Norepinephrine (NE) transporters (NETs) found in the neuronal plasma membrane mediate the removal of NE from the extracellular space, limiting the activation of adrenoceptors at noradrenergic synapses. Our previous studies with the noradrenergic neuroblastoma SK-N-SH have revealed a muscarinic receptor-triggered regulation of NET surface density and transport capacity, mediated in part by protein kinase C activation. Low abundance of NET proteins in this native cell model, however, preclude direct confirmation of altered trafficking of NET proteins. In our study, we monitored the activity and surface distribution of human NET proteins in transient and stably-transfected cell lines after application of kinase activators and inhibitors. Using hNET stably transfected HEK-293 and LLC-PK1 cells, as well as transiently transfected COS-7 cells, we demonstrate that PKC-activating phorbol esters, beta-PMA or beta-PDBu selectively diminish l-NE transport capacity (Vmax) with little change in NE Km. Effects of phorbol esters are rapid, stereospecific and blocked by protein kinase C inhibitors, staurosporine and bisindolylmaleimide I. As in SK-N-SH cells, beta-PMA induces a reduction in intact cell [3H]nisoxetine binding sites with no change in nisoxetine Kd or total membrane NET density. Cell-surface biotinylation and confocal immunofluorescence techniques confirm that protein kinase C-dependent reductions in NE transport capacity and whole-cell antagonist binding density are accompanied by reductions in cell-surface human NET protein expression. Together these findings argue for kinase-modulated protein trafficking as a potential route for acute regulation of antidepressant-sensitive NE clearance.

1. SB 200646A, N-(1-methyl-5-indolyl)-N'-(3-pyridyl) urea hydrochloride, the first reported selective 5-HT2C/2B over 5-HT2A receptor antagonist, (pK1 rat 5-HT2C receptor 6.9, pA2 rat 5-HT2B receptor 7.5, pK1 rat 5-HT2A receptor 5.2) dose-dependently blocked a putative rat model of 5-HT2C receptor activation; 1-(3-chlorophenyl)piperazine (mCPP, 5 mg kg-1, i.p. 20 min pretest)-induced hypolocomotion (estimated ID50 19.2 mg kg-1, p.o.). 2. SB 200646A also blocked another putative in vivo model of 5-HT2C receptor function; mCPP (5 mg kg-1, i.p. 20 min pretest)-induced hypophagia in 23 h food-deprived rats (estimated ID50 18.3 mg kg-1, p.o.). 3. SB 200646A did not antagonize 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced head shakes in rats at doses up to 200 mg kg-1, p.o., an effect thought to be mediated by 5-HT2A receptors for which SB 200646A has its next highest affinity (50 fold less) after the 5-HT2C and 5-HT2B sites. 4. SB 200646A (20, 40 mg kg-1, p.o., 1 h pretest) also reversed mCPP (0.5 mg kg-1, i.p., 30 min pretest)-induced anxiety in the social interaction test, under low light familiar conditions. 5. When given alone, under high light unfamiliar conditions, SB 200646A (2-40 mg kg-1, p.o.) increased active social interaction without affecting locomotor activity in the rat social interaction test. This is consistent with an anxiolytic action of SB 200646A. 6. These results indicate that SB 200646A has in vivo efficacy and that 5-HT2C or 5-HT2B receptors are indeed likely to mediate mCPP-induced hypolocomotion, hypophagia and anxiogenesis. They also suggest that 5-HT2C,2B receptor blockade induces anxiolysis.

