Inhibition Of Phosphate Uptake Research Articles

Effects of 1,25-Dihydroxyvitamin D 3 on intracellular Ca 2+ in cultured mesangial cells

The N-terminal regions of 1–34 parathyroid hormone (PTH) and 1–34 parathyroid hormone related protein (PTHrP) are thought to be required for full agonist activity of these molecules and for signal transduction by cyclic AMP (cAMP). The C-terminal regions are thought to be involved in receptor binding and protein kinase C activation. In this study, two analogs of PTH/PTHrP lacking the segment 1–14 exhibited agonist activity in opossum kidney (OK) 3B2 cells. Analogs cPTHrP(15–34) and ANA NPY(13–36), an analog of neuropeptide Y, which both have amphipathic α helices, inhibited phosphate uptake and stimulated cAMP production in a dose-dependent manner, with half maximal activity in the μM range, compared to the nM range for hPTHrP(1–34) and hPTH(1–34). They also exhibited proportionately lower receptor binding affinities. cAMP production by these analogs was suppressed by the antagonist hPTHrP(7–34). Inhibition of phosphate uptake in response to the analogs was partially suppressed by H-89, but not by bisindolylmaleimide. The analogs also inhibited phosphate uptake and stimulated cAMP in parent OK cells and stimulated cAMP production in UMR-106 cells. These studies present the novel finding that in these cell types, a C-terminal region encompassing PTH/PTHrP(24–31), with the α-helical structure maintained, is sufficient for full activity at reduced potency.

Pineal regulation of calcitonin in the lizard, [formula omitted

The N-terminal regions of 1–34 parathyroid hormone (PTH) and 1–34 parathyroid hormone related protein (PTHrP) are thought to be required for full agonist activity of these molecules and for signal transduction by cyclic AMP (cAMP). The C-terminal regions are thought to be involved in receptor binding and protein kinase C activation. In this study, two analogs of PTH/PTHrP lacking the segment 1–14 exhibited agonist activity in opossum kidney (OK) 3B2 cells. Analogs cPTHrP(15–34) and ANA NPY(13–36), an analog of neuropeptide Y, which both have amphipathic α helices, inhibited phosphate uptake and stimulated cAMP production in a dose-dependent manner, with half maximal activity in the μM range, compared to the nM range for hPTHrP(1–34) and hPTH(1–34). They also exhibited proportionately lower receptor binding affinities. cAMP production by these analogs was suppressed by the antagonist hPTHrP(7–34). Inhibition of phosphate uptake in response to the analogs was partially suppressed by H-89, but not by bisindolylmaleimide. The analogs also inhibited phosphate uptake and stimulated cAMP in parent OK cells and stimulated cAMP production in UMR-106 cells. These studies present the novel finding that in these cell types, a C-terminal region encompassing PTH/PTHrP(24–31), with the α-helical structure maintained, is sufficient for full activity at reduced potency.

Characterization of essential arginine residues implicated in the renal transport of phosphate and glucose

We have characterized the reaction of arginine-specific reagents with the phosphate and glucose carriers of the kidney brush-border membrane. The inhibition of phosphate and glucose transport by phenylglyoxal follows pseudo-first-order kinetics. The rate of inactivation of phosphate transport by 50 mM phenylglyoxal was about 3-fold higher than that for glucose transport ( k app was 0.052 s −1 for the uptake of phosphate and 0.019 s −1 for the uptake of glucose). The order of the reaction, n, with respect to phenylglyoxal was 1.25 and 1.31 for the inactivation of phosphate and glucose transport, respectively. The inactivation of phosphate flux by p-hydroxyphenylglyoxal also follows pseudo-first-order kinetics, but the inhibition rate ( k app = 0.0012 s −1) was slower than with phenylglyoxal. The inactivation increased with the alkalinity of the preincubation medium for both phosphate and glucose fluxes and was maximal at pH 9.0. The inactivation of phosphate flux by phenylglyoxal depends upon the presence of an alkaline intravesicular pH. Extravesicular pH does not affect the reaction. Phenylglyoxal does not interfere with the recycling of the protonated carrier since phosphate uptake is inhibited independently of the pH used for transport measurements. Moreover, phenylglyoxal completely abolished trans stimulation by phosphate. Trans sodium inhibited phosphate uptake and abolished the pH profile of phosphate uptake.

