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Na+-independent phosphate transport in Caco2BBE cells.

ABSTRACT: Pi transport in epithelia has both Na(+)-dependent and Na(+)-independent components, but so far only Na(+)-dependent transporters have been characterized in detail and molecularly identified. Consequently, in the present study, we initiated the characterization and analysis of intestinal Na(+)-independent Pi transport using an in vitro model, Caco2BBE cells. Only Na(+)-independent Pi uptake was observed in these cells, and Pi uptake was dramatically increased when cells were incubated in high-Pi DMEM (4 mM) from 1 day to several days. No response to low-Pi medium was observed. The increased Pi transport was mainly caused by Vmax changes, and it was prevented by actinomycin D and cycloheximide. Pi transport in cells grown in 1 mM Pi (basal DMEM) decreased at pH > 7.5, and it was inhibited with proton ionophores. Pi transport in cells incubated with 4 mM Pi increased with alkaline pH, suggesting a preference for divalent phosphate. Pi uptake in cells in 1 mM Pi was completely inhibited only by Pi and partially inhibited by phosphonoformate, oxalate, DIDS, SITS, SO4 (2-), HCO3 (-), and arsenate. This inhibition pattern suggests that more than one Pi transporter is active in cells maintained with 1 mM Pi. Phosphate transport from cells maintained at 4 mM Pi was only partially inhibited by phosphonoformate, oxalate, and arsenate. Attempts to identify the responsible transporters showed that multifunctional anion exchangers of the Slc26 family as well as members of Slc17, Slc20, and Slc37 and the Pi exporter xenotropic and polytropic retrovirus receptor 1 are not involved.

SUBMITTER: Candeal E

PROVIDER: S-EPMC4971830 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Na+-independent phosphate transport in Caco2BBE cells.

Candeal Eduardo E Caldas Yupanqui A YA Guillén Natalia N Levi Moshe M Sorribas Víctor V

American journal of physiology. Cell physiology 20141008 12

Pi transport in epithelia has both Na(+)-dependent and Na(+)-independent components, but so far only Na(+)-dependent transporters have been characterized in detail and molecularly identified. Consequently, in the present study, we initiated the characterization and analysis of intestinal Na(+)-independent Pi transport using an in vitro model, Caco2BBE cells. Only Na(+)-independent Pi uptake was observed in these cells, and Pi uptake was dramatically increased when cells were incubated in high-Pi ...[more]

PMID: 25298422

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Project description:The kinetics of sodium-stimulated phosphate flux and phosphate-stimulated sodium flux in human red cells have been previously described (Shoemaker, D.G., C.A. Bender, and R.B. Gunn. 1988. J. Gen. Physiol. 92:449-474). However, despite the identification of multiple isoforms in three gene families (Timmer, R.T., and R.B. Gunn. 1998. Am. J. Physiol. Cell Physiol. 274:C757-C769), the molecular basis for the sodium-phosphate cotransporter in erythrocytes is unknown. Most cells express multiple isoforms, thus disallowing explication of isoform-specific kinetics and function. We have found that erythrocyte membranes express one dominant isoform, hBNP-1, to which the kinetics can thus be ascribed. In addition, because the erythrocyte Na-PO(4) cotransporter can also mediate Li-PO(4) cotransport, it has been suggested that this transporter functions as the erythrocyte Na-Li exchanger whose activity is systematically altered in patients with bipolar disease and patients with essential hypertension. To determine the molecular basis for the sodium-phosphate cotransporter, we reasoned that if the kinetics of phosphate transport in a nucleated erythroid-like cell paralleled those of the Na-activated pathway in anucleated erythrocytes and yet were distinct from those known for other Na-PO(4) cotransporters, then the expressed genes may be the same in both cell types. In this study, we show that the kinetics of sodium phosphate cotransport were similar in anuclear human erythrocytes and K562 cells, a human erythroleukemic cell line. Although the erythrocyte fluxes were 750-fold smaller, the half-activation concentrations for phosphate and sodium and the relative cation specificities for activation of (32)PO(4) influx were similar. Na-activation curves for both cell types showed cooperativity consistent with the reported stoichiometry of more than one Na cotransported per PO(4). In K562 cells, external lithium activation of phosphate influx was also cooperative. Inhibition by arsenate, K(I) = 2.6-2.7 mM, and relative inhibition by amiloride, amiloride analogs, phosphonoformate, and phloretin were similar. These characteristics were different from those reported for hNaPi-3 and hPiT-1 in other systems. PCR analysis of sodium-phosphate cotransporter isoforms in K562 cells demonstrated the presence of mRNAs for hPiT-1, hPiT-2, and hBNP-1. The mRNAs for hNaPi-10 and hNaPi-3, the other two known isoforms, were absent. Western analysis of erythrocytes and K562 cells with isoform-specific antibodies detected the presence of only hBNP-1, an isoform expressed in brain neurons and glia. The similarities in the kinetics and the expression of only hBNP-1 protein in the two cell types is strong evidence that hBNP-1 is the erythrocyte and K562 cell sodium-phosphate cotransporter.

Project description:Phosphorus is essential in all cells' structural, metabolic and regulatory functions. For fungal cells that import inorganic phosphate (Pi) up a steep concentration gradient, surface Pi transporters are critical capacitators of growth. Fungi must deploy Pi transporters that enable optimal Pi uptake in pH and Pi concentration ranges prevalent in their environments. Single, triple and quadruple mutants were used to characterize the four Pi transporters we identified for the human fungal pathogen Candida albicans, which must adapt to alkaline conditions during invasion of the host bloodstream and deep organs. A high-affinity Pi transporter, Pho84, was most efficient across the widest pH range while another, Pho89, showed high-affinity characteristics only within one pH unit of neutral. Two low-affinity Pi transporters, Pho87 and Fgr2, were active only in acidic conditions. Only Pho84 among the Pi transporters was clearly required in previously identified Pi-related functions including Target of Rapamycin Complex 1 signaling, oxidative stress resistance and hyphal growth. We used in vitro evolution and whole genome sequencing as an unbiased forward genetic approach to probe adaptation to prolonged Pi scarcity of two quadruple mutant lineages lacking all 4 Pi transporters. Lineage-specific genomic changes corresponded to divergent success of the two lineages in fitness recovery during Pi limitation. Initial, large-scale genomic alterations like aneuploidies and loss of heterozygosity eventually resolved, as populations gained small-scale mutations. Severity of some phenotypes linked to Pi starvation, like cell wall stress hypersensitivity, decreased in parallel to evolving populations' fitness recovery in Pi scarcity, while severity of others like membrane stress responses diverged from Pi scarcity fitness. Among preliminary candidate genes for contributors to fitness recovery, those with links to TORC1 were overrepresented. Since Pi homeostasis differs substantially between fungi and humans, adaptive processes to Pi deprivation may harbor small-molecule targets that impact fungal growth, stress resistance and virulence.

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Na+-independent phosphate transport in Caco2BBE cells.

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Na+-independent phosphate transport in Caco2BBE cells.

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