The cytokine tumor necrosis factor-alpha (TNF) increases the frequency of apoptosis in confluent renal epithelial LLC-PK1 cells, an effect that can be blocked by an anti-TNFR1 monoclonal antibody. However, there were no visible "holes" in the cell sheet as a result of TNF-induced apoptosis. Instead a striking tissue remodeling occurred in response to the TNF-induced apoptosis. Apoptotic cells became surrounded and engulfed by repositioned neighboring cells distributed in a distinct "rosette" pattern. The cadherin-catenin cell-cell adhesion molecules, the tight junction-associated protein ZO-1, and actin accumulated at the sites of contact between apoptotic and neighboring cells. Pretreatment with cytochalasin B prevented the accumulation of cadherins-catenins and ZO-1 at the sites of apoptosis and resulted in microscopic holes in the TNF-treated cell sheet. Our results indicate that a renal epithelium can accommodate an increased frequency of apoptosis and still maintain its integrity by mechanisms of tissue remodeling involving the cadherin-catenin adhesion molecules, tight junctional proteins, and actin filaments.

Escherichia coli tmRNA (transfer-messenger RNA) facilitates a trans-translation reaction in which a stalled ribosome on a terminatorless mRNA switches to an internal coding sequence in tmRNA, resulting in the addition of an 11 amino acid residue tag to the truncated protein that is a signal for degradation and in recycling of the stalled ribosome. A tmRNA secondary structure model with a partial tRNA-like structure and several pseudoknots was recently proposed. This report describes an extensive mutational analysis of one predicted pseudoknot (PK1) located upstream of the E. coli tmRNA tag-encoded sequence. Both the extent of aminoacylation and the alanine incorporation into the tag sequence, reflecting the two functions of tmRNA, were measured in vitro for all the engineered RNA variants. To characterize structure-function relationships for the tmRNA mutants, their solution conformations were investigated by using structural probes and by measuring the temperature dependence of their UV absorbance. This analysis strongly supports the presence of a pseudoknot in E. coli tmRNA, and its involvement in trans-translation. Mutations disrupting the first stem of the pseudoknot inactivate function and promote stable alternative conformations. Mutations of the second stem of the pseudoknot also effect both functions. The nucleotide stretch between the two stems (loop 2) is required for efficient trans-translation, and nucleotides at positions 61 and 62 must be guanine residues. The probing data suggest the presence of magnesium ion(s) interacting with loop 2. The loops crossing the minor and major grooves can be mutated without significant effects on tmRNA function. Nucleotide insertion or deletion between the pseudoknot and the coding sequence do not change the mRNA frame of the tag-peptide sequence, suggesting that the pseudoknot structure is not a determinant for the resumption of translation.

Our previous kinetic analyses have shown that the transporter responsible for the renal uptake of pravastatin, an HMG-CoA reductase inhibitor, differs from that involved in its hepatic uptake. Although organic anion transporting polypeptides are now known to be responsible for the hepatic uptake of pravastatin, the renal uptake mechanism has not been clarified yet. In the present study, the involvement of rat organic anion transporter 3 (rOat3; Slc22a8) in the renal uptake of pravastatin was investigated. Immunohistochemical staining indicates the basolateral localization of rOat3 in the kidney. rOat1- and rOat3-expressed LLC-PK1 cells exhibited specific uptake of p-aminohippurate (PAH) and pravastatin, respectively, with the Michaelis-Menten constants (Km values) of 60 microM for rOat1-mediated PAH uptake and 13 microM for rOat3-mediated pravastatin uptake. Saturable uptake of PAH and pravastatin was observed in kidney slices with Km values of 69 and 11 microM, respectively. The difference in the potency of PAH and pravastatin in inhibiting uptake by kidney slices suggests that different transporters are responsible for their renal uptake. This was also supported by the difference in the degree of inhibition by benzylpenicillin, a relatively selective inhibitor of rOat3, for the uptake of PAH and pravastatin by kidney slices. These results suggest that rOat1 and rOat3 are mainly responsible for the renal uptake of PAH and pravastatin, respectively.

OBJECTIVE

Pancreatic cancer has a poor prognosis and few effective therapies are available. The oncolytic effect of reovirus has been observed in cancer cells with an activated Ras signaling pathway, and pancreatic cancer may be a candidate target for reovirus because K-ras mutation is frequently found in pancreatic cancer.

METHODS

In this study, we examined the feasibility of using reovirus (serotype 3) as an antihuman pancreatic cancer agent.

