The purpose of the study was to determine whether Hsp27 interacts with actin and could protect against selected manifestations of injury from energy depletion in renal epithelia. LLC-PK1 cells were stably transfected to overexpress human Hsp27 tagged with green fluorescence protein (GFP). Transfected expression of the labeled Hsp27 did not reduce endogenous Hsp25 levels in the cells compared with either nontransfected cells or cells transfected with GFP alone used as the transfectant control (G). By fluorescence energy transfer (FRET) between GFP-tagged Hsp27 and rhodamine phalloidin-decorated actin, minimal interaction was found in uninjured control cells. In ATP-depleted cells, Hsp27 was associated closely with F-actin at lateral cell boundaries and with aggregated actin within the cell body. Less Hsp27 interaction with actin was found during recovery; but when adjusted for total phalloidin fluorescence, FRET between Hsp27 and F-actin did not change between 2-h ATP depletion and 4-h recovery. Where Hsp27 association with actin persisted during recovery, it was principally with the residual aggregates of actin in the cell body. Detachment of Na,K-ATPase from the cytoskeleton at 2-h ATP depletion was significantly less in Hsp27 cells compared with transfectant control G cells but not at 4-h ATP depletion. Detachment of ezrin from the cytoskeleton during ATP depletion was nearly complete and was not prevented in the Hsp27 cells. Protection of the Hsp27 cells was not attributable to preservation of cellular ATP levels. Hsp27 appears to have specific actions in renal epithelia subjected to energy depletion, including interacting with actin to preserve architecture in specific intracellular domains.

An epithelial cell line from pig kidney (LLC-PK1) with properties of proximal tubular cells can be maintained indefinitely in hormone-supplemented serum-free medium. Continuous growth requires the presence of seven factors: transferrin, insulin, selenium, hydrocortisone, triiodothyronine, vasopressin, and cholesterol. The hormone-defined medium (a) supports growth of LLC-PK1 cells at a rate of approaching that observed in serum-supplemented medium; (b) allows vectorial transepithelial salt and fluid transport as measured by hemicyst formation; and (c) influences cell morphology. The vasopressin dependency for growth and morphology can be partially replaced by isobutylmethylxanthine or dibutyryl cyclic AMP. The medium has been used to isolate rabbit proximal tubular kidney epithelial cells free of fibroblasts.

Familial juvenile hyperuricemic nephropathy (FJHN) is an autosomal dominant genetic disorder that is characterized by hyperuricemia, gout, and tubulointerstitial nephritis. FJHN is caused by mutations in the UMOD gene, which encodes for uromodulin, the most abundant urinary protein. Herein is demonstrated that patients with FJHN and renal insufficiency exhibit a profound reduction in urinary uromodulin together with either elevated or decreased plasma uromodulin. One young patient with FJHN, however, had normal serum creatinine and normal urinary uromodulin with elevated plasma uromodulin. These observations suggest that there are different urinary and plasma uromodulin profiles in early and late disease and that there may be an altered direction of uromodulin secretion in the course of FJHN as a result of improper intracellular sorting of the mutated protein in the thick ascending limb. With the use of immunohistochemistry and a quantitative immunoassay, targeting and secretion of wild-type and mutant (C77Y and N128S) uromodulin were investigated in the polarized renal epithelial cell line LLC-PK1. In transfected cells, uromodulin mutants were targeted properly to the apical membrane but were secreted less efficiently to the apical compartment than wild-type protein. The expression of mutant uromodulin had no effect on caspase 3 activity. These results indicate that the mutations studied do not impair glycosyl-phosphatidylinositol-mediated apical targeting of the protein but do affect apical secretion. Because the mutant proteins are secreted as efficiently as wild type to the basolateral compartment, the possibility arises that interactions with the immune system at the site of secretion are a contributing factor to the development of tubulointerstitial nephritis in FJHN.

