Stimulated Phosphate Uptake Research Articles

Intestinal Cell Phosphate Uptake and the Targeted Knockout of the 1,25D3-MARRS Receptor/PDIA3/ERp57

We have crossed ERp57(flx/flx) mice with commercially available mice expressing villin-driven cre-recombinase. Enterocytes isolated from 3- to 4-wk-old littermate (LM) male mice responded to 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] with enhanced phosphate uptake relative to corresponding controls within 1 min of addition, whereas in cells from targeted knockout (KO) mice, the response was severely blunted. Unlike chick enterocytes, mouse enterocytes did not respond to phorbol ester with enhanced phosphate uptake. However, forskolin, which does not stimulate phosphate uptake in chick intestinal cells, did so in enterocytes isolated from either young male LM or KO mice. Intestinal cells isolated from young female LM mice also responded to 1,25(OH)₂D₃ with enhanced phosphate uptake within 5 min of hormone addition, whereas cells from KO mice did not. Forskolin also stimulated phosphate uptake in enterocytes from young female KO or LM mice. As with intestinal cells from adult male chickens or rats, cells from adult (8 wk) male LM mice lost the ability to respond to 1,25(OH)₂D₃) with enhanced phosphate uptake. In contrast, intestinal cells from adult female LM mice did respond with enhanced phosphate uptake within 1 min of steroid hormone addition relative to corresponding controls, and the magnitude of the effect was greater than that observed in enterocytes of young females. Cells isolated from young or adult male or female LM mice failed to respond to 1,25(OH)₂D₃ with enhanced protein kinase C activity. Finally, we have previously reported that mouse enterocytes have cell surface vitamin D receptor; however preincubation of such cells with anti-vitamin D receptor antibodies demonstrated that the classical receptor is not involved in the rapid 1,25(OH)₂D₃-stimulated uptake of phosphate.

The Effects of 24R, 25-dihydroxyvitamin D3 and 24S, 25-dihydroxyvitamin D3 on Phosphate Transport in Chicks In Vivo

Previous work in this laboratory has indicated that 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] inhibits rapid, 1,25-dihuydroxyvitamin D3 [1,25(OH)2D3] stimulated phosphate uptake in isolated intestinal cells and perfused duodenal loops. Studies were undertaken to determine if 24,25(OH)2D3 had a similar effect in vivo. 24,25(OH)2D3 has two isomers which are 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] and 24S,25-dihydroxyvitamin D3 [24S,25(OH)2D3]., which we studied separately and tested over a time course of 1, 5, 10, 15 and 18 hours after steroid using chicks on regular diet, but fasted, and chicks on a lower vitamin D diet. All chicks were anesthetized prior to surgical exposure of the duodenal loop and injection of a solution containing H3 32PO4 into the lumen. Blood was then collected and serum prepared to determine transport of the radionuclide by liquid scintillation counting. An initial time course study of phosphate transport determined that 3 to 9 min of absorption in vivo was in a linear range, as judged by serum levels of radioactivity. Chicks were then injected with either 200 μg 24R,25(OH)2D3, 20 μg 24S,25(OH)2D3 or vehicle for control groups within the same time course studies. We found that the isomers had different effects on phosphate absorption. 24R,25(OH)2D3 had a hypophosphatemic effect in vivo. The serum levels of radionuclide revealed hypophosphatemic effects at 1, 5, 15 and 18 hours time points with a decrease of 56%, 42%, 39% and 43%, respectively, (P< 0.05) compared with controls. In contrast, 24S,25(OH)2D3 stimulated intestinal phosphate absorption at the five hour time point by 64%, but had no other effects at the other time points tested. We conclude that 24R,25(OH)2D3, which is made when dietary vitamin D levels are high, inhibits phosphate transport from the intestine, while 24S,25(OH)2D3 lacks activity in vivo. Keywords: Chick, hypocalcem, hypophosphatemia, intestine, in vivo, phosphate absorption, phosphate transport, serum phosphate, vitamin D, 24S, 25-dihydroxyvitamin D, 24R, 25-dihydroxyvitamin D, phosphate transport, 25-dihydroxyvitamin D3.

