Ricardo Garay

Heterogenous effect of flavonoids on K+ loss and lipid peroxidation induced by oxygen-free radicals in human red cells

Abstract Treatment of fresh erythrocytes with phenazine methosulfate, an intracellular generator of oxygen-free radicals, and diethyldithiocarbamate an inhibitor of superoxide dismutase results in membrane damage consisting in lipid peroxidation and increase in passive K+ permeability. Various flavonoids which have previously been reported to act as oxygen-free radical scavengers were tested on this erythrocyte model. Surprisingly, flavonoids did not exhibit the same effect on the oxygen free radical-stimulated K+ permeability. It was possible to classify these agents into four groups: (i) protective (those decreasing the oxygen-free radical-stimulated K+ permeability): kaempferol, naringenin, apigenin, naringin; (ii) toxic (those increasing the deleterious effect of oxygen-free radicals): myricetin, delphinidin, quercetin; (iii) biphasic effective (characterized by opposite effects depending on the concentration): phloretin, cyanin, catechin, morin and (iv) inactive: rutin, phloridzin. In addition, a similar classification was observed when membrane lipid peroxidation was examined, i.e. kaempferol (i) decreased lipid peroxide formation whereas myricetin (ii) enhanced it, morin (iii) exhibited a biphasic effect and rutin (iv) has no effect. The previously reported metal chelating effect of flavonoids could not totally explain the protective effect of kaempferol as was demonstrated by the partial protective effect exhibited by desferrioxamine. Moreover, this study suggests that a generation of oxygen-free radicals in red cells induced a K+ loss which probably results from membrane lipid peroxidation.

Abnormal Na+,K+ cotransport function in a group of patients with essential hypertension.

Abstract. In 50 normotensive controls, the increase in erythrocyte Na+ concentration up to 12.4 ± 2.0 mmol/l cells (mean ± SD) ensures half‐maximal stimulation of outward Na+K+ cotransport fluxes. Forty‐six out of sixty‐five patients with essential hypertension required more than 16 mmol/l cells of internal Na+ concentration to obtain a similar effect, strongly suggesting an abnormal cotransport function.

An Na+-stimulated Mg2+-transport system in human red blood cells

The initial rate of net Mg2+ efflux was measured in human red blood cells by atomic absorption. In fresh erythrocytes incubated in Na+,K+-Ringers medium this rate was 7.3 +/- 2.8 mumol/l cells per h (mean +/- S.D. of 14 subjects) with an energy of activation of 13 200 cal/mol. Cells with total Mg2+ contents ([ Mg]i) ranging from 1.8 to 24 mmol/l cells were prepared by using a modified p-chloromercuribenzenesulphonate method. Mg2+ efflux was strongly stimulated by increases in [Mg]i and in external Na+ concentrations ([ Na]o). A kinetic analysis of Mg2+ efflux as a function of [Mg]i and [Na]o revealed the existence of two components: an Na+-stimulated Mg2+ efflux, which exhibited a Michaelian-like dependence of free internal Mg2+ content (apparent dissociation constant = 2.6 +/- 1.4 mmol/l cells; mean +/- S.D. of six subjects) and on external Na+ concentration (apparent dissociation constant = 20.5 +/- 1.9 mM; mean +/- S.D. of four subjects) and a variable maximal rate ranging from 35 to 370 mumol/l cells per h, and an Na+-independent Mg2+ efflux, which showed a linear dependence on internal Mg2+ content with a rate constant of (6.6 +/- 0.7) X 10(-3) h-1. Fluxes catalyzed by the Na+-stimulated Mg2+ carrier were partially dependent on the ATP content of the cells and completely inhibited by quinidine (IC50 = 50 microM) and by Mn2+ (IC50 = 0.5-1.0 mM).

Inhibition of the Na+/K+ cotransport system by cyclic AMP and intracellular Ca2+ in human red cells.

