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Biomedical subjects

M Tauc

Publications and source records attributed to M Tauc.

At least 37 records · Page 2Linked to original sources
PubMed1997

In vitro development and preservation of specific features of collecting duct epithelial cells from embryonic rabbit kidney are regulated by the electrolyte environment.

During kidney development the embryonic collecting duct (CD) epithelium changes its function. The capability for nephron induction is lost and the epithelium develops into functional principal (P) and intercalated (IC) cells. Aldosterone is able to modulate this differentiation. Consequently we investigated whether increased concentrations of extracellular NaCl or Na gluconate may also have an influence on the development of individual CD cell features. Embryonic CD epithelia were isolated from neonatal rabbit kidneys, placed on tissue carriers and cultured in gradient containers, which were constantly perfused with medium for 13 days. Isotonic culture conditions could be mimicked, when on both the luminal and basal side standard Iscove's Modified Dulbecco's Medium (IMDM) was used. In another set of experiments, gradient culture was performed. Standard IMDM was applied on the basal side and IMDM supplemented with 12 mM NaCl and 17 mM Na gluconate on the luminal side. This adaptation of IMDM led to the same Na concentrations as found in the serum of neonatal rabbits. The development of CD cell features was monitored by cellular markers such as the monoclonal antibodies (Mabs) 703 and 503 recognizing P and IC cell features respectively. Epithelia cultured under isotonic conditions showed less than 5% Mab 703- and 503-immunopositive cells. In contrast, epithelia cultured in a luminal-basal medium gradient revealed more than 80% positive cells. Immunoreactivity started to develop after a long lag period of 4 days, then increased continuously during the following 5 days and reached a maximum at day 14. When the medium gradient was then changed to an isotonic environment for another 5 days immunoreactivity for Mab 703 remained stable, while the number of Mab 503-positive cells was found to be decreased to 10%. Thus, the extra-cellular electrolyte environment not only induces but also preserves individual cell features.

Animals↗
PubMed1997

Chloride channels in primary cultures of seawater fish (Dicentrarchus labrax) gill.

Patch-clamp experiments were undertaken on primary cultures of respiratory cells originating from sea bass (Dicentrarchus labrax) gills. A small-conductance Cl- channel of 8 pS was characterized in cell-attached configuration with 140 mM N-methyl-D-glucamine-Cl in the pipette and bath solutions. No activity was recorded below a membrane holding potential of +20 mV (-Vp, referenced to the pipette solution), and the channel showed an inward rectification. In the inside-out configuration the Cl- channel was active at all membrane holding potentials. Its open probability strongly increased with membrane depolarization. The channel activity could be increased by the application of protein kinase A+ATP. This channel was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoic acid, diphenylamino-2-carboxylic acid, and I- and was insensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The presence of Cl- channels in the apical membrane of respiratory cells provides additional evidence for an important role of this cell type in the control of ion homeostasis of seawater fish.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

Cl- and K+ conductances activated by cell swelling in primary cultures of rabbit distal bright convoluted tubules.

Ionic currents induced by cell swelling were characterized in primary cultures of rabbit distal bright convoluted tubule (DCTb) by the whole cell patch-clamp technique. Cl- currents were produced spontaneously by whole cell recording with an isotonic pipette solution or by exposure to a hypotonic stress. Initial Cl- currents exhibited outwardly rectifying current-voltage relationship, whereas steady-state currents showed strong decay with depolarizing pulses. The ion selectivity sequence was I- = Br- > Cl- >> glutamate. Currents were inhibited by 0.1 mM 5-nitro-2-(3-phenylpropylamino) benzoic acid and 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and strongly blocked by 1 mM diphenylamine-2-carboxylate. Currents were insensitive to intracellular Ca2+ but required the presence of extracellular Ca2+. They were not activated in cells pretreated with 200 nM staurosporine, 50 microM LaCl3, 10 microM nifedipine, 100 microM verapamil, 5 microM tamoxifen, and 50 microM dideoxyforskolin. Staurosporine, tamoxifen, verapamil, or the absence of external Ca2+ was without effect on the fully developed Cl- currents. Osmotic shock also activated K+ currents in Cl- free conditions. These currents were time independent, activated at depolarized potentials, and inhibited by 5 mM BaCl2. The activation of Cl- and K+ currents by an osmotic shock may be implicated in regulatory volume decrease in DCTb cells.

