Parathyroid hormone inhibition of Na+/phosphate cotransport in OK cells: requirement of protein kinase C-dependent pathway

Parathyroid hormone (PTH) inhibits sodium/phosphate (Na+/Pi) cotransport across the apical membrane of opossum kidney (OK) cells principally through two pathways. First, cAMP stimulation and activation of protein kinase A; second, diacylglycerol release and stimulation of protein kinase C. Studies were designed to determine the importance of these regulatory cascades. Down-regulation of protein kinase C with prolonged phorbol ester (12-O-tetradecanoylphorbol 13-acetate (TPA] treatment leads to a refractory state in which the cells do not respond to PTH (10(-8) M), cAMP (10(-4) M) or rechallenge of TPA (200 nM) even though Na+/Pi cotransport is similar to control cells (8.1 +/- 0.1 nmol.mg-1 protein.5 min-1). Staurosporine, an inhibitor of protein kinase C, resulted in the complete inhibition of PTH, cAMP and TPA action in a dose-dependent manner. PTH, cAMP and TPA were additive below maximal concentrations, but had no further effect at maximal agonist concentrations. These results suggest that protein kinase C activity is important in PTH-mediated inhibition of Na+/phosphate cotransport in OK cells.     

Phorbol ester TPA inhibits the stimulation of bumetanide-sensitive Na+/K+/Cl- transporter by different mitogens in quiescent BALB/c 3T3 mouse fibroblasts

In this study we examined the effect of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the bumetanide-sensitive Na+/K+/Cl- transporter in quiescent BALB/c 3T3 cells. We have shown that exposure of quiescent BALB/c 3T3 cultures to phorbol ester did not inhibit the basal bumetanide-sensitive Rb+ influx or efflux. In fact, at high concentration (100 ng/ml), TPA slightly stimulated the bumetanide-sensitive Rb+ influx and efflux. However, when the quiescent cultures were stimulated by serum or by defined growth factors, the stimulated fraction of the bumetanide-sensitive Rb+ influx was drastically inhibited by exposure of the cells to the phorbol ester TPA. Based on the above findings, we propose that activation of protein kinase C by the phorbol ester TPA does not inhibit the Na+/K+/Cl- cotransport activity; however it does suppress only the growth-factors-stimulated fraction of the cotransport in quiescent BALB/c 3T3 cells. These data propose that activation of kinase C has a regulatory feedback effect on the stimulation of the Na+/K+/Cl- cotransport activity by growth factors.     

Endocytosis and Na+/solute cotransport in renal epithelial cells

Endocytic uptake of [3H]sucrose and lucifer yellow, markers for fluid-phase endocytosis, was studied in cultures of the renal epithelial cell lines LLC-PK1 and OK. Endocytosis in LLC-PK1 cells was inhibited when the cells were grown in the presence of gentamicin (1 mg/ml) for 4 days or when the cells were treated with concanavalin A (1 mg/ml) for 5 h. These changes occurred without perturbation of intracellular Na+ and K+ content, indicating that the cells maintained normal ion gradients. The inhibition of endocytosis was accompanied by marked increases in the apparent Vmax for Na+-dependent cell uptake of solutes such as Pi and L-alanine. The apparent Km was unchanged. In contrast, treatment of OK cells with concanavalin A produced marked stimulation of endocytosis and inhibition of the Na+-dependent uptake of Pi and L-glutamate. These changes occurred in the absence of changes in intracellular Na+ and K+ content. Neither gentamicin nor concanavalin A had a direct effect on Na+/solute cotransport in these cell lines. The changes in Na+/Pi cotransport induced by concanavalin A in both LLC-PK1 and OK cells were blocked by keeping the cells at 4 degrees C during exposure to the lectin, suggesting that endocytosis may be part of the mechanism which mediates the changes in solute uptake. The reciprocal relationship between the changes in endocytosis and the changes in Na+/solute cotransport is consistent with the possibility that the number of Na+/solute cotransporters present in the plasma membrane may be altered by an increase or decrease in the rate of membrane internalization by endocytosis. The Vmax changes in Na+/solute cotransport provide indirect support for this conclusion.     

