Differentiation-dependent redistribution of heparan sulfate in epithelial intestinal Caco-2 cells leads to basolateral entry of cytomegalovirus.

Human cytomegalovirus (HCMV) causes a broad spectrum of clinical manifestations in immunocompromised patients, including infection of the gastrointestinal tract. To investigate the role of epithelial cells in the gastrointestinal HCMV disease, we used the intestinal epithelial cell line Caco-2, which is permissive for HCMV replication. In differentiated Caco-2 cells, we showed previously that HCMV infection proceeds preferentially from the basolateral membrane, suggesting that receptors for HCMV may be contained predominantly in the basolateral membrane (A. Esclatine et al., 2000, J. Virol. 74, 513-517). Therefore, we examined expression and localization in Caco-2 cells of heparan sulfate (HS) proteoglycan and annexin II, previously implicated in initial events of HCMV infection. We observed that annexin II is expressed in Caco-2 cells, but is not essential for entry of HCMV. We showed that, during the differentiation process, HS, initially present on the entire surface of the membrane of undifferentiated cells, ultimately became sequestered at the basolateral cell surface of fully differentiated cells. We established by biochemical assays that membrane-associated HS proteoglycan mediates both viral attachment to, and subsequent infection of, Caco-2 cells, regardless of the cell differentiation state. Thus, the redistribution of HS is implicated in the basolateral entry of HCMV into differentiated Caco-2 cells.

Rotavirus infection induces cytoskeleton disorganization in human intestinal epithelial cells: implication of an increase in intracellular calcium concentration.

Rotavirus infection is the most common cause of severe infantile gastroenteritis worldwide. In vivo, rotavirus exhibits a marked tropism for the differentiated enterocytes of the intestinal epithelium. In vitro, differentiated and undifferentiated intestinal cells can be infected. We observed that rotavirus infection of the human intestinal epithelial Caco-2 cells induces cytoskeleton alterations as a function of cell differentiation. The vimentin network disorganization detected in undifferentiated Caco-2 cells was not found in fully differentiated cells. In contrast, differentiated Caco-2 cells presented Ca(2+)-dependent microtubule disassembly and Ca(2+)-independent cytokeratin 18 rearrangement, which both require viral replication. We propose that these structural alterations could represent the first manifestations of rotavirus-infected enterocyte injury leading to functional perturbations and then to diarrhea.

Rotavirus infection induces an increase in intracellular calcium concentration in human intestinal epithelial cells: role in microvillar actin alteration.

Rotaviruses, which infect mature enterocytes of the small intestine, are recognized as the most important cause of viral gastroenteritis in young children. We have previously reported that rotavirus infection induces microvillar F-actin disassembly in human intestinal epithelial Caco-2 cells (N. Jourdan, J. P. Brunet, C. Sapin, A. Blais, J. Cotte-Laffitte, F. Forestier, A. M. Quero, G. Trugnan, and A. L. Servin, J. Virol. 72:7228-7236, 1998). In this study, to determine the mechanism responsible for rotavirus-induced F-actin alteration, we investigated the effect of infection on intracellular calcium concentration ([Ca(2+)](i)) in Caco-2 cells, since Ca(2+) is known to be a determinant factor for actin cytoskeleton regulation. As measured by quin2 fluorescence, viral replication induced a progressive increase in [Ca(2+)](i) from 7 h postinfection, which was shown to be necessary and sufficient for microvillar F-actin disassembly. During the first hours of infection, the increase in [Ca(2+)](i) was related only to an increase in Ca(2+) permeability of plasmalemma. At a late stage of infection, [Ca(2+)](i) elevation was due to both extracellular Ca(2+) influx and Ca(2+) release from the intracellular organelles, mainly the endoplasmic reticulum (ER). We noted that at this time the [Ca(2+)](i) increase was partially related to a phospholipase C (PLC)-dependent mechanism, which probably explains the Ca(2+) release from the ER. We also demonstrated for the first time that viral proteins or peptides, released into culture supernatants of rotavirus-infected Caco-2 cells, induced a transient increase in [Ca(2+)](i) of uninfected Caco-2 cells, by a PLC-dependent efflux of Ca(2+) from the ER and by extracellular Ca(2+) influx. These supernatants induced a Ca(2+)-dependent microvillar F-actin alteration in uninfected Caco-2 cells, thus participating in rotavirus pathogenesis.

