Sodium-dependent Phosphate Transporter Research Articles

Unchanged expression of the sodium-dependent phosphate cotransporter NaPi-IIa despite diurnal changes in renal phosphate excretion

Renal phosphate excretion is subjected to circadian rhythmicity. The bulk of filtered inorganic phosphate (P(i)) is reabsorbed by the sodium-dependent phosphate cotransporter NaPi-IIa. The regulation of proximal tubular phosphate reabsorptive capacity is largely attributed to the altered abundance of NaPi-IIa residing in the brush border membrane (BBM) of proximal tubular cells. Therefore, we examined if the diurnal rise in renal phosphate excretion is accompanied by a corresponding change in NaPi-IIa expression. Renal phosphate excretion, creatinine clearance, and serum phosphate were determined at consecutive time points in rats, starting from 8 a.m. until 5 p.m. During this period, renal phosphate excretion (fractional P(i) excretion) increased more than eightfold until 5 p.m. compared to the morning values at 8 a.m. In addition, serum phosphate and creatinine clearance as well as the calculated tubular phosphate threshold increased. Neither immunoblot analysis of BBMs nor immunohistochemical staining for NaPi-IIa yielded evidence for a lower abundance of NaPi-IIa in kidneys collected in the afternoon compared to those in the morning. However, kidneys sampled in the afternoon showed a small decrease (14%) in (32)P uptakes into BBM vesicles (BBMVs). Thus, the diurnal rise in renal phosphate excretion was associated with a mild reduction in the sodium-dependent phosphate transport rate in proximal tubular BBMs. There was no apparent downregulation of NaPi-IIa abundance and only a small reduction in Na(+)-dependent Pi-transport activity. Thus, the diurnal changes in urinary phosphate excretion appear to be mainly related to changes in serum phosphate and tubular threshold but not to NaPi-IIa expression.

Adenoviral expression of NHERF-1 in NHERF-1 null mouse renal proximal tubule cells restores Npt2a regulation by low phosphate media and parathyroid hormone

Sodium-dependent phosphate transport in NHERF-1(-/-) proximal tubule cells does not increase when grown in a low phosphate media and is resistant to the normal inhibitory effects of parathyroid hormone (PTH). The current experiments employ adenovirus-mediated gene transfer in primary cultures of mouse proximal tubule cells from NHERF-1 null mice to explore the specific role of NHERF-1 on regulated Npt2a trafficking and sodium-dependent phosphate transport. NHERF-1 null cells have decreased sodium-dependent phosphate transport compared with wild-type cells. Infection of NHERF-1 null cells with adenovirus-GFP-NHERF-1 increased phosphate transport and plasma membrane abundance of Npt2a. Adenovirus-GFP-NHERF-1 infected NHERF-1 null proximal tubule cells but not cells infected with adenovirus-GFP demonstrated increased phosphate transport and Npt2a abundance in the plasma membrane when grown in low phosphate (0.1 mM) compared with high phosphate media (1.9 mM). PTH inhibited phosphate transport and decreased Npt2a abundance in the plasma membrane of adenovirus-GFP-NHERF-1-infected NHERF-1 null proximal tubule cells but not cells infected with adenovirus-GFP. Interestingly, phosphate transport is inhibited by activation of protein kinase A and protein kinase C in wild-type proximal tubule cells but not in NHERF-1(-/-) cells. Together, these results highlight the requirement for NHERF-1 for physiological control of Npt2a trafficking and suggest that the Npt2a/NHERF-1 complex represents a unique PTH-responsive pool of Npt2a in renal microvilli.

