Diurnal rhythmicity in intestinal SGLT-1 function, V max, and mRNA expression topography

2001 ◽  
Vol 280 (2) ◽  
pp. G209-G215 ◽  
Author(s):  
Ali Tavakkolizadeh ◽  
Urs V. Berger ◽  
K. Robert Shen ◽  
Lynne L. Levitsky ◽  
Michael J. Zinner ◽  
...  
Keyword(s):  
Short Circuit ◽  
Kinetic Studies ◽  
Short Circuit Current ◽  
Northern Blotting ◽  
Maximal Velocity ◽  
Ussing Chambers ◽  
Sugar Absorption ◽  
Circadian Time ◽  
Before And After ◽  
Absorptive Flux

Mechanisms underlying the circadian rhythmicity in intestinal sugar absorption remain unclear. To test whether this rhythmicity is caused by changes in Na+-glucose cotransporter 1 (SGLT-1) function, we measured phloridzin-inhibitable sugar fluxes as an index of SGLT-1 activity. Jejunum obtained from rats killed at 6-h intervals during a 12-h light-dark cycle (CT0 is circadian time 0 h, time of light onset) were mounted in Ussing chambers, and 3- O-methylglucose (3-OMG) fluxes were calculated before and after addition of phloridzin. 3-OMG-induced change in short-circuit current and absorptive flux were significantly greater at CT9 than at CT3. This increase was phloridzin inhibitable. Kinetic studies indicated a significant increase in SGLT-1 maximal velocity ( V max) at CT9. Food intake between CT3 and CT9 was <10% of the daily total, indicating that the increased SGLT-1 activity was anticipatory. Diurnicity of SGLT-1 mRNA was confirmed by Northern blotting. Expression topography analyzed by in situ hybridization revealed more intense labeling along the entire villus axis at CT9 and CT15 compared with CT3 and CT21. We conclude that diurnicity in intestinal sugar absorption is caused by periodicity in SGLT-1 V max.

Membrane site of action of CO2 on colonic sodium absorption

1989 ◽  
Vol 256 (3) ◽  
pp. C584-C590 ◽  
Author(s):  
A. N. Charney ◽  
R. W. Egnor
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Sodium Absorption ◽  
Ussing Chambers ◽  
Chloride Absorption ◽  
Before And After ◽  
36Cl Fluxes ◽  
Tension Increase ◽  
Ambient Co2

Increases in ambient CO2 tension increase colonic sodium absorption by increasing mucosal to serosal sodium flux. We examined the membrane site of CO2 action by utilizing the polyene antibiotic nystatin to create aqueous pores in the apical membrane. Under these conditions, the basolateral rather than the apical membrane is rate limiting for sodium absorption. Pairs of stripped rat distal colonic segments were mounted in modified Ussing chambers in a Ringer-HCO3 solution gassed with either 3% CO2-97% O2 or 11% CO2-89% O2. Mucosal-to-serosal 22Na and 36Cl fluxes were measured under short-circuited conditions, and ouabain-sensitive absorption was calculated before and after the addition of mucosal nystatin 300 U/ml. Ouabain-sensitive sodium absorption was fivefold greater at 11% CO2 than at 3% CO2 before nystatin addition. Nystatin increased short-circuit current (Isc), transcolonic conductance (Gt) and ouabain-sensitive sodium absorption at 3% CO2 but only increased Isc and Gt at 11% CO2. The levels of sodium absorption at 3% and 11% CO2 after nystatin were equal and identical to the level measured at 11% CO2 in the absence of nystatin. Ouabain-sensitive chloride absorption was similar at 3% and 11% CO2 in the absence of nystatin and was not affected by nystatin addition. These findings suggest that ambient CO2 tension affects colonic sodium absorption by a selective action at the apical membrane.

