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1.
Na+/K+-ATPase plays a key role in the transport of Na+ throughout the nephron, but ageing appears to be accompanied by changes in the regulation and localization of the pump. In the present study, we examined the effect of in vitro cell ageing on the transport of Na+ and K+ ions in opossum kidney (OK) cells in culture. Cells were aged by repeated passing, and Na+/K+-ATPase activity and K+ conductance were evaluated using electrophysiological methods. Na+K+-ATPase α1– and β1-subunit expression was quantified by Western blot techniques. Na+/H+ exchanger activity, changes in membrane potential, cell viability, hydrogen peroxide production and cellular proliferation were determined using fluorimetric assays. In vitro cell ageing is accompanied by an increase in transepithelial Na+ transport, which results from an increase in the number of Na+/K+-ATPase α1- and β1-subunits, in the membrane. Increases in Na+/K+-ATPase activity were accompanied by increases in K+ conductance as a result of functional coupling between Na+/K+-ATPase and basolateral K+ channels. Cell depolarization induced by both KCl and ouabain was more pronounced in aged cells. No changes in Na+/H+ exchanger activity were observed. H2O2 production was increased in aged cells, but exposure for 5 days to 1 and 10 μM of H2O2 had no effect on Na+/K+-ATPase expression. Ouabain (100 nM) increased α1-subunit, but not β1-subunit, Na+/K+-ATPase expression in aged cells only. These cells constitute an interesting model for the study of renal epithelial cell ageing.  相似文献   

2.
We have previously shown that epithelial Na+ channels in mouse mandibular gland duct cells are controlled by cytosolic Na+ and Cl, acting, respectively, via G o and G i proteins. Since we found no evidence for control of epithelial Na+ channels by extracellular Na+ ([Na+] o ), our findings conflicted with the long-held belief that Na+ channel activators, such as sulfhydryl reagents, like para-chloromercuriphenylsulfonate (PCMPS), and amiloride analogues, like benzimidazolylguanidinium (BIG) and 5-N-dimethylamiloride (DMA), induce their effects by blocking an extracellular channel site which otherwise inhibits channel activity in response to increasing [Na+] o . Instead, we now show that PCMPS acts by rendering epithelial Na+ channels refractory to inhibition by activated G proteins, thereby eliminating the inhibitory effects of cytosolic Na+ and Cl on Na+ channel activity. We also show that BIG, DMA, and amiloride itself, when applied from the cytosolic side of the plasma membrane, block feedback inhibition of Na+ channels by cytosolic Na+, while leaving inhibition by cytosolic Cl unaffected. Since the inhibitory effects of BIG and amiloride are overcome by the inclusion of the activated α-subunit of G o in the pipette solution, we conclude that these agents act by blocking a previously unrecognized intracellular Na+ receptor. Received: 1 October 1997/Revised: 24 December 1997  相似文献   

3.
P2U/2Y-receptors elicit multiple signaling in Madin-Darby canine kidney (MDCK) cells, including a transient increase of [Ca2+] i , activation of phospholipases C (PLC) and A2 (PLA2), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This study examines the involvement of these signaling pathways in the inhibition of Na+,K+,Cl cotransport in MDCK cells by ATP. The level of ATP-induced inhibition of this carrier (∼50% of control values) was insensitive to cholera and pertussis toxins, to the PKC inhibitor calphostin C, to the cyclic nucleotide-dependent protein kinase inhibitors, H-89 and H-8 as well as to the inhibitor of serine-threonine type 1 and 2A phosphoprotein phosphatases okadaic acid. ATP led to a transient increase of [Ca2+]i that was abolished by a chelator of Ca2+ i , BAPTA. However, neither BAPTA nor the Ca2+ ionophore A231287, or an inhibitor of endoplasmic reticulum Ca2+-pump, thapsigargin, modified ATP-induced inhibition of Na+,K+,Cl cotransport. An inhibitor of PLC, U73122, and an inhibitor of MAPK kinase (MEK), PD98059, blocked ATP-induced inositol-1,4,5-triphosphate production and MAPK phosphorylation, respectively. However, these compounds did not modify the effect of ATP on Na+,K+,Cl cotransport activity. Inhibitors of PLA2 (AACOCF3), cycloxygenase (indomethacin) and lypoxygenase (NDGA) as well as exogenous arachidonic acid also did not affect ATP-induced inhibition of Na+,K+,Cl cotransport. Inhibition of the carrier by ATP persisted in the presence of inhibitors of epithelial Na+ channels (amiloride), Cl channels (NPPB) and Na+/H+ exchanger (EIPA) and was insensitive to cell volume modulation in anisosmotic media and to depletion of cells with monovalent ions, thus ruling out the role of other ion transporters in purinoceptor-induced inhibition of Na+,K+,Cl cotransport. Our data demonstrate that none of the known purinoceptor-stimulated signaling pathways mediate ATP-induced inhibition of Na+,K+,Cl cotransport and suggest the presence of a novel P2-receptor-coupled signaling mechanism. Received: 29 July 1998/Revised: 19 October  相似文献   

