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1.
Nadezhda N. ZheleznovaPatricia D. Wilson Alexander Staruschenko 《生物化学与生物物理学报:疾病的分子基础》2011,1812(10):1301-1313
Members of the epidermal growth factor (EGF) family bind to ErbB (EGFR) family receptors which play an important role in the regulation of various fundamental cell processes including cell proliferation and differentiation. The normal rodent kidney has been shown to express at least three members of the ErbB receptor family and is a major site of EGF ligand synthesis. Polycystic kidney disease (PKD) is a group of diseases caused by mutations in single genes and is characterized by enlarged kidneys due to the formation of multiple cysts in both kidneys. Tubule cells proliferate, causing segmental dilation, in association with the abnormal deposition of several proteins. One of the first abnormalities described in cell biological studies of PKD pathogenesis was the abnormal mislocalization of the EGFR in cyst lining epithelial cells. The kidney collecting duct (CD) is predominantly an absorptive epithelium where electrogenic Na+ entry is mediated by the epithelial Na+ channel (ENaC). ENaC-mediated sodium absorption represents an important ion transport pathway in the CD that might be involved in the development of PKD. A role for EGF in the regulation of ENaC-mediated sodium absorption has been proposed. However, several investigations have reported contradictory results indicating opposite effects of EGF and its related factors on ENaC activity and sodium transport. Recent advances in understanding how proteins in the EGF family regulate the proliferation and sodium transport in normal and PKD epithelial cells are discussed here. This article is part of a Special Issue entitled: Polycystic Kidney Disease. 相似文献
2.
Kenneth R. Hallows Huamin Wang Robert S. Edinger Michael B. Butterworth Nicholas M. Oyster Hui Li Jochen Buck Lonny R. Levin John P. Johnson N��ria M. Pastor-Soler 《The Journal of biological chemistry》2009,284(9):5774-5783
Alkalosis impairs the natriuretic response to diuretics, but the underlying
mechanisms are unclear. The soluble adenylyl cyclase (sAC) is a chemosensor
that mediates bicarbonate-dependent elevation of cAMP in intracellular
microdomains. We hypothesized that sAC may be an important regulator of
Na+ transport in the kidney. Confocal images of rat kidney revealed
specific immunolocalization of sAC in collecting duct cells, and immunoblots
confirmed sAC expression in mouse cortical collecting duct
(mpkCCDc14) cells. These cells exhibit aldosterone-stimulated
transepithelial Na+ currents that depend on both the apical
epithelial Na+ channel (ENaC) and basolateral
Na+,K+-ATPase. RNA interference-mediated 60-70%
knockdown of sAC expression comparably inhibited basal transepithelial short
circuit currents (Isc) in mpkCCDc14 cells.
Moreover, the sAC inhibitors KH7 and 2-hydroxyestradiol reduced
Isc in these cells by 50-60% within 30 min.
8-Bromoadenosine-3′,5′-cyclic-monophosphate substantially rescued
the KH7 inhibition of transepithelial Na+ current. Aldosterone
doubled ENaC-dependent Isc over 4 h, an effect that was
abolished in the presence of KH7. The sAC contribution to
Isc was unaffected with apical membrane nystatin-mediated
permeabilization, whereas the sAC-dependent Na+ current was fully
inhibited by basolateral ouabain treatment, suggesting that the
Na+,K+-ATPase, rather than ENaC, is the relevant
transporter target of sAC. Indeed, neither overexpression of sAC nor treatment
with KH7 modulated ENaC currents in Xenopus oocytes. ATPase and
biotinylation assays in mpkCCDc14 cells demonstrated that sAC
inhibition decreases catalytic activity rather than surface expression of the
Na+,K+-ATPase. In summary, these results suggest that
sAC regulates both basal and agonist-stimulated Na+ reabsorption in
the kidney collecting duct, acting to enhance
Na+,K+-ATPase activity.Maintenance of intracellular pH depends in part on the extracellular to
intracellular Na+ gradient, and elevation of intracellular
[Na+] can lead to acidification of the cytoplasm. It has been shown
that acidification of the cytoplasm of cells from frog skin and toad bladder
by increased partial pressure of CO2 reduces Na+
transport and permeability (1,
2). Conversely, the rise in
plasma bicarbonate caused by metabolic alkalosis with chronic diuretic use has
been shown to increase net renal Na+ reabsorption independently of
volume status, electrolyte depletion, and/or increased aldosterone secretion
(3,
4). However, the underlying
mechanisms involved in these phenomena remain unclear.The soluble adenylyl cyclase
(sAC)2 is a
chemosensor that mediates the elevation of cAMP in intracellular microdomains
(5-7).
