scholarly journals Dynamics of PTH-induced disassembly of Npt2a/NHERF-1 complexes in living OK cells

2011 ◽  
Vol 300 (1) ◽  
pp. F231-F235 ◽  
Author(s):  
Edward J. Weinman ◽  
Deborah Steplock ◽  
Shirish Shenolikar ◽  
Thomas A. Blanpied
Keyword(s):  
Proximal Tubule ◽  
Time Course ◽  
Apical Surface ◽  
Adapter Protein ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Sequence Of Events ◽  
Kinase C ◽  
Sodium Hydrogen Exchanger ◽  
Rapid Activation

Parathyroid hormone (PTH) inhibits the reabsorption of phosphate in the renal proximal tubule by disrupting the binding of the sodium-dependent phosphate transporter 2A (Npt2a) to the adapter protein sodium-hydrogen exchanger regulatory factor-1 (NHERF-1), a process initiated by activation of protein kinase C (PKC). To gain additional insights into the dynamic sequence of events, the time course of these responses was studied in living opossum kidney (OK) cells. Using a FRET-based biosensor, we found that PTH activated intracellular PKC within seconds to minutes. In cells expressing GFP-Npt2a and mCherry-NHERF, PTH did not affect the relative abundance of NHERF-1 but there was a significant and time-dependent decrease in the Npt2a/NHERF-1 ratio. The half-time to maximal dissociation was 15 to 20 min. By contrast, PTH had no effect on the fluorescence ratio for GFP-ezrin compared with mCherry-NHERF-1 at the apical surface. These experiments establish that PTH treatment of proximal tubule OK cells leads to rapid activation of PKC with the subsequent dissociation of Npt2a/NHERF-1 complexes. The association of NHERF-1 with Ezrin and their localization at the apical membrane, however, was unperturbed by PTH, thereby enabling the rapid recruitment and membrane reinsertion of Npt2a and other NHERF-1 targets on termination of the hormone response.

PKC-α-mediated remodeling of the actin cytoskeleton is involved in constitutive albumin uptake by proximal tubule cells

2005 ◽  
Vol 288 (6) ◽  
pp. F1227-F1235 ◽  
Author(s):  
Deanne H. Hryciw ◽  
Carol A. Pollock ◽  
Philip Poronnik
Keyword(s):  
Proximal Tubule ◽  
Fluorescent Protein ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cell ◽  
Apical Surface ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Albumin Endocytosis ◽  
In Cells

One key role of the renal proximal tubule is the reabsorption of proteins from the glomerular filtrate by constitutive receptor-mediated endocytosis. In the opossum kidney (OK) renal proximal tubule cell line, inhibition of protein kinase C (PKC) reduces albumin uptake, although the isoforms involved and mechanisms by which this occurs have not been identified. We used pharmacological and molecular approaches to investigate the role of PKC-α in albumin endocytosis. We found that albumin uptake in OK cells was inhibited by the pan-PKC blocker bisindolylmaleimide-1 and the isoform-specific PKC blockers Gö-6976 and 2′,3,3′,4,4′-hexahydroxy-1,1′-biphenyl-6,6′-dimethanol dimethyl ether, indicating a role for PKC-α. Overexpression of a kinase deficient PKC-α(K368R) but not wild-type PKC-α significantly reduced albumin endocytosis. Western blot analysis of fractionated cells showed an increased association of PKC-α-green fluorescent protein with the membrane fraction within 10–20 min of exposure to albumin. We used phalloidin to demonstrate that albumin induces the formation of clusters of actin at the apical surface of OK cells and that these clusters correspond to the location of albumin uptake. These clusters were not present in cells grown in the absence of albumin. In cells treated either with PKC inhibitors or overexpressing kinase-deficient PKC-α(K368R) this actin cluster formation was significantly reduced. This study identifies a role for PKC-α in constitutive albumin uptake in OK cells by mediating assembly of actin microfilaments at the apical membrane.

Na+/H+ exchanger 3 is in large complexes in the center of the apical surface of proximal tubule-derived OK cells

2002 ◽  
Vol 283 (3) ◽  
pp. C927-C940 ◽  
Author(s):  
S. Akhter ◽  
O. Kovbasnjuk ◽  
X. Li ◽  
M. Cavet ◽  
J. Noel ◽  
...  
Keyword(s):  
Cell Surface ◽  
Proximal Tubule ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Insoluble Fraction ◽  
Proximal Tubule Cell ◽  
Apical Surface ◽  
Sucrose Density Gradient Centrifugation ◽  
Opossum Kidney ◽  
Ok Cells

