Lipid fluidity of hepatocyte plasma membrane subfractions and their differential regulation by calcium

Rat hepatocyte plasma membranes were subfractionated by several methods into canalicular, sinusoidal and mixed contiguous plus sinusoidal membranes. Assessment of lipid fluidity by steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and 12-(9-anthroyloxy)stearate indicates that the canalicular fraction is less fluid than the other membranes. Incubation with calcium decreases the fluidity of the sinusoidal and contiguous membranes by altering the lipid composition, an action which is not reversed by subsequent chelation of the cation. This effect of calcium is not observed in canalicular membranes.     

Tyrosine transport by membrane vesicles isolated from rat brain

Tyrosine uptake by membrane vesicles derived from rat brain has been investigated. The uptake is dependent on an Na⁺ gradient ([$\text{Na}{}^{\mathrm{+}}\text{]}\text{outside}\text{> [}\text{Na}{}^{\mathrm{+}}\text{]}\text{inside}$). The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. The process is stimulated by a membrane potential (negative inside) as demonstrated by the effect of the ionophores valinomycin and carbonyl cyanide $\text{m-}\text{chlorophenylhydrazone}$ and anions with different permeabilities. Kinetic data show that tyrosine is accumulated by two systems with different affinities. Tyrosine uptake is inhibited by the presence of phenylalanine and tryptophan.     

The respiration of brain mitochondria and its regulation by monovalent cation transport

The Na⁺ and K⁺ permeability properties of rat brain mitochondria were determined to explain the influences of these cations upon respiration. A new procedure for isolating exceptionally intact mitochondria with minimal contamination by synaptosomes was developed for this purpose.Respiration was uncoupled by Na⁺ and less so by K⁺. Uncoupling was maximal in the presence of EDTA plus P_i and was decreased by Mg²⁺. Maximal uncoupler-stimulated respiration rates were inhibited by Na⁺ but largely unaffected by K⁺. The inhibition by Na⁺ was relatively insensitive to Mg²⁺. Membrane Na⁺ and K⁺ conductances as well as neutral exchanges (Na⁺/H⁺ and K⁺/H⁺ antiport activities) were determined by swelling measurements and correlated with metabolic effects of the cations.Cation conductance, i.e. electrophoretic Na⁺ or K⁺ permeation, was increased by EDTA (Na⁺ > K⁺) and decreased by Mg²⁺. Magnesium preferentially suppressed Na⁺ conductance so as to reverse the cation selectivity (K⁺ > Na⁺). Neutral cation/H⁺ exchange rates (Na⁺ > K⁺) were not influenced by chelator or Mg²⁺.The extent of cation-dependent uncoupling of respiration correlated best with the inner membrane conductance of the ion according to an empirical relationship derived with the model K⁺ conductor valinomycin. The metabolic influences of Na⁺ and K⁺ can be explained in terms of coupled flow of these ions with protons and their effect upon the H⁺ electrochemical gradient although alternative possibilities are discussed. These in vitro studies are compared to previous observations in situ to assess their physiological significance.     

Uptake of asialoglycophorin-liposomes by the perfused rat liver

Asialoglycophorin-containing liposomes and their contents (¹²⁵I-labeled bovine serum albumin) were taken up by a perfused rat liver with subsequent digestion of their protein components. The uptake of these liposomes required Ca²⁺ as well as desialylation. The process was inhibited partially by asialofetuin and completely by further addition of asialoglycophorin to the perfusate.     

Large-scale preparation of plasma membrane vesicles from PC-12 Pheochromocytoma cells and their use in noradrenaline transport studies

Plasma membranes were isolated from rat pheochromocytoma cells (PC-12) grown in spinner culture. The rapid and simple isolation procedure consisted of a differential and isopycnic centrifugation (in a linear sucrose gradient) with the aid of a high capacity fixed angle rotor equipped with siliconized centrifuge tubes. The isolated membranes were closed and osmotically active vesicles (about 0.3 μm in diameter) with a mean intravesicular water space of 1.84 μl / mg protein. In the presence of an inward gradient of sodium chloride and an outward gradient of potassium, [³H]noradrenaline (50 nM) was taken up and accumulated 550-fold (at 31°C). The uptake and accumulation of [³H]noradrenaline was temperature-sensitive and inhibited by the tricyclic antidepressant desipramine. Membrane vesicles isolated from PC-12 cells represent a useful model for the investigation of the molecular mechanism of the neuronal noradrenaline transport system.     

