Na+-Coupled Nutrient Cotransport Induced Luminal Negative Potential and Claudin-15 Play an Important Role in Paracellular Na+ Recycling in Mouse Small Intestine

Many nutrients are absorbed via Na+ cotransport systems, and therefore it is predicted that nutrient absorption mechanisms require a large amount of luminal Na+. It is thought that Na+ diffuses back into the lumen via paracellular pathways to support Na+ cotransport absorption. However, direct experimental evidence in support of this mechanism has not been shown. To elucidate this, we took advantage of claudin-15 deficient (cldn15−/−) mice, which have been shown to have decreased paracellular Na+ permeability. We measured glucose-induced currents (ΔIsc) under open- and short-circuit conditions and simultaneously measured changes in unidirectional 22Na+ fluxes (ΔJNa) in Ussing chambers. Under short-circuit conditions, application of glucose resulted in an increase in ΔIsc and unidirectional mucosal to serosal 22Na+ (∆JNaMS) flux in both wild-type and cldn15−/− mice. However, under open-circuit conditions, ΔIsc was observed but ∆JNaMS was strongly inhibited in wild-type but not in cldn15−/− mice. In addition, in the duodenum of mice treated with cholera toxin, paracellular Na+ conductance was decreased and glucose-induced ∆JNaMS increment was observed under open-circuit conditions. We concluded that the Na+ which is absorbed by Na+-dependent glucose cotransport is recycled back into the lumen via paracellular Na+ conductance through claudin-15, which is driven by Na+ cotransport induced luminal negativity.

Expression of proximal tubular Na-Pi and Na-SO4 cotransporters in MDCK and LLC-PK1 cells by transfection

Two cD-NAs coding for proximal tubular Na-Pi cotransport (NaPi-2) and Na-SO4 cotransport (NaSi-1) have been transfected by the use of a dexamethasone-inducible vector (pLK-neo) into MDCK and LLC-PK1 cells. By reverse transcription-polymerase chain reaction, expression of corresponding mRNAs was observed after stimulation with dexamethasone only. Similarly, expression of the NaPi-2 protein was detected only after induction with dexamethasone. In transfected Madin-Darby canine kidney (MDCK) cells, dexamethasone induced a large increase of Na-Pi or Na-SO4 cotransport, whereas, in transfected LLC-PK1, cell transport was only minimally expressed. In MDCK cells grown on filter supports, transfected Na-Pi-cotransport activity was equally expressed at both cell surfaces; dual location of expressed NaPi-2 protein was also observed by immunohistochemistry. In contrast, transfected Na-SO4 cotransport activity was predominantly expressed at the apical cell surface of MDCK cells. The results demonstrate that 1), in MDCK cells, the sorting behavior of two proximal tubular cotransport systems seems to be different: apical for Na-SO4 cotransport (NaSi-1) and dual location for Na-Pi cotransport (NaPi-2); and 2) LLC-PK1 cells seem not to be a suitable system to functionally express sodium-dependent cotransport systems for phosphate and sulfate.

Na+, K+, and Cl- transport in resting pancreatic acinar cells.

To understand the role of Na+, K+, and Cl- transporters in fluid and electrolyte secretion by pancreatic acinar cells, we studied the relationship between them in resting and stimulated cells. Measurements of [Cl-]i in resting cells showed that in HCO3(-)-buffered medium [Cl-]i and Cl- fluxes are dominated by the Cl-/HCO3- exchanger. In the absence of HCO3-, [Cl-]i is regulated by NaCl and NaK2Cl cotransport systems. Measurements of [Na+]i showed that the Na(+)-coupled Cl- transporters contributed to the regulation of [Na+]i, but the major Na+ influx pathway in resting pancreatic acinar cells is the Na+/H+ exchanger. 86Rb influx measurements revealed that > 95% of K+ influx is mediated by the Na+ pump and the NaK2Cl cotransporter. In resting cells, the two transporters appear to be coupled through [K+]i in that inhibition of either transporter had small effect on 86Rb uptake, but inhibition of both transporters largely prevented 86Rb uptake. Another form of coupling occurs between the Na+ influx transporters and the Na+ pump. Thus, inhibition of NaK2Cl cotransport increased Na+ influx by the Na+/H+ exchanger to fuel the Na+ pump. Similarly, inhibition of Na+/H+ exchange increased the activity of the NaK2Cl cotransporter. The combined measurements of [Na+]i and 86Rb influx indicate that the Na+/H+ exchanger contributes twice more than the NaK2Cl cotransporter and three times more than the NaCl cotransporter and a tetraethylammonium-sensitive channel to Na+ influx in resting cells. These findings were used to develop a model for the relationship between the transporters in resting pancreatic acinar cells.

Cloning, genetic mapping, and expression analysis of a mouse renal sodium-dependent phosphate cotransporter

Renal tubular reabsorption of phosphate is critical to the maintenance of phosphate homeostasis in mammals, and the brush-border membrane Na-P(i) cotransport systems in proximal tubules play a major role in this process. We have isolated a cDNA encoding a mouse sodium-dependent phosphate transport protein (Npt1), which is expressed primarily in the kidney. This protein is highly similar to its human and rabbit homologues, based on nucleotide and amino acid comparisons. The presence of potential Asn-linked glycosylation and protein kinase C phosphorylation sites that are conserved among all three homologues suggests that these sites may be important in the function and regulation of this protein. The Npt1 gene was mapped to mouse chromosome 13, close to the Tcrg locus. By both in situ hybridization and reverse transcription-polymerase chain reaction, Npt1 mRNA was localized predominantly to the proximal tubule.

Insulin activates furosemide-sensitive K+ and Cl- uptake system in BC3H1 cells

Insulin augments the activity of Na(+)-K(+)-adenosinetriphosphatase (ATPase) in skeletal muscles. This study shows that when furosemide- and bumetanide-inhibitable 86Rb+ uptake is measured in the skeletal muscle-like BC3H1 cell line, insulin and insulin-like growth factor I (IGF-I) activate a loop diuretic-sensitive K+ and Cl- transport system but have no effect on Na(+)-K(+)-ATPase. The insulin-stimulated K+ transport system is extracellular Na+ concentration ([Na+]o) independent and extracellular Cl- concentration ([Cl-]o) dependent. Na(+)-independent K(+)-Cl- cotransport systems have been identified in other cells, but their sensitivity to insulin or growth factors has not been described. The affinities of the insulin-stimulated K+ uptake in BC3H1 cells for K+ (0.9 +/- 0.1 mM) and loop diuretics (5.9 x 10(-7) and 10(-7) M for furosemide and bumetanide, respectively) are higher than those of K(+)-Cl- cotransporters in other cells. Thus the insulin-stimulated K+ and Cl- transport system in BC3H1 seems kinetically different from K(+)-Cl- cotransporters in other cells. Insulin and IGF-I may activate a unique K(+)-Cl- cotransporter or activate a [Na+]o-independent K(+)-Cl- cotransport mode of Na(+)-K(+)-Cl- cotransporter in BC3H1 cells.