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.

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−].

Pathways for apical and basolateral membrane NH3 and NH4+ movement in rat proximal tubule

1990 ◽  
Vol 259 (4) ◽  
pp. F587-F593 ◽  
Author(s):  
P. A. Preisig ◽  
R. J. Alpern
Keyword(s):  
Proximal Tubule ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Basolateral Membranes ◽  
Ammonia Addition ◽  
Rat Proximal Tubule

To examine the mechanism of preferential luminal ammonia secretion in the proximal tubule the apical and basolateral membrane pathways for NH3 and NH4+ movement were studied in the in vivo microperfused rat proximal tubule. Na and Cl were absent from all perfusates. Changes in pHi in response to rapid addition of NH3-NH4+ to either the luminal or peritubular perfusates were measured microfluorimetrically and expressed as the H(+)-equivalent flux (JeqH in pmol.mm-1.min-1). After ammonia addition ([NH3] 0.3 mM; [NH4+] 14.7 mM) to the luminal or peritubular fluids, pHi increased, and JeqH = 1,713 +/- 181 and 1,040 +/- 132 pmol.mm-1.min-1, respectively. To determine whether the above difference was due to NH3- or NH4(+)-driven fluxes, the effect of a fivefold greater [NH4+] ([NH3] 0.3 mM; [NH4+] 74.5 mM) on JeqH was examined. With luminal addition of a fivefold greater [NH4+], JeqH increased to 3,299 +/- 292 pmol.mm-1.min-1, demonstrating a pathway for NH4(+)-driven H+ efflux. One millimolar luminal amiloride inhibited JeqH in response to luminal NH3-NH4+ addition, suggesting that the amiloride-sensitive Na(+)-H+ antiporter mediates the NH4(+)-driven H+ efflux. JeqH was unaffected by addition of a fivefold greater [NH4+] to the peritubular perfusate, demonstrating the absence of an NH4(+)-driven H+ flux on the basolateral membrane. From these data, the calculated NH3 permeabilities were 6.2 +/- 1.3 and 7.0 +/- 0.9 X 10(-2) cm/s for the apical and basolateral membranes, respectively (NS). We conclude that apical and basolateral membrane NH3 permeabilities are similar and large. The apical membrane can also transport NH4+ on the amiloride-sensitive Na(+)-H+ antiporter.

Gene ablation for PEPT1 in mice abolishes the effects of dipeptides on small intestinal fluid absorption, short-circuit current, and intracellular pH

2010 ◽  
Vol 299 (1) ◽  
pp. G265-G274 ◽  
Author(s):  
Mingmin Chen ◽  
Anurag Singh ◽  
Fang Xiao ◽  
Ulrike Dringenberg ◽  
Jian Wang ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Intracellular Ph ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Intestinal Fluid ◽  
Fluid Absorption

PEPT1 function in mouse intestine has not been assessed by means of electrophysiology and methods to assess its role in intracellular pH and fluid homeostasis. Therefore, the effects of the dipeptide glycilsarcosin (Gly-Sar) on jejunal fluid absorption and villous enterocyte intracellular pH (pHi) in vivo, as well as on enterocyte[14C]Gly-Sar uptake, short-circuit current ( Isc) response, and enterocyte pHi in vitro were determined in wild-type and PEPT1-deficient mice and in mice lacking PEPT1. Immunohistochemistry for PEPT1 failed to detect any protein in enterocyte apical membranes in Slc15a1−/− animals. Saturable Gly-Sar uptake in Slc15a1−/− everted sac preparations was no longer detectable. Similarly, Gly-Sar-induced jejunal Isc response in vitro was abolished. The dipeptide-induced increase in fluid absorption in vivo was also abolished in animals lacking PEPT1. Since PEPT1 acts as an acid loader in enterocytes, enterocyte pHi was measured in vivo by two-photon microscopy in SNARF-4-loaded villous enterocytes of exteriorized jejuni in anesthetized mice, as well as in BCECF-loaded enterocytes of microdissected jejunal villi. Gly-Sar-induced pHi decrease was no longer observed in the absence of PEPT1. A reversal of the proton gradient across the luminal membrane did not significantly diminish Gly-Sar-induced Isc response, whereas a depolarization of the apical membrane potential by high K+ or via Na+-K+-ATPase inhibition strongly diminished Gly-Sar-induced Isc responses. This study demonstrates for the first time that proton-coupled electrogenic dipeptide uptake in the native small intestine, mediated by PEPT1, relies on the negative apical membrane potential as the major driving force and contributes significantly to intestinal fluid transport.