4.
MDCK cells display several acid-base transport systems found in intercalated cells, such as Na+-H+ exchange, H+–K+ ATPase and Cl/HCO 3 exchange. In this work we studied the functional activity of a vacuolar H+-ATPase in MDCK cells and its chloride dependence. We measured intracellular pH (pHi) in monolayers grown on glass cover slips utilizing the pH sensitive probe BCECF. To analyze the functional activity of the H+ transporters we observed the intracellular alkalinization in response to an acute acid load due to a 20 mm NH+ 4 pulse, and calculated the initial rate of pHi recovery (dpHi/dt). The cells have a basal pHi of 7.17 ± 0.01 (n= 23) and control dpHi/dt of 0.121 ± 0.006 (n= 23) pHi units/min. This pHi recovery rate is markedly decreased when Na+ was removed, to 0.069 ± 0.004 (n= 16). It was further reduced to 0.042 ± 0.005 (n= 12) when concanamycin 4.6 × 10−8 m (a specific inhibitor of the vacuolar H+-ATPase) was added to the zero Na+ solution. When using a solution with zero Na+, low K+ (0.5 mm) plus concanamycin, pHi recovery fell again, significantly, to 0.023 ± 0.006 (n= 14) as expected in the presence of a H+–K+-ATPase. This result was confirmed by the use of 5 × 10−5 m Schering 28080. The Na+ independent pHi recovery was significantly reduced from 0.069 ± 0.004 to 0.042 ± 0.004 (n= 12) when NPPB 10−5 m (a specific blocker of Cl channels in renal tubules) was utilized. When the cells were preincubated in 0 Cl/normal Na+ solution for 8 min. before the ammonium pulse, the pHi recovery fell from 0.069 ± 0.004 to 0.041 ± 0.007 (n= 12) in a Na+ and Cl free solution. From these results we conclude that: (i) MDCK cells have two Na+-independent mechanisms of pHi recovery, a concanamycin sensitive H+-ATPase and a K+ dependent, Schering 28080 sensitive H+–K+ ATPase; and, (ii) pHi recovery in Na+-free medium depends on the presence of a chloride current which can be blocked by NPPB and impaired by preincubation in Cl–free medium. This finding supports a role for chloride in the function of the H+ ATPase, which might be electrical shunting or a biochemical interaction. Received: 24 October 1997/Revised: 19 February 1998  相似文献   

5.
Short-term (2–30 min) cyclic stretch activates the Na pump in cultured aortic smooth muscle cells (ASMCs). This effect of stretch involves the phosphotidylinositol 3-kinase (PI 3-kinase) participation. Presently, we investigated whether this stimulation is the result of translocation of Na+,K+-ATPase from endosomes to the plasma membrane. ASMCs were stretched 20% for 5 min using the Flexercell Strain Unit. The plasma membrane and endosome fractions were isolated and Western blotted to localize the Na+,K+-ATPase α-1-subunit protein. Membrane marker enzyme, 5′ nucleotidase activity, and the early and recycling endosome markers Rab4 and Rab11 were used to verify the enrichment of these fractions. Stretch increased Na+,K+-ATPase α-1 expression in plasma membrane fractions and decreased it in endosomes. PI 3-kinase inhibitors LY294002 and wortmannin blocked the stretch-induced translocation of the Na+,K+-ATPase α-1-subunit. Rab4 and Rab11 were enriched in the endosomal fraction, whereas 5′ nucleotidase activity was enriched in the plasma membrane fraction. We conclude that stimulation of the Na pump activity by shortterm cyclic stretch is the result, at least in part, of transport of the α-subunit of the enzyme from endosomes to the plasma membrane.  相似文献   