Effects of confluence on phosphate transport capacity in cultured renal cell lines

The LLC-PK1 cell line transports phosphate (Pi), glucose, and amino acids using carriers similar to those in proximal tubular cells. Others have reported that when monolayers reach confluence, hexose transport increases and activity of the A-amino acid transporter falls. The present study evaluates Pi uptake by two continuous cell lines derived from renal proximal tubule, and demonstrates that phosphate uptake falls sharply upon reaching confluence in LLC-PK1 cells but not in cultured opossum kidney (OK) cells. The fall in Pi uptake in LLC-PK1 cells at confluence represents a halving in Vmax for Na-dependent phosphate uptake (2.33 vs. 5.00 nmol/mg protein/5 min) without a change in Km (82 vs. 94 microM). Suppression of phosphate transport in confluent monolayers of LLC-PK1 cells is completely reversed by bringing the cells into suspension. As has been shown for the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), exposure of monolayers to serum stimulates phosphate uptake, but unlike phorbol ester, serum does so without stimulating alanine uptake. OK cells differ from LLC-PK1 in that no change occurs in Pi uptake at confluence, although they resemble LLC-PK1 cells in that sugar uptake rises and alanine uptake falls at confluence. The different temporal patterns for Pi uptake in the two cell lines indicates that developmental change in the uptake of Pi is not linked to that of glucose or alanine.     

Na+/K+/Cl- cotransport is stimulated by a Ca(++)-calmodulin-mediated pathway in BALB/c 3T3 fibroblasts.

In the present study, we investigated the role of intracellular Ca++ in the stimulation of the Na+/K+/Cl- cotransport in synchronized BALB/c 3T3 cells. The Na+/K+/Cl- cotransport was stimulated by the growth factors EGF, TGF-alpha, IGF-1, and IGF-2, which do not activate protein kinase C, but do induce a transient increase in free cytoplasmic Ca++. In addition, direct activation of protein kinase C by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) did not affect the Na+/K+/Cl- cotransport activity of quiescent cells. The Na+/K+/Cl- cotransport was also stimulated by the above mitogens in cells pretreated with the phorbol ester TPA. This treatment led to a progressive decline in the activity of cellular protein kinase C. This result implies that cells deficient in protein kinase C may still support stimulation of the Na+/K+/Cl- cotransport. Taken as a whole, these findings suggest that the Na+/K+/Cl- cotransport is stimulated predominantly by a protein kinase C-independent mechanism in BALB/c 3T3 fibroblasts. Both the intracellular Ca++ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) and two potent calmodulin antagonists, trifluoperazine (TFP) and chloropromazine (CP), blocked serum- and mitogen-stimulated Na+/K+/Cl- cotransport. These results suggest that the Na+/K+/Cl- cotransport is stimulated by an increase of intracellular Ca++ and subsequently by a Ca(++)-calmodulin-mediated pathway in the synchronized BALB/c 3T3 fibroblasts.     

Studies on the thermotropic behavior of aqueous phosphatidylethanolamines

Transport of phosphate has been studied in subconfluent monolayers of LLC-PK1 cells. It was found that this transport system shows similar characteristics to those observed in the kidney. Uptake of phosphate is mediated by a Na+-dependent, substrate-saturable process with an apparent Km value for phosphate of 96 +/- 15 mumol/l. Kinetic analysis of the effect of Na+ indicated that at (pH 7.4) two sodium ions are cotransported with one HOP4(2-) ion (Hill coefficient 1.5) with an apparent Km value for sodium of 56 mmol/l. Pi uptake is inhibited by metabolic inhibitors (ouabain and FCCP). In the pH range of 6.6 of 7.4 Pi uptake rate does not change significantly, indicating that both the monovalent and the divalent form of phosphate are accepted by the transport system. It is suggested that phosphate is transported by LLC-PK1 cells together with sodium (2 Na+:1 HPO4(2-) in an electroneutral manner down a favourable sodium gradient.     

