Cultured Madin-Darby Canine Kidney Cells Research Articles

Persistent Infection of MDCK Cells by Influenza C Virus: Initiation and Characterization

Persistent influenza C virus infection was readily initiated in Madin-Darby canine kidney (MDCK) cells at low m.o.i. and has been maintained for over 1 year. The persistently infected (p.i.) cultures were characterized by the following properties: virus infection was limited to a minority of cells, small amounts of infectious virus were produced together with low levels of interferon (IFN) and the cultures were resistant to superinfection by homologous virus and vesicular stomatitis virus, but not by influenza A and B viruses. These properties fluctuated cyclically with passage of the p.i. culture. When p.i. cultures were cured by cultivation in the presence of antiserum, the cultures lost their IFN-producing activity and became as susceptible to homologous virus as normal MDCK cell culture. The results suggest that persistent influenza C virus infection may be regulated by endogenously produced IFN. Under the condition of high m.o.i. a persistent influenza C virus infection could not be initiated in MDCK cells due to the development of cytopathic effects.

Pulmonary hemodynamics and blood parameters in rats exposed to long-term high-altitude hypoxia

A study was made of the effect of antiviral drugs remantadine and deitephorine on dynamics in the changes in structure-function parameters, mitotic activity, protein and nucleic acid synthesis in the Madin-Darby canine kidney cell culture. The application of methodical complex and regression analysis methods, the approximation of experimental data with analytical functions showed general regularity in the monolayer cell population response to the effect of drugs. In the response of cell populations three phases are distinguished: the first phase--transient inhibition; the second phase--relative compensation; the third phase--pathological cell destructive process.

Carbonic Anhydrase Activity in Madin Darby Canine Kidney Cells

Madin Darby canine kidney (MDCK) renal epithelial cell cultures have been investigated with respect to their potency to express carbonic anhydrase activity using histochemical methods. Acetazolamide inhibitable carbonic anhydrase activity could be detected in the cytoplasmic compartment as well as in the apical membrane of cells when grown on solid culture supports. Cells forming domes in MDCK monolayers exhibit the highest histochemically detectable enzyme activity. The attempt to subculture clonal cell lines from MDCK monolayer cultures resulted in the establishment of 5 clones, slightly different with respect to size and shape of cells and their potency to form domes. Scanning electron microscopy ensured the identification of one clone (1A4), which distinctly differed from the others with respect to the apical membrane architecture. Co-localization of peanut agglutinin and carbonic anhydrase activity at the plasma membrane always revealed a combined occurrence of enzyme reactivity and lectin binding in the apical membrane domain. Both, lectin binding and carbonic anhydrase activity were distinctly more intense in plasma membrane regions equipped with microvilli. From the results it is concluded that MDCK cells in tissue culture retained properties of intercalated cells of the nephron collecting duct segment.

Oxidants and conductance of cultured epithelial cell monolayers: inositol phospholipid hydrolysis.

Reactive oxidants contribute to the alterations in endothelial and epithelial permeability that characterize the inflammatory response. We previously noted that noncytolytic doses of oxidants reversibly decreased the electrical resistance across cultured monolayers of Madin-Darby canine kidney (MDCK) cells (J. Clin. Invest. 76: 1155-1168, 1985). In this investigation, we have found that similar doses of oxidants initiate inositol phospholipid hydrolysis by a phospholipase C in cultured MDCK cells, with resultant increases in inositol polyphosphates, phosphatidic acid, and 1,2 diglycerides. Activation of this pathway is linked to activation of protein kinase C in many cells. The addition of phorbol 12,13-dibutyrate (PDBU) and 1-oleoyl-2-acetyl-sn-glycerol, activators of protein kinase C, decreased the electrical resistance across MDCK monolayers cultured on micropore filters similar to the effects of hydrogen peroxide. In contrast, the addition of 4 alpha-phorbol 12,13-didecanoate, a chemically similar compound that does not activate protein kinase C, did not decrease the electrical resistance. When MDCK monolayers were exposed to PDBU, fixed, and stained with rhodamine phallicidin, the peripheral band of actin in the cells showed a loss of staining density and continuity similar to the changes in phallicidin staining we previously noted in cells exposed to hydrogen peroxide. These data are consistent with the hypothesis that some of the reversible effects of oxidants on epithelial barriers are mediated through phospholipase C hydrolysis of inositol phospholipids with consequent activation of protein kinase C.