We have identified a new putative transcription factor from the rat kidney, termed Kid-1 (for kidney, ischemia and developmentally regulated gene 1). Kid-1 belongs to the C2H2 class of zinc finger genes. Its mRNA accumulates with age in postnatal renal development and is detected predominantly in the kidney. Kid-1 mRNA levels decline after renal injury secondary to ischemia or folic acid administration, two insults which result in epithelial cell dedifferentiation, followed by regenerative hyperplasia and differentiation. The low expression of Kid-1 early in postnatal development, and when renal tissue is recovering after injury, suggests that the gene product is involved in establishment of a differentiated phenotype and/or regulation of the proliferative response. The deduced protein contains 13 C2H2 zinc fingers at the COOH end in groups of 4 and 9 separated by a 32-amino-acid spacer. There are consensus sites for phosphorylation in the NH2 terminus non-zinc finger region as well as in the spacer region between zinc fingers 4 and 5. A region of the deduced protein shares extensive homology with a catalytic region of Raf kinases, a feature shared only with TFIIE among transcription factors. To determine whether Kid-1 can modulate transcription, a chimeric construct encoding the Kid-1 non-zinc finger region (sense or antisense) and the DNA-binding region of GAL4 was transfected into COS and LLC-PK1 cells together with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing GAL4 binding sites, driven by either a minimal promoter or a simian virus 40 enhancer. CAT activity was markedly inhibited in cells transfected with the sense construct compared with the activity in cells transfected with the antisense construct. To our knowledge, this pattern of developmental regulation, kidney expression, and regulation of transcription is unique among the C2H2 class of zinc finger-containing DNA-binding proteins.

LLC-PK1 cells (a continuous epithelioid cell line with renal characteristics) are examined by microspectrofluorometry as single cells, in order to determine the mechanism of intracellular pH (pHi) recovery from an acid load imposed by ammonium preincubation and removal (NH4 prepulse). Initial experiments evaluate the intracellular K+ levels through a null point analysis of total cellular K+ with flame photometry. The response of BCECF (a pH-sensitive fluorescent dye) is then calibrated, using saturating concentrations of nigericin to cause defined changes in pH. For experiments with the microspectrofluorometer, LLC-PK1 cells were grown on either glass coverslips or filters (the latter attached to plastic coverslips with a hole under the filter). The cells on glass coverslips demonstrate a Na+-dependent recovery from an (NH4 prepulse) acid load which is sensitive to 1 microM ethylisopropylamiloride. They also demonstrate a 'set point' of activation of Na+/H+ exchange. When examined for changes in pH, due to changes in membrane potential, plasma membrane proton conductance could not be detected at resting pHi. Cells grown on filters also demonstrate a pHi recovery from an acid load which is Na+ dependent and ethylisopropylamiloride sensitive, but in this configuration, the majority of cells (22/23 preparations) require Na+ at the basolateral membrane for rapid pHi recovery. The morphology and polarity of the cells grown on permeable supports appears normal at the electron-microscopic level. The results are not affected by changes in cell seeding density or collagen treatment of the filters.

To study the membrane mobility of aquaporin water channels, clones of stably transfected LLC-PK1 cells were isolated with plasma membrane expression of GFP-AQP1 and GFP-AQP2, in which the green fluorescent protein (GFP) was fused upstream and in-frame to each aquaporin (AQP). The GFP fusion did not affect AQP tetrameric association or water transport function. GFP-AQP lateral mobility was measured by irreversibly bleaching a spot (diameter 0.8 microm) on the membrane with an Argon laser beam (488 nm) and following the fluorescence recovery into the bleached area resulting from GFP translational diffusion. In cells expressing GFP-AQP1, fluorescence recovered to >96% of its initial level with t(1/2) of 38 +/- 2 s (23 degrees C) and 21 +/- 1 s (37 degrees C), giving diffusion coefficients (D) of 5.3 and 9.3 x 10(-11) cm(2)/s. GFP-AQP1 diffusion was abolished by paraformaldehyde fixation, slowed >50-fold by the cholesterol-binding agent filipin, but not affected by cAMP agonists. In cells expressing GFP-AQP2, fluorescence recovered to >98% with D of 5.7 and 9.0 x 10(-11) cm(2)/s at 23 degrees C and 37 degrees C. In contrast to results for GFP-AQP1, the cAMP agonist forskolin slowed GFP-AQP2 mobility by up to tenfold. The cAMP slowing was blocked by actin filament disruption with cytochalasin D, by K(+)-depletion in combination with hypotonic shock, and by mutation of the protein kinase A phosphorylation consensus site (S256A) at the AQP2 C-terminus. These results indicate unregulated diffusion of AQP1 in membranes, but regulated AQP2 diffusion that was dependent on phosphorylation at serine 256, and an intact actin cytoskeleton and clathrin coated pit. The cAMP-induced immobilization of phosphorylated AQP2 provides evidence for AQP2-protein interactions that may be important for retention of AQP2 in specialized membrane domains for efficient membrane recycling.