Protein kinase C and membrane transport: divergent responses of Na+/K+/Cl- cotransport and sugar transport to exogenous diacylglycerol

Even though the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) is known to bind to and activate protein kinase C (PKC), it is still not certain that all cellular responses to phorbol esters are necessarily mediated by PKC. In BALB/c 3T3 preadipose cells, TPA has previously been shown to rapidly inhibit Na+K+Cl- -cotransport activity, stimulate 2-deoxyglucose uptake and induce ornithine decarboxylase activity. The cell-permeable diacylglycerol sn-1,2-dioctanoylglycerol (DiC8) was used in order to distinguish between PKC-dependent and -independent responses of BALB/c 3T3 cells. DiC8 modulated 86Rb+ fluxes in BALB/c 3T3 cells in the same manner as TPA: furosemide-sensitive 86Rb+ influx and efflux was inhibited, while in cotransport-defective cells no effect was observed. In contrast, DiC8 did not stimulate 2-deoxyglucose uptake in either parental or cotransport-defective cell lines, even though TPA is a very effective inducer of this transport system in both cell types. Pretreatment of cells with DiC8 did not substantially alter the subsequent induction of 2-deoxyglucose uptake by TPA, although a slight but reproducible reduction in the magnitude of the response was observed in DiC8-pretreated cells. The PKC-dependent phosphorylation of an acidic 80-kDa protein was stimulated by both TPA and DiC8 in parental and cotransport-defective cell lines, suggesting that a gross defect in the primary effector system used by both TPA and diacylglycerols cannot explain any of our results. Ornithine decarboxylase was induced by DiC8 and the K1/2 was approximately the same as that for inhibition of Na+/K+/Cl- cotransport in these cells. Thus, our results suggest that PKC is clearly essential for some phorbol ester membrane transport responses (such as inhibition of Na+/K+/Cl- cotransport), but our results do not allow us to conclude that other responses (such as stimulation of 2-deoxyglucose uptake) necessarily require PKC activation.     

Sodium-phosphate cotransport in human red blood cells. Kinetics and role in membrane metabolism

Orthophosphate (Pi) uptake was examined in human red blood cells at 37 degrees C in media containing physiological concentrations of Pi (1.0- 1.5 mM). Cells were shown to transport Pi by a 4,4'-dinitro stilbene- 2,2'-disulfonate (DNDS) -sensitive pathway (75%), a newly discovered sodium-phosphate (Na/Pi) cotransport pathway (20%), and a pathway linearly dependent on an extracellular phosphate concentration of up to 2.0 mM (5%). Kinetic evaluation of the Na/Pi cotransport pathway determined the K1/2 for activation by extracellular Pi ([Na]o = 140 mM) and extracellular Na [( Pi]o = 1.0 mM) to be 304 +/- 24 microM and 139 +/- 8 mM, respectively. The phosphate influx via the cotransport pathway exhibited a Vmax of 0.63 +/- 0.05 mmol Pi (kg Hb)-1(h)-1 at 140 mM Nao. Activation of Pi uptake by Nao gave Hill coefficients that came close to a value of 1.0. The Vmax of the Na/Pi cotransport varied threefold over the examined pH range (6.90-7.75); however, the Na/Pi stoichiometry of 1.73 +/- 0.15 was constant. The membrane transport inhibitors ouabain, bumetanide, and arsenate had no effect on the magnitude of the Na/Pi cotransport pathway. No difference was found between the rate of incorporation of extracellular Pi into cytosolic orthophosphate and the rate of incorporation into cytosolic nucleotide phosphates, but the rate of incorporation into other cytosolic organic phosphates was significantly slower. Depletion of intracellular total phosphorus inhibited the incorporation of extracellular Pi into the cytosolic nucleotide compartment; and this inhibition was not reversed by repletion of phosphorus to 75% of control levels. Extracellular 32Pi labeled the membrane-associated compounds that migrate on thin-layer chromatography (TLC) with the Rf values of ATP and ADP, but not those of 2,3-bisphosphoglycerate (2,3-DPG), AMP, or Pi. DNDS had no effect on the level of extracellular phosphate incorporation or on the TLC distribution of Pi in the membrane; however, substitution of extracellular sodium with N-methyl-D-glucamine inhibited phosphorylation of the membranes by 90% and markedly altered the chromatographic pattern of the membrane-associated phosphate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of Na+/K+/Cl- cotransport and [3H]bumetanide binding site density by phorbol esters in HT29 cells