6.
K+-Cl cotransporter-3 has two major amino terminal variants, KCC3a and KCC3b. In LLC-PK1 cells, exogenously expressed KCC3a co-immunoprecipitated with endogenous Na+,K+-ATPase α1-subunit (α1NaK), accompanying significant increases of the Na+,K+-ATPase activity. Exogenously expressed KCC3b did not co-immunoprecipitate with endogenous α1NaK inducing no change of the Na+,K+-ATPase activity. A KCC inhibitor attenuated the Na+,K+-ATPase activity in rat gastric mucosa in which KCC3a is predominantly expressed, while it had no effects on the Na+,K+-ATPase activity in rat kidney in which KCC3b is predominantly expressed. In these tissue samples, KCC3a co-immunoprecipitated with α1NaK, while KCC3b did not. Our results suggest that the NH2-terminus of KCC3a is a key region for association with α1NaK, and that KCC3a but not KCC3b can regulate the Na+,K+-ATPase activity.  相似文献   

7.
We demonstrated recently that in renal epithelial cells from collecting ducts of Madin-Darby canine kidneys (MDCK), Na+,K+,Cl cotransport is inhibited up to 50% by ATP via its interaction with P2Y purinoceptors (Biochim. Biophys. Acta 1998. 1369:233–239). In the present study we examined which type of renal epithelial cells possesses the highest sensitivity of Na+,K+,Cl cotransport to purinergic regulation. We did not observe any effect of ATP on Na+,K+,Cl cotransport in renal epithelial cells from proximal and distal tubules, whereas in renal epithelial cells from rabbit and rat collecting ducts ATP decreased the carrier's activity by ∼30%. ATP did not affect Na+,K+,Cl cotransport in C7 subtype MDCK cells possessing the properties of principal cells but led to ∼85% inhibition of this carrier in C11-MDCK cells in which intercalated cells are highly abundant. Both C7- and C11-MDCK exhibited ATP-induced IP3 and cAMP production and transient elevation of [Ca2+] i . In contrast to the above-listed signaling systems, ATP-induced phosphorylation of ERK and JNK MAP kinases was observed in C11-MDCK only. Thus, our results reveal that regulation of renal Na+,K+,Cl cotransport by P2Y receptors is limited to intercalated cells from collecting ducts and indicate the involvement of the MAP kinase cascade in purinergic control of this ion carrier's activity. Received: 10 June 1999/Revised: 23 August 1999  相似文献   

8.
To examine the involvement of Na+,K+,2Cl cotransport in monovalent ion fluxes in vascular smooth muscle cells (VSMC), we compared the effect of bumetanide on 86Rb, 36Cl and 22Na uptake by quiescent cultures of VSMC from rat aorta. Under basal conditions, the values of bumetanide-sensitive (BS) inward and outward 86Rb fluxes were not different. Bumetanide decreased basal 86Rb uptake by 70–75% with a K i of ∼0.2–0.3 μm. At concentrations ranging up to 1 μm, bumetanide did not affect 36Cl influx and reduced it by 20–30% in the range from 3 to 100 μm. In contrast to 86Rb and 36Cl influx, bumetanide did not inhibit 22Na uptake by VSMC. BS 86Rb uptake was completely abolished in Na+- or Cl-free media. In contrast to 86Rb, basal BS 36Cl influx was not affected by Na+ o and K+ o . Hyperosmotic and isosmotic shrinkage of VSMC increased 86Rb and 36Cl influx to the same extent. Shrinkage-induced increments of 86Rb and 36Cl uptake were completely abolished by bumetanide with a K i or ∼0.3 μm. Shrinkage did not induce BS 86Rb and 36Cl influx in (Na+ or Cl)- and (Na+ or K+)-depleted media, respectively. In the presence of an inhibitor of Na+/H+ exchange (EIPA), neither hyperosmotic nor isosmotic shrinkage activated 22Na influx. Bumetanide (1 μm) did not modify basal VSMC volume and intracellular content of sodium, potassium and chloride but abolished the regulatory volume increase in isosmotically-shrunken VSMC. These data demonstrate the absence of the functional Na+,K+,2Cl cotransporter in VSMC and suggest that in these cells basal and shrinkage-induced BS K+ influx is mediated by (Na+ o + Cl o )-dependent K+/K+ exchange and Na+ o -dependent K+,Cl cotransport, respectively. Received: 30 January 1996/Revised: 20 May 1996  相似文献   

