Increased anion permeability during volume regulation in human lymphocytes

Peripheral blood lymphocytes (p.b.ls) readjust their volumes after swelling in hypotonic media. An essential component of the regulatory response is an increase in K+ and Cl- permeability. No evidence was found for a tightly coupled co-transport of K+ and Cl-. The flux of either ion proceeds normally in the virtual absence of the transported counterion. Furthermore, alterations in membrane potential recorded during the phase of volume readjustment can be qualitatively accounted for by an increase in Cl- conductance. In tonsillar lymphocytes, a failure of the K+-permeability is nevertheless increased upon swelling. This further suggests that K+ and Cl- are transported during volume regulation through independent pathways. Cytoplasmic free Ca2+ appears to be involved in regulatory volume decrease. K+ and Cl-. Moreover, swelling and shrinking can be induced in isotonic K+-rich and K+-free media, respectively, by the Ca2+ ionophore. The ion flux and volume changes produced by either swelling or internal Ca2+ can be inhibited by similar concentrations of quinine and phenothiazines. The inhibitory activity of the latter drugs, which are powerful antagonists of calmodulin, suggests the participation of this Ca2+-regulator protein in volume regulation.     

The permeability of human lymphocytes to chloride

The normal amount of Cl in human lymphocytes is 82 mmoles/kg wet weight. Half of this undergoes rapid self-exchange with a half-time of 3 minutes, while the remainder exchanges slowly with a half-time of 180 minutes. The fast fraction of self-exchange of Cl also undergoes a rapid net loss into medium with a low concentration of Cl. Thus, exchange of Cl in lymphocytes has properties like that of K and Na with permeability constants on the order of 10(-6) cm/sec. The results are compatible with a simple model in which the fast fractions are dissolved within ordered cellular water at concentrations less than in the external medium and the slow fractions are adsorbed onto intracellular macromolecules.     

Mechanism of the increase in cation permeability of human erythrocytes in low-chloride media. Involvement of the anion transport protein capnophorin

When human erythrocytes are suspended in low-Cl- media (with sucrose replacing Cl-), there is a large increase in both the net efflux and permeability of K+. A substantial portion (greater than 70% with Cl- less than 12.5 mM) of this K+ efflux is inhibited by the anion exchange inhibitor DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). This inhibition cannot be explained as an effect of DIDS on net Cl- permeability (Pcl) and membrane potential, but rather represents a direct effect on the K+ permeability. When cells are reacted with DIDS for different times, the inhibition of K+ efflux parallels that of Cl- exchange, which strongly indicates that the band 3 anion exchange protein (capnophorin) mediates the net K+ flux. Since a noncompetitive inhibitor of anion exchange, niflumic acid, has no effect on net K+ efflux, the net K+ flow does not seem to involve the band 3 conformational change that mediates anion exchange. The data suggest that in low-Cl- media, the anion selectivity of capnophorin decreases so that it can act as a very low-conductivity channel for cations. Na+ and Rb+, as well as K+, can utilize this pathway.     

Glucocorticoids increase beta-adrenoceptors on human intact lymphocytes in vitro

A modification of beta-adrenoceptor binding has been observed in different human and animal tissues after glucocorticoid administration. In this study we observed that hydrocortisone increased human intact lymphocyte beta-adrenoceptors labelled with 3H-dyhydroalprenolol in vitro, in physiological and pharmacological concentrations. In saturation experiments hydrocortisone (10(-6)M) significantly raised beta-adrenoceptor Bmax and KD. Intact living lymphocytes seem to be a useful model for further investigation in man of the regulation of beta-adrenoceptors by glucocorticoids in physiological conditions.     

