Cyclic AMP-dependent protein phosphorylation in canine renal brush-border membrane vesicles is associated with decreased phosphate transport

It is known that the administration of parathyroid hormone to dogs results in phosphaturia and decreased phosphate transport in brush-border vesicles isolated from the kidneys of those dogs. Parathyroid hormone has been shown to activate adenylate cyclase at the basal-lateral membrane of the renal proximal tubular cell. It has been postulated that parathyroid hormone-induced phosphaturia is effected through phosphorylation of brush-border protein by membrane-bound cAMP-dependent protein kinase. An experimental system was designed such that phosphorylation of brush-border vesicles and Na+-stimulated solute transport could be studied in the same preparations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane vesicles revealed cAMP-dependent phosphorylation of 2 protein bands (Mr = 96,000 and 62,000), which was enhanced by exposure of the inside of the membrane vesicles to ATP and cAMP. Cyclic AMP-dependent phosphorylation of brush-border vesicles was accompanied by inhibition of Na+-stimulated Pi but not D-glucose transport or 22Na+ uptake. When renal brush-border vesicles from parathyroidectomized and normal dogs were phosphorylated in vitro in the presence and absence of cAMP, both the cAMP-dependent phosphorylation and inhibition of Na+-stimulated Pi transport were greater in vesicles isolated from kidneys of parathyroidectomized dogs relative to control animals. We conclude that the cAMP-dependent phosphorylation of brush-border membrane-vesicle proteins is associated with specific inhibition of Na+-stimulated Pi transport. The phosphaturic action of parathyroid hormone (PTH) could be mediated through the cAMP-dependent phosphorylation of specific brush-border membrane proteins.     

Reconstitution of the partially purified renal phosphate (Pi) transporter.

Proteins from rabbit kidney brush border membranes were solubilized with 1% Nonidet P-40 (crude membrane proteins) and fractionated according to their isoelectric points (pI) by chromatofocusing. The eluate was pooled into three fractions according to the pI of the samples (1, greater than 6.8; 2, 6.8-5.4; 3, 5.4-4.0). The crude membrane proteins as well as the three fractions were reconstituted into liposomes and transport of Pi was measured by a rapid filtration technique in the presence of an inwardly directed K+ or Na+ gradient. Arsenate-inhibitable Na+-dependent transport of Pi was reconstituted into an osmotically active intravesicular space from both the crude membrane proteins and Fraction 1. In contrast, Fractions 2 and 3 were inactive. Treatment of the crude membrane proteins and the three fractions with the method for extracting phosphorin (a Pi-binding proteolipid found in brush border membranes) yielded Mn2+-dependent binding of Pi characteristic of phosphorin only in the extracts from crude membrane proteins and Fraction 1, the same fractions in which Na+-dependent transport of Pi was found in the reconstituted system. When reconstituted into liposomes, phosphorin was, however, unable to yield Na+-dependent transport of Pi. Moreover, we cannot eliminate the possibility that Na+-Pi transport can occur in the absence of phosphorin, since complete recovery of Na+-Pi transport was not achieved. However, the present data showing localization of the recovered binding and transport systems for Pi in the same protein fraction lend support to the hypothesis that phosphorin might be a constituent of the renal Pi transport system. Whether the presence of phosphorin is necessary or accessory for Na+-dependent Pi transport in intact brush border membrane vesicles or in liposomes reconstituted with crude or purified membrane proteins requires further investigation.     