Animals↗
PubMed1996

Chloride currents activated by calcitonin and cAMP in primary cultures of rabbit distal convoluted tubule.

Chloride (Cl-) conductances were studied in primary cultures of the bright part of rabbit distal convoluted tubule (DCTb) by the whole cell patch clamp technique. The bath solution (33 degrees C) contained (in mM): 140 NaCl, 1 CaCl2, 10 N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.4 and the pipette solution 140 N-methyl-D-glucamine (NMDG)-Cl, 5 MgATP, 1 ethylene-glycol-bis(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 10 HEPES, pH 7.4. We identified a Cl- current activated by 10(-5) M forskolin, 10(-3) M 8-bromo adenosine 3',5'-cyclic monophophosphate (8 Br-cAMP), 10(-6) M phorbol 12-myristate 13-acetate (PMA), 10(-3) M intracellular adenosine 3',5'-cyclic monophophosphate (cAMP) and 10(-7) M calcitonin. The current-voltage relationship was linear and the relative ion selectivity was Br- > Cl- > > I- > glutamate. This current was inhibited by 10(-3) M diphenylamine-2-carboxylate (DPC) and 10(-4) M 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and was insensitive to 10(-3) M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). These characteristics are similar to those described for the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- conductance. In a few cases, forskolin and calcitonin induced an outwardly rectifying Cl- current blocked by DIDS. To determine the exact location of the Cl- conductance 6-methoxy-1-(3-sulfonatopropyl) quinolinium (SPQ) fluorescence experiments were carried out. Cultures seeded on collagen-coated permeable filters were loaded overnight with 5 mM SPQ and the emitted fluorescence analyzed by laser-scan cytometry. Cl- removal from the apical solution induced a Cl- efflux which was stimulated by 10(-5) M forskolin, 10(-7) calcitonin and inhibited by 10(-5) M NPPB. In 140 mM NaBr, forskolin stimulated an apical Br- influx through the Cl- pathway. Forskolin and calcitonin had no effect on the basolateral Cl- permeability. Thus in DCTb cultured cells, exposure to calcitonin activates a Cl- conductance in the apical membrane through a cAMP-dependent mechanism.

Animals↗
PubMed1996

Calcium-activated chloride currents in primary cultures of rabbit distal convoluted tubule.

Chloride (Cl-) conductances were studied in primary cultures of rabbit distal convoluted tubule (very early distal "bright" convoluted tubule, DCTb) by the whole cell patch-clamp technique. We identified a Cl- current activated by 2 microM extracellular ionomycin. The kinetics of the macroscopic current were time dependent for depolarizing potentials with a slow developing component. The steady state current presented outward rectification, and the ion selectivity sequence was I- > Br- > > Cl > glutamate. The current was inhibited by 0.1 mM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and 1 mM diphenylamine-2-carboxylate. To identify the location of the Cl- conductance, 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence experiments were carried out in confluent cultures developed on collagen-coated permeable filters. Cl- removal from the apical solution induced a Cl- efflux that was stimulated by 10 microM forskolin. Forskolin had no effect on the basolateral Cl- permeability Cl- substitution in the basolateral solution induced an efflux stimulated by 2 microM ionomycin or 50 microM extracellular ATP Ionomycin had no effect on the apical Cl- fluxes. Thus cultured DCTb cells exhibit Ca(2+)-activated Cl- channels located in the basolateral membrane. This Cl- permeability was active at a resting membrane potential and could participate in the Cl- reabsorption across the DCTb in control conditions.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

In young primary cultures of rabbit kidney cortical collecting ducts intercalated cells originate from principal or undifferentiated cells.