Human cytomegalovirus infects Caco-2 intestinal epithelial cells basolaterally regardless of the differentiation state.

Human cytomegalovirus (CMV) causes severe disease in immunosuppressed patients and notably infects the gastrointestinal tract. To understand the interaction of CMV with intestinal epithelial cells, which are highly susceptible to CMV infection in vivo, we used the intestinal epithelial cell line Caco-2 and demonstrated that CMV enters predominantly through the basolateral surface of polarized Caco-2 cells. As shown by expression of all three classes of CMV proteins and by visualization of nucleocapsids by transmission electron microscopy, both poorly and fully differentiated Caco-2 cells were permissive to CMV replication. However, infection failed to produce infectious particles in Caco-2 cells, irrespective of the state of differentiation.

Simultaneous production of IFN-gamma, IFN-alpha/beta and nitric oxide in peritoneal macrophages from TDM-treated mice.

Peritoneal macrophages (PM) were isolated from mice treated with Dimycolate of Trehalose (TDM), a glycolipid extracted from the cell wall of Mycobacterium tuberculosis. PM from TDM-treated mice (TDM-PM) were shown to secrete consistent amount of IFN-gamma, which was not detectable in control Resident-PM (Res-PM), as revealed by ELISA. In addition, biologically active IFN was detected in the supernatants of TDM-PM, whereas no IFN production was found in those of control Res-PM. The addition of specific antisera to PM cultures revealed the simultaneous production of both type I and II IFNs in TDM-PM cultures. No reciprocal regulation in the production of IFN-gamma and IFN-alpha/beta was found in these cultures. In parallel, nitric oxide (NO) production was measured in TDM-PM cultures by detecting nitrites (NO2-). TDM-PM cultures accumulated high amounts of NO2- which decreased to the level of Res-PM in the presence of NMMA, an inhibitor of NO-synthases. In vitro, neither type I nor type II IFNs were involved in the stimulation of NO production. The capacity of macrophages to simultaneously secrete IFN-gamma, IFN-alpha/beta and NO upon in vivo TDM-treatment could be of particular relevance for the defense process of innate immunity in which macrophages play a crucial role.

Activity of nitric oxide-generating compounds against encephalomyocarditis virus.

Nitric oxide (NO) generated by two NO donors (sodium nitroprusside or S-nitroso-L-glutathione) was shown to exert a dose-dependent inhibition of encephalomyocarditis virus growth in L-929 cells. This activity was not due to the cytotoxic or direct virucidal effects of NO donors. L-929 cells were shown to produce NO endogenously, but this low level of production did not counter encephalomyocarditis virus replication.

ANP-stimulated cGMP egression in renal principal cells: abrogation of polarity by SV40 large T.

Egression of atrial natriuretic peptide (ANP)-stimulated guanosine 3', 5'-cyclic monophosphate (cGMP) was compared with that of isoproterenol-stimulated adenosine 3', 5'-cyclic monophosphate (cAMP) in a rabbit collecting duct cell line transformed with a temperature-sensitive strain of simian virus 40 (SV40). At 39.5 degrees C (inactivated large T), cells exhibit major features of principal cells, whereas at 33 degrees C (functional large T) they lose most of their specific properties. When cells were grown on plastic at 39.5 degrees C, both cyclic nucleotides were predominantly released extracellularly via probenecid-sensitive carriers. Probenecid (3mM) reduced the ratios of extracellular cGMP and cAMP by 84 and 70%, respectively. The amount of extracellular cGMP or cAMP ws linearly correlated with the time integral of the intracellular cyclic nucleotide, suggesting first-order kinetics. The apparent first-order rate constant (k) was sixfold greater for cGMP (0.139 +/- 0.037 min-1, n = 3 experiments) than for cAMP (0.022 +/- 0.003(-1), n = 3 experiments). 3-Isobutyl-1-methylxanthine markedly inhibited extrusion of cGMP (k = 0.022 +/- 0.003 min-1), whereas that of cAMP was unchanged. When cells were grown on filters at 39.5 degrees C, both nucleotides were predominantly released in the apical medium but with a greater polarity for cGMP (83 +/- 4%, n = 6 experiments) than for cAMP (60 +/- 6%, n = 3 experiments) and a prevailing apical localization of the probenecid-sensitive carrier. Activation of SV40 large T at 33 degrees C did not alter cyclic nucleotide transport characteristics but abolished the polarity of probenecid-sensitive cyclic nucleotide extrusion. These results suggest a physiological role for luminal cGMP in the rabbit collecting duct and a specific effect of large T on the probenecid-sensitive carrier polarity.

