scholarly journals Inorganic Phosphate Modulates the Expression of the NaPi-2a Transporter in thetrans-Golgi Network and the Interaction with PIST in the Proximal Tubule

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Miguel A. Lanaspa ◽  
Yupanqui A. Caldas ◽  
Sophia Y. Breusegem ◽  
Ana Andrés-Hernando ◽  
Christina Cicerchi ◽  
...  
Keyword(s):  
Inorganic Phosphate ◽  
Culture Medium ◽  
Pdz Domain ◽  
Proximal Tubules ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Pi Homeostasis ◽  
Pi Transport ◽  
Endosomal Transport ◽  
Golgi Network

Inorganic phosphate (Pi) homeostasis is maintained by the tight regulation of renal Pi excretion versus reabsorption rates that are in turn modulated by adjusting the number of Pi transporters (mainly NaPi-2a) in the proximal tubules. In response to some hormones and a high dietary Pi content, NaPi-2a is endocytosed and degraded in the lysosomes; however, we show here that some NaPi-2a molecules are targeted to thetrans-Golgi network (TGN) during the endocytosis. In the TGN, NaPi-2a interacts with PIST (PDZ-domain protein interacting specifically with TC10), a TGN-resident PDZ-domain-containing protein. The extension of the interaction is proportional to the expression of NaPi-2a in the TGN, and, consistent with that, it is increased with a high Pi diet. When overexpressed in opossum kidney (OK) cells, PIST retains NaPi-2a in the TGN and inhibits Na-dependent Pi transport. Overexpression of PIST also prevents the adaptation of OK cells to a low Pi culture medium. Our data supports the view that NaPi-2a is subjected to retrograde trafficking from the plasma membrane to the TGN using one of the machineries involved in endosomal transport and explains the reported expression of NaPi-2a in the TGN.

Interaction of MAP17 with NHERF3/4 induces translocation of the renal Na/Pi IIa transporter to the trans-Golgi

2007 ◽  
Vol 292 (1) ◽  
pp. F230-F242 ◽  
Author(s):  
Miguel A. Lanaspa ◽  
Héctor Giral ◽  
Sophia Y. Breusegem ◽  
Nabil Halaihel ◽  
Goretti Baile ◽  
...  
Keyword(s):  
Pdz Domain ◽  
Phosphate Transport ◽  
Interacting Proteins ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Kinase C ◽  
Important Intermediate ◽  
Pdz Protein ◽  
Golgi Network ◽  
Two Hybrid

The function of the NaPiIIa renal sodium-phosphate transporter is regulated through a complex network of interacting proteins. Several PDZ domain-containing proteins interact with its COOH terminus while the small membrane protein MAP17 interacts with its NH2 end. To elucidate the function of MAP17, we identified its interacting proteins using both bacterial and mammalian two-hybrid systems. Several PDZ domain-containing proteins, including the four NHERF proteins, as well as NaPiIIa and NHE3, were found to bind to MAP17. The interactions of MAP17 with the NHERF proteins and with NaPiIIa were further analyzed in opossum kidney (OK) cells. Expression of MAP17 alone had no effect on the NaPiIIa apical membrane distribution, but coexpression of MAP17 and NHERF3 or NHERF4 induced internalization of NaPiIIa, MAP17, and the PDZ protein to the trans-Golgi network (TGN). This effect was not observed when MAP17 was cotransfected with NHERF1/2 proteins. Inhibition of protein kinase C (PKC) prevented expression of the three proteins in the TGN. Activation of PKC in OK cells transfected only with MAP17 induced complete degradation of MAP17 and NaPiIIa. When lysosomal degradation was prevented, both proteins accumulated in the TGN. When the dopamine D1-like receptor was activated with fenoldopam, both NaPiIIa and MAP17 also accumulated in the TGN. Finally, cotransfection of MAP17 and NHERF3 prevented the adaptive upregulation of phosphate transport activity in OK cells in response to low extracellular phosphate. Therefore, the interaction between MAP17, NHERF3/4, and NaPiIIa in the TGN could be an important intermediate or alternate path in the internalization of NaPiIIa.