Effects of metabolic inhibition on ion transport by dog bronchial epithelium

1988 ◽  
Vol 64 (1) ◽  
pp. 253-258 ◽  
Author(s):  
M. J. Stutts ◽  
J. T. Gatzy ◽  
R. C. Boucher
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Apical Cell ◽  
Short Circuit Current ◽  
Bronchial Epithelium ◽  
Metabolic Inhibition ◽  
Apical Cell Membrane ◽  
Solute Permeability ◽  
Ussing Chambers ◽  
Absorptive Flux

Mammalian bronchial epithelium absorbs Na+ under basal conditions, but Cl- secretion can be induced. We studied the effects of several modes of metabolic inhibition on the bioelectric properties and solute permeability of dog bronchial epithelium mounted in Ussing chambers. Net Na+ absorption and short-circuit current were inhibited by approximately 75% by hypoxia or by 10(-3) M NaCN. The reduced net Na+ absorption was characterized by a decrease in absorptive flux and an increase in backflux. The latter change was proportional to an increase in permeability to [14C]mannitol, implying that solute flow through a paracellular shunt was increased. In contrast, the reduction of conductance expected from exposure to amiloride (0.94 +/- 0.15 ms/cm2 or 12%) was abolished by NaCN pretreatment. Metabolic inhibition also decreased epithelial conductance and unidirectional Cl- fluxes by approximately 25%. NaCN rapidly and reversibly inhibited the hyperpolarization of potential difference (PD) induced by low luminal bath [Cl-]. This effect was mimicked by the Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino) benzoic acid. Because the transepithelial Cl- diffusion PD reflects, in part, the depolarization of the Cl- -conductive apical cell membrane, metabolic inhibition appears to affect this path. We conclude that metabolic inhibition not only decreased net ion transport by dog bronchial epithelium but also inhibited cellular Na+- and Cl- -conductive pathways and increased paracellular permeability.

Protein kinase C potentiates cAMP-stimulated mouse duodenal mucosal bicarbonate secretion in vitro

2004 ◽  
Vol 286 (5) ◽  
pp. G814-G821 ◽  
Author(s):  
Bi-Guang Tuo ◽  
Jimmy Y. C. Chow ◽  
Kim E. Barrett ◽  
Jon I. Isenberg
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Duodenal Mucosa ◽  
Bicarbonate Secretion ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ussing Chambers ◽  
Duodenal Bicarbonate Secretion

PKC has been shown to regulate epithelial Cl- secretion in a variety of models. However, the role of PKC in duodenal mucosal bicarbonate secretion is less clear. We aimed to investigate the role of PKC in regulation of duodenal mucosal bicarbonate secretion. Bicarbonate secretion by murine duodenal mucosa was examined in vitro in Ussing chambers using a pH-stat technique. PKC isoform expression and activity were assessed by Western blotting and in vitro kinase assays, respectively. PMA (an activator of PKC) alone had no effect on duodenal bicarbonate secretion or short-circuit current ( Isc). When PMA and dibutyryl-cAMP (db-cAMP) were added simultaneously, PMA failed to alter db-cAMP-stimulated duodenal bicarbonate secretion or Isc ( P > 0.05). However, a 1-h preincubation with PMA potentiated db-cAMP-stimulated duodenal bicarbonate secretion and Isc in a concentration-dependent manner (from 10-8 to 10-5M) ( P < 0.05). PMA preincubation had no effects on carbachol- or heat-stable toxin-stimulated bicarbonate secretion. Western blot analysis revealed that PKCα, -γ, -ϵ, -θ, -μ, and -ι/λ were expressed in murine duodenal mucosa. Ro 31–8220 (an inhibitor active against PKCϵ, -α, -β, and -γ), but not Gö 6983 (an inhibitor active against PKCα, -γ, -β, and -δ), reversed the potentiating effect of PMA on db-cAMP-stimulated bicarbonate secretion. PMA also time- and concentration-dependently increased the activity of PKCϵ, an effect that was prevented by Ro 31–8220 but not Gö 6983. These results demonstrate that activation of PKC potentiates cAMP-stimulated duodenal bicarbonate secretion, whereas it does not modify basal secretion. The effect of PKC on cAMP-stimulated bicarbonate secretion is mediated by the PKCϵ isoform.