The evolution of a primary culture of rabbit kidney cortical collecting tubule was followed over a period of 10 to 11 days. The cell types of this segment were characterized by using monoclonal antibodies, specifically directed against principal (Mab 703) and intercalated (Mab 503) cells of the apical membrane. The activity of a H+ pump ATPase was revealed in Mab 503-labeled cells, confirming that these cultured cells present characteristics of intercalated cells. The primary culture was also stained with peanut agglutinin (PNA), a specific ligand of beta intercalated cells. During the first two days, some cells, mainly Mab 503-labeled cells, disappeared, and cell division did not occur. At 2 days, the culture showed 80% and 18% of Mab 703-labeled and Mab 503-labeled cells, respectively. The first mitoses were observed at 2 days. From two to four days, cell division was nestly in Mab 703-labeled cells and only rarely seen in Mab 503-labeled cells, although during this period the proportion of Mab 703-labeled cells decreased to 44% of cells and that of Mab 503-labeled cells increased to 30%. The labeling with PNA was curious. Up to 2 days, PNA stained Mab 503-labeled cells, but from 4 days it stained other cells, probably dedifferentiated ones. In our culture conditions types of cells other than Mab 703-labeled and Mab 503-labeled cells occurred. First, throughout the life of the culture, some cells were not recognized by any monoclonal antibody; their number varied between 10 and 28% of the total cell number.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Zinc transport and metallothionein induction in primary cultures of rabbit kidney proximal cells.

Primary cultures of isolated rabbit renal proximal cells were grown on collagen-coated permeable supports. The confluent epithelia were polarized, making possible the measurement of uptakes and effluxes across the apical and the basolateral membranes. Uptakes of 65Zn were assessed under initial rate conditions, after 0.5 min incubation. The kinetic parameters of apical uptake were a Jmax of 25.1 +/- 5.3 pmol min-1 (micrograms DNA)-1, a Km of 43.3 +/- 7.3 microM and an unsaturable constant of 0.105 +/- 0.029 (n = 7) at 37 degrees C. Cadmium competitively inhibited the zinc uptake, with a Ki value of 24.5 +/- 7.3 microM. Basolateral uptake was characterized by a high capacity (Jmax = 227.9 +/- 46.6 pmol min-1 (micrograms DNA)-1) and an affinity similar to that of the apical uptake (Km = 35.4 +/- 14.2 microM). Cadmium had no effect on the basolateral zinc uptake. Effluxes across the basolateral face of the epithelium always exceeded those across the apical face. Excess zinc in the culture medium induced the synthesis of metallothionein in the epithelia, as judged by the rate of [35S]cysteine incorporation into a fraction of cytosolic proteins. Metallothionein induction did not appear to modify the kinetic parameters of the apical zinc uptake. These data suggest that separate saturable transport systems are responsible for the apical and basolateral zinc uptakes in proximal renal cells. Induction of metallothionein had no apparent effect on apical zinc uptake in this system.

Animals↗
PubMed1994

Activation of calcium influx by ATP and store depletion in primary cultures of renal proximal cells.

Cytoplasmic calcium changes and calcium influx evoked by adenosine triphosphate (ATP) were investigated in primary cultures of rabbit proximal convoluted tubule cells. Extracellular ATP (50 microM) induced a biphasic increase of [Ca2+]i measured with the calcium probe fura-2. In the early phase, the mobilization of intracellular pools resulted in a transient increase of [Ca2+]i from 106 +/- 11 nM (n = 36) to 1059 +/- 115% (n = 29) of the resting level within 10 s. In the presence of external calcium, [Ca2+]i then decreased within 3 min to a sustained level (398 +/- 38%, n = 8). Measurements of fura-2 quenching by external manganese revealed that this phase was the result of an increased Ca2+ uptake, blocked by lanthanum (10 microM) and verapamil (100 microM) but not by the nifedipin (25 microM). Internal calcium store depletion by ATP induced an increased calcium influx through lanthanum- and verapamil-sensitive, nifedipin-insensitive calcium channels, located on the apical membrane of the cells. As indicated by 86Rb+ efflux measurements, ATP activated a potassium efflux that was blocked by barium and Leiurus quinquestriatus hebraeus (LQH) venom (containing charybdotoxin) indicating the involvement of Ca(2+)-sensitive K+ channels. Moreover, in the presence of the LQH venom, the internal calcium stores were not replenished after being depleted by ATP. Our results indicate that an ATP-evoked hyperpolarization of the plasma membrane leads to increased Ca2+ influx, which facilitates the replenishment of the internal stores.