A class of N-substituted quinoline compounds has been introduced recently for the fluorescence measurement of Cl concentration in biological preparations. The most Cl-sensitive compound was 6-methoxy-N-[3-sulfopropyl] quinolinium with peak excitation and emission wavelengths of 350 and 442 nm and a Stern-Volmer constant for quenching by Cl of 118 M-1. Six water-soluble quinoline derivatives were synthesized and characterized for the purposes of increasing Cl sensitivity, adding ester functions for cell trapping, and red-shifting the fluorescence peak wavelengths. Acetic acid ester functions were added at the N-, 2-, and 6-positions of the quinoline ring. The best ester compound, N-(6-methoxyquinolyl)acetoethyl ester (MQAE), was water soluble (270 g/liter at 23 degrees C; octanol:H2O partition coefficient of 0.009), had a high Cl sensitivity (Stern-Volmer constant 200 M-1), peak excitation and emission wavelengths of 355 and 460 nm, a fluorescence lifetime of 21.6 ns, and a molar absorbance of 4850 M-1 cm-1 (320 nm). MQAE fluorescence was not altered by the physiological anions HCO3, SO4, and PO4, by cations, or by pH. MQAE was used to measure chloride transport in liposome membranes and in cultured LLC-PK1 cells in monolayer; MQAE leaked out of cells less than 20% in 60 min at 37 degrees C. The physical, optical, and anion quenching properties for the series of ester compounds were determined to establish a set of structure-activity correlates.

The two most frequently observed single nucleotide polymorphisms (SNPs) of the human multidrug resistance 1 (MDR1) gene are 2677G/T/A (893Ala/Ser/Thr) and 3435C/T (no amino acid substitution). In this study, six forms of MDR1 cDNAs with the SNPs were expressed in LLC-PK1 cells and their transport activities were determined. Nearly identical amounts of the recombinant MDR1 proteins were expressed in the established cell lines using the Flp recombinase, which integrates a gene of interest at a specific genomic location. Four structurally diverse compounds: verapamil, digoxin, vinblastine and cyclosporin A, were examined for transcellular transport activities and intracellular accumulation. No significant differences were observed between cells expressing five polymorphic types of the MDR1 cDNAs (2677G/3435T, 2677A/3435C, 2677A/3435T, 2677T/3435C, 2677T/3435T) and cells expressing the wild-type (2677G/3435C). These results suggested that the two frequently observed MDR1 SNPs had no effect on the transport activities of MDR1 proteins expressed in LLC-PK1 cells in vitro, and other genetic or environmental factors might control the expression of MDR1 and the in vivo activity of MDR1.

We have demonstrated that ouabain regulates protein trafficking of the Na/K-ATPase α1 subunit and NHE3 (Na/H exchanger, isoform 3) via ouabain-activated Na/K-ATPase signaling in porcine LLC-PK1 cells. To investigate whether this mechanism is species-specific, ouabain-induced regulation of the α1 subunit and NHE3 as well as transcellular (22)Na(+) transport were compared in three renal proximal tubular cell lines (human HK-2, porcine LLC-PK1, and AAC-19 originated from LLC-PK1 in which the pig α1 was replaced by ouabain-resistant rat α1). Ouabain-induced inhibition of transcellular (22)Na(+) transport is due to an ouabain-induced redistribution of the α1 subunit and NHE3. In LLC-PK1 cells, ouabain also inhibited the endocytic recycling of internalized NHE3, but has no significant effect on recycling of endocytosed α1 subunit. These data indicated that the ouabain-induced redistribution of the α1 subunit and NHE3 is not a species-specific phenomenon, and ouabain-activated Na/K-ATPase signaling influences NHE3 regulation.