RESULTS

Reovirus was able to infect five human pancreatic cancer cell lines (Panc1, MIApaca-2, PK1, PK9, and BxPC3) in vitro. We also confirmed that the Ras activity in these cancer cell lines was elevated compared with that in the normal cell line and that susceptibility to reovirus was associated with the Ras activity of these cells. In a unilateral murine xenograft model using Panc1 and BxPC3 cell lines, each tumor growth was suppressed by intratumoral injection of reovirus. Furthermore, local injection of reovirus also had systemic antitumor effects in a bilateral xenograft model using Panc1 cell line. Immunohistochemical examination revealed that reovirus replication was observed within the tumor but not in surrounding normal tissue.

CONCLUSIONS

These results suggest that reovirus can be considered for a novel therapy against pancreatic cancer.

BACKGROUND

Recent studies of Toxoplasma gondii isolates from animals in different regions of China have shown a limited genetic diversity and type China 1 was the dominant genotype of T. gondii prevalent in Chinese animals. However, little has been known concerning the isolation and genotyping of T. gondii circulating in chickens, pigs and rodents in China. The aim of the study was to characterize samples of T. gondii isolates obtained from naturally infected cats, pigs and free-range chickens slaughtered for human consumption in China.

METHODS

In the present study, brain tissues of 77 animals collected from different areas of China, including 24 free-range chickens (Gallus domesticus) , 13 voles (Rattus flavipectus), 23 pigs and 17 cats, were bioassayed in mice and viable T. gondii were isolated from the brains of eleven. These eleven T. gondii isolates were maintained in Kunming (KM) outbred mice and DNA isolated from tissues of infected mice was characterized using 11 PCR-restriction fragment length polymorphism (PCR-RFLP) markers: SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico, and CS3. Moreover, to determine mouse virulence of China 1 lineage of parasites, a TgCtgy5 genotype isolate was selected randomly and assessed in KM mice with different inoculation doses.

RESULTS

Results of genotyping revealed that ten isolates were type China 1 (ToxoDB PCR-RFLP genotype #9), and TgCksz1 was a new genotype that was reported for the first time designated here as ToxoDB PCR-RFLP #225. No clonal types I, II and III lineages were found. DNA sequencing of four introns (EF1, HP2, UPRT1 and UPRT7) and two genes (GRA6 and GRA7) from representative isolates confirmed the results of PCR-RFLP genotyping. The TgCtgy5 isolate was highly virulent in KM mice; all infected mice died of acute toxoplasmosis, irrespective of the inoculation dose. The results indicate that mouse virulent isolates of T. gondii are predominantly circulating in cats in China.

CONCLUSIONS

T. gondii isolated from chickens, pigs, cats and rodents in different locations in China were genotyped and the results reconfirmed the limited diversity of T. gondii in China and showed that type China 1 lineage was dominant in this country.

Cold acclimation is the major process that prepares plants for freezing tolerance. In addition to extensive transcription regulation by cold-inducible master transcription factors, oxidative stress signaling has been postulated to play a role in freezing tolerance. Activation of oxidative signaling through the expression of an active mitogen-activated protein kinase kinase kinase provided benefits in transgenic tobacco at freezing temperature bypassing cold acclimation. Because involvement of the mitogen-activated protein kinase cascade in oxidative stress signaling is evolutionarily conserved in eukaryotes from yeast to mammals, we tested the effect of expressing a heterologous tobacco mitogen-activated protein kinase kinase kinase (Nicotiana PK1), which can mimic H(2)O(2) signaling, in a major cereal crop. We demonstrate that low-level but constitutive expression of the Nicotiana PK1 gene enhances freezing tolerance in transgenic maize plants that are normally frost sensitive. Our results suggest that a new molecular approach can be designed to genetically enhance freezing tolerance in important crops.