Fluoride ion toxicity in human kidney collecting duct cells.

BACKGROUND: Several halogenated anesthetics induce a urinary concentrating defect, partly related to fluoride ion toxicity in collecting duct cells. The aim of this study was to investigate the effects of fluoride ion in human kidney cells. METHODS: Immortalized human collecting duct cells were used. In a first set of experiments, the toxicity threshold concentration was determined by exposing cell cultures for 24 h to increasing concentrations of fluoride ion in the medium: 0, 1, 5, and 10 mM. The second set of experiments was a time- effect study in which cells were exposed to 5 mM fluoride for 2, 6, and 24 h. Assessment of toxicity was based on several endpoints: cell number, protein content, (3)H-leucine incorporation in newly synthesized proteins, extracellularly released lactate dehydrogenase, Na-K-ATPase pump activity, and electron microscope studies. RESULTS: After 24 h of exposure, fluoride ion decreased cell number (-23%, P<0.05), total protein content (-30%, P<0.05) and increased lactate dehydrogenase release (+236%, P<0.05) at a threshold concentration of 5mM. Fluoride ion also inhibited Na-K- ATPase activity at 5 mM (-58%, P<0.05). Major morphologic alterations of mitochondria, including crystal formation, were detected from 1 mM fluoride concentration. Time-effect studies showed that, after only 6 h of exposure at 5 mM, fluoride decreased cell number (-13%, P<0.05), (3)H-leucine incorporation (-48%, P<0.05), and Na-K-ATPase activity (- 20%, P<0.05) and increased lactate dehydrogenase release (+145%, P<0.05). Crystal deposits in mitochondria again were a more sensitive marker of cell injury, detectable after only 2 h of exposure. CONCLUSIONS: these results suggest that the mitochondrion is a target of fluoride toxicity in human collecting duct cells, and its alteration is partly responsible for the sodium and water disturbances observed in patients.

Role of trehalose dimycolate-induced interferon-alpha/beta in the restriction of encephalomyocarditis virus growth in vivo and in peritoneal macrophage cultures.

Preventive intraperitoneal trehalose dimycolate (TDM) treatment of mice, inoculated with encephalomyocarditis (EMC) virus by the same route, caused restriction of virus growth in the peritoneum, which was correlated to IFN production in peritoneal fluids prior to infection. Peritoneal macrophages from TDM-treated mice (TDM-PM) spontaneously secreted IFN-alpha/beta in large amounts. By their supernatants, TDM-PM could transfer an antiviral state against EMC virus to permissive resident peritoneal macrophages from control mice. IFN-alpha/beta produced by TDM-PM was found to be involved in this transfer activity. TDM-PM also exerted a strong antiviral effect on EMC virus-infected L-929 cells, which increased with time and the macrophage-target cell ratio. This activity also occurred by an IFN-alpha/beta-dependent mechanism. These data point to the role of IFN-alpha/beta production prior to EMC virus infection in the antiviral activities of TDM-PM and, more generally, in the outcome of viral infection.

Immunoreactivity of endogenous digitalis-like substances in cord blood sera studied with antidigitoxin monoclonal antibodies.

