enhancing agents (1 nM vasopressin or 20 mM forskolin and 250 mM 3-isobutyl-1-methylxanthine) showed that these agents increased luciferase activity in a parallel fashion, suggesting that vasopressin-induced AQP2 gene transcription is mediated through increases in intracellular cAMP in at least one renal cell type, the LLC-PK1 cells. The mechanism of cAMP responsiveness of AQP2 gene transcription was further studied using a series of deletion mutants in renal epithelial cells and other cell types. The cAMP regulatory motifs were shown to exist in a 50-bp sequence between -340 and -290 (containing CRE) and a 65-bp sequence (containing an AP2 site) between -150 and the ATG start site in LLC-PK1 cells. In rat inner medullary collecting duct (IMCD) cells, the cAMP regulatory motifs also exist in a 50-bp sequence between -340 and -290 (containing CRE) and in a 10-bp sequence between -160 and -150 (containing an SP1 site). These separate regions may cooperate to confer full cAMP inducibility to the AQP2 gene in a cell-specific manner.

The antiepileptic drug valproic acid (VPA) is widely used in the treatment of epilepsy, bipolar disorders, and migraine. However, rather high doses are required for the clinical effects of VPA, which is due to its relatively inefficient delivery to the brain. The poor brain distribution of VPA is thought to reflect an asymmetric transport system at the blood-brain barrier (BBB). Based on recent data from in vitro experiments, multidrug resistance proteins (MRPs) have been proposed to be involved in the efflux transport of VPA at the BBB. In the present study, we used different experimental in vitro and in vivo strategies to evaluate whether VPA is a substrate for MRPs or the efflux transporter P-glycoprotein (Pgp). In contrast to known Pgp or MRP substrates, such as cyclosporin A or vinblastine, no directional transport of VPA was observed in cell monolayer efflux assays using the kidney cell lines Madin Darby canine kidney II and LLC-PK1, which had been transfected with either human or mouse cDNAs for the genes encoding Pgp, MRP1, or MRP2. Likewise, no indication for efflux transport of VPA was obtained in a rat microdialysis model, using inhibitors of either Pgp or MRPs. Furthermore, a significant role of MRP2 in brain efflux of VPA was excluded by using MRP2-deficient rats. Our data do not support the hypothesis that MRP1 or MRP2 is involved in the efflux of VPA from the brain. Thus, the molecular identity of the putative transporter(s) mediating the active efflux of VPA from the brain remains to be elucidated.

BACKGROUND

We have reported that digitalis-like substances (cardiotonic steroids), including marinobufagenin (MBG), induce endocytosis of the plasmalemmal Na/K-ATPase in LLC-PK1 cells. The current report addresses the potential relevance of plasmalemmal Na/K-ATPase redistribution to in vivo salt handling.

METHODS

Male Sprague-Dawley rats were given 1 week of a high salt (4.0% NaCl) or normal salt (0.4% NaCl) diet. Urinary sodium excretion, as well as MBG excretion, was monitored, and proximal tubules were isolated using a Percoll gradient method. Tubular (86)Rb uptake, Na/K-ATPase enzymatic activity, and Na/K-ATPase alpha1 subunit density were determined.

RESULTS

The high salt diet increased urinary sodium (17.8 +/- 1.8 vs. 2.5 +/- 0.3 mEq/day, P < 0.01) and MBG excretion (104 +/- 12 vs. 26 +/- 4 pmol/day), and decreased proximal tubular (86)Rb uptake (0.44 +/- 0.07 vs. 1.00 +/- 0.10, P < 0.01) and Na/K-ATPase enzymatic activity (5.1 +/- 1.1 vs. 9.9 +/- 1.6 micromol/mg pr/hr, P < 0.01) relative to the normal diet. Proximal tubular Na/K-ATPase alpha1 protein density was decreased in the plasmalemma fraction but increased in both early and late endosomes following the high salt diet. In rats fed a high salt diet, anti-MBG antibody caused a 60% reduction in urinary sodium excretion, substantial increases in proximal tubule (86)Rb uptake, and Na/K-ATPase enzymatic activity, as well as significant decreases in the early and late endosomal Na/K-ATPase alpha1 protein content.

CONCLUSIONS

These data suggest that redistribution of the proximal tubule Na/K-ATPase in response to endogenous cardiotonic steroids plays an important role in renal adaptation to salt loading.

