Albumin absorption by canine bronchial epithelium

1989 ◽  
Vol 66 (6) ◽  
pp. 2772-2777 ◽  
Author(s):  
L. G. Johnson ◽  
P. W. Cheng ◽  
R. C. Boucher
Keyword(s):  
Gel Filtration ◽  
Fluid Phase ◽  
Bronchial Epithelium ◽  
Bathing Solution ◽  
Michaelis Constant ◽  
Airway Epithelia ◽  
Ussing Chambers ◽  
Bronchial Epithelia ◽  
Surface Liquid ◽  
High Concentrations

Albumin concentrations in airway surface liquid are low compared with plasma. To investigate the mechanisms that generate albumin gradients across airway epithelia, we have investigated whether active albumin absorption is a feature of bronchial epithelia. Freshly excised canine bronchi were mounted in Ussing chambers and short-circuited. Permeability coefficients of 14C-labeled canine albumin (Palb) were measured in the mucosal-to-submucosal (M----S) and submucosal-to-mucosal (S----M) directions in conductance-matched tissues. Mean steady-state values for Palb in the absorptive (M----S) direction (5.97 +/- 1.89 x 10(-7) cm/s) were significantly greater than rates in the S----M direction (1.09 +/- 0.41 x 10(-7) cm/s). Simultaneous measurements detected no asymmetry of transport of the fluid phase marker [3H]inulin. Gel filtration chromatography demonstrated that the majority of the radiolabel released into the submucosal bathing solution represented albumin fragments. Albumin fragments per se were not transported because no asymmetries in permeabilities of albumin fragments isolated from spontaneous degradation of tracer were detected. Decreasing the temperature of the bathing solution from 37 to 4 degrees C completely inhibited net albumin absorption. [14C]albumin transport was saturated by addition of high concentrations of unlabeled albumin (estimated Michaelis constant = 1.6 x 10(-3) M). These results demonstrate that albumin is absorbed by a low-affinity process that may contribute to the maintenance of low albumin concentrations in secretions.

scholarly journals The human complement system: assembly of the classical pathway C3 convertase

1980 ◽  
Vol 189 (1) ◽  
pp. 173-181 ◽  
Author(s):  
M A Kerr
Keyword(s):  
Direct Evidence ◽  
Gel Filtration ◽  
Fluid Phase ◽  
Classical Pathway ◽  
Human Complement ◽  
Stabilizing Effect ◽  
Excess Substrate ◽  
High Concentrations ◽  
Low Ionic Strength

The assembly of the classical pathway C3 convertase in the fluid phase has been studied. The enzyme is assembled from C2 and C4 on cleavage of these proteins by C1s. Once assembled, the enzyme activity decays rapidly. Kinetic evidence has been obtained that this decay is even more rapid than previously suggested (kdecay is 2.0 min-1 at 37 degrees C). As a result, optimal C3 convertase activity is only observed with high C1s levels, which result in rapid rates of cleavage of C2 and increased rates of formation of the C3 convertase. Using high concentrations of C1s at lower temperatures (22 degrees C) in the presence of excess substrate we have demonstrated kinetically that the enzyme comprises an equimolar complex of C4b and cleaved C2. We have obtained direct evidence from gel-filtration experiments for the role of C2a as the catalytic subunit of the enzyme. C2b appears to mediate the interaction between C4 (or C4b) and C2 at pH 8.5 and at low ionic strength where the interactions can easily be detected. It may therefore be important in the assembly of the enzyme, though it is not involved in the catalytic activity. The decay of the C3 convertase reflects the release of C2a from the C4b x (C2b) x C2a complex, and the stabilizing effect of iodine on the C3 convertase is therefore apparently one of stabilizing the C4b-C2z interaction, which is otherwise weak. C1s is not a part of the C3 convertase enzyme.

