Active calcium absorption in primary cultures of cortical collecting duct cells

1993 ◽  
Vol 71 (7) ◽  
pp. 491-496 ◽  
Author(s):  
Lori G. Rochelle ◽  
Sangita Deveshwar ◽  
Anikó Náray-Fejes-Tóth ◽  
Géza Fejes-Tóth
Keyword(s):  
Cultured Cells ◽  
Calcium Absorption ◽  
Collecting Duct ◽  
Primary Cultures ◽  
Channel Blockers ◽  
Ca Channel ◽  
Cortical Collecting Duct ◽  
Cell Monolayers ◽  
Active Calcium ◽  
Plastic Surfaces

Primary cultures of rabbit cortical collecting duct (CCD) cells demonstrated accumulation of Ca at the basolateral (BL) side when cultured on either impermeable or permeable supports. Cell monolayers cultured on impermeable plastic surfaces absorbed Ca with such avidity that hydroxyapatite crystals formed. When cultured on a permeable matrix, the cells demonstrated significant net apical (A) to BL Ca transport against a lumen negative transepithelial potential difference. A steady-state BL/A [Ca] ratio of 120 developed across monolayers in 24 h on days 6 through 8 postseeding. Initial rates of unidirectional 45Ca fluxes on days 6 through 8 indicated a negligible BL to A flux (5.4 ± 2.6 nmol∙h−1∙cm−2) compared with A to BL 45Ca flux (99.4 ± 19.4 nmol∙h−1∙cm−2). Parathyroid hormone applied to the BL side had no significant effect on either unidirectional 45Ca flux, but the second messenger analog, 8-bromoadenosine cyclic monophosphate, increased the A to BL flux by 65%. Inhibiting the Na+–K+ ATPase with ouabain (10−4 M) reduced the A to BL flux by 77%; however, a significant net A to BL flux still remained. Apical addition of amiloride (2 × 10−5 M) did not affect either unidirectional 45Ca flux. In addition, the inorganic Ca channel blockers Ni2+ (100 μM and 1 mM), La3+ (100 μM and 1 mM), and Cd2+ (20 and 50 μM) did not significantly inhibit either unidirectional 45Ca flux. These results demonstrate that CCD monolayers actively absorb Ca and this can be stimulated by cyclic AMP, raising the possibility that apical Ca entry does not involve amiloride-sensitive channels, or typical Ca channels.Key words: calcium transport, cortical collecting duct, cultured cells.

AVP stimulates Na+ transport in primary cultures of rabbit cortical collecting duct cells

1992 ◽  
Vol 262 (3) ◽  
pp. F454-F461 ◽  
Author(s):  
C. M. Canessa ◽  
J. A. Schafer
Keyword(s):  
Cultured Cells ◽  
Collecting Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Cortical Collecting Duct ◽  
Na Transport ◽  
Permeable Membrane ◽  
Ussing Chambers ◽  
Principal And Intercalated Cells

Immunodissected rabbit cortical collecting duct (CCD) cells were grown in primary culture on permeable membrane supports. Transepithelial voltage, Na+, K+, and H+ gradients developed as expected for a mixed population of principal and intercalated cells. The amiloride-sensitive short-circuit current (Isc) was measured in Ussing chambers as an index of Na+ transport via apical membrane Na+ channels. Treatment of the cells in culture with 10 nM aldosterone for 48 h increased Isc from 7.4 +/- 1.4 to 19.3 +/- 3.2 microA/cm2. In contrast to the native rabbit CCD, 220 pM arginine vasopressin (AVP) produced a rapid and stable (greater than 60 min) increase in Isc to 15.8 +/- 2.0 and 29.0 +/- 3.8 microA/cm2 in untreated and aldosterone-treated cultures, respectively. Although prostaglandin E2 (PGE2) inhibits Na+ transport in the native rabbit CCD, it did not in the cultured cells, and it has previously been shown that PGE2 inhibition of AVP-dependent adenosine 3',5'-cyclic monophosphate production is lost in culture (W. K. Sonnenburg and W. L. Smith, J. Biol. Chem. 263: 6155-6160, 1988). We conclude that the development of a stable stimulation of Na+ transport by AVP is linked to the loss of the inhibitory effects of PGE2.

