Interaction of lysophospholipid/taurodeoxycholate submicellar aggregates with phospholipid bilayers.

The equilibrium partitioning and the rate of transfer of monoacylphosphatidylethanolamines (lysoPEs) between phospholipid bilayers and lysoPE/taurodeoxycholate submicellar aggregates (SMAs) were examined with a series of environment-sensitive fluorescent-labeled N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-1-monoacylphosphatidyletha nolamine (N-NBD-lysoPE) probes of differing acyl chain length. Our previous work has demonstrated the formation of SMAs between bile salts and lysophospholipids [Shoemaker & Nichols (1990) Biochemistry 29, 5837-5842]. The experiments in the current work demonstrate that SMAs can coexist with phospholipid vesicles and can function as shuttle carriers for the transfer of lysophospholipids between membranes. The formation of submicellar aggregates of N-NBD-lysoPE and taurodeoxycholate (TDC) in equilibrium with 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) vesicles was determined from the increase in fluorescence generated upon addition of TDC to POPC vesicles containing 3 mol% N-NBD-lysoPE and 3 mol% N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine (N-Rh-PE) as a nonextractable fluorescence energy-transfer quencher. The fraction of lysolipid extracted increased as a function of decreasing acyl chain length of the N-NBD-lysoPE molecule. The half-time for equilibration was independent of acyl chain length and averaged 44 ms at 10 degrees C. The delivery of N-NBD-lysoPE from preformed N-NBD-lysoPE/TDC SMAs into POPC vesicles containing the energy-transfer quencher N-Rh-PE was measured by the rate of fluorescence decline. The initial rate of insertion increased with decreasing acyl chain length of the N-NBD-lysoPE molecule and as a function of vesicle concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrophobic interaction of lysophospholipids and bile salts at submicellar concentrations.

A series of environment-sensitive, fluorescent-labeled N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-monoacylphosphatidylethano lamine (N-NBD-lysoPE) probes of differing acyl chain length (C12-C18) was used to demonstrate the hydrophobic interaction between lysophospholipids and two different bile salts at concentrations below their respective critical micelle concentrations (cmc's). Formation of submicellar aggregates in the presence of bile salt-phospholipid mixed micelles could facilitate lipid absorption in the intestine. To ensure the use of submicellar lysolipid concentrations in the experiments, the cmc of each fluorescent lysolipid probe was determined by concentration-dependent self-quenching. The cmc values obtained for the various N-NBD-lysoPE probes were as follows (microM): monolauroyl, greater than or equal to 40; monomyristoyl, 4; monopalmitoyl, 0.3; monostearoyl, 0.04. Probe concentrations well below their respective cmc's were used in all experiments. The fluorescence of a solution of each lysolipid probe was monitored as the concentration of bile salt was gradually increased. The increase in fluorescence was taken as a measure of the ability of the bile salt molecules to complex with the probe molecule, thereby increasing the fluorescent yield of the lysolipid probe molecule. Determination of the cmc of the bile salts in the presence of the lysolipid probe was made in parallel with the fluorescence measurement by monitoring the increase in light scattering of the solution. Both bile salts were shown to induce maximal increases in fluorescence of the N-NBD-lysoPE derivatives at concentrations of bile salt well below their respective cmc values, indicating the existence of submicellar lysolipid-bile salt aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of phospholipid transfer between mixed phospholipid-bile salt micelles.

Concentration-dependent self-quenching of the fluorescent phospholipid N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (N-NBD-PE) was used to measure the rate of N-NBD-PE transfer between phosphatidylcholine-bile salt mixed micelles. In a previous study using the same technique, the rate of N-NBD-PE transfer between phosphatidylcholine-taurocholate mixed micelles was found to be several orders of magnitude faster than its transfer between phosphatidylcholine vesicles as a result of an increased rate of transfer through the water at low micelle concentrations and an increased rate of transfer during transient micelle collisions at higher micelle concentrations [Nichols, J. W. (1988) Biochemistry 27, 3925-3931]. In this study we have determined the influence of bile salt structure, incorporation of cholesterol, and temperature on the rate and mechanism of phospholipid transfer between mixed micelles. We found that both transfer pathways were a common property of mixed micelles prepared from a series of different bile salts and that the rates of transfer by both pathways increased as a function of the degree of bile salt hydrophobicity. Cholesterol incorporation into phosphatidylcholine-taurocholate mixed micelles displaced taurocholate from the micelles and resulted in an increased rate of transfer through the water and a decreased rate of transfer during micelle collisions. The temperature dependence of the transfer rates was used to calculate the activation free energy, enthalpy, and entropy for both mechanisms. The activation enthalpy was the major barrier to transfer by both mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium-phosphate cotransport in human red blood cells. Kinetics and role in membrane metabolism.

