UNIPROT:P41181 - FACTA Search

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Query: UNIPROT:P41181 (collecting duct)
5,183 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Using equivalent short circuit current (ISC) measurements we examined the effect of extracellular ATP on transepithelial ion transport in M-1 mouse cortical collecting duct cells. Apical addition of ATP produced a rapid transient peak increase in ISC. This was followed by a fall below basal ISC due to a reduction in the amiloride-sensitive ISC component. 2. The ATP-induced ISC increase was preserved in the presence of apical amiloride while it was reduced in the absence of extracellular Cl- and in the presence of the apical Cl- channel blockers diphenylamine-2-carboxylic acid (DPC, 1 mM), DIDS (300 microM) and niflumic acid (100 microM). 3. The stimulatory effect of apical ATP on ISC was concentration dependent with an EC50 of about 0.6 microM. Basolateral ATP elicited a similar ISC response. Experiments using the ATP scavenger hexokinase demonstrated that the ATP effects were elicited via separate apical and basolateral receptors. 4. ATP and UTP applied to either the apical or the basolateral bath equi-potently stimulated ISC while 'purified' ADP and UDP had no effect consistent with P2Y2 purinoceptors, the expression of which was confirmed using RT-PCR. 5. Intracellular calcium concentration ([Ca2+]i) measurements using fura-2 demonstrated that ATP and UTP elicited a rise in [Ca2+]i with EC50 values of 1.1 and 0.6 microM, respectively. The shape and time course of the calcium response were similar to those of the ISC response. The peak ISC response was preserved in the nominal absence of extracellular calcium but was significantly reduced in cells pre-incubated with the calcium chelator BAPTA AM. 6. We conclude that in M-1 cells extracellular ATP reduces amiloride-sensitive Na+ absorption and stimulates Cl- secretion via calcium-activated Cl- channels through activation of P2Y2 purinoreceptors located in the apical and basolateral membrane.
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PMID:ATP stimulates Cl- secretion and reduces amiloride-sensitive Na+ absorption in M-1 mouse cortical collecting duct cells. 1074 85

Recent studies showed that coexpression of Kir6.1 or Kir6.2 with the sulfonylurea receptor (SUR1, SUR2A, or SUR2B) reconstituted an inwardly rectifying, ATP-sensitive K(+) channel that was inhibited by glibenclamide (2, 15-17). Here we report the isolation of a rat homolog of mouse SUR2B (denoted rSUR2B) from a rat kidney cDNA library. The rSUR2B sequence contains a 4,635-bp open reading frame that encodes a 1,545-amino acid polypeptide, showing 67% shared identity with SUR1 (a pancreatic beta-cell isoform) and 98% with both SUR2A (a brain isoform) and SUR2B (a vascular smooth muscle isoform). Consistent with the predicted structures of other members of the ATP-binding cassette (ABC) superfamily, the sequence of rSUR2B contains 17 putative membrane-spanning segments. Also, predicted Walker A and B consensus binding motifs, present in other ABC members, are conserved in the rSUR2B sequence. RT-PCR revealed that rSUR2B is widely expressed in various rat tissues including brain, colon, heart, kidney, liver, skeletal muscle, and spleen. The intrarenal distribution of the rSUR2B transcript was investigated using RT-PCR and Southern blot of microdissected tubules. The rSUR2B transcript was detected in proximal tubule, cortical thick ascending limb, distal collecting tubule, cortical collecting duct, and outer medullary collecting duct, but not medullary thick ascending limb. This distal distribution overlaps with that of ROMK. Coexpression of rSUR2B with ROMK2 cRNA (in 1:10 ratio) in Xenopus laevis oocytes resulted in whole cell Ba(2+)-sensitive K(+) currents that were inhibited by glibenclamide (50% inhibition with 0.2 mM glibenclamide). In contrast, rSUR2B did not confer significant glibenclamide sensitivity to oocytes coinjected with ROMK1 or ROMK3. The interaction between ROMK2 and rSUR2B was further studied by coimmunoprecipitation of in vitro translated rSUR2B and ROMK2. In agreement with the functional data, the rSUR2B protein was coimmunoprecipitated with ROMK2 in the ROMK2-rSUR2B cotranslated samples. Our data demonstrate that ROMK2, but not ROMK1 and ROMK3, can interact with rSUR2B to confer a sulfonylurea-sensitive K(+) channel, implicating SUR proteins in forming and regulating renal ATP-sensitive K(+) channels. The ROMK isoform specificity of glibenclamide effects suggests that the NH(2) terminus of the ROMK protein mediates rSUR2B-ROMK2 interactions.
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PMID:Rat homolog of sulfonylurea receptor 2B determines glibenclamide sensitivity of ROMK2 in Xenopus laevis oocyte. 1075 Dec 28

