scholarly journals Parsing apical oxalate exchange in Caco-2BBe1 monolayers: siRNA knockdown of SLC26A6 reveals the role and properties of PAT-1

2009 ◽  
Vol 297 (5) ◽  
pp. G918-G929 ◽  
Author(s):  
Robert W. Freel ◽  
Makoto Morozumi ◽  
Marguerite Hatch
Keyword(s):  
Ussing Chamber ◽  
Anion Transporter ◽  
Environment Control ◽  
Net Flux ◽  
Unidirectional Fluxes ◽  
The Difference ◽  
Sirna Knockdown ◽  
Interfering Rna ◽  
Oxalate Transport

The purpose of this investigation was to quantitate the contribution of the anion exchanger PAT-1 (putative anion transporter-1), encoded by SLC26A6, to oxalate transport in a model intestinal epithelium and to discern some characteristics of this exchanger expressed in its native environment. Control (Con) Caco-2 BBe1 monolayers, 6–8 days postseeding, were compared with those transfected with a small interfering RNA targeted to SLC26A6 (A6KD). Radiotracer and Ussing chamber techniques were used to determine the transepithelial unidirectional fluxes of Ox2−, Cl−, and SO42− whereas fluorometric/BCECF measurements of intracellular pH were used to assess HCO3− exchange. PAT-1 was functionally targeted to the apical membrane, and SLC26A6 knockdown reduced PAT-1 protein (>60%) and mRNA (>75%) expression in A6KD. No net flux of Ox2−, Cl−, or SO42− was detected in Con or A6KD monolayers, yet the unidirectional fluxes in A6KD were reduced 50, 35, and 15%, respectively. Cl−-dependent HCO3− efflux from A6KD was reduced 50% compared with Con. The difference between Con and A6KD properties represents that mediated solely by PAT-1, and by this approach we found that PAT-1-mediated oxalate influx and efflux are inhibited equally by mucosal DIDS (EC50 ∼5 μM) and that mucosal Cl− inhibits oxalate uptake with an EC50 < 20 mM. Transepithelial Cl− gradients supported large, DIDS-sensitive net absorptive or secretory fluxes of oxalate in a direction opposite that of the imposed Cl− gradient. The overall symmetry of PAT-1-mediated oxalate exchange suggests that vectorial oxalate transport observed in vivo is principally dependent on the magnitude and direction of counterion gradients.

Bile salts and ileal calcium transport in rats: a neglected factor in intestinal calcium absorption

1993 ◽  
Vol 264 (2) ◽  
pp. G319-G324
Author(s):  
M. S. Hu ◽  
L. H. Kayne ◽  
P. A. Willsey ◽  
A. B. Koteva ◽  
N. Jamgotchian ◽  
...  
Keyword(s):  
Calcium Transport ◽  
Bile Salts ◽  
Calcium Absorption ◽  
Ussing Chamber ◽  
Simultaneous Increase ◽  
Solute Permeability ◽  
Postprandial State ◽  
Net Flux ◽  
Intercellular Pathway

Ileum displays little active transcellular calcium (Ca2+) absorption but is credited with the bulk of Ca2+ absorbed in vivo. We examined the effect of taurodeoxycholic acid (TDC, 2 mM), a bile salt, on mannitol (MN, a marker of intercellular solute traffic) and Ca2+ fluxes in rat ileum. In the absence of electrochemical gradients between the mucosal (M) and serosal (S) bathing media in an Ussing chamber, net flux (Jnet) was observed in the S-to-M direction for both MN and Ca2+, i.e., the unidirectional secretory S-to-M flux (Js-->m) exceeded the absorptive M-to-S flux (Jm-->s). Mucosal TDC caused simultaneous increase in transepithelial conductance and Js-->m for both MN and Ca2+. This was followed by even greater increases in MN and Ca2+ Jm-->s, so that ultimately Jm-->s equaled Js-->m in each case. In control tissue, Js-->m for Ca2+ appeared to permeate exclusively through the intercellular MN pathway while part of Jm-->s for Ca2+ appeared to traverse through a non-MN route. After the TDC-induced increase in intercellular solute permeability, both Ca2+ fluxes appeared to traverse through the aqueous MN conduit. During the postprandial state, the presence of bile salts and the relative abundance of Ca2+ in ileal lumen can cause bulk Ca2+ absorption through the intercellular pathway.

