Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P41181 (collecting duct)
 5,183 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We studied the cellular pathways of K+ transport by the rabbit cortical collecting duct that was stimulated to absorb Na+ and to secrete K+. The vast majority of K+ secretion (into the lumen) was inhibited by benzamil, a blocker of epithelial Na+ channels. The residual K+ secretion was completely inhibited by ouabain. Thus all active K+ secretion was dependent on Na+ transport by the Na(+)-K+ pump. The passive pathways of K+ transport were further examined using tracer and electrophysiological measurements. K+ transfer across the apical membrane was predominantly or exclusively conductive; the apical K+ conductance was 31 mS/cm2. The basolateral membrane contained two pathways for K+ tracer translocation. The (barium-sensitive) conductive pathway accounted for a relatively small (12-20%) portion of the tracer permeation. A larger pathway appeared to be via K(+)-K+ exchange on the Na(+)-K+ pump. The magnitude of the Ba2(+)-sensitive (basolateral) K+ conductance predicted a substantially larger tracer flux than was actually measured. The best explanation for this difference is the presence of single-file diffusion through K+ channels on the apical and basolateral membranes. An analysis of the electrically silent K+ transport from lumen to bath suggests that the Na(+)-K+ pump can vary the ratio of its Na(+)-K+ and K(+)-K+ modes of operation. When the tubule is actively transporting Na+ and K+, the Na(+)-K+/K(+)-K+ turnover ratio is greater than 7. When Na+ transport is limited by inhibiting Na+ entry across the apical membrane, the ratio falls to less than 1. A major factor determining this ratio is probably the availability of Na+ to the cytoplasmic side of the pump.
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PMID:Analysis of K+ transport by rabbit CCD: conductive pathways and K(+)-K+ exchange by Na(+)-K+ pump. 131 Feb 30

Interference-contrast and fluorescent microscopy were used to differentiate between the two cell types--principal cells (PC) and intercalated cells (IC)--of the isolated perfused cortical collecting duct of the rabbit. Using Hoffman Modulation Contrast optics, two types of cell outlines could be identified: "hexagonal" and "circular" profiles. To characterize the cell types further, the binding of fluorescein-labeled peanut lectin, which has been shown to be specific for the luminal cell membrane of the IC, was monitored with epifluorescent techniques. The lectin was observed to bind to the circular cell type only, confirming it as the IC. With use of the fluorescent nuclear probe acridine orange to quantitate the total number of cells per millimeter of tubule length, the fraction of ICs (lectin-binding cells) was estimated to average 29%, and the fraction of PCs (non-lectin-binding cells) to average 71% of all cells. The studies were extended to functionally separate between the two cell types by monitoring cell swelling when a lumen-to-bath current pulse was passed. Current-induced swelling was observed only in the PC and could be inhibited by the luminal addition of both the Na+ channel blocker amiloride, and the K+ channel blocker barium, thereby implicating the PC in the process of Na+ absorption and K+ secretion in this tissue. It is concluded that optical techniques can be applied to the cortical collecting duct perfused in vitro to differentiate between and study functional properties of the cell types.
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PMID:Functional differentiation of cell types of cortical collecting duct. 257 83

Initial segments of the inner medullary collecting duct of the rat were perfused in vitro, and the electrophysiological properties of the apical and basolateral membranes were examined with KCl-filled microelectrodes. The fractional resistance of the apical membrane (FRa = Ra/Ra + Rbl) and the transepithelial resistance (RT) were estimated by cable analysis. In control tubules the transepithelial voltage (VT) averaged -2.2 mV, and the voltage across the basolateral membrane (Vbl) averaged -51.1 mV. RT was 11.9 k omega.cm (72.8 omega.cm2), and FRa was 0.94. Pretreatment of the rats with deoxycorticosterone (DOC)-pivalate for 7-10 days did not alter these electrophysiological properties. In control tubules, amiloride in the lumen (10(-5) M) changed VT from -3.0 to +1.4 mV and increased Vbl from -49.4 to -53.8 mV, RT from 12.5 to 13.6 k omega.cm, and FRa from 0.92 to 0.98. Thus the apical membrane is conductive to Na+. An increase of the bath K+ concentration from 4 to 15 mM caused an 18.8 mV depolarization of Vbl: barium in the bath also depolarized Vbl. A fivefold decrease in the [HCO3-] in the bath depolarized Vbl by 13.1 mV. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) blocked this depolarization. Thus the basolateral membrane is conductive to K+ and HCO3-. Experiments with ouabain revealed a Na+-K+-ATPase in the basolateral membrane. Taken together, the results support a model in which electrogenic Na+ absorption is driven by the Na+-K+-ATPase in the basolateral membrane, with passive movement of Na+ occurring through an amiloride-sensitive conductive pathway in the apical membrane.
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PMID:Characterization of apical and basolateral membrane conductances of rat inner medullary collecting duct. 271 19