Usher syndrome is the leading cause of genetic deaf-blindness. Monoallelic mutations in PDZD7 increase the severity of Usher type II syndrome caused by mutations in USH2A and GPR98, which respectively encode usherin and GPR98. PDZ domain-containing 7 protein (PDZD7) is a paralog of the scaffolding proteins harmonin and whirlin, which are implicated in Usher type 1 and type 2 syndromes. While usherin and GPR98 have been reported to form hair cell stereocilia ankle-links, harmonin localizes to the stereocilia upper tip-link density and whirlin localizes to both tip and ankle-link regions. Here, we used mass spectrometry to show that PDZD7 is expressed in chick stereocilia at a comparable molecular abundance to GPR98. We also show by immunofluorescence and by overexpression of tagged proteins in rat and mouse hair cells that PDZD7 localizes to the ankle-link region, overlapping with usherin, whirlin, and GPR98. Finally, we show in LLC-PK1 cells that cytosolic domains of usherin and GPR98 can bind to both whirlin and PDZD7. These observations are consistent with PDZD7 being a modifier and candidate gene for USH2, and suggest that PDZD7 is a second scaffolding component of the ankle-link complex.

The tobacco etch virus (TEV) 5'-leader promotes cap-independent translation in a 5'-proximal position and promotes internal initiation when present in the intercistronic region of a dicistronic mRNA, indicating that the leader contains an internal ribosome entry site. The TEV 143-nucleotide 5'-leader folds into a structure that contains two domains, each of which contains an RNA pseudoknot. Mutational analysis of the TEV 5'-leader identified pseudoknot (PK) 1 within the 5'-proximal domain and an upstream single-stranded region flanking PK1 as necessary to promote cap-independent translation. Mutations to either stem or to loops 2 or 3 of PK1 substantially disrupted cap-independent translation. The sequence of loop 3 in PK1 is complementary to a region in 18 S rRNA that is conserved throughout eukaryotes. Mutations within L3 that disrupted its potential base pairing with 18 S rRNA reduced cap-independent translation, whereas mutations that maintained the potential for base pairing with 18 S rRNA had little effect. These results indicated that the TEV 5'-leader functionally substitutes for a 5'-cap and promotes cap-independent translation through a 45-nucleotide pseudoknot-containing domain.

The highly conserved intraflagellar transport (IFT) proteins are essential for cilia formation in multiple organisms, but surprisingly, cilia form in multiple zebrafish ift mutants. Here, we detected maternal deposition of ift gene products in zebrafish and found that ciliary assembly occurs only during early developmental stages, supporting the idea that maternal contribution of ift gene products masks the function of IFT proteins during initial development. In addition, the basal bodies in multiciliated cells of the pronephric duct in ift mutants were disorganized, with a pattern suggestive of defective planar cell polarity (PCP). Depletion of pk1, a core PCP component, similarly led to kidney cyst formation and basal body disorganization. Furthermore, we found that multiple ift genes genetically interact with pk1. Taken together, these data suggest that IFT proteins play a conserved role in cilia formation and planar cell polarity in zebrafish.

In the present study, we have investigated the role of the multidrug resistance (mdr) P-glycoprotein (Pgp) at the blood-brain barrier in hampering the access of the synthetic glucocorticoid, prednisolone. In vivo, a tracer dose of [(3)H]prednisolone poorly penetrated the brain of adrenalectomised wild-type mice, but the uptake was more than threefold enhanced in the absence of Pgp expression in mdr1a (-/-) mice. In vitro, in stably transfected LLC-PK1 monolayers the human MDR1 P-glycoprotein was able to transport prednisolone present at a micromolar concentration. A specific Pgp blocker, LY 335979, could block this polar transport of [(3)H]prednisolone. Human Pgp does not transport all steroids, as cortexolone was not transported at all and aldosterone was only weakly transported. The ability of Pgp to export the synthetic glucocorticoid, prednisolone, suggests that uptake of prednisolone in the human brain is impaired, leading to a discrepancy between central and peripheral actions. Furthermore, the ensuing imbalance in activation of the two types of brain corticosteroid receptors may have consequences for cognitive performance and mood.