The effect of cyclic nucleotides and cholera toxin on in vivo and in vitro phosphorylation of small intestinal brush border membranes

The effect of cyclic nucleotides and cholera toxin on the phosphorylation of the brush border membrane proteins of the rat jejunum was studied. Phosphorylation was analyzed by autoradiography of brush border membrane proteins separated by SDS-polyacrylamide gel electrophoresis. Phosphorylation was performed either in vivo by perfusion of the jejunum with [³²P]orthophosphate followed by an analysis of the isolated membranes or in vitro by phosphorylation of isolated brush border membranes by [γ-³²P]ATP in the presence of saponin. The addition of cholera toxin (10 μg/ml) or dibutyryl-cAMP (5 mmol/l) to the perfusate was unable to produce significant changes in the phosphoprotein pattern. On the other hand, cAMP (at 5 μmol/l) induced an increase of the phosphorylation of a 86 kDa protein when freshly isolated brush border membranes were phosphorylated by [γ-³²P]ATP. However, the same effect could also be induced by low concentrations of cGMP (0.1 μmol/l). It is concluded that brush border membranes from rat jejunum do not contain cAMP-dependent protein kinase activity and that cAMP-dependent protein phosphorylation of this membrane does probably not represent the final event of cholera toxin-induced secretion.     

Renal brush border membrane phosphorylation: influence of pH, cAMP and ATP concentrations, parathyroid hormone status, and dietary phosphate

It is known that parathyroidectomy, administration of parathyroid hormone (PTH), and dietary phosphate depletion or excess result in variations in phosphaturia and in phosphate transport through brush border membrane vesicles isolated from the kidneys of various animals. Parathyroid hormone has been shown to ultimately phosphorylate some brush border membrane proteins and it has been postulated that the resulting phosphaturia is related to this phosphorylation. However, it is not known whether the regulation of phosphate transport by the diet is affected through similar pathways. Our experiments were designed to study the phosphorylation of brush border membrane with [gamma-32P]ATP using the intrinsic protein kinase of the membranes. Five groups of rats were used: normal, phosphate loaded, phosphate depleted, and thyroparathyroidectomized and acutely loaded with parathyroid hormone. In each series of animals, the proteins whose phosphorylation was cAMP dependent were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and their phosphorylation with various concentrations of ATP, in the presence or absence of cAMP in the incubation medium, was quantified. In the normal rat, 17 proteins were phosphorylated, the phosphorylation of two of them (Mr, 71 000 and 84 000) being cAMP dependent. Maximal response to cAMP for these two proteins was obtained with 10 microM cAMP. The peaks of phosphorylation were observed at pH 7 for protein 71 000 and pH 10 for protein 84 000. When brush border membranes from normal rats were incubated with 10-100 microM ATP, cAMP-dependent phosphorylation increased to reach a maximal phosphorylation of 4.44 +/- 0.90 pmol/mg protein for protein 71 000 and 1.32 +/- 0.15 pmol/mg protein for protein 84 000.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylated intermediates of (Ca2+ + Mg2+)-ATPase and alkaline phosphatase in renal plasma membranes

Renal basal-lateral and brush border membrane preparations were phosphorylated in the presence of [gamma-32P]ATP. The 32P-labeled membrane proteins were analysed on SDS-polyacrylamide gels. The phosphorylated intermediates formed in different conditions are compared with the intermediates formed in well defined membrane preparations such as erythrocyte plasma membranes and sarcoplasmic reticulum from skeletal muscle, and with the intermediates of purified renal enzymes such as (Na+ + K+)-ATPase and alkaline phosphatase. Two Ca2+-induced, hydroxylamine-sensitive phosphoproteins are formed in the basal-lateral membrane preparations. They migrate with a molecular radius Mr of about 130 000 and 100 000. The phosphorylation of the 130 kDa protein was stimulated by La3+-ions (20 microM) in a similar way as the (Ca2+ + Mg2+)-ATPase from erythrocytes. The 130 kDa phosphoprotein also comigrated with the erythrocyte (Ca2+ + Mg2+)-ATPase. In addition in the same preparation, another hydroxylamine-sensitive 100 kDa phosphoprotein was formed in the presence of Na+. This phosphoprotein comigrates with a preparation of renal (Na+ + K+)-ATPase. In brush border membrane preparations the Ca2+-induced and the Na+-induced phosphorylation bands are absent. This is consistent with the basal-lateral localization of the renal Ca2+-pump and Na+-pump. The predominant phosphoprotein in brush border membrane preparations is a 85 kDa protein that could be identified as the phosphorylated intermediate of renal alkaline phosphatase. This phosphoprotein is also present in basal-lateral membrane preparations, but it can be accounted for by contamination of those membranes with brush border membranes.     

