Pig Kidney Epithelial Cells Research Articles

Stimulation by serum of the Na+/H+ antiporter in quiescent pig kidney epithelial (LLC-PK1) cells and role of the antiporter in the reinitiation of DNA synthesis.

LLC-PK1 cells can be brought into a classical quiescent state by depriving them of serum for 6 days. At this time, pulse-labeling with [3H]-thymidine shows that only 3% of the cells are synthesizing DNA, but the quiescent cells can be stimulated with serum to re-enter the cell cycle at a point early in G1. The rate of amiloride-sensitive 22Na+ uptake (as a measure of the Na+/H+ antiporter) is relatively low during quiescence; it rises 2- to 3-fold within 4 h after serum addition. This increase in antiporter activity appears to be required for the resumption of DNA synthesis in the absence of bicarbonate, because ethylisopropylamiloride (EIPA) blocks [3H]-thymidine incorporation when serum is added to cells in bicarbonate-free medium. In the presence of bicarbonate, however, EIPA has no effect on [3H]-thymidine incorporation, indicating that another (bicarbonate-dependent) transport system can substitute for the antiporter under these conditions.

Parallel changes in amino acid transport and protein kinase C localization in LLC-PK1 cells treated with TPA or diradylglycerols.

Protein kinase C is considered to be a major target for tumor promoting phorbol esters such as 12-0-tetradecanoylphorbol-13-acetate (TPA). We have analyzed the correlation between A-system amino acid transport and the distribution of protein kinase C (PKC) between a membrane-rich fraction (100,000 g pellet) and cytosol (supernatant) from homogenized LLC-PK1 cells, a pig kidney epithelial cell line grown in culture. During log growth 1 day after seeding the cells onto culture plates, PKC activity is high in the membrane fraction and low in the cytosol. As the cells become confluent the PKC distribution shifts to a cytosolic pool. Concomitantly, A-system amino acid transport, as measured by methylaminoisobutyric acid [14C]MeAIB uptake, decreases. TPA (0.01-1.0 microM) induces a shift of PKC activity from the cytosol back to the membrane-rich fraction in post-confluent cells with a concomitant 2-3 fold stimulation of MeAIB uptake. The same responses can be achieved by treating cells with certain diradylglycerols, either diacylglycerols such as 1-oleyl-2-acetyl-sn-glycerol (OAG) or alkylacylglycerols such as 1-hexadecenyl-2-oleyl-sn-glycerol. Both responses to TPA are blocked by cytochalasin B, but cycloheximide inhibits the transport response without affecting PKC redistribution. It is suggested that the redistribution may be a necessary but not sufficient concomitant to the transport activation.

On the Mechanism of Sphingomyelin Interaction with Solubilized Membrane Proteins

Studies on the high-affinity receptor for IgE from rat basophilic leukemia cells (RBL-2H3) have shown that the phospholipid sphingomyelin remains attached to the protein complex during washing of the affinity immobilized complex under solubilizing conditions. Here we extended these findings and compared the species distribution patterns in sphingomyelin and phosphatidylcholine of the receptor-bound lipids to those of the plasma membrane lipids. FC epsilon-receptor-bound sphingomyelin but not phosphatidylcholine was enriched in long-chain fatty acids. We then examined other membrane proteins with respect to sphingomyelin enrichment. RBL-2H3 cell surface proteins, immobilized on concanavalin A-Sepharose and washed under solubilizing conditions, also showed a two- to six-fold enrichment in the associated sphingomyelin. Similar observations were also derived from other cell types, such as the mouse fibroblast cell line A-9 and the pig kidney epithelial cell line PK-1. Since this has been observed in all the three cell sources, it was suggested that sphingomyelin enrichment in FC epsilon-receptor preparations, although reproducible, was not specific for this protein. That this phenomenon was not specific for a particular protein might also be concluded from experiments that have shown nonhomogenous distribution of sphingomyelin in protein-free lipid-detergent mixtures. These results are compatible with a model whereby the interaction between sphingomyelin and soluble membrane proteins results from preference to nonmicellar phases or to structures with extended hydrophobic domains, probably due to the imperfect fitness of the detergent micelles to properly accomodate these lipids. This feature makes long-chain sphingomyelin a plausible candidate for the lipid responsible for the stabilizing effect that crude lipid preparations exert on the structural and functional properties of some membrane protein, e.g., FC epsilon R.