Cell biological approaches were used to examine the location and function of the brush border (BB) Na+/H+ exchanger NHE3 in the opossum kidney (OK) polarized renal proximal tubule cell line. NHE3 epitope tagged with the vesicular stomatitis virus glycoprotein epitope (NHE3V) was stably expressed and called OK-E3V cells. On the basis of cell surface biotinylation studies, these cells had 10–15% of total NHE3 on the BB. Intracellular NHE3V largely colocalized with Rab11 and to a lesser extent with EEA1. The BB location of NHE3V was examined by confocal microscopy relative to the lectins wheat germ aggluttinin (WGA) and phytohemagluttin E (PHA-E), as well as the B subunit of cholera toxin (CTB). The cells were pyramidal, and NHE3 was located in microvilli in the center of the apical surface. In contrast, PHA-E, WGA, and CTB were diffusely distributed on the BB. Detergent extraction showed that total NHE3V was largely soluble in Triton X-100, whereas virtually all surface NHE3V was insoluble. Sucrose density gradient centrifugation demonstrated that total NHE3V migrated at the same size as ∼400- and ∼900-kDa standards, whereas surface NHE3V was enriched in the ∼900-kDa form. Under basal conditions, NHE3 cycled between the cell surface and the recycling pathway through a phosphatidylinositol (PI) 3-kinase-dependent mechanism. Measurements of surface and intracellular pH were obtained by using FITC-WGA. Internalization of FITC-WGA occurred largely into the juxtanuclear compartment that contained Rab11 and NHE3V. pH values on the apical surface and in endosomes in the presence of the NHE3 blocker, S3226, were elevated, showing that NHE3 functioned to acidify both compartments. In conclusion, NHE3V in OK cells exists in distinct domains both in the center of the apical surface and in a juxtanuclear compartment. In the BB fraction, NHE3 is largely in the detergent-insoluble fraction in lipid rafts and/or in large heterogenous complexes ranging from ∼400 to ∼900 kDa.

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.

cAMP-dependent and -independent downregulation of type II Na-Pi cotransporters by PTH

1999 ◽  
Vol 276 (5) ◽  
pp. F720-F725 ◽  
Author(s):  
Markus F. Pfister ◽  
Jutka Forgo ◽  
Urs Ziegler ◽  
Jürg Biber ◽  
Heini Murer
Keyword(s):  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Phospholipase C ◽  
Type Ii ◽  
Peptide Fragments ◽  
Regulatory Pathways ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Kinase C ◽  
Proximal Tubular

Parathyroid hormone (PTH) leads to the inhibition of Na-Pi cotransport activity and to the downregulation of the number of type II Na-Pi cotransporters in proximal tubules, as well as in opossum kidney (OK) cells. PTH is known also to lead to an activation of adenylate cyclase and phospholipase C in proximal tubular preparations, as well as in OK cells. In the present study, we investigated the involvement of these two regulatory pathways in OK cells in the PTH-dependent downregulation of the number of type II Na-Pi cotransporters. We have addressed this issue by using pharmacological activators of protein kinase A (PKA) and protein kinase C (PKC), i.e., 8-bromo-cAMP (8-BrcAMP) and β-12- O-tetradecanoylphorbol 13-acetate (β-TPA), respectively, as well as by the use of synthetic peptide fragments of PTH that activate adenylate cyclase and/or phospholipase C, i.e., PTH-(1–34) and PTH-(3–34), respectively. Our results show that PTH signal transduction via cAMP-dependent, as well as cAMP-independent, pathways leads to a membrane retrieval and degradation of type II Na-Pi cotransporters and, thereby, to the inhibition of Na-Picotransport activity. Thereby, the cAMP-independent regulatory pathway leads only to partial effects (∼50%).

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.

Role of cAMP-PKA-PLC signaling cascade on dopamine-induced PKC-mediated inhibition of renal Na+-K+-ATPase activity

2002 ◽  
Vol 282 (6) ◽  
pp. F1084-F1096 ◽  
Author(s):  
Pedro Gomes ◽  
P. Soares-da-Silva
Keyword(s):  
Atpase Activity ◽  
Kinase Inhibitors ◽  
Short Circuit ◽  
Skf 38393 ◽  
Effector Proteins ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Sequence Of Events ◽  
System Activation ◽  
Stimulation Of

We studied the molecular events set into motion by stimulation of D1-like receptors downstream of Na+-K+-ATPase, while measuring apical-to-basal ouabain-sensitive, amphotericin B-induced increases in short-circuit current in opossum kidney (OK) cells. The D1-like receptor agonist SKF-38393 decreased Na+-K+-ATPase activity (IC50, 130 nM). This effect was prevented by the D1-like receptor antagonist SKF-83566, overnight cholera toxin treatment, the protein kinase A (PKA) antagonist H-89, or the PKC antagonist chelerythrine, but not the mitogen-activated PK inhibitor PD-098059 or phosphatidylinositol 3-kinase inhibitors wortmannin and LY-294002. Dibutyryl cAMP (DBcAMP) and phorbol 12,13-dibutyrate (PDBu) both effectively reduced Na+-K+-ATPase activity. PKA downregulation abolished the inhibitory effects of SKF-38393 and DBcAMP but not those of PDBu. PKC downregulation abolished inhibition by PDBu, SKF-38393, and DBcAMP. The phospholipase C (PLC) inhibitor U-73122 prevented inhibition by SKF-38393 and DBcAMP. However, DBcAMP increased PLC activity. Although OK cells express both Gsα and Gq/11α proteins, D1-like receptors are coupled to Gsα proteins only, as evidenced by studies in cells treated overnight with specific antibodies raised against Gsα and Gq/11α proteins. We conclude that PLC and Na+-K+-ATPase are effector proteins for PKA and PKC, respectively, after stimulation of D1-like receptors coupled to Gsα proteins, in a sequence of events that begins with adenylyl cyclase-PKA system activation followed by PLC-PKC system activation.