Adenosine Triphosphate↗
PubMed1994

Chloride channels in apical membrane of primary cultures of rabbit distal bright convoluted tubule.

Using the patch clamp technique on the apical membrane of primary cultures of rabbit distal bright convoluted tubule cells (DCTb), two types of Cl- channel were identified. A small channel of 9 pS was observed in 9% of the patches. Cells pretreated with 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) or 5 microM forskolin increased the expression of Cl- channels by 26 and 37%, respectively. In cell-attached and excised inside-out patches, the current-voltage (I-V) relationships of the 9-pS channel were linear. In only 1 out of 47 active patches was the small-conductance Cl- channel still active 1 h after membrane excision. The addition of 0.1 microM of the catalytic subunit protein kinase A with 2 mM ATP to the cytoplasmic side restored channel activity in 8 out of 15 excised membrane patches. In 5 out of 467 patches of stimulated or nonstimulated cells, a larger Cl- conductance of 30 pS was also recorded. In excised inside-out patches this channel outwardly rectified and was activated by strong depolarization. In cultured DCTb cells, the small-conductance, cAMP-activated Cl- channel shares many properties with the cystic fibrosis transmembrane conductance regulator. Our results suggest that at least the small-conductance channel may participate in Cl- secretion across the apical membrane of DCTb in primary culture. This secretion may increase the rate of the apical Cl-/HCO3- exchange indirectly by enhancing the inwardly-directed Cl- gradient.

Adenosine Triphosphate↗
PubMed1993

Synthesis of charybdotoxin and of two N-terminal truncated analogues. Structural and functional characterisation.

Charybdotoxin and two N-terminal truncated peptides, corresponding to the 2-37 and 7-37 sequences, were obtained by stepwise solid-phase synthesis using N alpha-t-butyloxycarbonyl and benzyltype side-chain protection. While this strategy was generally useful, the S-acetamidomethyl protecting group used for the six cysteines was not completely stable under HF treatment and its subsequent removal by mercury(II) treatment was neither complete nor devoid of side reactions. The completely deprotected native and truncated sequences were folded efficiently in the presence of glutathione and were finally purified by high-pressure liquid chromatography with overall yields of 4.0-5.0%. Each protein was characterised chemically, structurally and functionally. 1H-NMR spectroscopy was used and a complete assignment of all the protons of the three synthetic proteins was achieved. NMR data show that synthetic charybdotoxin is indistinguishable from the natural protein. The two truncated proteins contain the same elements of secondary structure and a similar overall three-dimensional structure, in agreement with circular dichroic measurements. The shortest analogue, however, may have local structural perturbations and/or higher flexibility. Biological activity on dog epithelial Ca(2+)-activated K+ channels and on rat brain synaptosomal voltage-dependent K+ channels show that synthetic charybdotoxin was as potent as the natural toxin on both channels. For both channels, deletion of the first amino acid, 5-oxoproline (pyroglutamic acid) decreased only slightly the potency of the inhibitor, while deletion of the entire 1-6 segment reduced potency much more. We conclude that the N-terminal region of charybdotoxin plays a functional role in tuning the toxin's biological activity but is not essential for the folding and stability of the structure. The structure of the shortest analogue represents an interesting example of how a well organised and stable alpha/beta fold can be engineered with only 31 amino acid residues.

Amino Acid Sequence↗
PubMed1993

Nucleotides mobilize intracellular calcium stores of renal proximal cells in primary culture: existence of a suramin-sensitive mechanism.