Unlike transmembrane adenylyl cyclases (tmACs), sAC is insensitive to
regulation by forskolin or heterotrimeric G proteins
(8) and is directly activated
by elevations of intracellular calcium
(9,
10) and/or bicarbonate ions
(11). Thus, sAC mediates
localized intracellular increases in cAMP in response to variations in
bicarbonate levels or its closely related parameters, partial pressure of
CO2 and pH. Mammalian sAC is more similar to bicarbonate-regulated
cyanobacterial adenylyl cyclases than to other mammalian nucleotidyl cyclases,
which may indicate that there is a unifying mechanism for the regulation of
cAMP signaling by bicarbonate across biological systems. Although sAC appears
to be encoded by a single gene, there is significant isoform diversity for
this ubiquitously expressed enzyme
(11,
12) generated by alternative
splicing (reviewed in Ref.
13). sAC has been shown to
regulate the subcellular localization and/or activity of membrane transport
proteins such as the vacuolar H+-ATPase (V-ATPase) and cystic
fibrosis transmembrane conductance regulator in epithelial cells
(14,
15). Functional activity of
sAC has been reported in the kidney
(16), and sAC has been
localized to epithelial cells in the distal nephron
(14,
17).Given that natriuresis is decreased during metabolic alkalosis, when
bicarbonate is elevated, and Na+ reabsorption is impaired by high
partial pressure of CO2, we hypothesized that bicarbonate-regulated
sAC may play a key role in the regulation of transepithelial Na+
transport in the distal nephron. Reabsorption of Na+ in the kidney
and other epithelial tissues is mediated by the parallel operation of apical
ENaC and basolateral Na+,K+-ATPase, and both transport
proteins can be stimulated by cAMP via the cAMP-dependent protein kinase (PKA)
(18,
53). The aims of this study
were to investigate the role of sAC in the regulation of transepithelial
Na+ transport in the kidney through the use of specific sAC
inhibitors and electrophysiological measurements. We found that sAC inhibition
blocks transepithelial Na+ reabsorption in polarized
mpkCCDc14 cells under both basal and hormone-stimulated conditions.
Selective membrane permeabilization studies revealed that although ENaC
activity appears to be unaffected by sAC inhibition, flux through the
Na+,K+-ATPase is sensitive to sAC modulation. Inhibiting
sAC decreases ATPase activity without affecting plasma membrane expression of
the pump; thus, tonic sAC activity appears to be required for Na+
reabsorption in kidney collecting duct. 相似文献
3.
This review summarizes the strategy of cellular immortalization based on the principle of targeted oncogenesis in transgenic
mice, used to establish models of transimmortalized renal proximal tubule cells, referred to as PKSV-PCT and PKSV-PR-cells,
and collecting duct principal cells, referred to as mpkCCDcl4 cells. These cell lines have maintained for long-term passages the main biochemical and functional properties of the parental
cells from which they were derived. Proximal tubule PKSV-PCT and PKSV-PR cells have been proved to be suitable cell systems
for toxicological and pharmacological studies. They also permitted the establishment of a model of multidrug-resistant (MDR)
renal epithelial tubule cells, PKSV-PRcol50, which have served for the study of both MDR-dependent extrusion of chemotherapeutic drugs and inappropriate accumulation
of weak base anthracyclines in intracellular acidic organelles. The novel collecting duct cell line mpkCCDcl4, which has maintained the characteristics of tight epithelial cells, in particular Na+ absorption stimulated by aldosterone, has been extensively used for pharmacological studies related to the regulation of
ion transport. These cells have permitted the identification of several aldosterone-induced proteins playing a key role in
the regulation of Na+ absorption mediated by the epithelial Na+ channel ENaC. Recent studies have also provided evidence that these cell lines represent valuable cell systems for the study
of host–pathogen interactions and the analysis of the role of renal tubule epithelial cells in the induction of inflammatory
response caused by uropathogens that may lead to severe renal damage. 相似文献
4.