The Na+ gradient-dependent transport of D-glucose in renal brush border membranes.

The Na+-dependent transport of D-glucose was studied in brush border membrane vesicles isolated from the rabbit renal cortex. The presence of a Na+ gradient between the external incubation medium and the intravesicular medium induced a marked stimulation of D-glucose uptake. Accumulation of the sugar in the vesicles reached a maximum and then decreased, indicating efflux. The final level of uptake of the sugar in the presence of the Na+ gradient was identical with that attained in the absence of the gradient, suggesting that equilibrium was established. At the peak of the overshoot the uptake of D-glucose was more than 10-fold the equilibrium value. These results suggest that the imposition of a large extravesicular to intravesicular gradient of Na+ effects the transient movement of D-glucose into renal brush border membranes against its concentration gradient. The stimulation of D-glucose uptake into the membranes was specific for Na+. The rate of uptake was enhanced with increased concentration of Na+. Increasing Na+ in the external medium lowered the apparent Km for D-glucose. The Na+ gradient effect on D-glucose transport was dissected into a stimulatory effect when Na+ and sugar were on the same side of the membrane (cis stimulation) and an inhibitory effect when Na+ and sugar were on opposite sides of the membrane (trans inhibition). The uptake of D-glucose, at a given concentration of sugar, reflected the sum of the contributions from a Na+-dependent transport system and a Na+-independent system. The relative stimulation of D-glucose uptake by Na+ decreased as the sugar concentration increased. It is suggested, however, that at physiological concentrations of D-glucose the asymmetry of Na+ across the brush border membrane might fully account for uphill D-glucose transport. The physiological significance of the findings is enhanced additionally by observations that the Na+-dependent D-glucose transport system in the membranes in vitro possessed the sugar specificities and higg phlorizin sensitivity characteristic of more intact preparations. These results provide strong experimental evidence for the role of Na+ in transporting D-glucose across the renal proximal tubule luminal membrane.     

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

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

Phenylalanine uptake in isolated renal brush border vesicles.

The uptake of L-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques. Brush border microvilli but not basolateral plasma membrane vesicles take up L-phenylalanine by an Na+-dependent, saturable transport system. The apparent affinity of the transport system for L-phenylalanine is 6.1 mM at 100 mM Na+ and for Na+ 13mM at 1 mM L-phenylalanine. Reduction of the Na+ concentration reduces the apparent affinity of the transport system for L-phenylalanine but does not alter the maximum velocity. In the presence of an electrochemical potential difference of Na+ across the membrane (etaNao greater than etaNai) the brush border microvilli accumulate transiently L-phenylalanine over the concentration in the incubation medium (overshoot pheomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K+ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient. These results indicate that the entry of L-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na+ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na+ and on the electrical potential difference. The exit of L-phenylalanine across the basolateral plasma membranes is Na+-independent and probably involves facilitated diffusion.     

Quantitation of the Na(+)-Pi cotransporter in renal cortical brush border membranes. [14C]phosphonoformic acid as a useful probe to determine the density and its change in response to parathyroid hormone.