Our studies of equilibrium solubilization of crystals of unconjugated bilirubin (UCB) in buffered aqueous NaCl (1988. J. Lipid Res. 29: 335-348) suggested that the two carboxylic pKa values were 6.8 and 9.3 and the solubility of UCB diacid was 0.1 microM. These data, however, were not ideal, due to possible effects of crystal size, metastability, 96-h incubation times with formation of polar derivatives, impurities in the bilirubin, and imprecision of analyses at low concentrations of UCB ([UCB]). In the present study, designed to determine the pKa values and self-association of UCB, these problems were minimized by solvent partition of UCB from solution in CHCl3 into buffered aqueous NaCl. There was no crystal phase. Equilibrium was attained rapidly (10 min); UCB and CHCl3 were highly purified; and accurate diazo assay of low [UCB] in the aqueous phase, [Bw], was achieved by concentrating the UCB through back-extraction into a small volume of CHCl3. By determining effects on partition rations of varying the [UCB] in the CHCl3 phase, [Bc], we could assess also the self-association of UCB species in the aqueous phase. Partition ratios (P = Bw/Bc) did not differ between initial and repeat extractions, indicating insignificant concentrations of polar UCB derivatives. Similar P ratios were obtained when equilibrium was approached from a supersaturated aqueous phase. At 21-25 degrees C, mu = 0.15, the data (n = 76) fit the equation: log P = log Po + log[1 + 10(pH-A) + 10(2pH-B) + Bc.10(4pH-D)]; the bracketed terms reflect P for H2Bo (diacid), HB- (monoanion), B= (dianion), and (B=)2 dimer, respectively. Computer-fitted values for constants (+/- SD) were: Po = P for H2Bo = 5.79 x 10(-5); A = pK1 = 8.12 +/- 0.23; B = pK1 + pK2 = 16.56 +/- 0.10; pK2 = 8.44 +/- 0.33; D = pk22 + 2(pK1 + pK2) -log(2Po) = 37.64 +/- 0.07, and k22 = 0.26 microM-1 [formation constant of (B=)2 dimer]. In ancillary studies, multiple cycles of direct dissolution of UCB crystals revealed a progressive decrease in aqueous solubility of UCB as fine crystals were removed; this effect was minimal in CHCl3. Unlike in water, moreover, varied UCB crystal forms had similar solubilities in CHCl3, with [Bc] = 1.14 mM at saturation. As determined from [Bc]sat.Po, the aqueous solubility of H2Bo was 66 nM.(ABSTRACT TRUNCATED AT 400 WORDS)

BACKGROUND

The multidrug resistance transporter, P-glycoprotein (P-gp), is involved in efflux transport of several antipsychotics in the blood-brain barrier (BBB).

OBJECTIVE

In the present study, we evaluated the inhibitory effect of the antipsychotics, i.e., risperidone, olanzapine, quetiapine, clozapine, haloperidol, chlorpromazine, a major metabolite of risperidone, 9-OH-risperidone, and a positive control inhibitor, PSC833, on the cellular uptake of a prototypic substrate of P-gp, rhodamine (Rhd) 123, in LLC-PK1 and L-MDR1 cells.

METHODS

After incubation of the antipsychotics (1-100 microM) and the positive (10 microM PSC833) or negative (1% dimethyl sulfoxide) controls with 5 microM Rhd 123 for 1 h, the effects of the antipsychotics on the intracellular accumulation of Rhd 123 were examined using a flow cytometric method.

RESULTS

All the antipsychotics showed various degrees of inhibitory effects on P-gp activity. The rank order of the concentration of inhibitor to cause 50% of the maximal increment of intracellular Rhd 123 fluorescence (EC(50)) was: PSC833 (0.5 microM) < olanzapine (3.9 microM) < chlorpromazine (5.8 microM) < risperidone (6.6 microM) < haloperidol (9.1 microM) < quetiapine (9.8 microM) < 9-OH-risperidone (12.5 microM) < clozapine (30 microM). Considering that the antipsychotics' plasma concentrations are generally lower than 1 microM, the present results suggest that olanzapine and risperidone are the only agents that may inhibit P-gp activity in the BBB. However, most of the antipsychotics are extensively accumulated in tissues. In addition, when given orally, the drug concentrations in the gastrointestinal tract are likely to be high.

CONCLUSIONS

Pharmacokinetic interactions due to inhibition of P-gp activity by the antipsychotics appear possible and warrant further investigation.