Recent studies have ascribed many non-pumping functions to the Na/K-ATPase. We show here that graded knockdown of cellular Na/K-ATPase alpha1 subunit produces a parallel decrease in both caveolin-1 and cholesterol in light fractions of LLC-PK1 cell lysates. This observation is further substantiated by imaging analyses, showing redistribution of cholesterol from the plasma membrane to intracellular compartments in the knockdown cells. Moreover, this regulation is confirmed in alpha1(+/-) mouse liver. Functionally, the knockdown-induced redistribution appears to affect the cholesterol sensing in the endoplasmic reticulum, because it activates the sterol regulatory element-binding protein pathway and increases expression of hydroxymethylglutaryl-CoA reductase and low density lipoprotein receptor in the liver. Consistently, we detect a modest increase in hepatic cholesterol as well as a reduction in the plasma cholesterol. Mechanistically, alpha1(+/-) livers show increases in cellular Src and ERK activity and redistribution of caveolin-1. Although activation of Src is not required in Na/K-ATPase-mediated regulation of cholesterol distribution, the interaction between the Na/K-ATPase and caveolin-1 is important for this regulation. Taken together, our new findings demonstrate a novel function of the Na/K-ATPase in control of the plasma membrane cholesterol distribution. Moreover, the data also suggest that the plasma membrane Na/K-ATPase-caveolin-1 interaction may represent an important sensing mechanism by which the cells regulate the sterol regulatory element-binding protein pathway.

Previous mechanisms describing how aminoglycosides exert their cellular toxicity, including lysosomal accumulation, rupture, and release, cannot account for the rapidity and extent of the observed subcellular and organ effects. Using immunoamplification techniques and colocalization with epitopes of the endoplasmic reticulum (ER), we report rapid retrograde transport of gentamicin to the ER. Additionally, exposure times of 2 and 4 h in LLC-PK1 cells produced cytosolic release and nuclear association. Cellular internalization and trafficking of aminoglycoside structural analogs, amine-containing cationic fluorescent dextrans of 3,000 molecular weight, corroborated these findings. However, identical anionic fluorescent dextrans, or larger cationic dextrans, of 10,000 molecular weight, failed to traverse from the ER into the cytosol or localize within the nucleus. These studies suggest that a pathway exists that transports internalized aminoglycosides, and other small-molecular-weight cationic compounds, in a retrograde manner through the Golgi complex and to the ER. From there, these compounds move into the cytosol for delivery throughout the cell. To quantify the potential toxic effects of cytosolic aminoglycoside release, experiments examining mitochondrial membrane potential in the continued presence of extracellular gentamicin were undertaken and demonstrated a significant reduction after 4 and 8 h. These observations provide a mechanism for the rapidly induced known cellular alterations, including aberrant vesicle fusion, mitochondrial toxicity/free radical generation, and decreased protein synthesis either by reduced transcription or translation after aminoglycoside exposure.

Tumour capillaries are frequently hyperpermeable compared with normal vasculature, and thus they offer a much sought-after gateway for targeted delivery of cancer chemotherapy. Phase I clinical trials reported recently describe the first synthetic polymer-drug conjugate to be tested in man. N-(2-Hydroxypropyl)methacrylamide copolymer-doxorubicin (PK1, FCE 28068) displayed antitumour activity in chemotherapy-refractory patients, considerably reduced toxicity compared with doxorubicin, and evidence of tumour-selective targeting. With increasing understanding of the vector- and tumour-related factors that govern vascular permeability, non-viral vectors are being designed for tumour-selective targeting and subsequent intracytoplasmic delivery of macromolecular medicines such as genes, antisense oligonucleotides, proteins and peptides.

