ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia

2001 ◽  
Vol 281 (2) ◽  
pp. C633-C648 ◽  
Author(s):  
Sasha Blaug ◽  
Kevin Hybiske ◽  
Jonathan Cohn ◽  
Gary L. Firestone ◽  
Terry E. Machen ◽  
...  
Keyword(s):  
Tight Junctions ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Equivalent Circuit Analysis ◽  
Mean Values ◽  
Apical Side

Mammary epithelial 31EG4 cells (MEC) were grown as monolayers on filters to analyze the apical membrane mechanisms that help mediate ion and fluid transport across the epithelium. RT-PCR showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na+ channel (ENaC) message, and immunomicroscopy showed apical membrane staining for both proteins. CFTR was also localized to the apical membrane of native human mammary duct epithelium. In control conditions, mean values of transepithelial potential (apical-side negative) and resistance ( R T) are −5.9 mV and 829 Ω · cm2, respectively. The apical membrane potential ( V A) is −40.7 mV, and the mean ratio of apical to basolateral membrane resistance ( R A/ R B) is 2.8. Apical amiloride hyperpolarized V A by 19.7 mV and tripled R A/ R B. A cAMP-elevating cocktail depolarized V A by 17.6 mV, decreased R A/ R B by 60%, increased short-circuit current by 6 μA/cm2, decreased R T by 155 Ω · cm2, and largely eliminated responses to amiloride. Whole cell patch-clamp measurements demonstrated amiloride-inhibited Na+ currents [linear current-voltage ( I-V) relation] and forskolin-stimulated Cl−currents (linear I-V relation). A capacitance probe method showed that in the control state, MEC monolayers either absorbed or secreted fluid (2–4 μl · cm−2 · h−1). Fluid secretion was stimulated either by activating CFTR (cAMP) or blocking ENaC (amiloride). These data plus equivalent circuit analysis showed that 1) fluid absorption across MEC is mediated by Na+ transport via apical membrane ENaC, and fluid secretion is mediated, in part, by Cl− transport via apical CFTR; 2) in both cases, appropriate counterions move through tight junctions to maintain electroneutrality; and 3) interactions among CFTR, ENaC, and tight junctions allow MEC to either absorb or secrete fluid and, in situ, may help control luminal [Na+] and [Cl−].

Forskolin-induced HCO3- current across apical membrane of the frog corneal epithelium

1990 ◽  
Vol 259 (2) ◽  
pp. C215-C223 ◽  
Author(s):  
O. A. Candia
Keyword(s):  
Corneal Epithelium ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Pump Current ◽  
Gas Composition ◽  
Apical Side ◽  
Disulfonic Stilbene ◽  
Stimulation Of

Forskolin (and other Cl- secretagogues) does not affect the very small Na(+)-originated short-circuit current (Isc) across frog corneal epithelium bathed in Cl- free solutions. However, forskolin in combination with increased PCO2 bubbling of the solutions (5-20% CO2) stimulated Isc proportionally to PCO2 to a maximum of approximately 8 microA/cm2. This current could be eliminated and reinstated by sequentially changing the gas composition of the bubbling to 100% air and 20% CO2-80% air. The same effects were observed when PCO2 changes were limited to the apical-side solution. Stroma-to-tear HCO3- movement was deemed unlikely, since the increase in Isc was observed with a HCO3(-)-free solution on the stromal side and CO2 gassing limited to the tear side. From the effects of ouabain and tryptamine, at least 80% of the Isc across the basolateral membrane can be accounted for by the Na+ pump current plus K+ movement from cell to bath. Methazolamide also inhibited Isc. Current across the apical membrane cannot be attributed to an electronegative Na(+)-HCO3- symport given the insensitivity of Isc to a disulfonic stilbene and the fact that stroma-to-tear Na+ fluxes did not increase on stimulation of Isc. The tear-to-stroma Na+ flux also remained unaltered, negating an increased apical bath-to-cell Na+ flow. The forskolin-20% CO2 manipulation produced a depolarization of the intracellular potential, a reduction in the apical-to-basolateral resistance ratio, and a decrease in transepithelial resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of K+ conductance in basolateral membrane of toad urinary bladder by oxytocin and cAMP

