Sodium transport in erythrocytes of the mollusc Anadara broughtonii: Evidence for inhibition by quinine

Sodium transport through the molluscan erythrocyte membrane was examined using ²²Na as a tracer. Incubation of the red cells in standard saline resulted in a rapid ²²Na uptake reaching steady state concentration (about 21.5 mmol/l cells) in the first 60 min. A similar pattern in the time course of ²²Na uptake was seen in the erythrocytes incubated in mantle fluid. The average value of unidirectional Na⁺ influx, measured as a 5-min ²²Na uptake, was 7.76 ± 0.36 mmol/1 cells/5 min or 93 ± 4.3 mmol/1 cells/hr. The initial rate of Na⁺ influx increased in a saturable fashion as a function of external Na⁺ concentration with apparent AT., of 380±12mM and V_max of 14.3 ± 2.4 mmol/1 cells/5 min. Amiloride (1 mM), furosemide (1 mM), and DIDS (0.1 mM) had no effect on either initial Na⁺ influx (5 min ²²Na uptake) or equilibrium Na⁺ concentration (60 min and 120min ²²Na uptake) in the molluscan red cells exposed to standard saline. Quinine (1 mM) caused a significant fall in the initial Na⁺ influx (by 48%) and in 60-min ²²Na uptake (by 32%) as compared with control levels. In the presence of 0.1 mM ouabain, ²²Na uptake into the red cells was enhanced by an average 27% and 44% during 60 min and 120 min of cell incubation, respectively. The ouabain-sensitive Na⁺ accumulation in the red cells reflected a contribution of the Na, K-pump to Na⁺ transport and the mean value was 5.6 ± 1.0 mmol/1 cells/hr.     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

Sodium channel activation does not alter lipid metabolism in cultured neuroblastoma cells

The interaction of voltage-sensitive Na⁺-channels and membrane lipid metabolism was examined by incubating cultured neuroblastoma cells with neurotoxins which alter the voltage-dependent relationship between the closed and open conformation of the channel protein. Guanidinium flux rate, a measure of Na⁺-channel activation, was increased 10-fold by the combined action of veratridine (100 M) and scorpion venom (28 g/ml). This response was completely blocked by tetrodotoxin (1 M). Under the same experimental conditions, the toxins did not increase the efflux of [³H]arachidonic acid from prelabeled cell membrane lipids or stimulate uptake of exogenous [³H]arachidonic acid. In addition, altering membrane fatty acid composition by incubating cells for 24 hr in a medium containing 50 M arachidonic or oleic acid did not alter guanidinium flux rates relative to that of control cultures. When cells were pulsed with³²Pi for 60 min and stimulated by veratridine plus scorpion venom for an additional 30 min, uptake of³²Pi into phosphatidylinositol as reduced; stimulating cells with bradykinin, a receptor agonist which activates the inositol cycle, promoted a 3.8 fold increase. Polyphosphoinositide turnover was not affected by Na⁺-channel activation, but was stimulated by bradykinin. These results suggest that voltage-sensitive Na⁺-channel activation in cultured neuroblastoma cells can function independent of membrane phospholipid and fatty acid metabolism.     

Facilitated Transport of Lactate by Rat Jejunal Enterocyte

L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles l-lactate uptake is stimulated by an inwardly directed H⁺ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K _m of 39.2 ± 4.8 mm and a J _max of 8.9 ± 0.7 nmoles mg protein⁻¹ sec⁻¹. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na⁺ and H⁺ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H⁺-lactate cotransporter, whereas in the apical membrane both H⁺-lactate and Na⁺-lactate cotransporters are present, even if they exhibit a low transport rate. Received: 22 October 1996/Revised: 11 March 1997     

A search for an ‘Ouabain-like’ substance from the electric organ of Electrophorus electricus which led to arachidonic acid and related fatty acids