Prions consist mainly of PrP(Sc), a pathogenic conformer of host-encoded PrP(C). Prion populations with distinct phenotypes but associated with PrP(Sc), having the same amino acid sequence, constitute distinct strains. Strain identity is thought to be encoded by the conformation of PrP(Sc) and to be maintained by seeded conversion. Prion strains can be distinguished by the cell panel assay, which measures their ability to infect distinct cell lines. Brain-derived 22L prions characteristically are able to infect R33 cells (i.e., are "R33 competent"), as well as PK1 cells in the presence of the inhibitor swainsonine (i.e. are "swa resistant"). Here we report that 22L prions retained their characteristic cell tropism and swa resistance when transferred from brain to R33 cells. However, when transferred from the R33 cells to PK1 cells, they gradually became R33 incompetent and swa sensitive, unless the transfer was in the presence of swa, in which case swa resistance and R33 competence were retained. PrP(Sc) associated with swa-resistant/R33-competent and swa-sensitive/R33-incompetent prions had different conformational stabilities. When cloned R33-incompetent/swa-sensitive prions were again propagated in brain, their properties gradually reverted to those of the original brain-derived 22L prions. Our results support the view that 22L prion populations are heterogeneous and that distinct prion variants are selected in different cellular environments.

OBJECTIVE

Oxalate and calcium oxalate (CaOx) crystals are injurious to renal epithelial cells. The injury is caused by the production of reactive oxygen species (ROS). Citrate is a well-known inhibitor of CaOx crystallization and as such it is one of the major therapeutic agents prescribed. Since citrate increases cellular reduced nicotinamide adenine dinucleotide phosphate and glutathione (GSH), we hypothesized that exogenously administered citrate should act as an antioxidant and protect cells from oxalate induced injury.

METHODS

We exposed LLC-PK1 and MDCK cells to 500 microM/ml oxalate or 150 mug/cm calcium oxalate crystals for 30, 60 and 180 minutes with or without 3 mg/ml citrate in the medium. We determined cell viability by lactate dehydrogenase release and trypan blue exclusion, ROS involvement by changes in hydrogen peroxide and GSH, and lipid peroxidation by quantifying 8-isoprostane.

RESULTS

The presence of citrate was associated with significant decrease in lactate dehydrogenase release (p <0.001) and staining with trypan blue (p <0.05). In addition, there was a significant increase in GSH (p <0.005) and a decrease in the production of hydrogen peroxide (p <0.05) and 8-isoprostane (p <0.0005) secretion into the culture medium when citrate was present in the medium.

CONCLUSIONS

Citrate protects cells from oxalate and CaOx crystal induced injury by preventing lipid peroxidation through a decrease in ROS production. The results provide additional data for the beneficial role of citrate therapy for CaOx nephrolithiasis.

Previously, we have shown that liposomal amphotericin B (L-AmpB) composed of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) was less nephrotoxic but equally as effective as Fungizone, which consists of amphotericin (AmpB) and deoxycholate. We have also observed that AmpB predominantly associates with high-density lipoproteins (HDL) in human serum and that the amount of AmpB associated with HDL increases when AmpB is incorporated into negatively charged liposomes. Furthermore, we observe that AmpB was less toxic in vitro to pig kidney cells when associated with HDL, but still toxic when associated with LDL. To further understand why HDL-associated AmpB causes reduced renal toxicity, we first examined LLC PK1 cells for the presence of LDL and HDL receptors and then the cytotoxic effects of HDL- and LDL-associated AmpB following trypsin treatment of LLC PK1 renal cells, which removed only the high-affinity LDL receptors. We found that LLC PK1 renal cells expressed high- and low-affinity LDL receptors but only low-affinity HDL receptors. Furthermore, when LLC PK1 cells were treated with trypsin, HDL- and LDL-associated AmpB were less toxic to the cells than was AmpB. The reduced renal cell toxicity of HDL-associated AmpB may be due to its lack of interaction with renal cells because of the absence of HDL receptors. Since AmpB interacts with cholesteryl esters (CE) whose transfer among lipoproteins is regulated by lipid transfer protein (LTP), the role of LTP on the distribution of AmpB to HDL and LDL was next investigated. We observed that LTP facilitated the transfer of AmpB, but not L-AmpB, from HDL to LDL.(ABSTRACT TRUNCATED AT 250 WORDS)