5.
Virginie Prulière-Escabasse Christine Clerici Grégoire Vuagniaux Andre Coste Estelle Escudier Carole Planès 《Respiratory research》2010,11(1):141
Background
Hyperactivity of the epithelial sodium (Na+) channel (ENaC) and increased Na+ absorption by airway epithelial cells leading to airway surface liquid dehydration and impaired mucociliary clearance are thought to play an important role in the pathogenesis of cystic fibrosis (CF) pulmonary disease. In airway epithelial cells, ENaC is constitutively activated by endogenous trypsin-like serine proteases such as Channel-Activating Proteases (CAPs). It was recently reported that ENaC activity could also be stimulated by apical treatment with human neutrophil elastase (hNE) in a human airway epithelial cell line, suggesting that hNE inhibition could represent a novel therapeutic approach for CF lung disease. However, whether hNE can also activate Na+ reabsorption in primary human nasal epithelial cells (HNEC) from control or CF patients is currently unknown.Methods
We evaluated by short-circuit current (Isc) measurements the effects of hNE and EPI-hNE4, a specific hNE inhibitor, on ENaC activity in primary cultures of HNEC obtained from control (9) and CF (4) patients.Results
Neither hNE nor EPI-hNE4 treatments did modify Isc in control and CF HNEC. Incubation with aprotinin, a Kunitz-type serine protease inhibitor that blocks the activity of endogenous CAPs, decreased Isc by 27.6% and 54% in control and CF HNEC, respectively. In control and CF HNEC pretreated with aprotinin, hNE did significantly stimulate Isc, an effect which was blocked by EPI-hNE4.Conclusions
These results indicate that hNE does activate ENaC and transepithelial Na+ transport in both normal and CF HNEC, on condition that the activity of endogenous CAPs is first inhibited. The potent inhibitory effect of EPI-hNE4 on hNE-mediated ENaC activation observed in our experiments highlights that the use of EPI-hNE4 could be of interest to reduce ENaC hyperactivity in CF airways. 相似文献6.
The relationship between active sodium transport and oxygen consumption was investigated in toad urinary bladder exposed to identical sodium-Ringer's solution at each surface, while controlling the transepithelial electrical potential difference Δψ. Rates of sodium transport and oxygen consumption were measured simultaneously, both in the short-circuited state (Δψ = 0) and when Δψ was varied. Under short-circuit conditions, when the rates of active sodium transport changed spontaneously or were depressed with amiloride, the ratio of active sodium transport to the estimated suprabasal oxygen consumption Na+/O2 was constant for each tissue, but varied among different tissues. Only when Δψ was varied did the ratio Na+/dO2 change with the rate of active sodium transport; under these circumstances dNa+/dO2 was constant but exceeded the ratio measured at short-circuit [(Na+/O2)Δψ=0]. This suggests that coupling between transport and metabolism is incomplete. The results are analyzed according to the principles of nonequilibrium thermodynamics, and interpreted in terms of a simple model of the transepithelial sodium transport system. 相似文献
7.