The presence of digitoxin-like immunoreactive substances, whose nature is yet unknown, has been demonstrated in the umbilical cord blood. We selected six antidigitoxin monoclonal antibodies (MAb) having different specificity profiles concerning digitoxin analogs and steroid hormones. These antibodies were tested in a digitoxin radioimmunoassay (RIA). With the help of this technique, we measured the concentrations of apparent digitoxin in the cord blood drawn either at birth or in utero from mothers not undergoing any digitalis treatment. In the cord blood of newborns, the concentrations of apparent digitoxin, measured by the two MAbs that have the highest cross-reactions with dehydroepiandrosterone (DHEA) (123A23 and 145A41), were two or three times higher than with the other antibodies. In the fetal cord blood, where the concentration of DHEA is five to seven times lower than that observed at birth, these antibodies revealed a threefold lower concentration of apparent digitoxin than that observed in blood drawn at birth. Furthermore, MAbs that had similar specificities towards digitoxin analogs and steroids showed different measurements of digitoxin-like concentrations. These observations suggest that digitoxin-like immunoreactive compounds detected by the RIA may constitute a group of different molecules, one of which would be the DHEA.

Principal cell-specific antigen and hormonal regulatory network in RC.SVtsA58 cell line.

We used a dual immunomorphological and physiological approach to demonstrate that the RC.SVtsA58 rabbit cortical cell line exhibits features of highly differentiated cortical collecting tubule (CCT) principal cells (PC). First, we raised monoclonal antibodies against RC.SVtsA58 cells and screened their reactivity with the rabbit kidney: three were specific for the basolateral domain of CCT PC and bound to 100% of RC.SVtsA58 cells. Second, we showed that bradykinin, atrial natriuretic peptide, and prostaglandin E2 increased intracellular Ca2+, guanosine 3',5'-cyclic monophosphate, and adenosine 3',5'-cyclic monophosphate (cAMP), respectively. In addition, 10 nM bradykinin inhibited desmopressin-elicited cAMP production by > or = 40%; this effect was suppressed by 10 microM of indomethacin and was reproduced with 1 nM of prostaglandin E2, indicating the conservation of arginine vasopressin-related regulatory loops described in microdissected CCT and freshly isolated cells. However, RC.SVtsA58 cells also express intercalated cell markers even after repeated cloning, which suggests that tsA58, a temperature-sensitive strain of simian virus-40, has transformed a multipotent type of PC in keeping with the cell interconversion hypothesis.

Antiviral action of trehalose dimycolate against EMC virus: role of macrophages and interferon alpha/beta.

Preventive treatment of mice with trehalose 6,6' dimycolate (TDM), an immunomodulator of bacterial origin, enhances their resistance to encephalomyocarditis (EMC) virus infection. The protective effect of TDM is totally abolished by the injection of silica particles in mice, demonstrating the role of macrophages in the antiviral action of TDM. In vitro, peritoneal macrophages from mice treated with TDM (TDM-PM) exhibit an intrinsic antiviral activity against EMC virus, while resident peritoneal macrophages (RES-PM) are permissive to this virus. Greater amounts of interferon are detected in supernatants of cultures of TDM-PM than of RES-PM. Neutralization of interferon (IFN) by addition in vitro of anti-IFN alpha/beta serum markedly reduces the antiviral activity of TDM-PM. These results indicate that interferon alpha/beta is involved in the intrinsic anti-EMC virus activity of peritoneal macrophages from mice treated with TDM.

Phenotypic effects of aldosterone and dexamethasone in a SV40-transformed mammalian cortical ascending limb cell line exhibiting mineralocorticoid receptors.