P-glycoprotein seems to be the most important factor limiting the oral absorption of paclitaxel. We have now explored the mechanisms responsible for the low oral bioavailability of docetaxel, a structurally related taxane drug. The recovery of 33% of oxidative metabolites and only 39% of unchanged drug in the feces of FVB wild-type mice receiving 10 mg/kg of oral docetaxel indicates that the major part of the oral dose has been absorbed. The feces and bile of mice receiving 10 mg/kg of i.v. docetaxel contained large amounts of metabolites and only minor quantities of unchanged drug, highlighting the importance of metabolism as an elimination route for this drug. In wild-type and P-glycoprotein knockout mice, dose escalation of p.o. administered docetaxel from 10 to 30 mg/kg resulted in a more than proportional increase in plasma levels, which suggested saturation of first-pass metabolism. Moreover, coadministration of 12.5 mg/kg of the HIV protease inhibitor ritonavir, also a strong inhibitor of cytochrome P4503A4 with only minor P-glycoprotein inhibiting properties, increased the plasma levels after oral docetaxel by 50-fold. In vitro transport studies across monolayers of LLC-PK1 cells (parental and transduced with MDR1 or Mdr1a) suggested that docetaxel is a weaker substrate for P-glycoprotein than paclitaxel is. In conclusion, docetaxel is well absorbed from the gut lumen in mice despite the presence of P-glycoprotein in the gut wall. Subsequent first-pass extraction is the most important factor determining its low bioavailability. The inhibition of docetaxel metabolism by ritonavir provides an interesting strategy to improve the systemic exposure of oral docetaxel.

Epithelial layers of LLC-PK1/PKE20 cells, a renal epithelial cell line which expresses Na+/H+ exchange activities in the apical as well as basolateral membrane domains, are examined in the single cell mode by microspectrofluorometry. We provide evidence that basolateral Na+/H+ exchange is more sensitive to amiloride inhibition than is apical Na+/H+ exchange. Furthermore, we demonstrate that the two exchange activities differ in their regulatory control: kinase A activation (forskolin, 8-Br-cAMP) leads to inhibition of both exchange activities, whereas kinase C activation (phorbol ester) stimulates basolateral and inhibits apical Na+/H+ exchange. Thus, renal epithelial cells may contain two Na+/H+ exchange activities: an apical ("epithelial") and basolateral ("housekeeping") which may serve different cellular functions and are under separate regulatory controls.

Incubation with L-DOPA induced a rise in GSH level in cultures of fetal rat mesencephalon, mouse neuroblastoma (Neuro-2A), human neuroblastoma (SK-N-MC), pig kidney epithelial cells (LLC-PK1), and glia from newborn rat brain, but not C6 glioma cells or neuronal cultures (no glia) from the mesencephalon. The pure neuronal cultures were destroyed by incubation with L-DOPA; added ascorbic acid or superoxide dismutase protected the cells. Washout of L-DOPA after 48 h amplified the rise in GSH content in mixed cultures (neurons plus glia). Examination of structure-activity relationships for elevating GSH levels in responsive cell types revealed that autooxidizable compounds (alpha-methyl-DOPA, dopamine, apomorphine, catechol, and hydroquinone) behaved similarly to L-DOPA, whereas structural analogues that cannot undergo autooxidation (3-O-methyl-DOPA, tyrosine, 2,4-dihydroxyphenylalanine, and resorcinol) failed to elevate GSH levels. Therefore, up-regulation of GSH appears to be a response to a mild oxidative stress. When mixed mesencephalic cultures were exposed to a strong oxidant stress by incubation with tert-butyl hydroperoxide, a loss in viability was seen. Cultures pretreated with L-DOPA or hydroquinone were protected from loss of viability. However, when cultures were pretreated with both L-DOPA and ascorbate, which prevents the rise in GSH level, protection was lost, in accord with the failure to up-regulate GSH. These results show that the up-regulation of cellular GSH evoked by autooxidizable agents is associated with significant protection of cells. Glia play an essential role in the response of mesencephalic cell cultures. An ability to up-regulate GSH may serve a protective role in vivo.