Na+-stimulated phosphate uptake system in Synechocystis sp. PCC 6803 with Pst1 as a main transporter

BackgroundMost living cells uptake phosphate, an indispensable nutrient for growth from their natural environment. In Synechocystis sp. PCC 6803, the cells lack phosphate-inorganic transport (Pit) system but contain two phosphate-specific transport (Pst) systems, Pst1 and Pst2. We investigated the kinetics of Pi uptake of these two Pst systems by constructing the two mutants, ΔPst1 and ΔPst2, and comparing their kinetic properties with those of the wild-type cells under both Pi-sufficient and deficient conditions. The effects of pH and Na+ on the uptake of phosphate in Synechocystis were also studied.ResultsGrowth rates of the two mutants and wild type were similar either under phosphate-sufficient or deficient condition. The Km for phosphate uptake was 6.09 μM in wild type and this was reduced to 0.13 μM in ΔPst1 cells and 5.16 μM in the ΔPst2 strain. The Vmax values of 2.48, 0.22, and 2.17 μmol • (min • mg of chlorophyll a)-1 were obtained for wild type, the ΔPst1 and ΔPst2 strains, respectively. A monophasic phosphate uptake was observed in wild-type cells. The uptake of phosphate was energy and pH-dependent with a broad pH optimum between pH 7-10. Osmolality imposed by NaCl stimulated phosphate uptake whereas that imposed by sorbitol decreased uptake, suggesting stimulation of uptake was dependent upon ionic effects.ConclusionThe data demonstrate that Pst2 system of Synechocystis has higher affinity toward phosphate with lower Vmax than Pst1 system. The Pst1 system had similar Km and Vmax values to those of the wild type suggesting that Pst1 is the main phosphate transporter in Synechocystis sp. PCC 6803. The Km of Pst1 of Synechocystis is closer to that of Pit system than to that of the Pst system of E. coli, suggesting that Synechocystis Pst1 is rather a medium/low affinity transporter whereas Pst2 is a high affinity transporter.

The 1,25D3‐MARRS Receptor is Required for Phosphate Uptake in Mouse Intestinal Cells

We have crossed ERp57lfx/flx mice with commercially available mice expressing villin-driven cre-recombinase (Nemere et al., J Biol Chem 285:31859). Enterocytes isolated from 3–4 wk old littermate (LM) male mice responded to 1,25D3 with enhanced phosphate uptake, relative to corresponding controls, whereas in cells from targeted knockout (KO) mice the response was severely blunted. Unlike chick enterocytes, mouse enterocytes did not respond to phorbol ester with enhanced phosphate uptake. However, forskolin, which does not stimulate phosphate uptake in chick intestinal cells did so in enterocytes isolated from either young male LM or KO mice. Intestinal cells isolated from young female LM mice also responded to 1,25D3 with enhanced phosphate uptake, whereas cells from KO mice did not. Forskolin also stimulated phosphate uptake in enterocytes from either young female KO or LM mice. As with intestinal cells from adult male chickens or rats, cells from adult (8wk) male LM mice lost the ability to respond to 1,25D3 with enhanced phosphate uptake. In contrast, intestinal cells from adult female LM mice did respond with enhanced phosphate uptake and the magnitude of the effect was greater than that observed in enterocytes of young females. Cells isolated from young or adult male or female LM mice failed to respond to 1,25D3 with enhanced PKC activity. Finally, we have previously reported that mouse enterocytes have cell surface VDR; however preincubation of such cells with anti-VDR antibodies demonstrated that the classical receptor is not involved in the rapid 1,25D3-stimulated uptake of phosphate.

Stimulation of phosphate uptake and polyphosphate accumulation by activated sludge microorganisms in response to sulfite addition

Enhanced phosphate removal from wastewaters is dependent on the synthesis and intracellular accumulation of polyphosphate by sludge microorganisms. However the role played by polyphosphate in microbial metabolism and the factors that trigger its formation remain poorly-understood. Many examples of the accumulation of the biopolymer by environmental microorganisms are documented; these include a recent report of the presence of large polyphosphate inclusions in sulfur-oxidizing marine bacteria. To investigate whether any link might exist outside the marine environment between the presence of reduced sulfur compounds and enhanced levels of microbial phosphate uptake and polyphosphate accumulation, activated sludge cultures were grown under laboratory conditions in media that contained sulfite, thiosulfate, hydrosulfite or tetrathionate. Only in the presence of sulfite was there any evidence of a stimulatory effect; in medium that contained 0.5 mM sodium sulfite some 17% more phosphate was removed by the sludge, whilst there was an almost two-fold increase in intracellular polyphosphate levels. No indications of sulfite toxicity were observed.