Comparison of the properties of active Ca2+ transport by the islet-cell endoplasmic reticulum and plasma membrane

The properties of active or ATP-dependent calcium transport by islet-cell endoplasmic reticulum and plasma membrane-enriched subcellular fractions were directly compared. These studies indicate that the active calcium transport systems of the two membranes are fundamentally distinct. In contrast to calcium uptake by the endoplasmic reticulum-enriched fraction, calcium uptake by islet-cell plasma membrane-enriched vesicles exhibited a different pH optimum, was not sustained by oxalate, and showed an approximate 30-fold greater affinity for ionized calcium. A similar difference in affinity for calcium was exhibited by the Ca²⁺-stimulated ATPase activities which are associated with these islet-cell subcellular fractions. Consistent with the effects of calmodulin on calcium transport, calmodulin stimulated Ca²⁺-ATPase in the plasma membranes, but did not increase calcium-stimulated ATPase activity in the endoplasmic reticulum membranes. The physiological significance of the differences observed in calcium transport by the endoplasmic reticulum and plasma membrane fractions relative to the regulation of insulin secretion by the islets of Langerhans is discussed.     

Properties and regulation of a trans-plasma membrane redox system of perfused rat heart

Ferricyanide was reduced to ferrocyanide by the perfused rat heart at a linear rate of 78 nmol/min per g of heart (non-recirculating mode). Ferricyanide was not taken up by the heart and ferrocyanide oxidation was minimal (3 nmol/min per g of heart). Perfusate samples from hearts perfused without ferricyanide did not reduce ferricyanide. A single high-affinity site (apparent K_m=22 μM) appeared to be responsible for the reduction. Perfusion of the heart with physiological medium containing 0.5 mM ferricyanide did not alter contractility, biochemical parameters or energy status of the heart. Perfusate flow rate and perfusate oxygen concentration exerted opposing effects on the rate of ferricyanide reduction. A net decreased reduction rate resulted from a decreased perfusion flow rate. Thus, the rate of supply of ferricyanide dominated over the stimulatory effect of oxygen restriction; the latter effect only becoming apparent when the oxygen concentration was lowered at a high perfusate flow rate. Whereas glucose (5 mM) increased the rate of ferricyanide reduction, pyruvate (2 mM), acetate (2 mM), lactate (2 mM) and 3-hydroxybutyrate (2 mM) each had no effect. Insulin (3 nM), glucagon (0.5 μM), dibutyryl cyclic AMP (0.1 mM) and the β-adrenergic agonist ritodrine (10 μM) also had no effect, however the α₁-adrenergic agonist, methoxamine (10 μM), produced a net increase in the rate of ferricyanide reduction. It is concluded that a trans-plasma membrane electron efflux occurs in perfused rat heart that is sensitive to oxygen supply, glucose, perfusion flow rate, and the α-adrenergic agonist methoxamine.     

The effects of general anaesthetics on glucose and phosphate transport across the membrane of the human erythrocyte

A study has been carried out into the effects of clinically important general anaesthetics, althesin, thiopentone and propanidid, on the transport of glucose and phosphate across the membrane of the human erythrocyte. In general these three substances all inhibit both transport processes but with characteristic inhibition profiles and varying degrees of efficacy. Glucose transport was more sensitive to the hydrophobic steroids and phosphate transport to propanidid. Some hydrophobic agents, e.g., iodobenzene and its azide, were not inhibitory. Removal of cholesterol to some extent augmented the inhibitory effects of most of these compounds (not propanidid). It is argued that these effects are due to the penetration of the anaesthetics into the lipid bilayer and either subsequent disruption of the lipid annuli surrounding the integral membrane proteins and/or direct anaesthetic-protein interaction.     