BACKGROUND

Angiotensin II is strongly incriminated in progressive renal injury. There is recent evidence that angiotensin II induces oxidative stress in vitro. We examined the capacity of angiotensin II to induce oxidative stress in vivo and the functional significance of such stress. The capacity of angiotensin II to induce the oxidant-sensitive gene heme oxygenase (HO) in vivo and in vitro was also examined.

METHODS

Angiotensin II was administered via mini-osmotic pumps to rats maintained on standard diets. Indices of oxidative stress, including thiobarbituric acid reactive substance, carbonyl protein content, and HO activity, were determined. Indices of oxidative stress and functional markers were also determined in the DOCA salt model. The effect of angiotensin II was studied in rats maintained on antioxidant-deficient diets so as to examine the functional significance of oxidative stress induced by angiotensin II. We also explored the inductive effect of angiotensin II on HO in vivo and whether such actions occur in vitro.

RESULTS

Angiotensin II administered in vivo increased kidney content of thiobarbituric acid reactive substances protein carbonyl content, and HO activity. These indices were not present in the kidney of rats treated with DOCA salt for three weeks. Such oxidative stress was functionally significant, since the administration of angiotensin II to rats maintained on a prooxidant diet demonstrated increased proteinuria and decreased creatinine clearance. The stimulatory effect on HO activity was due to induction of HO-1 mRNA, with HO-2 mRNA remaining unchanged. Expression of HO-1 was localized to the renal proximal tubules in vivo. We also demonstrate that angiotensin II at concentrations of 10-8 and 10-7 mol/L induces expression of HO-1 mRNA in LLC-PK1 cells.

CONCLUSIONS

Angiotensin II induces oxidative stress in vivo, which contributes to renal injury. This study also demonstrates that angiotensin II induces renal HO activity caused by up-regulation of HO-1 in renal proximal tubules. Finally, angiotensin II directly induces HO-1 in renal proximal tubular epithelial cells in vitro.

The renal epithelial cell line LLC-PK1 has topographically distinct regulatory roles for the alpha subunits of pertussis toxin-sensitive guanine nucleotide regulatory proteins (alpha i subunit); these include the inhibition of adenylyl cyclase at the basolateral membrane and the stimulation of Na+ channel activity at the apical membrane. We now report that LLC-PK1 cells contain two members of the alpha i protein family, alpha i-2 and alpha i-3, which have distinct cellular locations consistent with their diverse functional roles. By using specific alpha i antibodies and immunofluorescence, the alpha i-2 subunit was found to be localized to the basolateral membrane, whereas the alpha i-3 subunit was concentrated in the Golgi and was also detectable at low levels on apical membranes in some cells. Induction of a chimeric mouse metallothionein 1-rat or canine alpha i-2 gene stably transfected into the LLC-PK1 cells produced an increase in the content of the alpha i-2 subunit, which was targeted only to the basolateral membrane. These findings suggest that alpha i subunit specificity for effectors may be achieved in polarized renal epithelial cells by their geographic segregation to different cellular membranes. The LLC-PK1 cell stably transfected with the metallothionein-alpha i-2 fusion gene will provide a model for the study of guanine nucleotide regulatory protein function in epithelia.

LLC-PK1 cells have been stably transfected with cDNAs encoding the human norepinephrine transporter (NET), rat dopamine transporter (DAT), and rat serotonin transporter. Using these cell lines, the specificity of each transporter toward agents that inhibit substrate influx and stimulate substrate efflux across the plasma membrane was examined. With 1-methyl-4-phenylpyridinium as a substrate for DAT and NET and serotonin as a substrate for the serotonin transporter, each transporter demonstrated a distinct pattern of inhibition by a panel of amphetamine derivatives and analogs, including amphetamine, methamphetamine (also known as "ecstasy"), p-chloroamphetamine, 3,4-methylenedioxymethamphetamine, methylphenidate (ritalin), and 5-methoxy-6-methyl-2-aminoindan. For each cell line expressing a single biogenic amine transporter, efflux of the accumulated substrate was stimulated by amphetamine derivatives, and this efflux was blocked by mazindol, an inhibitor of all three transporters. Of the amphetamine derivatives tested, some caused efflux at concentrations similar to those that inhibited transport. Other derivatives were much less effective at stimulating efflux than at inhibiting uptake. Methylphenidate caused little or no efflux, although it blocked uptake mediated by both NET and DAT. Other inhibitors of transport, such as cocaine, mazindol, citalopram, and nisoxetine, failed to stimulate efflux from these cells at concentrations that inhibited influx. The results suggest that potency toward individual plasma membrane biogenic amine transporters and the ability to release accumulated amine substrates are independent properties of each amphetamine derivative.

