Hypotonic Hemolysis Research Articles

Transport of sodium and protons and hypotonic haemolysis in the valinomycin-treated erythrocytes of rats with spontaneous hypertension

After the addition of valinomycin into the incubation medium, the potassium content of rat erythrocytes rapidly decreases. The rate-limiting step of this reaction is a unidirectional efflux of anions through band 3 protein. The rate of this efflux in erythrocytes of spontaneously hypertensive rats (SHR) of the Wistar-Kyoto strain, is not altered. The loss of KCl by rat erythrocytes is accompanied by a decrease in intracellular water, cell shrinking and activation of Na+-H+i exchange. The rate of Na+-H+ exchange in the erythrocytes of SHR in the pre-hypertensive stage (4 weeks old) was decreased by 30%. There were no differences between 14-week-old and 28-week-old SHR and normotensive Wistar-Kyoto (WKY) rats. The half-maximal increase of the valinomycin-induced Na+-H+ exchange in erythrocytes of 14-week-old WKY and SHR was observed at KCl concentrations in the incubation medium of 25 and 40 mmol, respectively. The addition of activators of protein kinase A (dibutyryl-cAMP) or protein kinase C (beta-phorbol ester) resulted in an increase in the maximal rate of Na+-H+ exchange, and did not modify its dependence on K+o concentration. In all groups of SHR, the rate of valinomycin-induced H+ efflux from erythrocytes in the sodium-free medium was 1.5-2.5-fold higher than in age-matched WKY. Under these conditions (addition of valinomycin and inhibition of Na+-H+ exchange), haemoglobin release from erythrocytes of SHR, treated with hypotonic solution, was significantly decreased. We conclude that these differences are due to the alteration of the skeleton protein organization in the erythrocyte membranes of SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Conversion of encapsulated 5-fluoro-2'-deoxyuridine 5'-monophosphate to the antineoplastic drug 5-fluoro-2'-deoxyuridine in human erythrocytes.

The fluoropyrimidine deoxyribonucleotide 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) was encapsulated in human erythrocytes by a procedure based on hypotonic hemolysis and isotonic resealing. Encapsulated FdUMP (up to 9 mumol/ml of packed erythrocytes) did not affect erythrocyte metabolism or morphology. Hemolysates were found to catalyze efficient dephosphorylation of FdUMP to yield nearly stoichiometric amounts of the corresponding deoxyribonucleoside 5-fluoro-2'-deoxyuridine (FdUrd), an antineoplastic drug showing selective cytotoxicity toward liver metastases from colorectal carcinomas. The dephosphorylation reaction had an apparent Km of 7.7 +/- 1.2 mM FdUMP at pH 7.4 and was remarkably slower at pH 8.2. ATP, GTP, and UTP inhibited both the disappearance of FdUMP and the formation of FdUrd in hemolysates. The enzyme responsible for the FdUMP-to-FdUrd conversion was identified with the deoxyribonucleotide-specific isozyme of erythrocyte pyrimidine 5'-nucleotidase (EC 3.1.3.5). Intracellular formation and subsequent release of FdUrd were observed in intact erythrocytes loaded with FdUMP. Inhibition of FdUrd release from these erythrocytes was obtained by raising the pH intracellularly and, alternatively, by coencapsulation of ATP. Autologous FdUMP-loaded erythrocytes might be used as endogenous bioreactors designed for time-programmed and liver-targeted delivery of FdUrd.

Osmotic stability of red cells in renal circulation requires rapid urea transport.

Urea transport by the human erythrocyte occurs via an asymmetric-facilitated diffusion system with high Michaelis constants and high maximal velocities; the equivalent permeability in the limit of zero urea concentration is approximately 10(-3) cm/s (J. Gen. Physiol. 81: 221-237, 239-253, 1983). A physiological role for this system is revealed by numerical integration of the appropriate equations that show that rapid urea transport is essential for red cell stability in passing through the renal medulla. The calculation compares two cells. Cell A transports urea with permeability characteristics of normal red cells; cell B has urea permeability similar to lipid bilayers. On entering the hypertonic medulla, both cells shrink, but only B swells on leaving the medulla. The osmotic stress for cell B is greater than for A. Cell B is close to hypertonic hemolysis in the medulla and to hypotonic hemolysis in the cortex. Cell B remains swollen for some time after its exit; the resulting decreased deformability presents a hazard if B reenters the microcirculation. Furthermore, cell B removes a significant fraction of the filtered load of urea and compromises the osmotic gradients in the medulla.