BMP-2 promotes phosphate uptake, phenotypic modulation, and calcification of human vascular smooth muscle cells

Vascular calcification is associated with increased risk of cardiovascular events that are the most common cause of death in patients with end-stage renal disease. Clinical and experimental studies indicate that hyperphosphatemia is a risk factor for vascular calcification and cardiovascular mortality in these patients. Our previous studies demonstrated that phosphate transport through the type III sodium-dependent phosphate cotransporter, Pit-1, was necessary for phosphate-induced calcification and osteochondrogenic phenotypic change in cultured human smooth muscle cells (SMC). BMP-2 is a potent osteogenic protein required for osteoblast differentiation and bone formation that has been implicated in vascular calcification. In the present study, we have examined the effects of BMP-2 on human SMC calcification in vitro. We found that treatment of SMC with BMP-2 enhanced elevated phosphate-induced calcification, but did not induce calcification under normal phosphate conditions. mRNAs for BMP receptors, including ALK2, ALK3, ALK6, BMPR-II, ActR-IIA and ActR-IIB were all detected in human SMCs. Mechanistically, BMP-2 dose-dependently stimulated phosphate uptake in SMC (200 ng/ml BMP-2 vs. vehicle: 13.94 vs. 7.09 nmol/30 min/mg protein, respectively). Real-time PCR and Western blot revealed the upregulation of Pit-1 mRNA and protein levels, respectively, by BMP-2. More importantly, inhibition of phosphate uptake by a competitive inhibitor of sodium-dependent phosphate cotransport, phosphonoformic acid, abrogated BMP-2-induced calcification. These results indicate that phosphate transport via Pit-1 is crucial in BMP-2-regulated SMC calcification. In addition, BMP-2-induced Runx2 and inhibited SM22 expression, indicating that it promotes osteogenic phenotype transition in these cells. Thus, BMP-2 may promote vascular calcification via increased phosphate uptake and induction of osteogenic phenotype modulation in SMC.

English

Phosphorous (P) starved cells of the cyanobacterium&nbsp;Anabaena oryzae&nbsp;showed higher phosphate uptake rates than P-sufficient cells. The P-uptake obeyed saturation kinetics. The Km value for P-deficient cells was lower (54.34&nbsp;mM) than P-sufficient cells (82.64mM) while Vmax&nbsp;was higher in P-deficient and lower in P-sufficient cells. Salinity (NaCl) stimulated phosphate uptake significantly in the cyanobacterium which is followed by greater amount of P-accumulation in the form of polyphosphate bodies. Inhibition of P-uptake in P-deficient cells was 45% in dark grown compared to light grown cells. P-uptake was inhibited 52 and 85% in culture treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; 10&nbsp;mM) and carbonyl cyanide m-chloro phenylhydrazone (CCCP; 100&nbsp;mM), respectively, suggesting that energy for uptake could be derived from oxidative phosphorylation and photophosphorelation. &nbsp; Key words:&nbsp;Anabaena oryzae, metabolic inhibitors, phosphorus-uptake, salinity.

Mechanism of 24,25‐dihydroxyvitamin D3‐mediated inhibition of rapid, 1,25‐dihydroxyvitamin D3‐induced responses: Role of reactive oxygen species