Nifedipine-activated Ca2+ permeability in newborn rat cortical collecting duct cells in primary culture

2001 ◽  
Vol 280 (5) ◽  
pp. C1193-C1203 ◽  
Author(s):  
Laura Valencia ◽  
Michel Bidet ◽  
Sonia Martial ◽  
Elsa Sanchez ◽  
Estela Melendez ◽  
...  
Keyword(s):  
Primary Culture ◽  
Transient Receptor Potential ◽  
Collecting Duct ◽  
Primary Cultures ◽  
Receptor Potential ◽  
Cortical Collecting Duct ◽  
Newborn Rat ◽  
Adult Rat ◽  
Intracellular Ca ◽  
Transient Receptor

To characterize Ca2+ transport in newborn rat cortical collecting duct (CCD) cells, we used nifedipine, which in adult rat distal tubules inhibits the intracellular Ca2+concentration ([Ca2+]i) increase in response to hormonal activation. We found that the dihydropyridine (DHP) nifedipine (20 μM) produced an increase in [Ca2+]i from 87.6 ± 3.3 nM to 389.9 ± 29.0 nM in 65% of the cells. Similar effects of other DHP (BAY K 8644, isradipine) were also observed. Conversely, DHPs did not induce any increase in [Ca2+]i in cells obtained from proximal convoluted tubule. In CCD cells, neither verapamil nor diltiazem induced any rise in [Ca2+]i. Experiments in the presence of EGTA showed that external Ca2+ was required for the nifedipine effect, while lanthanum (20 μM), gadolinium (100 μM), and diltiazem (20 μM) inhibited the effect. Experiments done in the presence of valinomycin resulted in the same nifedipine effect, showing that K+ channels were not involved in the nifedipine-induced [Ca2+]i rise. H2O2also triggered [Ca2+]i rise. However, nifedipine-induced [Ca2+]i increase was not affected by protamine. In conclusion, the present results indicate that 1) primary cultures of cells from terminal nephron of newborn rats are a useful tool for investigating Ca2+transport mechanisms during growth, and 2) newborn rat CCD cells in primary culture exhibit a new apical nifedipine-activated Ca2+ channel of capacitive type (either transient receptor potential or leak channel).

Modulation of aldosterone-induced stimulation of ENaC synthesis by changing the rate of apical Na+ entry

2001 ◽  
Vol 281 (4) ◽  
pp. F687-F692 ◽  
Author(s):  
Lisette Dijkink ◽  
Anita Hartog ◽  
Carel H. Van Os ◽  
René J. M. Bindels
Keyword(s):  
Feedback Inhibition ◽  
Collecting Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Inhibitory Effect ◽  
Cortical Collecting Duct ◽  
Entry Rate ◽  
Protein Levels ◽  
Stimulation Of

Primary cultures of immunodissected rabbit connecting tubule and cortical collecting duct cells were used to investigate the effect of apical Na+ entry rate on aldosterone-induced transepithelial Na+ transport, which was measured as benzamil-sensitive short-circuit current ( I sc). Stimulation of the apical Na+ entry, by long-term short-circuiting of the monolayers, suppressed the aldosterone-stimulated benzamil-sensitive I sc from 320 ± 49 to 117 ± 14%, whereas in the presence of benzamil this inhibitory effect was not observed (335 ± 74%). Immunoprecipitation of [35S]methionine-labeled β-rabbit epithelial Na+ channel (rbENaC) revealed that the effects of modulation of apical Na+ entry on transepithelial Na+ transport are exactly mirrored by β-rbENaC protein levels, because short-circuiting the monolayers decreased aldosterone-induced β-rbENaC protein synthesis from 310 ± 51 to 56 ± 17%. Exposure to benzamil doubled the β-rbENaC protein level to 281 ± 68% in control cells but had no significant effect on aldosterone-stimulated β-rbENaC levels (282 ± 68%). In conclusion, stimulation of apical Na+ entry suppresses the aldosterone-induced increase in transepithelial Na+transport. This negative-feedback inhibition is reflected in a decrease in β-rbENaC synthesis or in an increase in β-rbENaC degradation.