Resistance to parathyroid hormone-induced inhibition of inorganic phosphate transport in opossum kidney cells cultured in low inorganic phosphate medium

1992 ◽  
Vol 134 (3) ◽  
pp. 361-368 ◽  
Author(s):  
J. Caverzasio ◽  
J.-P. Bonjour
Keyword(s):  
Inorganic Phosphate ◽  
Kidney Cells ◽  
Camp Production ◽  
Cellular Resistance ◽  
Opossum Kidney ◽  
Pi Transport ◽  
Camp Generation ◽  
Opossum Kidney Cells ◽  
Cells Cultured ◽  
In Cells

ABSTRACT Renal resistance to the phosphaturic action of parathyroid hormone (PTH) is observed during dietary deprivation of inorganic phosphate (Pi) in vivo. In the present work, the influence of short (3 h)- or long (72 h)-term deprivation of Pi on the effect of bovine PTH (bPTH(1–34)) on both Na-dependent Pi transport and cyclic AMP(cAMP) production was examined in cultured opossum kidney epithelium. Na-dependent Pi transport increased by 100% in cells exposed to low Pi medium containing no Pi (LPM) for 3 h, as compared with transport in high Pi medium containing 2 mmolPi/l (HPM). In response to a submaximal dose (1 nmol/l) of bPTH(1–34), Na-dependent Pi transport was similarly inhibited by about 40% in LPM and HPM. This inhibition was preceded by increased cAMP production which was identical in LPM and HPM. In opossum kidney cells exposed for 72 h to LPM, Na-dependent Pi transport was also increased by 100% compared with that in HPM. However, bPTH(1–34) added at 1 nmol/l did not induce any significant change in Na-dependent Pi transport or cAMP. Stimulation of cAMP could only be elicited at bPTH(1–34) concentrations higher than 1 nmol/l. Such a reduced cAMP response was also observed with forskolin in cells incubated for 72 h in LPM. The cellular resistance to the generation of cAMP was associated with a significantly lower level of ATP in cells cultured for 72 h in LPM compared with ATP levels in HPM. There was a good relationship between cellular ATP level and expression of the inhibitory effect of bPTH on Pi transport in culture media containing various concentrations of Pi. In conclusion, long-term Pi depletion in opossum kidney cells leads to cellular resistance to PTH-induced cAMP generation and inhibition of Pi transport. The lower level of cAMP in Pi-depleted cells is probably due to insufficient substrate for cAMP generation. It is likely that other ATP-dependent processes involved in cellular Pi transport regulation might also be affected in the severely Pi-depleted cells and contribute to the resistance of PTH action on Pi transport inhibition. Journal of Endocrinology (1992) 134, 361–368

Cell-matrix interactions modulate transepithelial phosphate transport in Pi-deprived OK cells

2007 ◽  
Vol 293 (4) ◽  
pp. C1272-C1277 ◽  
Author(s):  
Mario Barac-Nieto ◽  
Edward J. Weinman ◽  
Adrian Spitzer
Keyword(s):  
Phosphate Transport ◽  
Proximal Tubules ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Cell Matrix ◽  
Matrix Interactions ◽  
Enhanced Expression ◽  
Altered Cell ◽  
Cell Matrix Interactions ◽  
Induced Changes

In opossum kidney (OK) cells as well as in kidney proximal tubules, Pi depletion increases apical (A) and basolateral (B) Na+-dependent Pi cell influxes. In OK cells' monolayers in contrast to proximal tubules, there is no increase in transepithelial Pi transport. This limitation may be due to altered cell-matrix interactions. A and B cell 32Pi uptakes and transepithelial 32Pi and [14C]mannitol fluxes were measured in OK cells grown on uncoated or on Matrigel-coated filter inserts. Cells were exposed overnight to solution of either low (0.25 mM) or high (2.5 mM) Pi. When grown on Matrigel, immunofluorescence of apical NaPi4 (an isoform of the sodium-phosphate cotransporter) transporters increased and A and B 32Pi uptakes into Pi depleted cells were five and threefold higher than in Pi replete cells ( P < 0.001). Pi deprivation resulted in larger increase in A to B (4.6×, P < 0.001) than in B to A (3.5×, P < 0.001) Pi flux and net Pi transport from A to B increased 10-fold ( P < 0.001). With Pi depletion increases in B to A (3.4×) and A to B (3.3×) paracellular [14C]mannitol fluxes were similar, and its net flux was opposite to that of Pi. In cells grown on uncoated filters, transepithelial and paracellular unidirectional and net Pi fluxes decreased or did not change with Pi depletion, despite twofold increases in apical and basolateral Pi cell influxes. In summary, Matrigel-OK cell interactions, particularly in Pi-depleted cells, led to enhanced expression of apical NaPi4 transporters resulting in higher Pi transport rates across cell boundaries; apical Pi readily entered the transcellular transport pool and paracellular fluxes were smaller fractions of transepithelial Pi fluxes. These Matrigel-induced changes led to an increase in net transepithelial apical to basolateral Pi transport.