9.
The aim of this study was to clarify the mechanism of isotonic fluid transport in frog skin glands. Stationary ion secretion by the glands was studied by measuring unidirectional fluxes of 24Na+, 42K+, and carrier-free 134Cs+ in paired frog skins bathed on both sides with Ringer's solution, and with 10−5 m noradrenaline on the inside and 10−4 m amiloride on the outside. At transepithelial thermodynamic equilibrium conditions, the 134Cs+ flux ratio, J out Cs/J in Cs, varied in seven pairs of preparations from 6 to 36. Since carrier-free 134Cs+ entering the cells is irreversibly trapped in the cellular compartment (Ussing & Lind, 1996), the transepithelial net flux of 134Cs+ indicates that a paracellular flow of water is dragging 134Cs+ in the direction from the serosal- to outside solution. From the measured flux ratios it was calculated that the force driving the secretory flux of Cs+ varied from 30 to 61 mV among preparations. In the same experiments unidirectional Na+ fluxes were measured as well, and it was found that also Na+ was subjected to secretion. The ratio of unidirectional Na+ fluxes, however, was significantly smaller than would be predicted if the two ions were both flowing along the paracellular route dragged by the flow of water. This result indicates that Na+ and Cs+ do not take the same pathway through the glands. The flux ratio of unidirectional K+ fluxes indicated active secretion of K+. The time it takes for steady-state K+ fluxes to be established was significantly longer than that of the simultaneously measured Cs+ fluxes. These results allow the conclusion that — in addition to being transported between cells — K+ is submitted to active transport along a cellular pathway.Based on the recirculation theory, we propose a new model which accounts for stationary Na+, K+, Cl and water secretion under thermodynamic equilibrium conditions. The new features of the model, as compared to the classical Silva-model for the shark-rectal gland, are: (i) the sodium pumps in the activated gland transport Na+ into the lateral intercellular space only. (ii) A barrier at the level of the basement membrane prevents the major fraction of Na+ entering the lateral space from returning to the serosal bath. Thus, Na+ is secreted into the outside bath. It has to be assumed then that the Na+ permeability of the basement membrane barrier (P BM Na) is smaller than the Na+ permeability of the junctional membrane (P JM Na), i.e., P JM Na/P BM Na > 1. The secretory paracellular flow of water further requires that the Na+ reflection coefficients (σNa) of the two barriers are governed by the conditions, σBM Na > 0, and σBM Na > σJM Na. (iii) Na+ channels are located in the apical membrane of the activated gland cells, so that a fraction of the Na+ outflux appearing downstream the lateral intercellular space is recirculated by the gland cells. Based on measured unidirectional fluxes, a set of equations is developed from which we estimate the ion fluxes flowing through major pathways during stationary secretion. It is shown that 80% of the sodium ions flowing downstream the lateral intercellular space is recycled by the gland cells. Our calculations also indicate that under the conditions prevailing in the present experiments 1.8 ATP molecule would be hydrolyzed for every Na+ secreted to the outside bath. Received: 30 January 1996/Revised: 12 March 1996  相似文献   