Membrane proteins related to anion permeability of human red blood cells

Summary (³H)DIDS (4,4-diisothiocyano-2,2-ditritiostilbene-disulfonate) was used as a convalent label for membrane sites involved in anion permeability. The label binds to a small, superficially located population of sites, about 300,000 per cell, resulting in almost complete inhibition of anion exchange. The relationship of biding to inhibition is linear suggesting that binding renders each site nonfunctional. In the inhibitory range less than 1% of the label is associated with lipids but at higher concentrations of DIDS, the fraction may be as high as 4%. In ghosts, however, treatment with (³H)DIDS results in extensive labeling of lipids. In cells, a protein fraction that behavens on SDS acrylamide gels as thought its molecular weight is 95,000 daltons (95K) is predominatly labeled by (³H)DIDS. The only other labeled protein is the major sialoglycoprotein which contains less than, 5% of the total bound (³H)DIDS. Because of the linear relationship of binding to inhibition and the unique architecture of the site, it is suggested that the (³H)DIDS-binding site of the 95K protein is the substrate binding site of the anion transport system. The 95K protein is asymmetrically arranged in the membrane with the sites arranged on the outer face accessible to agent in the medium. In leaky ghost, only a few additional binding sites can be reached from the inside of the membrane in the 95K protein, in contrast to the extensive labeling of other membrane proteins in ghosts as compared to cells.Abbreviations DADS 4,4-Diamino-2,2-dihydrostilbene disulfonic acid - DIDS 4,4-Diisothiocyano-2,2-stilbene disulfonic acid - (³H)DADS 4,4-Diamino-2,2-ditritiostilbene disulfonic acid - (³H)DIDS 4,4-Diisothiocyano-2,2-ditritiostilbene disulfonic acid     

Adenovirus-dependent increase in cell membrane permeability

When KB cells were labeled with either 51Cr (1 microCi/ml) or [35S]methionine (5 microCi/ml) and treated with 10 micrograms/ml of adenovirus type 2 (Ad2) at pH 6.0 for 60 min at 37 degrees C, about 25% of the cell-associated 51Cr and 5% of the [35S]methionine were released into the medium. The 51Cr was mainly associated with molecules of 1500 Da or less. When KB cells were labeled with either [3H] choline, alpha-[3H]aminobutyric acid, or [3H]deoxy-2-fluoro-D-glucose and exposed to Ad2, these molecules were released in amounts much higher than 51Cr. The Ad2-dependent release of choline was found to be dependent on Ad2 concentration, with maximum release (nearly 60%) at 10 micrograms/ml of Ad2, on the length of the incubation with Ad2, with maximum release at about 90 min, and on the medium pH with maximum activity at pH 6.0 to 6.5. Greater than 95% of the choline released was water-soluble and identified as choline phosphate. Less than 5% of the choline released was associated with lipids, and none was released as a phospholipid vesicle or micelle. The ability of Ad2 to release choline was abolished by incubating Ad2 for 10 min at 45 degrees C, whereas the binding of Ad2 to the cells was not affected. Fetal calf serum also blocked Ad2-dependent choline release.     

Protonophore anion permeability of the human red cell membrane determined in the presence of valinomycin

Summary A transport model for translocation of the protonophore CCCP across the red cell membrane has been established and cellular CCCP binding parameters have been determined. The time course of the CCCP redistribution across the red cell membrane, following a jump in membrane potential induced by valinomycin addition, has been characterized by fitting values of preequilibrium extracellular pHvs. time to the transport model. It is demonstrated, that even in the presence of valinomycin, the CCCP-anion is well behaved, in that the translocation can be described by simple electrodiffusion. The translocation kinetics conform to an Eyring transport model, with a single activation energy barrier, contrary to translocation across lipid bilayers, that is reported to follow a transport model with a plateau in the activation energy barrier. The CCCP anion permeability across the red cell membrane has been calculated to be close to 2.0×10^–4 cm/sec at 37°C with small variations between donors. Thus the permeability of CCCP in the human red cell membrane deviates from that found in black lipid membranes, in which the permeability is found to be a factor of 10 higher.     

The anion permeability of vesicles reconstituted with intrinsic proteins from the human erythrocyte membrane