Pseudomonas aeruginosainduces changes in fluid transport across airway surface epithelia

1998 ◽  
Vol 275 (5) ◽  
pp. C1284-C1290 ◽  
Author(s):  
D. J. Evans ◽  
P. S. Matsumoto ◽  
J. H. Widdicombe ◽  
C. Li-Yun ◽  
A. A. Maminishkis ◽  
...  
Keyword(s):  
Fluid Transport ◽  
Bacterial Colonization ◽  
Organic Anion ◽  
Ionic Composition ◽  
Basolateral Membrane ◽  
Fluid Secretion ◽  
Fluid Absorption ◽  
Gene Encoding ◽  
Transport Inhibitors

Fluid transport across cultures of bovine tracheal epithelium was measured with a capacitance probe technique. Baseline fluid absorption ( Jv) across bovine cells of 3.2 μl ⋅ cm−2⋅ h−1was inhibited by ∼78% after 1 h of exposure to suspensions of Pseudomonas aeruginosa, with a concomitant decrease in transepithelial potential (TEP) and increase in transepithelial resistance ( Rt). Effects of P. aeruginosa were blocked by amiloride, which decreased Jvby 112% from baseline of 2.35 ± 1.25 μl ⋅ cm−2⋅ h−1, increased Rtby 101% from baseline of 610 ± 257 Ω ⋅ cm2, and decreased TEP by 91% from baseline of −55 ± 18.5 mV. Microelectrode studies suggested that effects of P. aeruginosa on amiloride-sensitive Na absorption were due in part to a block of basolateral membrane K channels. In the presence of Cl transport inhibitors [5-nitro-2-(3-phenylpropylamino)-benzoic acid, H2-DIDS, and bumetanide], P. aeruginosa induced a fluid secretion of ∼2.5 ± 0.4 μl ⋅ cm−2⋅ h−1and decreased Rtwithout changing TEP. However, these changes were abolished when the transport inhibitors were used in a medium in which Cl was replaced by an impermeant organic anion. Filtrates of P. aeruginosa suspensions had no effect on Jv, TEP, or Rt. Mutants lacking exotoxin A or rhamnolipids or with defective lipopolysaccharide still inhibited fluid absorption and altered bioelectrical properties. By contrast, mutations in the rpoN gene encoding a ς factor of RNA polymerase abolished actions of P. aeruginosa. In vivo, changes in transepithelial salt and water transport induced by P. aeruginosa may alter viscosity and ionic composition of airway secretions so as to foster further bacterial colonization.

Dibutyryl cAMP activates bumetanide-sensitive electrolyte transport in Malpighian tubules

1991 ◽  
Vol 261 (3) ◽  
pp. C521-C529 ◽  
Author(s):  
J. L. Hegarty ◽  
B. Zhang ◽  
T. L. Pannabecker ◽  
D. H. Petzel ◽  
M. D. Baustian ◽  
...  
Keyword(s):  
Yellow Fever ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Fluid Secretion ◽  
Ringer Solution ◽  
Malpighian Tubules ◽  
Membrane Voltage ◽  
Dibutyryl Camp ◽  
K Secretion ◽  
Transepithelial Voltage

