Transport of d-glucose by brush border membranes isolated from the renal cortex

The transport of d-glucose by brush border membranes isolated from the rabbit renal cortex was studied. At concentrations less than 2 mM, the rate of d-glucose uptake increased linearly with the concentration of the sugar. No evidence was found for a “high-affinity” (μM) saturable site. Saturation was indicated at concentrations of d-glucose greater than 5 mM. The uptake of d-glucose was stereospecific and selectively inhibited by d-galactose and other sugars. Phlorizin inhibited the uptake of d-glucose in the presence and absence of Na⁺. The glycoside was a potent inhibitor of the efflux of d-glucose. Preloading the brush border membrane vesicles with d-glucose, but not with l-glucose, accelerated exchange diffusion of d-glucose. These results demonstrate that the uptake of d-glucose by renal brush borders represents transport into an intravesicular space rather than solely binding. The rate of d-glucose uptake was increased when the Na⁺ in the extravesicular medium was high and the membranes were preloaded with a Na⁺-free medium. The rate of d-glucose uptake was inhibited by preloading the brush border membranes with Na⁺. These results are consistent with the Na⁺ gradient hypothesis for d-glucose transport in the kidney. Thus, the presence of a Na⁺-dependent facilitated transport of d-glucose in isolated renal brush border membranes is indicated. This finding is consistent with what is known of the transport of the sugar in more physiologically intact preparations and suggests that the membranes serve as an effective model system in examining the mechanism of d-glucose transport in the kidney.     

A calcium-stat method for the measurement of calcium transport rates in isolated sarcoplasmic reticulum vesicles

Calcium transport by isolated sarcoplasmic reticulum vesicles has been measured by means of a calcium-stat method, utilizing a calcium-specific electrode as sensor. Free calcium ion levels were maintained between 10^?7 and 10^?4m during assay, without the use of calcium buffering agents. The method may be used at temperatures between 5 and 40°C and in the pH range 5.0 to 8.5. Measured initial rates of ATP-dependent calcium transport at 10^?5m free calcium, 20°C, pH 7.2, and 100 μg sarcoplasmic reticulum protein per milliliter were between 1.5 and 2.3 μmol min^?1 mg^?1, with a coefficient of variation of 2%.     

Diuretic effects on renal brush border membrane transport and metabolism

Previous studies have indicated that the thiazide diuretics exert effects on proximal electrolyte transport. To determine whether the locus of these effects is at the brush border membrane (BBM) and if renal metabolism is affected, adult female Sprague-Dawley rats were acutely treated with either 1 mg/kg metolazone, 20 mg/kg chlorothiazide followed by a 20 mg/kg/hr maintenance infusion, 10 mg/kg acetazolamide followed by a 10 mg/kg/hr maintenance infusion, or the vehicles only. Administration of these agents resulted in an approximately tenfold increase in sodium excretion. Neither urinary phosphate nor inulin excretion changed significantly in any group. Sodium dependent BBM vesicle phosphate transport was examined at 0.15, 0.5, and 1 and 120 minute incubation periods in the diuretic treated groups and their respective control groups. Decreased uptake was seen in all pre-equilibrium time points in rats treated with metolazone: 0.15 minutes: 221 +/- 24 pmoles/mg protein (pmol/mg prot) in control rats versus (vs) 185 +/- 23 pmoles/mg prot in metolazone-treated animals (P less than .05) ; 0.5 minutes: 463 +/- 54 vs 369 +/- 49 pmol/mg prot (P less than .005); 1 minute: 549 +/- 74 vs 460 +/- 61 pmol/mg prot (P less than .05); no significant difference in phosphate transport was noted at the two hour equilibrium time point. No significant differences in sodium dependent phosphate transport existed between chlorothiazide or acetazolamide treated rats and control animals. Substrate-stimulated renal gluconeogenesis did not differ between metolazone treated and control animals. We therefore conclude that metolazone inhibits phosphate transport through an effect on the BBM and does not affect renal gluconeogenesis in the rat.     

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.     