We have used the patch-clamp technique to study the effects of changing extracellular ATP concentration on the activity of the small-conductance potassium channel (SK) on the apical membrane of the mouse cortical collecting duct. In cell-attached patches, the channel conductance and kinetics were similar to its rat homologue. Addition of ATP to the bathing solution of split-open single cortical collecting ducts inhibited SK activity. The inhibition of the channel by ATP was reversible, concentration dependent (K(i) = 64 microM), and could be completely prevented by pretreatment with suramin, a specific purinergic receptor (P(2)) blocker. Ranking of the inhibitory potency of several nucleotides showed strong inhibition by ATP, UTP, and ATP-gamma-S, whereas alpha, beta-Me ATP, and 2-Mes ATP failed to affect channel activity. This nucleotide sensitivity is consistent with P(2)Y(2) purinergic receptors mediating the inhibition of SK by ATP. Single channel analysis further demonstrated that the inhibitory effects of ATP could be elicited through activation of apical receptors. Moreover, the observation that fluoride mimicked the inhibitory action of ATP suggests the activation of G proteins during purinergic receptor stimulation. Channel inhibition by ATP was not affected by blocking phospholipase C and protein kinase C. However, whereas cAMP prevented channel blocking by ATP, blocking protein kinase A failed to abolish the inhibitory effects of ATP. The reduction of K channel activity by ATP could be prevented by okadaic acid, an inhibitor of protein phosphatases, and KT5823, an agent that blocks protein kinase G. Moreover, the effect of ATP was mimicked by cGMP and blocked by L-NAME (N(G)-nitro-l-arginine methyl ester). We conclude that the inhibitory effect of ATP on the apical K channel is mediated by stimulation of P(2)Y(2) receptors and results from increasing dephosphorylation by enhancing PKG-sensitive phosphatase activity.
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PMID:Extracellular ATP inhibits the small-conductance K channel on the apical membrane of the cortical collecting duct from mouse kidney. 1091 72

It has previously been shown that osmotic cell shrinkage activates a nonselective cation (NSC) channel in M-1 mouse cortical collecting duct cells [54] and in a variety of other cell types [20]. In the present study we further characterized the shrinkage-activated NSC channel in M-1 cells and its mechanism of activation using whole-cell current recordings. Osmotic cell shrinkage induced by addition of 100 mm sucrose to the bath solution caused a 20-fold increase in whole-cell inward currents from -10.8 +/- 1.5 pA to -211 +/- 10.2 pA (n = 103). A similar response was observed when cell shrinkage was elicited using a hypo-osmotic pipette solution. This indicates that cell shrinkage and not extracellular osmolarity per se is the signal for current activation. Cation substitution experiments revealed that the activated channels discriminate poorly between monovalent cations with a selectivity sequence NH(4) (1.2) > or = Na(+) (1) approximately K(+) (0.9) approximately Li(+) (0.9). In contrast there was no measurable permeability for Ca(2+) or Ba(2+) and the cation-to-anion permeability ratio was about 14. The DPC-derivatives flufenamic acid, 4-methyl-DPC and DCDPC were the most effective blockers followed by LOE 908, while amiloride and bumetanide were ineffective. The putative channel activator maitotoxin had no effect. Current activation was dependent upon the presence of intracellular ATP and Mg(2+) and was inhibited by staurosporine (1 microm) and calphostin C (1 microm). Moreover, cytochalasin D (10 microm) and taxol (2 microm) reduced the current response to cell shrinkage. These findings suggest that the activation mechanism of the shrinkage-activated NSC channel involves protein kinase mediated phosphorylation steps and cytoskeletal elements.
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PMID:Mechanism of shrinkage activation of nonselective cation channels in M-1 mouse cortical collecting duct cells. 1101 61