The effects of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and bumetanide (both 10(-4) M) on transepithelial Na+, K+, Cl-, and fluid secretion and on tubule electrophysiology were studied in isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti. Peritubular DBcAMP significantly increased Na+, Cl-, and fluid secretion but decreased K+ secretion. In DBcAMP-stimulated tubules, bumetanide caused Na+, Cl-, and fluid secretion to return to pre-cAMP control rates and K+ secretion to decrease further. Peritubular bumetanide significantly increased Na+ secretion and decreased K+ secretion so that Cl- and fluid secretion did not change. In bumetanide-treated tubules, the secretagogue effects of DBcAMP are blocked. In isolated Malpighian tubules perfused with symmetrical Ringer solution, DBcAMP significantly hyperpolarized the transepithelial voltage (VT) and depolarized the basolateral membrane voltage (Vbl) with no effect on apical membrane voltage (Va). Total transepithelial resistance (RT) and the fractional resistance of the basolateral membrane (fRbl) significantly decreased. Bumetanide also hyperpolarized VT and depolarized Vbl, however without significantly affecting RT and fRbl. Together these results suggest that, in addition to stimulating electroconductive transport, DBcAMP also activates a nonconductive bumetanide-sensitive transport system in Aedes Malpighian tubules.

Luminal flow rate regulates proximal tubule H-HCO3 transporters

1992 ◽  
Vol 262 (1) ◽  
pp. F47-F54 ◽  
Author(s):  
P. A. Preisig
Keyword(s):  
Proximal Tubule ◽  
Flow Rate ◽  
Initial Rate ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Rate Dependence ◽  
Unstirred Layer ◽  
Disulfonic Acid ◽  
Luminal Flow

In vivo microperfusion was used to examine the mechanism of luminal flow rate dependence of proximal tubule acidification. Luminal flow rate was acutely changed between 5 and 40 nl/min, while luminal and peritubular capillary composition were held constant. With inhibition of basolateral membrane base transport by peritubular 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), cell pH (pHi) provides a sensitive index of apical membrane H secretory activity. At a luminal perfusate [HCO3] of 25 mM, progressive increases in luminal flow rate (5----15----25----40 nl/min) caused progressive increases in pHi. This effect was of a smaller magnitude with a luminal perfusate [HCO3] of 60 mM and was further decreased at a luminal perfusate [HCO3] of 100 mM. This pattern of diminished flow rate dependence at higher luminal [HCO3] is consistent with the presence of a luminal unstirred layer, whose composition can be modified by luminal flow rate. The activity of the apical membrane Na-H antiporter, assayed as the initial rate of pHi recovery from an acid load in the presence of peritubular DIDS, was faster at 40 compared with 5 nl/min. Basolateral membrane Na-3HCO3 symporter activity, assayed as the initial rate of pHi recovery from an alkali load in the absence of luminal and peritubular chloride, was faster at 40 compared with 5 nl/min. This effect was eliminated by luminal amiloride, suggesting an indirect effect of flow mediated by changes in pHi secondary to flow rate-dependent changes in apical membrane Na-H antiporter activity. In summary, increases in luminal flow rate directly increase apical membrane H secretion, possibly by modification of a luminal unstirred layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Convergence of apical and basolateral endocytic pathways at apical late endosomes in absorptive cells of suckling rat ileum in vivo

1990 ◽  
Vol 97 (2) ◽  
pp. 385-394
Author(s):  
M. Fujita ◽  
F. Reinhart ◽  
M. Neutra
Keyword(s):  
Basolateral Membrane ◽  
Hydrolytic Enzymes ◽  
Membrane Domains ◽  
Rat Ileum ◽  
Absorptive Cells ◽  
Apical Side ◽  
Late Endosomes ◽  
Membrane Components ◽  
Endocytic Pathways

Absorptive cells of the intestinal epithelium endocytose proteins from both apical and basolateral membrane domains. In absorptive cells of suckling rat ileum, luminal protein tracers first enter an apical tubulovesicular endosomal system, then enter larger apical endosomal vesicles and multivesicular bodies (MVB), and finally are delivered to a giant supranuclear lysosomal vacuole. To determine whether proteins endocytosed from the basolateral domain in vivo enter the same endosomal or lysosomal compartments as those taken up from the apical side, we simultaneously applied cationized ferritin (CF) apically (by intra-luminal injection) and horseradish peroxidase (HRP) basally (by intravenous injection), and examined absorptive cells after 3 min to 60 min using light, electron and fluorescence microscopy. At early times, CF and HRP entered separate endosomal compartments at apical and basolateral poles. At no time did HRP enter the apical tubulovesicular system, and CF never entered early basolateral endosomes. After 15 min, however, both tracers appeared together in large late endosomes and MVB located apically, above the giant vacuole. From 15 to 60 min both tracers accumulated in the giant vacuole. Membranes of some apical late endosomes, all apical MVB, the giant vacuole, and occasional sub-nuclear vesicles contained immunoreactive Igp120, a glycoprotein specific to late compartments of the endosome-lysosome system. These results show that highly polarized intestinal epithelial cells have separate apical and basolateral early endosomal compartments, presumably to maintain distinct membrane domains while allowing endocytosis and recycling of membrane from both surfaces. Apical and basolateral endocytic pathways, and presumably vesicles delivering hydrolytic enzymes and lysosomal membrane components, converge at the apical late endosome.