Induction of mitochondrial metabolism and pH-modulated ammoniagenesis by rocking LLC-PK1 cells.

In an effort to find a model system in which the regulation of renal ammoniagenesis could be delineated, the LLC-PK1 line of cultured pig kidney epithelial cells was examined. Ammonia production by normally cultured LLC-PK1 cells (monolayers on 75-cm2 flasks, under 6 ml still serum-containing medium) was found to be neither modulated by direct (3 h) nor adapted by chronic (3 day) manipulation of medium pH (production at pH 7.05, 7.40, or 7.60 not significantly different). Considering that the mitochondrial glutamine to alpha-ketoglutarate pathway is the major regulatory site of ammoniagenesis, and that mitochondrial metabolism might be restricted under the normal lactate-generating or hypoxic culture conditions, we examined ammonia production by rocked flasks of LLC-PK1 cells. Flask were rocked continually at a rate of 2.5 oscillations/min, thereby exposing the cells to the atmosphere 40-50% of the time and also maximizing medium O2 tension and nutrient-waste exchange. Mitochondrial enzyme activities in rocked LLC-PK1 cells were shown to be consistently 1.5-fold greater than those in normally cultured cells. Ammonia production by rocked cells was not only directly modulated by short incubations with low and high pH media (production at pH 7.05 greater than 7.40 greater than 7.60) but was adapted by chronic (3 day) manipulations of medium pH (16 h after return to pH 7.40 medium production by pH 7.05- greater than 7.40- greater than 7.60-adapted cells). Thus rocker culture of the LLC-PK1 cell line both induces mitochondrial metabolism and converts cellular ammoniagenesis to a pH-sensitive phenomenon.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid alterations in LLC-PK1 cells exposed to mercuric chloride

We have studied the effects of HgCl2 on the lipids of LLC-PK1 (pig kidney) epithelial cells. Our results show that treatment of cells with HgCl2 caused a rapid accumulation of unesterified fatty acids (particularly arachidonic acid) and lysophospholipids. A 27-fold increase in unesterified arachidonic acid and a 17-fold increase in lysophosphatidylethanolamine (LPE) was accompanied by a 26% decline in the mass of phosphatidylethanolamine as determined by gas chromatography and lipid phosphorus assay. Similar changes were seen following HgCl2 treatment of cells whose lipids were labelled with 14C stearic acid, 3H arachidonic acid, or 14C acetate, but the radiolabelling techniques also identified an increased content of label in lysophosphatidylcholine (LPC) and a corresponding decrease in phosphatidylcholine. These alterations were accompanied by the formation of blebs on the plasma membrane and irreversible injury as indicated by electron microscopy. The possible role of unesterified fatty acids in the pathogenesis of injury was studied by adding fatty acids to the cells. The addition of unsaturated fatty acids (oleic, linoleic, or arachidonic acids) to the cells caused plasma membrane blebbing and loss of viability. Similarly, the addition of LPC or LPE to the cells resulted in cell death; however, plasma membrane blebbing did not result.

Na +/H + exchanger activity in the pig kidney epithelial cell line, LLC-PK 1: Inhibition by amiloride and its derivatives

Rapidly growing pig-kidney-derived epithelial cells, LLC-PK1, lack detectable amiloride-sensitive Na+/H+ exchange activity when assayed directly. A large 22Na uptake is induced when the cells are acid-loaded prior to assay by incubation with buffer containing ammonium chloride or nigericin. The acid-stimulated sodium uptake is sensitive to amiloride, with half-maximal inhibition at 3.5-4.5 microM in buffer containing 15 mM sodium ion. There is simple competitive interaction between amiloride and sodium ion when the amiloride concentration is below 25 microM and the sodium ion concentration is above 20 mM. Derivatives of amiloride which carry substituents on the 5-amino group are 35- to 175-fold more inhibitory than amiloride itself.

Cytotoxic effect of Pasteurella haemolytica on ovine bronchoalveolar macrophages in vitro

Live Pasteurella haemolytica A1 was shown to have a cytotoxic effect on suspensions of sheep bronchoalveolar macrophages. Cytotoxic activity was also demonstrable in bacteria-free supernatants from suspensions containing P. haemolytica. Heat-killed and ultraviolet killed organisms of P. haemolytica and live Staphylococcus aureus were not toxic to sheep BAM. These results suggest that a bacterial cell-free cytotoxin is produced by metabolically active P. haemolytica. Guinea-pig peritoneal macrophages, McCoy and pig kidney epithelial cell suspensions were unaffected by live P. haemolytica and supernatant from P. haemolytica cultures, indicating that the cytotoxin may only affect phagocytic cells of ovine or bovine origin.