Effect of metabolic acidosis on phosphate transport by the renal brush-border membrane

Metabolic acidosis produces a phosphaturia which is independent of parathyroid hormone or dietary phosphorus intake. To study the underlying mechanism, inorganic phosphate (P_i) and glucose transport were studied in brush-border membrane vesicles prepared from the renal cortex of parathyroidectomized rats gavaged for three days with either 7.5 ml of 1.6% NaCl (control) or 1.5% NH₄Cl (acidosis). At killing, blood pH and plasma bicarbonate were $\text{7.36 ± 0.01}$ and $\text{21.8 ± 0.8}\text{mequiv./l}$, respectively, in control and $\text{7.12 ± 0.03}$ ($\text{P < 0.01}$) and $\text{11.1 ± 1.2}$ ($\text{P < 0.01}$) in acidotic rats. Serum P_i was similar in both groups, while 24 h urine P_i excretion was higher in the acidotic group ($\text{P < 0.01}$). Peak sodium-dependent uptake of P_i, measured after 1.5 min of incubation, was higher in controls than acidotic rats ($\text{4442 ± 464}$ vs. $\text{2412 ± 259}\text{pmol/mg}$ protein, $\text{P < 0.01}$), whereas peak glucose uptake at 1.5 min was not significantly different between the groups. Equilibrium values for P_i and glucose uptake were similar in the two groups. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for P_i uptake in the control and acidotic animals were not different, 0.036 and 0.040 mM, respectively. By contrast, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ was higher in controls than in the acidotic group, 3.13 vs. 1.15 nmol/mg protein per 15 s. These results suggest that metabolic acidosis directly inhibits P_i uptake by the brush border of the proximal tubule by decreasing the availability of P_i carriers of the renal brush-border membrane.     

Kinetics of mitochondrial phosphate transport and rates of respiration and phosphorylation during greening of etiolated Avena leaves

Using the technique of silicone oil filtration of organelles and the inhibitor stop method, the kinetics of transport of inorganic phosphate across the inner mitochondrial membrane were tested in relation to different stages of greening (0 to 24 h) of etiolated laminae of Avena sativa L., and compared to the rates of oxygen consumption and ATP formation. The results demonstrate that there is a pronounced increase in phosphate transport after 3 h of greening, reaching values for V_max (about 17 mol mg protein^-1 h^-1) that are three times as high as those measured with mitochondria from etiolated tissue. This is also mirrored by the rates of respiration and oxidative phosphorylation. After 24 h of light treatment (4 Klx), respiration and ATP formation, as well as V decreased again to levels below those of the etiolated stage. In contrast to V, there was no change in the affinity between inorganic phosphate and the binding sites of the transporting systems involved, as indicated by a rather constant K_m (0.23 mM) for phosphate transport. Of the inhibitors of phosphate transport tested, mersalyl and methyl mercuric iodide were most efficient with identical characteristics of inhibition; but compared to animal mitochondria, the concentrations needed to result in similar amounts of inhibition, were more than ten times higher. The results are discussed with respect to plastid development.Abbreviations BSA bovine serum albumine - CH₃HgJ methyl mercuric iodide - Cyt cytochrome - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid - MDH malate dehydrogenase - NEM N-ethylmaleimide     

Mitochondrial membrane potential, transmembrane difference in the NAD redox potential and the equilibrium of the glutamate-aspartate translocase in the isolated perfused rat heart

The distribution of glutamate and aspartate and the mitochondrial membrane potential (Δψ) were studied in isolated rat heart mitochondria and in the intact perfused rat heart. The diffusion potential imposed by the glutamate-aspartate exchange through mediation of the electrogenic glutamate-aspartate translocator attained a value close to the mitochondrial Δψ measured from the distribution of triphenylmethylphosphonium ion (TPMP⁺) both in isolated mitochondria and in intact myocardium. Distributions of the Δψ probe and metabolites were determined by subcellular fractionation of the heart muscle in a non-aqueous medium. The results indicate that the glutamate-aspartate translocator is in near equilibrium in the myocardium. The diffusion potential of the glutamate-aspartate exchange, and the mitochondrial/cytosolic difference in the redox potentials of the free NAD⁺/NADH pools are equal allowing for experimental error. These data obtained from intact tissue can therefore be interpreted as supporting the notion of the transmembrane uphill transport of reducing equivalent from the cytosolic free NAD⁺/NADH pool being driven by the malate-aspartate cycle energized by the mitochondrial Δψ.     