Reduced parasitemia observed with erythrocytes containing inositol hexaphosphate.

Chemicals entrapped in erythrocytes by hypotonic hemolysis can be assessed for possible antiparasitic activity both in vivo and in vitro, regardless of whether they are able to diffuse into erythrocytes readily. Inositol hexaphosphate, a highly charged compound, produced a dramatic lowering of the percentage of cells infected by Babesia microti in vivo and both B. microti and Plasmodium falciparum in vitro. Several possible mechanisms for this observation are discussed.

Felodipine-induced inhibition of polymorphonuclear leukocyte functions

Felodipine inhibits fMet-Leu-Phe or ionophore A23187-induced exocytosis in rabbit peritoneal polymorphonuclear leukocytes (PMNs), in the concentration range 1–50 μM. Activation of the metabolic burst, and migration of PMNs towards fMet-Leu-Phe are equally inhibited by felodipine in the same concentration range. The effect is not due to blocking of calcium channels in the plasma membrane, because the degree of inhibition remains the same when Ca 2+ is omitted from the medium. Felodipine interferes with ionophore A23187-induced association of 45Ca with the PMN but this interference occurs at lower concentrations than the inhibition of exocytosis. Hypotonic hemolysis of erythrocytes is inhibited by felodipine; maximal protection against hemolysis occurs at a concentration of 50 μM felodipine. It is suggested that at least a part of the inhibiting effect on PMN functions might be due to an anesthetic-like membrane effect of felodipine.

Dopamine-depleting activity of L-3,4-(dioxyphenylacetyl)-phenylalanine.

Dioxyphenylacetyl-L-DOPA was synthesized and shown to be resistant toward oxidative cyclization in the presence of plasma and brain extracts. This new DOPA derivative is slightly inhibitory towards monoamine oxidase (IC50 2.5 mM) and the synaptosomal uptake of dopamine (IC50 0.35 mM) and acts as an antagonist of haloperidol (IC50 0.6 microM) in displacing 3H-spiroperidol from isolated dopamine receptors. The substance, although more hydrophobic than DOPA, overcomes the blood-brain barrier to a lower extent than DOPA and lowers the levels of dopamine and norepinephrine in the left and right hemisphere of mice treated with it for 10 days at a dose of 10 mg/kg/day. Dioxyphenylacetyl-L-DOPA inhibits red cell hypotonic hemolysis and brain Na/K+-ATPase activity in vitro and potentiates the barbiturate-induced sleep.

Inhibition of calmodulin stimulation of phosphodiesterase and Ca 2+Mg 2+-ATPase activities and shape change of erythrocyte ghosts by chloroquine

The effects of chloroquine on calmodulin (CaM)-related enzyme activities and the shape of human erythrocytes have been studied. It was found that the CaM activation of rat brain phosphodiesterase was abolished by the addition of chloroquine. CaM was included in the assay of phosphodiesterase activity at the concentration that gave half-maximal activation. The concentration of chloroquine that caused 50% inhibition of CaM stimulation of phosphodiesterase was 7 × 10 −5 M. The type of inhibition was competitive with respect to CaM. The CaM-stimulated Ca 2+, Mg 2+-ATPase in erythrocyte membrane was also inhibited by chloroquine, the 50% inhibitory concentration of which was about 2 × 10 −4 M. Its mode of action was also competitive with respect to CaM. The shapes of erythrocyte ghosts prepared by hypotonic hemolysis were examined in a solution consisting of 2 mM MgCl 2, 154 mM NaCI and 10 mM Tris-HCl (pH7.4); they were discocytic in the presence of 2mM ATP and in its absence. They were converted to the invaginated form by the addition of chloroquine in the concentration range of 1 x 10 −4–5 × 10 −4 M. This concentration is similar to that which caused the inhibition of CaM activation of Ca 2+,Mg 2+-ATPase.

Movements of fluorescent probes in the mechanism of cell fusion induced by poly(ethylene glycol).