Inhibition of NaCl absorption from perfused rat ileum by furosemide

1976 ◽  
Vol 230 (6) ◽  
pp. 1517-1523 ◽  
Author(s):  
MH Humphreys
Keyword(s):  
Carbonic Anhydrase ◽  
Electrolyte Solution ◽  
Perfusion Fluid ◽  
Nacl Transport ◽  
Rat Ileum ◽  
Carbonic Anhydrase Inhibition ◽  
Neutral Process ◽  
Net Flux ◽  
Unidirectional Fluxes

The effect of furosemide on intestinal absorption of water and electrolytes was studied using segments of rat ileum perfused in vivo. Furosemide (1 mM) in the perfusion fluid reduced absorption of Na, Cl, and water by 50% from a balanced electrolyte solution without changing the transepithelial potential difference (PD). This effect was also observed in the absence of luminal glucose and was largely reversible. Substitution of all Na in perfusion fluid with choline produced secretion of Na and water and abolished Cl absorption; substitution of all Cl with SO4 reduced Na absorption to 20% of control values. Under both these conditions, furosemide had only trivial effects on electrolyte absorption and exerted no effect on PD. Measurements of unidirectional fluxes of Na and Cl showed that furosemide decreased net flux by reducing lumen-to-blood flux of these ions rather than increasing blood-to lumen flux. These results resemble those obtained in this tissue following exposure to acetazolamide, and suggest that furosemide inhibits a coupled, neutral process of NaCl transport from lumen to blood. Although this effect could be a result of carbonic anhydrase inhibition it more likely occurs from a separate action of furosemide on ileal transport.

scholarly journals Physiological roles of renal anion transporters NaS1 and Sat1

2011 ◽  
Vol 300 (6) ◽  
pp. F1267-F1270 ◽  
Author(s):  
Daniel Markovich
Keyword(s):  
Current Knowledge ◽  
Targeted Disruption ◽  
Sulfate Anion ◽  
Calcium Oxalate Urolithiasis ◽  
Anion Transporters ◽  
Anion Transporter ◽  
Proximal Tubular ◽  
Renal Proximal Tubular ◽  
Oxalate Transport

This review will briefly summarize current knowledge on the renal anion transporters sodium-sulfate cotransporter-1 (NaS1; Slc13a1) and sulfate-anion transporter-1 (Sat1; Slc26a1). NaS1 and Sat1 mediate renal proximal tubular sulfate reabsorption and thereby regulate blood sulfate levels. Sat1 also mediates renal oxalate transport and controls blood oxalate levels. Targeted disruption of murine NaS1 and Sat1 leads to hyposulfatemia and hypersulfaturia. Sat1 null mice also exhibit hyperoxalemia, hyperoxaluria, and calcium oxalate urolithiasis. NaS1 and Sat1 null mice also have other phenotypes that result due to changes in blood sulfate and oxalate levels. Experimental data indicate that NaS1 is essential for maintaining sulfate homeostasis, whereas Sat1 controls both sulfate and oxalate homeostasis in vivo.

scholarly journals Adenosinergic signaling inhibits oxalate transport by human intestinal Caco2-BBE cells through the A2B adenosine receptor

2018 ◽  
Vol 315 (5) ◽  
pp. C687-C698 ◽  
Author(s):  
Daniel Jung ◽  
Altayeb Alshaikh ◽  
Sireesha Ratakonda ◽  
Mohamed Bashir ◽  
Ruhul Amin ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Adenosine Receptor ◽  
Surface Expression ◽  
Oxalate Secretion ◽  
Urine Oxalate ◽  
Inflammatory Bowel ◽  
Sirna Knockdown ◽  
Oxalate Transport ◽  
Exchange Activity