Duramycin enhances chloride secretion in airway epithelium

1990 ◽  
Vol 259 (3) ◽  
pp. C450-C454 ◽  
Author(s):  
M. M. Cloutier ◽  
L. Guernsey ◽  
P. Mattes ◽  
B. Koeppen
Keyword(s):  
Airway Epithelium ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Bathing Solution ◽  
Maximum Increase ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Polypeptide Antibiotic ◽  
High Concentrations

The effect of duramycin, a polypeptide antibiotic, on Cl- transport in canine tracheal epithelium mounted in Ussing chambers was studied. Over a narrow concentration range, duramycin increased short-circuit current (Isc) and net Cl- secretion and had no effect on mannitol flux when added to the mucosal bathing solution. The maximum increase in Isc was observed at a duramycin concentration of 2 X 10(-6) M and was associated with an increase in both unidirectional Cl- fluxes. Higher duramycin concentrations produced a decrease in Isc. Submucosal addition of duramycin had no effect on Isc except at high concentrations. Pretreatment of tissues with mucosal amiloride (10(-4) M) to reduce basal Na+ transport had no effect on the subsequent response to duamycin. In other tissues pretreated with 10(-3) M dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP), duramycin produced a further increase in Isc and net Cl- secretion similar to its effect in nonpretreated tissues. In all instances the increase in Isc was entirely accounted for by an increase in net Cl- secretion. We conclude that duramycin increases Isc and Cl- secretion in airway epithelium. Although the mechanism of activation is not known, these data demonstrate that duramycin increases Cl- secretion by a pathway other than cAMP. An understanding of the mechanism of action of duramycin may further our understanding of Cl- secretion regulation in airway epithelium.

Volume transport across tracheal and bronchial airway epithelia in a tubular culture system

1997 ◽  
Vol 273 (1) ◽  
pp. C21-C29 ◽  
Author(s):  
B. R. Grubb ◽  
F. R. Schiretz ◽  
R. C. Boucher
Keyword(s):  
Culture System ◽  
Culture Medium ◽  
Electrical Potential ◽  
Volume Transport ◽  
Electrical Potential Difference ◽  
Airway Epithelial ◽  
Airway Epithelia ◽  
Bronchial Epithelia ◽  
Surface Liquid ◽  
Insight Into

Airway epithelia are thought to play an important role in maintaining the depth (volume) and composition of airway surface liquid (ASL). However, due to the difficulty in measuring airway epithelial volume flow (Jv) and ASL composition, our knowledge of ASL homeostasis is limited. We have developed a permeable tubular culture system (biofiber) suitable for growing airway epithelia on the biofiber luminal surface, which allows measurements of bioelectric properties and Jv. Canine tracheal and bronchial epithelia readily attach, grow to confluence, and develop an electrical potential difference (-10 to -40 mV) across the biofiber. Using a six-hormone-supplemented medium, we detected a significant basal absorptive Jv across both the tracheal cells (0.65 +/- 0.08 microliter.cm-2.h-1) and bronchial cells (2.21 +/- 0.42 microliters.cm-2.h-1), which was significantly reduced by amiloride. Forskolin stimulated a net secretory Jv in tracheal biofibers (-0.56 +/- 0.19 microliter.h-1.cm-2) only. When the culture medium was supplemented with cholera toxin (CT), the basal absorptive Jv was significantly reduced in the bronchial biofibers and the tracheal biofibers exhibited net secretion. The forskolin-stimulated secretory Jv in the tracheal biofibers was significantly greater in the presence of CT than in its absence (-1.30 +/- 0.29 microliters.h-1.cm-2), whereas bronchial biofibers exhibited no significant Jv response to forskolin. We conclude that the Jv measured in tubular culture is highly dependent on the region from which the cells originated as well as the composition of the culture medium. Use of the biofiber culture system to study airway epithelia should give further insight into factors regulating Jv and composition of ASL.

Effect of pH on chloride absorption in the flounder intestine

1988 ◽  
Vol 255 (2) ◽  
pp. G247-G252 ◽  
Author(s):  
A. N. Charney ◽  
J. I. Scheide ◽  
P. M. Ingrassia ◽  
J. A. Zadunaisky
Keyword(s):  
Small Intestine ◽  
Short Circuit ◽  
Ph Effect ◽  
Short Circuit Current ◽  
Bathing Solution ◽  
Solution Ph ◽  
Transport Pathway ◽  
Ussing Chambers ◽  
Chloride Absorption ◽  
In Series