10.
Transport Pathways for Therapeutic Concentrations of Lithium in Rat Liver   总被引:1,自引:0,他引:1  
Although both amiloride- and phloretin-sensitive Na+/Li+ exchange activities have been reported in mammalian red blood cells, it is still unclear whether or not the two are mediated by the same pathway. Also, little is known about the relative contribution of these transport mechanisms to the entry of therapeutic concentrations of Li+ (0.2–2 mm) into cells other than erythrocytes. Here, we describe characteristics of these transport systems in rat isolated hepatocytes in suspension. Uptake of Li+ by hepatocytes, preloaded with Na+ and incubated in the presence of ouabain and bumetanide, comprised three components. (a) An amiloride-sensitive component, with apparent K m 1.2 mm Li+, V max 40 μmol · (kg dry wt · min)−1, showed increased activity at low intracellular pH. The relationship of this component to the concentration of intracellular H+ was curvilinear suggesting a modifier role of [H+] i . This system persisted in Na+-depleted cells, although with apparent K m 3.8 mm. (b) A phloretin-sensitive component, with K m 1.2 mm, V max 21 μmol · (kg · min)−1, was unaffected by pH but was inactive in Na+-depleted cells. Phloretin inhibited Li+ uptake and Na+ efflux in parallel. (c) A residual uptake increased linearly with the external Li+ concentration and represented an increasing proportion of the total uptake. The results strongly suggest that the amiloride-sensitive and the phloretin-sensitive Li+ uptake in rat liver are mediated by two separate pathways which can be distinguished by their sensitivity to inhibitors and intracellular [H+]. Received: 8 April 1999/Revised: 19 July 1999  相似文献   

11.
Epithelial cells from the anterior and equatorial surfaces of the frog lens were isolated and used the same day for studies of the Na/K ATPase. RNase protection assays showed that all cells express α1- and α2-isoforms of the Na/K pump but not the α3-isoform, however the α2-isoform dominates in anterior cells whereas the α1-isoform dominates in equatorial cells. The whole cell patch-clamp technique was used to record functional properties of the Na/K pump current (I P ), defined as the current specifically inhibited by dihydro-ouabain (DHO). DHO-I P blockade data indicate the α1-isoform has a dissociation constant of 100 μm DHO whereas for the α2-isoform it is 0.75 μm DHO. Both α1- and α2-isoforms are half maximally activated at an intracellular Na+-concentration of 9 mm. The α1-isoform is half maximally activated at an extracellular K+-concentration of 3.9 mm whereas for the α2-isoform, half maximal activation occurs at 0.4 mm. Lastly, transport by the α1-isoform is inhibited by a drop in extracellular pH, which does not affect transport by the α2-isoform. Under normal physiological conditions, I P in equatorial cells is approximately 0.23 μA/μF, and in anterior cells it is about 0.14 μA/μF. These current densities refer to the area of cell membrane assuming a capacitance of around 1 μF/cm2. Because cell size and geometry are different at the equatorial vs. anterior surface of the intact lens, we estimate Na/K pump current density per area of lens surface to be around 10 μA/cm2 at the equator vs. 0.5 μA/cm2 at the anterior pole. Received: 17 May 2000/Revised: 11 August 2000  相似文献   

12.
The Na+,K+-ATPase isolated from shark rectal gland or pig kidney was inserted into liposomes and phosphorylated with cAMP-dependent protein kinase without detergent. Stoichiometry of phosphorylation of α-subunit of the enzyme was 0.9 and 0.2 mol Pi/mol α-subunit of the pig kidney and shark gland, respectively. The phosphorylation of the shark Na+,K+-ATPase led to an increase in maximum of hydrolytic activity dependent on the cytoplasmic sodium concentration and the extracellular activation with potassium ions. On the contrary, the phosphorylation of the sodium pump of the pig kidney did not produce any significant functional effect.  相似文献   

13.
The present experiments were designed to examine the function of Na/K pumps from Dahl salt-sensitive (S) and salt-resistant (R) rats. Previous reports have suggested that there is a difference in primary sequence in the α1 subunit, the major Na/K pump isoform in the kidney. This sequence difference might contribute to differences in NaCl excretion in these two strains which in turn could influence the systemic blood pressure. Using ``back-door' phosphorylation of pumps isolated from basolateral membranes of kidney cortex, we found no differences between S and R strains. We also examined the Na/K pumps from cultured inner medullary collecting duct (IMCD) cells. This approach takes advantage of the fact that monolayers cultured from S rats transport about twice as much Na+ as monolayers cultured from R rats. In cells whose apical membrane was made permeable with amphotericin B, comparison of the affinities for ouabain, Na+, and K+, respectively, showed only small or no differences between S and R monolayers. Ouabain binding showed no difference in the number of Na/K pumps on the basolateral membrane of cultured cells, despite a 2-fold difference in Na+ transport rates. The analysis of the steady-state Na+ transport indicates that Na/K pumps in IMCD monolayers from S rats operate at a higher fraction of their maximum capacity than do pumps in monolayers from R rats. The results, taken together, suggest that the major reason for the higher rate of Na+ transport in S monolayers is because of a primary increase in the conductive permeability of the apical membrane to Na+. They suggest that the epithelial Na+ channel is intrinsically different or differently regulated in S and R rats. Received: 6 May 1996/Revised: 16 October 1996  相似文献   