Two H(+)-K(+)-ATPase isoforms are present in kidney: the gastric, highly sensitive to Sch-28080, and the colonic, partially sensitive to ouabain. Upregulation of Sch-28080-sensitive H(+)-K(+)-ATPase, or "gastric" H(+)-K(+)-ATPase, has been demonstrated in hypokalemic rat inner medullary collecting duct cells (IMCDs). Nevertheless, only colonic H(+)-K(+)-ATPase mRNA and protein abundance increase in this condition. This study was designed to determine whether Sch-28080 inhibits transporters other than the gastric H(+)-K(+)-ATPase. In the presence of bumetanide, Sch-28080 (200 microM) and ouabain (2 mM) inhibited (86)Rb(+) uptake (>90%). That (86)Rb(+) uptake was almost completely abolished by Sch-28080 indicates an effect of this agent on the Na(+)-K(+)-ATPase. ATPase assays in membranes, or lysed cells, demonstrated sensitivity to ouabain but not Sch-28080. Thus the inhibitory effect of Sch-28080 was dependent on cell integrity. (86)Rb(+)-uptake studies without bumetanide demonstrated that ouabain inhibited activity by only 50%. Addition of Sch-28080 (200 microM) blocked all residual activity. Intracellular ATP declined after Sch-28080 (200 microM) but recovered after removal of this agent. In conclusion, high concentrations of Sch-28080 inhibit K(+)-ATPase activity in mouse IMCD-3 (mIMCD-3) cells as a result of ATP depletion.
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PMID:Sch-28080 depletes intracellular ATP selectively in mIMCD-3 cells. 1102 78

Phospholipase D (PLD) is an enzyme involved in signal transduction and widely distributed in mammalian cells. The signal transduction pathways and role for phospholipid metabolism during hormonal response in cortical collecting duct remain partly undefined. It has been reported that dexamethasone increases transepithelial transport in M-1 cells that are derived from the mouse cortical collecting duct. We investigated the expression and activity of PLD in M-1 cells. Basal PLD activity of M-1 cells cultured in the presence of dexamethasone (5 microM) was higher than in the absence of dexamethasone. Dexamethasone and ATP activated PLD in M-1 cells but phorbol ester did not stimulate PLD activity. Vasopressin, bradykinin, dibutyryl cyclic AMP, and ionomycin were ineffective in activating PLD of the cells. The PLD2 isotype was detected by immunoprecipitation but PLD1 was not detected in M-1 cells. Addition of GTPgammaS and ADP-ribosylation factor or phosphatidylinositiol 4,5-bisphosphate to digitonin-permeabilized cells did not augment PLD activity. In intact cells PLD activity was increased by sodium oleate but there was no significant change between dexamethasone treated- and untreated cells by oleate. These results suggest that at least two types of PLD are present in M-1 cells and PLD plays a role in the corticosteroid-mediated response of cortical collecting duct cells.
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PMID:Dexamethasone enhances phospholipase D activity in M-1 cells. 1104 49