Band 3 protein was reconstituted with lipid vesicles consisting of 94:6 (molar ratio) egg phosphatidylcholine-bovine heart phosphatidylserine in a 2500:1 phospholipid:protein molar ratio by means of a Triton X-100/beads method. The SO2-4 permeability of the resulting vesicles was measured using an influx assay procedure in which the vesicles were sampled and subsequently eluted over Sephadex columns at appropriate time intervals. The accuracy of the assay was greatly increased by using an internal standard in order to correct for vesicle recovery. In agreement with previous work, it could be demonstrated that incorporation of band 3 in the vesicles caused an increase in SO2-4 permeability, which could be (partially) inhibited by high concentrations of DIDS or a competitive anion such as thiocyanate. However, the magnitude of the increased SO2-4 permeability was highly variable, even when vesicles were reconstituted using band 3 isolated from one batch of ghosts. In addition, the SO2-4 influx curves showed complex kinetics. These results are related to the existence of vesicle heterogeneity with respect to protein content and vesicle size as revealed by stractan density gradient centrifugation and freeze-fracture electron microscopy. Band 3 incorporation also increased the L-glucose permeability of the vesicles which could also be inhibited by DIDS. Glycophorin, which has no known transport function, reconstituted with lipid vesicles consisting of 94:6 (molar ratio) egg phosphatidylcholine-bovine heart phosphatidylserine in a 400:1 phospholipid:protein molar ration increased the bilayer permeability towards SO2-4 as well as towards L-glucose. Surprisingly, the SO2-4 permeability in the vesicles could also be inhibited by DIDS and thiocyanate. It is concluded that the use of DIDS and a competitive anion, thiocyanate, in order to prove that band 3 is functionally reconstituted, is highly questionable. The increased SO2-4 and L-glucose permeability of band 3-lipid as well as glycophorin-lipid vesicles and the inhibitory action of DIDS are discussed in the light of the presence of defects at the lipid/protein interface and protein aggregation, which may induce the formation of pores. Since the band 3-lipid vesicles are more permeable for SO2-4 than for L-glucose, in contrast to the glycophorin-containing vesicles, it is suggested that some anion specificity of the increased bilayer permeability in the band 3-lipid vesicles is still preserved.     

Determination of the permeability of human lymphocytes with a microscope diffusion chamber

A diffusion chamber similar to that proposed by J.J. McGrath (J. Microsc., in press) was constructed which allows microscopic observation of osmotically induced volume changes of individual cells in small (microliter) sample volumes. The cells are kept fixed in position in the upper compartment of the chamber by means of a highly permeable membrane and exposed to a step-like change in concentration generated in the lower compartment. An electrical conductivity probe in the upper compartment was used to monitor the temporal change of salt concentration as experienced by the cells. The rise from isotonic to hypertonic can be approximated by an exponential function. Its time constant of tau = 2.08 sec seems to be mainly determined by the change in flushing solution as tau = 1.48 sec was measured with no membrane installed. With human lymphocytes, no loss of cell volume was detected before 5 sec, i.e., when 95% of the final concentration was reached extracellularly. A step change can hence be assumed when modeling exosmosis for determining the lymphocyte membrane permeability. The equations for coupled transport of water and salt were solved numerically and fitted to the experimental data. The results were also compared to various other transport models described in the literature. Human lymphocytes are almost ideally semipermeable with a hydraulic reference permeability of Lp = 4.23 X 10(-4) cm/sec (3.13 X 10(-3) micron X atm-1 X sec-1) at T = 23 degrees C. The temperature and concentration dependence are described by an activation energy Ea = 14.3 kJ/mol and a concentration coefficient alpha 2 = 0.261 osmol/kg. An osmotically inactive volume fraction of 36.9% was determined from the final cell volumes reached asymptotically after shrinkage.     

Histamine alters E-cadherin cell adhesion to increase human airway epithelial permeability.

During the immediate response to an inhaled allergen, there is an increase in the paracellular permeability of the airway epithelium.1 Histamine is an important agonist released during the immediate response to inhaled allergen. We hypothesized that histamine would increase human airway epithelial paracellular permeability and that it would do this by interrupting E-cadherin-based cell adhesion. Histamine, applied to the basolateral surface, increased the paracellular permeability of cultured human airway epithelia, and this effect of histamine was blocked by the histamine receptor antagonist promethazine. ECV304 cells express a histamine receptor, N-cadherin, and elements of the tight junction, including claudins, but they do not express E-cadherin. Histamine increased the paracellular permeability of ECV304 cells transfected with a vector and expressing E-cadherin but not ECV304 cells expressing lac-Z in the same vector. L cells do not express the histamine receptor, cadherins, or claudins. Histamine decreased adhesion of L cells expressing the human histamine receptor and E-cadherin to an E-cadherin-Fc fusion protein. Histamine did not alter the adhesion to the E-cadherin fusion protein of L cells expressing either the histamine receptor or E-cadherin alone. When applied to the apical surface, adenovirus poorly infects airway epithelial cells because its receptor, CAR, is restricted to the basolateral surface of the cells. When histamine was applied to the basolateral surface of airway epithelial cells, infection of the cells by adenovirus increased by approximately one log. This effect of histamine was also blocked by promethazine. Histamine increases airway paracellular permeability and increases susceptibility of airway epithelial cells to infection by adenovirus by interrupting E-cadherin adhesion.     