14.
The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits, α and β, and tissue-specific isoforms exist for each of these, α1, α2 and α3 and β1, β2 and β3. We have proposed that an additional α isoform, α4, exists based on genomic and cDNA cloning. The mRNA for this gene is expressed in rats and humans, exclusively in the testis, however the expression of a corresponding protein has not been demonstrated. In the current study, the putative α4 isoform has been functionally characterized as a novel isoform of the Na,K-ATPase in both rat testis and in α4 isoform cDNA transfected 3T3 cells. Using an α4 isoform-specific polyclonal antibody, the protein for this novel isoform is detected for the first time in both rat testis and in transfected cell lines. Ouabain binding competition assays reveal the presence of high affinity ouabain receptors in both rat testis and in transfected cell lines that have identical K D values. Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na+ and K+. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPase in testis. Received: 4 December 1998/Revised: 1 February 1999  相似文献   

15.
Amiloride-sensitive, Na+-dependent, DIDS-insensitive cytoplasmic alkalinization is observed after hypertonic challenge in Ehrlich ascites tumor cells. This was assessed using the fluorescent pH-sensitive probe 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). A parallel increase in the amiloride-sensitive unidirectional Na+ influx is also observed. This indicates that hypertonic challenge activates a Na+/H+ exchanger. Activation occurs after several types of hypertonic challenge, is a graded function of the osmotic challenge, and is temperature-dependent. Observations on single cells reveal a considerable variation in the shrinkage-induced changes in cellular pH i , but the overall picture confirms the results from cell suspensions. Shrinkage-induced alkalinization and recovery of cellular pH after an acid load, is strongly reduced in ATP-depleted cells. Furthermore, it is inhibited by chelerythrine and H-7, inhibitors of protein kinase C (PKC). In contrast, Calyculin A, an inhibitor of protein phosphatases PP1 and PP2A, stimulates shrinkage-induced alkalinization. Osmotic activation of the exchanger is unaffected by removal of calcium from the experimental medium, and by buffering of intracellular free calcium with BAPTA. At 25 mm HCO 3, but not in nominally HCO 3-free medium, Na+/H+ exchange contributes significantly to regulatory volume increase in Ehrlich cells. Under isotonic conditions, the Na+/H+ exchanger is activated by ionomycin, an effect which may be secondary to ionomycin-induced cell shrinkage. Received: 2 March 1995/Revised: 29 September 1995  相似文献   

16.
Extracellular nucleotides modulate renal ion transport. Our previous results in M-1 cortical collecting duct cells indicate that luminal and basolateral ATP via P2Y2 receptors stimulate luminal Ca2+-activated Cl channels and inhibit Na+ transport. Here we address the mechanism of ATP-mediated inhibition of Na+ transport. M-1 cells had a transepithelial voltage (V te ) of −31.4 ± 1.3 mV and a transepithelial resistance (R te ) of 1151 ± 28 Ωcm2. The amiloride-sensitive short circuit current (I sc ) was −28.0 ± 1.1 μA/cm2. The ATP-mediated activation of Cl channels was inhibited when cytosolic Ca2+ increases were blocked with cyclopiazonic acid (CPA). Without CPA the ATP-induced [Ca2+]i increase was paralleled by a rapid and transient R te decrease (297 ± 51 Ωcm2). In the presence of CPA, basolateral ATP led to an R te increase by 144 ± 17 Ωcm2 and decreased V te from −31 ± 2.6 to −26.6 ± 2.5 mV. I sc dropped from −28.6 ± 2.4 to −21.6 ± 1.9 μA/cm2. Similar effects were observed with luminal ATP. In the presence of amiloride, ATP was without effect. This reflects ATP-mediated inhibition of Na+ absorption. Lowering [Ca2+]i by removal of extracellular Ca2+ did not alter the ATP effect. PKC inhibition or activation were without effect. Na+ absorption was activated by pHi alkalinization and inhibited by pHi acidification. ATP slightly acidified M-1 cells by 0.05 ± 0.005 pH units, quantitatively not explaining the ATP-induced effect. In summary this indicates that extracellular ATP via luminal and basolateral P2Y2 receptors inhibits Na+ absorption. This effect is not mediated via [Ca2+]i, does not involve PKC and is to a small part mediated via intracellular acidification. Received: 9 February 2001/Revised: 17 May 2001  相似文献   