The electric organ of Electrophorus electricus contains substances which inhibit (Na⁺ + K⁺)-ATPase activity, the specific binding of [³H]ouabain to purified (Na⁺ + K⁺)-ATPase and ⁸⁶Rb⁺ uptake by chick cardiac cells in culture. The active organic material was extracted from microsomal membranes. Its purification was carried out by chromatography on Sep-Pak C-18 and thin-layer chromatography. Reverse-phase liquid chromatography and mass spectrometry identified the active material as a mixture of unsaturated fatty acids. Linoleic (18:2), arachidonic (20:4), linolenic (18:3) and docosahexaenoic acids (22:6) contributed to about 60% of the total activity of the active material. The other active substances could be arachidonic analogs, since they have both a lipophilic and carboxylic character. Pure unsaturated fatty acids have been shown to be active in the different biological assays used to analyze the endogenous ‘ouabain-like’ activity. Linolenic, arachidonic and docosahexaenoic acids were the most active, whereas saturated fatty acids and glyceryl esters or methyl esters of unsaturated fatty acids were inactive. It is possible that in pathological situations in which the level of unsaturated fatty acids increases, these molecules may then act as physiological inhibitors of the sodium pump.     

Taurine Stimulation of Isolated Hamster Brain Na+, K+-ATPase: Activation Kinetics and Chemical Specificity

Epileptic foci are associated with locally reduced taurine (2-aminoethanesulfonic acid) concentration and Na⁺, K⁺-ATPase (EC 3.6.1.3) specific activity. Topically applied and intraperitoneally administered taurine can prevent the development and/or spread of foci in many animal models. Taurine has been implicated as a possible cytosolic modulator of monovalent ion distribution, cytosolic “free” calcium activity, and neuronal excitability. Taurine may act in part by modulating Na⁺, K⁺-ATPase activity of neuronal and glial cells. We characterized the requirements for in vitro modulation of Na⁺, K⁺-ATPase by taurine. Normal whole brain homogenate Na⁺, K⁺-ATPase activity is 5.1 ± 0.4 (4) μmol P_i± h^?1± mg^?1 Lowry protein. Partial purification of the plasma membrane fraction to remove cytosolic proteins and extrinsic proteins and to uncouple cholinergic receptors yields a membrane-bound Na⁺, K⁺-ATPase activity of 204.6 ± 5.8 (4) mol P_i± h^?1± mg^?1 Lowry protein. Taurine activates the Na⁺, K⁺-ATPase at all levels of purification. The concentration dependence of activation follows normal saturation kinetics (K_1/2= 39 mM taurine, activation maximum =+87%). The activation exhibits chemical specificity among the taurine analogues and metabolites: taurine = isethionic acid > hypotaurine > no activation =β-alanine = methionine = choline = leucine. Taurine can act as an endogenous activator/modulator of Na⁺, K⁺-ATPase. Its action is mediated by a membrane-bound protein.     

Renal cortical basolateral Na+/HCO 3 − cotransporter III. Evidence for a regulatory protein in the inhibitory effect of protein kinase A