A simple method for the isolation of basolateral plasma membrane vesicles from rat kidney cortex Enzyme activities and some properties of glucose transport

A procedure for preparing basolateral membrane vesicles from rat renal cortex was developed by differential centrifugation and Percoll density gradient centrifugation, and the uptake of d-[³H]glucose into these vesicles was studied by a rapid filtration technique. (Na⁺ + K⁺)-ATPase, the marker enzyme for basolateral membranes, was enriched 22-fold compared with that found in the homogenate. The rate of d-glucose uptake was almost unaffected by Na⁺ gradient (no overshoot).     

Plasma membrane redox and regulation of cell growth

Summary Gene expression can be activated by external oxidants which are reduced at the cell surface by plasma membrane electron transport. The signals generated in response to the plasma membrane electron transport include activation of proton release, internal calcium changes, and change in reductant/oxidant ratio in the cytosol. H₂O₂ generated in response to ligands which bind to plasma membrane receptors can also activate protein tyrosine kinases and gene expression. Inhibition of oxygen radical generation at the cell surface in response to the mitogen, phorbol myristate acetate by retinoic acid is consistent with a role for the plasma membrane electron transport as the source for H₂O₂ in Balb 3T3 cells. Agents which affect the binding of coenzyme Q to redox sites in the plasma membrane electron transport may increase formation of semiquinone radicals in the membrane which can be a source of oxygen radicals and H₂O₂. The generation of H₂O₂ by transformed cells indicates that oncogene product expression in the plasma membrane may also increase quinone-based oxygen radical generation.     

Plasma membrane NADH dehydrogenase and Ca2+-dependent potassium transport in erythrocytes of several animal species

Ca²⁺-dependent K⁺ transport and plasma membrane NADH dehydrogenase activities have been studied in several ‘high-K⁺’ (human, rabbit and guinea pig) and ‘low-K⁺’ (dog, cat and sheep) erythrocytes. All the species except sheep showed Ca²⁺-dependent K⁺ transport. NADH-ferricyanide reductase was detected in all the species and showed positive correlation with the flavin contents of the membranes. NADH-cytochrome $\text{c}$ reductase was very low or absent in dog, sheep and guinea pig membranes. No correlation was found between NADH dehydrogenase and Ca²⁺-dependent K⁺ channel activities in the species studied. Nor were any of the above activities correlated with $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity.     

Characterization of amino-acid transport in Ricinus communis roots using isolated membrane vesicles

The mechanism and specificity of amino-acid transport at the plasma membrane of Ricinus communis L. roots was investigated using membrane vesicles isolated by phase partitioning. The transport of glutamine, isoleucine, glutamic acid and aspartic acid was driven by both a pH gradient and a membrane potential (internally alkaline and negative), created artificially across the plasma membrane. This is consistent with transport via a proton symport. In contrast, the transport of the basic amino acids, lysine and arginine, was driven by a negative internal membrane potential but not by a pH gradient, suggesting that these amino acids may be taken up via a voltage-driven uniport. The energized uptake of all of the amino acids tested showed a saturable phase, consistent with carrier-mediated transport. In addition, the membrane-potential-driven transport of all the amino acids was greater at pH 5.5 than at pH 7.5, which suggests that there could be a direct pH effect on the carrier. Several amino-acid carriers could be resolved, based on competition studies: a carrier with a high affinity for a range of neutral amino acids (apart from asparagine) but with a low affinity for basic and acidic amino acids; a carrier which has a high affinity for a range of neutral amino acids except isoleucine and valine, but with a low affinity for basic and acidic amino acids; and a carrier which has a higher affinity for basic and some neutral amino acids but has a lower affinity for acidic amino acids. The existence of a separate carrier for acidic amino acids is discussed.Abbreviations PM plasma membrane - TPP⁺ tetraphenylphosphonium ion - pH pH gradient - membrane potential This work was supported by the Agricultural and Food Research Council and The Royal Society. We would like to thank Mrs. Sue Nelson for help with some of the membrane preparations.     