To determine the density of Na(+)-Pi symporters in brush border membranes (BBM) from rat renal cortex, [14C] phosphonoformic acid [( 14C] PFA), a competitive inhibitor of Na(+)-Pi cotransport, was employed as a probe. The [14C]PFA binding was measured in BBM vesicles (BBMV) under equilibrated conditions (extra-vesicular Na+, K+, and H+ = intravesicular Na+, K+, and H+) to avoid modulatory effects of these solutes. BBMV were preincubated in media without or with addition of molar excess of Pi (greater than 20 times) to determine the Pi-protectable PFA-binding sites, and then [14C] PFA binding was determined. Only the [14C]PFA binding in the presence of Na+ displaceable by an excess of Pi was saturated and was independent of intravesicular volume of BBMV. This value denoted as "Pi-protectable Na(+)-[14C]PFA binding," was analyzed by Scatchard plot showing BmaxPFA = 375 +/- 129 pmol of PFA/mg protein, KDPFA = 158 +/- 18 microM; the Hill coefficient was congruent to 1. For Na(+)-dependent binding of [3H]phlorizin, in the same BBMV, Bmax = 310 +/- 37 pmol/mg protein and KD V 2.2 +/- 0.5 microM. BBMV prepared from cortex of thyroparathyroidectomized rats infused with phosphaturic doses of parathyroid hormone (PTH) were compared with vehicle-infused controls. Administration of PTH resulted in decrease of BmaxPFA (-38%) and of Na(+)-gradient-dependent uptake of 32Pi (-35%), but KDPFA was not changed. Neither BmaxPhl and KDPhl for Na(+)-phlorizin binding, nor the Na(+)-gradient-dependent uptake of [3H]D-glucose differed between PTH-treated and control rats. We conclude: (a) measurement of Pi-protectable Na(+)-[14C]PFA binding determines numbers and affinity of Na(+)-Pi symporters in renal BBMV; (b) the affinity of PFA for Na(+)-Pi symporter is similar to apparent affinity for Pi (KmPi), as determined from measurements of Na(+)-gradient-dependent 32Pi uptake by BBMV; (c) both Na(+)-Pi symporter and [Na+]D-glucose symporters are present within renal BBM in a similar range of density; (d) PTH decreases the number of Na(+)-Pi cotransporters in BBMV commensurate with the parallel decrease of Na(+)-gradient-dependent Pi transport, whereas the affinity of Na(+)-Pi symporters for Pi is not changed. These observations support the hypothesis that PTH decreases capacity for Na(+)-dependent Pi reabsorption by internalization of Na(+)-Pi symporters in BBM of renal proximal tubules.     

Phosphorylation of phospholamban in intact myocardium. Role of Ca2+-calmodulin-dependent mechanisms

Phospholamban, a putative regulator of cardiac sarcoplasmic reticulum Ca2+ transport, has been shown to be phosphorylated in vitro by cAMP-dependent protein kinase and an intrinsic Ca2+-calmodulin-dependent protein kinase activity. This study was conducted to determine if Ca2+-calmodulin-dependent phosphorylation of phospholamban occurs in response to physiologic increases in intracellular Ca2+ in intact myocardium. Isolated guinea pig and rat ventricles were perfused with 32Pi after which membrane vesicles were isolated from individual hearts by differential centrifugation. Administration of isoproterenol (10 nM) to perfused hearts stimulated 32P incorporation into phospholamban, Ca2+-ATPase activity, and Ca2+ uptake of sarcoplasmic reticulum isolated from these hearts. These biochemical changes were associated with increases in contractility and shortening of the t 1/2 of relaxation. Elevated extracellular Ca2+ produced comparable increases in contractility but failed to stimulate phospholamban phosphorylation or Ca2+ transport and did not alter the t 1/2 of relaxation. Inhibition of trans-sarcolemmal Ca2+ influx by perfusing the ventricles with reduced extracellular Ca2+ (50 microM) attenuated the increases in 32P incorporation produced by 10 nM isoproterenol. Trifluoperazine (10 microM) also attenuated isoproterenol-induced increases in 32P incorporation into phospholamban. In both cases, Ca2+ transport was reduced to a degree comparable to the reduction in phospholamban phosphorylation. These results suggest that direct physiologic increases in intracellular Ca2+ concentration do not stimulate phospholamban phosphorylation in intact functioning myocardium. Ca2+-calmodulin-dependent phosphorylation of phospholamban may occur in response to agents which stimulate cAMP-dependent mechanisms in intact myocardium.     