The activity of the Na-H antiporter is inhibited by cyclic AMP-dependent protein kinase A (cAMP.PKA). The inhibitory effect of PKA on the Na-H antiporter is mediated through a regulatory protein that can be dissociated from the antiporter by limited protein digestion. PKA also inhibits the activity of the Na⁺/ HCO ₃ ^? cotransporter. We investigated whether the activity of Na⁺/HCO ₃ ^? cotransporter and the effect of PKA on this transporter may also be regulated by limited protein digestion. In rabbit renal cortical basolateral membranes (BLM) and in solubilized BLM reconstituted in liposomes (proteoliposomes), trypsin (100 μg) increased ²²Na uptake in the presence of HCO₃ but not in the presence of gluconate, indicating that trypsin does not alter diffusive ²²Na uptake but directly stimulates the Na⁺/HCO ₃ ^? cotransporter activity. In proteoliposomes phosphorylated with ATP, the catalytic subunit (CSU) of cAMP-PKA decreased the activity of the Na⁺/HCO ₃ ^? cotransporter (expressed as nanomoles/mg protein/3s) from 23 ± 10 to 14 ± 6 (P < 0.01). In the presence of trypsin, the inhibitory effect of CSU of cAMP-PKA on the activity of Na⁺/HCO ₃ ^? cotransporter was blunted. To identify a fraction that was responsible for the inhibitory effect of the CSU on the Na⁺/HCO ₃ ^? cotransporter activity, solubilized proteins were separated by size exclusion chromatography. The effect of CSU of cAMP-PKA on the Na⁺/HCO ₃ ^? cotransporter activity was assayed in proteoliposomes digested with trypsin with the addition of a fraction containing the 42 kDa protein (fraction S+) or without the 42 kDa protein (fraction S?). With the addition of fraction S?, the CSU of cAMP-PKA failed to inhibit the Na⁺/HCO ₃ ^? cotransporter activity (control 27 ± 6, CSU 27 ± 3) while the addition of fraction S+ restored the inhibitory effect of CSU (27 ± 6 to 3 ± 0.3 P < 0.01). The CSU of cAMP-PKA phosphorylated several proteins in solubilized protein including a 42 kDa protein. Fluorescein isothiocyanate (FITC) labels components of the Na⁺/HCO ₃ ^? cotransporter including the 56 kDa and 42 kDa proteins. In trypsin-treated solubilized protein the 42 kDa protein was not identified with FITC labeling. The results demonstrate that the activity of the Na⁺/HCO ₃ ^? cotransporter is regulated by protein(s) which mediates the inhibitory effect of PKA. Limited protein digestion can dissociate this protein from the cotransporter.     

Angiotensin II directly increases rabbit renal brush-border membrane sodium transport: Presence of local signal transduction system

Summary In the present study, we have examined the direct actions of angiotensin II (AII) in rabbit renal brush border membrane (BBM) where binding sites for AII exist. Addition of AII (10^–11–10^–7 m) was found to stimulate²²Na^– uptake by the isolated BBM vesicles directly. AII did not affect the Na⁺-dependent BBM glucose uptake, and the effect of AII on BBM²²Na⁺ uptake was inhibited by amiloride, suggesting the involvement of Na⁺/H⁺ exchange mechanism. BBM proton permeability as assessed by acridine orange quenching was not affected by AII, indicating the direct effect of AII on Na⁺/H⁺ antiport system.In search of the signal transduction mechanism, it was found that AII activated BBM phospholipase A₂ (PLA) and that BBM contains a 42-kDa guanine nucleotide-binding regulatory protein (G-protein) that underwent pertussis toxin (PTX)-catalyzed ADP-ribosylation. Addition of GTP potentiated, while GDP-ßS or PTX abolished, the effects of AII on BBM PLA and²²Na⁺ uptake, suggesting the involvement of G-protein in AII's actions. On the other hand, inhibition of PLA by mepacrine prevented AII's effect on BBM²²Na⁺ uptake, and activation of PLA by mellitin or addition of arachidonic acid similarly enhanced BBM²²Na⁺ uptake, suggesting the role of PLA activation in mediating AII's effect on BBM²²Na⁺ uptake.In summary, results of the present study show a direct stimulatory effect of AII on BBM Na⁺/H⁺ antiport system, and suggest the presence of a local signal transduction system involving G-protein mediated PLA activation.     

Transport of l-proline by the proton-coupled amino acid transporter PAT2 in differentiated 3T3-L1 cells

Mechanism and substrate specificity of the proton-coupled amino acid transporter 2 (PAT2, SLC36A2) have been studied so far only in heterologous expression systems such as HeLa cells and Xenopus laevis oocytes. In this study, we describe the identification of the first cell line that expresses PAT2. We cultured 3T3-L1 cells for up to 2 weeks and differentiated the cells into adipocytes in supplemented media containing 2 μM rosiglitazone. During the 14 day differentiation period the uptake of the prototype PAT2 substrate l-[³H]proline increased ~5-fold. The macro- and microscopically apparent differentiation of 3T3-L1 cells coincided with their H⁺ gradient-stimulated uptake of l-[³H]proline. Uptake was rapid, independent of a Na⁺ gradient but stimulated by an inwardly directed H⁺ gradient with maximal uptake occurring at pH 6.0. l-Proline uptake was found to be mediated by a transport system with a Michaelis constant (K_t) of 130 ± 10 μM and a maximal transport velocity of 4.9 ± 0.2 nmol × 5 min^?1 mg of protein^?1. Glycine, l-alanine, and l-tryptophan strongly inhibited l-proline uptake indicating that these amino acids also interact with the transport system. It is concluded that 3T3-L1 adipocytes express the H⁺-amino acid cotransport system PAT2.     