In intestine, 24,25(OH)(2)D(3), which is made under conditions of calcium-, phosphate-, and 1,25(OH)(2)D(3) sufficiency, inhibits the stimulatory actions of 1,25(OH)(2)D(3) on phosphate and calcium absorption. In the current work, we provide evidence that 24,25(OH)(2)D(3)-mediated signal transduction occurs mechanistically through increased H(2)O(2) production which involves binding of 24,25(OH)(2)D(3) to catalase and resultant decreases in enzyme activity. Physiological levels of H(2)O(2) mimicked the action of 24,25(OH)(2)D(3) on inhibiting 1,25(OH)(2)D(3)-stimulated phosphate uptake in isolated enterocytes. Moreover, the molecular basis of such inhibition was suggested by the presence of two thioredoxin domains in the 1,25D(3)-MARRS protein/ERp57: Exposure of cells to either 24,25(OH)(2)D(3) or H(2)O(2) gradually reduced 1,25(OH)(2)D(3) binding to 1,25D(3)-MARRS protein, between 10 and 20 min of incubation, but not to VDR. Feeding studies with diets enriched in the antioxidants vitamins C and E showed that net phosphate absorption in vivo nearly doubled relative to chicks on control diet. Antioxidant diets also resulted in increased [(3)H]1,25(OH)(2)D(3) binding to both 1,25D(3)-MARRS and VDR, suggesting benefits to both transcription- and membrane-initiated signaling pathways. Intriguingly, phosphorous content of bones from birds on antioxidant diets was reduced, suggesting increased osteoclast activity. Because mature osteoclasts lack VDR, we analyzed a clonal osteoclast cell line by RT-PCR and found it contained the 1,25D(3)-MARRS mRNA. The combined data provide mechanistic details for the 1,25(OH)(2)D(3)/24,25(OH)(2)D(3) endocrine system, and point to a role for the 1,25D(3)-MARRS protein as a redox-sensitive mediator of osteoclast activity and potential therapeutic target.

Biological Phosphate Uptake and Release: Effect of pH and Magnesium Ions

Enhanced biological phosphorus removal (EBPR) is based on poly-phosphate accumulating organisms' (PAOs) unique features of "luxury" phosphate uptake during aerobic conditions and phosphate release in anaerobic conditions. It is believed that poly-phosphate accumulation is accompanied by the uptake and accumulation of potassium ions (K+) and magnesium ions (Mg2+). The release of phosphate under anaerobic conditions is also accompanied by the release of both cations. The objective of this research was to evaluate the effect of pH and Mg2+ on the biological phosphate uptake and release behavior of activated sludge mixed liquor during aeration and sedimentation. Research results indicate that Mg2+, supplied either by magnesium chloride (MgCl2) or magnesium hydroxide [Mg(OH)2], stimulated phosphate uptake during the aeration period, while pH increase, caused by the application of Mg(OH)2, enhanced phosphate release during the sedimentation period. It is also noted in our experiments with MgCl2 that Mg2+ slightly inhibited anaerobic phosphate release.

Ribozyme knockdown functionally links a 1,25(OH)2D3 membrane binding protein (1,25D3-MARRS) and phosphate uptake in intestinal cells.

We used a ribozyme loss-of-function approach to demonstrate that the protein product of a cDNA encoding a multifunctional membrane-associated protein binds the seco-steroid 1,25(OH)(2)D(3) and transduces its stimulatory effects on phosphate uptake. These results are paralleled by studies in which the ability of the hormone to stimulate phosphate uptake in isolated chick intestinal epithelial cells is abolished by preincubation with Ab099 directed against the amino terminus of the protein. We now report the complete sequence of the cloned chicken cDNA for the 1,25D(3)-MARRS (membrane-associated, rapid-response steroid-binding) protein and reveal it to be identical to the multifunctional protein ERp57. Functional studies showed that active ribozyme, but not a scrambled control, decreased specific membrane-associated 1,25(OH)(2)D(3) binding, but did not affect binding to the nuclear receptor for 1,25(OH)(2)D(3). Seco-steroid-dependent stimulation of protein kinase C activity was diminished as 1,25D(3)-MARRS protein levels were reduced in the presence of the ribozyme, as judged by Western blot analyses. Phosphate uptake in isolated cells is an index of intestinal phosphate transport that occurs during growth and maturation. Whereas cells and perfused duodena robustly responded to 1,25(OH)(2)D(3) in preparations from young birds, older animals no longer responded with stimulated phosphate uptake or transport. The age-related decline was accompanied by a decrease in 1,25D(3)-MARRS mRNA that was apparent up to 1 year of age. Together, these studies functionally link phosphate transport in the chick duodenum with the 1,25D(3)-MARRS protein and point to a previously uncharacterized role for this multifunctional protein class.