Characterization of anion permeability in AH-66 hepatoma ascites cells

The sulfate transport in AH-66 hepatoma ascites cells was examined under various controlled conditions using 35SO42- as a tracer. The sulfate efflux rate was dependent on temperature, pH and anion species of the cell suspending medium. The efflux rate became saturated as the concentration of extracellular anions was increased. The efflux of anion was inhibited by some chemical reagents specifically reactive with amino or sulfhydryl groups. The results obtained in this study suggest that sulfate anions were transported by a facilitated transport system(s), and that some membrane protein(s) is involved in the anion transport system(s) of AH-66 cells. Both amino and sulfhydryl groups are thought to play a determinant role at the sulfate transport site in AH-66 cells.     

Enhancement of conductive anion permeability in cultured cells by cetiedil

Cetiedil, a drug that is reported to block K+-channels, substantially increases the conductive C1- permeability of Chinese hamster ovary (CHO) cells. The permeability was monitored by volume changes in cells treated with gramicidin to increase the cation permeability. Under this circumstance, increases in Cl- conductances result in volume changes detectable by electronic sizing, with the direction determined by the gradients of the permeating ions. In NaCl or KCl media, swelling occurs, but in N-methylglucamine chloride, shrinking. The increases in Cl- conductance could also be measured as an increased 36Cl- flux or by changes in membrane potential (measured by fluorescence of a potential-sensitive dye) toward the Cl- equilibrium potential. The effect of cetiedil was concentration dependent, with maximal effect at 50 microM. The anion specificity for the conductance was NO3- greater than Cl- = Br- much greater than SO4-2 or isethionate. A number of other drugs that influence transport activities had no effect on Cl- conductance. The cetiedil effect on Cl- conductance was observed in one other cell line, but was absent in several other cell types. The cetiedil-induced Cl- conductance in CHO cells appears to involve a different pathway than that induced by exposure to hypotonic medium.     

On the ATP dependence of the Ca-induced increase in K permeability observed in human red cells

When red cells are starved or incubated in the presence of metabolic poisons, with or without substrates, a large increase in K⁺ permeability is observed which depends on the presence of Ca²⁺ in the medium. The production or removal of a metabolite which controls the K⁺ permeability has been proposed to explain these effects. In the present experiments, a parallelism is found to exist between the rate of ATP depletion and the increase in Ca²⁺ uptake and K⁺ loss when red cells are depleted by different methods. The results support the view that the intracellular concentration of ATP may be the main factor on which the rate of Ca²⁺ uptake and the subsequent increase in K⁺ permeability depend.     

The release of superoxide anion from granulocytes: effect of inhibitors of anion permeability.

In vivo administration of ecdysterone produced a decrease in cyclic AMP levels and cyclic AMP-binding protein activity in mouse liver 40 min after injection. These changes were accompanied by a concomitant decrease in cyclic AMP-dependent protein kinase. The effect on phosphoprotein phosphatase was the opposite pattern of that on protein kinase. These results support the idea that the cyclic AMP-protein kinase system may be involved in the heterophylic action of ecdysterone.     