It has been claimed that purified poly(ethylene glycol) (PEG) is able only to aggregate cells and not to fuse them. In our hands, purified PEG 6000 (recrystallized/dialysed) induces both aggregation and fusion of human erythrocytes, and the mechanism of fusion by the purified polymer has been investigated with fluorescent probes. No movement of a carbocyanine probe or of octadecyl rhodamine B chloride from labelled to unlabelled cells occurred in the absence of PEG or with cells treated with concanavalin A, protamine or spermine. With 40% PEG, however, both probes immediately started to diffuse into the membranes of unlabelled cells. This indicates that continuity between the phospholipid bilayer membranes of adjacent erythrocytes (i.e. membrane fusion) is established within seconds in concentrated solutions of the polymer, and precedes the cell fusion event that is induced by purified PEG. These observations are consistent with the idea that micro-regions of shared phospholipid bilayer may be formed in the membranes of cells when they are forced together as a consequence of the dehydrating action of PEG. Intact erythrocytes were cytoplasmically labelled with 6-carboxyfluorescein to avoid the possibility that loading the cells with a cytoplasmic marker by hypotonic haemolysis might modify their response to PEG. Unlike the lipid probes, carboxyfluorescein did not diffuse from labelled to unlabelled cells in the presence of 40% PEG, and there was little diffusion on subsequent dilution of the polymer solution to 13%. However, after the PEG solution had been replaced by an isotonic buffer, a rapid transfer of the cytoplasmic fluorophore to unlabelled cells often occurred. This is considered to be more consistent with the osmotic rupture of a membranous barrier, such as a shared bilayer, between the labelled and unlabelled cells than with the return of cytoplasmic viscosity to normal when the PEG is removed. Possible reasons are discussed for the reported inability of purified PEG to fuse fibroblasts with hypotonically loaded human erythrocytes.

Hepatic or splenic targeting of carrier erythrocytes: a murine model.

Carrier mouse erythrocytes, i.e., red cells, subjected to a dialysis technique involving transient hypotonic hemolysis and isotonic resealing were treated in vitro in three different ways: (a) energy depletion by exposure for 90 min at 42 degrees C; (b) desialylation by incubation with neuroaminidase; and (c) oxidative stress by incubation with H2O2 and NaN3. Procedure (c) afforded maximal damage, as shown by analysis of biochemical properties of the treated erythrocytes. Reinfusion in mice of the variously manipulated erythrocytes following their 51Cr labeling showed extensive fragilization as indicated by rapid clearance of radioactivity from the circulation. Moreover, both the energy-depleted and the neuraminidase-treated erythrocytes showed a preferential liver uptake, reaching 50 and 75%, respectively, within 2 h. On the other hand, exposure of erythrocytes to the oxidant stress triggered a largely splenic removal, accounting for almost 40% of the reinjected cells within 4 h. Transmission electron microscopy of liver from mice receiving energy-depleted erythrocytes demonstrated remarkable erythrocyte congestion within the sinusoids, followed by hyperactivity of Kupffer cells and by subsequent thickening of the perisinusoidal Disse space. Concomitantly, levels of serum transaminase activities were moderately increased. Each of the three procedures of manipulation of carrier erythrocytes may prove applicable under conditions where selective targeting of erythrocyte-encapsulated chemicals and drugs to either the liver or the spleen has to be achieved.

Effects of purines and forskolin on haemolysis.

Effects of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine on the antihaemolytic action of procaine, and the effect of ATP on the antihaemolytic actions of lidocaine, dibucaine, tetracaine, pentobarbitone, and chlorpromazine were investigated in rat erythrocytes. The effects of adenosine and its analogues D-N6-phenylisopropyladenosine (D-PIA), L-N6-phenylisopropyladenosine (L-PIA), N6-cyclohexyladenosine (CHA), 2-chloroadenosine, and N6-methyladenosine, as well as the effects of cyclic-AMP (cAMP), dibutyryl cAMP, and forskolin on haemolysis were also investigated in rat erythrocytes. ATP, ADP, and AMP, but not adenosine, antagonized in a concentration-dependent manner the antihaemolytic action of procaine, and ATP was ineffective against the antihaemolytic actions of lidocaine, dibucaine, tetracaine, pentobarbitone, and chlorpromazine. Adenosine and its analogues, but not N6-methyladenosine, protected erythrocytes against hypotonic haemolysis in a concentration-dependent manner. The order of potencies was: D-PIA greater than CHA greater than L-PIA greater than adenosine greater than 2-chloroadenosine. The effect of adenosine on haemolysis was not prevented by theophylline, 8-phenyltheophylline, or 8-sulfophenyltheophylline. Dibutyryl cAMP, a stable analogue of cAMP, and forskolin, a specific activator of adenylate cyclase, mimicked the antihaemolytic action of adenosine. cAMP was less efficient than adenosine. Adenine nucleotides antagonized the antihaemolytic action of procaine, probably due to calcium-chelating properties. In contrast, adenine nucleosides have antihaemolytic properties. The possibility that the antihaemolytic effects of these substances relate to cAMP and/or to lipophilicity is discussed.