Functional expression of a stably transfected parathyroid hormone/parathyroid hormone related protein receptor complementary DNA in CHO cells

Chinese hamster ovary (CHO) cells were stably transfected with OK-O complementary DNA encoding the parathyroid hormone/parathyroid hormone related protein (PTH/PTHrP) receptor derived from opossum kidney (OK) cells (Jüppner et al., 1991). A subclone of transfected CHO cells, CHO-E 2, presented high affinity binding of 125I-labeled [Tyr 36]chickenPTHrP(1–36)amide ([ 125I]chPTHrP(1–36)) ( K d 1.28 ± 0.10nM) similar to that of wildtype OK cells ( K d 2.23 ± 0.16nM) (P< 0.01). Photoaffmity labeling of the PTH/PTHrP receptors using N-hydroxysuccinimidyl-4-azidobenzoate modified [ 125I]chPTHrP(1–36) revealed the same specifically labeled 90kDa protein in CHO-E 2 and OK cells. In CHO-cells, chPTHrP(1–36) stimulated cyclic AMP accumulation in dose-dependent fashion (EC 50 0.15±0.04nM) and raised peak cytosolic free calcium concentration (EC 50 2.90 ± 0.36 nM) independent of extracellular calcium, and stimulated phosphate uptake (EC 50 0.21 ± 0.07nM). Both, chPTHrP(1–36) and 12-0-tetradecanoylphorbol-13-acetate stimulated phosphate uptake were suppressed by staurosporine. But, Sp-cyclic adenosine-3',5'-monophosphothioate did not affect phosphate uptake in CHO-E 2 cells. In conclusion, a PTH/PTHrP receptor stably expressed in CHO cells is linked to stimulation of phosphate uptake. Receptor coupling presumably occurred through the protein kinase C rather than the protein kinase A pathway.

Characterization and expression of a novel Na+-inorganic phosphate transporter at the liver plasma membrane of the rat

Background: Phosphate transport across the plasma membrane of the intestine and kidney occur by an Na+-dependent process. The mechanism by which phosphate enters the hepatocytes across the basolateral membrane is not known. Therefore, our study was designed to investigate whether the plasma membranes of the liver possess a specialized transport system for translocation of phosphate into the hepatocyte. Methods: Liver plasma membrane vesicles and expression of liver poly(A)+ RNA into Xenopus laevis oocytes was used. Results: Phosphate was driven into the intravesicular space as depicted by the equation y = 493.96x + 0.001, r2 = 0.96. Inwardly directed Na+ and pH gradients stimulated phosphate uptake, with a Vmax of 0.58 ± 0.03 and 0.22 ± 0.03 nmol/mg protein/10 s, at pH 6.1 and 7.4, respectively (P < 0.05). Km values were 0.39 ± 0.07 and 0.25 ± 0.1 mmol/L, respectively. To confirm the presence of a phosphate carrier, the liver Na+ phosphate transporter was expressed in X. laevis oocytes. The size-selected messenger RNA encoding for the phosphate transporter was 1.6 kilobases (kb) with kinetic parameters of Vmax 6.75 ± 0.9 pmol/mg protein/5 min and Km of 0.29 ± 0.1 mmol/L, compared with a Vmax of 0.41 ± 0.03 pmol/mg protein/5 min and a Km of 0.085 ± 0.022 mmol/L for water injection. Colonic poly(A)+ RNA did not stimulate phosphate uptake in the oocytes. Conclusion: The combined studies of plasma membrane vesicles and the expression system into X. laevis oocytes confirm the presence of an Na+-phosphate transporter at the liver plasma membranes.

Multiple modulation of Na-dependent Pi uptake by cellular Ca in MDCK cells.