The intracellular pharmacokinetics and pharmacodynamics of oritavancin (LY333328) were studied in cultured cells. Oritavancin was avidly accumulated by J774 and THP-1 macrophages and rat fibroblasts and to a lesser extent by LLC-PK1 and Caco-2 cells. In J774 macrophages, the level of accumulation reached a plateau (at 370-fold the extracellular concentration) within 24 h and was partly defeated by a rise in serum protein levels. Efflux was incomplete (with a plateau at two-thirds of the original level at 6 h). In short-term kinetic studies, oritavancin uptake was linear for up to 4 h (as was the case for horseradish peroxidase and small latex beads, used as markers of the fluid phase and adsorptive endocytosis, respectively), which was in contrast to azithromycin and chloroquine uptake (which accumulate in cells by diffusion and segregation). The rates of clearance of oritavancin and latex beads were comparable (150 and 120 microl x mg of protein(-1) x h(-1), respectively) and were approximately 200 times higher than that of horseradish peroxidase. Oritavancin accumulation was partially reduced by monensin but was unaffected by acidic pH (these conditions abolished chloroquine accumulation). Cell-associated oritavancin was found in lysosomal fractions after homogenization of J774 macrophages and fractionation by isopycnic centrifugation. Oritavancin was bactericidal against intracellular Staphylococcus aureus (phagolysosomal infection) but was unable to control the intracellular growth of Listeria monocytogenes (cytosolic infection), even though its cellular concentration largely exceeded the MIC (0.02 mg/liter) and minimal bactericidal concentration (2 mg/liter). We conclude that oritavancin enters cells by adsorptive endocytosis (favored by its lipophilic side chain and/or the presence of three protonatable amines), which drives it to lysosomes, where it exerts antibiotic activity.

The human multidrug-resistance (MDR1) P-glycoprotein (Pgp) is an ATP-binding-cassette transporter (ABCB1) that is ubiquitously expressed. Often its concentration is high in the plasma membrane of cancer cells, where it causes multidrug resistance by pumping lipophilic drugs out of the cell. In addition, MDR1 Pgp can transport analogues of membrane lipids with shortened acyl chains across the plasma membrane. We studied a role for MDR1 Pgp in transport to the cell surface of the signal-transduction molecule platelet-activating factor (PAF). PAF is the natural short-chain phospholipid 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine. [(14)C]PAF synthesized intracellularly from exogenous alkylacetylglycerol and [(14)C]choline became accessible to albumin in the extracellular medium of pig kidney epithelial LLC-PK1 cells in the absence of vesicular transport. Its translocation across the apical membrane was greatly stimulated by the expression of MDR1 Pgp, and inhibited by the MDR1 inhibitors PSC833 and cyclosporin A. Basolateral translocation was not stimulated by expression of the basolateral drug transporter MRP1 (ABCC1). It was insensitive to the MRP1 inhibitor indomethacin and to depletion of GSH which is required for MRP1 activity. While efficient transport of PAF across the apical plasma membrane may be physiologically relevant in MDR1-expressing epithelia, PAF secretion in multidrug-resistant tumours may stimulate angiogenesis and thereby tumour growth.

Selenium, an essential trace element, showed the significant protective effects against kidney damage induced by some heavy metals. Our previous research have found that the protection effects of selenium on ROS mediated-apoptosis by mitochondria dysfunction in cadmium (Cd)-induced LLC-PK1 cells. The present study as a continuation of our earlier one to investigate the protective effects and mechanism of selenium on Cd-induced apoptosis of kidney in vivo. Cadmium exposure increased the production of reactive oxygen species (ROS) and altered the levels of oxidative stress related biomarkers in kidney tissue. A concomitant by the loss of mitochondrial membrane potential, cytochrome c release and regulation of VDAC, Bcl-2 and Bax were observed. Apoptotic nature of cell death is confirmed by activation of caspase-3, which is also supported by histological examination. During the process, selenium played a beneficial role against Cd-induced renal damage. Pretreatment with selenium partially blocked Cd-induced ROS generation, inhibited Cd induced mitochondrial membrane potential collapse, prevented cytochrome c release, inhibited caspase activation and changed the level of VDAC, Bcl-2 and Bax. Combining all, results suggest that selenium has an ability to inhibit mitochondrial apoptotic pathway in oxidative stress mediated kidney dysfunction caused by cadmium.

The mdr1 gene product P-glycoprotein (P-gp) is involved in the bioavailability and pharmacokinetics of various drugs. Racemic [(11)C]verapamil has been used to image P-gp expression in vivo. A racemic tracer, however, is not suitable for quantification. The purpose of the present study was to identify the most appropriate enantiomer of [(11)C]verapamil as a potential PET-tracer for quantifying P-gp function. The two enantiomers, (R)- and (S)-[(11)C]verapamil, were synthesized and studied in vivo. For the in vivo model mdr1a/1b double gene knock-out and wild type mice were used. The in vitro study made use of the LLC-PK1 MDR cell line to examine the P-gp mediated transport of both enantiomers. The biodistribution of (R)- and (S)-[(11)C]verapamil in dKO and WT mice demonstrated no stereoselectivity of verapamil for P-gp in the blood-brain barrier and in the testes. In addition, no significant differences in P-gp transport for both enantiomers were observed in the in vitro experiments. Previous studies have shown that (R)-verapamil is metabolized less in man and that it has lower affinity for calcium channels. Since (R)- and (S)-verapamil have equal transport for P-gp, the (R)-enantiomer seems to be the best and safest candidate as PET-tracer for measuring P-gp function in vivo.