1988 ◽  
Vol 254 (6) ◽  
pp. C816-C821 ◽  
Author(s):  
W. Van Driessche ◽  
D. Erlij
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Potassium Conductance ◽  
Short Circuit Current ◽  
Membrane Properties ◽  
Toad Urinary Bladder ◽  
Electrical Behavior ◽  
Apical Side ◽  
Camp Analogue

We incubated toad urinary bladders with Na+-free, isotonic K+ solutions on the apical side and increased the cationic conductance of the apical membrane with nystatin (150 U/ml). Under these conditions, the short-circuit current is mostly carried by K+ flowing from mucosa to serosa. Impedance measurements showed that in nystatin-treated preparations, the electrical behavior of the tissue is dominated by the basolateral membrane properties. Oxytocin (0.1 U/ml) produced an increase of the current and the conductance of the basolateral membrane. Both the resting and the oxytocin-stimulated current were rapidly and reversibly blocked by serosal Ba2+. Addition of the adenosine 3',5'-cyclic monophosphate (cAMP) analogue [8-(4-chloropheylthio)-cAMP] to the basolateral solution mimicked the effects of oxytocin. These results show that oxytocin and cAMP stimulate a potassium conductance in the basolateral membrane and that the stimulation is not related to an increase in sodium entry through the apical membrane. Addition of ouabain (10(-3) M) to the serosal solution did not modify the stimulation by oxytocin, indicating that the activated pathway is not linked to the rate of turnover of the Na+ pump.

P2 purinoceptors regulate calcium-activated chloride and fluid transport in 31EG4 mammary epithelia

2003 ◽  
Vol 284 (4) ◽  
pp. C897-C909 ◽  
Author(s):  
Sasha Blaug ◽  
Jodi Rymer ◽  
Stephen Jalickee ◽  
Sheldon S. Miller
Keyword(s):  
Membrane Potential ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Fluid Secretion ◽  
Extracellular Nucleotides ◽  
Basolateral Membranes ◽  
Apical Side ◽  
Mammary Epithelia

It has been reported that secretory mammary epithelial cells (MEC) release ATP, UTP, and UDP upon mechanical stimulation. Here we examined the physiological changes caused by ATP/UTP in nontransformed, clonal mouse mammary epithelia (31EG4 cells). In control conditions, transepithelial potential (apical side negative) and resistance were −4.4 ± 1.3 mV (mean ± SD, n = 12) and 517.7 ± 39.4 Ω · cm2, respectively. The apical membrane potential was −43.9 ± 1.7 mV, and the ratio of apical to basolateral membrane resistance ( R A/ R B) was 3.5 ± 0.2. Addition of ATP or UTP to the apical or basolateral membranes caused large voltage and resistance changes with an EC50 of ∼24 μM (apical) and ∼30 μM (basal). Apical ATP/UTP (100 μM) depolarized apical membrane potential by 17.6 ± 0.8 mV ( n = 7) and decreased R A/ R B by a factor of ≈3. The addition of adenosine to either side (100 μM) had no effect on any of these parameters. The ATP/UTP responses were partially inhibited by DIDS and suramin and mediated by a transient increase in free intracellular Ca2+ concentration (427 ± 206 nM; 15–25 μM ATP, apical; n = 6). This Ca2+ increase was blocked by cyclopiazonic acid, by BAPTA, or by xestospongin C. 31EG4 MEC monolayers also secreted or absorbed fluid in the resting state, and ATP or UTP increased fluid secretion by 5.6 ± 3 μl · cm−2 · h−1( n = 10). Pharmacology experiments indicate that 31EG4 epithelia contain P2Y2 purinoceptors on the apical and basolateral membranes, which upon activation stimulate apical Ca2+-dependent Cl channels and cause fluid secretion across the monolayer. This suggests that extracellular nucleotides could play a fundamental role in mammary gland paracrine signaling and the regulation of milk composition in vivo.