The events accounting for the adaptation of the sodium-dependent phosphate cotransport (Na-Pi) to phosphate deprivation other than genomic regulation remain unknown. The involvement of changes in intracellular calcium concentration was investigated in Madin-Darby canine kidney (MDCK) cells. Calcium concentration was decreased by 15 h of phosphate deprivation (-24 to -35%) or low-calcium medium (calcium deprivation) (-45%), or 8-(N,N'-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB8) (-32%). Calcium deprivation stimulated Na-Pi (2-fold at 1 h and up to 15 h) by increasing the affinity for phosphate. Combined calcium and phosphate deprivation had more than additive effects on phosphate uptake. The effect of a 15-h calcium deprivation, but not of a 2-h one, was dependent on gene transcription and protein synthesis. TMB8 stimulated phosphate uptake similarly to phosphate deprivation (increase in maximum velocity dependent on gene transcription). The ionophore A23187 decreased basal Na-Pi as well as its stimulation by phosphate or calcium deprivation or by TMB8. Calcium deprivation stimulated (3.2-fold increase) the sodium-coupled alanine transport, whereas phosphate deprivation and TMB8 did not. We conclude that 1) phosphate deprivation decreases intracellular calcium concentration, 2) low intracellular calcium concentration is instrumental in the stimulation by prolonged calcium or phosphate deprivation of Na-Pi, and 3) phosphate or calcium deprivation modulates Na-Pi through different cellular pathways.

PKC and Pi deprivation modulate differently the ubiquitous Na-dependent Pi uptake in MDCK cells

The role of protein kinase C (PKC) in the modulation of the ubiquitous sodium-dependent phosphate transport and in adaptation of that transport to phosphate deprivation was investigated in MDCK cells. Phorbol myristate acetate (PMA) had a biphasic effect on sodium-dependent phosphate uptake characterized by early inhibition (-25% at 1 h) followed by late stimulation (2.3-fold at 15 h). Late stimulation was related to a decreased apparent affinity (Km) with unchanged maximal velocity (Vmax). The 15-h stimulation of phosphate uptake was also induced by an initial 1-h PMA treatment followed by a 14-h washout of PMA or by R59 022. The stimulation was inhibited by PKC downregulation. PMA stimulation was dependent on protein synthesis but not on transcription, as shown by the respective effects of cycloheximide, 3'-deoxyadenosine, and actinomycin D. In phosphate-deprived cells PMA had also a biphasic effect. A potentiation of PMA stimulation of phosphate uptake with phosphate deprivation was observed. Adaptation to phosphate deprivation was not prevented by PKC downregulation. Cytosolic and membranous PKC activities were not changed by 15-h phosphate deprivation. We conclude that 1) PKC modulates sodium-dependent phosphate uptake in MDCK cells, and 2) phosphate deprivation and PKC modulation of sodium-dependent phosphate uptake involve different cellular pathways; that is, phosphate deprivation acts through gene regulation, and PKC acts through translation regulation.

5-HT1A and histamine H1 receptors in HeLa cells stimulate phosphoinositide hydrolysis and phosphate uptake via distinct G protein pools.

Regulation of phosphate uptake was studied in a HeLa cell line after transfection with DNA encoding the human 5-HT1A receptor. In these cells, 5-HT stimulates sodium-dependent phosphate uptake via protein kinase C activation. Endogenous histamine H1 receptors (739 +/- 20 fmol/mg protein) were identified with [3H]pyrilamine. Histamine (i) stimulated phosphoinositide hydrolysis (EC50 = 8.6 +/- 4.1 microM), (ii) activated protein kinase C (2.4-fold increase in activity), and (iii) increased phosphate uptake (EC50 = 3.2 +/- 1.8 microM) by increasing maximal transport (Vmax(basal) = 6.2 +/- 0.3 versus Vmax(histamine) = 9.1 +/- 0.4) without changing the affinity of the transport process for phosphate. Prolonged treatment with 16 microM phorbol 12-myristate 13-acetate completely blocked protein kinase C activation and markedly attenuated the stimulation of phosphate uptake induced by histamine, establishing that 5-HT and histamine stimulate phosphate uptake through the common pathway of protein kinase C activation. The linkages of the histamine H1 and 5-HT1A receptors to G protein pools were assessed in two ways. (i) The stimulation of phosphoinositide hydrolysis, protein kinase C activity, and phosphate uptake associated with histamine were insensitive to pertussis toxin, whereas those associated with 5-HT were very sensitive to pertussis toxin. (ii) The stimulation of phosphoinositide hydrolysis, protein kinase C activity, and phosphate uptake induced by histamine and 5-HT were additive. These findings suggest that distinct receptor types can stimulate phosphoinositide hydrolysis, protein kinase C, and phosphate uptake in an additive fashion through distinct pools of G proteins in a single cell type.