Microelectrode techniques were applied to the rabbit isolated perfused cortical collecting duct to provide an initial quantitation and characterization of the cell membrane and tight junction conductances. Initial studies demonstrated that the fractional resistance (ratio of the resistance of the apical cell membrane to the sum of the resistances of the apical and basolateral membranes) was usually independent of the point along the tubule of microelectrode impalement--implicating little cell-to-cell coupling--supporting the application of quantitative techniques to the cortical collecting duct. It was demonstrated that in the presence of amiloride, either reduction in the luminal pH or the addition of barium to the perfusate selectively reduced the apical membrane potassium conductance. From the changes in Gte and fractional resistance upon reducing the luminal pH or addition of barium to the perfusate, the transepithelial, apical membrane, basolateral membrane and tight junction conductances were estimated to be 9.3, 6.7, 8.1 and 6.0 mS cm-2, respectively. Ninety to ninety-five percent of the apical membrane conductance reflected the barium-sensitive potassium conductance in the presence of amiloride with an estimated potassium permeability of 1.1 X 10(-4) cm sec-1. Reduction in the perfusate pH to 4.0 caused a 70% decrease in the apical membrane potassium conductance, implying a blocking site with an acidic group having a pKa near 4.4. It is concluded that both the transcellular and paracellular pathways of the cortical collecting tubule have high ionic conductances, and that the apical membrane conductance primarily reflects a high potassium conductance. Furthermore, both reduction in the perfusate pH and addition of barium to the perfusate selectively block the apical potassium channels, although the site of inhibition likely differs since the two ions display markedly different voltage-dependent blocks of the channel.
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PMID:Electrophysiological properties of cellular and paracellular conductive pathways of the rabbit cortical collecting duct. 609 25

On isolated inner medullary collecting duct (IMCD) cells of the rat kidney the capability of osmoregulatory adaptation was investigated in vitro. IMCD cells were isolated by differential centrifugation at 600 mOsm (268 mM NaCl) and subsequently exposed to hypotonic buffers (300 mOsm, 118 mM NaCl). The alterations of ion content and cell volume following this change in extracellular osmolarity were studied by electron probe microanalysis and determination of intracellular water. After swelling within 40 seconds to 152 +/- 15% of control (P < 0.001; N = 9) cell volume was restored after 15 minutes. This regulatory volume decrease (RVD) was observed irrespective whether extracellular osmolarity was changed by using NaCl or mannitol as the major osmolyte. During RVD the cells lost sodium (48 +/- 11%) and chloride (14 +/- 5%), and the potassium content remained nearly unchanged. Correspondingly, sodium and chloride concentrations were progressively lowered, whereas the potassium concentration changed only transiently. RVD was diminished by 10(-4) M NPPB, 10(-3) M SITS and in the absence of HCO3-. Twenty millimoles of ouabain or 5 mM barium also inhibited RVD with little additive effect. A total of 10(-3) M amiloride and 10(-4) M bumetanide showed no effect on the hypoosmotic volume response. The experiments show that in isolated IMCD cells exposed to hypotonic conditions, rapid reversible changes in cell volume and sustained alterations in cell inorganic ion content occur, and thereby transmembrane sodium and potassium gradients are maintained. Since the loss in inorganic electrolytes does not account for RVD, the major part of volume regulation seems to occur via changes in organic osmolytes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ion content and cell volume in isolated collecting duct cells: effect of hypotonicity. 823 Oct 23

Excised patches of apical membranes from immunodissected rabbit cortical collecting duct cells in primary culture were studied by the patch-clamp technique. Barium (1 mM) and tetraethylammonium chloride (5 mM) were added to all solutions to block potassium channel activity. A unique channel was observed that exhibited inward rectification under symmetrical ionic conditions with a measured chord conductance of 54.0 +/- 2.5 pS at -80 mV (n = 11) and 22.1 +/- 1.7 pS at +80 mV (n = 5). This channel was chloride selective, with a PNa:PCl of 0.16 (n = 3). Kinetic analysis revealed a voltage-independent open-time probability of 0.80 +/- 0.07 (n = 6). Open-time probability within bursts was 0.96 +/- 0.01. Addition of ATP to the cytosolic surface of the channel resulted in a dose-dependent decrease in open probability, with a threshold effect at 10(-4) M, due to a reduction in burst open time. The effect of ATP was immediate, rapidly reversible at room temperature, and mimicked by GTP, adenosine 5'-O-(3-thiotriphosphate), and guanosine 5'-O-(3-thiotriphosphate). This channel may link epithelial chloride permeability to cellular ATP content in the rabbit cortical collecting duct.
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PMID:ATP-inhibitable Cl- channel in apical membranes of cultured rabbit cortical collecting duct cells. 823 21