Vectorial efflux of cGMP and its dependence on sodium in the cortical collecting duct

1996 ◽  
Vol 271 (6) ◽  
pp. R1676-R1681 ◽  
Author(s):  
B. A. Stoos ◽  
J. L. Garvin
Keyword(s):  
Cultured Cells ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Transport Mechanisms ◽  
Cortical Collecting Duct ◽  
Phosphodiesterase Inhibition ◽  
Luminal Membrane ◽  
Sodium Dependent ◽  
Independent Mechanism ◽  
Presence And Absence

Guanosine 3',5'-cyclic monophosphate (cGMP) is an important second messenger that regulates transport in the nephron. We propose that the transport mechanisms that remove cGMP from the cell are different in the luminal and basolateral membranes of the cortical collecting duct (CCD). We examined efflux of cGMP from cultured and isolated perfused CCDs in response to atrial natriuretic factor (ANF) and nitric oxide (NO). In the presence of phosphodiesterase inhibition, these compounds resulted in preferential efflux of cGMP across the basolateral membrane in both cultured and isolated CCDs. In the presence of ANF, efflux was five times higher across the basolateral than the luminal membrane in cultured CCD cells (n = 14). In isolated CCDs, effluxes across the basolateral and luminal membranes were 1.02 +/- 0.2 and 0.03 +/- 0.01 fmol.mm-1.min-1, respectively, in the presence of ANF (n = 6; P < 0.007) and 0.87 +/- 0.21 and 0.02 +/- 0.01 fmol.mm-1.min-1, respectively, in the presence of NO (n = 6; P < 0.011). Efflux across the basolateral membrane in the presence and absence of sodium was 37 +/- 7.3 and 19.9 +/- 5 fmol.cm-2.min-1, respectively, in cultured cells (n = 12; P < 0.044) and 1.02 +/- 0.2 (n = 6) and 0.41 +/- 0.12 (n = 5) fmol.mm-1.min-1 in isolated perfused tubules (P < 0.042). There was no difference in luminal transport in the presence and absence of sodium in either model. We conclude that there are at least two different mechanisms involved in the removal of cGMP from the cell, one sodium dependent and the other sodium independent. The basolateral membrane appears to contain both, whereas the luminal membrane contains only the sodium-independent mechanism.

Characteristics of papillary collecting duct cells in primary culture

1988 ◽  
Vol 255 (6) ◽  
pp. F1160-F1169 ◽  
Author(s):  
R. F. Husted ◽  
M. Hayashi ◽  
J. B. Stokes
Keyword(s):  
Cultured Cells ◽  
Collecting Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Calf Serum ◽  
Morphological Characteristics ◽  
Short Circuit Current ◽  
Papillary Collecting Duct ◽  
Collecting Duct Cells ◽  
Na Uptake

We examined the electrophysiological and Na+ transport characteristics of rat papillary collecting duct (PCD) cells grown in primary cultures. Grown as monolayers on polycarbonate filters, the cells displayed similar morphological characteristics to native epithelia. They also bound Dolichus biflorus lectin, a property shared by native cells. Monolayers developed a peak electrical resistance of 100-200 omega.cm2 and a transmonolayer voltage of less than 2 mV. Similar values were measured in the perfused, native PCD of the same species as well as PCD cells cultured from rabbit and bovine kidneys. Hamster cells did not readily develop confluent monolayers under the same conditions. Exposure of the cultured cells to 10% fetal calf serum for 24 h caused the Na+ uptake across the apical membrane to double, an effect not reproduced by indomethacin, insulin, vasopressin, aldosterone, dexamethasone, or hexamethylene bisacetamide (an inducer of differentiation). Amiloride (1 mM) inhibited Na+ uptake by 50-80%. The measured short-circuit current did not correlate with Na+ uptake and was clearly dissociated by exposure to serum. The results suggest that there is more than one mechanism of ion transport by the rat PCD.