The current study was based on the uncertainty as to how well monolayer cell cultures growing in customary polystyrene dishes are supplied with O2. For dishes maintained in an air-5% CO2 atmosphere at 37 degrees C, microelectrode measurements revealed that the pericellular steady-state PO2 was 78 mm Hg in confluent bovine endothelial, 110 mm Hg in rat renal mesangial, and 0 (< 0.2) mm Hg in renal (LLC-PK1 and LLC-MK2) or hepatic (HepG2, Hep3B) epithelial cell cultures. These measured PO2 values were in good agreement with those calculated from Fick's law of gas diffusion, applied for the present culture conditions (one-dimensional O2 diffusion, 0.52 cm medium height), the individual cell layer density and the tissue-specific rate of O2 utilization. Our results provide reasons to speculate that conventional monolayer cultures are often hypoxic when incubated in an air-5% CO2 atmosphere. Diffusion-limitations of cellular O2 availability are to be taken into consideration when tissue cultures are used to study PO2-dependent processes.

We examined two potential intracellular targets in the glycerol model of acute renal failure, namely, the mitochondrion and the nucleus. Within three hours, alterations in mitochondrial function are already apparent. With either glutamate/malate or succinate/rotenone, state 3 and uncoupled respirations were decreased at three hours, and at 24 hours, such decrements were quite pronounced; in the presence of glutamate/malate, state 2 respiration was also depressed at 24 hours, while with succinate/rotenone state 2 was increased. Marked ultrastructural changes were observed in mitochondria studied at three hours, including the novel finding of degenerate mitochondria in autophagic vacuoles. Since the heme content in mitochondria was increased some tenfold within three hours, mitochondrial function was studied after exposure to concentrations of heme that reproduced such contents of heme: mitochondria initially displayed increased respiration, and subsequently, a persistent decline in oxygen consumption until oxygen consumption was virtually undetectable. With higher concentrations of heme, the early increase in oxygen consumption was blunted and the progressive decline in oxygen consumption was hastened. The antioxidant iron chelator, deferoxamine, prevented the early rise in oxygen consumption but did not prevent or delay the subsequent decline. We also assessed nuclear damage as a potential lesion in the glycerol model. DNA laddering was not observed at any time point. At 3 and 24 hours there was DNA injury by the TUNEL technique in the distal nephron but not in the proximal nephron. The 8-hydroxydeoxyguanosine/deoxyguanosine content was increased in the glycerol kidneys at 24 hours but not at three hours. At neither time point was evidence of apoptosis observed by light or electron microscopy. In studies undertaken in cell culture models, heme, at concentrations of 10 microM, failed to evince any such changes in LLC-PK1 cells, a cell line from the proximal tubule, or in MDCK cells, a cell line derived from the distal tubule. At concentrations of 50 microM, heme induced approximately 20% positivity in MDCK cells but none in LLC-PK1 cells by the TUNEL technique. We conclude that mitochondria and nuclei are prominent targets for injury in the glycerol model of acute renal failure. The presence of TUNEL-positive cells in the distal nephron but not at proximal sites in vivo underscores the increasing appreciation of the distinct responses of these nephron sites to nephrotoxic insults.

The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV40. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and C11 cells (CV-1 cell lines transformed with UV-irradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC11. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys----Arg at amino acid 516. pC11 has two mutations, one predicting a change of Pro----Ser at residue 522, and another predicting a Pro----Arg change at amino acid 549. The two C11 mutations were separated from each other to form two distinct viral genomes in pC11A and pC11B. pC2, pC11, pC11A, and pC11B are able to transform both primary and established rodent cell cultures. The C11 and C11A T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen.