Effects of Essential Oils on Erythrocytes and Hepatocytes from Rats and Dipalmitoyl Phosphatidylcholine-Liposomes

The effect of essential oils, eugenol, thymol and menthol, on erythrocytes, hepatocytes, dipalmitoyl phosphatidylcholine (DPPC)-liposomes and surface tension were studied at various concentrations. Maximal inhibition of eugenol, thymol and menthol on the hypotonic hemolysis in rat erythrocytes were observed at a concentration of 2 mM, 1 mM and 1 mM, respectively. Eugenol at 4 mM and thymol at 2 mM caused an acceleration of hypotonic hemolysis. In isolated rat hepatocytes, thymol caused an increase in GOT leakage, but eugenol at 4 mM and menthol at 0.1 and 0.4 mM inhibited the GOT leakage. The leakage of GPT from hepatocytes was inhibited by eugenol at 0.1 mM and 0.4 to 4 mM and menthol at 0.1 to 0.6 mM. The inhibition of eugenol and menthol on the LDH leakage in hepatocytes were observed at a concentration of 0.001 to 4 mM and 0.1, 0.4 and 0.6 mM, respectively. Thymol caused no change in GPT and LDH leakage. Eugenol, thymol and menthol indicated a depression of surface tension at a concentration of 0.1 mM. The rank by order of surface activity was eugenol>thymol. Eugenol, thymol and menthol depressed the phase-iransition temperature of DPPC-liposomes. The depression of phase-transition temperature by thymol was greater than that by eugenol and menthol. These results suggest the periapical tissue damage produced by essential oils may be related to membrane lysis and surface activity and that the their tissue penetration may be related to membrane affinity and lipid solubility.

Effect of sho-saiko-to and mecobalamin on the biological responses in methotrexate treated rats. Especially from biorheological view points

It was found that the subcutaneous treatment with 0.6 mg/kg of methotrexate (MTX) twice a week for 7 weeks in rat led to the increases in blood viscosity-adjusted hematocrit and serum lipidperoxide level and a decrease in the expansibility of erythrocyte during the course of hypotonic hemolysis. The effect of sho-saiko-to, one of the useful kampo prescriptions used in traditional Chinese system of medicine, on biological responses in MTX-treated rats was examined mainly from the biorheological view points. The combined use of sho-saiko-to and MTX inhibited the blood flow properties affected by MTX treatment. It was also found that the administration of sho-saiko-to inhibited the decrease of body weight and the increase of liver lipidperoxide induced by MTX treatment. The MTX-induced increase of liver lipidperoxide level was also restored by the administration of mecobalamin which showed no effect on the blood viscosity and body weight affected by MTX treatment.

１‐（２‐Ｅｔｈｏｘｙｅｔｈｙｌ）‐２‐（４‐ｍｅｔｈｙｌ‐１‐ｈｏｍｏｐｉｐｅｒａｚｉｎｙｌ）ｂｅｎｚｉｍｉｄａｚｏｌｅ ｄｉｆｕｍａｒａｔｅ（ＫＢ‐２４１３）の抗アレルギー作用

The effects of KB-2413 on four types of allergic reactions classified by Coombs and Gell were investigated. KB-2413 inhibited homologous passive cutaneous anaphylaxis and passive anaphylactic bronchoconstriction in guinea pigs mediated by IgE-like antibody, and ED50 values were 0.0017 mg/kg, p.o., and 0.022 mg/kg, p.o., respectively. KB-2413 also inhibited IgG-mediated anaphylactic bronchoconstriction in guinea pigs actively sensitized with egg albumin. Both complement-dependent immune hemolysis and complement-independent hypotonic hemolysis were inhibited by KB-2413 in a concentration-dependent manner. KB-2413 had no effect on the Forssman systemic reaction. The passive Arthus reaction in guinea pigs sensitized with anti-egg albumin rabbit serum was unaffected by KB-2413. However, the early stage of the active Arthus reaction in rabbits sensitized with egg albumin was inhibited. KB-2413 had an inhibitory effect on the efferent phase of delayed-type hypersensitivity induced by picryl chloride (PC-DTH) in mice. On the other hand, the afferent phase of PC-DTH in mice was unaffected. These results suggest that KB-2413 strongly suppresses type I allergic reactions, and it slightly suppresses type II, III and IV allergic reactions.