Amphotropic murine leukemia virus is preferentially attached to cholesterol-rich microdomains after binding to mouse fibroblasts

BackgroundWe have recently shown that amphotropic murine leukemia virus (A-MLV) can enter the mouse fibroblast cell line NIH3T3 via caveola-dependent endocytosis. But due to the size and omega-like shape of caveolae it is possible that A-MLV initially binds cells outside of caveolae. Rafts have been suggested to be pre-caveolae and we here investigate whether A-MLV initially binds to its receptor Pit2, a sodium-dependent phosphate transporter, in rafts or caveolae or outside these cholesterol-rich microdomains.ResultsHere, we show that a high amount of cell-bound A-MLV was attached to large rafts of NIH3T3 at the time of investigation. These large rafts were not enriched in caveolin-1, a major structural component of caveolae. In addition, they are rather of natural occurrence in NIH3T3 cells than a result of patching of smaller rafts by A-MLV. Thus cells incubated in parallel with vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped MLV particles showed the same pattern of large rafts as cells incubated with A-MLV, but VSV-G pseudotyped MLV particles did not show any preference to attach to these large microdomains.ConclusionThe high concentration of A-MLV particles bound to large rafts of NIH3T3 cells suggests a role of these microdomains in early A-MLV binding events.

Inorganic phosphate inhibits growth of human osteosarcoma U2OS cells via adenylate cyclase/cAMP pathway

In order to elucidate how phosphate regulates cellular functions, we investigated the effects of inorganic phosphate (Pi) on adenylate cyclase (AC)/cyclic AMP (cAMP) axis. Here we describe that Pi treatment of human osteosarcoma U2OS cells results in a decrease of both intracellular cAMP levels and AC activity, and in a cell growth inhibition. The phosphate-triggered effects observed in U2OS cells are not a widespread phenomenon regarding all cell lines, since other cell lines screened respond differently to parallel Pi treatments. In U2OS cell line, the AC activity/cAMP downregulation is accompanied by significant variations in the levels of some membrane proteins belonging to the AC system. Remarkably, the above effects are blunted by pharmacological inhibition of sodium-dependent phosphate transport. Moreover, 8-Br-cAMP and other cAMP-elevating agents, such as IBMX and forskolin, interestingly, prevent the cell growth inhibition in response to phosphate. Our results enforce the increasing evidences of phosphate as a signaling molecule, identifying in U2OS cell line the AC/cAMP axis, as a novel-signaling pathway modulated by phosphate to ultimately affect cell growth.

Role of the Sodium-Dependent Phosphate Cotransporter, Pit-1, in Vascular Smooth Muscle Cell Calcification

Vascular calcification is associated with cardiovascular morbidity and mortality. Hyperphosphatemia is an important contributor to vascular calcification. Our previous studies demonstrated that elevated phosphate induces calcification of smooth muscle cells (SMC) in vitro. Inhibition of phosphate transport by phosphonoformic acid blocked phosphate-induced calcification, implicating sodium-dependent phosphate cotransporters in this process. In the present study, we have investigated the role of the type III sodium-dependent phosphate cotransporter, Pit-1, in SMC calcification in vitro. Human SMC stably expressing Pit-1 small interfering double-stranded RNA (SMC-iRNA) were established using a retroviral system. SMC-iRNA had decreased Pit-1 mRNA and protein levels and sodium-dependent phosphate transport activity compared with the control transduced cells (SMC-CT) (2.9 versus 9.78 nmol/mg protein per 30 minutes, respectively). Furthermore, phosphate-induced SMC calcification was significantly inhibited in SMC-iRNA compared with SMC-CT at all time points examined. Overexpression of Pit-1 restored phosphate uptake and phosphate-induced calcification in Pit-1 deficient cells. Mechanistically, although Pit-1-mediated SMC calcification was not associated with apoptosis or cell-derived vesicles, inhibition of phosphate uptake in Pit-1 knockdown cells blocked the induction of the osteogenic markers Cbfa-1 and osteopontin. Our results indicate that phosphate uptake through Pit-1 is essential for SMC calcification and phenotypic modulation in response to elevated phosphate.