Little information is available concerning the presence of viable Toxoplasma gondii in tissues of goats worldwide. In the present study, hearts of 234 goats obtained from a local USA grocery store were examined for T. gondii infection. Blood clot or fluid removed from each heart was tested for antibodies to T. gondii by using the modified agglutination test (MAT). Antibodies to T. gondii were found in 125 (53.4%) of 234 goats, with titers of 1:5 in 20, 1:10 in 44, 1:20 in 16, 1:40 in five, 1:160 in five, 1:320 in five, and 1:640 or higher in 30 goats. Hearts of 112 goats (46 goats <1:5, and 66 goats 1:10 or higher) were used for isolation of viable T. gondii by bioassays in mice. For bioassays, 50 g of the myocardium were digested in an acid pepsin solution and the digest inoculated into mice; the recipient mice were examined for T. gondii infection. Toxoplasma gondii was isolated from 29 goats; from hearts of one of 46 with titers of <1:5, one of nine with titers of 1:10, one of three with titers of 1:40, and 26 of 40 with titers of 1:160 or higher. Two isolates were highly virulent to outbred Swiss Webster mice; all infected mice died of toxoplasmosis, irrespective of the dose. All T. gondii isolates were subsequently grown in cell cultures. Genotyping of the 29 T. gondii isolates using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) from DNA obtained from cell culture grown tachyzoites revealed 12 genotypes. Nine isolates were clonal Type II lineage, four isolates had type II alleles at all loci except a type I allele at the Apico locus, and four isolates were clonal Type III. The remaining 12 strains were divided into nine atypical genotypes, including five new and four previously identified genotypes. DNA sequences of four introns (EF1, HP2, UPRT1 and UPRT7) and two genes (GRA6 and GRA7) were generated for the five new genotypes. Comparing these sequences with previously published data revealed no unique sequences in these goat strains. Taken together, these results indicate high parasite prevalence and moderate genetic diversity of T. gondii in goats, which have important implications in public health. We believe this is the first genetic analysis of T. gondii isolates from goats in the USA.

Murine prions transferred from brain to cultured cells gradually adapt to the new environment. Brain-derived 22L prions can infect neuroblastoma-derived PK1 cells in the presence of swainsonine (swa); that is, they are 'swa resistant'. PK1 cell-adapted 22L prions are swa sensitive; however, propagation in swa results in selection of swa-resistant substrains. Cloned, PK1 cell-adapted 22L prions were initially unable to develop swa resistance ('swa incompetent'); however, after serial propagation for 30-90 doublings, four of nine clones became swa competent, showing that swa-resistant 'mutants' arose during replication. Mutations in the case of prions are attributed to heritable changes in PrP(Sc) conformation. One clone remained swa incompetent even after 10(35)-fold expansion; surprisingly, after propagation in brain, it yielded swa-resistant prions, indistinguishable from the original 22L population. Thus, cell-adapted 22L prions assumed either mutable or virtually immutable conformations; however, when passaged through the brain all became mutable. Mutability is thus a substrain-specific attribute.