Human erythrocytes are able to incorporate cyclic AMP (cAMP) in amounts larger than those required to saturate cAMP-dependent protein kinase. In contrast to previous observations in avian red blood cells in which cAMP stimulates the Na+/K+ cotransport system, we demonstrate that cAMP inhibits this system in human erythrocytes. The cotransport inhibition is enhanced by addition of phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine to the incubation medium. The cAMP concentration giving half-maximal cotransport inhibition showed a wide variation among different individuals (from 0.1 to 5 mM external cAMP concentration). In contrast to cAMP, cyclic GMP showed little effect on the cotransport system. Ca2+ introduced into the cell interior was an inhibitor of the Na+/K+ cotransport system. These results suggest that in human cells in which endogeneous levels of cAMP and Ca2+ are modulated by hormones, the Na+/K+ cotransport system may be under hormonal regulation.

A kinetic approach to the immunology of cancer: Stationary states properties of efffector-target cell reactions

We present a kinetic model of the onset of a tumor which involves the coupling of three principal phenomena: 1. (1) the transformation of normal into neoplastic cells, 2. (2) the replication of transformed cells, 3. (3) the immunological interaction of the host organism with transformed cells and/or oncogenic viruses. We assume that the immunological component has two opposite (destructing and facilitating) functions, each of which is regulated by activators and inhibitors. In this paper our analysis is devoted to the cell-mediated neoplastic cell destruction and its negative regulation by blocking factors or by other immunosuppressive agents. We assume that the immunological system may reach different stationary states (nonactivated, activated, suppressed, etc.). In each of these immunological stationary states, Michaelian-like kinetics for the cell-mediated cytotoxic reaction allow to describe the homogeneous stationary states of the neoplastic cell population in term of three adimensional parameters: (1) α, which represents the relative rate of cellular transformation; (2) β, which characterizes the relative rate of neoplastic cell destruction, and (3) θ, α parameter whose value determines the presence of a metastable region for the neoplastic transition of the tissue. The application of our model to the kinetic analysis of the immunological rejection of MSV-M induced tumors confirms our kinetic hypotheses and predicts the minimum ratio of aggressor to target cells that must be reached within the tumor mass to obtain a rejection. Since during rejection this level is not attained with the immune T-cells, we suggest additional co-operative mechanisms involving antibodies and other effector cell types. In the framework of our model we propose the following conclusions: 1. (1) Under physiological conditions tissues contain at least, one neoplastic cell to 1014–1018 normal cells, suggesting that they are exempted from neoplastic cells. 2. (2) The cell to cell cytotoxic activity of the different types of effectors seems to increase in the order: activated macrophages < immune T lymphocytes and natural activity of T lymphocytes. 3. (3) Whereas the cytotoxic activity of T lymphocytes is sufficiently high to cope with allogenic tumor rejection, in syngeneic tumor systems a complex interaction between differejnt effector cells and humoral factors must certainly take place in order to obtain a successful surveillance against cancer. 4. (4) The transition of normal to neoplastic tissues may bear some analogy with metastability phenomena.

The significance of the relative effects of loop diuretics and anti-brain edema agents on the Na+, K+, Cl− cotransport system and the Cl−/NaCO3− anion exchanger

Summary3-Amino-5-sulfamoylbenzoic acids and several series of (aryloxy)alkanoic acids were evaluated for their inhibitory effects on two human erythrocyte ion transport systems — the Na+, K+ cotransport system and the DIDS-sensitive anion carrier.Several classic loop diuretics, including the (aryloxy)alkanoic acid-ethacrynic acid and several 3-amino-5-sulfamoylbenzoic acids, like bumetanide and furosemide, displayed relatively strong inhibitory activity versus the cotransport system with relatively weaker action versus the anion carrier. Furthermore, diuretic potency correlated with cotransport inhibitory potency.Another class of (aryloxy)alkanoic acids, namely the [(2,3-dihydro-1 H-inden-5-yl)oxy]acetic acids, such as indacrinone and MK-473, which exhibit less potent loop dacrinone and MK-473, which exhibit less potent loop diuretic activity, were less potent cotransport inhibitors and more effective inhibitors of the anion carrier.Still other (aryloxy)alkanoic acids, with little saliuretic activity, namely a sub-class of [(2,3-dihydro-1 H-inden-5-yl)oxy]alkanoic acids and a series of [(2,3,9,9a-tetrahydro-1 H-fluoren-7-yl)oxy]acetic acids displayed little or no inhibitory action on the cotransport system but enhanced inhibitory action on the anion carrier. Most interestingly, the relative anion carrier inhibitory potency correlated well with the relative inhibitory activity of each compound on bicarbonate-stimulated cell swelling in cat cerebrocortical slices.