CFTR disruption impairs cAMP-dependent Cl−secretion in primary cultures of mouse cortical collecting ducts

2001 ◽  
Vol 281 (3) ◽  
pp. F434-F442 ◽  
Author(s):  
Marcelle Bens ◽  
Jean-Paul Duong Van Huyen ◽  
Françoise Cluzeaud ◽  
Jacques Teulon ◽  
Alain Vandewalle
Keyword(s):  
Collecting Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Similar Degree ◽  
Transmembrane Conductance Regulator ◽  
Cortical Collecting Duct ◽  
Transmembrane Conductance ◽  
Nacl Transport ◽  
Cystic Fibrosis Transmembrane

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in the renal cortical collecting duct (CCD) has not yet been fully elucidated. Here, we investigated the effects of deamino-8-d-arginine vasopressin (dDAVP) and isoproterenol (ISO) on NaCl transport in primary cultured CCDs microdissected from normal [CFTR(+/+)] and CFTR-knockout [CFTR(−/−)] mice. dDAVP stimulated the benzamyl amiloride (BAm)-sensitive transport of Na+ assessed by the short-circuit current ( I sc) method in both CFTR(+/+) and CFTR(−/−) CCDs to a very similar degree. Apical addition of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) or glibenclamide partially inhibited the rise in I sc induced by dDAVP and ISO in BAm-treated CFTR(+/+) CCDs, whereas dDAVP, ISO, and NPPB did not alter I sc in BAm-treated CFTR(−/−) CCDs. dDAVP stimulated the apical-to-basal flux and, to a lesser extent, the basal-to-apical flux of 36Cl− in CFTR(+/+) CCDs. dDAVP also increased the apical-to-basal36Cl− flux in CFTR(−/−) CCDs but not the basal-to-apical 36Cl− flux. These results demonstrate that CFTR mediates the cAMP-stimulated component of secreted Cl− in mouse CCD.

scholarly journals Cyclic AMP Increases Cell Surface Expression of Functional Na,K-ATPase Units in Mammalian Cortical Collecting Duct Principal Cells

2001 ◽  
Vol 12 (2) ◽  
pp. 255-264 ◽  
Author(s):  
Sandrine Gonin ◽  
Georges Deschênes ◽  
Frank Roger ◽  
Marcelle Bens ◽  
Pierre-Yves Martin ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Cell Surface ◽  
Atpase Activity ◽  
Cultured Cells ◽  
Collecting Duct ◽  
Brefeldin A ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Cortical Collecting Duct ◽  
Expression And Activity

Cyclic AMP (cAMP) stimulates the transport of Na+ and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to investigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity in microdissected rat CCDs and cultured mouse mpkCCDc14 collecting duct cells. db-cAMP (10−3 M) stimulated by 2-fold the activity of Na,K-ATPase from rat CCDs as well as the ouabain-sensitive component of 86Rb+ uptake by rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinylation procedure revealed that db-cAMP increased the amount of Na,K-ATPase at the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1.3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20°C) prevented both the db-cAMP-dependent increase in cell surface expression and activity of Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca2+chelator bis-(o-aminophenoxy)-N,N,N′,N′-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly suggest that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD results from the translocation of active pump units from an intracellular compartment to the plasma membrane.

Cellular calcium transport in renal epithelia: measurement, mechanisms, and regulation

1995 ◽  
Vol 75 (3) ◽  
pp. 429-471 ◽  
Author(s):  
P. A. Friedman ◽  
F. A. Gesek
Keyword(s):  
Plasma Membranes ◽  
Single Channel ◽  
Calcium Absorption ◽  
Vital Role ◽  
Channel Blockers ◽  
Calcium Efflux ◽  
Cellular Calcium ◽  
Wide Range ◽  
Active Calcium ◽  
Convoluted Tubules

The kidneys play a vital role in mineral homeostasis. In this review, the handling of calcium and the methods currently applied to measuring its intracellular concentration are discussed. The bulk of calcium absorption proceeds in proximal tubules, with smaller fractions recovered by thick ascending limbs, distal convoluted tubules, and connecting tubules. Hormonally regulated transcellular calcium absorption is essentially limited to distal convoluted and connecting tubules. At physiological concentrations, parathyroid hormone, calcitonin, and vitamin D increase net calcium absorption. Calcium absorption by polarized epithelial cells is a two-step process wherein calcium enters the cell across apical plasma membranes and exits across basolateral membranes. Recent electrophysiological and pharmacological experiments demonstrate that apical entry is mediated by calcium channels, which are modestly calcium selective, sensitive to dihydropyridine-type calcium channel blockers, and exhibit a wide range of single-channel conductances. Cellular calcium efflux is mediated by Ca(2+)-ATPase and by Na+/Ca2+ exchange. Ca(2+)-ATPase activity is highest in segments that exhibit significant rates of active calcium absorption. Multiple plasma membrane Ca(2+)-ATPase isoforms have been found in the kidney. Several renal Na+/Ca2+ exchange isoforms have been identified, and their role in effecting calcium efflux is under investigation.