Thrombin-induced increase in albumin permeability across the endothelium

We studied the effect of thrombin on albumin permeability across the endothelial monolayer in vitro. Bovine pulmonary artery endothelial cells were grown on micropore membranes. Morphologic analysis confirmed the presence of a confluent monolayer with interendothelial junctions. Albumin permeability was measured by the clearance of 125I-albumin across the endothelial monolayer. The control 125I-albumin clearance was 0.273 +/- 0.02 microliter/min. The native enzyme, alpha-thrombin (10(-6) to 10(-10) M), added to the luminal side of the endothelium produced concentration-dependent increases in albumin clearance (maximum clearance of 0.586 +/- 0.08 microliter/min at 10(-6) M). Gamma (gamma) thrombin (10(-6) M and 10(-8) M), which lacks the fibrinogen recognition site, also produced a concentration-dependent increase in albumin clearance similar to that observed with alpha-thrombin. Moreover, the two proteolytically inactive forms of the native enzyme, i-Pr2 P-alpha-thrombin and D-Phe-Pro-Arg-CH2-alpha-thrombin, increased the 125I-albumin clearance (0.610 +/- 0.09 microliter/min and 0.609 +/- 0.02 microliter/min for i-Pr2 P-alpha-thrombin and D-Phe-Pro-Arg-CH2-alpha-thrombin at 10(-6) M, respectively). Since the modified forms of thrombin lack the fibrinogen recognition and active serine protease sites, the results indicate that neither site is required for increased albumin permeability. The increase in albumin clearance with alpha-thrombin was not secondary to endothelial cell lysis because lactate dehydrogenase concentration in the medium following thrombin was not significantly different from baseline values. There was also no morphological evidence of cell lysis. Moreover, the increase in 125I-albumin clearance induced by alpha-thrombin was reversible by washing thrombin from the endothelium. The basis for the increased albumin permeability following the addition of alpha-thrombin appears to be a reversible change in endothelial cell shape with formation of intercellular gaps.     

Plasmid genes increase membrane permeability in Escherichia coli

The membrane permeability to o-nitrophenyl beta-D-galactoside is increased in the presence of rifampicin in Escherichia coli cells carrying srnB+ or pnd+ plasmids, but not in the cells carrying srnB- or pnd- mutant plasmids. The same permeability alteration was also observed at 42 degrees C when a rpoC4- mutant strain was used as a host strain in the absence of rifampicin. These results and the blockage of the effects by action of chloramphenicol suggest that the increase of permeability to o-nitrophenyl galactoside was caused by the expression of srnB+ or pnd+ gene, respectively. srnB+ gene expression leads to massive RNA degradation, probably through the activation of the rna+ gene product. In an rna- strain carrying the srnB+ plasmid, the extent of RNA degradation was reduced, whereas the permeability to o-nitrophenyl galactoside was increased to the same level as in the rna+ strain. Also, the increase in permeability to o-nitrophenyl galactoside was observed at 30 degrees C, although high-temperature incubation (42 degrees C) was necessary for the induction of RNA degradation. These results suggest that the alteration in permeability is a more direct effect of the expression of srnB+ or pnd+ gene and that the RNA degradation is a secondary phenomenon caused by the alteration in the membrane.     

Role of superoxide anion radicals in microvascular permeability and leukocyte behaviour

Inflammation is associated with the accumulation and activation of phagocytic cells, such as polymorphonuclear leukocytes, and with the subsequent release and generation of a group of activated oxygen species, some of which are free radicals. These studies were carried out to assess the influence of enzymatically generated free radicals on both microvascular permeability and leukocyte adhesion. The extravasation of fluorescein-labelled dextran mean molecular weight (MW) 150 000 was used to assess microvascular permeability and methodology was developed to measure in vivo leukocyte endothelial interactions. Enzymatically generated superoxide anion radical (O-.2) was associated with an increase in macromolecular extravasation (seen primarily from postcapillary venules) and an increase in leukocyte adhesion. Macromolecular extravasation was found to be dependent on the generation of a hydroxyl radical related interaction while leukocyte adhesion was dependent on the presence of O-.2. It is suggested that the permeability alterations and increased polymorphonuclear leukocyte adhesion seen during inflammation may be partially related to the release of free radicals from inflammatory cells.     

Glucocorticoids increase insulin binding and the amount of insulin-receptor mRNA in human cultured lymphocytes.

The effect of steroid hormones on insulin binding and the amount of insulin-receptor mRNA was examined in IM-9 lymphocytes. Cortisol and cortexolone, but not oestrogen, increased both the binding of insulin and the amount of insulin-receptor mRNA in a time- and dose-dependent manner. Cortisol was most potent, and induced a 2-fold increase in insulin binding and a 4-fold increase in mRNA. The elevation in binding was due to an increased number of insulin receptors at the cell surface. The increase in mRNA involved all four of the insulin-receptor mRNAs and could not be inhibited by cycloheximide. The cortisol-induced increase in mRNA was associated with a 3-4-fold increase in the synthesis of pro-receptor. The relative potency of the three steroids indicated that these effects were mediated by an interaction with the glucocorticoid receptor. The results of this study suggest that cortisol can increase the number of insulin receptors at the cell surface by increasing the amounts of insulin-receptor mRNA and the synthesis de novo of insulin receptors.