Citrate transport in proximal cell line

1992 ◽  
Vol 263 (1) ◽  
pp. C220-C225 ◽  
Author(s):  
D. Law ◽  
K. S. Hering-Smith ◽  
L. L. Hamm
Keyword(s):  
Proximal Tubule ◽  
Cell Line ◽  
Transport Process ◽  
Competitive Inhibition ◽  
Basolateral Membrane ◽  
Opossum Kidney ◽  
Cell Monolayers ◽  
Citrate Transport ◽  
Sodium Dependent ◽  
A Cell

Citrate uptake into kidney proximal tubules occurs via an apical dicarboxylate transporter and a poorly characterized process in the basolateral membrane. We used OK cells, a cell line derived from opossum kidney, to study citrate transport in proximal tubule-like cells. Citrate uptake into cell monolayers was studied using [14C]citrate with [3H]mannitol as a volume marker. Citrate uptake into these cells was sodium dependent and saturable with increasing concentrations of citrate. In contrast to previous models, citrate transport was altered minimally by changes in pH from 6.2 to 7.0 and increased at pH 7.4 to 7.8. A variety of di- and tricarboxylates were tested for interaction with citrate transport. The dicarboxylates succinate, malate, and oxaloacetate at 1 mM concentration inhibited citrate uptake minimally (uptake at least 80% of control); one dicarboxylate, alpha-ketoglutarate, did inhibit citrate uptake significantly. In contrast, the tricarboxylates isocitrate and tricarballylate inhibited citrate uptake significantly, indicating probable competitive inhibition with the transport process. These characteristics are distinctly different from those of the apical membrane dicarboxylate transporter. 1,2,3-Benzenetricarboxylic acid, an inhibitor of the mitochondrial tricarboxylate transporter, did not alter citrate uptake. In conclusion, the OK proximal cell line exhibits a novel citrate transport process compared with the apical transport of citrate described in most proximal systems. This transport process probably involves the trivalent species of citrate in contrast to the usual predominant transport of divalent citrate. This transport process may represent a process similar to that in the basolateral membrane of the proximal tubule.

HeLa cells express cAMP-inhibitable sodium-dependent phosphate uptake

1990 ◽  
Vol 258 (2) ◽  
pp. F433-F437 ◽  
Author(s):  
J. R. Raymond ◽  
J. P. Middleton ◽  
V. W. Dennis
Keyword(s):  
Cell Line ◽  
Hela Cells ◽  
Phosphate Uptake ◽  
Opossum Kidney ◽  
Opossum Kidney Cells ◽  
Sodium Dependent ◽  
A Cell ◽  
Dose Dependent Decrease ◽  
Camp Levels ◽  
Dose Dependent

Receptor-mediated regulation of the sodium-phosphate symporter, and hence sodium-dependent phosphate uptake, typically relates to epithelial cells of renal origin. In this study we have characterized sodium-dependent phosphate uptake and aspects of its receptor-mediated regulation in the HeLa cell line, a cell line derived from a human epithelioid carcinoma. Phosphate uptake (greater than 90% sodium dependent; Vmax = 4.02 +/- 0.24 nmol.mg and Km = 0.11 +/- 0.02 mM phosphate at 140 mM sodium) was kinetically similar to that observed in opossum kidney cells. Incubation with vasoactive intestinal peptide (VIP) resulted in a dose-dependent (50% maximal dose of 8.8 +/- 3.6 nM) approximately fivefold increase in basal adenosine 3',5'-cyclic monophosphate (cAMP) levels (basal = 14.6 +/- 1.7 pmol.mg protein-1.15 min-1; VIP stimulated = 72.7 +/- 13.2 pmol.mg protein-1.15 min-1), as well as a dose-dependent maximal 32.6 +/- 5.5% decrease in sodium-dependent phosphate uptake (50% maximal decrease of 46.2 +/- 21.2 nM). The VIP-induced decrease in phosphate uptake was due to decrease in maximal transport (VmaxVIP = 2.78 +/- 0.16 nmol.mg protein-1.3 min-1) and not to a change in the affinity of the transporter for phosphate (KmVIP = 0.11 +/- 0.01 mM phosphate). Preincubation of HeLa cells with forskolin and cholera toxin, which stimulate adenylate cyclase, resulted in dose-dependent decreases in sodium-dependent phosphate uptake. Incubation with 8-bromo-cAMP and dibutyryl cAMP, permeant analogues of cAMP, similarly resulted in a dose-dependent decrease in sodium-dependent phosphate uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