Maintenance of a negative membrane potential in the cortical collecting duct (CCD) principal cell depends on a small-conductance, inward-rectifying basolateral membrane K+ (Kir) channel. In the present study, a candidate cDNA encoding this K+ channel, CCD-IRK3, was isolated from a mouse collecting duct cell line, M1. CCD-IRK3 shares a high degree of homology with a human brain inward-rectifier K+ channel (Kir 2.3). By Northern analysis, CCD-IRK3 transcript (2.9 kb) was readily detected in M1 CCD cells but not in Madin-Darby canine kidney, LLC-PK1, Chinese hamster ovary, or monkey kidney fibroblast cell lines. CCD-IRK3-specific reverse transcription-polymerase chain reaction confirmed bonafide expression in the kidney. Functional expression studies in Xenopus oocytes revealed that CCD-IRK3 operates as strongly inward-rectifying K+ channel. The cation selectivity profile of CCD-IRK3 [ionic permeability values (PK/Pi), Tl>> or = Rb>> or = K+>>) NH4>> Na; inward-slope conductance (GK/Gi), Tl>> or = K+>>) NH4>> Na>> Rb] is similar to the macroscopic CCD basolateral membrane K+ conductance (GK/Gi, K+>>) NH4>> Rb; PK/Pi, Rb approximately equal to K+>>) NH4). CCD-IRK3 also exhibits the pharmacological features of the native channel. Patch-clamp analysis reveals that CCD-IRK3 functions as a high open probability, voltage-independent, small-conductance channel (14.5 pS), consistent with the native channel. Based on these independent lines of evidence, CCD-IRK3 is a possible candidate for the small-conductance basolateral Kir channel in the CCD.

Ouabain, a cardiotonic steroid and a specific inhibitor of the Na(+)-K(+)-ATPase, has been shown to significantly inhibit transcellular Na(+) transport without altering the intracellular Na(+) concentration ([Na(+)](i)) in the epithelial cells derived from the renal proximal tubules. We therefore studied whether ouabain affects the activity and expression of Na(+)/H(+) exchanger isoform 3 (NHE3) representing the major route of apical Na(+) reabsorption in LLC-PK(1) cells. Chronic basolateral, but not apical, exposure to low-concentration ouabain (50 and 100 nM) did not change [Na(+)](i) but significantly reduced NHE3 activity, NHE3 protein, and mRNA expression. Inhibition of c-Src or phosphoinositide 3-kinase (PI3K) with PP2 or wortmannin, respectively, abolished ouabain-induced downregulation of NHE3 activity and mRNA expression. In caveolin-1 knockdown LLC-PK(1) cells, ouabain failed to downregulate NHE3 mRNA expression and NHE3 promoter activity. Ouabain response elements were mapped to a region between -450 and -1,194 nt, where decreased binding of thyroid hormone receptor (TR) and Sp1 to their cognate cis-elements was documented in vitro and in vivo by protein/DNA array analysis, EMSA, supershift, and chromatin immunoprecipitation. These data suggest that, in LLC-PK(1) cells, ouabain-induced signaling through the Na(+)-K(+)-ATPase-Src pathway results in decreased Sp1 and TR DNA binding activity and consequently in decreased expression and activity of NHE3. These novel findings may represent the underlying mechanism of cardiotonic steroid-mediated renal compensatory response to volume expansion and/or hypertension.

Tumor promoting phorbol esters and mezerein strongly induced plasminogen activator (urokinase, uPA) synthesis in porcine kidney cell cultures (LLC-PK1). Induction was due to increased uPA-mRNA levels which rose from 10 to 300 molecules/cell within 2 h of exposure to 16 nM phorbol myristate acetate. We have compared the action of tumor promoters with that of 8-bromo-cAMP, another potent inducer of uPA; the similarities between the two kinds of induction were: both involved transcriptional activation of the uPA gene; both were rapid in onset, changes in transcription rate being detectable within 10-20 min; the initial rates of transcription and uPA-mRNA accumulation were substantial and in the same order of magnitude; neither class of inducer required protein synthesis to stimulate uPA transcription. The main contrast between the two types of agents was that the uPA response to tumor promoters was transient whereas that to cAMP compounds was sustained: cultures rapidly lost their response to tumor promoters within 2 h after initial exposure while retaining responsiveness to cAMP-related agents. The cells developed a specific drug-induced desensitization which was slowly reversed after tumor promoters were removed from the culture medium. Since protein kinase C is now well established as the receptor for phorbol-derived and several other tumor promoters it will be of interest to determine whether desensitization occurs at the level of receptor.