Energy-dependent sodium efflux and sodium-dependent α-aminoisobutyrate transport in purple photosynthetic bacteria

Uptake of alanine and its nonmetabolizable analog α-aminoisobutyric acid (AIB) by the photosynthetic purple sulfur bacterium Chromatium vinosum is stimulated fivefold by Na⁺. Neither Li⁺ nor K⁺ have any stimulatory effect. AIB uptake can be supported by a Na⁺ gradient in the absence of other energy sources. AIB uptake is also accompanied by Na⁺ uptake. These results suggest that AIB is taken up by C. vinosum via a sodium symport. Cells of C. vinosum and the purple nonsulfur bacterium Rhodospirillum rubrum show energy-dependent Na⁺ efflux and Na⁺ uptake can be demonstrated with chromatophores prepared from these bacteria.     

Arachidonic Acid Activates Cation Channels in Bovine Adrenal Chromaffin Cells

Abstract— Microscopic fluorescence analysis of fura-2-loaded bovine adrenal chromaffin cells demonstrates that ～70% of the cells responded to arachidonic acid in increasing the intracellular Ca²⁺ concentration. Because this increase was markedly less in the absence of external Ca²⁺, we examined the effect of arachidonic acid on Ca²⁺ influx electrophysiologically. Bath application of 10 μM arachidonic acid induced a long-lasting inward current when the cell was clamped at -50 mV. Other fatty acids, such as oleic acid, linoleic acid, eicosatrienoic acid, and eicosa-pentaenoic acid, were all ineffective. The current-voltage relationships suggest that arachidonic acid may activate voltage-insensitive channels. Arachidonic acid (2μM) activated a single-channel current in the inside-out patch, even in the presence of inhibitors of cyclooxygenase and lipoxygenase, possibly suggesting that arachidonic acid could activate channels directly. The onset delay of the inward channel current in the outside-out patch configuration (54.02 ± 63.5 s; mean SD) was significantly shorter than that in the inside-out patch one (197.3 ± 177.7 s). Washout of arachidonic acid decreased the probability of channel openings in the outside-out patch but not in the inside-out one. These results suggest that arachidonic acid activates channels reversibly from outside of the plasma membrane. The unitary conductarce for Ca²⁺ of arachidonic acid-activated channel was ～17 pS. The arachidonic acid-activated channel was permeable to Ba²⁺, Ca²⁺, and Na⁺ but not to Cl^?. The opening probability of the arachidonic acid-activated channel did not depend on membrane potential. These results demonstrate that arachidonic acid activates cation-selective, Ca²⁺-permeable channels in bovine adrenal chromaffin cells.     

COUPLED TRANSPORT OF GLUTAMATE AND SODIUM IN A CEREBELLAR NERVE CELL LINE

The cerebellar nerve cell line ε¹ has a very effective active transport system for glutamate. Glutamate uptake is dependent on extracellular Na⁺ and furthermore, ²²Na⁺ uptake is stimulated by glutamate, indicating that glutamate uptake and Na⁺ uptake are coupled. Two molecules of Na ⁺ are transported for each molecule of glutamate. The K_m for glutamate is found to be 5 × 10^?5M in both the glutamate uptake assay and the ²²Na⁺ uptake assay, providing additional evidence for glutamate-Na⁺ coupling. Pre-incubation with ouabain, which inhibits the Na⁺-K⁺ ATPase, results in a gradual inhibition of glutamate uptake due to the deterioration of the Na⁺ gradient. Tetrodotoxin, however, has no effect on glutamate-induced ²²Na⁺ uptake, showing that this Na⁺ flux does not occur via voltage-dependent Na⁺ channels. Studies on the specificity of the ε¹ glutamate transport system show that it is distinct from systems that transport alanine and glycine. l -Glutamate, d -aspartate, l -cysteate, and l -cysteine sulfinate are able to utilize the transport system efficiently. d -Glutamate, l -homocysteate, N-methyl-d , l -aspartate, and kainic acid are very poor substrates for the glutamate transport system, and in addition do not stimulate ²²Na⁺ uptake. These data allow us to distinguish the glutamate transport system from the glutamate receptor which is known to mediate depolarization in response to all nine of the above compounds. Thus, ε¹ does not have an excitatory glutamate receptor.     