Bradykinin-activated membrane-associated phospholipase C in Madin-Darby canine kidney cells.

Previous studies have demonstrated that bradykinin stimulates the rapid release of inositol 1,4,5 trisphosphate (IP3) from membrane phosphatidylinositol 4,5 bisphosphate (PIP2) in Madin-Darby canine kidney (MDCK) cells. Since current evidence would suggest that the activation of phospholipase C (PLC) is mediated through a guanine nucleotide-binding protein in receptor-mediated activation of PLC, we evaluated the role of guanine nucleotide proteins in receptor-mediated (bradykinin-stimulated) activation of PLC in MDCK cells. Bradykinin at 10(-7) M produced a marked increase in IP3 formation within 10 s increasing from a basal level of 46.2 to 686.6 pmol/mg cell protein a 15-fold increase. Pretreatment of MDCK cells in culture with 200 ng/ml of pertussis toxin for 4 h reduced the bradykinin-stimulated response to 205.8 pmol/mg protein. A 41-kD protein substrate in MDCK membranes was ADP ribosylated in vitro in the presence of pertussis toxin. The ADP ribosylation in vitro was inhibited by pretreatment of the cells in culture with pertussis toxin. Membranes from MDCK cells incubated in the presence of [3H]PIP2/phosphatidyl ethanolamine liposomes demonstrated hydrolysis of [3H]PIP2 with release of [3H]IP3 when GTP 100 microM or GTP gamma S 10 microM was added. Bradykinin 10(-7) M added with GTP 100 microM markedly increased the rate of hydrolysis within 10 s, thus demonstrating a similar time course of PLC activation as intact cells. These results demonstrate that bradykinin binds to its receptor and activates a membrane-associated PLC through a pertussis toxin-sensitive, guanine nucleotide protein.

The lateral mobility of the (Na+,K+)-dependent ATPase in Madin-Darby canine kidney cells.

Fluorescence microphotolysis (recovery after photobleaching) was used to determine the lateral mobility of the (Na+,K+)ATPase and a fluorescent lipid analogue in the plasma membrane of Madin-Darby canine kidney (MDCK) cells at different stages of development. Fluorescein-conjugated Fab' fragments prepared from rabbit anti-dog (Na+,K+)ATPase antibodies (IgG) and 5-(N-hexadecanoyl)aminofluorescein (HEDAF) were used to label the plasma membrane of confluent and subconfluent cultures of MDCK cells. Fractional fluorescence recovery was 50% and 80-90% for the protein and lipid probes, respectively, and was independent of developmental stage. The estimated diffusion constants of the mobile fraction were approximately 5 X 10(-10) cm2/s for the (Na+,K+)ATPase and approximately 2 X 10(-9) cm2/s for HEDAF. Only HEDAF diffusion showed dependency on developmental stage in that D for confluent cells was approximately twice that for subconfluent cells. These results indicate that (Na+,K+)ATPase is 50% immobilized in all developmental stages, whereas lipids in confluent MDCK cells are more mobile than in subconfluent cells. They suggest, furthermore, that the degree of immobilization of the (Na+,K+)ATPase is insufficient to explain its polar distribution, and they support restricted mobility of the ATPase through the tight junctions as the likely mechanism for preventing the diffusion of this protein into the apical domain of the plasma membrane in confluent cell cultures.