These studies examined the effects of oxalate, a constituent of renal stones, on the growth of LLC-PK1 cells. Exposure to oxalate resulted in an initiation of DNA synthesis in serum-starved, growth-arrested cells as measured by 3H-thymidine incorporation. The effects of oxalate were comparable to those observed in response to 10% serum. Moreover, exposure to oxalate plus 10% serum stimulated DNA synthesis to a greater extent than oxalate or serum alone. These studies indicate that oxalate promotes the progression of cells from the G0/G1 to the S phase of the cell cycle. However, the increase in DNA synthesis was not always followed by an increase in cell number since high concentrations of oxalate led to a reduction in cell number.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) activate the PTH/PTHrP receptor to trigger parallel increases in adenylyl cyclase (AC) and phospholipase C (PLC). The amino (N)-terminal region of PTH-(1-34) is essential for AC activation. Ligand domains required for activation of PLC, PKC, and other effectors have been less well-defined, although some studies in rodent systems have identified a core region [hPTH-(29-32)] involved in PKC activation. To determine the critical ligand domain(s) for PLC activation, a series of truncated hPTH-(1-34) analogues were assessed using LLC-PK1 cells that stably express abundant transfected human or rat PTH/PTHrP receptors. Phospholipase C signaling and ligand-binding affinity were reduced by carboxyl (C)-terminal truncation of hPTH-(1-34) but were coordinately restored when a binding-enhancing substitution (Glu(19) ->> Arg(19)) was placed within hPTH-(1-28), the shortest hPTH peptide that could fully activate both AC and PLC. Phospholipase C, but not AC, activity was reduced by substituting Gly(1) for Ser(1) in hPTH-(1-34) and was eliminated entirely by removing either residue 1 or the alpha-amino group alone. These changes did not alter binding affinity. These findings led to design of an analogue, [Gly(1),Arg(19)]hPTH-(1-28), that was markedly signal-selective, with full AC but no PLC activity. Thus, the extreme N-terminus of hPTH constitutes a critical activation domain for coupling to PLC. The C-terminal region, especially hPTH-(28-31), contributes to PLC activation through effects upon receptor binding but is not required for full PLC activation. The N-terminal determinants of AC and PLC activation in hPTH-(1-34) overlap but are not identical, as subtle modifications in this region may dissociate activation of these two effectors. The [Gly(1),Arg(19)]hPTH-(1-28) analogue, in particular, should prove useful in dissociating AC- from PLC-dependent actions of PTH.

Ferric nitrilotriacetate (Fe-NTA) induces oxidative renal damage leading to a high incidence of renal cell carcinoma (RCC) in rats. Differential display analysis of such RCCs revealed elevated expression of annexin 2 (Anx2), a substrate for kinases and a receptor for tissue-type plasminogen activator and plasminogen. We conducted this study to clarify the significance of Anx2 in Fenton reaction-based carcinogenesis. Messenger RNA and protein levels of Anx2 were increased time-dependently in the rat kidney after Fe-NTA administration as well as in LLC-PK1 cells after exposure to H2O2. The latter was inhibited by pretreatment with N-acetylcysteine, pyrrolidine dithiocarbamate or catalase. Immunohistochemistry revealed negligible staining in the normal renal proximal tubules, but strong staining in regenerating proximal tubules, karyomegalic cells and RCCs. Metastasizing RCCs showed higher Anx2 protein levels. Anx2 was phosphorylated at serine and tyrosine residues in these cells and coimmunoprecipitated with phosphorylated actin. Overexpression of Anx2 induced a higher cell proliferation rate in LLC-PK1 cells. In contrast, a decrease in proliferation leading to apoptosis was observed after Anx2 antisense treatment to cell lines established from Fe-NTA-induced RCCs. These results suggest that Anx2 is regulated by redox status, and that persistent operation of this adaptive mechanism plays a role in the proliferation and metastasis of oxidative stress-induced cancer.