Most kidney stones (KS) are composed of calcium oxalate, and small increases in urine oxalate affect the stone risk. Intestinal oxalate secretion mediated by anion exchanger SLC26A6 (PAT1) plays a crucial role in limiting net absorption of ingested oxalate, thereby preventing hyperoxaluria and related KS, reflecting the importance of understanding regulation of intestinal oxalate transport. We previously showed that ATP and UTP inhibit oxalate transport by human intestinal Caco2-BBE cells (C2). Since ATP is rapidly degraded to adenosine (ADO), we examined whether intestinal oxalate transport is regulated by ADO. We measured [14C]oxalate uptake in the presence of an outward Cl gradient as an assay of Cl-oxalate exchange activity, ≥49% of which is PAT1-mediated in C2 cells. We found that ADO significantly inhibited oxalate transport by C2 cells, an effect completely blocked by the nonselective ADO receptor antagonist 8- p-sulfophenyltheophylline. ADO also significantly inhibited oxalate efflux by C2 cells, which is important since PAT1 mediates oxalate efflux in vivo. Using pharmacological antagonists and A2B adenosine receptor (A2B AR) siRNA knockdown studies, we observed that ADO inhibits oxalate transport through the A2B AR, phospholipase C, and PKC. ADO inhibits oxalate transport by reducing PAT1 surface expression as shown by biotinylation studies. We conclude that ADO inhibits oxalate transport by lowering PAT1 surface expression in C2 cells through signaling pathways including the A2B AR, PKC, and phospholipase C. Given higher ADO levels and overexpression of the A2B AR in inflammatory bowel disease (IBD), our findings have potential relevance to pathophysiology of IBD-associated hyperoxaluria and related KS.

Enhancement of ADP-induced Platelet Aggregation by Adrenaline in Vivo and Its Prevention

1973 ◽  
Vol 29 (02) ◽  
pp. 490-498 ◽  
Author(s):  
Hiroh Yamazaki ◽  
Itsuro Kobayashi ◽  
Tadahiro Sano ◽  
Takio Shimamoto
Keyword(s):  
Platelet Count ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
The Other ◽  
Endothelial Surface ◽  
Important Interaction ◽  
Blood Coagulability ◽  
The Difference

SummaryThe authors previously reported a transient decrease in adhesive platelet count and an enhancement of blood coagulability after administration of a small amount of adrenaline (0.1-1 µg per Kg, i. v.) in man and rabbit. In such circumstances, the sensitivity of platelets to aggregation induced by ADP was studied by an optical density method. Five minutes after i. v. injection of 1 µg per Kg of adrenaline in 10 rabbits, intensity of platelet aggregation increased to 115.1 ± 4.9% (mean ± S. E.) by 10∼5 molar, 121.8 ± 7.8% by 3 × 10-6 molar and 129.4 ± 12.8% of the value before the injection by 10”6 molar ADP. The difference was statistically significant (P<0.01-0.05). The above change was not observed in each group of rabbits injected with saline, 1 µg per Kg of 1-noradrenaline or 0.1 and 10 µg per Kg of adrenaline. Also, it was prevented by oral administration of 10 mg per Kg of phenoxybenzamine or propranolol or aspirin or pyridinolcarbamate 3 hours before the challenge. On the other hand, the enhancement of ADP-induced platelet aggregation was not observed in vitro, when 10-5 or 3 × 10-6 molar and 129.4 ± 12.8% of the value before 10∼6 molar ADP was added to citrated platelet rich plasma (CPRP) of rabbit after incubation at 37°C for 30 second with 0.01, 0.1, 1, 10 or 100 µg per ml of adrenaline or noradrenaline. These results suggest an important interaction between endothelial surface and platelets in connection with the enhancement of ADP-induced platelet aggregation by adrenaline in vivo.

Reliability of a Single β-Thromboglobulin Measurement in a Diabetic Population : Importance of PGE1 in Anticoagulant Mixture

1987 ◽  
Vol 57 (02) ◽  
pp. 201-204 ◽  
Author(s):  
P Y Scarabin ◽  
L Strain ◽  
C A Ludlam ◽  
J Jones ◽  
E M Kohner
Keyword(s):  
In Vitro Release ◽  
Mean Value ◽  
Reliable Estimate ◽  
Diabetic Patients ◽  
Single Measurement ◽  
Diabetic Population ◽  
The Difference ◽  
Vitro Release