Monoclonal antibodies that bind the renal Na+/glucose symport system. 1. Identification

Phlorizin is a specific, high-affinity ligand that binds the active site of the Na+/glucose symporter by a Na+-dependent mechanism but is not itself transported across the membrane. We have isolated a panel of monoclonal antibodies that influence high-affinity, Na+-dependent phlorizin binding to pig renal brush border membranes. Antibodies were derived after immunization of mice either with highly purified renal brush border membranes or with apical membranes purified from LLC-PK1, a cell line of pig renal proximal tubule origin. Antibody 11A3D6, an IgG2b, reproducibly stimulated Na+-dependent phlorizin binding whereas antibody 18H10B12, an IgM, strongly inhibited specific binding. These effects were maximal after 30-min incubation and exhibited saturation at increased antibody concentrations. Antibodies did not affect Na+-dependent sugar uptake in vesicles but significantly prevented transport inhibition by bound phlorizin. Antibodies recognized a 75-kDa antigen identified by Western blot analysis of brush border membranes, and a 75-kDa membrane protein could be immunoprecipitated by 18H10B12. These properties, taken together with results in the following paper [Wu, J.-S.R., & Lever, J.E. (1987) Biochemistry (following paper in this issue)], provide compelling evidence that the 75-kDa antigen recognized by these antibodies is a component of the renal Na+/glucose symporter.     

Cyclic adenosine monophosphate-dependent and -independent protein kinase activity of renal brush border membranes.

Kinase(s) in brush border membranes, isolated from rabbit renal proximal tubules, phosphorylated proteins intrinsic to the membrane and exogenous proteins. cAMP stimulated phosphorylation of histone; phosphorylation of protamine was cAMP independent. cAMP-dependent increases in phosphorylation of endogenous membrane protein were small, but highly reproducible. Most of the ³²P incorporated into membranes represented phosphorylation of serine residues, with phosphorylthreonine comprising a minor component. cAMP did not alter the electrophoretic pattern of ³²P-labeled membrane polypeptides. The small cAMP-dependent phosphorylation of brush border membrane proteins was not due to membrane phosphodiesterase or adenylate cyclase activities. Considerable cAMP was found “endogenously” bound to the membranes as prepared. However, this did not result in preactivation of the kinase since activity was not inhibited by a heat-stable protein inhibitor of cAMP-dependent protein kinases. With intrinsic membrane protein as phosphate acceptor, the relationship between rate of phosphorylation and ATP concentration appeared to follow Michaelis-Menton kinetics. With histone the relationship was complex. cAMP did not affect the apparent K_m for histone. One-half maximal stimulation of the rate of histone phosphorylation was obtained with 7 × 10^?8m cAMP. The K_a values for dibutyryl cAMP, cIMP, and cGMP were one to two orders of magnitude greater. Treatment of brush border membranes with detergent greatly increased the dependency of histone phosphorylation on cAMP. Phosphorylations of intrinsic membrane protein and histone were nonlinear with time, due in part to the lability of the protein kinase, the hydrolysis of ATP, and minimally to the presence of phosphoprotein phosphatase in the border membrane. The membrane phosphoprotein phosphatase was unaffected by cyclic nucleotides. Protein kinase activity was also found in cytosolic and crude particulate fractions of the renal cortex. Activity was enriched in the brush border membrane relative to that in the crude membrane preparation. The kinase activities in the different loci were distinct both in relative activities toward different substrates and in responsiveness to cAMP.     

On the mechanism of sugar and amino acid interaction in intestinal transport.

The influence of amino acids on D-glucose transport was studied in isolated vesicles of brush border membrane from rat small intestine. It is demonstrated that: (a) Uptake of D-glucose by the membranes is inhibited by simultaneous flow of L- and D-alanine into the vesicles. (b) Addition of L-alanine to membranes pre-equilibrated with D-glucose causes efflux of this sugar. (c) The influence of amino acids on D-glucose is dependent on the presence of Na+. (d) The ionophorous agents monactin and valinomycin are able to prevent the transport interaction of D-glucose and amino acids. Monactin is effective in the presence of Na+ without further addition of other cations, while valinomycin is effective only with added K+, in accordance with the known specificity of these antibiotics. (e) The inhibitory effect increases with L-alanine concentration up to about 50 mM after which it levels off. The experiments provide evident that the Na+-dependent sugar and amino acid fluxes across the brush border membrane are coupled electrically.     

