Ion pathways in renal brush border membranes

The absorbance change of the weak base dye probe, Acridine orange, was used to monitor alterations of pH gradients across renal brush border membrane vesicles. The presence of Na⁺/H⁺ or Li⁺/H⁺ exchange was demonstrated by diluting Na₂SO₄ or Li₂SO₄ loaded vesicles into Na⁺- or Li⁺-free solutions, which caused dye uptake. About 20% of the uptake was abolished by lipid permeable cations such as valinomycin-K⁺ or tetraphenylphosphonium, indicating perhaps the presence of a finite Na⁺ conductance smaller than electroneutral Na⁺/H⁺ exchange. The protonophore tetrachlorosalicylanilide raised the rate of dye uptake under these conditions, hence the presence of an Na⁺ conductance greater than the H⁺ conductance was suggested. K⁺ gradients also induced changes of pH, at about 10% of the Na⁺ or Li⁺ rate. Partial inhibition (21%) was seen with 0.1 mM amiloride indicating that K⁺ was a low affinity substrate for the Na⁺/H⁺ exchange. Acceleration both by tetrachlorosalicylanilide (2-fold) and valinomycin (4-fold) suggested the presence of 2 classes of vesicles, those with high and those with low K⁺ conductance. The larger magnitude of the valinomycin dependent signal suggested that 75% of the vesicles had a low K⁺ conductance. Inward Cl^? gradients also induced acidification, partially inhibited by the presence of tetraphenylphosphonium, and accelerated by tetrachlorosalicylanilide. Thus both a Cl^? conductance greater than the H⁺ conductance and a Cl^?/OH^? exchange were present. The rate of Na⁺/H⁺ exchange was amiloride sensitive with a pH optimum of 6.5 and an apparent K_m for Na⁺ or Li⁺ of about 10 mM and an E_A of 14.3 kcal per mol. A 61-fold Na₂SO₄ gradient resulted in a pH gradient of 1.64 units which increased to 1.8 with gramicidin. An equivalent NaCl gradient gave a much lower ΔpH even in the presence of gramicidin showing that the H⁺ and Cl^? pathways could alter the effects of the Na⁺/H⁺ exchange.     

Substrate specificity of urate transporter in rat renal brush border membranes

To further demonstrate the substrate specificity of urate-anion exchanger in rat renal brush border membrane vesicles, the hydroxyl ion gradient-dependent [2-14C] urate uptake was studied by a rapid filtration technique. The [2-14C] urate uptake was more sensitive to unlabeled urate than to unlabeled xanthine and hypoxanthine. In addition, urate derivatives which are methylated at the positions 3 and 9 hardly inhibited the urate uptake. Because of the substrate specificity, the urate-anion exchanger in brush border membranes appears to selectively use urate as the endogenous substrate.     

Identification and partial purification of a band 3-like protein from rabbit renal brush border membranes

A monoclonal antibody against the membrane domain of human erythrocyte band 3 was tested for its ability to bind to rabbit renal brush border membranes. A single brush border protein with a molecular mass of 43 kDa was recognized by the band 3 antibody. Using DNase I coupled to an agarose-bead support this 43-kDa protein was partially purified by removing actin and a number of actin-bound proteins from the brush border membranes. The partially purified 43 kDa-band was eluted from sodium dodecyl sulfate-polyacrylamide gels and used to make a highly sensitive and specific guinea pig antiserum. This antiserum, but not serum from control guinea pigs, cross-reacts with purified band 3 from human, rabbit, and bovine erythrocytes confirming the immunologic similarity among these proteins. The 43-kDa protein can be stained by the periodic acid-Schiff base method and binds wheat germ agglutinin and concanavalin A, demonstrating that it is a glycoprotein. Furthermore, in the absence of dithiothreitol, the immunoreactive brush border protein migrates with a molecular mass of 86 kDa on an sodium dodecyl sulfate-polyacrylamide gel suggesting that under nonreducing conditions it exists as a dimer. The 43-kDa protein could be solubilized in octyl glucoside and was further purified using gel filtration chromatography. The amino acid composition of the 43-kDa brush border protein was obtained, and its similarity with erythrocyte band 3 is discussed.     

The binding of cyclic AMP to renal brush border membranes.