Within the renal cortical collecting duct (CCD), transepithelial Na(+) absorption and K(+) secretion are linked to basolateral Na(+)-K(+)-ATPase activity. Our purpose was to examine the developmental changes in basolateral Na(+)-K(+)-ATPase-mediated (86)rubidium (Rb) uptake, its inhibitor sensitivity and relationship to pump hydrolytic activity and Na(+) transport. Multiple CCDs ( approximately 6 mm) from maturing rabbits were affixed to coverslips, preincubated at 37 degrees C for 10 min (+/-1-2.5 mM ouabain or 10 or 100 micro M Schering-28080, an inhibitor of H(+)-K(+)-ATPase), and then transferred to prewarmed incubation solution containing tracer amounts of (86)Rb (+/-inhibitors). After 1 min at 37 degrees C, tubular samples were rinsed and permeabilized and isotope counts were measured to calculate basolateral Rb uptake. Ouabain-inhibitable Rb uptake, an index of basolateral Na(+)-K(+) pump activity, increased approximately 3-fold during the 1st 8 wk of postnatal life (P < 0.03). The approximately 2-fold increase in absolute rate of Rb uptake between 1 and 6 wk (2.64 +/- 0.45 to 5.02 +/- 0.32 pmol. min(-1). mm(-1)) did not reach statistical significance. The rate of basolateral Rb uptake increased further after the 6th wk of life to 7.29 +/- 0.53 pmol. min(-1). mm(-1) in adult animals (P < 0.03 vs. 6 wk). Schering-28080 failed to inhibit Rb uptake, implying that functional H(+)-K(+)-ATPase is absent at the basolateral membrane. Na(+)-K(+)-ATPase hydrolytic activity, determined by using a microassay that measured inorganic phosphate release from [gamma-(32)P]ATP under maximum velocity (V(max)) conditions, also increased in the differentiating CCD (from 316.2 +/- 44.4 pmol. h(-1). mm(-1) at 2 wk to 555.9 +/- 105.1 at 4 wk to 789.7 +/- 145.0 at 6 wk; r = 1.0 by linear regression analysis; P < 0.005). The parallel approximately 2.5-fold increases in Na(+)-K(+)-ATPase activity and ouabain-sensitive Rb uptake between 2- and 6-wk postnatal age suggest that the developmental increase in basolateral transport capacity is due predominantly to an increase in enzyme abundance. The signals mediating the developmental increase in Na(+)-K(+)-ATPase activity in the CCD remain to be defined.
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PMID:Na(+)-K(+)-ATPase-mediated basolateral rubidium uptake in the maturing rabbit cortical collecting duct. 1109 35

The mechanism by which bradykinin regulates renal epithelial salt transport has been investigated using a mouse inner medullary renal collecting duct cell-line mIMCD-K2. Using fura-2 loaded mIMCD-K2 cells bradykinin (100 nM) has been shown to induce a transient increase in intracellular Ca(2+) via activation of bradykinin B2 receptors localized to both the apical and basolateral epithelial cell surfaces. In mIMCD-K2 epithelial cell-layers clamped in Ussing chambers, 100 nM bradykinin via apical and basolateral bradykinin B2 receptors stimulated a transient increase in inward short-circuit current (I:(sc)) of similar duration to the increase in intracellular Ca(2+). Replacements of the bathing solution Na(+) by the impermeant cation, N-methyl-D-glucamine and of Cl(-) and HCO(3)(-) by the impermeant anion gluconate at either the apical (no reduction) or basal bathing solutions (abolition of the response) are consistent with the bradykinin-stimulated increase in inward I:(sc) resulting from basal to apical Cl(-) (anion) secretion. Using the slow whole cell configuration of the patch-clamp technique, bradykinin was shown to activate a transient Cl(-) selective whole cell current which showed time-dependent activation at positive membrane potentials and time-dependent inactivation at negative membrane potentials. These currents were distinct from those activated by forskolin (CFTR), but identical to those activated by exogenous ATP and are therefore consistent with bradykinin activation of a Ca(2+)-dependent Cl(-) conductance. The molecular identity of the Ca(2+)-dependent Cl(-) conductance has been investigated by an RT - PCR approach. Expression of an mRNA transcript with 96% identity to mCLCA1/2 was confirmed, however an additional but distinct mRNA transcript with only 81% of the identity to mCLCA1/2 was identified.
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PMID:Bradykinin regulation of salt transport across mouse inner medullary collecting duct epithelium involves activation of a Ca(2+)-dependent Cl(-) conductance. 1113 48