In the present study we investigated the effect of extracellular gadolinium on amiloride-sensitive Na+ current across Xenopus alveolar epithelium by Ussing chamber experiments and studied its direct effect on epithelial Na+ channels with the patch-clamp method. As observed in various epithelia, the short-circuit current (I
sc) and the amiloride-sensitive Na+ current (I
ami) across Xenopus alveolar epithelium was downregulated by high apical Na+ concentrations. Apical application of gadolinium (Gd3+) increased I
sc in a dose-dependent manner (EC
50 = 23.5 µM). The effect of Gd3+ was sensitive to amiloride, which indicated the amiloride-sensitive transcellular Na+ transport to be upregulated. Benz-imidazolyl-guanidin (BIG) and p-hydroxy-mercuribenzonic-acid (PHMB) probably release apical Na+ channels from Na+-dependent autoregulating mechanisms. BIG did not stimulate transepithelial Na+ currents across Xenopus lung epithelium but, interestingly, it prevented the stimulating effect of Gd3+ on transepithelial Na+ transport. PHMB increased I
sc and this stimulation was similar to the effect of Gd3+. Co-application of PHMB and Gd3+ had no additive effects on I
sc. In cell-attached patches on Xenopus oocytes extracellular Gd3+ increased the open probability (NP
o) of Xenopus epithelial sodium channels (ENaC) from 0.72 to 1.79 and decreased the single-channel conductance from 5.5 to 4.6 pS. Our data indicate that Xenopus alveolar epithelium exhibits Na+-dependent non-hormonal control of transepithelial Na+ transport and that the earth metal gadolinium interferes with these mechanisms. The patch-clamp experiments indicate that Gd3+ directly modulates the activity of ENaCs. 相似文献
8.
9.
The present study investigates the role of small G-proteins of the Ras family in the epidermal growth factor (EGF)-activated cellular signalling pathway that downregulates activity of the epithelial Na+ channel (ENaC). We found that H-Ras is a key component of this EGF-activated cellular signalling mechanism in M1 mouse collecting duct cells. Expression of a constitutively active H-Ras mutant inhibited the amiloride-sensitive current. The H-Ras-mediated signalling pathway that inhibits activity of ENaC involves c-Raf, and that the inhibitory effect of H-Ras on ENaC is abolished by the MEK1/2 inhibitor, PD98059. The inhibitory effect of H-Ras is not mediated by Nedd4-2, a ubiquitin protein ligase that regulates the abundance of ENaC at the cell surface membrane, or by a negative effect of H-Ras on proteolytic activation of the channel. The inhibitory effects of EGF and H-Ras on ENaC, however, were not observed in cells in which expression of caveolin-1 (Cav-1) had been knocked down by siRNA. These findings suggest that the inhibitory effect of EGF on ENaC-dependent Na+ absorption is mediated via the H-Ras/c-Raf, MEK/ERK signalling pathway, and that Cav-1 is an essential component of this EGF-activated signalling mechanism. Taken together with reports that mice expressing a constitutive mutant of H-Ras develop renal cysts, our findings suggest that H-Ras may play a key role in the regulation of renal ion transport and renal development. 相似文献
10.
The effect of l-arginine on transepithelial ion transport was examined in cultured M-1 mouse renal cortical collecting duct (CCD) cells using
continuous short circuit current (I
SC
) measurements in HCO3
−/CO2 buffered solution. Steady state I
SC
averaged 73.8 ± 3.2 μA/cm2 (n= 126) and was reduced by 94 ± 0.6% (n= 16) by the apical addition of 100 μm amiloride. This confirms that the predominant electrogenic ion transport in M-1 cells is Na+ absorption via the epithelial sodium channel (ENaC). Experiments using the cationic amino acid l-lysine (radiolabeled) as a stable arginine analogue show that the combined activity of an apical system y+ and a basal amino acid transport system y+L are responsible for most cationic amino acid transport across M-1 cells. Together they generate net absorptive cationic
amino acid flux. Application of l-arginine (10 mm) either apically or basolaterally induced a transient peak increase in I
SC
averaging 36.6 ± 5.4 μA/cm2 (n= 19) and 32.0 ± 7.2 μA/cm2 (n= 8), respectively. The response was preserved in the absence of bath Cl− (n= 4), but was abolished either in the absence of apical Na+ (n= 4) or by apical addition of 100 μm amiloride (n= 6). l-lysine, which cannot serve as a precursor of NO, caused a response similar to that of l-arginine (n= 4); neither L-NMMA (100 μm; n= 3) nor L-NAME (1 mm; n= 4) (both NO-synthase inhibitors) affected the I
SC
response to l-arginine. The effects of arginine or lysine were replicated by alkalinization that mimicked the transient alkalinization
of the bath solution upon addition of these amino acids. We conclude that in M-1 cells l-arginine stimulates Na+ absorption via a pH-dependent, but NO-independent mechanism. The observed net cationic amino acid absorption will counteract
passive cationic amino acid leak into the CCD in the presence of electrogenic Na+ transport, consistent with reports of stimulated expression of Na+ and cationic amino acid transporters by aldosterone.