M-1 cells, derived from a microdissected cortical collecting duct of a transgenic mouse, grown to confluence on a permeable support, develop a lumen-negative amiloride-sensitive transepithelial potential, reabsorb sodium, and secrete potassium. Electron micrographs show morphological features typical of principal cells in vivo. Using the patch clamp technique distinct differences are detected in whole-cell membrane current and voltage (Vm) between single M-1 cells 24 h after seeding vs cells grown to confluence. (a) Under control conditions (pipette: KCl-Ringer; bath: NaCl-Ringer) Vm averages -42.7 +/- 3.4 mV in single cells vs -16.8 +/- 4.1 mV in confluent cells. Whole-cell conductance (Gcell) in confluent cells is 2.6 times higher than in single cells. Cell capacitance values are not significantly different in single vs confluent M-1 cells, arguing against electrical coupling of confluent M-1 cells. (b) In confluent cells, 10(-4)-10(-5) M amiloride hyperpolarizes Vm to -39.7 +/- 3.0 mV and the amiloride-sensitive fractional conductance of 0.31 shows a sodium to potassium selectivity ratio of approximately 15. In contrast, single cells express no significant amiloride-sensitive conductance. (c) In single M-1 cells, Gcell is dominated by an inwardly rectifying K-conductance, as exposure to high bath K causes a large depolarization and doubling of Gcell. The barium-sensitive fraction of Gcell in symmetrical KCl-Ringer is 0.49 and voltage dependent. (d) In contrast, neither high K nor barium in the apical bath affect confluent M-1 cells, showing that confluent cells lack a significant apical K conductance. (e) Application of 500 microM glibenclamide reduces whole-cell currents in both single and confluent M-1 cells with a glibenclamide-sensitive fractional conductance of 0.71 and 0.83 in single and confluent cells, respectively. Glibenclamide inhibition occurs slower in confluent M-1 cells than in single cells, suggesting a basolateral action of this lipophilic drug on ATP-sensitive basolateral K channels in M-1 cells. (f) A component of the whole-cell conductance in M-1 cells appears as a deactivating outward current during large depolarizing voltage pulses and is abolished by extracellular chloride removal. The deactivating chloride current averages 103.6 +/- 16.1 pA/cell, comprises 24% of the outward current, and decays with a time constant of 179 +/- 13 ms. The outward to inward conductance ratio obtained from deactivating currents and tail currents is 2.4, indicating an outwardly rectifying chloride conductance.
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PMID:Whole-cell currents in single and confluent M-1 mouse cortical collecting duct cells. 827 Sep 12

This study describes the establishment of a rat kidney cortical collecting duct (CCD) clonal cell line (RCCD1 cells) that maintains high transepithelial resistance and specific hormonal sensitivities. Immortalized cells were obtained by infection of primary cultured CCD cells with the wild-type simian virus 40. Grown on Petri dishes, RCCD1 cells are organized as monolayers of cuboid cells separated by tight junctions and form domes. Grown on permeable filters, confluent RCCD1 cells exhibit high transepithelial resistance (Rt: 2390 +/- 140 omega. cm2), transepithelial potential difference (PD) of -10.5 +/- 1.2 mV lumen negative, an associated short-circuit current (Isc) of 4.3 +/- 0.5 microA/cm2, and generated significant Na+, K+, H+ and HCO3- gradients, reflecting Na+ and H+ reabsorption and K+ and HCO3- secretion. RCCD1 cells exhibit features of both principal (PC) and intercalated (IC) cells. Consistent with PC phenotype, about 50% of the cells were positively stained by a PC-specific agglutinin. In situ hybridization studies revealed the presence of alpha, beta and gamma subunit mRNAs of the amiloride-sensitive epithelial Na+ channel and alpha 1 and beta 1 subunits of Na(+)-K(+)-ATPase. Moreover, Na(+)-K(+)-ATPase was immunolocalized at the basolateral side of the cells. Arginine vasopressin (AVP) induced a significant increase in both cellular cAMP content and Isc. Amiloride decreased in a dose-dependent manner Isc from untreated and AVP-treated RCCD1 cells. In addition, a barium-sensitive K+ conductance was evidenced in the apical side of the cells. Consistent with IC phenotype, isoproterenol (ISO) provoked a large increase in cellular cAMP and stimulated Isc. The effect of ISO on Isc was blocked by 5 x 10(-3) M DPC, a chloride channel blocker. Finally, AVP plus ISO had additive effect on Isc. Taken together, these results provide evidence that the RCCD1 cell line has maintained many of the original properties of rat CCD from which they were derived.
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PMID:Characteristics of a rat cortical collecting duct cell line that maintains high transepithelial resistance. 884 Feb 62