OKP cells express the Na-dicarboxylate cotransporter NaDC-1

2004 ◽  
Vol 287 (1) ◽  
pp. C64-C72 ◽  
Author(s):  
Seiji Aruga ◽  
Ana M. Pajor ◽  
Kiyoshi Nakamura ◽  
Liping Liu ◽  
Orson W. Moe ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Xenopus Oocytes ◽  
Fluorescent Protein ◽  
Stone Formation ◽  
Chronic Metabolic Acidosis ◽  
Opossum Kidney ◽  
Citrate Transport ◽  
Increased Risk ◽  
Green Fluorescent ◽  
Urinary Citrate

Urinary citrate concentration, a major factor in the formation of kidney stones, is primarily determined by its rate of reabsorption in the proximal tubule. Citrate reabsorption is mediated by the Na-dicarboxylate cotransporter-1 (NaDC-1). The opossum kidney (OKP) cell line possesses many characteristics of the renal proximal tubule. The OKP NaDC-1 (oNaDC-1) cDNA was cloned and encodes a 2.4-kb mRNA. When injected into Xenopus oocytes, the cotransporter is expressed and demonstrates Na-coupled citrate transport with a stoichiometry of ≥3 Na:1 citrate, specificity for di- and tricarboxylates, pH-dependent citrate transport, and pH-independent succinate transport, all characteristics of the other NaDC-1 orthologs. Chronic metabolic acidosis increases proximal tubule citrate reabsorption, leading to profound hypocitraturia and an increased risk for stone formation. Under the conditions studied, endogenous OKP NaDC-1 mRNA abundance is not regulated by changes in media pH. In OKP cells transfected with a green fluorescent protein-oNaDC-1 construct, however, media acidification increases Na-dependent citrate uptake, demonstrating posttranscriptional acid regulation of NaDC-1 activity.

Clonal sublines that are morphologically and functionally distinct from parental OK cells

1989 ◽  
Vol 256 (4) ◽  
pp. F672-F679 ◽  
Author(s):  
J. A. Cole ◽  
L. R. Forte ◽  
W. J. Krause ◽  
P. K. Thorne
Keyword(s):  
Cell Line ◽  
Phosphate Transport ◽  
Parental Line ◽  
Apical Surface ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Sodium Dependent ◽  
Clonal Lines ◽  
P Cells ◽  
Camp Formation

Three clonal subpopulations of opossum kidney (OK) cells were derived from the parental line. The distribution of apical microvilli suggested that the OK cell line was heterogeneous. The clonal OK sublines appeared homogeneous as reflected by microvilli, which were uniformly distributed on the apical surface. Parathyroid hormone (PTH), forskolin (FSK), and prostaglandin E1 (PGE1) increased adenosine 3',5'-cyclic monophosphate (cAMP) formation in OK cells and all of the clones. PTH inhibited sodium-dependent phosphate transport in parental cells and in OK/B and OK/P clones with maximal effects appearing at 4, 2, and 1 h, respectively. PTH had no effect on phosphate transport in OK/H cells. FSK inhibited phosphate transport in parental cells and OK/B and OK/P clones but was relatively ineffective in OK/H cells. PGE1 decreased phosphate transport in OK/B and OK/P cells but was ineffective in the parental line and in OK/H cells. Phorbol 12-myristate 13-acetate, a potent inhibitor of phosphate transport in the parental OK cell line, had little effect in the clonal sublines. These clonal lines have remained phenotypically stable for 10 passages and should prove useful in studying the regulation of phosphate transport by PTH as well as addressing the question of whether PTH receptor subclasses exist which couple to cAMP and/or calcium effector systems in kidney cells.