SummaryDuring the collection of samples for plasma β-thromboglobulin (β-TG) determination, it is well established that artificially high values can be observed due to in-vitro release. To estimate the reliability of a single β-TG measurement, blood samples were collected simultaneously from both arms on two separate occasions in 56 diabetic patients selected for a clinical trial. From each arm, blood was taken into two tubes containing an anticoagulant mixture with (tube A) and without (tube B) PGE!. The overall mean value of B-TG in tube B was 1.14 times higher than in tube A (p <0.01). The markedly large between-arms variation accounted for the most part of within-subject variation in both tubes and was significantly greater in tube B than in tube A. Based on the difference between B-TG values from both arms, the number of subjects with artifically high B-TG values was significantly higher in tube B than in tube A on each occasion (overall rate: 28% and 14% respectively). Estimate of between-occasions variation showed that B-TG levels were relatively stable for each subject between two occasions in each tube. It is concluded that the use of PGEi decreases falsely high B-TG levels, but a single measurement of B-TG does not provide a reliable estimate of the true B-TG value in vivo.

SiRNA technology, the gene therapy of the future?

2008 ◽  
Vol 149 (4) ◽  
pp. 153-159 ◽  
Author(s):  
Zsuzsanna Rácz ◽  
Péter Hamar
Keyword(s):  
Gene Therapy ◽  
Short Interfering Rna ◽  
The Future ◽  
Interfering Rna

A genetikában új korszak kezdődött 17 éve, amikor a petúniában felfedezték a koszuppressziót. Később a koszuppressziót azonosították a növényekben és alacsonyabb rendű eukariótákban megfigyelt RNS-interferenciával (RNSi). Bár a növényekben ez ősi vírusellenes gazdaszervezeti védekezőmechanizmus, emlősökben az RNSi élettani szerepe még nincs teljesen tisztázva. Az RNSi-t rövid kettős szálú interferáló RNS-ek (short interfering RNA, siRNS) irányítják. A jelen cikkben összefoglaljuk az RNSi történetét és mechanizmusát, az siRNS-ek szerkezete és hatékonysága közötti összefüggéseket, a célsejtbe való bejuttatás virális és nem virális módjait. Az siRNS-ek klinikai alkalmazásának legfontosabb akadálya az in vivo alkalmazás. Bár a hidrodinamikus kezelés állatokban hatékony, embereknél nem alkalmazható. Lehetőséget jelent viszont a szervspecifikus katéterezés. A szintetizált siRNS-ek ismert mellékhatásait szintén tárgyaljuk. Bár a génterápia ezen új területén számos problémával kell szembenézni, a sikeres in vitro és in vivo kísérletek reményt jelentenek emberi betegségek siRNS-sel történő kezelésére.

scholarly journals Force Generation on the Hallux Is More Affected by the Ankle Joint Angle than the Lesser Toes: An In Vivo Human Study

Biology ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 48
Author(s):  
Junya Saeki ◽  
Soichiro Iwanuma ◽  
Suguru Torii
Keyword(s):  
Repeated Measures ◽  
Joint Angle ◽  
Plantar Flexion ◽  
Human Study ◽  
Force Generation ◽  
Functional Difference ◽  
Multiple Comparison Test ◽  
The Difference ◽  
Metatarsophalangeal Joints

The structure of the first toe is independent of that of the other toes, while the functional difference remains unclear. The purpose of this study was to investigate the difference in the force generation characteristics between the plantar-flexion of the first and second–fifth metatarsophalangeal joints (MTPJs) by comparing the maximal voluntary plantar-flexion torques (MVC torque) at different MTPJs and ankle positions. The MVC torques of the first and second–fifth MTPJs were measured at 0°, 15°, 30°, and 45° dorsiflexed positions of the MTPJs, and at 20° plantar-flexed, neutral, and 20° dorsiflexed positions of the ankle. Two-way repeated measures analyses of variance with Holm’s multiple comparison test (MTPJ position × ankle position) were performed. When the MTPJ was dorsiflexed at 0°, 15°, and 30°, the MVC torque of the first MTPJ when the ankle was dorsiflexed at 20° was higher than that when the ankle was plantar-flexed at 20°. However, the ankle position had no significant effect on the MVC torque of the second–fifth MTPJ. Thus, the MVC torque of the first MTPJ was more affected by the ankle position than the second–fifth MTPJs.

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