Central role of the apical membrane H+-ATPase in electrogenesis and epithelial transport in Malpighian tubules

2000 ◽  
Vol 203 (9) ◽  
pp. 1459-1468 ◽  
Author(s):  
K.W. Beyenbach ◽  
T.L. Pannabecker ◽  
W. Nagel
Keyword(s):  
Epithelial Transport ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Electrical Coupling ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Malpighian Tubules ◽  
Proton Channel ◽  
Transepithelial Transport

The effects of bafilomycin A(1), a blocker of V-type H(+)-ATPases, were investigated in Malpighian tubules of Aedes aegypti. Bafilomycin A(1) reduced rates of transepithelial fluid secretion and the virtual short-circuit current (vI(sc)) with an IC(50) of approximately 5 micromol l(−)(1). As vI(sc) decreased, the electrical resistance increased across the whole epithelium and across the apical membrane, indicating effects on electroconductive pathways. Bafilomycin A(1) had no effect when applied from the tubule lumen, pointing to the relative impermeability of the apical membrane to bafilomycin A(1). Thus, bafilomycin A(1) must take a cytoplasmic route to its blocking site in the proton channel of the H(+)-ATPase located in the apical membrane of principal cells. The inhibitory effects of bafilomycin A(1) were qualitatively similar to those of dinitrophenol in that voltages across the epithelium (V(t)), the basolateral membrane (V(bl)) and the apical membrane (V(a)) depolarized towards zero in parallel. Moreover, V(bl)always tracked V(a), indicating electrical coupling between the two membranes through the shunt. Electrical coupling allows the H(+)-ATPase to energize not only the apical membrane, but also the basolateral membrane. Furthermore, electrical coupling offers a balance between electroconductive entry of cations across the basolateral membrane and extrusion across the apical membrane to support steady-state conditions during transepithelial transport.

Histamine-induced Cl- secretion in human nasal epithelium: responses of apical and basolateral membranes

1992 ◽  
Vol 263 (6) ◽  
pp. C1190-C1199 ◽  
Author(s):  
L. L. Clarke ◽  
A. M. Paradiso ◽  
R. C. Boucher
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Secretory Response ◽  
Bathing Solution ◽  
Equivalent Circuit Analysis ◽  
Cl Secretion ◽  
Influx Pathways ◽  
Cl Conductance

The mechanism by which receptors coupled to phospholipase C (PLC) induce Cl- secretion in amiloride-pretreated cultures of human nasal epithelial (HNE) cultures was investigated. Histamine (10(-4) M, basolateral administration) stimulated a rapid increase in equivalent short-circuit current, an index of Cl- secretion, that returned to baseline within 5 min. Intracellular recordings with double-barreled Cl(-)-selective microelectrodes showed that the apical and basolateral membrane potentials rapidly hyperpolarized, the fractional resistance of the apical membrane increased, and the transepithelial resistance decreased in response to histamine. Intracellular Cl- activity remained constant. Equivalent circuit analysis revealed that the early portion (< 0.9 min) of the Cl- secretory response was driven by an activation of a hyperpolarizing basolateral conductance, likely K+, whereas the later (> 0.9 min) phase of Cl- secretion reflects activation of the apical membrane Cl- conductance. Histamine raised intracellular Ca2+ (Ca2+i) measured by fura-2 in HNE with a potency similar to that observed for induction of Cl- secretion. Both intracellular release and plasma membrane influx pathways were identified, typical of receptor-mediated activation of PLC. The intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (15 microM), coupled with reduced bathing solution Ca2+, blunted the rise in Ca2+i and the net transepithelial Cl- secretory response to histamine. We conclude that 1) histamine induced Cl- secretion in HNE by a sequential mechanism: the rapid initial component reflects activation of the basolateral K+ conductance, and the later component reflects activation of an apical Cl- conductance; and 2) the level of Ca2+i may participate in the activation of both the basolateral and apical conductances.

Effects of bradykinin on Na+ and Cl- transport in human nasal epithelium

1992 ◽  
Vol 262 (3) ◽  
pp. C644-C655 ◽  
Author(s):  
L. L. Clarke ◽  
A. M. Paradiso ◽  
S. J. Mason ◽  
R. C. Boucher
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Nasal Epithelium ◽  
Induced Response ◽  
Equivalent Circuit Analysis ◽  
Human Nasal Epithelium ◽  
Cl Conductance