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.

Active electrolyte transport in mammalian buccal mucosa

1988 ◽  
Vol 255 (3) ◽  
pp. G286-G291 ◽  
Author(s):  
R. C. Orlando ◽  
N. A. Tobey ◽  
V. J. Schreiner ◽  
R. D. Readling
Keyword(s):  
Buccal Mucosa ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Electrolyte Transport ◽  
Electrical Potential Difference ◽  
Ussing Chambers ◽  
Net Fluxes

The transmural electrical potential difference (PD) was measured in vivo across the buccal mucosa of humans and experimental animals. Mean PD was -31 +/- 2 mV in humans, -34 +/- 2 mV in dogs, -39 +/- 2 mV in rabbits, and -18 +/- 1 mV in hamsters. The mechanisms responsible for this PD were explored in Ussing chambers using dog buccal mucosa. After equilibration, mean PD was -16 +/- 2 mV, short-circuit current (Isc) was 15 +/- 1 microA/cm2, and resistance was 1,090 +/- 100 omega.cm2, the latter indicating an electrically "tight" tissue. Fluxes of [14C]mannitol, a marker of paracellular permeability, varied directly with tissue conductance. The net fluxes of 22Na and 36Cl were +0.21 +/- 0.05 and -0.04 +/- 0.02 mueq/h.cm2, respectively, but only the Na+ flux differed significantly from zero. Isc was reduced by luminal amiloride, serosal ouabain, or by reducing luminal Na+ below 20 mM. This indicated that the Isc was determined primarily by active Na+ absorption and that Na+ traverses the apical membrane at least partly through amiloride-sensitive channels and exits across the basolateral membrane through Na+-K+-ATPase activity. We conclude that buccal mucosa is capable of active electrolyte transport and that this capacity contributes to generation of the buccal PD in vivo.

scholarly journals Acid-Base Transport and Control in Locust Hindgut: Artefacts Caused by Short-Circuit Current

1991 ◽  
Vol 155 (1) ◽  
pp. 455-467
Author(s):  
R. BRENT THOMSON ◽  
N. AUDSLEY ◽  
JOHN E. PHILLIPS
Keyword(s):  
Cyclic Amp ◽  
Acid Secretion ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Salt Bridges ◽  
Electrical Parameters ◽  
Acid Base ◽  
Before And After ◽  
And Control ◽  
Stimulation Of

The commonly used method of passing short-circuit current (Isc) across insect epithelia through Ag-AgCl electrodes, without the use of salt bridges, leads to significant OH− production at the cathode (lumen side) when high currents are applied. The alkalization of the lumen previously reported when cyclic AMP was added to short-circuited locust hindgut is a result of this phenomenon rather than cyclic-AMP-mediated stimulation of acid-base transport in the hindgut. When salt bridges are used to pass short-circuit current across locust hindgut, acid secretion (JH) into the lumen equals alkaline movement (JOH) to the haemocoel side, and JH is similar under both open- and short-circuit conditions. JH is similar (1.5 μequiv cm−2 h−1) in recta and ilea. Addition of cyclic AMP inhibits JH across the rectum by 42–66%, but has no effect on the ileum when salt bridges are used. Electrical parameters (Isc, Vt, Rt) reflecting hindgut Cl− transport (JCL) before and after stimulation with cyclic AMP are the same whether or not salt bridges are used. We found no evidence of any coupling between JCl and JH/JOH.