Dipeptide metabolism in the isolated perfused rat kidney

Renal handling of glycyl-proline was studied in the isolated perfused rat kidney. Glycyl-proline disappeared from the perfusate as a function of time. The dipeptide was freely filtered at the glomerulus but only 6% of the filtered load was excreted in the urine as the intact peptide. More than 90% of the filtered dipeptide was reabsorbed as the intact peptide and/or its hydrolytic products. Non-filtration mechanisms were also involved to a significant extent in the clearance of the peptide. Hydrolysis at intratubular, intracellular and peritubular sites all contribute to the disappearance of the dipeptide from the perfusate, though the relative contributions of each mechanism are not known. Significant metabolic conversions, especially the conversion of glycine to serine, were also observed during perfusion.     

Role of ATP and beta-gamma-iminoadenosinetriphosphate in the stimulation of epinephrine and protein release from isolated adrenal secretory vesicles.

ATP-stimulated release of epinephrine and protein from isolated chromaffin granules of the bovine adrenal medulla has been characterized with respect to pH optimum, substrate requirements, and temperature. Chromaffin granules incubated at 37 °C under optimal conditions released virtually all of their epinephrine and soluble protein within 10 min. ATP-stimulated epinephrine release was optimal at pH 5.8–6.2 and was five times greater than at pH 7.0–7.4. Magnesium and chloride were absolutely required for this process. Magnesium stimulated release at concentrations up to 1.0 mm; however, it was moderately inhibitory at higher concentrations. The dependence of release on Cl^? exhibited positive cooperativity and was nonsaturable. At 90 mm Cl^? and 1.0 mm Mg²⁺, ATP-stimulated epinephrine release followed Michaelis-Menten kinetics with a $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 0.2 mm. The release of soluble chromaffin granule proteins closely paralleled epinephrine release under all conditions tested, while membrane components were not released. Analogs of ATP substituted at the β-γ position with methylene or imino groups were also capable of stimulating release from granules. These ATP analogs had reduced affinity and lower activity than ATP itself. ATP analogs substituted at the α-β position were essentially inactive but were potent inhibitors of ATP-stimulated release. We conclude that the regulation of release from granules by ATP is rapid and specific and may not depend on hydrolysis of ATP at the β-γ position. This ATP-dependent reaction may be involved in the cellular process of exocytosis.     

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.     

Effects of calcitonin and parathyroid hormone on the metabolism of chondrocytes in culture

Rabbit articular chondrocytes in suspension culture synthesize Type II colagen [3α₁(II)] in the absence of extracellular Ca²⁺ and Type Icollagen [2α₁?(I)·α₂] in the complete medium. As a result of pre-treatment in monolayer culture with calcitonin or parathyroid hormone in the complete medium, an influx of Ca²⁺ into the cells occurs. These cells produce mainly Type I collagen when transferred to suspension cultures in the medium devoid of CaCl₂. If added directly to the suspension culture medium containing no CaCl₂, calcitonin stimulates an active efflux of Ca²⁺ from the cells into the medium and leads the cells to synthesize Type I collagen. Under similar conditions, parathyroid hormone does not change the collagen-phenotype.     

Amino acid transport in kidney epithelial cell line (MDCK): characteristics of Na+/amino acid symport in membrane vesicles and basolateral localization in cell monolayers

Na+-stimulated amino acid transport was investigated in MDCK kidney epithelial cell monolayers and in isolated membrane vesicles. When transport polarity was assessed in confluent polarized epithelial cell monolayers cultured on Nucleopore filters and mounted between two lucite chambers, Na+-stimulated transport of 2-(methylamino)isobutyric acid (MeAIB), a substrate specific for the A system, was predominantly localized on the basolateral membrane. Na+-stimulated amino acid transport activity was maximal in subconfluent cultures, and was substantially reduced after confluence. A membrane vesicle preparation was isolated from confluent MDCK cell cultures which was enriched in Na+-stimulated MeAIB transport activity and Na+,K+,ATPase activity, a basolateral marker, but was not enriched in apical marker enzyme activities or significantly contaminated by mitochondria. Na+-coupled amino acid transport activity assayed in vesicles exhibited a marked dependence on external pH, with an optimum at pH 7.4. The pattern of competitive interactions among neutral amino acids was characteristic of A system transport. Na+-coupled MeAIB and AIB transport in vesicles was electrogenic, stimulated by creation of an interior-negative membrane potential. The Na+ dependence of amino acid transport in vesicles suggested a Na+ symport mechanism with a 1:1 stoichiometry between Na+ and amino acid.     