We have analyzed the functional and morphological effects of corticosteroid hormones in a SV40-transformed rabbit cortical-ascending-limb (CAL) cell line (RC.SV2, Vandewalle et al., 1989) having mineralocorticoid (MR) and glucocorticoid (GR) receptors (Rafestin-Oblin et al., 1993). Both aldosterone and dexamethasone (5 x 10(-8) M) induced a marked increase in (3H)ouabain binding (used to quantify membrane Na(+)-K+ ATPase) detectable as early as 6 hours and maximal at 24 hours (+56-57%) (due to a 1.6-1.8-fold increase in cell membrane binding sites without Kd alteration), and significantly augmented the ouabain-sensitive component of Rb+ influx. Triiodothyronine (T3, 10(-9) M) also stimulated ouabain binding by 21% but was not permissive for steroid action, whereas 5 micrograms/ml insulin had no effect. Both steroid hormones, T3 and insulin induced the formation of domes that was tightly correlated with ouabain binding (r = 0.949) except for insulin. The effects of aldosterone and dexamethasone on cell monolayers and cell ultrastructure were, however, strikingly different as aldosterone induced a marked amplification of basolateral areas with appearance of large intercellular spaces, reminiscent of the changes observed in deoxycorticosterone-treated rats, whereas dexamethasone predominantly influenced cell height. This discrepancy might be due to specific occupancy of MR and GR by aldosterone and dexamethasone, respectively, and/or to nongenomic effects of dexamethasone. We have thus characterized a cell culture model making it possible to analyze the actions of mineralocorticoid and glucocorticoid hormones in the mammalian kidney.

Major influence of cell differentiation status on characteristics of proteoglycans synthesized by cultured rabbit renal proximal tubule cells: role of insulin and dexamethasone.

To analyze the influence of epithelial cell differentiation and the effects of hormones on the characteristics of cell-associated and secreted proteoglycans (PGs), we studied their distribution, synthesis, and biochemical features in a model of renal proximal tubule cells in primary culture in which cell differentiation could be controlled by medium composition. In cells cultured in serum-free, hormonally defined medium supplemented with insulin and dexamethasone that exhibited a high degree of morphological and functional proximal differentiation (Ronco et al., 1990), cell-associated PGs were similar to those extracted in vivo by their size estimated by Sepharose CL-6B chromatography (Kav = 0.27, vs. 0.26), composition (heparan-sulfate), and localization in a continuous basal layer of extra-cellular matrix (ECM). In contrast, major quantitative and qualitative anomalies of cell-associated PGs were observed in poorly differentiated cells grown in 1% fetal calf serum-supplemented medium (FCS). PGs alterations included: (1) reduced and irregular expression of PGs at the cell basal pole, (2) a 2.8-fold decrease in [35S]-sulfate incorporation into cell-associated PGs, (3) a 3.1-fold increase in trypsin-releasable PGs, and (4) the emergence of a high MW PG composed exclusively of chondroitin-sulfate (CS) (Kav = 0.09 on Sepharose CL-6B) as well as of putative free CS-glycosaminoglycan (GAG) chains (Kav = 0.49 on Sepharose CL-6B). The same alterations were identified in the basal defined medium devoid of hormones but were partially or totally abolished by addition of insulin and dexamethasone, respectively. At variance with cell-associated PGs, production and biochemical features of secreted PGs were not influenced by cell differentiation status and medium composition.

Regulation by calcitonin of Na(+)-K(+)-Cl- cotransport in a rabbit thick ascending limb cell line.

The hormonal regulation of a Na(+)-K(+)-Cl- cotransport was investigated in a renal tubule cell line (RC.SV2 cells) transformed by the simian virus 40. This cell line has the main characteristics of cells from the thick ascending limb of Henle, including the presence of Tamm-Horsfall protein and stimulation of adenosine 3',5'-cyclic monophosphate (cAMP) production by calcitonin (CT). Kinetic studies with 22Na+, 36Cl-, and 86Rb+ indicated the existence of a Na(+)-K(+)-Cl- cotransport with a stoichiometry of 1Na+:1K+: 2Cl-. All compounds stimulating cAMP production enhanced the ouabain-resistant bumetanide-sensitive (Or-Bs) Rb+ influx mediated by Na(+)-K(+)-Cl- cotransport. CT (100 ng/ml) increased the Or-Bs influx twofold by enhancing maximum velocity without changing the apparent Michaelis constant. The K(+)-channel blocker barium blunted the CT-stimulated Or-Bs influx by 64-74%, whereas the Cl(-)-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate reduced the CT-stimulated influx by 28-40%. These results suggest that CT stimulates the Na(+)-K(+)-Cl- cotransport by a cAMP-dependent mechanism and that K+ recycling through K+ membrane channels is an important modulator of cotransporter-mediated ion fluxes.