Mechanisms of sodium and chloride transport across equine tracheal epithelium

1990 ◽  
Vol 259 (6) ◽  
pp. L459-L467 ◽  
Author(s):  
G. J. Tessier ◽  
T. R. Traynor ◽  
M. S. Kannan ◽  
S. M. O3'Grady
Keyword(s):  
Chloride Transport ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Tracheal Epithelium ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Cotransport System ◽  
Ethanesulfonic Acid

Equine tracheal epithelium, stripped of serosal muscle, mounted in Ussing chambers, and bathed in plasmalike Ringer solution generates a serosa-positive transepithelial potential of 10–22 mV and a short-circuit current (Isc) of 70–200 microA/cm2. Mucosal amiloride (10 microM) causes a 40–60% decrease in Isc and inhibits the net transepithelial Na flux by 95%. Substitution of Cl with gluconate resulted in a 30% decrease in basal Isc. Bicarbonate substitution with 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid decreased the Isc by 21%. The Cl-dependent Isc was inhibited by serosal addition of 1 mM amiloride. Bicarbonate replacement or serosal amiloride (1 mM) inhibits the net Cl flux by 72 and 69%, respectively. Bicarbonate replacement significantly reduces the effects of serosal amiloride (1 mM) on Isc, indicating its effect is HCO3 dependent. Addition of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 100 microM) causes a 40% increase in Isc. This effect is inhibited by subsequent addition of 10 microM serosal bumetanide. Bumetanide (10 microM) reduces net Cl secretion following stimulation with 8-BrcAMP (100 microM). Serosal addition of BaCl2 (1 mM) causes a reduction in Isc equal to that following Cl replacement in the presence or absence of 100 microM cAMP. These results suggest that 1) Na absorption depends on amiloride-inhibitable Na channels in the apical membrane, 2) Cl influx across the basolateral membrane occurs by both a Na-H/Cl-HCO3 parallel exchange mechanism under basal conditions and by a bumetanide-sensitive Na-(K?)-Cl cotransport system under cAMP-stimulated conditions, and 3) basal and cAMP-stimulated Cl secretion depends on Ba-sensitive K channels in the basolateral membrane.

Alterations in capsaicin-evoked electrolyte transport during the evolution of guinea pig TNBS ileitis

2002 ◽  
Vol 282 (6) ◽  
pp. G972-G980 ◽  
Author(s):  
Paula Miceli ◽  
Gerald P. Morris ◽  
Wallace K. MacNaughton ◽  
Stephen Vanner
Keyword(s):  
Substance P ◽  
Guinea Pig ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Electrolyte Transport ◽  
Secretory Function ◽  
Trinitrobenzenesulfonic Acid ◽  
Ussing Chambers ◽  
Circulating Cytokines

The efferent secretomotor activity of capsaicin-sensitive nerves was monitored during the evolution of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced ileitis in the guinea pig by recording changes in short-circuit current (Δ I sc) in response to capsaicin, substance P (SP), and carbachol. Submucosal-mucosal preparations mounted in standard Ussing chambers were studied at time 0, at 8 h, and 1, 3, 5, 7, 14, and 30 days following the intraluminal instillation of TNBS or saline. Maximal Δ I scresponses to capsaicin were dramatically attenuated (54%) by 24 h. By day 7, SP- and TTX-insensitive carbachol-stimulated Δ I sc were also significantly reduced. Similar attenuation in capsaicin and carbachol responses was observed in jejunal tissue 20 cm proximal to the inflamed site at day 7. These studies demonstrate that efferent secretomotor function of capsaicin-sensitive nerves is maintained early in TNBS ileitis but significantly reduced by 24 h. By day 7, defects in enterocyte secretory function at inflamed and noninflamed sites also occurred, an effect that may be mediated by circulating cytokines.