Distribution of vesicular glutamate transporters in rat and human retina

Central nervous system neurons have traditionally been thought to express exclusively membrane transporters and/or vesicular transporters for their transmitter. Three vesicular glutamate transporters have recently been cloned: BNPI/VGLUT1 (a brain-specific sodium-dependent inorganic phosphate (Pi) transporter), and its homologs DNPI/VGLUT2 (differentiation-associated sodium-dependent Pi transporter) and VGLUT3. We investigated the subcellular distributions of these three vesicular transporters in rat and human retina. VGLUT1 was present in the outer and inner plexiform layers (OPL and IPL), as shown by punctate staining in both human and rat retina. In the OPL, it was colocalized with synaptophysin, consistent with its expression in glutamatergic photoreceptor terminals, and it was present in PKC-α-labeled glutamatergic bipolar cell terminals in the IPL. By contrast, VGLUT2 was present in horizontal cells and ganglion cells in rat and human retina. In human retina, VGLUT2 was also found in some amacrine cells, including GAD-immunopositive amacrine cells. VGLUT3 was present in glycine-releasing amacrine cells in rat retina but was restricted to a few ganglion cells in human retina. The distribution of VGLUT1 in excitatory synaptic terminal was consistent with its involvement in glutamate release at excitatory synapses, whereas the cellular distributions of VGLUT2 and VGLUT3 suggested that these molecules may be involved in functions other than glutamate release, such as glutamate storage for GABA synthesis in non-glutamatergic neurons.

Vascular calcification in chronic kidney disease

Vascular calcification is often encountered in advanced atherosclerotic lesions and is a common consequence of aging. Calcification of the coronary arteries has been positively correlated with coronary atherosclerotic plaque burden, increased risk of myocardial infarction, and plaque instability. Chronic kidney disease (CKD) patients have two to five times more coronary artery calcification than healthy age-matched individuals. Vascular calcification is a strong prognostic marker of cardiovascular disease mortality in CKD patients. Vascular calcification has long been considered to be a passive, degenerative, and end-stage process of atherosclerosis and inflammation. However, recent evidence indicates that bone matrix proteins such as osteopontin, matrix Gla protein (MGP), and osteocalcin are expressed in calcified atherosclerotic lesions, and that calcium-regulating hormones such as vitamin D3 and parathyroid hormone-related protein regulate vascular calcification in in vitro vascular calcification models based on cultured aortic smooth muscle cells. These findings suggest that vascular calcification is an actively regulated process similar to osteogenesis, and that bone-associated proteins may be involved in the development of vascular calcification. The pathogenesis of vascular calcification in CKD is not well understood and is almost multifactorial. In CKD patients, several studies have found associations of both traditional risk factors, such as hypertension, hyperlipidemia, and diabetes, and uremic-specific risk factors with vascular calcification. Most patients with progressive CKD develop hyperphosphatemia. An elevated phosphate level is an important risk factor for the development of calcification and cardiovascular mortality in CKD patients. Thus, it is hypothesized that an important regulator of vascular calcification is the level of inorganic phosphate. In order to test this hypothesis, we characterized the response of human smooth muscle cell (HSMC) cultures to inorganic phosphate levels. Our findings indicate that inorganic phosphate directly regulates HSMC calcification through a sodium-dependent phosphate transporter mechanism. After treatment with elevated phosphate, there is a loss of smooth muscle lineage markers, such as alpha-actin and SM-22alpha, and a simultaneous gain of osteogenic markers such as cbfa-1 and osteocalcin. Elevated phosphate may directly stimulate HSMC to undergo phenotypic changes that predispose to calcification, and offer a novel explanation of the phenomenon of vascular calcification under hyperphosphatemic conditions. Furthermore, putative calcification inhibitory molecules have been identified using mouse mutational analyses, including MGP, beta-glucosidase, fetuin-A, and osteoprotegerin. Mutant mice deficient in these molecules present with enhanced cardiovascular calcification, demonstrating that specific molecules are normally important in suppressing vascular calcification. These findings suggest that the balance of inducers, such as phosphate, and inhibitors, such as MGP, fetuin-A, and others, are likely to control whether or not calcification occurs under pathological conditions.