21] Hypotonic hemolysis methods for entrapment of agents in resealed erythrocytes

This chapter describes the hypotonic hemolysis methods for entrapment of agents in resealed erythrocytes. Erythrocytes have a remarkable capacity for reversible shape changes with or without accompanying volume changes and for reversible deformation under stress. Increases in volume initially lead to the conversion of the normal biconcave discocytes to spherocytes. When erythrocytes are hemolyzed and washed in large volumes of hypotonic medium, they lose nearly all their hemoglobin and hence, the very pale or transparent cells formed after resealing are referred to as erythrocyte ghosts. These ghosts have little or no capacity for survival in vivo, but would be useful for the delivery of substances to lysosomes of erythrophagocytic cells. Resealed erythrocytes containing, as nearly as possible, the normal levels of intracellular substances circulate for prolonged period of time, and sometimes for the normal length of time. Two procedures in particular, the preswell dilutional and the dialysis method, were devised to minimize losses of intracellular substances during lysis. Resealing with a solution made from lysed erythrocytes might be useful as well.

Effects of phenol and related compounds on erythrocytes and hepatocytes from rats and dipalmitoyl phosphatidylcholine-liposomes

The effects of phenol, guaiacol and m-cresol on erythrocytes, hepatocytes, dipalmitoyl phosphatidylcholine (DPPC)-liposomes and surface tension were studied at various concentrations. Phenol at 10 mM caused a slight inhibition of hypotonic hemolysis in rat erythrocytes. Guaiacol at 4 and 10 mM and m-cresol at 0.6 to 10 mM caused a significant inhibition of hypotonic hemolysis. In the enzyme leakage from isolated rat hepatocytes, phenol at 0.001 to 0.4 mM and 2 to 10 mM, guaiacol at 2 to 10 mM and m-cresol at 0.001 to 4 mM caused an inhibition in GOT leakage. The leakage of GPT from hepatocytes was inhibited by phenol at 0.4 to 10 mM, guaiacol at 2 to 10 mM, and m-cresol at 0.001 to 4 mM. m-Cresol at 10 mM caused increases in GOT and GPT leakage. The inhibition of phenol and m-cresol on the LDH leakage in hepatocytes were observed at a concentration of 0.001 mM and 0.1 to 1 mM, respectively. Guaiacol or m-cresol at 10 mM caused an increase in LDH leakage. Phase-transition temperature of DPPC-liposomes was depressed by phenol and m-cresol at 1 to 10 mM and by guaiacol at 5 and 10 mM. Guaiacol at 1 and 10 mM and m-cresol at 10 mM caused a depression of surface tension, but phenol caused no change in surface tension. The order of effects on erythrocyte, hepatocyte and DPPC-liposome membranes was m-cresol greater than phenol greater than or equal to guaiacol. In the present study, phenol and its related compound showed a positive correlation between their effects on various membranes and germicidal effects as evaluated by the phenol coefficient, but the effects were not related to a depression of surface tension.

Permeability alterations and antihaemolysis induced by amphiphiles in human erythrocytes

In an attempt to define the parameters in amphiphilic molecules important for their interaction with the erythrocyte membrane, the effects of cationic, anionic, zwitterionic and nonionic amphiphilic agents (C 10-C 16) on osmotic fragility and transport of potassium and phosphate in human erythrocytes were studied. (1) All the amphiphiles protected the erythrocytes against hypotonic haemolysis. Half-maximum protection occurred at a concentration which was about 15% of that inducing 50% haemolysis. The concentrations of amphiphiles required to induce protection or haemolysis were related to the length of the alkyl chain in a way indicating that a membrane/aqueous phase partition is the mechanism whereby the amphiphile monomers intercalate into the membrane. (2) At antihaemolytic concentrations all the amphiphiles increased potassium efflux and passive potassium influx. The increase in the fluxes was about the same in both directions through the membrane and there were no clear differences in the effects of the different amphiphilic derivatives at equi-protecting concentrations. (3) Active potassium influx was decreased by cationic, zwitterionic and non-ionic amphiphiles. The ability of the amphiphiles to inhibit the influx was not related to the length of the alkyl chain. Anionic amphiphiles had no or only a weak stimulatory effect on the influx. (4) Phosphate efflux was reduced by all the amphiphiles. The inhibitory potency of the different amphiphiles decreased in the following order; anionic > zwitterionic, non-ionic > cationic. Short-chained amphiphiles were more potent inhibitors than long-chained. (5) The possible participation of non-bilayer phases (mixed inverted micelles) in the intercalation of amphiphiles into the membrane is discussed.