scholarly journals Coexpression of MAST205 inhibits the activity of Na+/H+ exchanger NHE3

2006 ◽  
Vol 290 (2) ◽  
pp. F428-F437 ◽  
Author(s):  
Dongsheng Wang ◽  
Hye Jeong Lee ◽  
Deborah S. Cooper ◽  
Ludmila Cebotaro ◽  
Paul D. Walden ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Pdz Domain ◽  
Kinase Domain ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Apical Region ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Immunohistochemical Studies

Recent studies have shown that accessory proteins that interact with the apical Na+/H+ exchanger NHE3 are a vital part of the dynamic nature of the Na+/H+ exchanger regulation. We have identified MAST205, a microtubule-associated serine/threonine kinase with a molecular mass of 205 kDa that interacts with NHE3. MAST205 contains a S/T kinase domain and a PDZ domain that mediates interaction with NHE3. Northern blot analysis showed that MAST205 is highly expressed in human and rat kidney. Expression in opossum kidney (OK) cells showed that MAST205 is predominantly expressed in the apical membrane of the cells. Immunohistochemical studies demonstrated the presence of MAST205 at the apical region of the renal proximal tubules. Heterologous expression of MAST205 in OK cells inhibited endogenous NHE3 activity, and this inhibition required the presence of the kinase domain of MAST205, since deletion of the kinase domain or a dominant-negative mutant of MAST205 did not affect the activity of NHE3. Consistent with these results, we found that MAST205 phosphorylated NHE3 under in vitro conditions. However, overexpression of MAST205 did not affect expression of NHE3 proteins, suggesting that the effect of MAST205 was not mediated by a decrease in NHE3 expression. These findings suggest that MAST205 regulates NHE3 activity and, although the precise mechanism is yet to be determined, MAST205 appears to inhibit NHE3 activity through a phosphorylation-dependent mechanism.

Expression of the angiotensinogen gene is synergistically stimulated by 8-BrcAMP and Dex in opossum kidney cells

1995 ◽  
Vol 268 (1) ◽  
pp. R105-R111 ◽  
Author(s):  
M. Ming ◽  
T. T. Wang ◽  
S. Lachance ◽  
A. Delalandre ◽  
S. Carriere ◽  
...  
Keyword(s):  
Fusion Gene ◽  
Dependent Manner ◽  
Fusion Genes ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Angiotensinogen Gene ◽  
Opossum Kidney Cells ◽  
Dependent Protein ◽  
Flanking Region ◽  
Responsive Element

We transiently transfected fusion genes with the 5'-flanking region of the angiotensinogen gene linked to a bacterial chloramphenicol acetyltransferase (CAT) coding sequence as a reporter into opossum kidney (OK) cells. The addition of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) (10(-3)-10(-7) M) or forskolin (10(-9)-10(-5) M) stimulated the expression of the plasmid pOCAT [angiotensinogen nucleotide (N) -1498/+18] fusion gene in OK cells in a dose-dependent manner. The addition of dexamethasone (Dex) (10(-6) M) further enhanced the stimulatory effect of 8-BrcAMP or forskolin, whereas the addition of (R)-p-adenosine 3',5'-cyclic monophosphorothioate [(Rp)-cAMP[S], an inhibitor of cAMP-dependent protein kinase A, I and II] blocked the stimulatory effect of 8-BrcAMP. Furthermore, the addition of 8-BrcAMP (10(-3) M) or Dex (10(-6) M) or a combination of both stimulated the expression of pOCAT (angiotensinogen N -1138/+18), pOCAT (angiotensinogen N -960/+18), pOCAT (angiotensinogen N -814/+18), and pOCAT (angiotensinogen N -688/+18), but had no effect on the expression of pOCAT (angiotensinogen N -280/+18), pOCAT (angiotensinogen N -198/+18), pOCAT (angiotensinogen N -110/+18), pOCAT (angiotensinogen N -53/+18), and pOCAT (angiotensinogen N -35/+18). To further localize the putative cAMP-responsive element (CRE) in the angiotensinogen gene, we constructed fusion genes by inserting the DNA fragments angiotensinogen N -814 to N -689, angiotensinogen N -814 to N -761, and angiotensinogen N -760 to N -689 of the 5'-flanking region of the angiotensinogen gene upstream of the thymidine kinase (TK) promoter fused to a CAT gene and introduced them into OK cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Prior heat stress enhances survival of renal epithelial cells after ATP depletion