NaPi-mediated Transcellular Permeation is the Dominant Route in Intestinal Inorganic Phosphate Absorption in Rats

Inorganic phosphate in food is absorbed two ways, the transcellular route via the brush border membrane and the paracellular route via tight junctions. NaPi, a sodium-dependent inorganic phosphate transporter, is expressed in rat and human intestine. However, the relative contribution of NaPi to total carrier-mediated transport of physiological concentrations of inorganic phosphate in rat intestine is not clear. Here, we characterized inorganic phosphate transport across the rat small intestine using a voltage-clamp analysis which allowed the diffrentiation of inorganic phosphate permeation through these two (transcellular and paracellular) routes. Results showed that, under a physiologically normal transmucosal electrical potential difference (about 2 mV), permeation of inorganic phosphate by the transcellular route was greater than that by the paracellular route. Further, transport was significantly decreased by the addition to the incubation medium of phosphonoformic acid, a sodium-dependent phosphate transporter inhibitor, and severely inhibited under sodium-free conditions. Similar results were obtained without the voltage-clamp. Together, these results suggest that NaPi-mediated transcellular permeation is the dominant route in the absorption of inorganic phosphate across the small intestine.

A High Inorganic Phosphate Diet Perturbs Brain Growth, Alters Akt-ERK Signaling, and Results in Changes in Cap-Dependent Translation

Inorganic phosphate (Pi) plays a key role in diverse physiological functions. Recently, considerable progress has been made in our understanding of the function and regulation of the brain-specific sodium-dependent inorganic phosphate transporter 1 (NPT1), which is found to exist principally in cerebrum and cerebellum. The potential importance of Pi as a novel signaling molecule and the poor prognosis of diverse neurodegenerative diseases that involve brain-specific NPT1 have prompted us to define the pathways by which Pi affects mouse brain growth. A high phosphate diet caused an increase in serum Pi accompanied by a decrease in calcium, and a decrease in body weight coupled with a decreased relative weight of cerebellum. A high phosphate diet caused a significant increase in protein expression of NPT1, both in cerebrum and cerebellum. Additionally, the high phosphate diet increased Homo sapiens v-akt murine thymoma viral oncogene homolog 1 (Akt) phosphorylation at Ser473 in cerebrum and cerebellum, whereas suppression of Akt phosphorylation at Thr308 was observed only in cerebellum. Selective suppression of eukaryotic translation initiation factor-binding protein (eIF4E-BP1) in cerebrum was induced by high levels of Pi, which induced cap-dependent and cap-independent protein translation in cerebrum and cerebellum, respectively. Phosphorylation of extracellular regulated kinase 1 (ERK1) in comparison with that of ERK2 was significantly reduced in both cerebrum and cerebellum. High levels of Pi reduced protein expressions of proliferating cell nuclear antigen (PCNA) and cyclin D1 in cerebrum and cerebellum. In conclusion, the results indicate that high dietary Pi can perturb normal brain growth, possibly through Akt-ERK signaling in developing mice.

Isolation and characterization of a sodium-dependent phosphate transporter gene in Dunaliella viridis

A sodium-dependent phosphate transporter gene, DvSPT1, was isolated from a cDNA library using a probe derived from a subtracted cDNA library of Dunaliella viridis. Sequencing analyses revealed a cDNA sequence of 2649 bp long and encoded an open-reading frame consisting of 672 amino acids. The deduced amino acid sequence of DvSPT1 exhibited 31.2% identity to that of TcPHO from Tetraselmis chui. Hydrophobicity and secondary structure prediction revealed 11 conserved transmembrane domains similar to those found in PHO89 from Saccharomyces cerevisiae and PHO4 from Neurospora crassa. Northern blot analysis indicated that the DvSPT1 expression was induced upon NaCl hyperosmotic stress or phosphate depletion. Functional characterization in yeast Na + export pump mutant G19 suggested that DvSPT1 encoded a Na + transporter protein. The gene sequence of GDvSPT1 (7922 bp) was isolated from a genomic library of D. viridis. Southern blot analysis indicated that there exist at least two homologous genes in D. viridis.