Microtubules and microfilaments during cell spreading and colony formation in PK 15 epithelial cells.

We have studied the distribution of microtubules and microfilaments during the cell spreading and subsequent colony formation in PK 15 pig kidney epithelial cells using indirect immunofluorescence. During the cell spreading on a solid substratum, microtubules grew out from the region around the nucleus, and a collar of microfilament bundles formed around the cell periphery. Although virtually all well-spread cells showed a complex microtubular network, distinctly different patterns of stress fibers were observed. In small colonies, the most commonly observed pattern was a ring of microfilament bundles that appeared to be in register between adjacent cells and encircled the entire colony in a fashion similar to that seen in single cells. In large colonies (more than 50 cells), approximately 60% of the cells displayed clearly stained microfilament bundles, either at the cell periphery or throughout their cytoplasm, whereas in the remaining 40%, no microfilament bundles were observed and only the outline of the cells was delineated by interaction with anti-actin. Such "negative" cells were seen in groups alongside "positive" cells (i.e., cells possessing extensive stress fiber networks) within the same colony. Independent of their stress fiber phenotype, all cells maintained a flattened shape and an extensive network of microtubules. We suggest that dense microfilament bundles are not a uniform feature of well-spread PI 15 cells in culture and that a loss of microfilament bundle occurs in some cells.

The necessity for cellular RNA and protein synthesis for viral inhibition resulting from interferon

Experimental evidence indicated that interferon-inducible guanylate-binding proteins (GBPs) are similar with genes in the myxovirus (Mx) resistance protein subfamily of the large GTPases protein family and play important roles in the resistance to intracellular pathogens. As more diseases exert significant influence on pig industry, it is anticipated that more candidate disease-resistance genes could be found in future strategies aimed at improving genetic resistance to infectious diseases. In this study, we cloned cDNA sequences and analyzed the genomic structure of porcine GBP1 (poGBP1) and GBP2 (poGBP2). The two genes were mapped to SSC4q21-q23 and SSC4q24 by the SCHP panel respectively, further IMpRH panel analysis showed both genes were most closely linked to the marker SWR153. The deduced amino acid sequences of these two genes share the same three classical GTP-binding motifs at the amino terminus and are less conserved at the carboxyl termini except for a CaaX motif, compared with human and mouse counterparts. The reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that poGBP1 and poGBP2 were both widely expressed in many tissues, and transient transfection indicated that poGBP1 and poGBP2 proteins were both located in cytoplasms within Pig Kidney Epithelial cells (PK15). Quantitative real-time PCR (Q-RT-PCR) analyses showed poGBP1 and poGBP2 had very similar expression patterns in PK15 cells at different time points after poly I:C stimulation, suggesting that ISRE (interferon-stimulated response element) plays a crucial role in the transcriptional regulation of these two genes. Four single nucleotide polymorphisms (SNPs) and three SNPs were detected in the cDNA sequences of poGBP1 and poGBP2, respectively. Association analyses revealed that the poGBP1 Eco81I and poGBP2 SspI polymorphisms both had significant associations (p < 0.05) with red blood cell count (RBC), haemoglobin concentration (HGB) and hematocrit (HCT) of 17-day-old pigs.

Studies on Enteroviruses of the Pig—VI: The Relationship of the T80 Strain of a Swine Polio-encephalomyelitis Virus to some other Viruses as Shown by Neutralization Tests in Tissue Cultures

SUMMARY Cross-neutralization tests were made in monolayer cultures of pig kidney epithelial cells with the T80 strain of a swine polio-encephalomyelitis virus against antisera to the following viruses: poliovirus types 1, 2 and 3, Coxsackie virus types A9 and B1, B2, B3, B4 and B5, Echo virus types 1 to 14 inclusive, canine hepatitis virus, and louping ill virus. None of the antisera to these viruses neutralized the T80 virus. Both the T80 and T52A strains of a swine polio-encephalomyelitis virus, which are closely related antigenically, were tested against antisera to the viruses of Talfan and Teschen diseases. Neither strain was neutralized by these antisera: nor did antisera to the T80 and T52A viruses neutralize the viruses of Talfan and Teschen diseases. These results indicate that at least 2 immunologically distinct viruses can produce polio-encephalomyelitis in pigs.