Regulation of reconstituted renal Na+/H+ exchanger by calcium-dependent protein kinases

Summary Studies were performed to determine the effect of protein phosphorylation mediated by calcium-calmodulin-dependent multifunctional protein kinase II and calcium-phospholipid-dependent protein kinase on Na⁺/H⁺ exchange activity. Proteins from the apical membrane of the proximal tubule of the rabbit kidney were solubilized in octyl glucoside and incubated in phosphorylating solutions containing the protein kinase.²²Na⁺ uptake was determined subsequently after reconstitution of the proteins into proteoliposomes. Calcium-calmodulin-dependent multifunction protein kinase II inhibited the amiloride-sensitive component of proton gradient-stimulated Na⁺ uptake in a dose-dependent manner. The inhibitory effect of this kinase had an absolute requirement for calmodulin, Ca²⁺, and ATP. Calcium-phospholipid-dependent protein kinase stimulated the amiloride-sensitive component of proton gradient-stimulated Na⁺ uptake in a dose-dependent manner. The stimulating effect of this kinase had an absolute requirement for ATP, Ca²⁺, and an active phorbol ester. These experiments indicate that Na⁺/H⁺ exchange activity of proteoliposomes reconstituted with proteins from renal brush-border membranes are inhibited by protein phosphorylation mediated by calcium-calmodulin-dependent multifunctional protein kinase II and stimulated by that mediated by calcium-calmodulin-dependent protein kinase.     

Growth,pigments, and biochemical composition of marine red alga Gracilaria crassa

The marine red alga Gracilaria crassa was investigated for its proximate composition, minerals, fatty acids, amino acids, and agar content to decipher its nutritional implications. The growth performance and pigments were studied under different combinations of temperature and salinity. On a dry weight basis the total lipid content was 1.30?±?0.05 %, protein was 5.18?±?0.64 %, carbohydrate was 42.0?±?1.2 %, ash was 43.18?±?1.15 %, and agar content was 21.52?±?0.73 %. Appreciable amounts of macro-, micro-nutrients (K?>?Na, Ca, Mg, and Fe), and essential amino acids (Ileu, His, Thr, Leu, and Lys) were found. Palmitic, stearic acid, and arachidonic acid were major fatty acids detected. The alga showed maximum daily growth rate (DGR %) 5.8?±?0.09 % at 25 °C, 35 ‰ salinity. The highest content of pigment R-phycoerythrin (444.7?±?1.9 μg g^?1 fresh weight (FW) basis) was obtained at 25 ‰ salinity at 35 °C while that of R-phycocyanin (476.3?±?2.3 μg g^?1 DW) at 30 ‰ salinity at 30 °C. This study revealed that this alga can be utilized as a potential source for food and feed. The data generated on best growth conditions will be very useful for farming of G. crassa in open sea. This alga could be used for production of natural colorants at defined control condition.     