Evidence from 23Na NMR studies for the existence of sodium-channels in the brush border membrane of the renal proximal tubule

A fast Na+-exchange is shown to be present in isolated renal brush border membranes. The lower limit of the rate constant for this process, calculated from the 23Na-NMR spectrum is 580 sec-1. The actual exchange rate may be higher. A fast 7Li exchange is also shown to be present in the isolated membrane vesicles. The characteristic overshoot of the Na+ dependent D-glucose cotransport and Na+/H+ antiport can be demonstrated. The fact that neither treatment with papain, nor lowering of the temperature to 5 degrees C affected the 23Na-NMR spectra obtained in the renal brush border membrane vesicles is consistent with the possibility that the fast Na+-exchange occurs through a channel mechanism.     

Energetics of the Na+-dependent transport of D-glucose in renal brush border membrane vesicles.

The energetics of the Na+-dependent transport of D-glucose into osmotically active membrane vesicles, derived from the brush borders of the rabbit renal proximal tubule, was studied by determining how alterations in the electrochemical potential of the membrane induced by anions, ionophores, and a proton conductor affect the uptake of the sugar. The imposition of a large NaCl gradient (medium is greater than vesicle) resulted in the transient uptake of D-glucose into brush border membranes against its concentration gradient. In the presence of Na+ salts of isethionate or sulfate, both relatively impermeable anions, there was no accumulation of D-glucose above the equilibrium value. With Na+ salts of two highly permeable lipophilic anions, NO3- and SCN-, the transient overshoot was enhanced relative to that with Cl-. With Na+ salts whose mode of membrane translocation is electroneutral, i.e. acetate, bicarbonate, and phosphate, no overshoot was found. These findings suggest that only anions which penetrate the brush border membrane and generate an electrochemical potential, negative on the inside, permit the uphill Na+-dependent transport of D-glucose.     

Different molecular sizes for Na(+)-dependent phosphonoformic acid binding and phosphate transport in renal brush border membrane vesicles

We compared several features of Na(+)-dependent phosphono[14C]formic acid (PFA) binding and Na(+)-dependent phosphate transport in rat renal brush border membrane vesicles. From kinetic analyses, we estimated an apparent Km for PFA binding of 0.86 mM, an order of magnitude greater than that for phosphate and the high-affinity phosphate transport system. A hyperbolic Na(+)-saturation curve for PFA binding and a sigmoidal Na(+)-saturation curve for phosphate transport were demonstrated; based on these data, we estimated stoichiometries of 1:1 for Na+/PFA and 2:1 for Na+/phosphate. By radiation inactivation analysis, target sizes for brush border membrane protein(s) mediating Na(+)-dependent PFA binding and Na(+)-dependent phosphate transport corresponded to molecular masses of 555 +/- 32 kDa and 205 +/- 36 kDa, respectively. Similar analysis of the phosphate-inhibitable component of Na(+)-dependent PFA binding gave a target size of 130 +/- 28 kDa. We also demonstrated that phosphate deprivation, which elicits a 2.6-fold increase in brush border membrane Na(+)-dependent phosphate transport, had no effect on either Na(+)-dependent PFA binding or on the target size for PFA binding. However, phosphate deprivation appeared to increase the target size for phosphate transport (from 255 +/- 32 to 335 +/- 75 kDa (P less than 0.01]. In summary, we present evidence for several differences between Na(+)-dependent PFA binding and Na(+)-dependent phosphate transport in rat renal brush border membrane vesicles and suggest that PFA may not interact exclusively with the proteins mediating Na(+)-phosphate co-transport.     