The involvement of protein kinase C in the regulation of Na+/K+/Cl- cotransport was investigated in cultured HT29 human colonic adenocarcinoma cells. We have demonstrated previously the presence of a Na+/K+/Cl- cotransport pathway in HT29 cells (Kim, H.D., Tsai, Y-S., Franklin, C.C., and Turner, J.T. (1989) Biochim. Biophys. Acta 946, 397-404). Treatment of cells with the phorbol esters phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) caused an increase in membrane-associated protein kinase C activity that was accompanied by a concomitant decrease in cytosolic protein kinase C activity. PMA also produced a rapid transient increase in cotransport to 137% of control values by 5 min followed by a progressive decrease to 19% of control values by 2 h. To determine the underlying mechanism for the reduction in Na+/K+/Cl- cotransport, changes in cotransporter number and/or affinity were determined in radioligand binding studies using [3H]bumetanide. PMA and PDBu produced essentially identical time- and dose-dependent decreases in specific [3H]bumetanide binding that were similar to the observed decreases in cotransport. Analysis of saturation and competition binding data indicated that the decrease in binding was due to a lowered Bmax with no change in affinity. Both the decrease in binding and the changes in cotransport elicited by PMA were prevented by the protein kinase inhibitor H7. These findings suggest that phorbol esters cause a decrease in the number of cotransporters in HT29 cells, resulting in a reduction in Na+/K+/Cl- cotransport activity.     

A phorbol ester-nonproliferative variant of Swiss 3T3 cells is deficient in Na+K+Cl- cotransport activity

The identity of the genetic defect(s) in Swiss 3T3 TNR-2 and TNR-9 that confers nonresponsiveness to the proliferative effect of 12-0-tetradecanoylphorbol-13-acetate (TPA) is not known. In BALB/c 3T3 cells, loss (via mutation) of a specific membrane ion transport system, the furosemide-sensitive Na+K+Cl- cotransporter, is associated with decreased responsiveness to TPA. In this study, the transport properties of parental Swiss 3T3 cells and the TPA-nonresponsive lines TNR-2 and TNR-9 were determined in the presence and absence of TPA. When the rate of 86Rb+ efflux (as a tracer for K+) was measured from each of the three cell lines, a furosemide- and TPA-inhibitable component of efflux was clearly evident in parental and TNR-9 cells but was virtually absent in TNR-2 cells. 86Rb+ influx measurements indicated the presence in parental 3T3 cells and the TNR-9 line of a substantial furosemide-sensitive flux that could be inhibited by TPA. In contrast, much less furosemide-sensitive influx was present in 3T3-TNR-2 cells and it was relatively unaffected by TPA. In both parental 3T3 and 3T3-TNR-2 cells, most of the furosemide-sensitive 86Rb+ influx is dependent on extracellular Na+ and Cl-. The apparent affinities of the transporter for these two ions, as well as for K+, were similar in both cell lines. In parental cells, the inhibition of furosemide-sensitive 86Rb+ influx was quite sensitive to TPA (K1/2 approximately equal to 1 nM) and occurred very rapidly after phorbol ester exposure. As expected because of its volume-regulatory role, inhibition of Na+K+Cl- cotransport by TPA in parental cells caused a substantial reduction in cell volume (25%). In contrast, because of the reduced level of cotransport activity in TNR-2 cells, TPA had only a slight effect on cell volume. These results suggest that the genetic defect in 3T3-TNR-2 cells (but not TNR-9 cells) responsible for nonresponsiveness to phorbol esters may be the reduction of Na+K+Cl- cotransport activity. Thus this membrane transport system may be an important component of the signal transduction pathway used by phorbol esters in 3T3 cells.     