Received: 11 September 2000/Revised: 6 December 2000 相似文献
11.
A study was performed to correlate regional morphology and amiloride inhibitable Na+‐transport in the coprodeal epithelium in hens, Gallus domesticus, on low‐NaCl diet and in controls. Proximal (close to colon), mid and distal (close to urodeum) regions were examined using light microscopy, transmission‐ and scanning electron microscopy. Na+‐transport was measured electrophysiologically in Ussing‐chambers in the proximal and distal regions. The epithelium, simple and columnar, is composed of absorptive intestinal epithelial cells, goblet cells, brush cells, migrating lymphoid cells, and entero‐endocrine cells. Brush cells, identified in avians for the first time, occur in highest number in the proximal part of the coprodeum in low‐NaCl hens. Na+‐transport is high in the low‐NaCl hens, ranging from 347μA/cm2 (proximal) to 187μA/cm2 (distal). In control hens, which correspond to hens on high‐NaCl diet, it is low in all regions (0–4 μA/cm2). Absorptive intestinal epithelial cells as well as brush cells adapt to variations in transepithelial Na+‐transport by regulating height and packing density of their microvilli, number, size, and localization of apical vesicles, and the width of the intercellular space. Regional differences in the epithelial cell composition and ultrastructure are closely correlated to transepithelial Na+‐transport but only in low‐NaCl hens, as controls do not show these variations. J. Morphol. 239:75–86, 1999. © 1999 Wiley‐Liss, Inc. 相似文献
12.
Zohreh Hosseinzadeh Dong Luo Mentor Sopjani Shefalee K. Bhavsar Florian Lang 《The Journal of membrane biology》2014,247(4):331-338
Janus kinase-2 (JAK2), a signaling molecule mediating effects of various hormones including leptin and growth hormone, has previously been shown to modify the activity of several channels and carriers. Leptin is known to inhibit and growth hormone to stimulate epithelial Na+ transport, effects at least partially involving regulation of the epithelial Na+ channel ENaC. However, no published evidence is available regarding an influence of JAK2 on the activity of the epithelial Na+ channel ENaC. In order to test whether JAK2 participates in the regulation of ENaC, cRNA encoding ENaC was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, gain-of-function V617FJAK2 or inactive K882EJAK2. Moreover, ENaC was expressed with or without the ENaC regulating ubiquitin ligase Nedd4-2 with or without JAK2, V617FJAK2 or K882EJAK2. ENaC was determined from amiloride (50 μM)-sensitive current (I amil) in dual electrode voltage clamp. Moreover, I amil was determined in colonic tissue utilizing Ussing chambers. As a result, the I amil in ENaC-expressing oocytes was significantly decreased following coexpression of JAK2 or V617FJAK2, but not by coexpression of K882EJAK2. Coexpression of JAK2 and Nedd4-2 decreased I amil in ENaC-expressing oocytes to a larger extent than coexpression of Nedd4-2 alone. Exposure of ENaC- and JAK2-expressing oocytes to JAK2 inhibitor AG490 (40 μM) significantly increased I amil. In colonic epithelium, I amil was significantly enhanced by AG490 pretreatment (40 μM, 1 h). In conclusion, JAK2 is a powerful inhibitor of ENaC. 相似文献
13.