Chloride absorption in the small intestine of the winter flounder, Pseudopleuronectes americanus, is reported to be sensitive to ambient pH. We studied this sensitivity in isolated stripped intestinal mucosa mounted in modified Ussing chambers. Unidirectional 36Cl fluxes (JClm----s, JCls----m) were measured under short-circuited conditions in bathing solutions containing various combinations of HCO3- (0-20 mM), partial pressure of CO2 (0-36 mmHg), and pH (6.77-7.85). We found that JClm----s, net 36Cl flux (JClnet), and short-circuit current (Isc) increased and JCls----m decreased predominately in response to increases in bathing solution pH. There was a linear relationship between pH and both JClnet (r = 0.92, P less than 0.01) and Isc (r = 0.96, P less than 0.005) between pH 6.77 and 7.74. The pH effect was completely reversible, did not require either CO2 or HCO3-, and was not affected by the presence of mucosal barium at 1 mM. Mucosal bumetanide (0.1 mM) completely inhibited the pH effect. These data suggest that the process by which Cl- is absorbed in the flounder intestine is sensitive to pH. The data do not indicate whether pH affects Na+-K+-2Cl- cotransport or a Cl- transport pathway in series with this process. The direction of Cl- absorption in response to pH contrasts with inverse relation of pH and Cl- absorption in mammalian small intestine.

scholarly journals The CFTR trafficking mutation F508del inhibits the constitutive activity of SLC26A9

2017 ◽  
Vol 312 (6) ◽  
pp. L912-L925 ◽  
Author(s):  
Carol A. Bertrand ◽  
Shalini Mitra ◽  
Sanjay K. Mishra ◽  
Xiaohui Wang ◽  
Yu Zhao ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Family Member ◽  
Pdz Domain ◽  
Protein S ◽  
Constitutive Activity ◽  
Airway Epithelia ◽  
Anion Secretion ◽  
Anion Transporters ◽  
Bronchial Epithelia ◽  
Domain Protein

Several members of the SLC26A family of anion transporters associate with CFTR, forming complexes in which CFTR and SLC26A functions are reciprocally regulated. These associations are thought to be facilitated by PDZ scaffolding interactions. CFTR has been shown to be positively regulated by NHERF-1, and negatively regulated by CAL in airway epithelia. However, it is unclear which PDZ-domain protein(s) interact with SLC26A9, a SLC26A family member found in airway epithelia. We have previously shown that primary, human bronchial epithelia (HBE) from non-CF donors exhibit constitutive anion secretion attributable to SLC26A9. However, constitutive anion secretion is absent in HBE from CF donors. We examined whether changes in SLC26A9 constitutive activity could be attributed to a loss of CFTR trafficking, and what role PDZ interactions played. HEK293 coexpressing SLC26A9 with the trafficking mutant F508del CFTR exhibited a significant reduction in constitutive current compared with cells coexpressing SLC26A9 and wt CFTR. We found that SLC26A9 exhibits complex glycosylation when coexpressed with F508del CFTR, but its expression at the plasma membrane is decreased. SLC26A9 interacted with both NHERF-1 and CAL, and its interaction with both significantly increased with coexpression of wt CFTR. However, coexpression with F508del CFTR only increased SLC26A9’s interaction with CAL. Mutation of SLC26A9’s PDZ motif decreased this association with CAL, and restored its constitutive activity. Correcting aberrant F508del CFTR trafficking in CF HBE with corrector VX-809 also restored SLC26A9 activity. We conclude that when SLC26A9 is coexpressed with F508del CFTR, its trafficking defect leads to a PDZ motif-sensitive intracellular retention of SLC26A9.

Taurine secretion in primary monolayer cultures of flounder renal epithelium: stimulation by low osmolality

2000 ◽  
Vol 279 (2) ◽  
pp. R704-R712 ◽  
Author(s):  
Siribhinya Benyajati ◽  
J. Larry Renfro
Keyword(s):  
Transport Systems ◽  
Disulfonic Acid ◽  
Renal Epithelium ◽  
Ussing Chambers ◽  
Monolayer Cultures ◽  
High Concentrations ◽  
The One ◽  
Secretory System ◽  
Taurine Efflux

Transepithelial taurine fluxes determined in short-circuited monolayer cultures of flounder renal proximal cells in Ussing chambers revealed net taurine secretion. Both unidirectional secretory and reabsorptive taurine fluxes exhibited saturation kinetics contributed by two distinct saturable transepithelial taurine transport systems operating at different taurine concentration ranges. The taurine secretory system operating below 0.5 mM had lower affinity but higher capacity than the reabsorptive system, whereas the one operating at high concentrations (0.5–3.0 mM) had higher affinity but the same capacity as the corresponding reabsorptive system. Exposure (2 h) of the cultures to hyposmotic medium in the presence of taurine increased taurine secretory flux twofold with no effect on the reabsorptive flux. The hyposmolality-induced increase in taurine secretion was associated with a decreased peritubular taurine efflux and a concurrent increased luminal taurine efflux; the latter occurred via a pathway that was not affected by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid but inhibited by probenecid. The culture response in hyposmotic medium mimics the in vivo response of the intact marine fish kidney to dilution.