The mechanism of NH4+ transport in inner medulla is not known. The purpose of these experiments was to study the process that is involved in ammonium (NH4+) transport in cultured inner medullary collecting duct (mIMCD-3) cells. Cells grown on coverslips were exposed to NH4+ and monitored for pHi changes by the use of the pH-sensitive dye BCECF. The rate of cell acidification following the initial cell alkalinization was measured as an index of NH4+ transport. The rate of NH4+ transport was the same in the presence or absence of sodium in the media (0.052 +/- 0.003 vs 0.048 +/- 0.004 pH/min. P > 0.05), indicating that NH4+ entry into the cells was independent of sodium. The presence of ouabain, bumetanide, amiloride, barium, or 4,4'-di-isothiocyanostilbene-2-2'-disulfonic acid (DIDS) did not block the NH4(+)-induced cell acidification, indicating lack of involvement of Na+:K(+)-ATPase, Na+:K+:2Cl- transport, Na+:H+ exchange, K+ channel, or Cl-/base exchange, respectively, in NH4+ transport. The NH4(+)-induced cell acidification was significantly inhibited in the presence of high external [K+] as compared to low external [K+] (0.018 +/- 0.001 vs. 0.049 +/- 0.003 pH/min for 140 mM K+ vs. 1.8 mM K+ in the media, respectively, P < 0.001). Inducing K+ efflux by imposing an outward K+ gradient caused intracellular acidification by approximately 0.3 pH unit in the presence but not the absence of NH4+. This K+ efflux-induced NH4+ entry increased by extracellular NH4+ in a saturable manner with a Km of approximately 5 mM, blocked by increasing extracellular K+ and was not inhibited by barium. The K+ efflux-coupled NH4+ entry was electroneutral as monitored by the use of cell membrane potential probe 3,3'-dipropylthiadicarbocyanine. These results are consistent with the exchange of internal K+ with external NH4+ in a 1:1 ratio. The K(+)-NH4+ antiporter was inhibited by verapamil and Schering 28080 in a dose-dependent manner, was able to work in reverse mode, and did not show any affinity for H+ as a substrate, indicating that it is distinct from other NH4(+)-carrying transporters. We conclude that a unique transporter, a potassium-ammonium (K+/NH4+) antiport, is responsible for NH4+ transport in renal inner medullary collecting duct cells. This antiporter is sensitive to verapamil and Schering 28080, is electroneutral, and is selective for NH4+ and K+ as substrates. The K+/NH4+ antiporter may play a significant role in acid-base regulation by excretion of ammonium and elimination of acid.
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PMID:K+/NH4+ antiporter: a unique ammonium carrying transporter in the kidney inner medulla. 904 54

We recently cloned an inward-rectifying K channel (Kir) cDNA, CCD-IRK3 (mKir 2.3), from a cortical collecting duct (CCD) cell line. Although this recombinant channel shares many functional properties with the "small-conductance" basolateral membrane Kir channel in the CCD, its precise subcellular localization has been difficult to elucidate by conventional immunocytochemistry. To circumvent this problem, we studied the targeting of several different epitope-tagged CCD-IRK3 in a polarized renal epithelial cell line. Either the 11-amino acid span of the vesicular stomatitis virus (VSV) G glycoprotein (P5D4 epitope) or a 6-amino acid epitope of the bovine papilloma virus capsid protein (AU1) was genetically engineered on the extreme N terminus of CCD-IRK3. As determined by patch-clamp and two-microelectrode voltage-clamp analyses in Xenopus oocytes, neither tag affected channel function; no differences in cation selectivity, barium block, single channel conductance, or open probability could be distinguished between the wild-type and the tagged constructs. MDCK cells were transfected with tagged CCD-IRK3, and several stable clonal cell lines were generated by neomycin-resistance selection. Immunoprecipitation studies with anti-P5D4 or anti-AU1 antibodies readily detected the predicted-size 50-kDa protein in the transfected cells lines but not in wild-type or vector-only (PcB6) transfected MDCK cells. As visualized by indirect immunofluorescence and confocal microscopy, both the tagged CCD-IRK3 forms were exclusively detected on the basolateral membrane. To assure that the VSV G tag was not responsible for the targeting, the P5D4 epitope modified by a site-directed mutagenesis (Y2F) to remove a potential basolateral targeting signal contained in this tag. VSV(Y2F) was also detected exclusively on the basolateral membrane, confirming bona fide IRK3 basolateral expression. These observations, with our functional studies, suggest that CCD-IRK3 may encode the small-conductance CCD basolateral K channel.
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PMID:Basolateral membrane targeting of a renal-epithelial inwardly rectifying potassium channel from the cortical collecting duct, CCD-IRK3, in MDCK cells. 937 45


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