L. Schild 《Reviews of Physiology, Biochemistry and Pharmacology》2004,151(1):92-106
Genetic analysis has demonstrated that Na absorption in the aldosterone-sensitive distal nephron (ASDN) critically determines extracellular blood volume and blood pressure variations. The epithelial sodium channel (ENaC) represents the main transport pathway for Na+ absorption in the ASDN, in particular in the connecting tubule (CNT), which shows the highest capacity for ENaC-mediated Na+ absorption. Gain-of-function mutations of ENaC causing hypertension target an intracellular proline-rich sequence involved in the control of ENaC activity at the cell surface. In animal models, these ENaC mutations exacerbate Na+ transport in response to aldosterone, an effect that likely plays an important role in the development of volume expansion and hypertension. Recent studies of the functional consequences of mutations in genes controlling Na+ absorption in the ASDN provide a new understanding of the molecular and cellular mechanisms underlying the pathogenesis of salt-sensitive hypertension. 相似文献
14.
Intracellular [Na+] ([Na+]i) modulates the activity of the epithelial Na channel (ENaC) to help prevent cell swelling and regulate epithelial Na+ transport, but the underlying mechanisms remain unclear. We show here that short-term (60–80 min) incubation of ENaC-expressing oocytes in high Na+ results in a 75% decrease in channel activity. When the β subunit was truncated, corresponding to a gain-of-function mutation found in Liddle''s syndrome, the same maneuver reduced activity by 45% despite a larger increase in [Na+]i. In both cases the inhibition occurred with little to no change in cell-surface expression of γENaC. Long-term incubation (18 hours) in high Na+ reduced activity by 92% and 75% in wild-type channels and Liddle''s mutant, respectively, with concomitant 70% and 52% decreases in cell-surface γENaC. In the presence of Brefeldin A to inhibit forward protein trafficking, high-Na+ incubation decreased wt ENaC activity by 52% and 88% after 4 and 8 hour incubations, respectively. Cleaved γENaC at the cell surface had lifetimes at the surface of 6 hrs in low Na+ and 4 hrs in high Na+, suggesting that [Na+]i increased the rate of retrieval of cleaved γ ENaC by 50%. This implies that enhanced retrieval of ENaC channels at the cell surface accounts for part, but not all, of the downregulation of ENaC activity shown with chronic increases in [Na+]i. 相似文献
15.
《Channels (Austin, Tex.)》2013,7(5):444-451
Intracellular [Na+] ([Na+]i) modulates the activity of the epithelial Na channel (ENaC) to help prevent cell swelling and regulate epithelial Na+ transport, but the underlying mechanisms remain unclear. We show here that short-term (60–80 min) incubation of ENaC-expressing oocytes in high Na+ results in a 75% decrease in channel activity. When the β subunit was truncated, corresponding to a gain-of-function mutation found in Liddle's syndrome, the same maneuver reduced activity by 45% despite a larger increase in [Na+]i. In both cases the inhibition occurred with little to no change in cell-surface expression of γENaC. Long-term incubation (18 hours) in high Na+ reduced activity by 92% and 75% in wild-type channels and Liddle's mutant, respectively, with concomitant 70% and 52% decreases in cell-surface γENaC. In the presence of Brefeldin A to inhibit forward protein trafficking, high-Na+ incubation decreased wt ENaC activity by 52% and 88% after 4 and 8 hour incubations, respectively. Cleaved γENaC at the cell surface had lifetimes at the surface of 6 hrs in low Na+ and 4 hrs in high Na+, suggesting that [Na+]i increased the rate of retrieval of cleaved γ ENaC by 50%. This implies that enhanced retrieval of ENaC channels at the cell surface accounts for part, but not all, of the downregulation of ENaC activity shown with chronic increases in [Na+]i. 相似文献
16.