Short-term regulation of Na+/K+ adenosine triphosphatase by recombinant human serotonin 5-HT1A receptor expressed in HeLa cells.

Agonist occupancy of the cloned human serotonin (5-HT)1A receptor expressed in HeLa cells stimulates Na+/K+ ATPase activity as assessed by rubidium uptake. The purpose of the study was to determine which of the receptor-associated signaling mechanisms was responsible for this effect. 5-HT stimulated Na+/K+ ATPase 38% at 2 mM extracellular potassium, an effect characterized by a decrease in apparent K0.5 from 2.8 +/- 0.3 to 1.8 +/- 0.3 mM potassium without a significant change in apparent Vmax. The EC50 for the transport effect was approximately 3 microM 5-HT. The response was pertussis toxin-sensitive but did not involve inhibition of adenylate cyclase, as stimulation of Na+/K+ ATPase by 5-HT was observed in the presence of excess dibutyryl cAMP. Protein kinase C was not required for the response since short-term incubation with the phorbol esters phorbol 12 myristate, 13 acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) did not mimic the 5-HT effect. Moreover, 5-HT increased Na+/K+ ATPase activity after inactivation of protein kinase C by overnight incubation with PMA. 5-HT and the sesquiterpene lactone thapsigargin increased cytosolic calcium in this cell model, and the EC50 for 5-HT corresponded with that for stimulation of Na+/K+ ATPase. Both thapsigargin and A23187, a calcium ionophore, also increased Na+/K+ ATPase activity in a dose-responsive fashion. The response to 5-HT, thapsigargin, and A23187 was blocked by conditions that removed the cytosolic calcium response. By two-dimensional gel electrophoresis, we established evidence for a calcium-sensitive but protein kinase C-independent signaling pathway. We conclude that the 5-HT1A receptor, which we have previously shown to stimulate phosphate uptake via protein kinase C, stimulates Na+/K+ ATPase via a calcium-dependent mechanism. This provides evidence for regulation of two separate transport processes by a single receptor subtype via different signaling mechanisms.

The Human 5-HT1A Receptor Expressed in HeLa Cells Stimulates Sodium-dependent Phosphate Uptake via Protein Kinase C

Regulation of phosphate uptake was studied in HeLa cell lines after transfection with DNA encoding the human 5-HT1A receptor. Phosphate uptake was saturable and greater than 90% sodium-dependent, with Vmax approximately 30-35% without changing Km. Treatment with 5-HT or the 5-HT1A-specific agonist 8-OH-2-(di-n-propylamino)1,2,3,4-tetrahydronaphthalene increased Vmax approximately 40% without affecting Km. This effect was blocked by pretreatment with the 5-HT1 antagonists, methiothepine and spiperone, or pertussis toxin. Surprisingly, the stimulation was not secondary to an inhibition of adenylyl cyclase because 5-HT stimulated phosphate uptake approximately 20% in the presence of 1 mM 8-Br-cAMP. Rather, the primary pathway linked to the stimulation of phosphate uptake involved activation of protein kinase C because (i) 5-HT measurably activated protein kinase C in these cells, (ii) activators of protein kinase C (phorbol esters and diacylglycerol analogues) stimulated phosphate uptake in these cells (iii) the half-maximal doses for 5-HT-induced phosphatidylinositol hydrolysis and stimulation of phosphate uptake were virtually equivalent, and both effects were equally sensitive to pertussis toxin, and (iv) the stimulation was markedly attenuated in cells made deficient in protein kinase C. These results demonstrate that the stimulation of phosphatidylinositol hydrolysis by the 5-HT1A receptor can generate physiologically measurable effects on cellular transport and suggest that such accessory pathways may play a prominent role in signal transduction.

Intestinal maturation: characterization of mitochondrial phosphate transport in the rat and its regulation by 1,25-(OH)2 vitamin D3.