Orthophosphate (Pi) uptake was examined in human red blood cells at 37 degrees C in media containing physiological concentrations of Pi (1.0-1.5 mM). Cells were shown to transport Pi by a 4,4'-dinitro stilbene-2,2'-disulfonate (DNDS) -sensitive pathway (75%), a newly discovered sodium-phosphate (Na/Pi) cotransport pathway (20%), and a pathway linearly dependent on an extracellular phosphate concentration of up to 2.0 mM (5%). Kinetic evaluation of the Na/Pi cotransport pathway determined the K1/2 for activation by extracellular Pi ([Na]o = 140 mM) and extracellular Na [( Pi]o = 1.0 mM) to be 304 +/- 24 microM and 139 +/- 8 mM, respectively. The phosphate influx via the cotransport pathway exhibited a Vmax of 0.63 +/- 0.05 mmol Pi (kg Hb)-1(h)-1 at 140 mM Nao. Activation of Pi uptake by Nao gave Hill coefficients that came close to a value of 1.0. The Vmax of the Na/Pi cotransport varied threefold over the examined pH range (6.90-7.75); however, the Na/Pi stoichiometry of 1.73 +/- 0.15 was constant. The membrane transport inhibitors ouabain, bumetanide, and arsenate had no effect on the magnitude of the Na/Pi cotransport pathway. No difference was found between the rate of incorporation of extracellular Pi into cytosolic orthophosphate and the rate of incorporation into cytosolic nucleotide phosphates, but the rate of incorporation into other cytosolic organic phosphates was significantly slower. Depletion of intracellular total phosphorus inhibited the incorporation of extracellular Pi into the cytosolic nucleotide compartment; and this inhibition was not reversed by repletion of phosphorus to 75% of control levels. Extracellular 32Pi labeled the membrane-associated compounds that migrate on thin-layer chromatography (TLC) with the Rf values of ATP and ADP, but not those of 2,3-bisphosphoglycerate (2,3-DPG), AMP, or Pi. DNDS had no effect on the level of extracellular phosphate incorporation or on the TLC distribution of Pi in the membrane; however, substitution of extracellular sodium with N-methyl-D-glucamine inhibited phosphorylation of the membranes by 90% and markedly altered the chromatographic pattern of the membrane-associated phosphate.(ABSTRACT TRUNCATED AT 400 WORDS)

Amiloride fluxes across erythrocyte membranes.

Amiloride is known to inhibit both the influx of Na+ and the activation of mitogenesis in many cultured cell lines. This paper describes experiments in which the permeability coefficient of amiloride was determined from measurements of tracer fluxes across human erythrocytes and resealed ghosts. From an analysis of these fluxes, a permeability coefficient of 10(-7) cm/s for the uncharged form of amiloride was deduced. Based upon this measured permeability value, we present calculations of intracellular accumulation times of amiloride in cells of differing surface-to-volume ratio.

[3H]Ouabain binding and Na+, K+-ATPase in resealed human red cell ghosts.

The interaction of the cardiac glycoside [3H]ouabain with the Na+, K+ pump of resealed human erythrocyte ghosts was investigated. Binding of [3H]ouabain to high intracellular Na+ ghosts was studied in high extracellular Na+ media, a condition determined to produce maximal ouabain binding rates. Simultaneous examination of both the number of ouabain molecules bound per ghost and the corresponding inhibition of the Na+, K+-ATPase revealed that one molecule of [3H]ouabain inhibited one Na+, K+-ATPase complex. Intracellular magnesium or magnesium plus inorganic phosphate produced the lowest ouabain binding rate. Support of ouabain binding by adenosine diphosphate (ADP) was negligible, provided synthesis of adenosine triphosphate (ATP) through the residual adenylate kinase activity was prevented by the adenylate kinase inhibitor Ap5A. Uridine 5'-triphosphate (UTP) alone did not support ouabain binding after inhibition of the endogenous nucleoside diphosphokinase by trypan blue and depletion of residual ATP by the incorporation of hexokinase and glucose. ATP acting solely at the high-affinity binding site of the Na+, K+ pump (Km approximately 1 microM) promoted maximal [3H]ouabain binding rates. Failure of 5'-adenylyl-beta-gamma-imidophosphate (AMP-PNP) to stimulate significantly the rate of ouabain binding suggests that phosphorylation of the pump was required to expose the ouabain receptor.