Chloride Currents Activated by Calcitonin and cAMP in Primary Cultures of Rabbit Distal Convoluted Tubule

International audienceChloride (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

Toxin Sensitivity of the Calcium-Dependent Rubidium Efflux in Madin-Darby Canine Kidney Cells

International audience86Rb+ 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

Chloride currents in primary cultures of rabbit proximal and distal convoluted tubules.

Cl- conductances were studied in cultured rabbit proximal convoluted tubule (PCT) epithelial cells and compared with those measured in cultured distal bright convoluted tubule (DCTb) epithelial cells. Using the whole cell patch-clamp technique, three types of Cl- conductances were identified in DCTb cultured cells. These consisted of volume-sensitive, Ca2+-activated, and forskolin-activated Cl- currents. In PCT cultured cells, only volume-sensitive and Ca2+-activated Cl- currents were recorded. The characteristics of Ca2+-activated currents in PCT cells closely resembled those in DCTb cells. Volume-sensitive Cl- currents could be elicited both in PCT and in DCTb cells by hypotonic stress. The pharmacological profile of this conductance was established for both cell types. Forskolin activated a linear Cl- current in DCTb cells but not in PCT cells. This conductance was insensitive to DIDS and corresponds to cystic fibrosis transmembrane conductance regulator (CFTR)-like channels. Quantitative measurements of SPQ fluorescence showed that only the apical membrane of DCTb cells possessed a Cl- pathway that was sensitive to forskolin. RT-PCR experiments showed the presence of CFTR mRNA in both cultures, whereas immunostaining experiments revealed the expression of CFTR in DCTb cells only. The physiological role of the different types of channels is discussed

Characterization of monoclonal antibodies specific for rabbit renal brush-border hydrolases: application to immunohistological localization.

International audienceBy use of immunodepletion studies, we characterized four monoclonal antibodies reactive with rabbit brush-border (BB) as specific for aminopeptidase N (AP), dipeptidylpeptidase IV (DPPIV), neutral endopeptidase (EP), and angiotensin-converting enzyme (ACE), and we used these antibodies for immunohistochemical detection of these four hydrolases. Expression within the kidney was studied by light and electron microscopy. All four hydrolases are expressed on the various segments of the proximal tubule. In addition, EP and DPPIV are detectable on visceral epithelial cells of the glomerulus and AP on the cells of Bowman's capsule. Outside the kidney, the four hydrolases are expressed within the digestive and genital tracts, where AP, EP, and DPPIV predominate on epithelial structures, whereas ACE is essentially located in vascular structures. The latter localization is also characteristic of ACE in the other organs studied, where clear-cut systematic distribution of the other hydrolases was often difficult to demonstrate. In addition, AP, DPPIV, and EP were detected on lymphoid cells. As compared to reports of data obtained essentially by enzymatic or immunoradiometric assays, these observations suggest considerable interspecies variations of extrarenal expression of the major BB hydrolases. This should be taken into account in attempting to define a general physiological role for a given enzyme

Evolution of a cortical collecting tubule primary culture.

International audienceThe 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