Substrate specificity of the mammary tissue anionic amino acid carrier operating in the cotransport and exchange modes

The substrate specificity of the rat mammary tissue high affinity, Na⁺-dependent anionic amino acid transport system has been investigated using explants and the perfused mammary gland. d-Aspartate appears to be transported via the high affinity, Na⁺-dependent l-glutamate carrier. Thus, d-aspartate transport by rat mammary tissue was Na⁺-dependent and saturable with respect to extracellular d-aspartate with a K_m and V_max of 32.4 μM and 49.0 nmol/2 min per g of cells respectively. The uptake of d-aspartate by mammary explants was cis-inhibited by l-glutamate and l-aspartate, but not by d-glutamate. l-glutamate uptake by mammary tissue explants was cis-inhibited by β-glutamate, l-cysteate, l-cysteine sulfinate and dihydrokainate but not by dl-α-aminoadipate. In addition, dihydrokainate, but not dl-α-aminoadipate inhibited d-aspartate and l-glutamate uptake by the perfused gland. d-Aspartate efflux from mammary tissue explants was trans-accelerated by external l-glutamate in a dose-dependent fashion (50-500 μM). The effect of l-glutamate on d-aspartate efflux was dependent on the presence of extracellular Na⁺. d-Aspartate, l-aspartate and l-cysteine sulfinate (at 500 μM) also markedly trans-stimulated d-aspartate efflux from mammary tissue explants. In contrast, l-cysteine, d-glutamate, l-leucine, dihydrokainate and dl-α-aminoadipate were either weak stimulators of d-aspartate efflux or were without effect. d-Aspartate efflux from the perfused mammary gland was trans-stimulated by l-glutamate but not by d-glutamate and only weakly by l-cysteine (all at 500 μM). It appears that the mammary tissue high affinity anionic amino acid carrier can operate in the exchange mode with a similar substrate specificity to that of the co-transport mode.     

Osmoregulation of amino acid transport activity in cultured fibroblasts

The effect of exposure of chick embryo cells to increasing concentrations of Na⁺ in the culture medium on the subsequent amino acid transport as determined at physiological osmolarity was investigated in detail. It was found that the hyperosmolar treatment stimulated amino acid transport in a dose-dependent manner up to 200 mM Na⁺. Changes were measurable as early as 1 h after altering Na⁺ and reached a maximum after 4 h, remaining constant thereafter. The maintenance of this effect required continuous exposure of the cell to high Na⁺ in the culture medium. Hyperosmolarity-mediated increases in amino acid transport activity by system A have been detected with l-proline and l-alanine. Transport activities of systems ASC and L did not change appreciably after exposure of the cells to high Na⁺. Inhibition of protein synthesis by cycloheximide or RNA synthesis by actinomycin D (actD) prevented these uptake changes. Kinetic analysis indicated that the stimulation of the activity of transport system A by high Na⁺ treatment occurred through a mechanism affecting V_max rather than K_m.     

Alterations in erythrocyte membrane fluidity and Na+/K+-ATPase activity in chronic alcoholics

Ethanol disorders biological membranes causing perturbations in the bilayer and also by altering the physicochemical properties of membrane lipids. But, chronic alcohol consumption also increases nitric oxide (NO) production. There was no systemic study was done related to alcohol-induced production of NO and consequent formation of peroxynitrite mediated changes in biophysical and biochemical properties, structure, composition, integrity and function of erythrocyte membranes in chronic alcoholics. Hence, keeping all these conditions in mind the present study was undertaken to investigate the role of over produced nitric oxide on red cell membrane physicochemical properties in chronic alcoholics. Human male volunteers aged 44 ± 6 years with similar dietary habits were divided into two groups, namely nonalcoholic controls and chronic alcoholics (~125 g of alcohol at least five times per week for the past 10–12 years). Elevated nitrite and nitrate levels in plasma and lysate, changes in erythrocyte membrane individual phospholipid composition, increased lipid peroxidation, protein carbonyls, cholesterol and phospholipids ratio (C/P ratio) and anisotropic value (γ) with decreased sulfhydryl groups and Na⁺/K⁺-ATPase activity in alcoholics was evident from this study. RBC lysate NO was positively correlated with C/P ratio (r = 0.547) and anisotropic (γ) value (r = 0.428), Na⁺/K⁺-ATPase activity was negatively correlated with RBC lysate NO (r = ?0.372) and anisotropic (γ) value (r = ?0.624) in alcoholics. Alcohol-induced overproduction of nitric oxide reacts with superoxide radicals to produce peroxynitrite, which appears to be responsible for changes in erythrocyte membrane lipids and the activity of Na⁺/K⁺-ATPase.     