The binding of cyclic AMP to the proximal tubule luminal (brush border) membrane isolated from the rabbit renal cortex was studied. The rate of binding was dependent on temperature; at 37 degrees equilibrium was attained in 45 min, whereas at 0 degrees 120 min was required. The final levels of binding were identical. The binding of 3H-cyclic AMP was reversed by dilution or addition of unlabeled cyclic nucleotide. Debinding was markedly temperature sensitive. Binding was only partially saturable with respect to cyclic AMP concentration, apparently with more than one binding site. The cyclic AMP bound to the membrane was recovered unchanged. When bound to the membrane cyclic AMP was resistant to hydrolysis by endogenous membrane or exogenously added phosphodiesterase. The binding to the membranes was relatively specific for cyclic AMP, although other cyclic purine nucleotides inhibited, cyclic IMP greater than dibutyryl cyclic AMP greater than cyclic GMP. The renal membranes did bind cyclic GMP, but this binding was relatively non-specific. Hormones and drugs, that mediate cyclic AMP generation or renal function, as well as other compounds common to the proximal tubule were without significant effect on cyclic AMP binding. Binding was inhibited by sulfhydryl reacting agents and this inhibition could be blocked and partially reversed by mercaptoethanol.     

Lipid-phase structure in epithelial cell membranes: comparison of renal brush border and basolateral membranes

The lipid-phase structures of brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) isolated from rabbit renal cortex were compared by steady-state and phase-modulation measurements of diphenylhexatriene (DPH) and trans- and cis-parinaric acid (tPnA and cPnA) fluorescence. A temperature-scanning system was used which gave reproducible temperature profiles of steady-state and dynamic fluorescence parameters with a resolution of 0.1 degrees C. Steady-state anisotropy of DPH showed a triphasic dependence on temperature with slope discontinuities at 22 +/- 4 and 47 +/- 3 degrees C (BBMV) and at 23 +/- 2 and 48 +/- 1 degrees C (BLMV). At all temperatures, DPH anisotropy in BBMV was greater than that in BLMV. Ground-state heterogeneity analysis of tPnA and cPnA fluorescence lifetime data demonstrated the presence of long (greater than 12 ns) and short (less than 5 ns) lifetime components, interpreted in terms of solid-phase and fluid-phase lipid domains. The fraction of solid-phase phospholipid decreased from 0.9 to 0.1 for BBMV and from 0.7 to 0.3 in BLMV with increasing temperature (10-50 degrees C). In both membranes, tryptophan-PnA fluorescence energy-transfer measurements showed that membrane proteins were surrounded by a fluidlike phospholipid phase. These results demonstrate the inadequacy of steady-state DPH anisotropy data in defining the structural characteristics of complex biological membranes. Results obtained with the phase-sensitive parinaric acid probes demonstrate major differences in the phase structure of the two opposing cell membranes in both the bulk lipid and the lipid microenvironment around membrane proteins.     

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 sodium-dependent phosphate transport in renal brush border membranes

The effect of ischemia induced acute renal failure (ARF) on the transport of phosphate (Pi) after early (15-30 min) and prolonged (60 min) ischemia in the brush border membrane vesicles (BBMV) from rat renal cortex was studied. Sodium-dependent transport of Pi declined significantly and progressively due to ischemia. Western blot analysis of BBM from ischemic rats showed decreased expression of NaPi-2. A compensatory increase was observed in Pi uptake in BBMV from contralateral kidneys. There was no significant difference in NaPi-2 expression between BBMV from sham and contralateral kidneys. Early blood reperfusion for 15 min after 30 min ischemia caused further decline in Pi uptake. Prolonged reperfusion for 120 min caused partial reversal of transport activities in 30-min ischemic rats. However, no improvement in the transport of Pi was observed in 60-min ischemic rats after 120 min of blood reperfusion. Kinetic studies showed that the effect of ischemia and blood reperfusion was dependent on the V_max of the Na-Pi transporter. Western blot analysis showed increased expression of NaPi-2 in the BBMs from ischemia-reperfusion animals. Further, a shift in the association of Na ions to transport one molecule of Pi was observed under different extracellular Na concentrations [Na]_o. Feeding rats with low Pi diet and/or treatment with thyroid hormone (T3) prior to ischemia resulted in increased basal Pi transport. Ischemia caused similar decline in Pi transport in BBM from LPD and/or T3 animals. However, recovery in these animals was faster than the normal Pi diet fed (NPD) animals. The study suggests a change in the intrinsic properties of the Na-Pi transporter in rat kidneys due to ischemia. The study also indicates that treatment with T3 and feeding LPD prior to ischemia caused faster recovery of phosphate uptake due to ischemia-reperfusion injury.     

Evidence for a dipeptide transport system in renal brush border membranes from rabbit

Papain treatment of renal brush border vesicles was carried out as a successful first step towards the purification of the membrane components involved in dipeptide transport. The treated vesicles exhibited increased specific transport activity of glycyl-l-proline. In contrast, the specific transport activity of l-alanine in the treated vesicles was less than that in the control vesicles. Papain treatment resulted in the solubilization of 38% of protein, 55% of alkaline phosphatase, 90% of γ-glutamyltransferase and 95% of leucine aminopeptidase. There was no change in the intravesicular volume nor was there any increase in vesicular permeability. Glycyl-l-proline transport was Na⁺-independent in the control and papain-treated vesicles. Diamide reduced the Na⁺-dependent l-alanine transport while glycyl-l-proline transport remained unaffected in the presence of Na⁺. Many dipeptides inhibited glycyl-l-proline transport both in the presence and absence of Na⁺. The inhibition by dipeptides was greater than the inhibition by equivalent concentrations of free amino acids. These data demonstrate that renal brush border vesicles can efficiently handle dipeptides by a mechanism completely different from that of amino acid transport.     