Changes of intracellular calcium concentrations [Ca2+]i were measured in primary cultured rabbit proximal convoluted tubules (PCT). A dual-excitation, digital-imaging inverted microscope was used to monitor the fura-2 fluorescence. The basal calcium level was 106 +/- 11 nM (n = 36). The stimulatory effects of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine were studied. ATP and ADP induced transient increases of [Ca2+]i (1059 +/- 115% of the resting level (n = 29), and 659 +/- 134% (n = 10), respectively) by releasing calcium from cytoplasmic stores. Adenosine had less effect (279 +/- 48% of the resting level, n = 3). In the same conditions the ATP antagonist suramin (100 microM) inhibited the action of ATP and ADP to 231 +/- 52% (n = 3), and 308 +/- 29% (n = 4) of the resting level, respectively, but did not modify that of adenosine (281 +/- 72%, n = 3). A pretreatment (500 ng/ml for 2 h at 37 degrees C) of the culture with the toxin of Bordetella pertussis completely blocked the ATP response. Our results are evidence for the presence of a functional suramin-sensitive ATP and ADP puriceptor in cultured renal proximal cells. A pertussis-toxin-sensitive G protein is linked to the transduction mechanism. This receptor is distinct from an adenosine puriceptor also found in the proximal monolayer.

Adenosine Diphosphate↗
PubMed1993

Toxin sensitivity of the calcium-dependent rubidium efflux in Madin-Darby canine kidney cells.

86Rb+ efflux was measured on polarized Madin-Darby canine kidney cells under A23187 or ATP stimulation. This efflux, inhibited by barium, Leiurus quinquestriatus hebraeus venom and charybdotoxin was attributed to the stimulation of Ca(++)-activated maxi K+ channels. Snake venom from Dendroaspis polylepis did not alter the stimulation as well as did apamine. ATP was effective on both the apical and basolateral membranes and the Ca(++)-activated maxi K+ channels were predominantly found on the basolateral membrane. This study presents the physiological evidence that dendrotoxin is ineffective on the epithelial Ca(++)-activated maxi K+ channel present in MDCK cells.

Adenosine Triphosphate↗
PubMed1993

Toxin pharmacology of the large-conductance Ca(2+)-activated K+ channel in the apical membrane of rabbit proximal convoluted tubule in primary culture.

The patch-clamp technique was used to study the toxin pharmacology of the large-conductance Ca(2+)-activated K+ channel (BKCa) present in the apical membrane of rabbit proximal convoluted tubules (PCT) in primary culture. Experiments were performed with the inside-out configuration. This channel was very selective for K+ against Na+ and had a conductance of 180 pS with 140 mmol/l in the pipette and the bath. The action of toxins was studied on the extracellular side of the channel by using the pipette perfusion technique. Experimental conditions were 140 mmol/l KCl in the pipette and 140 mmol/l NaCl in the bath. Pipette potential was maintained at 0 mV. Perfusion of crude venom from Leiurus quinquestriatus hebraeus inhibited reversibly the open probability (Po) in a concentration-dependent fashion (IC50 = 0.8 mg/l; n = 3). The following synthetic or purified toxins were tested: synthetic charybdotoxin (ChTX) IC50 = 7.3 x 10(-9) M (n = 5); iberiotoxin (IbTX) IC50 = 5.5 x 10(-7) mol/l (n = 3); and kaliotoxin (KTX) IC50 = 4.8 x 10(-7) mol/l (n = 3). The suppression of the six first N-terminal amino-acids slightly reduced the affinity of ChTX (IC50 = 1.2 x 10(-8) mol/l, n = 4). Neither Dendroaspis polylepis venom nor purified alpha dendrotoxin modified Po even at high concentrations (20 mg/l and 10(-6) mol/l respectively). Apamin, which blocked the small-conductance K+ channel in cultured PCT, did not act on BKCa. These results indicate that ChTX is the most efficient known toxin against the epithelial BKCa in primary cultures of PCT.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Evolution of a cortical collecting tubule primary culture.

The evolution of a primary culture of rabbit kidney cortical collecting duct (CCT) was followed with the electron microscope using two monoclonal antibodies directed against the principal (Mab 703) and intercalated (Mab 503) cells, respectively. As a result of the loss of the basement membrane surrounding the seeded tubule, the intercalated cells showed a tendency to be eliminated while the basal cytoplasm of the remaining cells consisting mainly of principal cells, quickly spread out at the surface of the filter. Between the first and the seventh hour, cells underwent rapid processes of both dedifferentiation and redifferentiation. At 48 h and later on, they started to proliferate with the production of many multinucleated cells. Normal mitotic divisions, in contrast, were rarely encountered. Whereas the number of intercalated cells as recognized by Mab 503 increased from the fourth day up to the tenth day corresponding to a fully mature culture, culture cells at all time intervals rather resembled principal cells found in the internal part of the cortex or in the outer stripe of the external medulla. It is suggested that in our experimental conditions, dedifferentiated principal cells give rise to both principal and intercalated cells as recognized by immunocytochemistry in the fully developed cell culture.