Bioavailability of talinolol, a beta(1)-adrenergic receptor antagonist, was enhanced by coadministration with grapefruit juice (GFJ) in rats, whereas GFJ ingestion markedly reduced the absorption of talinolol in humans. Because our recent study indicated that the inhibitory effect of GFJ on organic anion-transporting polypeptide (Oatp)- and P-gp-mediated talinolol absorption depends on the concentration of naringin in ingested GFJ, the apparent inconsistent findings may be explained by the species difference in the affinity of naringin for OATP/Oatp and P-gp multidrug resistance 1 (MDR1/Mdr1) between humans and rats. Although human MDR1-mediated talinolol transport was not inhibited by 2000 microM naringin, naringin inhibited human OATP1A2-, rat Oatp1a5-, and rat Mdr1a-mediated talinolol transport with IC(50) values of 343, 12.7, and 604 microM, respectively, in LLC-PK1 cell and Xenopus laevis oocyte systems. Because the naringin concentration in commercially prepared GFJ was found to be approximately 1200 microM, these results suggested that GFJ would reduce the intestinal absorption of talinolol through inhibition of OATP1A2-mediated talinolol uptake in humans, whereas an increase of talinolol absorption is mainly through inhibition of Mdr1a-mediated efflux in rats. The rat intestinal permeability of talinolol measured by the in situ closed loop method was indeed significantly increased in the presence of GFJ, whereas a significant decrease was observed with 6-fold diluted GFJ, in which the naringin concentration was approximately 200 microM. The present study indicated that the species difference in the effect of GFJ on intestinal absorption of talinolol between humans and rats may be due to differences in the affinity of naringin for OATP/Oatp and MDR1/Mdr1 transporters between the two species.

Oxidant-induced cell injury has been implicated in the pathogenesis of several forms of acute renal failure. The present studies examined whether activation of poly(ADP-ribose)polymerase (PARP) by oxidant-induced DNA damage contributes to oxidant injury of renal epithelial cells. H2O2 exposure resulted in an increase in PARP activity and decreases in cell ATP and NAD content. These changes were significantly inhibited by 10 mM 3-aminobenzamide (3-ABA), a PARP inhibitor. In contrast, H2O2-induced DNA damage was not prevented by 3-ABA. Exposure of LLC-PK(1) cells to 1 mM H2O2 for 2 h induced necrotic cell death as measured by increased lactate dehydrogenase (LDH) release. 3-ABA completely prevented the H2O2-induced LDH release. Live/dead fluorescent staining confirmed the protection by 3-ABA. These results are consistent with the view that oxidant-induced DNA damage activates PARP and that the subsequent ATP and NAD depletion contribute to necrotic cell death. Of note, although protected from necrosis, cells treated with H2O2 and 3-ABA underwent apoptosis as evidenced by DNA fragmentation and bis-benzimide staining. In conclusion, activation of PARP contributes to oxidant-induced ATP depletion and necrosis in LLC-PK1 cells. However, PARP inhibition may target cells toward an apoptotic form of cell death.

The solvent-accessibility-modified, Tanford-Kirkwood, discrete charge model for electrostatic effects is applied to both ribonuclease A and ribonuclease S. The behavior of individual titratable sites and the pH-dependent free energy of denaturation are correctly predicted. The use of the solvent-accessibility factor in reducing charge-site interactions introduces a higher Coulombic shielding for solvent-exposed sites. This shielding is interpreted as a higher local strength or alternatively a higher effective dielectric constant. Specific anion binding sites are determined by locating areas of high positive electrostatic potential at the protein solvent interface. The potential and thus the anion affinity of a given site are calculated and shown to vary with the pH-dependent charge array. pH-dependent anion binding constants are calculated for the ribonuclease S active site. These binding constants and the predicted response of the active-site histidine pK1/2 values to anion binding are shown to agree with experimental determinations.

Calcium oxalate (CaOx) nephrolithiasis was induced in male Sprague-Dawley rats by administration of 0.75% ethylene glycol. Urinary excretion of lactate dehydrogenase (LDH) was used as a marker of cellular injury. Lipid peroxides (LP), as marker for free radical injury, were measured as malondialdehyde (MDA) in urine and the kidneys. Urinary oxalate (Ox), LDH, LP, CaOx crystals, and renal LP and CaOx crystal deposits were examined on day 0, 5, 30 and 60 of the experiment. There were significant differences between control and experimental rats in all the parameters except LDH which did not show a significant increase after 15 days. Subconfluent cultures of MDCK and LLCPK1 cells were exposed to various concentrations of oxalate and/or 500 fg/ml CaOx crystals. Cell viability was assayed by trypan blue exclusion, cellular injury was determined by measuring LDH in the media, and free radical injury was measured as MDA contents of the cells. On exposure to both Ox and/or CaOx crystals trypan blue exclusion decreased and LDH and MDA increased significantly in both tissue cultures. LLC-PK1 appeared more sensitive. The results indicate that both oxalate and calcium oxalate crystals are injurious to renal epithelial cells in the kidneys as well as in culture.