Calcium uptake into renal brush border membranes in vitamin B6 deficient rats

The calcium uptake into renal brush border membrane vesicles, which has been purified from normal or vitamin B6 deficient rat renal cortex by calcium precipitation, was investigated. The values of Km and Vmax were determined to be 1.89 mM and 4.26 nmol of Ca2+/mg of protein per 20s in vitamin B6 deficient rats, respectively. This Vmax was lower than that of normal rats. The chemical compositions of renal brush border membranes did not display a difference in normal and vitamin B6 deficient rats. The amount of brush border membranes isolated from 1 gram of renal cortex in vitamin B6 deficient rats was less than in normal rats.     

Ubiquinone,nonheme iron,and flavins in the renal brush border plasma membranes

The specific levels of ubiquinone, nonheme iron, and flavins have been estimated in renal brush border membrane preparations. In all cases, the levels were clearly lower than those of kidney mitochondria, on the basis of both protein and cytochrome content. These results suggest that kidney mitochondria and brush border membranes differ in the composition of their electron transfer systems.     

Effect of pH on the transport of Krebs cycle intermediates in renal brush border membranes

Lowering extravesicular pH stimulated Na⁺-dependent citrate transport in renal brush border membrane vesicles: e.g., at pH_out = 5.5, the initial rate of citrate uptake was increased 10-fold compared to parallel control experiments at pH 7.5. The same experimental conditions had little effect on succinate uptake. The influence of pH on citrate transport is a product of the extravesicular H⁺ concentration; pH gradients did not potentiate the effects nor were proton gradients capable of driving transport in the absence of Na⁺. The effect of pH is adequately explained if only the mono- and divalent species of citrate (Cit^1?, Cit^2?) are considered acceptable substrates for transport. The stimulatory influence of pH on transport correlated quite well with pH-related increases in the concentrations of Cit^1? and Cit^2?, and over the same pH range [Cit^3?] was inversely related to citrate uptake. A model of the Na⁺-dependent dicarboxylate transport system is discussed in which three sodium ions are translocated per molecule of dicarboxylic acid.     

Temperature-induced transitions of porcine intestinal brush border membranes

Thermotropic transitions of the membrane components in porcine intestinal brush border membranes were studied by means of fluorimetry using a fluorogenic thiol reagent, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM), and a lipophilic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). 1. The reactivity of the sulfhydryl groups of the membrane proteins with DACM was dependent on temperature, with a transition point at about 33°C. A conspicuous transition was also observed in the relation between temperature and the fluorescence intensity of DACM-labeled membranes at 35°C. 2. Temperature dependence profiles of the solubilization of DPH in the membranes and of the fluorescence polarization of DPH-membrane complex suggested that the phase transition of the lipid from gel to liquid-crystalline state occurs over a temperature range of 30 to 35°C. 3. Efficient fluorescence energy transfer was observed from tryptophan residues of the membrane proteins to DPH located in the lipid phase of the membranes, and its efficiency was extremely enhanced, dependent on temperature, above 35°C. The intensity of the tryptophan fluorescence of the membrane proteins decreased with increasing temperature and a discontinuity was observed at about 33°C. Based on these results, it may be concluded that there are co-operative interactions between proteins and lipids in the membranes and that the temperature-induced conformational changes of the membrane proteins are closely related to the dynamics of the hydrocarbon cores of the lipid.     

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.     

Partial purification of the Na+-dependentd-glucose transport system from renal brush border membranes

Summary A membrane extract enriched with the Na⁺-dependentd-glucose transport system was obtained by differential cholate solubilization of rat renal brush border membranes in the presence of 120mm Na⁺ ions. Sodium ions were essential in stabilizing the transport system during cholate treatment. This membrane extract was further purified with respect to its Na⁺-coupledd-glucose transport activity and protein content by the use of asolectin-equilibrated hydroxylapatite. The reconstituted proteoliposomes prepared from this purified fraction showed a transient accumulation ofd-glucose in response to a Na⁺ gradient. The observed rate of Na⁺-coupledd-glucose uptake by the proteoliposomes represented about a sevenfold increase as compared to that of the reconstituted system derived from an initial 1.2% cholate extract of the membranes. Other Na⁺-coupled transport systems such asl-alanine, -ketoglutarate and phosphate were not detected in these reconstituted proteoliposomes.