Moesin, a member of the talin-4.1 superfamily, is a linking protein of the submembraneous cytoskeleton. It is expressed in variable amounts in cells of different phenotypes such as macrophages, lymphocytes, fibroblastic, endothelial, epithelial, and neuronal cell lines. In this report we show that moesin is not randomly distributed throughout the cortical cytoskeleton, but rather that it is concentrated in specialized microdomains. It is localized in the intracellular core of microextensions known as filopodia, microvilli, microspikes, and retraction fibers. This subcellular distribution follows closely the dynamic changes in cell shape that take place when cells attach, spread, and move spontaneously or in response to extracellular signals. This suggests a similar function for moesin in diverse cell types related to the dynamic restructuring of domains of the plasma membrane and underlying membrane skeleton. Support for this comes from studies on PC-12 cells, which respond to NGF by extending neurites and moesin is redistributed from a diffuse localization to growth cone filopodia. In fibroblastic (NIH3T3) or macrophage (RAW264.7) cell lines, moesin is found in filopodia appearing at random on the cell surface soon after the cells are placed in culture, begin to attach, and spread. In polarized epithelial cells (LLC-PK1), moesin is associated with peripheral filopodia and apical microvilli. The cellular microextensions containing moesin are devoid of microtubules, focal contact proteins such as vinculin, and cortical cytoskeletal elements such as protein 4.1, but they do contain varying amounts of actin microfilaments. This localization of moesin in microextensions is not influenced by cytochalasin B. Treatment of cells with phorbolester (PMA) causes rapid cell spreading, disappearance of filopodia and retraction fibers, and moesin does not accumulate in the actin-rich lamellae that form at the cellular edges. After removal of PMA, cells retract and moesin again becomes concentrated in filopodia and retraction fibers. These studies support the hypothesis that filopodia, retraction fibers, and other microextensions of the plasma membrane are unique cellular microdomains with characteristic submembraneous components. Moesin could be involved in the dynamic restructuring of such microdomains by regulating binding interactions between the plasma membrane and the actin cytoskeleton.

Data on the synthesis, physicochemical characterisation and in vitro and in vivo biological properties of the new, nontargeted or antibody-targeted polymer-doxorubicin conjugates designed as anticancer drugs are presented. In the conjugates, the anticancer drug doxorubicin (DOX) is attached to the polymer carrier via a simple hydrolytically labile spacer containing either a hydrazone bond or cis-aconitic acid residue. In vitro incubation of the conjugates in buffers led to a fast DOX release from the polymer at pH 5 (modelling intracellular environment) while at pH 7.4 (modelling blood) the conjugates are relatively stable. Cytotoxicity of the conjugates to T cell lymphoma EL4 depended on the detailed structure of the spacer and the method used for antibody attachment and was much higher compared with the effect of similar classic conjugates (DOX attached to the polymer via enzymatically degradable spacer). In both protective and therapeutic regimes of drug administration, the in vivo anti-tumor activity of the hydrazone conjugates containing only DOX was significantly enhanced (T cell lymphoma EL4, C57BL/10 mice) in comparison with free DOX or classic PK1, the PHPMA-DOX conjugate clinically tested at present. Increasing the molecular weight of the polymer carrier resulted in a more pronounced in vivo antitumor effect. Antibody-targeted conjugates with DOX bound via hydrazone bond exhibited even more extensive inhibition of the tumor growth with some long-term survivors. No survivors were observed after treatment of mice with free DOX or the nontargeted PHPMA-DOX conjugate.

BACKGROUND

Crystal formation and retention are critical events for the formation of kidney stones. Oxalate and calcium oxalate (CaOx) crystals are injurious to renal epithelium, and membranes of injured cells promote crystal adherence and retention. Calcium phosphate (CaP) is the most common crystal in both urine and stones, most likely to form in the early segments of the nephron and can nucleate CaOx in a metastable solution. We hypothesized that CaP can also injure the renal epithelial cells.

METHODS

We exposed proximal tubular origin line derived from pig proximal tubules (LLC-PK1), and collecting duct origin Madin-Darby canine kidney (MDCK) cell lines to various concentrations of Brushite (Br) crystals and investigated staining with Trypan Blue and the release of lactate dehydrogenase (LDH) into the medium as an indicator of injury. In order to determine the involvement of reactive oxygen species, we also measured LDH release in the presence of superoxide dismutase (SOD) and production of hydrogen peroxide (H2O2) and 8-isoprostane (8-IP) in the presence of the catalase.

RESULTS

Exposure to Br crystals was associated with LDH release by both cell types, induced the production of H2O2 and 8-IP. Presence of SOD and catalase reduced LDH release as well as staining with trypan blue. Catalase was also associated with reduced production of H2O2 and 8-IP.

CONCLUSIONS

Brushite crystals are injurious to cells of both the proximal tubules as well as collecting ducts. Injury is mediated by reactive oxygen species. We propose that CaP crystals can independently interact with renal epithelium, promote sites for crystal attachment, and then either grow into mature CaP stones or create sites for CaOx crystal nucleation, retention, and stone development.