Increase of (Ca2++Mg2+)-ATPase activity of renal basolateral membrane by parathyroid hormone via cyclic AMP-dependent membrane phosphorylation

Studies were made on the mechanism of the effect of parathyroid hormone (PTH) on the activity of (Ca2++Mg2+)-ATPase, a membrane bound Ca2+-extrusion pump enzyme from the basolateral membranes (BLM) of canine kidney (Km for free Ca2+ = 1.3 X 10(-7) M, Vmax = 200 nmol Pi/mg/min). At 1 X 10(-7) M free Ca2+, both PTH (10(-7)-10(-6) M) and cAMP (10(-6)-10(-4) M) stimulated (Ca2++Mg2+)-ATPase activity dose-dependent and their stimulatory effects were inhibited completely by 5 microM H-8, an inhibitor of cAMP-dependent protein kinase. PTH (10(-7) M) also caused 40% increase in 32P incorporation into the BLM and 5 microM H-8 inhibited this increase too. PTH (10(-7) M) was found to stimulate phosphorylation of a protein of Mr 9000 by cAMP dependent protein kinase and 5 microM H-8 was found to block this stimulation also. From these results, it is proposed that PTH stimulates (Ca2++Mg2+)-ATPase activity by enhancing its affinity for free Ca2+ via cAMP-dependent phosphorylation of a BLM protein of Mr 9000.     

CAMP-dependent protein kinase inhibits proline transport across the rat renal tubular brush border membrane

Very little is known about the cellular mechanisms controlling renal tubular amino acid transport. cAMP-dependent protein kinase (cAK) modulates the activity of several ion channels and pumps in biological membranes. The direct influence of cAK on transmembrane amino acid transport has not been investigated. We studied the effect the cAK-mediated phosphorylation on Na+- and Cl–-linked proline transport across the rat renal brush border membrane (BBM). cAK bioassay and Western hybridization analysis using cAK subunit-specific antibodies demonstrated the presence of the enzyme in the BBM. Brush border membrane vesicles (BBMV) were phosphorylated using the hyposmotic shock technique. cAMP, by activating endogenous cAK,and exogenous, highly purified catalytic subunit of cAK inhibited NaCl-dependent proline transport by phosphorylated, lysed/resealed BBMV compared with control vesicles. The cAK-mediated inhibition of proline uptake was completely abolished when phosphorylation at the cytoplasmic (inner side) of the membrane was prevented by isosmotic, rather than hyposmotic, phosphorylation. The cAK-induced inhibition of proline transport was reversed by the specific cAK inhibitor peptide, PKl. These data suggest that cAMP-dependent protein kinase-mediated phosphorylation modulates Na+- and Cl–-linked proline transport across the tubular luminal membrane.     

Uptake of L-(+)lactate by cell membrane (luminal and contraluminal) isolated from rat small intestine microvilli

L-lactate uptake was measured in vesicles formed by intestinal brush border and baso-lateral membranes, using a rapid filtration technique. In the presence of a Na+ gradient directed into the vesicle, L-lactate can be transiently accumulated in brush border vesicles, but not in baso-lateral ones. The transient L-lactate accumulation does not occur in the presence of a KCl gradient. alpha-cyanocinammic acid strongly inhibits L-lactate uptake in brush border vesicles, but not in baso-lateral ones. These results support the existence of a carrier mediated, Na+ dependent, transport of L-lactate across the brush border membrane.     

Isolation and partial characterization of a lectin from a false brome grass (Brachypodium sylvaticum).

Purified rat renal brush-border membrane vesicles possess a heat-labile enzyme activity which hydrolyses NAD+. A reciprocal relationship exists between the disappearance of NAD+ and the appearance of adenosine; 2 mol of Pi are liberated from each mol of NAD+ incubated with brush-border membrane vesicles. Freezing and thawing brush-border membrane vesicles does not enhance the initial rate of NAD+ hydrolysis. Preincubation of brush-border membrane vesicles with NAD+ results in inhibition of Na+-dependent Pi-transport activity, whereas Na+-dependent glucose transport is not affected. EDTA, which prevents the release of Pi from NAD+ and which itself has no direct effect on brush-border membrane Pi transport, reverses the NAD+ inhibition of Na+-dependent Pi transport. These results suggest that it is the Pi liberated from NAD+ and not NAD+ itself that inhibits Na+-dependent Pi transport.