Protein kinase C activation has dissimilar effects on sodium-coupled uptakes in renal proximal tubular cells in primary culture

Protein kinase C (Ca2+/phospholipid-dependent enzyme) was shown to be present in renal brush border membranes. To evaluate the influence of protein kinase C activation on three apical transport systems, we studied the effect of phorbol myristate acetate (PMA) and of two diacylglycerol analogs, oleoylacetylglycerol and dioctanoylglycerol, on sodium-dependent uptakes of phosphate (Pi), L-alanine, and alpha-methyl-D-glucopyranoside (MGP), as well as on specific phlorizin binding, in cultured rabbit proximal tubular cells. PMA, at 100 ng/ml, decreased the Vmax of Pi and MGP uptake by 30 and 17%, respectively, but not that of alanine uptake. None of the Km values were affected. PMA also decreased the number of phlorizin binding sites by 40%. PMA-induced inhibition of Pi and MGP uptake was time- and concentration-dependent, was mimicked by oleoylacetylglycerol, dioctanoylglycerol, and the diacylglycerol kinase inhibitor R59022, and was reversed by the protein kinase C antagonist 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7). The effects of PMA persisted in the presence of amiloride and dimethyl amiloride, and were potentiated by Ca2+ ionophore A23187. Opening of tight junctions blunted subsequent PMA-induced decrease of MGP uptake, but not of Pi uptake. It is concluded that: (i) activation of protein kinase C does not affect similarly Na-Pi, Na-hexose, and Na-alanine cotransport; and (ii) different pathways are likely to be involved in the observed effects.     

Sodium-dependent phosphate transport by apical membrane vesicles from a cultured renal epithelial cell line (LLC-PK1)

Apical membrane vesicles were prepared from confluent monolayers of LLC-PK1 cells grown upon microcarrier beads. The final membrane preparation, obtained by a modified divalent cation precipitation technique, was enriched in alkaline phosphatase, leucine aminopeptidase and trehalase (8-fold compared to the initial homogenate). Analysis of phosphate uptake into the vesicles identified a specific sodium-dependent pathway. Lithium and other cations were unable to replace sodium. At 100 mmol/l sodium and pH 7.4, an apparent Km for phosphate of 99 +/- 19 mumol/l and an apparent Ki for arsenate of 1.9 mmol/l were found. Analysis of the sodium activation of phosphate uptake gave an apparent Km for sodium of 32 +/- 12 mmol/l and suggested the involvement of two sodium ions in the transport mechanism. Sodium modified the apparent Km of the transport system for phosphate. The rate of sodium-dependent phosphate uptake was higher at pH 6.4 than at pH 7.4. At both pH values, an inside negative membrane potential (potassium gradient plus valinomycin) had no stimulatory effect on the rate of the sodium-dependent component of phosphate uptake. It is concluded that the apical membrane of LLC-PK1 cells contains a sodium-phosphate cotransport system with a stoichiometry of 2 sodium ions: 1 phosphate anion.     