The mitochondria play a major role in the regulation of oxidative phosphorylation within the cell. Despite the fact that the enterocytes receive the majority of absorbed phosphate and their high metabolic turnover rate, the role of the intestinal mitochondria in phosphate transport system during maturation is not known. Therefore, the current studies were designed to characterize phosphate transport by jejunal mitochondria of rats during maturation (suckling, weanling, and adolescent rats). The functional integrity of the intestinal mitochondria of suckling and adolescent rats was determined by oxygen consumption studies demonstrating respiratory control ratios of more than 3 when succinate was used as a test substrate. Phosphate uptake was significantly stimulated by the presence of 3 mM ATP at all age groups studied. Maximal phosphate uptake in the presence of 3 mM ATP and 2 mM succinate was 16.5 +/- 1.0, 20.5 +/- 1 and 28.7 +/- 0.4 nmol/mg protein (mean +/- SE) in suckling, weanling, and adolescent rats respectively. ATP-dependent phosphate uptake was inhibited by 80% with 100 microM p-MB. Kinetic parameters for ATP stimulated phosphate uptake at 10 s revealed a Km of 4 +/- 0.9, 2.8 +/- 0.4, and 0.9 +/- 0.1 mM and Vmax of 5 +/- 0.7, 9.5 +/- 1, and 11 +/- 0.7 nmol/mg protein per 10 s in suckling, weanling, and adolescent rats, respectively. Phosphate uptake was also stimulated by an inwardly directed pH gradient (pH out less than pH inside) compared to no pH gradient condition suggesting the presence of PO4-/OH- exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation by thyroid hormone of phosphate transport in primary cultured renal cells

The regulation by thyroid hormone of phosphate transport in primary cultured chick renal cells was examined. The more physiologically active L-analogs of triiodothyronine and thyroxine, but not the D-analogs of the hormones, stimulated the Na+-dependent phosphate uptake system. Na+-independent phosphate uptake and Na+-dependent uptakes of alpha-methylglucoside and L-proline were unaffected. The increase in Na+-dependent phosphate uptake was concentration dependent, exhibited an induction period, and was blocked by inhibitors of RNA and protein synthesis. The stimulation of phosphate uptake by triiodothyronine was due to an increased Vmax rather than to an altered affinity for phosphate. These findings demonstrate that thyroid hormone acts directly on renal cells to modulate phosphate transport and suggest that the renal cell system may serve as a model to examine the mechanism by which thyroid hormone controls gene expression and regulates plasma membrane transport function.

Electrogenicity of phosphate transport by renal brush-border membranes.

Phosphate uptake by rat renal brush-border membrane vesicles was studied under experimental conditions where transmembrane electrical potential (delta psi) could be manipulated. Experiments were performed under initial rate conditions to avoid complications associated with the dissipation of ion gradients. First, phosphate uptake was shown to be strongly affected by the nature of Na+ co-anions, the highest rates of uptake being observed with 100 mM-NaSCN (1.010 +/- 0.086 pmol/5 s per micrograms of protein) and the lowest with 50 mM-Na2SO4 (0.331 +/- 0.046 pmol/5 s per micrograms of protein). Anion substitution studies showed that potency of the effect of the co-anions was in the order thiocyanate greater than nitrate greater than chloride greater than isethionate greater than gluconate greater than sulphate, which correlates with the known permeability of the membrane to these anions and thus to the generation of transmembrane electrical potentials of decreasing magnitude (inside negative). The stimulation by ion-diffusion-induced potential was observed from pH 6.5 to 8.5, indicating that the transport of both monovalent and divalent phosphate was affected. In addition, inside-negative membrane potentials were generated by valinomycin-induced diffusion of K+ from K+-loaded vesicles and showed a 57% stimulation of phosphate uptake, at pH 7.5. Similar experiments with H+-loaded vesicles, in the presence of carbonyl cyanide m-chlorophenylhydrazone gave a 50% stimulation compared with controls. Inside-positive membrane potentials were also induced by reversal of the K+ gradient (outside greater than inside) in the presence of valinomycin and gave 58% inhibition of phosphate uptake. The membrane-potential dependency of phosphate uptake was finally analysed under thermodynamic equilibrium, and a stimulation by inside-negative potential was observed. The transport of phosphate was thus driven against a concentration gradient by a membrane potential, implicating the net transfer of a positive charge during the translocation process. These results indicate a major contribution of electrical potential to phosphate uptake in renal brush-border membranes.