“Phosphatonins” and the regulation of phosphorus homeostasis

Phosphate ions are critical for normal bone mineralization, and phosphate plays a vital role in a number of other biological processes such as signal transduction, nucleotide metabolism, and enzyme regulation. The study of rare disorders associated with renal phosphate wasting has resulted in the discovery of a number of proteins [fibroblast growth factor 23 (FGF-23), secreted frizzled related protein 4 (sFRP-4), matrix extracellular phosphoglycoprotein, and FGF 7 (FGF-7)] that decrease renal sodium-dependent phosphate transport in vivo and in vitro. The "phosphatonins," FGF-23 and sFRP-4, also inhibit the synthesis of 1alpha,25-dihydroxyvitamin D, leading to decreased intestinal phosphate absorption and further reduction in phosphate retention by the organism. In this review, we discuss the biological properties of these proteins, alterations in their concentrations in various clinical disorders, and their possible physiological role.

Effect of fibroblast growth factor-23 on phosphate transport in proximal tubules

Fibroblast growth factor-23 (FGF-23) has been implicated in the renal phosphate wasting in tumor-induced osteomalacia, X-linked hypophosphatemia, and autosomal-dominant hypophosphatemic rickets. In this in vitro microperfusion study we examined if FGF23R176Q, a stable mutant of FGF-23, impairs phosphate transport in rabbit proximal convoluted and proximal straight tubules perfused in vitro. We also examined if heparin, a molecule that is known to facilitate binding of FGFs to their receptor was necessary for the action of FGF23R176Q on transport. In the presence of heparin, FGF23R176Q reduced phosphate transport from 10.8 +/- 2.0 to 9.9 +/- 1.9 pmol/mm/min in proximal convoluted tubules and 1.0 +/- 0.2 to 0.8 +/- 0.2 pmol/mm/min in proximal straight tubules (both P < 0.05). There was no effect of FGF23R176Q in the absence of heparin. Incubation of finely minced mouse renal cortical tissue in tissue culture media for 3 hours resulted in a reduction in brush border membrane vesicles (BBMV) sodium-dependent phosphate transport (NaPi-2A) protein abundance in the presence but not in the absence of heparin. These data demonstrate that the inhibition of phosphate transport by FGF23R176Q in vitro requires heparin. The action of FGF23R176Q is associated with a reduction in BBMV NaPi-2A protein abundance.

Role of membrane microdomains in PTH-mediated down-regulation of NaPi-IIa in opossum kidney cells

Parathyroid hormone (PTH) rapidly down-regulates type IIa sodium-dependent phosphate transporter (NaPi-IIa) via an endocytic pathway. Since the relationship between PTH signaling and NaPi-IIa endocytosis has not been explored, we investigated the role of membrane microdomains in this process. We examined the submembrane localization of NaPi-IIa in opossum kidney (OK-N2) cells that stably expressed human NaPi-IIa, and searched for a PTH-induced specific phosphorylating substrate on their membrane microdomains by immunoblotting with specific antibody against phospho substrates of protein kinases. We found that NaPi-IIa was primarily localized in low-density membrane (LDM) domains of the plasma membrane; PTH reduced the levels of immunoreactive NaPi-IIa in these domains. Furthermore, PTH activated both protein kinase A (PKA) and protein kinase Calpha (PKCa) and increased the phosphorylation of 250 kD and 80 kD substrates; this latter substrate was identified as ezrin, which a member of the ezrin-radixin-moesin (ERM) protein family. In response to PTH, ezrin was phosphorylated by both PKA and PKC. Dominant negative ezrin blocked the reduction in NaPi-IIa expression in the LDM domains that was induced by PTH. These data suggest that NaPi-IIa and PTH-induced phosphorylated proteins that include ezrin are compartmentalized in LDM microdomains. This compartmentalization may play an important role in the down-regulation of NaPi-IIa via endocytosis.

