Piotr Gmaj

Ca2+-stimulated, Mg2+-independent ATP hydrolysis and the high affinity Ca2+-pumping ATPase. Two different activities in rat kidney basolateral membranes

The Mg2+-dependency of Ca2+-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg2+- and Ca2+-buffering ligands. ATP hydrolysis is strongly stimulated by Mg2+ with a Km of 13 microM in the absence or presence of 1 microM free Ca2+. At free Mg2+ concentrations of 1 microM and lower, ATP hydrolysis is Mg2+-independent, but is strongly stimulated by submicromolar Ca2+ concentrations (Km = 0.25 microM, Vmax = 24 mumol Pi/h per mg protein). The Ca2+-stimulated ATP hydrolysis strongly decreases at higher Mg2+ concentrations. The Ca2+-stimulated Mg2+-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg2+ concentrations calmodulin and trifluoperazine affect the high affinity Ca2+-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca2+-pumping in renal basolaterals and on Ca2+-ATPase in erythrocyte ghosts suggest that the Ca2+-pumping enzyme requires Mg2+. In contrast, a role of the Ca2+-stimulated Mg2+-independent ATP hydrolysis in active Ca2+ transport across basolateral membranes is rather unlikely.

[20] Transport studies with renal proximal tubular and small intestinal brush border and basolateral membrane vesicles: Vesicle heterogeneity, coexistence of transport systems

Publisher Summary The membrane mechanisms involved in small intestinal and renal proximal tubular transcellular transport of various solutes can be explored in isolated brush border and basolateral membrane vesicles. This chapter discusses the properties of a transport system. Studies with isolated membrane vesicles are useful for the determination of the asymmetric distribution of transport pathways between a brush border and a basolateral membrane. However, the heterogeneity of vesicle preparations presents problems that should be considered in the interpretation of experiments on the localization of specific transport pathways. Preparations of membrane vesicles are heterogeneous—that is, membranes with different transport pathways even for the same substrate might be present. This heterogeneity can be related to (1) the cross contamination of basolateral membranes with brush border membranes and vice versa and (2) a heterogeneous epithelial cell population used for vesicle isolation. The chapter provides recommendations regarding the problems related to tissue heterogeneity and/or cross contaminations and presents some experimental approaches that allow the documentation of different transport pathways coexisting in one membrane vesicle.

Calcium uptake into rat small intestinal brush border membrane vesicles: Characterization of transmembrane calcium transport at short initial incubation times

Calcium transport into brush border vesicles from rat small intestine was investigated by determining uptake rates at very short incubation periods. At incubation times up to 1 second a linear relationship between calcium uptake and time was observed at free calcium concentrations ranging from 1 microM to 5 mM. At time points above 1 second calcium uptake deviates progressively from linearity. Several lines of evidences (EGTA-wash, dependency on membrane potential, temperature sensitivity and effect of the calcium ionophore A23187) suggest transmembrane transport rather than extravesicular binding of calcium as being responsible for calcium uptake. Saturation experiments performed under initial linear and curvilinear uptake conditions show a saturable transport component in the mu molar and only a tendency to saturate in the molar concentration range. It is concluded that uptake values far from equilibrium are characteristic for transmembrane flux of calcium. Transmembrane flux of calcium is mediated by multiple and potential-sensitive mechanisms.

Intravesicular NAD has no effect on sodium-dependent phosphate transport in isolated renal brush border membrane vesicles

The effects of intravesicular NAD on Na+-dependent32Pi uptake were investigated in isolated rat kidney brush border membrane vesicles (BBMV). NAD was introduced into the vesicles by osmotic shock, and extravesicular NAD was removed by passing the vesicles through a anion exchange column. The effectiveness of the osmotic shock procedure and the hydrolysis of extra- and intravesicular NAD were controlled by enzymatic analysis and thin layer chromatography. ADP-ribosylation of the membrane proteins was analyzed in vesicles osmotically shocked in the presence of either [adenylate-32P]-NAD or [adenine-2,8-3H]-NAD by SDS-polyacrylamide gel electrophoresis.It was found that the Na+-dependent Pi uptake was inhibited when the BBMV were incubated with NAD at alkaline pH, which resulted in rapid NAD hydrolysis. When NAD was present in the intravesicular space only, the Na+-dependent Pi uptake was not inhibited.32P from NAD was rapidly incorporated into a number of brush border membrane proteins, but no incorporation of3H-adenine could be detected.The results provide evidence that NAD does not inhibit Pi transport by a direct interaction with the cytoplasmic side of the brush border membrane. No evidence of ADP-ribosylation of the brush border membrane protein(s) was found.

Calcium transport in basolateral plasma membranes from kidney cortex of Milan hypertensive rats

Ca2+ transport was investigated in basolateral plasma membranes (BLM) isolated from kidney cortex of the Milan strain of genetically hypertensive rats (MHS) and their normotensive controls (MNS) during a pre-hypertensive stage (age 3-4 weeks). It was found that the Vmax of ATP-dependent Ca2+ transport (in the presence of calmodulin) was about 16% lower in MHS than in control rats. In membranes from MNS rats which had been isolated in the presence of EGTA, the ATP-dependent Ca2+ transport showed a hyperbolic Ca2+ concentration dependence, a high Km (Ca2+) and a low Vmax; upon addition of exogenous calmodulin, the kinetics became sigmoidal, the Km (Ca2+) was decreased and the Vmax was increased. In membranes from MHS rats, the Ca2+ concentration dependence of ATP-driven Ca2+ transport was sigmoidal and the Ca2+ affinity was high in the absence of added calmodulin. Addition of exogenous calmodulin to these membranes resulted in an increase in Vmax, but no change in other kinetic parameters. Low-affinity hyperbolic kinetics of Ca2+ transport could only be obtained in MHS rats if the membranes were extracted with hypotonic EDTA and hypertonic KCl. These data suggest that the plasma membrane Ca2+-ATPase, which catalyses the ATP-dependent Ca2+ transport, exists in BLM of pre-hypertensive MHS rats predominantly in an activated, high-affinity form.

Role of c-AMP and NAD in the Regulation of Na+-Dependent Phosphate Transport Across Rat Renal Brush Border Membranes

The activity of the sodium-dependent Pi-transport system in the brush border membrane changes in concordance with the tubular phosphate reabsorption under a variety of physiological conditions (Dousa and Kempson, 1982). Two mechanisms were postulated to be involved in these adaptations: a cyclic-AMP dependent phosphorylation of brush border membrane protein(s) (Hammerman and Hruska, 1982) and a direct interaction of NAD with the membrane (Kempson et al., 1981), apparently via a ADP-ribosylation of certain membrane proteins (Hammerman et al., 1982).