Toxic effects on algal phosphate uptake

The inhibitory effects on growth and phosphate uptake by two toxicants, 3,5-dichloro-phenol and potassium dichromate, were studied in batch culture experiments with the freshwater green alga Selenastrum capricornutum. The EC50 figures for inhibition of phosphate uptake were considerably smaller than corresponding figures for growth inhibition, but threshold toxicities did not differ as much. Toxic inhibition of phosphate uptake may potentially influence interspecies competition among phytoplankton.

Effect of Dexamethasone on Parathyroid Hormone (PTH) and PTH-Related Protein Regulated Phosphate Uptake in Opossum Kidney Cells*

Effects of dexamethasone (DEX) on receptor binding of bovine PTH (1-34) [bPTH (1-34)] and human PTH-related protein (1-34) [hPTHrP(1-34)] and cAMP accumulation, and on the inhibition of Na(+)-dependent phosphate uptake were studied in opossum kidney (OK) cells. Maximal specific binding of [125I]hPTHrP(1-34) was reduced by 40% in cells treated with 100 nM DEX, but half-maximal inhibition of binding of [125I]hPTHrP(1-34) by bPTH(1-34) or hPTHrP(1-34) was unaffected by DEX (0.5 nM and 1.4 nM, in the absence and presence of DEX, respectively). Moreover, the EC50 of bPTH(1-34) and hPTHrP(1-34)-evoked cAMP accumulation ranged from 3-7 nM and was the same with and without DEX. The EC50 of the inhibition of phosphate uptake by bPTH(1-34) and hPTHrP(1-34) ranged from 0.2-0.5 nM. Treatment with 100 nM DEX increased phosphate uptake 1.5-fold (EC50 3 nM), and the inhibition of phosphate uptake by bPTH(1-34) and hPTHrP(1-34) was suppressed. The inhibition of phosphate uptake by forskolin was also suppressed in cells treated with 100 nM DEX. DEX, on the other hand, enhanced forskolin-stimulated cAMP accumulation 1.6- to 1.8-fold. 12-O-Tetradecanoylphorbol-13-acetate did not affect cAMP production, but phosphate uptake was inhibited both in the absence and the presence of 100 nM DEX (EC50 3 nM). In conclusion, treatment of OK cells with DEX only minimally affected PTH receptor binding, and cAMP accumulation evoked by bPTH(1-34) and hPTHrP(1-34) remained unaltered. The inhibition of phosphate uptake by PTH, however, was suppressed.

Human PTH-(3–34) inhibited the effects of human parathyroid hormone-related protein on phosphate uptake in a cultured renal cell line (OK cells)

The action mechanism of hPTH and hPTHrP-(1-34) on phosphate uptake in opossum kidney (OK) cells was studied using [Nle8,18Tyr34]hPTH-(3-34)-NH2, a potent competivie inhibitor of adenylate cyclase-coupled PTH receptor. We examined the effects of hPTH-(1-34), hPTHrP-(1-34), and hPTH-(3-34) separately or in combination on the change in renal cyclic AMP production and phosphate uptake in OK cells. Both hPTH-(1-34) and hPTHrP-(1-34) stimulated intracellular cyclic AMP production to the same degree at concentrations between 10(-10) and 10(-7) M and inhibited phosphate uptake equipotently on a molar basis (27.5 +/- 2.0 and 33.2 +/- 1.2% inhibition at 10(-7) M, respectively). Both exogenous addition of (Bu)2cAMP and endogenous stimulation of cAMP by forskolin inhibited phosphate uptake in a dose-dependent manner. Cyclic AMP production induced by either hPTH-(1-34) or hPTHrP-(1-34) was inhibited by both [Nle8,18Tyr34]-hPTH-(3-34)-NH2 and [Tyr34]-hPTH-(7-34)-NH2. However, [Nle8,18Tyr34]hPTH-(3-34)-NH2 and [Tyr34]-hPTH-(7-34)-NH2 inhibited hPTH-induced cAMP production more strongly. The inhibitory action of phosphate uptake by hPTH-(1-34) and hPTHrP-(1-34) was prevented in the presence of a 100-fold greater concentration of [Nle8,18Tyr34]hPTH-(3-34)-NH2. The antagonistic action of [Nle8,18Tyr34]hPTH-(3-34)-NH2 on the inhibition of phosphate uptake induced by hPTH-(1-34) and hPTHrP-(1-34) became weaker with time (0-120 minutes), and [Nle8,18Tyr34]hPTH-(3-34)-NH2 did not antagonize the inhibition of phosphate uptake induced by hPTHrP-(1-34) at 120 minutes of incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Dissociation of cAMP accumulation and phosphate uptake in opossum kidney (OK) cells with parathyroid hormone (PTH) and parathyroid hormone related protein (PTHrP)