Nitric oxide activates PKCα and inhibits Na+-K+-ATPase in opossum kidney cells

1999 ◽  
Vol 277 (6) ◽  
pp. F859-F865 ◽  
Author(s):  
Mingyu Liang ◽  
Franklyn G. Knox
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Proximal Tubule ◽  
Cell Line ◽  
Soluble Guanylate Cyclase ◽  
Inhibitory Effect ◽  
Proximal Tubule Cell ◽  
Tubule Cell ◽  
Opossum Kidney ◽  
Ok Cells

Nitric oxide (NO) reduces the molecular activity of Na+-K+-ATPase in opossum kidney (OK) cells, a proximal tubule cell line. In the present study, we investigated the cellular mechanisms for the inhibitory effect of NO on Na+-K+-ATPase. Sodium nitroprusside (SNP), a NO donor, inhibited Na+-K+-ATPase in OK cells, but not in LLC-PK1cells, another proximal tubule cell line. Similarly, phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, inhibited Na+-K+-ATPase in OK, but not in LLC-PK1, cells. PKC inhibitors staurosporine or calphostin C, but not the protein kinase G inhibitor KT-5823, abolished the inhibitory effect of NO on Na+-K+-ATPase in OK cells. Immunoblotting demonstrated that treatment with NO donors caused significant translocation of PKCα from cytosolic to particulate fractions in OK, but not in LLC-PK1, cells. Furthermore, the translocation of PKCα in OK cells was attenuated by either the phospholipase C inhibitor U-73122 or the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. U-73122 also blunted the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. The phospholipase A2inhibitor AACOCF3 did not blunt the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. AACOCF3 alone, however, also decreased Na+-K+-ATPase activity in OK cells. In conclusion, our results demonstrate that NO activates PKCα in OK, but not in LLC-PK1, cells. The activation of PKCα in OK cells by NO is associated with inhibition of Na+-K+-ATPase.

scholarly journals Endocytosis of the somatostatin analogue, octreotide, by the proximal tubule-derived opossum kidney (OK) cell line

2005 ◽  
Vol 67 (3) ◽  
pp. 969-976 ◽  
Author(s):  
Raffaella Barone ◽  
Patrick Van Der Smissen ◽  
Olivier Devuyst ◽  
Viviane Beaujean ◽  
Stanislas Pauwels ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Cell Line ◽  
Somatostatin Analogue ◽  
Opossum Kidney ◽  
Analogue Octreotide

scholarly journals Characterization of the regulation of renal Na+/H+ exchanger NHE3 by insulin

2007 ◽  
Vol 292 (2) ◽  
pp. F577-F585 ◽  
Author(s):  
Daniel G. Fuster ◽  
I. Alexandru Bobulescu ◽  
Jianning Zhang ◽  
James Wade ◽  
Orson W. Moe
Keyword(s):  
Proximal Tubule ◽  
Insulin Receptors ◽  
Acute Effect ◽  
Dominant Negative ◽  
Insulin Effect ◽  
Opossum Kidney ◽  
Homologous Protein ◽  
Culture Model ◽  
Activation Mechanisms ◽  
A Cell