The gamma subunit of the Na,K-ATPase is a member of the FXYD family of type 2 transmembrane proteins that probably function as regulators of ion transport. Rat gamma is present primarily in the kidney as two main splice variants, gamma(a) and gamma(b), which differ only at their extracellular N termini (TELSANH and MDRWYL, respectively; Kuster, B., Shainskaya, A., Pu, H. X., Goldshleger, R., Blostein, R., Mann, M., and Karlish, S. J. D. (2000) J. Biol. Chem. 275, 18441-18446). Expression in cultured cells indicates that both variants affect catalytic properties, without a detectable difference between gamma(a) and gamma(b). At least two singular effects are seen, irrespective of whether the variants are expressed in HeLa or rat alpha1-transfected HeLa cells, i.e. (i) an increase in apparent affinity for ATP, probably secondary to a left shift in E(1) <--> E(2) conformational equilibrium and (ii) an increase in K(+) antagonism of cytoplasmic Na(+) activation. Antibodies against the C terminus common to both variants (anti-gamma) abrogate the first effect but not the second. In contrast, gamma(a) and gamma(b) show differences in their localization along the kidney tubule. Using anti-gamma (C-terminal) and antibodies to the rat alpha subunit as well as antibodies to identify cell types, double immunofluorescence showed gamma in the basolateral membrane of several tubular segments. Highest expression is in the medullary portion of the thick ascending limb (TAL), which contains both gamma(a) and gamma(b). In fact, TAL is the only positive tubular segment in the medulla. In the cortex, most tubules express gamma but at lower levels. Antibodies specific for gamma(a) and gamma(b) showed differences in their cortical location; gamma(a) is specific for cells in the macula densa and principal cells of the cortical collecting duct but not cortical TAL. In contrast, gamma(b) but not gamma(a) is present in the cortical TAL only. Thus, the importance of gamma(a) and gamma(b) may be related to their partially overlapping but distinct expression patterns and tissue-specific functions of the pump that these serve.
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PMID:Functional role and immunocytochemical localization of the gamma a and gamma b forms of the Na,K-ATPase gamma subunit. 1127 61

We have used perforated patch clamp and Fura-2 microfluorescence measurements to study Ca(2+)-activated Cl- currents in cultured mouse renal inner medullary collecting duct cells (mIMCD-3). The conductance was spontaneously active under resting conditions and whole cell currents were time and voltage-independent with an outwardly rectifying current-voltage relationship. The channel blockers DIDS, niflumic acid, glybenclamide and NPPB reversibly decreased the basal currents, whereas the sulfhydryl agent, DTT produced an irreversible inhibition. Increasing or decreasing extracellular calcium produced parallel changes in the size of the basal currents. Variations in external Ca2+ were associated with corresponding changes in free cytosolic Ca2+ concentration. Increasing cytosolic Ca2+ by extracellular ATP or ionomycin, further enhanced Cl- conductance, with whole cell currents displaying identical biophysical properties to the basal currents. However, the agonist-stimulated currents were now increased by DTT exposure, but still inhibited by the other channel blockers. Using RT-PCR, three distinct mRNA transcripts belonging to the CLCA family of Ca(2+)-activated Cl- channel proteins were identified, two of which represent novel sequences. Whether different channels underlie the basal and agonist-stimulated currents in mIMCD-3 cells is unclear. Our findings establish a novel link between alterations in external and internal Ca2+ and the activity of Ca(2+)-activated Cl- transport in these cells.
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PMID:Regulation of an outwardly rectifying chloride conductance in renal epithelial cells by external and internal calcium. 1128 3

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