Angiogenesis is a key process of dynamic tissue remodeling occurring during placentation. Compelling evidence indicates that vascular endothelial growth factor (VEGF) is an important mediator of placental angiogenesis and appears to be deregulated in preeclampsia. Recently a new angiogenic factor, endocrine gland-derived VEGF (EG-VEGF), also known as prokineticin 1 (PK1), has been identified, and its expression was shown to be restricted to endocrine glands, including the placenta. In this study we investigated the pattern of expression of EG-VEGF, its related factor Bv8/PK2, and their common receptors, PKR1 and PKR2, in human placenta during the first trimester of pregnancy. We also examined EG-VEGF and PKR1 regulation by oxygen tension in isolated trophoblast cells (TCs). Our results show that EG-VEGF, but not Bv8/PK2, is expressed in human placenta. EG-VEGF is mainly localized to the syncytiotrophoblast layer with the highest expression detected between the 8th and 10th wk of gestation. EG-VEGF expression within placental villi is different from that of VEGF, which is mainly localized in the cytotrophoblast and extravillous trophoblast cells. In TCs, PKR1 mRNA is about 80 times more abundant than PKR2 mRNA. Both EG-VEGF and PKR1 mRNAs appear to be regulated by hypoxia. These findings suggest that EG-VEGF has a direct effect on TCs via its receptor PKR1 and is likely to play an important role in human placentation. The expression pattern of EG-VEGF, its regulation by oxygen tension, and its complementary localization to that of VEGF suggest that this new factor might also be deregulated in preeclampsia.

The importance of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) as a receptor determinant for bovine coronavirus (BCV) on cultured cells was analysed. Pretreatment of MDCK I (Madin Darby canine kidney) cells with neuraminidase or acetylesterase rendered the cells resistant to infection by BCV. The receptors on a human (CaCo-2) and a porcine (LLC-PK1) epithelial cell line were also found to be sensitive to neuraminidase treatment. The susceptibility to infection by BCV was restored after resialylation of asialo-MDCK I cells with Neu5,9Ac2. Transfer of sialic acid lacking a 9-O-acetyl group was ineffective in this respect. These results demonstrate that 9-O-acetylated sialic acid is used as a receptor determinant by BCV to infect cultured cells. The possibility is discussed that the initiation of a BCV infection involves the recognition of different types of receptors, a first receptor for primary attachment and a second receptor to mediate the fusion between the viral envelope and the cellular membrane.

We investigated the expression of ERK, p38 mitogen-activated protein kinase (p38), and JNK in renal tubules of diabetic rats following 3 wk after streptozotocin injection (DM). Although the expression of ERK was not different between controls and DM, phosphorylated ERK was expressed more intensely in DM. p38 And phosphorylated p38 were detected only in the diabetic kidney and were localized in all tubular segments. JNK and phosphorylated JNK were expressed similarly in controls and DM. Transforming growth factor (TGF)-beta was expressed in all tubular segments of DM, coinciding with the localization of p38. In LLC-PK1 cells, phosphorylation of ERK and p38 increased after 24- to 72-h exposure to high glucose (HG). Coincubation with a p38 inhibitor SB-203580 or a MEK inhibitor, PD-98059, suppressed the HG-induced increases in protein content, [3H]leucine incorporation, and the protein-to-DNA ratio. SB-203580 or PD-98059 also abolished the HG-stimulated expression of TGF-beta protein. These results demonstrate that ERK and p38 are activated in renal tubular cells of DM and may mediate HG-induced cellular hypertrophy and TGF-beta expression.