Involvement of K+ movements in the membrane signal induced by PAF-acether

We investigated the effects of PAF-acether and its specific antagonist BN 52021 on Na+ and K+ transport systems in human red cells and mouse macrophages. PAF-acether and BN 52021 exhibited specific and opposite effects on a Cs+-stimulated, K+ efflux in human red cells. PAF-acether increased and BN 52021 decreased the apparent dissociation constant for external Cs+ without significant effects on the maximal rate of K+ translocation. In mouse macrophages, PAF-acether stimulated a quinidine-sensitive K+ efflux. In the presence of the Ca2+-ionophore A 23187, PAF-acether and BN 52021 showed opposite effects (stimulation and inhibition respectively). For most cells, membrane potential is dependent on K+-permeability. In addition, opening of potential-dependent Ca2+ channels appears to be associated with cell activation in several models. We thus propose that the specific interaction of PAF-acether with a K+:K+ exchange increases Ca2+ uptake through transitory changes in membrane potential. This in turn may lead to a more permanent membrane hyperpolarization through to opening of Ca2+ dependent, K+ channels.

Modulation of erythrocyte Na transport pathways by excess Na intake

Different Na transport pathways were studied in the erythrocytes of 10 normotensive subjects who received 240 meq/day of Na in excess of their usual diet. In most of these subjects the maximal rate (Vmax) of the Na,K pump and the Na,K-cotransport system was markedly decreased on the first day of the diet. In some of these subjects, excess Na intake induced an increase in the apparent affinity for internal Na for the Na,K pump and the Na,K-cotransport system. The decrease in the Na,K pump fluxes was not concomitant to that of the co-transport system and not accompanied with an increase in blood pressure or cation concentration in the plasma. Interestingly, the apparent affinity for internal Li of the Li-Na exchange was markedly increased without alteration of the Vmax. The passive permeability for Na and the cellular Na content were not altered by excess Na intake. Ouabain and bumetanide at low concentrations respectively induced an increase in the apparent affinity for internal Na of the Na,K pump and the Na,K- cotransport system. These results are similar to those observed after excess Na intake. These later agree with the hypothesis that Na homeostasis regulates some endogenous factors with ouabain-like and furosemide-like properties that might contribute to the regulation of cellular Na handling.

Inhibition of the erythrocyte Na+, K+-pump by mammalian lignans.

Several mammalian lignans, particularly enterolactone, 3-oxy-methyl enterolactone and prestegane B are able to inhibit Na+, K+-pump activity in human red cells with IC50 of about 1 mM. The inhibition of Na+, K+-pump activity by mammalian lignans have the following properties: the IC50 for ouabain remains unchanged suggesting a noncompetitive inhibition. The apparent affinity for internal Na+ and maximal rate of cation translocation are both diminished. The above inhibition of the Na+, K+-pump was obtained at doses 2-3 orders of magnitude higher than those required for ouabain. However we cannot exclude that a glycosyl- (and/or butenolide)-derivative of enterolactone could be one endogenous ouabain-like factor.

Action of azelastine on intracellular Ca2+ in cultured airway smooth muscle

Azelastine, a novel antiasthmatic/antiallergic agent, was tested for Ca2+ antagonistic properties in cultured rabbit airway smooth muscle, vascular smooth muscle and cardiocytes. In airway smooth muscle cells, the basal cytosolic free calcium content was 195 +/- 72 nM (mean +/- S.D., n = 18). These basal values were decreased by azelastine with an IC50 value of 1.1 +/- 0.3 x 10(-4) M. Endothelin-1 (10(-7) M) induced a rapid increase in free cytosolic calcium up to 806 +/- 314 nM, which returned to normal levels in 3-5 min. This was fully blocked by azelastine in a concentration-dependent manner, with an IC50 value of 6.7 +/- 2.9 x 10(-5) M. Moreover, azelastine fully blocked histamine-induced calcium mobilization (IC50 = 7 x 10(-5) M). In cultured vascular smooth muscle cells and cardiocytes, azelastine was unable to decrease the basal cytosolic free calcium content or inhibit agonist-induced calcium mobilization. Therefore, at therapeutic levels, a specific, mild inhibition of calcium mobilization in airway smooth muscle may be one component of the antiasthmatic action of azelastine.