1996 ◽  
Vol 270 (6) ◽  
pp. F1057-F1065 ◽  
Author(s):  
Y. H. Wang ◽  
S. C. Borkan
Keyword(s):  
Heat Stress ◽  
Epithelial Cells ◽  
Recovery Period ◽  
Atp Depletion ◽  
Protective Effects ◽  
Atp Content ◽  
Renal Epithelial Cells ◽  
Opossum Kidney ◽  
Ok Cells

The 72-kDa heat stress protein (HSP-72) is an inducible cytoprotectant protein. Although transient renal ischemia in vivo induces HSP-72, it is not known whether prior heat stress protects renal epithelial cells from injury mediated by ATP depletion. To evaluate this hypothesis, opossum kidney (OK) cells were exposed to sodium cyanide and 2-deoxy-D-glucose in the absence of medium glucose, a maneuver that reduced cell ATP content to < 10% of the control value within 10 min and decreased cell survival. One day after 2 h of ATP depletion, OK cells previously exposed to heat stress (to induce accumulation of HSP-72) exhibited marked improvement in survival (a > 4-fold increase in total DNA), less uptake of vital dye, and less release of lactate dehydrogenase (LDH) than cells subjected to ATP depletion alone (23.0 +/- 1.6 vs. 34.1 +/- 1.2% of total LDH, respectively). Enhanced clonogenicity post-heat stress was completely prevented by cycloheximide and positively correlated with the steady-state content of HSP-72. In the recovery period after ATP depletion, cell ATP content, maximum mitochondrial ATP production rate, and total LDH activity were all significantly higher in cells with abundant HSP-72. Although the protective effects associated with heat stress are likely to be multifactoral, preserved cell metabolism and higher ATP content could enhance cellular repair processes after ATP depletion.

Intracellular compartmentation of organic anions within renal cells

1993 ◽  
Vol 264 (5) ◽  
pp. R882-R890 ◽  
Author(s):  
D. S. Miller ◽  
D. E. Stewart ◽  
J. B. Pritchard
Keyword(s):  
Organic Anion ◽  
Basolateral Membrane ◽  
Organic Anions ◽  
Epifluorescence Microscopy ◽  
Renal Cells ◽  
Bladder Epithelium ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Intracellular Compartments ◽  
Energy Dependent

Epifluorescence microscopy and video-image analysis were used to measure the distribution of the monovalent organic anion fluorescein (FL) within the cells of three organic anion-secreting renal epithelia: crab urinary bladder (a proximal tubule analogue), opossum kidney (OK) cells in culture, and intact teleost proximal tubules. In all three preparations the intracellular FL distribution was nonuniform. Two distinct intracellular compartments were detected, one being diffuse and cytoplasmic and the other punctate. With low FL concentrations in the medium (1 microM and below) dye accumulation in the punctate compartment exceeded that of the cytoplasm. In crab bladder epithelium FL uptake into both compartments was inhibited by external probenecid, p-aminohippurate (PAH), and LiCl and stimulated by 10-50 microM external glutarate, suggesting that the punctate compartment loaded by a two-step mechanism: transport into the cytoplasm at the basolateral membrane, followed by accumulation at specific intracellular sites. Experiments in which FL was microinjected into OK cells directly demonstrated movement of FL from the cytoplasmic to the punctate compartment. Accumulation in the latter was specific, i.e., inhibitable by coinjected PAH and probenecid, and energy dependent. Together, these findings indicate that during secretion organic anions are sequestered within renal cells. The role of sequestration in overall transport remains to be determined.