Bovine parathyroid hormone (PTH) 1–34 [bPTH(1–34)] and human PTH related protein [hPTHrP(1–34)] stimulated cAMP accumulation in opossum kidney (OK) cells with K m of 5 × 10 −9 M , but inhibition of phosphate uptake was obtained with 17-fold lower K m of 3 × 10 −10 M . Phosphate uptake was partially inhibited with [Nle 8,18Tyr 34]bPTH(3–34)NH 2 without concomitant cAMP stimulation. With hPTHrP(7–34)NH 2, cAMP accumulation was increased in parallel to inhibition of phosphate uptake. [D-Trp 12Tyr 34]bPTH(7–34)NH 2 and [Tyr 34]hPTH(7–34)NH 2 had no agonist activity on cellular cAMP and inhibition of phosphate uptake. bPTH(1–34)-stimulated cAMP accumulation was antagonized by [Nle 8,18Tyr 34]bPTH(3–34)NH 2, [D-Trp 12Tyr 34]bPTH (7–34)NH 2, hPTHrP(7–34)NH 2 and [Tyr 34]hPTH(7–34)NH 2 with K i of 1.4 × 10 −7 , 2 × 10 −7 , 4.7 × 10 −7 and 3.7 × 10 −6 M , respectively. But [Nle 8,18Tyr 34]bPTH(3–34)NH 2 and [D-Trp 12Tyr 34]bPTH(7–34)NH 2 reversed the inhibition of phosphate uptake only marginally, and hPTHrP(7–34)NH 2 and [Tyr 34]hPTH(7–34)NH 2 were inactive. With hPTHrP(1–34) the K i for cAMP accumulation of [Nle 8,18Tyr 34]bPTH(3–34)NH 2 and hPTHrP(7–34)NH 2 were 1.9 × 10 −7 and 7.2 × 10 −7 M , and inhibition of phosphate uptake was partially reversed with [Nle 8,18Tyr 34]bPTH(3–34)NH 2, but not with hPTHrP(7–34)NH 2. The present results indicate that truncated hPTHrP(7–34)NH 2, unlike [Tyr 34]hPTH(7–34)NH 2 and [D-Trp 12Tyr 34]bPTH(7–34)NH 2, elevates cellular cAMP and inhibits phosphate uptake. bPTH(1–34)- and hPTHrP(1–34)-evoked cAMP accumulation is suppressed by PTH and PTHrP fragments while inhibition of phosphate uptake remains largely unaltered.

Sodium‐dependent phosphate transport in primary cultures of renal tubule cells from young and adult rats

The transport of phosphate by primary cultures of renal cells from young (5-6 weeks) and adult (10-12 months) rats was studied. Renal tubule cells isolated from young and adult groups exhibited typical epithelial morphology and similar growth rates. The Na-dependent phosphate uptake was saturable with a Km of 5-7 microM over a substrate range of 1-500 microM. A decrease in Na-dependent phosphate uptake in adult cells (30%) was found compared to that of young cells. The Na-independent component of phosphate uptake did not vary with age. In addition, the inhibition of phosphate uptake by a variety of compounds (ouabain, gramicidin, 2,4-dinitrophenol, KCN, and arsenate) were similar in both age groups. Kinetic analysis showed that a significant reduction in Vmax (4.4 +/- 0.4 vs. 3.1 +/- 0.2 nmol Pi/mg protein/10 min in young and adult cells, respectively), but not Km, resulted in this decreased uptake of phosphate in adult groups. There was no difference in the efflux of phosphate from both age groups. When cells were preincubated in a phosphate-free medium for 24 hours, the uptake of phosphate was increased to 46% and 24% of their corresponding controls in young and adult cells, respectively. The decreased phosphate uptake and limited adaptation to a phosphate-free medium by the adult renal cells may account for the hypophosphatemia and phosphaturia seen in adult and old animals in vivo.