Effects of bile acids on dog pancreatic duct epithelial cell secretion and monolayer resistance

2002 ◽  
Vol 283 (5) ◽  
pp. G1042-G1050 ◽  
Author(s):  
Charles Okolo ◽  
Thomas Wong ◽  
Mark W. Moody ◽  
Toan D. Nguyen
Keyword(s):  
Pancreatic Duct ◽  
Bile Acids ◽  
Short Circuit ◽  
Bile Reflux ◽  
Short Circuit Current ◽  
Cell Secretion ◽  
Luminal A ◽  
Transport Pathways ◽  
Ussing Chambers ◽  
Lactate Dehydrogenase Release

Pancreatic duct epithelial cells (PDEC) mediate the secretion of fluid and electrolytes and are exposed to refluxed bile. In nontransformed cultured dog PDEC, which express many ion transport pathways of PDEC, 1 mM taurodeoxycholic acid (TDCA) stimulated an125I−efflux inhibited by DIDS and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and a86Rb+efflux inhibited by charybdotoxin. Inhibition by 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA)-AM suggests mediation via increased intracellular Ca2+concentration, whereas the absence of lactate dehydrogenase release excludes cellular toxicity. At 1 mM, TDCA stimulated a larger125I−efflux than glycodeoxycholate; two dihydroxy bile acids, taurochenodeoxycholate and TDCA, were similarly effective, whereas a trihydroxy bile acid, taurocholate, was ineffective. In Ussing chambers, 1 mM serosal or 2 mM luminal TDCA stimulated an Iscincrease from confluent PDEC monolayers. TDCA also stimulated 1) a short-circuit current ( Isc) increase from basolaterally permeabilized PDEC subject to a serosal-to-luminal Cl−gradient that was inhibited by BAPTA-AM, DIDS, and NPPB and 2) an Iscincrease from apically permeabilized PDEC subject to a luminal-to-serosal K+gradient inhibited by BAPTA-AM and charybdotoxin. Along with the efflux studies, these findings suggest that TDCA interacts directly with PDEC to stimulate Ca2+-activated apical Cl−channels and basolateral K+channels. Monolayer transepithelial resistance was only minimally affected by 1 mM serosal and 2 mM luminal TDCA but decreased after exposure to higher TDCA concentrations (2 mM serosal and 4 mM luminal). A secretory role for bile acids should be considered in pancreatic diseases associated with bile reflux.

Apical acidification induces paracellular injury in canine gastric mucosal monolayers

1994 ◽  
Vol 267 (6) ◽  
pp. G1012-G1020 ◽  
Author(s):  
M. C. Chen ◽  
A. Chang ◽  
T. Buhl ◽  
M. Tanner ◽  
A. H. Soll
Keyword(s):  
Short Circuit ◽  
Phase 1 ◽  
Short Circuit Current ◽  
Paracellular Permeability ◽  
Ussing Chambers ◽  
Low Concentrations ◽  
Three Phase ◽  
Mucosal Cells ◽  
Primary Monolayer ◽  
Primary Monolayer Cultures

We used primary monolayer cultures of enzyme-dispersed canine oxyntic mucosal cells mounted in Ussing chambers to characterize the apical barrier to H+. [3H]mannitol flux (MF) and [14C]inulin flux (IF) were used as size probes for tight junctions. Apical H+ produced a three-phase effect. In phase 1, as the apical pH was decreased from 7 to about 2.5, resistance (R) increased, but short-circuit current (Isc) did not change. In phase 2, an increased paracellular permeability developed at pH below 2.5-1.7, evidenced by decreased R and increased MF but not IF. Size sieving and monolayer integrity were preserved, and this paracellular leak was either fully reversed or stabilized by apical neutralization, depending on the duration of the paracellular leak. In phase 3, after sustained exposure to an apical pH below approximately 2, transepithelial integrity was lost; R decreased to fluid R, and both MF and IF increased. Basolateral acidification below pH 5.5 produced rapid monolayer disruption. Low concentrations of cytochalasin D (CD) decreased R and increased MF but not IF; apical acidification to pH 4 after CD increased R and decreased the MF, indicating reduced paracellular permeability by apical H+. Apical amiloride did not alter Isc; however, after 48 h of treatment with hydrocortisone and insulin, an amiloride-sensitive Isc component became evident. Our data indicate that the increase in R observed with apical acidification reflects decreased paracellular permeability and that the earliest injury with apical acidification is a selective paracellular leak.

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