Insulin receptors are widely distributed in the kidney and affect multiple aspects of renal function. In the proximal tubule, insulin regulates volume and acid-base regulation through stimulation of the Na+/H+ exchanger NHE3. This paper characterizes the signaling pathway by which insulin stimulates NHE3 in a cell culture model [opossum kidney (OK) cell]. Insulin has two distinct phases of action on NHE3. Chronic insulin (24 h) activates NHE3 through the classic phosphatidylinositol 3-kinase-serum- and glucocorticoid-dependent kinase 1 (PI3K-SGK1) pathway as insulin stimulates SGK1 phosphorylation and the insulin effect can be blocked by the PI3K inhibitor wortmannin or a dominant-negative SGK1. We showed that SGK1 transcript and protein are expressed in rat proximal tubule and OK cells. We previously showed that glucocorticoids augment the effect of insulin on NHE3 (Klisic J, Hu MC, Nief V, Reyes L, Fuster D, Moe OW, Ambuhl PM. Am J Physiol Renal Physiol 283: F532–F539, 2002). Part of this can be mediated via induction of SGK1 by glucocorticoids, and indeed the insulin effect on NHE3 can also be amplified by overexpression of SGK1. We next addressed the acute effect of insulin (1–2 h) on NHE3 by systematically examining the candidate signaling cascades and activation mechanisms of NHE3. We ruled out the PI3K-SGK1-Akt and TC10 pathways, increased surface NHE3, NHE3 phosphorylation, NHE3 association with calcineurin homologous protein 1 or megalin as mechanisms of acute activation of NHE3 by insulin. In summary, insulin stimulates NHE3 acutely via yet undefined pathways and mechanisms. The chronic effect of insulin is mediated by the classic PI3K-SGK1 route.

scholarly journals Calcium sensitivity of dicarboxylate transport in cultured proximal tubule cells

2011 ◽  
Vol 300 (2) ◽  
pp. F425-F432 ◽  
Author(s):  
Kathleen S. Hering-Smith ◽  
Faith R. Schiro ◽  
Ana M. Pajor ◽  
L. Lee Hamm
Keyword(s):  
Proximal Tubule ◽  
Transport Process ◽  
Urinary Calcium ◽  
Extracellular Calcium ◽  
Calcium Excretion ◽  
Proximal Tubule Cells ◽  
Citrate Transport ◽  
Dicarboxylate Transport ◽  
Tubule Cells ◽  
Urinary Citrate

Urinary citrate is an important inhibitor of calcium nephrolithiasis and is primarily determined by proximal tubule reabsorption. The major transporter to reabsorb citrate is Na+-dicarboxylate cotransporter (NaDC1), which transports dicarboxylates, including the divalent form of citrate. We previously found that opossum kidney (OK) proximal tubule cells variably express either divalent or trivalent citrate transport, depending on extracellular calcium. The present studies were performed to delineate the mechanism of the effect of calcium on citrate and succinate transport in these cells. Transport was measured using isotope uptake assays. In some studies, NaDC1 transport was studied in Xenopus oocytes, expressing either the rabbit or opossum ortholog. In the OK cell culture model, lowering extracellular calcium increased both citrate and succinate transport by more than twofold; the effect was specific in that glucose transport was not altered. Citrate and succinate were found to reciprocally inhibit transport at low extracellular calcium (<60 μM), but not at normal calcium (1.2 mM); this mutual inhibition is consistent with dicarboxylate transport. The inhibition varied progressively at intermediate levels of extracellular calcium. In addition to changing the relative magnitude and interaction of citrate and succinate transport, decreasing calcium also increased the affinity of the transport process for various other dicarboxylates. Also, the affinity for succinate, at low concentrations of substrate, was increased by calcium removal. In contrast, in oocytes expressing NaDC1, calcium did not have a similar effect on transport, indicating that NaDC1 could not likely account for the findings in OK cells. In summary, extracellular calcium regulates constitutive citrate and succinate transport in OK proximal tubule cells, probably via a novel transport process that is not NaDC1. The calcium effect on citrate transport parallels in vivo studies that demonstrate the regulation of urinary citrate excretion with urinary calcium excretion, a process that may be important in decreasing urinary calcium stone formation.