A hyperpolarization-activated, cyclic nucleotide-gated, (Ih-like) cationic current and HCN gene expression in renal inner medullary collecting duct cells

2008 ◽  
Vol 294 (4) ◽  
pp. C893-C906 ◽  
Author(s):  
Juan J. Bolívar ◽  
Dagoberto Tapia ◽  
Gabina Arenas ◽  
Mauricio Castañón-Arreola ◽  
Haydee Torres ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Collecting Duct ◽  
Primary Cultures ◽  
Inner Medullary Collecting Duct ◽  
Cationic Current ◽  
Duct Cells ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct

The cation conductancein primary cultures of rat renal inner medullary collecting duct was studied using perforated-patch and conventional whole cell clamp techniques. Hyperpolarizations beyond −60 mV induced a time-dependent inward nonselective cationic current ( Ivti) that resembles the well-known hyperpolarization-activated, cyclic nucleotide-gated Ih and If currents. Ivti showed a half-maximal activation around −102 mV with a slope factor of 25 mV. It had a higher conductance (but, at its reversal potential, not a higher permeability) for K+ than for Na+ ( gK+/ gNa+ = 1.5), was modulated by cAMP and blocked by external Cd2+ (but not Cs+ or ZD-7288), and potentiated by a high extracellular K+ concentration. We explored the expression of the Ih channel genes (HCN1 to -4) by RT-PCR. The presence of transcripts corresponding to the HCN1, -2, and -4 genes was observed in both the cultured cells and kidney inner medulla. Western blot analysis with HCN2 antibody showed labeling of ∼90- and ∼120-kDa proteins in samples from inner medulla and cultured cells. Immunocytochemical analysis of cell cultures and inner medulla showed the presence of HCN immunoreactivity partially colocalized with the Na+-K+-ATPase at the basolateral membrane of collecting duct cells. This is the first evidence of an Ih-like cationic current and HCN immunoreactivity in either kidney or any other nonexcitable mammalian cells.

scholarly journals Effect of luminal flow on doming of mpkCCD cells in a 3D perfusable kidney cortical collecting duct model

2020 ◽  
Vol 319 (1) ◽  
pp. C136-C147
Author(s):  
Joshua L. Rein ◽  
Szilvia Heja ◽  
Daniel Flores ◽  
Rolando Carrisoza-Gaytán ◽  
Neil Y. C. Lin ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Three Dimensional ◽  
Transepithelial Transport ◽  
Cortical Collecting Duct ◽  
Acid Base ◽  
Cell Monolayers ◽  
Luminal Flow ◽  
Total Body ◽  
Epithelial Sheets

The cortical collecting duct (CCD) of the mammalian kidney plays a major role in the maintenance of total body electrolyte, acid/base, and fluid homeostasis by tubular reabsorption and excretion. The mammalian CCD is heterogeneous, composed of Na+-absorbing principal cells (PCs) and acid-base-transporting intercalated cells (ICs). Perturbations in luminal flow rate alter hydrodynamic forces to which these cells in the cylindrical tubules are exposed. However, most studies of tubular ion transport have been performed in cell monolayers grown on or epithelial sheets affixed to a flat support, since analysis of transepithelial transport in native tubules by in vitro microperfusion requires considerable expertise. Here, we report on the generation and characterization of an in vitro, perfusable three-dimensional kidney CCD model (3D CCD), in which immortalized mouse PC-like mpkCCD cells are seeded within a cylindrical channel embedded within an engineered extracellular matrix and subjected to luminal fluid flow. We find that a tight epithelial barrier composed of differentiated and polarized PCs forms within 1 wk. Immunofluorescence microscopy reveals the apical epithelial Na+ channel ENaC and basolateral Na+/K+-ATPase. On cessation of luminal flow, benzamil-inhibitable cell doming is observed within these 3D CCDs consistent with the presence of ENaC-mediated Na+ absorption. Our 3D CCD provides a geometrically and microphysiologically relevant platform for studying the development and physiology of renal tubule segments.

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