Human nasal epithelium (HNE) is a Na+ absorptive epithelium but establishes a baseline Cl- secretory current in the presence of amiloride (10(-4) M, luminal). We compared the effects of an inflammatory mediator, bradykinin (BK), on ion transport in primary cultures of HNE using double-barreled Cl(-)-selective microelectrodes. In untreated HNE, BK (10(-5) M) transiently increased the equivalent short-circuit current (Ieq). Maximal Ieq occurred with hyperpolarization of the transepithelial potential difference (Vt), which was associated with hyperpolarization and decreased resistance of the basolateral membrane; a subsequent depolarization of Vt was observed that was associated with depolarization and decreased resistance of the apical membrane. Removal of bath Cl- did not affect the BK-induced Ieq response. In amiloride-treated HNE, the electrical pattern of the BK-induced response was identical, but the magnitude of the Ieq was reduced by 54% and the change in Ieq could be abolished by removal of bath Cl-. Equivalent-circuit analysis of the response in amiloride-treated tissues indicated activation of a hyperpolarizing conductance in the basolateral membrane, followed 20-30 s later by activation of an apical Cl- conductance. We conclude that BK stimulates both Na+ absorption in untreated HNE and Cl- secretion in amiloride-treated HNE by activating a basolateral (K+) conductance. Analysis of the entire Ieq response under both conditions also suggested that BK induces a delayed activation of apical membrane Na+ and Cl- conductances.

Electrolyte and fluid secretion by cultured human inner medullary collecting duct cells

2002 ◽  
Vol 283 (6) ◽  
pp. F1337-F1350 ◽  
Author(s):  
Darren P. Wallace ◽  
Marcy Christensen ◽  
Gail Reif ◽  
Franck Belibi ◽  
Brantley Thrasher ◽  
...  
Keyword(s):  
Fluid Transport ◽  
Collecting Duct ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Transport Studies ◽  
Collecting Ducts ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct

Inner medullary collecting ducts (IMCD) are the final nephron segments through which urine flows. To investigate epithelial ion transport in human IMCD, we established primary cell cultures from initial (hIMCDi) and terminal (hIMCDt) inner medullary regions of human kidneys. AVP, PGE2, and forskolin increased cAMP in both hIMCDi and hIMCDt cells. The effects of AVP and PGE2 were greatest in hIMCDi; however, forskolin increased cAMP to the same extent in hIMCDi and hIMCDt. Basal short-circuit current ( I SC) of hIMCDi monolayers was 1.4 ± 0.5 μA/cm2 and was inhibited by benzamil, a Na+ channel blocker. 8-Bromo-cAMP, AVP, PGE2, and forskolin increased I SC; the current was reduced by blocking PKA, apical Cl− channels, basolateral NKCC1 (a Na+-K+-2Cl−cotransporter), and basolateral Cl−/HCO[Formula: see text]exchangers. In fluid transport studies, hIMCDi monolayers absorbed fluid in the basal state and forskolin reversed net fluid transport to secretion. In hIMCDt monolayers, basal current was not different from zero and cAMP had no effect on I SC. We conclude that AVP and PGE2stimulate cAMP-dependent Cl− secretion by hIMCDi cells, but not hIMCDt cells, in vitro. We suggest that salt secretion at specialized sites along human collecting ducts may be important in the formation of the final urine.

Campylobacter jejuni inhibits the absorptive transport functions of Caco-2 cells and disrupts cellular tight junctions

Microbiology ◽  
2005 ◽  
Vol 151 (7) ◽  
pp. 2451-2458 ◽  
Author(s):  
Amanda MacCallum ◽  
Simon P. Hardy ◽  
Paul H. Everest
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Campylobacter Jejuni ◽  
Fluid Transport ◽  
Cell Function ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Ussing Chambers ◽  
Cell Monolayers ◽  
Junction Protein