Defective PTH regulation of sodium-dependent phosphate transport in NHERF-1−/−renal proximal tubule cells and wild-type cells adapted to low-phosphate media

The present experiments using primary cultures from renal proximal tubule cells examine two aspects of the regulation of sodium-dependent phosphate transport and membrane sodium-dependent phosphate transporter (Npt2a) expression by parathyroid hormone (PTH). Sodium-dependent phosphate transport in proximal tubule cells from wild-type mice grown in normal-phosphate media averaged 4.4 +/- 0.5 nmol.mg protein(-1).10 min(-1) and was inhibited by 30.5 +/- 8.6% by PTH (10(-7) M). This was associated with a 32.7 +/- 5.2% decrease in Npt2a expression in the plasma membrane. Proximal tubule cells from Na(+)/H(+) exchanger regulatory factor-1 (NHERF-1)(-/-) mice had a lower rate of phosphate transport compared with wild-type cells and a significantly reduced inhibitory response to PTH. Wild-type cells incubated in low-phosphate media for 24 h had a higher rate of phosphate transport compared with wild-type cells grown in normal-phosphate media but a significantly blunted inhibitory response to PTH. These data indicate a role for NHERF-1 in mediating the membrane retrieval of Npt2a and the subsequent inhibition of phosphate transport in renal proximal tubules. These studies also suggest that there is a blunted phosphaturic effect of PTH in cells adapted to low-phosphate media.

Distinctive features of dietary phosphate supply

Dietary phosphate has profound effects on growth and renal handling of the compound. On the basis of changes in growth rate and food intake, after alterations in phosphate load, our laboratory previously suggested that these effects are mediated by intestinal signals (Landsman A, Lichtstein D, Bacaner M, and Ilani A. Br J Nutr 86: 217-223, 2001). The aim of this study was to further evaluate the role of dietary phosphate on food intake and appetite and specific organ growth, and to test for the presence of a serum factor that may affect renal phosphate handling in phosphate-resupplied rats. The experimental design was based on a comparison between groups of rats receiving identical low-phosphate diets but drinking water containing either phosphate or chloride. We show that 1) changes in food intake after alterations in phosphate load occurred in parallel with variations in digestive system distention, suggesting that dietary phosphate has also a direct effect on appetite; 2) dietary phosphate-dependent growth has a specific effect on the growth of liver and epididymal fat; and 3) serum of rats supplied with phosphate contains a factor that inhibits sodium-dependent phosphate transport in a model of renal proximal tubule cells. Collectively, these observations are in accord with the hypothesis that factor(s) emanating from the digestive system in response to dietary phosphate load may be involved in growth, appetite and renal handling of phosphate.

Nicotinamide prevents the development of hyperphosphataemia by suppressing intestinal sodium-dependent phosphate transporter in rats with adenine-induced renal failure

Nicotinamide has been shown to inhibit intestinal sodium-dependent phosphate transport activity in normal rats. It was reported recently that type IIb sodium-dependent phosphate co-transporter (NaPi-2b) is a carrier of intestinal phosphate absorption, and that its expression level is regulated by serum 1,25-dihydroxyvitamin D [1,25(OH)(2)D] and Pi levels in normal rats. However, in chronic renal failure (CRF), serum 1,25(OH)(2)D and Pi levels are often abnormal. In a rat model of CRF, we investigated whether short-term nicotinamide administration was effective in reducing intestinal phosphate absorption and, if so, whether the effect was mediated by intestinal NaPi-2b. Adenine-induced CRF rats were given a single daily intrapenitoneal administration of nicotinamide or vehicle solution for 6 days, and time course changes in serum Pi, Ca, blood urea nitrogen (BUN) and creatinine levels were monitored. Intestinal phosphate absorption was examined by oral administration of radiolabelled phosphate on the final day. In addition, NaPi-2b protein content in jejunum brush border membranes was determined. Nicotinamide prevented the progressive increase in serum Pi associated with renal failure and significantly inhibited intestinal Pi absorption as assessed by the influx of orally administered radiolabelled phosphate into the circulation. This effect was accompanied by a decrease in NaPi-2b expression in jejunum brush border membranes. In addition, nicotinamide treatment was also associated with less marked elevations in BUN and serum creatinine and a higher creatinine clearance. Nicotinamide inhibited intestinal Pi absorption in a rat model of CRF, at least in part by inhibiting the expression of NaPi-2b, and appeared to protect against the deterioration of renal function.