Transformation of renal tubule epithelial cells by simian virus-40 is associated with emergence of Ca(2+)-insensitive K+ channels and altered mitogenic sensitivity to K+ channel blockers.

We compared the pattern of K+ channels and the mitogenic sensitivity to K+ channel blocking agents in primary cultures of rabbit proximal tubule cells (PC.RC) (Ronco et al., 1990) and two derived SV-40-transformed cell lines exhibiting specific functions of proximal (RC.SV1) and more distal (RC.SV2) tubule cells (Vandewalle et al., 1989). First, K+ channel equipment surveyed by the patch-clamp technique was modified after SV-40 transformation in both cell lines; although a high conductance Ca(2+)-activated K+ channel [K+200 (Ca2+)] remained the most frequently recorded K+ channel, the transformed state was characterized by emergence of three Ca(2+)-insensitive K+ channels (150, 50, and 30 pS), virtually absent from primary culture, contrasting with reduced frequency of two Ca(2+)-sensitive K+ channels (80 and 40 pS). Second, quinine (Q), tetraethylammonium ion (TEA) and charybdotoxin (CTX), at concentrations not affecting cell viability, all decreased 3H-TdR incorporation and cell growth in PC.RC cultures, but only TEA had similar effects in transformed cells. The latter were further characterized by paradoxical effects of Q that induced a marked increase in thymidine incorporation. Q also exerted contrasting effects on channel activity: it inhibited the [K+200 (Ca2+)] when the channel was highly active, with a Ki (0.2 mM) similar to that measured for 3H-TdR incorporation in PC.RC cells (0.3 mM), but increased the mean current through poorly active channels. TEA blocked all K+ channels with conductance greater than or equal to 50 pS, including the [K+200 (Ca2+)], in a range of concentrations that substantially affected cell proliferation. The unique effect of TEA on SV-40-transformed cells might be related to broad inhibition of K+ channels.

Activation of the simian virus 40 (SV40) genome abrogates sensitivity to AVP in a rabbit collecting tubule cell line by repressing membrane expression of AVP receptors.

To analyze the role of SV40 genome in the phenotypic alterations previously observed in SV40-transformed cell lines, we infected rabbit renal cortical cells with a temperature-sensitive SV40 mutant strain (tsA58) and compared the cell phenotypes at temperatures permissive (33 degrees C) and restrictive (39.5 degrees C) for SV40 genome expression. At both temperatures, the resulting cell line (RC.SVtsA58) expresses cytokeratin and uvomorulin, but epithelial differentiation is more elaborate at 39.5 degrees C as shown by the formation of a well-organized cuboidal monolayer with numerous tight junctions and desmosomes. Functional characteristics are also markedly influenced by the culture temperature: cells grown at 33 degrees C respond only to isoproterenol (ISO, 10(-6) M) by a sevenfold increase in cAMP cell content above basal values; in contrast, when transferred to 39.5 degrees C, they exhibit increased sensitivity to ISO (ISO/basal: 19.1) and a dramatic response to 10(-7) M dDarginine vasopressin (dDAVP/basal: 18.2, apparent Ka: 5 X 10(-9) M) which peaks 48 h after the temperature shift. The latter is associated with membrane expression of V2-type AVP receptors (approximately 50 fmol/10(6) cells) which are undetectable when SV40 genome is activated (33 degrees C). Clonal analysis, additivity studies, and desensitization experiments argue for the presence of a single cell type responsive to both AVP and ISO. The characteristics of the RC. SVtsA58 cell line at 39.5 degrees C (effector-stimulated cAMP profile, lack of expression of brush-border hydrolases and Tamm-Horsfall protein) suggest that it originates from the cortical collecting tubule, and probably from principal cells.

Polarized membrane expression of brush-border hydrolases in primary cultures of kidney proximal tubular cells depends on cell differentiation and is induced by dexamethasone.