An investigation into the feasibility of using yeast protoplasts to study the ion transport properties of the plasma membrane

A method for studying ion uptake in enzymatically isolated protoplasts from the yeast, Saccharomyces cerevisiae, is described. The kinetics of K⁺ and Rb⁺ uptake, metabolic proton extrusion and cell electrophoretic mobility bave been determined. Enzymic removal of the cell wall does not significantly alter the above-mentioned properties of the yeast cells. It is concluded that studies of these properties can be performed equally well with intact yeast cells or protoplasts. However, in studies aimed at determining effects of complex organic substances, e.g., antibiotics, on plasma membrane function the use of protoplasts is recommended. The effectiveness of the antibiotic, Dio-9, for example, in reversing the metabolic proton extrusion into a net proton influx is at least 50 times higher after enzymic removal of the yeast cell wall.     

Heterogeneous distribution of the sodium-dependent alanine transport activity in the rat hepatocyte plasma membrane

The localization of the sodium-dependent alanine uptake activity in rat liver cells was studied. Fractions representative of the canalicular, the contiguous (lateral) and the blood-sinusoidal surface of the hepatocyte were isolated by means of centrifugal fractionation and density gradient centrifugation. The distribution of various marker-enzyme activities in conjunction with the occurrence of alanine transport activity was studied both in fractions obtained after zonal density gradient centrifugation, and in the subcellular fractions mentioned above.It is concluded that the sodium-dependent alanine transport activity is primarily located in the blood-sinusoidal plasma membrane of the hepatocyte.     

Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart: 31P-NMR studies

Fluxes catalyzed by soluble creatine kinase (MM) in equilibrium in vitro and by the creatine kinase system in perfused rat hearts were studied by ³¹P-NMR saturation transfer method. It was found that in vitro both forward and reverse fluxes through creatine kinase at equilibrium were almost equal and very stable to changes in $\text{phosphocreatine}\text{creatine}$ ratio (from 0.2 to 3.0) as well as to changes in pH (from 7.4 to 6.5 or 8.1), free Mg²⁺ concentration and 2-fold decrease of total adenine nucleotides and creatine pools (from 8.0 to 4.0 mM and from 30 to 14 mM, respectively). In the rat hearts perfused by the Langendorff method the creatine kinase-catalyzed flux from phosphocreatine to ATP was increased by 50% when oxygen consumption grew from 8 to 55 μmol/min per g of dry wt. due to transition from rest to high workload. These changes could not be exclusively explained on the basis of the equilibrium model by activation of heart creatine kinase due to some decrease in $\text{[}\text{phosphocreatine}\text{]}\text{[}\text{creatine}\text{]}$ ratio (from 1.8 to 0.8) observed during transition from rest to high workload. Analysis of our data showed that an increase in the flux via creatine kinase is correlated with an increase in the rate of ATP synthesis with a linearity coefficient higher than 1.0. These data are more consistent with the concept of energy channeling by phosphocreatine shuttle than with that of the creatine kinase equilibrium in the heart.     

Interaction of phenylisothiocyanates with the mitochondrial phosphate carrier. I. Covalent modification and inhibition of phosphate transport

The effects of phenylisothiocyanate (PITC) and of the polar analogue p-sulfophenylisothiocyanate (p-sulfoPITC) on the phosphate carrier of bovine heart mitochondria have been investigated. Incubation of mitochondria with the two phenylisothiocyanates leads to inhibition of the phosphate carrier protein. The inhibition of phosphate transport by PITC is unaffected by the addition of dithioerythritol (DTE) or by variation of the pH. The inhibition by p-sulfoPITC is in part removed by DTE; the remaining inactivation of the phosphate carrier, which can be attributed to the reaction with NH₂ groups, is temperature and pH-dependent. Inhibition of phosphate transport by both p-sulfoPITC and PITC depends on the time of incubation and the concentration of the inhibitor. Preincubation with mersalyl protects the carrier protein against the inactivation by p-sulfoPITC but not against PITC. Other SH reagents tested do not show any protective effect. It can thus be concluded that two types of lysine residues are essential for the activity of the phosphate carrier. Lysine(s) of the former type are located at the surface of the membrane and are topologically related to the functional SH groups of the protein. Lysine residue(s) of the latter type are buried in the hydrophobic phase of the membrane.