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)

Hormone-controlled cAMP-mediated fluid secretion in yellow-fever mosquito

1987 ◽  
Vol 253 (5) ◽  
pp. R701-R711 ◽  
Author(s):  
D. H. Petzel ◽  
M. M. Berg ◽  
K. W. Beyenbach
Keyword(s):  
Yellow Fever ◽  
Natriuretic Peptides ◽  
Basolateral Membrane ◽  
Fluid Secretion ◽  
Malpighian Tubules ◽  
In Vivo Studies ◽  
Intracellular Camp ◽  
Yellow Fever Mosquito ◽  
Water Fraction

Evidence is presented for hormone-controlled adenosine 3',5'-cyclic monophosphate (cAMP)-mediated NaCl diuresis in Malpighian tubules of the blood-feeding yellow-fever mosquito Aedes aegypti. Studies in isolated Malpighian tubules reveal that cAMP added to the peritubular bath selectively stimulates NaCl secretion and not KCl secretion by increasing the Na conductance of the basolateral membrane of primary cells. These effects are duplicated by forskolin and theophylline in parallel with increased intracellular concentrations of endogenous cAMP. Two natriuretic peptides that we have isolated by high-pressure liquid chromatography (HPLC) methods from mosquito heads also increase NaCl and fluid secretion in isolated Malpighian tubules together with increased intracellular levels of cAMP. These results are consistent with a mechanism of NaCl diuresis in which the natriuretic peptides and cAMP are respectively the primary and secondary messengers that couple the ingestion of a blood meal to the excretion of the unwanted salt and water fraction of the meal. This hypothesis is supported by in vivo studies that reveal elevated intracellular cAMP levels in Malpighian tubules at the time of maximum NaCl diuresis.

scholarly journals Water and solute permeabilities of medullary thick ascending limb apical and basolateral membranes

1998 ◽  
Vol 274 (3) ◽  
pp. F453-F462 ◽  
Author(s):  
Rickey Rivers ◽  
Anne Blanchard ◽  
Dominique Eladari ◽  
Francois Leviel ◽  
Michel Paillard ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Basolateral Membrane ◽  
Thick Ascending Limb ◽  
Membrane Structures ◽  
Membrane Unit ◽  
Small Surface ◽  
Basolateral Membranes ◽  
Medullary Thick Ascending Limb ◽  
Osmotic Water

The medullary thick ascending limb (MTAL) reabsorbs solute without water and concentrates [Formula: see text] in the interstitium without a favorable pH gradient, activities which require low water and NH3 permeabilities. The contributions of different apical and basolateral membrane structures to these low permeabilities are unclear. We isolated highly purified apical and basolateral MTAL plasma membranes and measured, by stopped-flow fluorometry, their permeabilities to water, urea, glycerol, protons, and NH3. Osmotic water permeability at 20°C averaged 9.4 ± 0.8 × 10−4 cm/s for apical and 11.9 ± 0.5 × 10−4cm/s for basolateral membranes. NH3 permeabilities at 20°C averaged 0.0023 ± 0.00035 and 0.0035 ± 0.00080 cm/s for apical and basolateral membranes, respectively. These values are consistent with those obtained in isolated perfused tubules and can account for known aspects of MTAL function in vivo. Because the apical and basolateral membrane unit permeabilities are similar, the ability of the apical membrane to function as the site of barrier function arises from its very small surface area when compared with the highly redundant basolateral membrane.

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