Chemotactic factors activate differentiable permeation pathways for sodium and calcium in rabbit neutrophils. Effect of amiloride

The effects of the potassium-sparing diuretic amiloride on the chemotactic factor stimulated Na⁺ and Ca²⁺ fluxes in rabbit peritoneal neutrophils were investigated. Amiloride inhibits in a dose-dependent fashion the f-Met-Leu-Phe stimulated Na⁺ uptake (IC₅₀:1.1 × 10^?6 M) but did not affect the stimulated rate of Na⁺ efflux from preloaded cells. Amiloride did not inhibit the f-Met-Leu-Phe stimulated Ca²⁺ uptake. These results allow, for the first time, the differentiation between the Na⁺ and the Ca²⁺ permeation pathways and the investigation into the functional role of the stimulated Na⁺ uptake.     

Solubilization of active (H+ + K+)-ATPase from gastric membrane

(H⁺ + K⁺)-ATPase-enriched membranes were prepared from hog gastric mucosa by sucrose gradient centrifugation. These membranes contained Mg²⁺-ATPase and p-nitrophenylphosphatase activities (68 ± 9 μmol P_i and 2.9 ± 0.6 μmol p-nitrophenol/mg protein per h) which were insensitive to ouabain and markedly stimulated by 20 mM KCl (respectively, 2.2- and 14.8-fold). Furthermore, the membranes autophosphorylated in the absence of K⁺ (up to 0.69 ± 0.09 nmol P_i incorporated/mg protein) and dephosphorylated by 85% in the presence of this ion. Membrane proteins were extracted by 1–2% (w/v) n-octylglucoside into a soluble form, i.e., which did not sediment in a 100 000 × g × 1 h centrifugation. This soluble form precipitated upon further dilution in detergent-free buffer. Extracted ATPase represented 32% (soluble form) and 68% (precipitated) of native enzyme and it displayed the same characteristic properties in terms of K⁺-stimulated ATPase and p-nitrophenylphosphatase activities and K⁺-sensitive phosphorylation: Mg²⁺-ATPase (μmol P_i/mg protein per h) 32 ± 9 (basal) and 86 ± 20 (K⁺-stimulated); Mg²⁺-p-nitrophenylphosphatase (μmol p-nitrophenol/mg protein per h) 2.6 ± 0.5 (basal) and 22.2 ± 3.2 (K⁺-stimulated); Mg²⁺-phosphorylation (nmol P_i/mg protein) 0.214 ± 0.041 (basal) and 0.057 ± 0.004 (in the presence of K⁺). In glycerol gradient centrifugation, extracted enzyme equilibrated as a single peak corresponding to an apparent 390 000 molecular weight. These findings provide the first evidence for the solubilization of (H⁺ + K⁺)-ATPase in a still active structure.     

Facilitated diffusion and active transport systems for glucose in metacercariae of Clinostomum marginatum (Digenea)

Metacercariae of Clinostomum marginatum excysted from yellow perch, Perca flavescens, appear to have two systems for transporting glucose across the tegument, facilitated diffusion and active transport. These systems were distinguished by their differential sensitivities to Na⁺, phlorizin and phloretin. In Ringer's saline for cold-blooded vertebrates, 0.1 mm phlorizin and phloretin were incomplete, but similarly effective inhibitors of glucose uptake in 3 min incubations; worms accumulated in 1 h nonmetabolized 3-O-methylglucose against an apparent concentration difference demonstrating the active transport component. In Na⁺-free saline, phlorizin sensitivity and active transport capacity disappeared, but a phloretin sensitive, Na⁺-independent component remained. The V_max and K₁ of the Na⁺-independent system were 3.0 ± 0.54 μmol/g ethanol-extracted dry wt/h, and 0.8 ± 0.36 mm, respectively. V_max and K₁ of the Na⁺-dependent system, estimated by subtracting the Na⁺-independent values from those obtained in Ringer's saline, were 1.3 ± 0.27 μ mol/g ethanol-extracted dry wt/h, and0.7 ± 0.36mm, respectively.