Na+/H+ exchanger-regulatory factor 1 mediates inhibition of phosphate transport by parathyroid hormone and second messengers by acting at multiple sites in opossum kidney cells

The opossum kidney (OK) line displays PTH-mediated activation of adenylyl cyclase and phospholipase C and inhibition of phosphate (Pi) uptake via regulation of the type IIa sodium-phosphate cotransporter, consistent with effects in vivo. OKH cells, a subclone of the OK cell line, robustly activates PTH-mediated activation of adenylyl cyclase, but is defective in PTH-mediated inhibition of sodium-phosphate cotransport and signaling via phospholipase C. Compared with wild-type OK cells, OKH cells express low levels of the Na+/H+ exchanger regulatory factor 1 (NHERF-1). Stable expression of NHERF-1 in OKH cells (OKH-N1) rescues the PTH-mediated inhibition of sodium-phosphate cotransport. NHERF-1 also restores the capacity of 8-bromo-cAMP and forskolin to inhibit Pi uptake, but the PTH dose-response for cAMP accumulation and inhibition of Pi uptake differ by 2 orders of magnitude. NHERF-1, in addition, modestly restores phorbol ester-mediated inhibition of Pi uptake, which is much weaker than that elicited by PTH. A poor correlation exists between the inhibition of Pi uptake mediated by PTH ( approximately 60%) and the inhibition mediated by phorbol 12-myristate 13-acetate ( approximately 30%) and the ability of these molecules to activate the protein kinase C-responsive reporter gene. Furthermore, we show that NHERF-1 directly interacts with type IIa cotransporter in OK cells. Although, PTH-mediated inhibition of Pi uptake in OK cells is largely NHERF-1 dependent, the signaling pathway(s) by which this occurs is still unclear. These pathways may involve cooperativity between cAMP- and protein kinase C-dependent pathways or activation/inhibition of an unrecognized NHERF-1-dependent pathway(s).     

Activation of protein kinase C by phorbol esters induces DNA synthesis and protein phosphorylations in glomerular mesangial cells

The tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) is shown to be mitogenic for quiescent glomerular mesangial cells cultured in serum-free conditions. TPA induces DNA synthesis measured by [3H]thymidine incorporation in a dose-dependent manner with an ED50 of 7 ng/ml and an optimal response for 50 ng/ml. The phorbol ester action is potentiated by insulin with an increase of the maximal effect from 232 +/- 15% for TPA alone to 393 +/- 96% for TPA plus insulin. Down-regulation of protein kinase C by prolonged exposure to TPA completely abolishes the mitogenic effect of the phorbol ester. Using a highly resolutive 2D electrophoresis, we have shown that TPA is able to stimulate the phosphorylation of 2 major proteins of Mr 80,000, pl 4.5 (termed 80K) and Mr 28,000, pI 5.7-5.9 (termed 28K). The 80K protein phosphorylation is time- and dose-dependent with an ED50 of 8 ng/ml TPA. Exposure of mesangial cells to heat-shock induces synthesis of a 28K protein among a set of other proteins suggesting that the 28K protein kinase C substrate belongs to the family of low molecular mass stress proteins. Mitogenic concentrations of TPA and phorbol 12,13-dibutyrate inhibit [125 I]epidermal growth factor binding and stimulate the 80K protein phosphorylation with the same order of potency. The inactive tumor-promoter 4 alpha-phorbol was found to be ineffective both on these 2 parameters and on DNA synthesis. These results suggest a positive role for protein kinase C on mesangial cell proliferation and indicate the existence in this cell line of 2 major protein kinase C substrates.     

Phorbol esters and mitogenesis: comparison of the proliferative response of parental and Na+K+Cl- -cotransport-defective BALB/c 3T3 cells to 12-0-tetradecanoylphorbol-13-acetate

The ability of the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) to stimulate the growth of quiescent BALB/c 3T3 cell lines lacking Na+K+Cl- cotransport activity was tested. We have previously isolated and characterized two mutant cell lines defective in this important ion transport system by mutagenesis and selection in medium containing low K+. To test our hypothesis that loss of this transport activity might abrogate the proliferative response to TPA, two kinds of mitogenesis assays were performed. First, the effect of 0.16 microM TPA on the saturation density of parental vs. mutant cell lines was determined. TPA caused a small but reproducible 30-35% increase in the saturation density of mutant cells compared to the 100-120% increase seen in parental cell lines. Second, the effect of TPA on the incorporation of 3H-thymidine into cell nuclei (labeling index) was measured. While some variability from experiment to experiment in the extent and time course of the response of mutant cells was noted, TPA either had no effect or only a small effect on the labeling index when compared to the response of parental cells. When a range of concentrations of TPA (0.016-1.6 microM) was tested, neither cell line exhibited a large response to any concentration. These results suggest that loss of Na+K+Cl- cotransport activity decreases the response of these cells to the mitogenic action of TPA.     