The distribution and dynamics of the membranous organelles in two cell types were investigated during cell division. Live cells (either PtK2 or LLC-PK1) labeled with the vital dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] were observed via serial optical sectioning with the laser-scanning confocal microscope. Z-series of labeled, dividing cells were collected every 1-2 minutes throughout mitosis, beginning at prophase and extending to the spreading of the daughter cells. Membrane distribution began to change from the onset of prophase in both cell types. When the mitotic spindle formed in prometaphase, fine tubular membranes, similar to those extending out to the edges of interphase cells aligned along the kinetochore spindle fibers. The lacy polygonal network typical of interphase cells persisted beneath the spindle, and a membrane network was also associated with the dorsal layer of the cell. As PtK2 cells reached metaphase, their spindles were nearly devoid of membrane staining, whereas the spindles of LLC-PK1 cells contained many tubular and small vesicular membranous structures. X-Z series of the LLC-PK1 metaphase spindle revealed a small cone of membranes that was separated from the rest of the cytoplasm by kinetochore MTs. In both cell types, as chromosome separation proceeded, the interzone remained nearly devoid of membranes until the onset of anaphase B. At this time the elongating interzonal microtubules were closely associated with the polygonal network of endoplasmic reticulum. Cytokinesis caused a compression, and then an exclusion of organelles from the midbody. Immunofluorescence staining with anti-tubulin antibodies suggested that spindle membranes were associated with microtubules throughout mitosis. In addition, taxol induced a dense and extensive collection of small vesicles to collect at the spindle poles of both cell types. Nocodazole treatment induced a distinct loss of organization of the membranous components of the spindles. Together these results suggest that microtubules organize the membrane distribution in mitotic cells, and that this organization may vary in different cell types depending on the quantity of microtubules within the spindle.

This study investigated the possible role of the newly discovered endocrine gland-derived vascular endothelial growth factors and their cognate receptors in the human endometrium during the menstrual cycle. Endocrine gland-derived vascular endothelial growth factors are also known as prokineticin (PK) 1 and PK2 and their receptors as PKR1 and PKR2. Expression of PK1 was elevated in the secretory compared with the proliferative phase of the menstrual cycle (P < 0.05). There was no temporal variation in expression of PK2, PKR1, or PKR2. PK1 and PK2 and their receptors were localized to multiple cellular compartments, including glandular epithelial, stromal, and endothelial cells in the endometrium and endothelial and smooth muscle cells in the myometrium. The elevation in PK1 expression in the secretory phase of the menstrual cycle indicated potential regulation of PK1 by progesterone. To investigate this, endometrial tissue was treated with 1 microM (micromol/liter(-1)) progesterone for 24 h, and PK1 expression was assessed by quantitative RT-PCR. Treatment with 1 microM ( micromol/liter(-1)) progesterone resulted in 2.91 +/- 0.75-fold elevation in PK1 expression, compared with controls (P < 0.05). These data identify a paracrine role for the PKs and their receptors in endometrial vascular function.

The contribution of organic anion transporters to the total efflux of 17beta-estradiol-D-17beta-glucuronide (E(2)17betaG) through the blood-brain barrier (BBB) was investigated using the Brain Efflux Index method by examining the inhibitory effects of probenecid, taurocholate (TCA), p-aminohippurate (PAH), and digoxin. E(2)17betaG was eliminated through the BBB with a rate constant of 0.037 min(-1) after the microinjection into the brain. Probenecid and TCA inhibited this elimination with an IC50 value of 34 and 1.8 nmol/0.5 microl of injectate, respectively, whereas PAH and digoxin reduced the total efflux to about 80 and 60% of the control value, respectively. The selectivity of these inhibitors was confirmed by examining their inhibitory effects on the transport via organic anion transporting polypeptide 1 (Oatp1), Oatp2, organic anion transporter 1 (Oat1), and Oat3 transfectants using LLC-PK1 cells as hosts. Digoxin specifically inhibited the transport via Oatp2 (K(i) = 0.037 microM). The K(i) values of TCA for Oatp1 and Oatp2 (11 and 39 microM, respectively) were about 20 times lower than those for Oat1 and Oat3 (2.8 and 0.8 mM, respectively). PAH did not affect the transport via the Oatp family, but had a similar affinity for Oat1 and Oat3 (85 and 300 microM, respectively). Probenecid had a similar affinity for these transporters (Oatp1, Oatp2, Oat1, and Oat3) examined in this study. Taking the selectivity of these inhibitors into consideration, the maximum contribution made by the Oatp2 and Oat family to the total efflux of E(2)17betaG from the brain appears to be about 40 and 20%, respectively.

Antigenic variation of malaria parasites was discovered in P. knowlesi, using a schizont-infected cell agglutination (SICA) assay to detect variant antigens expressed at the surface of infected erythrocytes. Later studies utilizing stable clones, Pk1(A+) and its direct derivative, Pk1(B+)1+, showed that SICA[+] clones express distinct parasite-encoded antigens of approximately 200 kDa. Here we identify a P. knowlesi variant antigen gene and cDNA and demonstrate that it encodes the 205 kDa variant antigen expressed by B+ parasites. This gene belongs to a multigene family, which we term SICAvar. Its ten-exon structure with seven cysteine-rich coding modules is unique compared to P. falciparum var genes. Further, we highlight a 3' genomic alteration that we predict is related to SICAvar gene switching.