Nataliya Pidkovka Reena Rao Shaojun Mei Yan Gong Raymond C. Harris Wen-Hui Wang Jorge H. Capdevila 《The Journal of biological chemistry》2013,288(7):5223-5231
The epithelial sodium channel (ENaC) participates in the regulation of plasma sodium and volume, and gain of function mutations in the human channel cause salt-sensitive hypertension. Roles for the arachidonic acid epoxygenase metabolites, the epoxyeicosatrienoic acids (EETs), in ENaC activity have been identified; however, their mechanisms of action remain unknown. In polarized M1 cells, 14,15-EET inhibited amiloride-sensitive apical to basolateral sodium transport as effectively as epidermal growth factor (EGF). The EET effects were associated with increased threonine phosphorylation of the ENaC β and γ subunits and abolished by inhibitors of (a) mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal regulated kinases 1 and 2 (MEK/ERK1/2) and (b) EGF receptor signaling. CYP2C44 epoxygenase knockdown blunted the sodium transport effects of EGF, and its 14,15-EET metabolite rescued the knockdown phenotype. The relevance of these findings is indicated by (a) the hypertension that results in mice administered cetuximab, an inhibitor of EGF receptor binding, and (b) immunological data showing an association between the pressure effects of cetuximab and reductions in ENaCγ phosphorylation. These studies (a) identify an ERK1/2-dependent mechanism for ENaC inhibition by 14,15-EET, (b) point to ENaC as a proximal target for EET-activated ERK1/2 mitogenic kinases, (c) characterize a mechanistic commonality between EGF and epoxygenase metabolites as ENaC inhibitors, and (d) suggest a CYP2C epoxygenase-mediated pathway for the regulation of distal sodium transport. 相似文献
17.
Na+,K+-ATPase activity was monitored in MDCK kidney epithelial cell monolayers and in cell extracts as a function of cell density, cAMP elevation, and exposure to hexamethylene bisacetamide (HMBA) and dimethylsulfoxide (Me2SO). Ouabain-sensitive Na+,K+-ATPase and 86Rb+ uptake activities, and the number of [3H]-ouabain binding sites were maximal in subconfluent cultures and decreased accompanying the development of a confluent monolayer. A sodium pump density of 8 × 107 pumps/cell was estimated for subconfluent cultures, declining to 9 × 105 pumps/cell at confluence. Previous studies have shown that dibutyryl cyclic AMP (Bt2cAMP), 1-methyl-3-isobutylxanthine (IBMX), or the differentiation inducers HMBA and Me2SO, which also caused cAMP elevation, all stimulated dome formation, a visible manifestation of active transepithelial Na+ and water transport (Lever, 1979). In the present study, all of these inducers were found to elevate intracellular Na+ content, implicating this variable in control of induction of dome formation. Operationally, inducers could be divided into two classes. HMBA and Me2SO partially inhibited ouabain-sensitive 86Rb+ influx. Ouabain, at a concentration that caused partial sodium pump inhibition and increased intracellular Na+ content, was also effective as an inducer. The second class, exemplified by IBMX and Bt2cAMP caused a furosemide-sensitive increase in intracellular Na+ content. This class of inducers stimulated ouabain-sensitive 86Rb+ uptake, presumably by substrate effects due to increased Na+ levels. The Na+ or ATP activation of Na+,K+-ATPase activity assayed in cell-free extracts, the affinity of the transport system for Rb+ in intact cells and intracellular ATP levels were unchanged by inducer treatment. Elevation of intracellular Na+ concentration, either by cAMP-stimulated, furosemide-sensitive mechanisms or by partial inhibition of the sodium pump may stimulate the induction of dome formation in MDCK cells. 相似文献
18.