Cell Signaling through the Protein Kinases cAMP-dependent Protein Kinase, Protein Kinase Cϵ, and RAF-1 Regulates Amphotropic Murine Leukemia Virus Envelope Protein-induced Syncytium Formation

Amphotropic murine leukemia virus (A-MuLV) utilizes the PiT2 sodium-dependent phosphate transporter as its cell surface receptor to infect mammalian cells. The process of A-MuLV infection requires cleavage of the R peptide from the envelope protein. This occurs within virions thereby rendering them competent to fuse with target cells. Envelope proteins lacking the inhibitory R peptide (e.g. envelope (R-) proteins) induce viral envelope-mediated cell-cell fusion (syncytium). Here we have performed studies to determine if cell signaling through protein kinases is involved in the regulation of PiT2-mediated A-MuLV envelope (R-)-induced syncytium formation. Truncated A-MuLV retroviral envelope protein lacking the inhibitory R peptide (R-) was used to induce viral envelope-mediated cell-cell fusion. Signaling through cyclic AMP to activate PKA was found to inhibit envelope-induced cell-cell fusion, whereas treatment of cells with PKA inhibitors H89, KT5720, and PKA Catalpha siRNA all enhanced this cell fusion process. It was noted that activation of PKC, as well as overexpression of PKCepsilon, up-regulated A-MuLV envelope protein-induced cell-cell fusion, whereas exposure to PKC inhibitors and expression of a kinase-inactive dominant-negative mutant of PKCepsilon (K437R) inhibited syncytium formation. v-ras transformed NIH3T3 cells were highly susceptible to A-MuLV envelope-induced cell-cell fusion, whereas expression of a dominant-negative mutant of Ras (N17Ras) inhibited this cell fusion process. Importantly, activation of Raf-1 protein kinase also is required for A-MuLV envelope-induced syncytium formation. Expression of constitutively active BXB Raf supported, whereas expression of a dominant-negative mutant of Raf-1 (Raf301) blocked, A-MuLV-induced cell-cell fusion. These results indicate that specific cell signaling components are involved in regulating PiT2-mediated A-MuLV-induced cell-cell fusion. Selective pharmacological modulation of these signaling components may be an effective means of altering cell susceptibility to viral-mediated cytopathic effects.

Physiological evidence for a sodium-dependent high-affinity phosphate and nitrate transport at the plasma membrane of leaf and root cells of Zostera marina L.

Zostera marina L. is an angiosperm that grows in a medium in which inorganic phosphate (P(i)) and nitrate (NO(3)(-)) are present in micromolar concentrations and must be absorbed against a steep electrochemical potential gradient. The operation of a Na(+)-dependent NO(3)(-) transport was previously demonstrated in leaf cells of this plant, suggesting that other Na(+)-coupled systems could mediate the uptake of anions. To address this question, P(i) transport was studied in leaves and roots of Z. marina, as well as NO(3)(-) uptake in roots. Electrophysiological studies demonstrated that micromolar concentrations of P(i) induced depolarizations of the plasma membrane of root cells. However, this effect was not observed in leaf cells. P(i)-induced depolarizations showed Michaelis-Menten kinetics (K(m)=1.5+/-0.6 microM P(i); D(max)=7.8+/-0.8 mV), and were not observed in the absence of Na(+). However, depolarizations were restored when Na(+) was resupplied. NO(3)(-) additions also evoked depolarizations of the plasma membrane of root cells only in the presence of Na(+). Both NO(3)(-)- and P(i)-induced depolarizations were accompanied by an increase in cytoplasmic Na(+) activity, detected by Na(+)-sensitive microelectrodes. P(i) net uptake (measured in depletion experiments) was stimulated by Na(+). These results strongly suggest that P(i) uptake in roots of Z. marina is mediated by a high-affinity Na(+)-dependent transport system. Both NO(3)(-) and P(i) transport systems exploit the steep inwardly directed electrochemical potential gradient for Na(+), considering the low cytoplasmic Na(+) activity (10.7+/-3.3 mM Na(+)) and the high external Na(+) concentration (500 mM Na(+)).