Parathyroid hormone inhibition of Na+-H+ antiporter activity in a cultured renal cell line

1986 ◽  
Vol 250 (2) ◽  
pp. F217-F225 ◽  
Author(s):  
A. S. Pollock ◽  
D. G. Warnock ◽  
G. J. Strewler
Keyword(s):  
Steady State ◽  
Parathyroid Hormone ◽  
Proximal Tubule ◽  
Cell Line ◽  
Intracellular Ph ◽  
Proximal Tubule Cells ◽  
Opossum Kidney ◽  
Renal Cell Line ◽  
Monensin A ◽  
22Na Uptake

The renal effects of parathyroid hormone (PTH) include a decreased rate of acidification by the proximal tubule. To determine whether this effect represented a PTH action on the Na+-H+ antiporter, we investigated the effect of PTH on the established opossum kidney (OK) cell line. This cell line retains several features characteristic of proximal tubule cells, including an amiloride-sensitive Na+-H+ antiporter and high-affinity PTH receptors with a coupled cAMP response. We measured steady-state intracellular pH and amiloride-sensitive 22Na+ uptake as a reflection of the activity of the Na+-H+ antiporter. Under bicarbonate and CO2-free conditions, the steady-state intracellular pH of OK cell cultures was modified by altering the rate of Na+-H+ exchange. When Na+-H+ exchange was inhibited by amiloride, intracellular pH fell. Conversely, augmenting antiporter activity by addition of monensin, a Na+-H+ exchange ionophore, raised intracellular pH. PTH (2.5 X 10(-8) M) lowered intracellular pH by up to 0.17 pH units, and half of the maximum PTH effect was present at a concentration of 10(-12) M. This effect was not seen in the presence of amiloride or in the absence of sodium, suggesting that a functional Na+-H+ antiporter is necessary for its expression. The decrease in intracellular pH was reproduced by forskolin and 8-bromo-cAMP, suggesting that this is a cAMP-mediated effect. PTH, forskolin, and 8-bromo-cAMP also decreased the amiloride-sensitive component of 22Na+ uptake in OK cells by up to 64%, whereas the amiloride-insensitive component was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasopressin and cAMP stimulate electrogenic chloride secretion in an IMCD cell line

1995 ◽  
Vol 268 (5) ◽  
pp. F854-F861 ◽  
Author(s):  
N. L. Kizer ◽  
D. Vandorpe ◽  
B. Lewis ◽  
B. Bunting ◽  
J. Russell ◽  
...  
Keyword(s):  
Cell Line ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Second Messenger ◽  
Chloride Secretion ◽  
Disulfonic Acid ◽  
Cellular Mechanisms ◽  
Cl Secretion ◽  
Cl Channels ◽  
A Cell

Previously, we demonstrated that the mIMCD-K2 cell line, derived from the inner medullary collecting duct (IMCD) of a transgenic mouse, secretes Cl- by an electrogenic mechanism [N. L. Kizer, B. Lewis, and B. A. Stanton, Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F347-F355, 1995]. The objective of the present study was to characterize the cellular mechanisms of electrogenic Cl- secretion (IscCl) and to determine whether arginine vasopressin (AVP) and adenosine 3',5'-cyclic monophosphate (cAMP) stimulate IscCl. To this end, we measured IscCl across monolayers of mIMCD-K2 cells mounted in Ussing-type chambers. AVP increased IscCl with a Michaelis constant (Km) of 2.1 +/- 0.7 x 10(-12) M. 1-Desamino-8-D-AVP, a specific V2 receptor agonist, increased IscCl from 3.3 +/- 0.4 to 17.4 +/- 1.3 microA/cm2, 8-(4-Chlorophenylthio)-cAMP, a cell-permanent analogue of cAMP, a second messenger of AVP, increased IscCl from 1.4 +/- 0.3 to 15.2 +/- 1.2 microA/cm2. Furosemide and bumetanide, inhibitors of Na(+)-2Cl(-)-K+ cotransport, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of Cl-/HCO3- exchange, reduced IscCl when added to the basolateral solution. Our data suggest that AVP, via V2 receptors, and the second messenger cAMP stimulate IscCl and that Cl- secretion by mIMCD-K2 cells involves uptake of Cl- across the basolateral membrane by Na(+)-2Cl(-)-K+ cotransport and Cl-/HCO3- exchange and diffusion out of the cells across the apical membrane by cystic fibrosis transmembrane conductance regulator Cl- channels.

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