Encapsulation of adriamycin in human erythrocytes.

Adriamycin (doxorubicin) was encapsulated in human erythrocytes by means of a dialysis technique involving transient hypotonic hemolysis followed by isotonic resealing. Up to 1.6 mg of the drug was entrapped per ml of packed erythrocytes, with the efficiency of encapsulation being 60-80%. In vitro incubation of the Adriamycin-loaded erythrocytes in autologous plasma was accompanied by progressive release of unaltered Adriamycin in the medium. The efflux was still evident after 50 hr. The metabolism of encapsulated Adriamycin was restricted to limited formation of the C-13 hydroxylated metabolite, adriamycinol, in the normal erythrocytes but not in erythrocytes from individuals deficient in glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) activity. Reductive bioactivation of encapsulated Adriamycin to yield the corresponding aglycones was not observed in a variety of conditions. However, when NADPH ferredoxin reductase and ferredoxin, both purified from spinach leaves, were co-entrapped within erythrocytes and allowed to catalyze electron transfer to Adriamycin intracellularly under N2, a quantitative conversion to 7-deoxyadriamycin aglycone was obtained. Adriamycin-loaded erythrocytes did not show any significant oxidative damage, except for a variable increase of methemoglobin, suggesting some redox cycling between native Adriamycin and its semiquinone radical. Encapsulation of Adriamycin in autologous human erythrocytes may represent a therapeutic strategy for the slow release in circulation of this antineoplastic drug in order to reduce or prevent its adverse effects and especially the delayed cardiotoxicity that limits its use in patients with neoplastic disease.

Isolation and labelling of human leukocytes with 99mTc

The role of the leukocyte isolation procedure on cell labelling with 99mTc has been evaluated. Separation of leukocytes was performed by two procedures: sedimentation on methyl cellulose, followed by discontinuous gradient centrifugation; methyl cellulose sedimentation and hypotonic haemolysis of residual red blood cells. After washing the cells in saline and incubation with a stannous pyrophosphate agent, the leukocytes were labelled with 5-10 mCi 99mTc. Procedure 1 gave a higher purity but lower recovery of polymorphonuclear leukocytes, and a minor contamination of red blood cells. 99mTc labelling of cells was slightly more efficient with this method, probably due to the presence of red blood cells. Procedure 1 is recommended for in vitro studies on cell kinetics and procedure 2 is recommended for clinical use.

Membrane phospholipid asymmetry as a determinant of erythrocyte recognition by macrophages.

To assess the role of transbilayer phospholipid distribution in the recognition and phagocytosis of erythrocytes by macrophages, human erythrocytes with either a symmetric or asymmetric distribution of membrane phospholipids were prepared by hypotonic hemolysis and then incubated with cultures of human monocyte-derived macrophages. Erythrocytes with an abnormal, symmetric distribution were phagocytosed 4 times more readily than their counterparts with an asymmetric distribution or than normal, asymmetric intact erythrocytes. This enhanced phagocytosis correlated with two biophysical properties of the membrane: the spacing of phospholipids, as assessed by binding of the dye merocyanine 540, and the relative hydrophobicity, as measured by aqueous two-phase polymer partitioning. These results suggest a mechanism by which loss of membrane asymmetry is translated into recognition by macrophages and provide guidelines in loading erythrocytes that may be useful in manipulating the mode of delivery when erythrocytes are used as drug carriers in vivo.

Dibucaine stimulation of p-aminohippurate accumulation in rat kidney cortical slices.

We studied the effects of dibucaine on p-aminohippurate (PAH) transport using rat kidney cortical slices. Addition of dibucaine in concentrations from 0.01 to 0.1 mM stimulated PAH accumulation in a concentration-dependent way; at higher concentrations, accumulation was inhibited. Kinetic studies showed that dibucaine increased the apparent maximal activity of PAH transport, whereas the apparent Km remained essentially constant. The efflux of PAH from the slices was significantly depressed by the drug. Dibucaine had no significant effect on the sodium gradient, medium to intracellular fluid. The drug had biphasic effects on hypotonic hemolysis; at lower concentrations (up to 0.2 mM) it protected erythrocytes, and at much higher concentrations, it has a lytic effect. The pattern of the membrane-stabilizing action of dibucaine was similar to that of the stimulation of PAH accumulation. These results indicate that dibucaine markedly stimulated accumulation of PAH. Stimulation by this drug of PAH transport cannot be explained by the increase in the sodium gradient; it may be due at least in part to membrane stabilization.