17.
We characterized the signaling and ion transport pathways that mediate epidermal growth factor receptor physiological control in SV40-immortalized rabbit corneal epithelial cells (tRCEC). Our evaluation employed single-cell fluorescence imaging to measure the intracellular [Na+]i in these cells loaded with the Na+ sensitive dye, SBFI. EGF (1 to 5 ng/ml) transiently increased [Na+]i from 10 mm to as much as 35 mm after 25 min, which was followed by a decline towards its control value. These increases waned at higher EGF concentrations up to 50 ng/ml. Both inhibition of EGF receptor-linked tyrosine kinase activity (50 μm RG-13022) and cPLA2 activity (10 μm AACOCF3) obviated EGF-induced increases in [Na+]i. In contrast, PGE2 (10 μg/ml) and cAMP (2 mm) increased [Na+]i by 25 mm. Inhibition of NKCC activity through exposure to either Cl-free Ringers or 300 μm furosemide in NaCl Ringers eliminated EGF-induced increases in [Na+]i. Similarly, EGF failed to increase [Na+]i following inhibition of: 1) PKA activity (10 μm H-89); 2) Erk1/2 (15 μm PD98059) or 3) p38 (15 μm SB203580) activity. Stimulation protein kinase C activity (0.1 μm PMA) transiently increased [Na+]i followed by a decline towards its baseline value. EGF-induced increases in [Na+]i were unaltered by inhibition of K+ conductance (100 μm 4-AP). Taken together, EGF stimulates Erk1/2; p38 and cPLA2 activity. Their stimulation increases PGE2 and cAMP levels resulting in PKA and NKCC activation. Received: 18 December 2000/Revised: 24 May 2001  相似文献   

18.
+ and Na+ transport in RBCs from control mice (C57Bl/6J) and a transgenic (αHβSMDD]) mouse line that expresses high levels of human αH and βS-chains and has a small percent dense cells but does not exhibit anemia. In transgenic mouse RBCs (n= 5) under oxygenated conditions, K+ efflux was 0.22 ± 0.01 mmol/L cell × min and Na+ influx was 0.17 ± 0.02 mmol/L cell × min. Both fluxes were stimulated by 10 min deoxygenation in transgenic but not in control mice. The deoxy-stimulated K+ efflux from transgenic mouse RBCs was about 55% inhibited by 5 nm charybdotoxin (CTX), a blocker of the calcium activated K+-channel. To compare the fluxes between human and mouse RBCs, we measured the area of mouse RBCs and normalized values to area per liter of cells. The deoxy-simulated CTX-sensitive K+ efflux was larger than the CTX-sensitive K+ efflux observed in RBCs from SS patients. These results suggest that in transgenic mice, deoxygenation increases cytosolic Ca2+ to levels which open Ca2+-activated K+ channels. The presence of these channels was confirmed in both control and transgenic mice by clamping intracellular Ca2+ at 10 μm with the ionophore A23187 and measuring Ca2+-activated K+ efflux. Both types of mouse had similar maximal rates of CTX-sensitive, Ca2+-activated K+ efflux that were similar to those in human SS cells. The capacity of the mouse red cell membrane to regulate cytosolic Ca2+ levels was examined by measurements of the maximal rate of calmodulin activated Ca2+-ATPase activity. This activity was 3-fold greater than that observed in human RBCs thus indicating that mouse RBC membranes have more capacity to regulate cytosolic Ca2+ levels. In summary, transgenic mouse RBCs exhibit larger values of deoxy-stimulated K+ efflux and Na+ influx when compared to human SS cells. They have a similar Ca2+-activated K+ channel activity to human SS cells while expressing a very high Ca2+ pump activity. These properties may contribute to the smaller percent of very dense cells and to the lack of adult anemia in this animal model. Received: 23 October/Revised: 15 May 1997  相似文献   