To analyze the influence of cell differentiation and the effects of hormones on the subcellular distribution of apical antigens in polarized epithelial cells, we have compared the localization of three brush border (BB) hydrolases [neutral endopeptidase (ENDO), aminopeptidase N (APN), and dipeptidylpeptidase IV (DPPIV)] in primary cultures of renal proximal tubule cells grown in various culture media. The degree of cell differentiation modulated by medium composition was estimated by measuring proximal functions, including glucose transport, specific enzymatic activities, and PTH responsiveness. In the dedifferentiated state observed in cells grown in 1% fetal calf serum (FCS)-supplemented medium, the three hydrolases are abnormally concentrated in a cytoplasmic vesicle compartment with weak expression on both membrane domains. By contrast, in serum-free hormonally defined medium (DM: insulin, 5 microgram/ml; dexamethasone, 5 x 10(-8) M), which markedly enhances morphological and functional cell differentiation, the distribution of hydrolases parallels that observed in the normal tubule. When added to the DM devoid of hormones, insulin has little polarizing effect, whereas dexamethasone dramatically increases the apical expression of the hydrolases, which then almost disappear from the basolateral membrane and cytoplasmic vesicular compartments. This glucocorticoid hormone augments the amount of immunoreactive antigen detectable on the apical domain in paraformaldehyde-fixed cells but does not change the total enzymatic activity. This suggests the presence in tubular cells of a dexamethasone-dependent polarizing machinery that requires de novo RNA and protein synthesis, and probably acts mainly by targeting a storage cytoplasmic pool of enzyme to the apical domain.

Maintenance of proximal and distal cell functions in SV40-transformed tubular cell lines derived from rabbit kidney cortex.

This paper reports the preparation and describes the properties of three renal tubular cell lines derived using SV40 infection of primary cultures of rabbit kidney cortical cells, enriched in proximal cells. RC.SV1 was initially derived from cultures grown in the presence of fetal calf serum exhibiting a low degree of proximal differentiation. The cells were subsequently adapted to grow in serum-free hormonally defined medium and display basic properties of proximal tubule cells including well-developed apical microvilli, strong expression of brush-border hydrolases, Na+-coupled glucose uptake, and increased cyclic AMP production when exposed to PTH. The other two cell lines were derived from cultures in serum-free hormonally defined medium and propagated in the same medium. They are characterized by some common properties including rare and short microvilli, low expression of apical hydrolases, and low or undetectable Na+-dependent glucose uptake, but differ by their abilities to respond by an increase in cAMP to various hormonal stimuli. RC.SV2 cells are sensitive to calcitonin and to a lesser extent to isoproterenol and PTH, suggesting that they may originate from the thick ascending limb of Henle's loop and the bright portion of the distal tubule. RC.SV3 responds essentially to isoproterenol and arginine vasopressin, suggesting a more distal origin (late distal and initial collecting tubule). Emergence of distal cell lines from cultures exhibiting proximal characteristics may be related to distal cell overgrowth as suggested by analysis of growth kinetics and increased Na+/H+ exchanger activity in RC.SV2 compared with RC.SV1.

Specific binding characteristics of high affinity monoclonal antidigitoxin antibodies.

The specificity of various monoclonal antidigitoxin antibodies was characterized using 6 cardiac glycoside analogs. Spleen cells from BALB/c mice, immunized with BSA- or KLH-digitoxin conjugates, were fused with NS1 myeloma cells, and antibody-producing hybrids were identified by radioimmunoassay. Twenty-one monoclonal antidigitoxin-specific antibodies were obtained, 10 of which were cloned and characterized for affinity and specificity. All the antibodies had a high affinity constant, ranging from 8.10(8) to 2.5.10(10) 1/M. On the basis of their binding specificities, the antibodies could be classified into 3 groups: the first contained 7 antibodies exhibiting high cross reactivity (42-100%) with digitoxigenin, whereas the second and third groups did not recognize this analog (cross-reactivity of 1%). In the former group, the absence of the sugar moiety only slightly affected the binding reaction, although for the two other groups, this structure did appear to be involved in antibody recognition. Changes in the functional groups of the hapten molecule led to considerable changes in the antibody-antigen reaction. For all the antibodies except one, saturation of the lactone ring considerably affected binding. These results demonstrated that monoclonal antibodies of different specificities with respect to both the steroid backbone and the sugar moiety of digitoxin can be induced using a digitoxin-protein conjugate.