Caco-2 cells are models of absorptive enterocytes. The net transport of fluid from apical to basolateral surfaces results in ‘domes' forming in differentiated monolayers. Here, the effect of Campylobacter jejuni on this process has been examined. C. jejuni caused no changes in short-circuit current upon infection of Caco-2 cell monolayers in Ussing chambers. Thus, no active secretory events could be demonstrated using this model. It was therefore hypothesized that C. jejuni could inhibit the absorptive function of enterocytes and that this may contribute to diarrhoeal disease. C. jejuni infection of fluid-transporting (‘doming’) Caco-2 cells resulted in a significant reduction in dome number, which correlated with a decrease in tight junction integrity in infected monolayers, when measured as transepithelial electrical resistance. Defined mutants of C. jejuni also reduced dome numbers in infected monolayers. C. jejuni also altered the distribution of the tight junction protein occludin within cell monolayers. The addition to monolayers of extracellular gentamicin prevented these changes, indicating the contribution of extracellular bacteria to this process. Thus, tight junction integrity is required for fluid transport in Caco-2 cell monolayers as leaky tight junctions cannot maintain support of transported fluid at the basolateral surface of infected cell monolayers. Inhibition of absorptive cell function, changes in epithelial resistance and rearrangement of tight junctional proteins such as occludin represent a potential diarrhoeal mechanism of C. jejuni.

Aspects of electrolyte transport across isolated dog retinal pigment epithelium

1986 ◽  
Vol 250 (5) ◽  
pp. F781-F784 ◽  
Author(s):  
S. Tsuboi ◽  
R. Manabe ◽  
S. Iizuka
Keyword(s):  
Retinal Pigment Epithelium ◽  
Apical Membrane ◽  
Short Circuit ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Short Circuit Current ◽  
Bathing Solution ◽  
Apical Side ◽  
Net Flux ◽  
Net Fluxes

Transport of Na and Cl across the isolated dog retinal pigment epithelium (RPE) choroid was investigated. Under the short-circuit condition, a net Na flux was observed from choroid to retina and a net Cl flux was determined in the opposite direction. The current created by the net flux of these two ions was larger than the short-circuit current (SCC). Addition of 10(-5) M ouabain to the apical side inhibited net fluxes of both Na and Cl, whereas it reduced the SCC 84%. Addition of 10(-4) M furosemide to the apical side inhibited net Cl flux but had no effect on the net Na transport. The 10(-4) M furosemide reduced the SCC 38%. These drugs had no effect when applied to the basal side. Thus the transport of both Na and Cl depends on the Na-K-ATPase in the apical membrane of the dog RPE. A furosemide-sensitive neutral carrier at the apical membrane is suggested for the transport of Cl. Replacement of HCO3 with SO4 in the bathing solution caused an increase in the SCC, indicating the choroid-to-retina movement of HCO3 across the short-circuited dog RPE choroid.

scholarly journals Interaction between the basolateral K+ and apical Na+ conductances in Necturus urinary bladder.

1987 ◽  
Vol 89 (4) ◽  
pp. 563-580 ◽  
Author(s):  
J R Demarest ◽  
A L Finn
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
K Channel ◽  
Channel Blockers ◽  
Na Transport ◽  
Transepithelial Na Transport ◽  
Pump Activity ◽  
Relationship Of

Experimental modulation of the apical membrane Na+ conductance or basolateral membrane Na+-K+ pump activity has been shown to result in parallel changes in the basolateral K+ conductance in a number of epithelia. To determine whether modulation of the basolateral K+ conductance would result in parallel changes in apical Na+ conductance and basolateral pump activity, Necturus urinary bladders stripped of serosal muscle and connective tissue were impaled through their basolateral membranes with microelectrodes in experiments that allowed rapid serosal solution changes. Exposure of the basolateral membrane to the K+ channel blockers Ba2+ (0.5 mM/liter), Cs+ (10 mM/liter), or Rb+ (10 mM/liter) increased the basolateral resistance (Rb) by greater than 75% in each case. The increases in Rb were accompanied simultaneously by significant increases in apical resistance (Ra) of greater than 20% and decreases in transepithelial Na+ transport. The increases in Ra, measured as slope resistances, cannot be attributed to nonlinearity of the I-V relationship of the apical membrane, since the measured cell membrane potentials with the K+ channel blockers present were not significantly different from those resulting from increasing serosal K+, a maneuver that did not affect Ra. Thus, blocking the K+ conductance causes a reduction in net Na+ transport by reducing K+ exit from the cell and simultaneously reducing Na+ entry into the cell. Close correlations between the calculated short-circuit current and the apical and basolateral conductances were preserved after the basolateral K+ conductance pathways had been blocked. Thus, the interaction between the basolateral and apical conductances revealed by blocking the basolateral K+ channels is part of a network of feedback relationships that normally serves to maintain cellular homeostasis during changes in the rate of transepithelial Na+ transport.

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