A dual mechanism for regulation of kidney phosphate transport by parathyroid hormone

1987 ◽  
Vol 253 (2) ◽  
pp. E221-E227 ◽  
Author(s):  
J. A. Cole ◽  
S. L. Eber ◽  
R. E. Poelling ◽  
P. K. Thorne ◽  
L. R. Forte
Keyword(s):  
Protein Kinase C ◽  
Parathyroid Hormone ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Rank Order ◽  
Phosphate Transport ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Kinase C ◽  
Camp Formation

Regulation of phosphate transport by parathyroid hormone (PTH) was investigated in continuous lines of kidney cells. Phosphate transport was reduced by PTH-(1-34) at physiological concentrations (EC50 5 X 10(-11) M), whereas much higher concentrations were required to stimulate cAMP formation (EC50 1 X 10(-8) M) in opossum kidney (OK) cells. The PTH analogue [Nle]PTH-(3-34) also inhibited phosphate transport but did not enhance cAMP formation. Instead, [Nle]PTH-(3-34) was a competitive antagonist of PTH-(1-34) at cyclase-coupled receptors. PTH-(7-34) had no effect on phosphate transport or cAMP formation. Phorbol esters or mezerein were potent inhibitors of phosphate transport but did not affect cAMP synthesis. Their potencies paralleled the rank-order potency of these agents as activators of protein kinase c in other systems. Maximally effective concentrations of PTH-(1-34) and mezerein did not produce additive inhibition of phosphate transport in OK cells. Phorbol esters stimulated phosphate transport in JTC-12 cells, but PTH-(1-34) had no effect. We concluded that PTH regulates OK cell phosphate transport by interacting with two classes of receptors, and transmembrane-signaling mechanisms. Physiological levels of PTH-(1-34) may regulate phosphate transport by activation of protein kinase c, whereas higher concentrations appear to activate adenylate cyclase.

Production and control of extracellular enzymes in Micrococcus sodonensis

1974 ◽  
Vol 20 (1) ◽  
pp. 81-90 ◽  
Author(s):  
Cecily Mills ◽  
J. N. Campbell
Keyword(s):  
Alkaline Phosphatase ◽  
Inorganic Phosphate ◽  
Culture Medium ◽  
Extracellular Enzymes ◽  
Synthetic Medium ◽  
Culture Conditions ◽  
The Other ◽  
Organic Nutrients ◽  
And Control ◽  
Logarithmic Growth

Micrococcus sodonensis has been shown to produce several extracellular enzymes: an alkaline phosphatase, at least two forms of phosphodiesterase, a 5′-nucleotidase, and an alkaline proteinase. The quantitative release of these enzymes into the culture medium during logarithmic growth under all the various culture conditions tested indicates that these enzymes are truly extracellular in nature. Inorganic phosphate repressed the production of the alkaline phosphatase in synthetic as well as in complex media, whereas, the repression of the production of active diesterase and 5′-nucleotidase by inorganic phosphate was partly reversed by the addition of supplemental organic nutrients to the culture medium. Proteinase production was independent of the culture conditions used. A mutant strain of M. sodonensis with an altered production of diesterase was obtained; the other extracellular enzymes were unaffected. These results suggest that the extracellular enzymes of M. sodonensis are not produced in a pleiotropic fashion since the level of one of the enzymes can be changed without affecting a corresponding change in the levels of the other enzymes. An extracellular high molecular weight carbohydrate fraction was shown to be produced by M. sodonensis in synthetic medium. The fraction was also shown to contain glycoprotein.

Differences between OK and LLC-PK1 cells: cystine handling

1991 ◽  
Vol 261 (1) ◽  
pp. C8-C16 ◽  
Author(s):  
B. States ◽  
D. Harris ◽  
S. Segal
Keyword(s):  
Cell Lines ◽  
Apical Membrane ◽  
Reduced Glutathione ◽  
Basolateral Membrane ◽  
Growth Period ◽  
Transport Systems ◽  
Amino Acid Transport System ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Dibasic Amino Acid

Cultured opossum kidney (OK) and porcine kidney (LLC-PK1) cells were compared for biochemical characteristics and cystine transport systems. The cell lines differ in amount of protein per cell, with OK cells having approximately one-half the amount found in LLC-PK1. Both cell lines contain 19 micrograms DNA/10(6) cells. As cells reach confluence, cystine uptake increases in OK and decreases in LLC-PK1 cells. Throughout the growth period, only lysine inhibits cystine uptake in OK, whereas glutamate is the inhibitor in LLC-PK1. The predominant site of cystine transport in OK cells is across the apical membrane, and the basolateral membrane is the corresponding site of transport in LLC-PK1 cells. Although the intracellular reduced glutathione pool is the same, the cysteine pool in OK cells is approximately one-fourth that found in LLC-PK1 cells. The ability of OK cells to reflect the shared cystine-dibasic amino acid transport system and LLC-PK1 to exhibit the cystine-glutamate antiporter system makes available two models for investigation of the development and structure of cystine transport systems.

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