Thyroid hormones stimulate Na+?Pi transport activity in rat renal brush-border membranes: Role of membrane lipid composition and fluidity

Antecedent studies have suggested that lipid composition and fluidity of cellular membranes of various organs are altered in response to thyroid hormone status. To date, the effects of thyroid hormone status on these parameters have not been examined in rat renal apical membrane in regard to sodium-dependent phosphate transport. In the present study, we determined the potential role of alterations in cortical brush-border membrane lipid composition and fluidity in modulation of Na+-Pi transport activity in response to thyroid hormone status. Thyroid hormone status influences the fractional excretion of Pi, which is associated with alteration in renal brush-border membrane phosphate transport. The increment in Na+-Pi transport in renal BBMV isolated from Hyper-T rats is manifested as an increase in the maximal velocity (Vmax) of Na+-Pi transport. Further, the cholesterol content was significantly increased in renal BBM of Hypo-T rats and decreased in Hyper-T rats as compared to the Eu-T rats. The molar ratio of cholesterol/phospholipids was also higher in renal BBM from hypo-T rats. Subsequently, fluorescence anisotropy of diphenyl hexatriene (rDPH) and microviscosity were significantly decreased in the renal BBM of the Hyper-T rats and increased in the Hypo-T rats as compared to Eu-T rats. The result of this study, therefore, suggest that alteration in renal BBM cholesterol, cholesterol/phospholipid molar ratio, and membrane fluidity play an important role in the modulation of renal BBM Na+-Pi transport in response to thyroid hormone status of animals.

Defective Parathyroid Hormone Regulation of NHE3 Activity and Phosphate Adaptation in Cultured NHERF-1-/- Renal Proximal Tubule Cells

The present experiments using primary cultures of renal proximal tubule cells derived from wild-type and NHERF-1 knockout animals examines the regulation of NHE3 by phenylthiohydantoin (PTH) and the regulation of phosphate transport in response to alterations in the media content of phosphate. Forskolin (34.8 +/- 6.2%) and PTH (29.7 +/- 1.8%) inhibited NHE3 activity in wild-type proximal tubule cells but neither forskolin (-3.2 +/- 3.3%) nor PTH (-16.6 +/- 8.1%) inhibited NHE3 activity in NHERF-1(-/-) cells. Using adenovirus-mediated gene transfer, expression of NHERF-1 in NHERF-1(-/-) proximal tubule cells restored the inhibitory response to forskolin (28.2 +/- 3.0%) and PTH (33.2 +/- 3.9%). Compared with high phosphate media, incubation of wild-type cells in low phosphate media resulted in a 36.0 +/- 6.3% higher rate of sodium-dependent phosphate transport and a significant increase in the abundance of Npt2a and PDZK1. NHERF-1(-/-) cells, on the other hand, had lower rates of sodium-dependent phosphate uptake and low phosphate media did not stimulate phosphate transport. Npt2a expression was not affected by the phosphate content of the media in NHERF-1 null cells although low phosphate media up-regulated PDZK1 abundance. Primary cultures of mice proximal tubule cells retain selected regulatory pathways observed in intact kidneys. NHERF-1(-/-) proximal tubule cells demonstrate defective regulation of NHE3 by PTH and indicate that reintroduction of NHERF-1 repairs this defect. NHERF-1(-/-) cells also do not adapt to alterations in the phosphate content of the media indicating that the defect resides within the cells of the proximal tubule and is not dependent on systemic factors.