Inhibition of renal Na(+)-Pi cotransporter by mercuric chloride: role of sulfhydryl groups.

We studied the role of sulfhydryl groups in Na(+)-Pi cotransport across the renal brush border membrane (BBM), using HgCl2, an agent which penetrates membranes freely. HgCl2 inhibited the initial Na(+)-dependent 32Pi transport in a dose-dependent manner (IC50 = 54 microM). Na(+)-independent transport was not affected. The inhibitory effect persisted under Na+ equilibrium-exchange conditions. Additionally, HgCl2 had no effect on the diffusional uptake of 22Na up to 1 min incubation. Exposure to HgCl2 had no effect on vesicle integrity as determined by osmotic shrinking experiments. BBM vesicle (BBMV) volume, determined by D-glucose equilibrium uptake, was not affected at low HgCl2 concentrations, but decreased at higher concentrations (greater than 100 microM). Vesicle volumes, determined by flow cytometry, were not changed after exposure to HgCl2. Kinetic studies showed a reduction in the apparent Vmax for Pi transport from 1.40 +/- 0.13 to 0.75 +/- 0.19 nmoles/mg protein/5 sec, without a significant change in the apparent Km. In protection studies, dithiothreitol (DTT) completely protected against inhibition, but Pi, phosphonoformic acid (PFA), and Na+ gave no protection. The data suggest that sulfhydryl groups are essential for the function of Na(+)-Pi cotransporter of renal BBM.     

Phorbol diesters promote beta-adrenergic receptor phosphorylation and adenylate cyclase desensitization in duck erythrocytes

Preincubation of duck erythrocytes with tumor promoting phorbol diesters or catecholamines leads to attenuation of adenylate cyclase activity. 12-0-Tetradecanoyl phorbol-13-acetate (TPA) and phorbol 12,13-dibutyrate treatment induced a 38% and 30% desensitization of isoproterenol-stimulated adenylate cyclase activity, respectively. In contrast, the inactive phorbol diester, 4 alpha-phorbol 12,13-didecanoate, was without effect in promoting adenylate cyclase desensitization. The catecholamine isoproterenol induced a 51% desensitization. Incubation of 32Pi labeled erythrocytes with TPA promoted a 3- to 4-fold increase in phosphorylation of the beta-adrenergic receptor as did incubation with isoproterenol. Treatment of the cells with both TPA and isoproterenol together resulted in desensitization and receptor phosphorylation which were no greater than those observed with either agent alone. These data suggest a potential role for protein kinase C in regulating beta-adrenergic receptor function.     

Phorbol esters and gene expression: the role of rapid changes in K+ transport in the induction of ornithine decarboxylase by 12-O- tetradecanoylphorbol-13-acetate in BALB/c 3T3 cells and a mutant cell line defective in Na+K+Cl- cotransport