19.
Previous studies in expression systems have found different ion activation of the Na+/K+-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na+,K+-ATPase activity, and the Na+ affinity of Na+,K+-ATPase was studied in total membranes from rat muscle and purified membranes from muscle with different fiber types. The Na+ affinity was higher (K m lower) in oxidative muscle compared with glycolytic muscle and in purified membranes from oxidative muscle compared with glycolytic muscle. Na+,K+-ATPase isoform analysis implied that heterodimers containing the β1 isoform have a higher Na+ affinity than heterodimers containing the β2 isoform. Immunoprecipitation experiments demonstrated that dimers with α1 are responsible for approximately 36% of the total Na,K-ATPase activity. Selective inhibition of the α2 isoform with ouabain suggested that heterodimers containing the α1 isoform have a higher Na+ affinity than heterodimers containing the α2 isoform. The estimated K m values for Na+ are 4.0, 5.5, 7.5 and 13 mM for α1β1, α2β1, α1β2 and α2β2, respectively. The affinity differences and isoform distributions imply that the degree of activation of Na+,K+-ATPase at physiological Na+ concentrations differs between muscles (oxidative and glycolytic) and between subcellular membrane domains with different isoform compositions. These differences may have consequences for ion balance across the muscle membrane.  相似文献   

20.
The aim of the present study was to investigate the roles of Ca2+ and protein tyrosine kinase (PTK) in the insulin action on cell volume in fetal rat (20-day gestational age) type II pneumocytes. Insulin (100 nm) increased cell volume in the presence of extracellular Ca2+ (1 mm), while cell shrinkage was induced by insulin in the absence of extracellular Ca2+ (<1 nm). This insulin action in a Ca2+-containing solution was completely blocked by co-application of bumetanide (50 μm, an inhibitor of Na+/K+/2Cl cotransporter) and amiloride (10 μm, an inhibitor of epithelial Na+ channel), but not by the individual application of either bumetanide or amiloride. On the other hand, the insulin action on cell volume in a Ca2+-free solution was completely blocked by quinine (1 mm, a blocker of Ca2+-activated K+ channel), but not by bumetanide and/or amiloride. These observations suggest that insulin activates an amiloride-sensitive Na+ channel and a bumetanide-sensitive Na+/K+/2Cl cotransporter in the presence of 1 mm extracellular Ca2+, that the stimulatory action of insulin on an amiloride-sensitive Na+ channel and a bumetanide-sensitive Na+/K+/2Cl cotransporter requires Ca2+, and that in a Ca2+-free solution insulin activates a quinine-sensitive K+ channel but not in the presence of 1 mm Ca2+. The insulin action on cell volume in a Ca2+-free solution was almost completely blocked by treatment with BAPTA (10 μm) or thapsigargin (1 μM, an inhibitor of Ca2+-ATPase which depletes the intracellular Ca2+ pool). Further, lavendustin A (10 μm, an inhibitor of receptor type PTK) blocked the insulin action in a Ca2+-free solution. These observations suggest that the stimulatory action of insulin on a quinine-sensitive K+ channel is mediated through PTK activity in a cytosolic Ca2+-dependent manner. Lavendustin A, further, completely blocked the activity of the Na+/K+/2Cl cotransporter in a Ca2+-free solution, but only partially blocked the activity of the Na+/K+/2Cl cotransporter in the presence of 1 mm Ca2+. This observation suggests that the activity of the Na+/K+/2Cl cotransporter is maintained through two different pathways; one is a PTK-dependent, Ca2+-independent pathway and the other is a PTK-independent, Ca2+-dependent pathway. Further, we observed that removal of extracellular Ca2+ caused cell shrinkage by diminishing the activity of the amiloride-sensitive Na+ channel and the bumetanide-sensitive Na+/K+/2Cl cotransporter, and that removal of extracellular Ca2+ abolished the activity of the quinine-sensitive K+ channel. We conclude that the cell shrinkage induced by removal of extracellular Ca2+ results from diverse effects on the cotransporter and Na+ and K+ channels. Received: 2 September 1998/Revised: 30 November 1998  相似文献   

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