Binding of bovine parathyroid hormone to surface receptors of cultured B-lymphocytes

Binding of parathyroid hormone onto B-lymphocytes is detected by the utilization of the labelled antibody membrane assay. The amount of parathyroid hormone bound to the receptor sites was depending on the quantity of cells in the incubation milieu. Each cell line showed typical characteristics in time course of parathyroid hormone binding and maximal receptor capacity. Fragmentation of intact parathyroid hormone, also varying with the cell line tested, was very rapid, even at 24°C. Within 20 min most of the cell lines destroyed 50% of the native hormone in the incubation mixture, indicating a fragmentation rate of up to 2.25 ng/min at 37°C. B_max and K_D for the different lymphocytes was 5.3–19 · 10¹¹ M and 1.8–18.5 · 10¹¹ M, respectively. These values are in the range of reported plasma concentrations and may therefore represent more physiological values for the capacity and affinity of membrane receptors.     

Activation of a Na+-dependent amino acid transport system in preimplantation mouse embryos

The uptake of l-methionine-methyl-³H and l-leucine-³H from completely defined medium into acid-soluble fractions of preimplantation mouse embryos has been studied. Late four-cell embryos and early blastocysts raised in vitro can concentrate both amino acids by processes which exhibit saturable, Michaelis-Menten type kinetics, characteristic of carrier-mediated active transport systems. This uptake is temperature-sensitive and inhibited by certain amino acids which compete for the same uptake sites. Methionine uptake seems to be mediated by a single transport system (K_m = 6.25 × 10^?5M) at the four-cell stage. Complex kinetics suggest that two distinct transport systems exist at the early blastocyst stage (K_m = 6.25 × 10^?5M; 8.9 × 10^?4M). V_max values (mg/embryo/15 min) for methionine and leucine transport increase significantly from the late four-cell stage to the blastocyst stage, suggesting that additional carriers are produced or activated during development.Most importantly, leucine and methionine transport is Na⁺-independent at the four-cell stage, methionine transport is partially dependent at the morula stage, and both amino acids are completely Na⁺-dependent at the blastocyst stage. The cumulative results suggest that preimplantation embryos accumulate leucine and methionine by specific, chemically mediated, active transport systems. The qualitative and quantitative developmental changes in cell membrane function may represent preparatory steps for subsequent growth of embryonic and/or trophoblastic cells.     

Developmental changes in the endogenous Ca2+-stimulated proteolysis of mouse lung cAMP-dependent protein kinases

Regulatory subunits (R subunits) of mouse lung cAMP-dependent protein kinases undergo age-dependent changes in endogenous proteolysis, with the greatest amount of the major M_r = 37,000 proteolytic fragment detectable during fetal and neonatal development. Homogenization of lung in the presence of various protease inhibitors does not affect this age-related difference, suggesting that the observed quantitative change in R subunit proteolysis occurs in vivo. Mechanisms were sought to account for this age-dependent change. The production of a M_r = 37,000 proteolytic fragment can be stimulated in lung extracts by the addition of exogenous calcium and is due to the action of an endogenous Ca²⁺-stimulated protease. Neonatal lung extracts show more Ca²⁺-stimulated proteolysis of R subunits than adult extracts, although only slight agerelated differences in either the Ca²⁺-stimulated protease or its specific endogenous inhibitor were observed. Age-dependent differences in R subunits which may affect sensitivity to proteases were also examined. Analysis of the two-dimensional patterns of adult and neonatal 8-N₃-[³²P]cAMP-labeled R subunits before or after limited proteolysis with trypsin suggests that the R subunits are structurally similar. Differences are found, however, in the relative proportions of adult and neonatal Type I R subunits (R_I) in the holoenzyme or dissociated forms. An increased proportion of neonatal R subunits exist in the dissociated state, whereas adult R subunits exist primarily in the holoenzyme form. Dissociated R subunits from mouse lung are more susceptible than the holoenzyme to limited proteolysis by the partially purified lung Ca²⁺-stimulated protease. Dissociation of the holoenzyme in vivo may be a major factor in the age-dependent proteolytic changes observed in mouse lung protein kinases.     