Animals↗
PubMed1992

Toxin pharmacology of the ATP-induced hyperpolarization in Madin-Darby canine kidney cells.

The effects of Leiurus quinquestriatus hebraeus (LQH) venom, mamba venom, Buthus tamulus (BT) venom, purified apamin and synthetic charybdotoxin on the membrane hyperpolarization induced by extracellular ATP were examined in Madin-Darby canine kidney cells. For this we used a membrane potential probe (bisoxonol) to determine the potential variations. The relation between bisoxonal fluorescence and membrane potential was established by treating Madin-Darby canine kidney cells suspended in solutions containing various external sodium concentrations with gramicidin. Extracellular ATP induced a rapid hyperpolarization that was blocked by LQH venom and synthetic charybdotoxin. BT venom also blocked the response but at a much higher concentration than that of LQH. Mamba venom (Dendroaspis polylepis) and apamin did not modify the ATP-induced hyperpolarization. We concluded that the ATP induced hyperpolarization was due to the augmentation of the potassium conductance probably through Ca(2+)-activated K+ channels sensitive to charybdotoxin but not to mamba venom. The interaction previously described between charybdotoxin and dendrotoxin (the main toxin of mamba venom) was not observed in our case.

Adenosine Triphosphate↗
PubMed1992

Effect of calcitonin on the regulation of intracellular pH in primary cultures of rabbit early distal tubule.

To examine the intracellular pH (pHi) regulation in primary cultures of rabbit distal convoluted tubules (DCTb) we used the pH-sensitive dye 2,7-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF/AM) and a video-microscopy technique. DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The culture epithelia were grown on semi-transparent permeable supports. Before pHi measurement, DCTb primary cultures were maintained for 48-96 h in growth-factor-free medium to obtain quiescent cells. We had previously shown that two mechanisms are involved in the regulation of intracellular pH: a basolateral Na+/H+ exchanger and an apical Cl-/HCO3- exchanger. The pHi of DCTb cells was significantly decreased by the addition of 60 nM human calcitonin (from 7.30 +/- 0.04 to 7.08 +/- 0.04). This response to calcitonin was dose-dependent and mimicked by both forskolin and permeant cyclic AMP derivatives. An initial acidification (of 0.25 pH unit in 7-8 min) was observed after the addition of basolateral amiloride (1 mM). The persistence of the effect induced by human calcitonin in these conditions, suggests that the Na+/H+ exchanger is not involved in the response. However, the acidification response was blocked in both the absence of chloride at the apical side and by the apical addition of 0.1 mM 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). These experiments suggest that the target for the human calcitonin effect on pHi is the Cl-/HCO3- exchanger. This study confirms the importance of this transporter in pHi regulation within the physiological pHi range and the influence of calcitonin in the regulation of DCTb cell function.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Principal and intercalated cells in primary cultures of rabbit renal collecting tubules revealed by monoclonal antibodies.

In this study we describe the production and characterization of two monoclonal antibodies (mAb 503 and mAb 703) raised against the apical membrane of rabbit cortical collecting tubule (CCT) cells. The specificity of the two monoclonal antibodies was studied by immunoelectron microscopy on kidney sections. These antibodies were used to identify principal and intercalated cells in primary cultures of CCT. To assess the maintenance of the basic characteristics of the cortical collecting cells during the growth process we determined the biochemical and electrophysiological properties of cultured CCT. Of the monoclonal antibodies produced mAb 503 was specifically directed against the luminal membrane of intercalated cells as shown by immunoelectron microscopy. mAb 703 bound specifically the apical membrane of the principal cells. In primary cultures of CCT mAb 503 and mAb 703 bound antigens present on the apical membrane of different cells and permitted the study of the distribution of the two cell types. Results showed the maintenance of the epithelial polarity of cultured CCT and the expression of specific antigens.

Animals↗