Protein kinase A, cytosolic calcium, and phosphate uptake in human proximal renal cells

Phosphate uptake by proximal renal cells derived from the human kidney occurs by a saturable process that is approximately 85% dependent on the presence of sodium. Kinetic analysis is consistent with two distinct transport events with Km of 0.08 and 0.63 mM and Vmax of 3.4 and 11.0 nmol.mg-1.3 min-1, respectively. Parathyroid hormone (PTH), isoproterenol, and prostaglandin E2 (PGE2) increased cellular adenosine 3',5'-cyclic monophosphate (cAMP). PTH-stimulated cAMP prevented binding of the photolabel 8-azido[32P]cAMP with a half-maximal effective concentration (EC50) of 1 nM PTH, 30-fold lower than the EC50 for intracellular cAMP accumulation. These data are qualitatively similar to those observed in OK cells. PTH did not inhibit phosphate uptake in human cells, although it activated cAMP-dependent protein kinase and increased cytosolic calcium. Thus phosphate uptake in human proximal renal cells maintained in short-term culture is unresponsive to PTH in spite of increased cytosolic calcium and activation of the cAMP pathway.

Sodium gradient-dependent phosphate transport in placental brush border membrane vesicles

Abstract We recently described a sodium gradient-dependent transport of phosphate through the brush border membrane vesicles from human placenta. In order to characterize this transport carrier further, we studied the influence of temperature and membrane potential on the transport of this electrolyte, the stoichiometry of the sodium-phosphate interaction, and the interrelationship between phosphate uptake and other sodium-dependent systems. Temperature influenced phosphate uptake by changing the maximal velocity and the affinity of the carrier for the substrate. The Arrhenius plot for uptake velocity exhibited an abrupt breakpoint at 28.6°C, suggesting that membrane fluidity is a factor in phosphate uptake. Increasing the sodium concentration in the incubation medium augmented the phosphate uptake according to a sigmoid curve, and the Hill plot analysis of these data indicates that at least two sodium ions are transported with each phosphate radical. The effect of membrane potential on phosphate uptake was studied by inducing potassium diffusion with vafinomycin and by using various sodium salts with different anion conductance in the incubation medium. In both series of experiments, the inside-negative potential significantly enhanced phosphate uptake. We concluded that the phosphate-sodium cotransport is an electrogenic process, a conclusion which is compatible with the observation that at least two sodium ions accompany each phosphate radical. Glycine, alanine and proline all inhibited phosphate uptake according to an uncompetitive type of inhibition. In contrast, the addition of glucose to the incubation medium had no effect.

Rat Atrial Natriuretic Factor (ANP-III) Inhibits Phosphate Transport in Brush Border Membrane from Superficial and Juxtamedullary Cortex

Previous studies have shown that administration of synthetic atrial natriuretic factor (ANF, 101-126) decreases sodium-dependent phosphate transport across renal brush border membrane vesicles (BBMV) in rats fed a normal or low phosphate diet. In the present study, infusion of rat ANF (atriopeptin III (ANP-III), 103-126 rat ANF) to rats fed a normal phosphate diet caused natriuretic and phosphaturic effects similar to those of ANF (101-126), but unlike ANF (101-126) did not increase the glomerular filtration rate. The effect of ANP-III infusion on sodium-dependent transport of phosphate was also determined in BBM vesicles isolated from the superficial cortex (BBMV-SC) and juxtamedullary cortex (BBMV-JM). The results indicate that ANP-III decreases phosphate transport across BBMV-SC and BBMV-JM similarly (20-24%). However, it had no effect on sodium-dependent transport of proline in these vesicles. The infusion of ANP-III to rats fed a normal phosphate diet inhibits phosphate uptake both in BBMV-SC and BBMV-JM and causes phosphaturia without increments in glomerular filtration rate.

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.

Atrial natriuretic factor inhibits phosphate uptake in opossum kidney cells: As a model of renal proximal tubules

The cultured renal cell, an opossum kidney (OK) cell line, which contains several features characteristic of proximal tubular cells, was utilized to examine the direct effects of atrial natriuretic factor (ANF) and cyclic GMP (cGMP) on phosphate uptake. ANF at 2 x 10(-7) M significantly inhibited phosphate uptake by 10.1% of control (P less than 0.01). Incubation of the cells with ANF (10(-8) to 10(-6) M) resulted in an increment of intracellular cGMP in a dose dependent fashion. Exogenous addition of 8-bromo-cGMP (10(-4) M) also significantly inhibited phosphate uptake by 14.6%. These results suggest that ANF directly inhibits phosphate transport in renal proximal tubular cells, probably through stimulation of cGMP production.