Simon A. Lewis A. Grant Butt M. Joanne Bowler John P. Leader Anthony D. C. Macknight 《The Journal of membrane biology》1985,83(1-2):119-137
Summary The effects of complete substitution of gluconate for mucosal and/or serosal medium Cl– on transepithelial Na+ transport have been studied using toad urinary bladder. With mucosal gluconate, transepithelial potential difference (V
T) decreased rapidly, transepithelial resistance (R
T) increased, and calculated short-circuit current (I
sc) decreased. CalculatedE
Na was unaffected, indicating that the inhibition of Na+ transport was a consequence of a decreased apical membrane Na+ conductance. This conclusion was supported by the finding that a higher amiloride concentration was required to inhibit the residual transport. With serosal gluconateV
T decreased,R
T increased andI
sc fell to a new steady-state value following an initial and variable transient increase in transport. Epithelial cells were shrunken markedly as judged histologically. CalculatedE
Na fell substantially (from 130 to 68 mV on average). Ba2+ (3mm) reduced calculatedE
Na in Cl– Ringer's but not in gluconate Ringer's. With replacement of serosal Cl– by acetate, transepithelial transport was stimulated, the decrease in cellular volume was prevented andE
Na did not fall. Replacement of serosal isosmotic Cl– medium by a hypo-osmotic gluconate medium (one-half normal) also prevented cell shrinkage and did not result in inhibition of Na+ transport. Thus the inhibition of Na+ transport can be correlated with changes in cell volume rather than with the change in Cl– per se. Nystatin virtually abolished the resistance of the apical plasma membrane as judged by measurement of tissue capacitance. With K+ gluconate mucosa, Na+ gluconate serosa, calculated basolateral membrane resistance was much greater, estimated basolateral emf was much lower, and the Na+/K+ basolateral permeability ratio was much higher than with acetate media. It is concluded the decrease in cellular volume associated with substitution of serosal gluconate for Cl– results in a loss of highly specific Ba2+-sensitive K+ conductance channels from the basolateral plasma membrane. It is possible that the number of Na+ pump sites in this membrane is also decreased. 相似文献
19.
JIAN SUN MEI‐JUAN WANG MING‐QUAN DING SHU‐RONG DENG MEI‐QIN LIU CUN‐FU LU XIAO‐YANG ZHOU XIN SHEN XIAO‐JIANG ZHENG ZENG‐KAI ZHANG JIN SONG ZAN‐MIN HU YUE XU SHAO‐LIANG CHEN 《Plant, cell & environment》2010,33(6):943-958
Using confocal microscopy, X‐ray microanalysis and the scanning ion‐selective electrode technique, we investigated the signalling of H2O2, cytosolic Ca2+ ([Ca2+]cyt) and the PM H+‐coupled transport system in K+/Na+ homeostasis control in NaCl‐stressed calluses of Populus euphratica. An obvious Na+/H+ antiport was seen in salinized cells; however, NaCl stress caused a net K+ efflux, because of the salt‐induced membrane depolarization. H2O2 levels, regulated upwards by salinity, contributed to ionic homeostasis, because H2O2 restrictions by DPI or DMTU caused enhanced K+ efflux and decreased Na+/H+ antiport activity. NaCl induced a net Ca2+ influx and a subsequent rise of [Ca2+]cyt, which is involved in H2O2‐mediated K+/Na+ homeostasis in salinized P. euphratica cells. When callus cells were pretreated with inhibitors of the Na+/H+ antiport system, the NaCl‐induced elevation of H2O2 and [Ca2+]cyt was correspondingly restricted, leading to a greater K+ efflux and a more pronounced reduction in Na+/H+ antiport activity. Results suggest that the PM H+‐coupled transport system mediates H+ translocation and triggers the stress signalling of H2O2 and Ca2+, which results in a K+/Na+ homeostasis via mediations of K+ channels and the Na+/H+ antiport system in the PM of NaCl‐stressed cells. Accordingly, a salt stress signalling pathway of P. euphratica cells is proposed. 相似文献
20.
Alexey V. Karpushev Daria V. Ilatovskaya Tengis S. Pavlov Yuri A. Negulyaev Alexander Staruschenko 《PloS one》2010,5(1)