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 Regulation of Megalin Expression in Cultured Proximal Tubule Cells by Angiotensin II Type 1A Receptor- and Insulin-Mediated Signaling Cross Talk

Endocrinology ◽  
2009 ◽  
Vol 150 (2) ◽  
pp. 871-878 ◽  
Author(s):  
Michihiro Hosojima ◽  
Hiroyoshi Sato ◽  
Keiko Yamamoto ◽  
Ryohei Kaseda ◽  
Taeko Soma ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Proximal Tubule ◽  
Cross Talk ◽  
Proximal Tubule Cells ◽  
Opossum Kidney ◽  
Receptor Associated Protein ◽  
Ok Cells ◽  
Proximal Tubular ◽  
Tubule Cells ◽  
Proximal Tubular Function

Impairment of proximal tubular endocytosis of glomerular-filtered proteins including albumin results in the development of proteinuria/albuminuria in patients with chronic kidney disease. However, the mechanisms regulating the proximal tubular function are largely unknown. This study aimed to investigate the role of angiotensin II type 1A receptor (AT1AR)- and insulin-mediated signaling pathways in regulating the expression of megalin, a multiligand endocytic receptor in proximal tubule cells (PTCs). Opossum kidney PTC-derived OK cells that stably express rat AT1AR but are deficient in endogenous angiotensin II receptors (AT1AR-OK cells) were used for this study. Treatment of the cells with angiotensin II suppressed mRNA and protein expression of megalin at 3- and 24-h incubation time points, respectively. Cellular uptake and degradation of albumin and receptor-associated protein, megalin’s endocytic ligands were suppressed 24 h after angiotensin II treatment. The AT1AR-mediated decrease in megalin expression was partially prevented by ERK inhibitors. Insulin competed with the AT1AR-mediated ERK activation and decrease in megalin expression. Inhibitors of phosphatidylinositol 3-kinase (PI3K), a major component of insulin signaling, also suppressed megalin expression, and activation of the insulin receptor substrate (IRS)/PI3K system was prevented by angiotensin II. Collectively the AT1AR-mediated ERK signaling is involved in suppressing megalin expression in the OK cell line, and insulin competes with this pathway. Conversely, the insulin-IRS/PI3K signaling, with which angiotensin II competes, tends to stimulate megalin expression. In conclusion, there is AT1AR- and insulin-mediated competitive signaling cross talk to regulate megalin expression in cultured PTCs. Angiotensin II type 1A receptor- and insulin-mediated competitive signaling cross-talk regulates the expression of megalin, a multi-ligand endocytic receptor, in cultured proximal tubule cells.

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.

scholarly journals Osmotic and phorbol ester-induced activation of Na+/H+ exchange: possible role of protein phosphorylation in lymphocyte volume regulation.

1985 ◽  
Vol 101 (1) ◽  
pp. 269-276 ◽  
Author(s):  
S Grinstein ◽  
S Cohen ◽  
J D Goetz ◽  
A Rothstein
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Time Course ◽  
Phorbol Esters ◽  
Underlying Mechanism ◽  
Atp Depletion ◽  
Sequence Of Events ◽  
Kinase C ◽  
Stimulation Of

The Na+/H+ antiport is stimulated by 12-O-tetradecanoylphorbol-13, acetate (TPA) and other phorbol esters in rat thymic lymphocytes. Mediation by protein kinase C is suggested by three findings: (a) 1-oleoyl-2-acetylglycerol also activated the antiport; (b) trifluoperazine, an inhibitor of protein kinase C, blocked the stimulation of Na+/H+ exchange; and (c) activation of countertransport was accompanied by increased phosphorylation of specific membrane proteins. The Na+/H+ antiport is also activated by osmotic cell shrinking. The time course, extent, and reversibility of the osmotically induced and phorbol ester-induced responses are similar. Moreover, the responses are not additive and they are equally susceptible to inhibition by trifluoperazine, N-ethylmaleimide, and ATP depletion. The extensive analogies between the TPA and osmotically induced effects suggested a common underlying mechanism, possibly activation of a protein kinase. It is conceivable that osmotic shrinkage initiates the following sequence of events: stimulation of protein kinase(s) followed by activation of the Na+/H+ antiport, resulting in cytoplasmic alkalinization. The Na+ taken up through the antiport, together with the HCO3- and Cl- accumulated in the cells as a result of the cytoplasmic alkalinization, would be followed by osmotically obliged water. This series of events could underlie the phenomenon of regulatory volume increase.

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