Sodium gradient-dependent phosphate transport in placental brush border membrane vesicles.

We recently described a sodium gradient-dependent transport of phosphate through the brush border membrane vesicles from human placenta. In order to characterize this transport carrier further, we studied the influence of temperature and membrane potential on the transport of this electrolyte, the stoichiometry of the sodium-phosphate interaction, and the interrelationship between phosphate uptake and other sodium-dependent systems. Temperature influenced phosphate uptake by changing the maximal velocity and the affinity of the carrier for the substrate. The Arrhenius plot for uptake velocity exhibited an abrupt breakpoint at 28.6 degrees C, suggesting that membrane fluidity is a factor in phosphate uptake. Increasing the sodium concentration in the incubation medium augmented the phosphate uptake according to a sigmoid curve, and the Hill plot analysis of these data indicates that at least two sodium ions are transported with each phosphate radical. The effect of membrane potential on phosphate uptake was studied by inducing potassium diffusion with valinomycin and by using various sodium salts with different anion conductance in the incubation medium. In both series of experiments, the inside-negative potential significantly enhanced phosphate uptake. We concluded that the phosphate-sodium cotransport is an electrogenic process, a conclusion which is compatible with the observation that at least two sodium ions accompany each phosphate radical. Glycine, alanine and proline all inhibited phosphate uptake according to an uncompetitive type of inhibition. In contrast, the addition of glucose to the incubation medium had no effect.

Phorbol esters inhibit phosphate uptake in opossum kidney cells: A model of proximal renal tubular cells

The effects of phorbol esters and diacylglycerol on phosphate uptake in opossum kidney (OK) cells were investigated to assess the possible role of Ca 2+-activated, phospholipid dependent protein kinase (protein kinase C) on renal phosphate handling. OK cells are widely used as a model of proximal renal tubular cells and are reported to possess a Na +-dependent phosphate transport system. Phorbol-12,13-dibutyrate (PDBu) inhibited phosphate uptake. This inhibitory effect was synergistically enhanced with A23187. 4β-phorbol 12,13-didecanoate inhibited phosphate uptake, while 4α-phorbol 12,13-didecanoate did not. 1-oleoyl-2-acetyl-glycerol (OAG), a synthetic diacylglycerol, also exhibited an inhibitory effect on phosphate uptake. These data suggest the possible involvement of protein kinase C in proximal renal tubular phosphate transport.

Light History, Phosphorus Status, and the Occurrence of Light Stimulation or Inhibition of Phosphate Uptake in Lake Superior Phytoplankton and Bacteria

Phosphate uptake in Lake Superior was stimulated by light on 17 out of 34 occasions, inhibited on 10, and unaffected on 7. In a stepwise multiple regression model the variables explaining most of the variance in the light effect on phosphate uptake were, in decreasing order of importance, the light history as estimated by hours of sunshine in previous 3 d, phosphate turnover time, phytoplankton biovolume, and time of day. Stimulation was most common in spring at inshore stations, while inhibition occurred mainly in spring at offshore stations. We suggest that light-limited but phosphorus-sufficient phytoplankton show the former, but extremely low light adapted populations, the latter response. We discovered an apparent diurnal pattern in the response, with maximal stimulation by light during daylight hours and inhibition predominating at sunrise and sunset.

Phosphate Transport across the Plasma Membrane of Wheat Leaf Protoplasts

The kinetics and inhibitor specificities of phosphate transport across the plasma membrane of wheat leaf mesophyll protoplasts have been examined. Studies were also carried out on the effects of light and pH on phosphate transport and the plasma membrane electropotential. At pH 5.8 (30 degrees C), protoplasts accumulated phosphate at the rate of 3.9 +/- 0.2 nanomoles per milligram protein per hour. Phosphate uptake rates and inhibitor specificities for the leaf cell plasma membrane phosphate transporter were qualitatively similar to those observed with root protoplasts. Neither picrylsulfonic acid, or p-chloromercuribenzene sulfonate affected phosphate uptake significantly at 0.1 millimolar. Of all compounds tested, carbonyl cyanide-p-trifluoromethoxy phenylhydrazone was the most effective inhibitor of phosphate uptake (60% at 0.1 millimolar). Tribenzylphosphate inhibited uptake by 34% while dibenzylphosphate had no effect. The plasma membrane electropotential was found to be -37 +/- 3 millivolts. Initiation of photosynthesis lowered the membrane potential to -39 +/- 3 millivolts. Inhibition of phosphate uptake by 34% with the substrate analog tribenzylphosphate resulted in a measured membrane potential of -33 +/- 3 millivolts. These changes in potential were not significant at the 5% probability level. Phosphate uptake rates remained constant under photosynthetic and nonphotosynthetic conditions. The utility of tribenzylphosphate as an inhibitor in plant systems is demonstrated.