A BALB/c 3T3 preadipose cell line defective in Na+K+Cl- cotransport (3T3-E12a cells) has been used to study the relationship between phorbol ester-induced rapid changes in cation fluxes and changes in expression of a gene known to be modulated by this agent. In contrast to its effect on parental 3T3 cells, 12-O-tetradecanoylphorbol-13-acetate (TPA) did not inhibit either furosemide-sensitive 86Rb+ influx or the rate of 86Rb+ efflux from preloaded mutant cells. TPA-induced changes in intracellular K+ content were diminished in 3T3-E12a cells as compared with parental cells. Thus, mutation of the Na+K+Cl- cotransport system renders overall potassium transport in mutant cells largely insensitive to modulation by TPA. The morphological and functional responses of 3T3 and 3T3-E12a cells to TPA were also compared. In contrast to the extensive and long-lasting changes in morphology of 3T3 cells after 0.16 microM TPA addition, only slight and shorter-lived morphological effects of TPA were observed in 3T3-E12a cells. The transport properties of mutant cells were not totally unresponsive to TPA since hexose transport (2-deoxyglucose uptake) could be stimulated in both cell types. To establish a possible link between early changes in cation fluxes and activation of gene expression by TPA, the induction of the enzyme ornithine decarboxylase (ODC) was studied in detail. Addition of fresh medium containing serum or exposure to hypoosmotic conditions resulted in the induction of ODC in both 3T3 and 3T3-E12a cells. However, TPA failed to cause an increase in ODC activity in mutant cells, although a substantial induction of the enzyme was seen in parental cells. These results suggest that rapid changes in ion fluxes mediated by the Na+K+Cl- cotransport system are necessary for at least one of the phorbol ester-induced changes in gene expression in responsive cells.     

Parathyroid hormone inhibition of Na+/phosphate cotransport in OK cells: intracellular [Ca2+] as a second messenger

Parathyroid hormone increases cellular cAMP, 1,2-diacylglycerol, inositol 1,4,5-trisphosphate and cytosolic Ca2+ concentration ([Ca2+]i) in OK cells. In the present study, we determined the importance of the PTH-dependent increase in [Ca2+]i in the control of sodium-dependent phosphate (Na+/Pi) cotransport. PTH (10(-7) M) results in a transient increase in [Ca2+]i from basal levels of 67 +/- 4 nM to maximal concentrations of 190 +/- 9 nM. The increase in [Ca2+]i was dose-dependent with half-maximal increases at about 5.10(-8) M PTH. These hormone levels were 10(3)-fold higher than that required for half-maximal inhibition of Na+/Pi cotransport. Clamping [Ca2+]i with either intracellular Ca2+ chelators or by ionomycin in the presence of high concentrations of extracellular Ca2+ did not alter PTH-dependent inhibition of Na/Pi cotransport. Nor did indomethacin, an inhibitor of the cyclooxygenase pathway, influence the hormonal inhibition of cotransport. Accordingly, these data suggest that changes in [Ca2+]i and/or activation of the phospholipase A2 and the cyclooxygenase pathways are not involved in signal induction of the PTH-mediated control of Na+/Pi cotransport.     

Effect of ischemia-reperfusion on the renal brush-border membrane sodium-dependent phosphate cotransporter NaPi-2

To understand the mechanisms underlying ischemia-reperfusion-induced renal proximal tubule damage, we analyzed the expression of the Na+-dependent phosphate (Na+/Pi) cotransporter NaPi-2 in brush border membranes (BBM) isolated from rats which had been subjected to 30 min renal ischemia and 60 min reperfusion. Na+/Pi cotransport activities of the BBM vesicles were also determined. Ischemia caused a significant decrease (about 40%, P < 0.05) in all forms of NaPi-2 in the BBM, despite a significant increase (31+/-3%, P < 0.05) in the Na+/Pi cotransport activity. After reperfusion, both NaPi-2 expression and Na+/Pi cotransport activity returned to control levels. In contrast with Na+/Pi cotransport, ischemia significantly decreased Na+-dependent glucose cotransport but did not affect Na+-dependent proline cotransport. Reperfusion caused further decreases in both Na+/glucose (by 60%) and Na+/proline (by 33%) cotransport. Levels of NaPi-2 were more reduced in the BBM than in cortex homogenates, suggesting a relocalization of NaPi-2 as a result of ischemia. After reperfusion, NaPi-2 levels returned to control values in both BBM and homogenates. These data indicate that the NaPi-2 protein and BBM Na+/Pi cotransport activity respond uniquely to reversible renal ischemia and reperfusion, and thus may play an important role in maintaining and restoring the structure and function of the proximal tubule.