scholarly journals Properties of Plasminogen Activators in Circulating Human Blood and Human Cadaveric Veins

1977 ◽  
Author(s):  
Michael Mackie ◽  
Bruce Bennett ◽  
Derek Ogston
Keyword(s):  
Plasminogen Activator ◽  
Protease Inhibitors ◽  
Human Blood ◽  
Gel Filtration ◽  
Molecular Size ◽  
Plasminogen Activators ◽  
Venous Occlusion ◽  
Dextran Sulphate ◽  
Ammonium Sulphate Precipitation ◽  
High Concentrations

The properties of 3 partially purified plasminogen activators prepared from human blood have been compared with activator prepared from cadaveric veins and urokinase.Circulating plasminogen activator present in blood after venous occlusion of the arm has been partially purified by gel filtration on Sephadex G-200 in phosphate buffer containing 0.15 M NaCl followed by gel filtration of active fractions on Sephadex G-200 in buffer containing 1 M NaCl.Plasminogen activator generated from lysine-sepharose treated plasma after activation of Hageman factor by kaolin has been partially purified by elution of the activator from kaolin by 0.5 M NaCl and gel filtration on Sephadex G-200; the activator precipitated by dextran sulphate from the euglobulin supernatant prepared from lysine-sepharose treated plasma has been partially purified by similar gel filtration techniques.Cadaveric plasminogen activator has been prepared by ammonium sulphate precipitation of the washout from cadaveric veins followed by sequentail filtration on Sephadex G-200 in (a) low and (b) high concentrations of NaCl. The properties of these 4 plasminogen activators are compared with those of urokinase in terms of their stability, susceptibility to protease inhibitors and molecular size.

Acid secretion in isolated guinea pig colon

1987 ◽  
Vol 253 (2) ◽  
pp. G155-G164 ◽  
Author(s):  
Y. Suzuki ◽  
K. Kaneko
Keyword(s):  
Carbonic Anhydrase ◽  
Guinea Pig ◽  
Acid Secretion ◽  
Proximal Part ◽  
Bathing Solution ◽  
Maximal Effect ◽  
Dose Dependency ◽  
Ussing Chambers ◽  
Mucosal Acidification ◽  
Ph Stat

Isolated guinea pig distal colons secreted acid into the mucosal bathing solution at a rate of 1.0-1.5 mumol X cm-2 X h-1 when the preparations were mounted in Ussing chambers and bathed with HCO3(-)-CO2-free solution. The rates of the acidification and alkalinization of the solutions were measured by a pH stat system or calculated from changes in the pH of the solution. The acid secretion was localized in the middle and distal parts of the colon but absent in the proximal part of the colon and the cecum. The mucosal acidification was accompanied by serosal alkalinization, the rate of the latter being approximately 60% of the former. A carbonic anhydrase inhibitor, methazolamide (10(-4) M), reduced both the mucosal acidification and serosal alkalinization rates by a similar magnitude. The mucosal acidification was completely abolished by mucosal K+-free conditions but unaffected by mucosal Na+-free conditions. Ouabain added to the mucosal solution promptly inhibited the acid secretion. Dose dependency of the inhibition conformed to the Michaelis-Menten equation with a half-maximal effect at 4 X 10(-6) M. When the pH of the mucosal solution was reduced to 4.3, the rate of the mucosal acidification remained essentially the same as that at pH = 7.4. Vanadate (10(-4) M) added to both the mucosal and serosal solutions significantly reduced the mucosal acidification rate. These results suggest that CO2 derived from the epithelial metabolism is hydrated by carbonic anhydrase in the cell and released H+ enters the mucosal solution while HCO3- enters the serosal solution. H+ exit across the mucosal membrane may be mediated by H+-ATPase that is sensitive to ouabain.

scholarly journals Paracellular bicarbonate flux across human cystic fibrosis airway epithelia tempers changes in airway surface liquid pH

2020 ◽  
Vol 598 (19) ◽  
pp. 4307-4320 ◽  
Author(s):  
Ian M. Thornell ◽  
Tayyab Rehman ◽  
Alejandro A. Pezzulo ◽  
Michael J. Welsh
Keyword(s):  
Cystic Fibrosis ◽  
Airway Surface Liquid ◽  
Airway Epithelia ◽  
Cystic Fibrosis Airway ◽  
Surface Liquid
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