Regulation of the Ca2+-dependent neutral proteinases from rabbit liver by an endogenous inhibitor

An endogenous inhibitor of neutral Ca2+-dependent proteinases has been isolated from rabbit liver cytosol. The inhibitor is a heat-stable, 240-kDa, tetrameric protein. It is dissociated into its 60-kDa subunits by high concentrations of Ca2+ (0.1-1 mM), but not by lower concentrations in the physiological range. Inhibition of the 150-kDa proteinase of rabbit liver [Melloni, E., Pontremoli, S., Salamino, F., Sparatore, B., Michetti, M. and Horecker, B.L. (1984) Arch. Biochem. Biophys. 232, 505-512] requires the monomeric form of the inhibitor, and occurs only at the high concentrations of Ca2+ which also cause dissociation of the dimeric 150-kDa proteinase into its 80-kDa subunits. The molecular weight of the inactive proteinase-inhibitor complex was estimated by the equilibrium gel penetration method to be 140 kDa, suggesting that it contains one subunit of proteinase and one of inhibitor. The mechanism of interaction of the inhibitor with the 200-kDa proteinase at high concentrations of Ca2+ is identical to that observed for the 150-kDa proteinase, namely dissociation of both proteinase and inhibitor into subunits and formation of an inactive 160-kDa proteinase-inhibitor complex. However, unlike the 150-kDa proteinase, which does not interact with the inhibitor at low Ca2+ concentrations, the 200-kDa proteinase is also inhibited at low concentrations of Ca2+. Under these conditions, the high-molecular-weight complex (greater than 400 kDa) formed between the tetrameric inhibitor and the dimeric proteinase prevents conversion of the 200-kDa proenzyme to the active, low-Ca2+-requiring form.     

Effect of adrenocorticotrophic hormone on the activity of sterol ester hydrolase from rat brain

Adrenocorticotrophic hormone (ACTH) stimulates in vitro the hydrolysis of oleoylcholesterol by a sterol ester hydrolase from rat brain synaptosomes. The stimulatory effect involves an alkaline shift of the pH optimum of catalysis and culminates at pH 6 with a 15 to 20-fold increase in lipolytic rates. The effect requires trace amount of organic solvent in the substrate emulsion and is dependent on the NH₂-terminal sequence extending through the basic amino acid residues at positions 15–18. The hormonal stimulation is decreased when a lipid-depleted preparation is used as enzyme source, and fully restored upon addition of lecithin. The results raise the possibility that ACTH may have a neuro-hormonal role in brain via modulation of local lipolytic processes.     

Influence of membrane fluidity on transport mediated by ubiquinones through phospholipid vesicles

Coenzymes Q₁₀ and Q₃ are incorporated into dipalmitoylphosphatidylcholine and egg yolk lecithin liposomes. Dithionite reduction of ferricyanide trapped inside these phospholipid vesicles is taken as a measure of ubiquinone-mediated transport of reducing equivalents. The reaction shows complex pattern with a high order for CoQ. The initial transport rates are very sensitive to the membrane physical state, being considerably reduced at temperatures below the phase transition of the pure dipalmitoylphosphatidylcholine, both for CoQ₁₀ and CoQ₃ reconstituted with this phospholipid. It is suggested that a different reaction mechanism operates in fluid and rigid membranes. This suggestion is related to the possible organization of CoQs in phospholipid membranes.     

Mg2+- or Mn2+-dependent p-nitrophenylphosphatase activity is present in Ehrlich ascites tumor cells

The presence of a soluble, Mg2+- or Mn2+-dependent p-nitrophenylphosphatase activity in Ehrlich ascites tumor cell homogenates is reported. The crude homogenate was fractionated over Sephadex G-150 gel-filtration and DEAE-Sephacel anion-exchange columns, and two p-nitrophenylphosphatase activities were resolved. The most active fraction, Peak I, was characterized and found to be similar to phosphotyrosyl-protein phosphatases characterized elsewhere in that it has optimal activity at neutral pH; it is inhibited by phosphate, Zn2+, and vanadate; and it is not inhibited by levamisole. However, Peak I differs from phosphotyrosyl-protein phosphatases in that Mg2+ or Mn2+ is required for activity, fluoride is an inhibitor, and pyrophosphate is not inhibitory. Inhibition by the phosphorylated compounds phosphotyrosine, phosphoserine, phosphothreonine, ATP, CTP, GTP, ITP, NADP, fructose 6-phosphate, glucose 1-phosphate, galactose 1-phosphate, 2-phosphogluconic acid, and 6-phosphogluconic acid was also observed. Ehrlich ascites tumor cell p-nitrophenylphosphatase is shown to be sensitive to inactivation by trypsin, N-ethylmaleimide, or heat treatments.