Studies on the mechanism of phospholipid storage induced by amantadine and chloroquine in Madin Darby canine kidney cells

Previous studies suggested that hepatic lipidosis caused by cationic amphiphilic drugs in rats is related to the capacity of these drugs to concentrate in liver lysosomes. These drugs inhibit lysosomal phospholipases, causing phospholipid accumulation. Amantadine, an inhibitor of influenza A virus replication, is a cationic amphiphilic drug which concentrates in the lysosomes of the Madin Darby canine kidney (MDCK) cell. In the present study, amantadine and chloroquine were shown to inhibit soluble lysosomal phospholipases isolated from MDCK cell in. vitro. Both amantadine and chloroquine concentrated in MDCK cell lysosomes. These drugs caused phospholipid storage in cultured MDCK cells, and the amounts of the respective agents required to cause phospholipid storage correlated with the capacities of the agents to inhibit lysosomal phospholipases. The mechanisms involved in this phenomenon are discussed, and a three-step hypothesis is presented predicting which agents will cause phospholipidosis.

Adherence of staphylococcus aureus to influenza A virus-infected Madin-Darby canine kidney cell cultures.

Madin-Darby canine kidney cells infected with influenza A virus (strains PR8, FM1, Jap 305, and Tex 1) were tested with nine strains of Staphylococcus aureus and group B Streptococcus type Ic to determine whether mammalian cells become susceptible to bacterial adherence as a result of virus infection. Bacterial adherence to virus-infected cells varied depending on the virus strain and on the strain of bacteria tested. A quantitative radioassay was developed to study the parameters of adherence. Attachment of 3H-labeled S. aureus grown in chemically defined or biologically complex medium to FM1 virus-infected cells was significantly increased (P less than 0.0005) compared with attachment to control cells. Adherence coincided with the appearance of hemadsorption, which is a marker of the presence of virus-induced glycoproteins on the cell surface. Adherence was temperature dependent, increased with a decrease in hydrogen ion concentration, and was not affected by the presence of K+, Mg2+, or Ca2+. Adherence was blocked when 3H-labeled S. aureus was pretreated with trypsin but not when cells were pretreated with neuraminidase.

Studies with a cold-recombinant A/Victoria/3/75 (H3N2) virus. I. biologic, genetic, and biochemical characterization.

A cold-recombinant virus CR 22, was derived from an attenuated cold-adapted parent strain. A/Ann Arbor/6/60 (H2N2), and a wild-type parent strain, A/Victoria/3/75 (H3N2). Antigenic analysis showed that CR 22 possesses the hemagglutinin and neruaminidase surface antigens derived from the A/Victoria/3/75 (H3N2) parent. From studies of virus-induced polypeptides using polyacrylamide gel electrophoresis, it was deduced that a polymerase protein, P1, is coded and by an RNA segment derived from the wild-type parent; all other genetic elements are derived from the attenuated parent. The attenuated parent and the recombinant CR 22 both possess temperature-sensitive (ts) lesions, evident by restriction of multiplication in fertile chicken eggs or in Madin-Darby canine kidney cell cultures at greater than or equal to 38 C. Genetic analysis of CR 22 by complementation tests using Hong Kong and Wilson Smith neurotropic ts mutants gave evidence for a ts lesion in the genetic elements coding for complementary RNA.

3-Deazauridine: inhibition of ribonucleic acid virus-induced cytopathogenic effects in vitro.

3-Deazauridine, a synthetic analogue of uridine and a potential antitumor agent, was found to possess antiviral activity against a number of ribonucleic acid-containing animal viruses in mammalian cell culture. Inhibition of virus-induced cytopathogenic effects by 3-deazauridine was observed in cells infected with rhinovirus types 1A and 13, coxsackievirus type A21, and PR-8 influenza virus, but not in cells infected with poliovirus type 2 or echovirus type 12. The extracellular yield of progeny influenza virus was found to be significantly reduced in titer in 3-deazauridine-treated Madin-Darby canine kidney cell cultures at 12 to 24 hr after infection. Although the precise biochemical mechanism of action of this antimetabolite's antiviral activity is not known, the compound does not appear to exert a direct virucidal effect on the influenza virion itself.