Effect of phosphorus supply on phosphate uptake and alkaline phosphatase activity in Rhizobia

The effect of P nutrition on phosphate uptake and alkaline phosphatase activity was studied in chemostat culture for four rhizobial and three bradyrhizobial species. Phosphate-limited cells took up phosphate 10- to 180-fold faster than phosphate-rich cells. The four fast-growing rhizobial strains contained high levels of alkaline phosphatase activity under P-limited conditions compared to the repressed levels found in P-rich cells; alkaline phosphatase activity could not be detected in three slow-growing rhizobial strains, regardless of their P-status. Glycerol 1-phosphate-uptake in the cowpea Rhizobium NGR234 was derepressed over 50-fold under P-limited conditions, and appeared to be co-regulated with phosphate uptake. The phosphate-uptake system appeared similar in all strains with apparent K m values ranging from 1.6 μM to 6.0 μM phosphate and maximum activities from 17.2 to 126 nmol · min-1 · (mg dry weight of cells)-1. Carbonyl cyanide m-chlorophenyl hydrazone strongly inhibited phosphate uptake in all strains and a number of other metabolic inhibitors also decreased phosphate uptake in the cowpea Rhizobium NGR234. The phosphate uptake system in all strains failed to catalyse exchange of 32P label in preloaded cells or efflux of phosphate. The results suggest a single, repressible, unidirectional and energy-dependent system for the transport of phosphate into rhizobia.

Effect of arginine modification on kidney brush-border-membrane transport activity.

The effect of phenylglyoxylation on brush-border-membrane functions was studied with membrane vesicles from rat kidney cortex. Na+-gradient-dependent uptake of phosphate, glucose and alanine was inhibited by 65, 88 and 70% by pre-incubation of vesicles with 50 mM-phenylglyoxal for 2 min. The inhibition showed a dependency for alkaline pH. Borate co-operativity in butanedione inactivation was used to prove that inhibition was caused by arginine modification. Intravesicular volumes, alkaline phosphatase, aminopeptidase M and Na+-H+ exchange were not affected by phenylglyoxal treatment. Inhibition of phosphate uptake was studied in more detail and showed that the chemical modification introduced by phenylglyoxal inhibited the overshoot of phosphate uptake caused by the Na+ gradient, and decreased the apparent maximal velocity of the phosphate-transport system in its interaction with Na+. Phosphate uptake measured in the absence of Na+ was not affected by phenylglyoxal. Shunting of the transmembrane electrical potential with K+ and valinomycin had no effect on phenylglyoxal inhibition, proving that the alteration of transmembrane electrical potential could not be responsible for this effect. Phenylglyoxal had no ionophoric effect on the Na+ gradients studied (1-100 mM). Na+ efflux was also unaffected by phenylglyoxal treatment. Na+, harmaline and amiloride were ineffective in protecting against phenylglyoxal inhibition, suggesting that the site modified was not an Na+-binding site. These results indicate the involvement of highly reactive arginine residues in phosphate, glucose and alanine uptake.

Phosphate uptake by kidney epithelial (LLC-PK 1) cells

Phosphate uptake by the cultured kidney epithelial cell (LLC-PK 1) was studied. The uptake was Na + dependent, saturable with respect to phosphate and Na +, and energy dependent. The characteristics of the cell uptake system resembled the properties of phosphate transport in the kidney. Parathyroid hormone, dibutyryl cyclic AMP, and forskolin decreased Na +-dependent phosphate uptake. These agonists did not affect Na +-dependent α-methylglucoside uptake. Vasopressin and isoproterenol, which do not affect renal phosphate transport, did not inhibit phosphate uptake by the cell. These findings suggest that the cultured cell system may be a useful experimental model for studies of renal phosphate transport and its regulation.