In cultured mammalian cells, how dynein/dynactin contributes to spindle positioning is poorly understood. To assess the role of cortical dynein/dynactin in this process, we generated mammalian cell lines expressing localization and affinity purification (LAP)-tagged dynein/dynactin subunits from bacterial artificial chromosomes and observed asymmetric cortical localization of dynein and dynactin during mitosis. In cells with asymmetrically positioned spindles, dynein and dynactin were both enriched at the cortex distal to the spindle. NuMA, an upstream targeting factor, localized asymmetrically along the cell cortex in a manner similar to dynein and dynactin. During spindle motion toward the distal cortex, dynein and dynactin were locally diminished and subsequently enriched at the new distal cortex. At anaphase onset, we observed a transient increase in cortical dynein, followed by a reduction in telophase. Spindle motion frequently resulted in cells entering anaphase with an asymmetrically positioned spindle. These cells gave rise to symmetric daughter cells by dynein-dependent differential spindle pole motion in anaphase. Our results demonstrate that cortical dynein and dynactin dynamically associate with the cell cortex in a cell cycle-regulated manner and are required to correct spindle mispositioning in LLC-Pk1 epithelial cells.

Ezrin, a membrane-cytoskeleton linker, is required for cell morphogenesis, motility, and survival through molecular mechanisms that remain to be elucidated. Using the N-terminal domain of ezrin as a bait, we found that p125 focal adhesion kinase (FAK) interacts with ezrin. We show that the two proteins coimmunoprecipitate from cultured cell lysates. However, FAK does not interact with full-length ezrin in vitro, indicating that the FAK binding site on ezrin is cryptic. Mapping experiments showed that the entire N-terminal domain of FAK (amino acids 1-376) is required for optimal ezrin binding. While investigating the role of the ezrin-FAK interaction, we observed that, in suspended kidney-derived epithelial LLC-PK1 cells, overproduction of ezrin promoted phosphorylation of FAK Tyr-397, the major autophosphorylation site, creating a docking site for FAK signaling partners. Treatment of the cells with a Src family kinase inhibitor reduced the phosphorylation of Tyr-577 but not that of Tyr-397, indicating that ezrin-mediated FAK activation does not require the activity of Src kinases. Altogether, these observations indicate that ezrin is able to trigger FAK activation in signaling events that are not elicited by cell-matrix adhesion.

Norepinephrine (NE) contracts smooth muscle cells within the human lower urinary tract (LUT) (bladder neck, prostate, and urethra). Receptor distribution and pharmacological evidence have implicated activation of alpha 1A-adrenoceptors. We disclose the pharmacological properties of the novel, selective alpha 1A-adrenoceptor antagonist N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro- alpha,alpha-dimethyl-1H-indole-3-ethanamine hydrochloride (RS-17053) and examine critically the pharmacological identity of the alpha 1-adrenoceptor mediating contractions to NE in human LUT tissues. In several tissues from rat and cloned adrenoceptors, RS-17053 displayed high affinity for the alpha 1A-adrenoceptor (pKi and pA2 estimates of 9.1-9.9) and a 30-100-fold selectivity over the alpha 1B- and the alpha 1D-adrenoceptor subtypes (pK1 and pA2 estimates of 7.7-7.8). However, in isolated smooth muscle preparations from human LUT tissues, RS-17053 antagonized responses to NE only at high concentrations. Estimates of affinity (pA2) at alpha 1-adrenoceptors mediating NE-induced contractions were 7.5 in prostatic periurethral longitudinal smooth muscle (compared with 8.6 for prazosin), 6.9 in anterior fibromuscular stroma (prazosin, 8.9), and 7.1 in bladder neck (prazosin, 8.5). These findings indicate that contractile responses to NE in human LUT tissues are mediated by a receptor displaying pharmacological properties that are clearly different from those of the defined alpha 1A-adrenoceptor and raise the possibility that multiple forms of the alpha 1A-adrenoceptor may exist in human LUT that are discriminated by RS-17053. In this regard, the affinity estimates obtained with RS-17053 and other alpha 1-adrenoceptor antagonists in human LUT tissues are identical to those described for the putative alpha 1L-adrenoceptor.