Biomedical subjects

R Motais

Publications and source records attributed to R Motais.

At least 55 records · Page 3Linked to original sources

PubMed1986

Catecholamine-induced transport systems in trout erythrocyte. Na+/H+ countertransport or NaCl cotransport?

It has previously been shown (Baroin, A., F. Garcia-Romeu, T. Lamarre, and R. Motais. 1984a, b. Journal of Physiology. 350:137, 356:21; Mahé, Y., F. Garcia-Romeu, and R. Motais. 1985. European Journal of Pharmacology. 116:199) that the addition of catecholamines to an isotonic suspension of nucleated red blood cells of the rainbow trout first stimulates a cAMP-dependent, amiloride-sensitive Na+/H+ exchange. This stimulation seems to be transient. It is followed by a more permanent activation of a coupled entry of Na+ and Cl-, which is inhibited by amiloride but also by inhibitors of band 3 protein (DIDS, furosemide, niflumic acid). The coupled entry of Na+ and Cl- could therefore result from the parallel and simultaneous exchange of Na+out for H+in (via the cAMP-dependent Na+/H+ antiporter) and Cl- out for HCO3- in (via the anion exchange system located in band 3 protein). However, in view of the following arguments, it had been proposed that NaCl uptake does not proceed by the double-exchanger system but via an NaCl cotransport: (a) Na+ entry requires Cl- as anion (in NO3- medium, the Na uptake is strongly inhibited, whereas NO3- is an extremely effective substitute for Cl- in the anion exchange system); (b) Na uptake is not significantly affected by the presence of HCO3- in the suspension medium despite the fact that in red cells, Cl-/HCO3- exchange occurs more readily than the exchanges of Cl- for basic equivalents in a theoretically CO2-free medium (the so-called Cl-/OH- exchanges). The purpose of the present paper was a reassessment of the two models by using monensin, an ionophore allowing Na+/H+ exchange. From this study, it appears that NaCl entry results from the simultaneous functioning of the Na+/H+ antiporter and the anion exchange system. The apparent Cl dependence is explained by the fact that, in these erythrocytes, NO3- clearly inhibits the turnover rate of the Na+/H+ antiporter. As Na+/H+ exchange is the driving component in the salt uptake process, this inhibition explains the Cl requirement for Na entry. The lack of stimulation of cell swelling by bicarbonate is explained by the fact that the rate of anion exchange in a CO2-free medium (Cl-/OH- exchange) is roughly equivalent to that of Na+/H+ exchange and thus in practice is not limiting to the net influx of NaCl through the two exchangers.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1986

Effect of deep hypoxia on acid-base balance in trout: role of ion transfer processes.

Continuous recordings were made of pH, Po2, and Pco2 of arterial blood in an extracorporeal circulation during a 20-min period of deep hypoxia (inspired Po2 = 40 Torr) and subsequent normoxia in rainbow trout. Blood concentrations of lactate, Na+, K+, and Cl- and net fluxes of electrolytes across gills and kidney were also continuously monitored. Deep hypoxia induced a short respiratory alkalosis followed by a marked metabolic acidosis. The acidification is biphasic in nature. The first rapid phase of acidification is not related to lactic acid production. On the other hand, the second phase of acidification is associated with lactic acid diffusion into the blood. However, a considerable proportion of the lactate ions is balanced by Na+ ions coming from a shift in the distribution of this cation between intra- and extracellular compartments. Most of the H+ ions formed from the dissociation of lactic acid are buffered in the intracellular space. The acidification period is also characterized by a very small increase in plasma Cl- content, much less than that of Na+, and by the apparent accumulation of an unknown anion in the blood. Recovery from hypoxia is associated with a pH readjustment, a large and identical decline of blood Na+ and Cl- contents without modification of the concentrations of K+, lactate, and the unknown anion. During this period the branchial ionic exchanges are slightly stimulated, but in the acid-base regulation as a whole, the branchial regulatory processes only play a minor part, the ionic movements being mainly performed by transfer between intra- and extracellular spaces.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-Base Equilibrium↗

PubMed1985

Inhibition by amiloride of both adenylate cyclase activity and the Na+/H+ antiporter in fish erythrocytes.

In fish erythrocytes isoproterenol stimulates cellular accumulation of cyclic adenosine 3':5'-monophosphate (cyclic AMP) and produces a large increase in sodium permeability which corresponds to the activation of Na+/H+ exchanges and chloride-dependent sodium uptake. The stimulation of sodium transport by isoproterenol was reproduced by adding cyclic AMP or forskolin to the medium and was blocked by propranolol. This increase in sodium permeability was completely inhibited by amiloride at the relatively high levels (0.1-1 mM) of the diuretic required to inhibit the activity of the Na+/H+ exchanger under physiological conditions in various biological systems. It was shown that amiloride inhibited cyclic AMP accumulation. This effect, which was reversible and dose-dependent (ED50 6 X 10(-6) M-maximal effect 0.5 mM), resulted from the inhibition of the catalytic unit of adenylate cyclase. Amiloride also directly inhibited the sodium entry system but the Na transporter was less sensitive than adenylate cyclase to amiloride (ED50 6 X 10(-5) M). It appears from the data presented in this report that the inhibition of sodium permeability observed in fish erythrocytes in the presence of amiloride can result either from the effect of the diuretic on the adenylate cyclase system or from the effect on the sodium transport system, depending on the conditions in which amiloride is used. Thus, caution is required when interpreting amiloride action in terms of inhibition of specific transport processes.

Adenylyl Cyclase Inhibitors↗

PubMed1984

Hormone-induced co-transport with specific pharmacological properties in erythrocytes of rainbow trout, Salmo gairdneri.

On the addition of isoprenaline to an isotonic suspension of red blood cells of rainbow trout (Salmo gairdneri), the cell volume increases. This increase in volume is the result of net uptake of Na+ and osmotically obligated water. Two different pathways are involved in the salt uptake. The minor component of Na+ entry (about 20%) corresponds to a Na+ uptake independent of Cl- and is inhibited by amiloride, yet is insensitive to DIDS, furosemide and niflumic acid. It could result from Na+/H+ countertransport. The major component of salt uptake is due to Na+ entry which requires Cl- as anion, and is electroneutral, independent of extracellular K+, sensitive to amiloride, DIDS, niflumic acid and furosemide, but insensitive to other loop diuretics such as piretanide or bumetanide. These characteristics, as well as the response of valinomycin-treated cells to isoprenaline and some other properties (ionic selectivity, drug sensitivity) of the anion exchange system of volume-static trout red cells, permit the definition of the nature of this Cl--dependent pathway. The findings are inconsistent with the electrically silent double antiporter model (proposed in amphibian red cells by Cala, 1980) and with the co-migration of Cl- with Na+ through parallel conductive pathways, but strongly suggest a symport mechanism. Striking differences, mainly pharmacological, exist between this NaCl co-transport and the duck red blood cell Na+/K+/2Cl- co-transport (Kregenow, 1977, 1978; McManus & Schmidt, 1978).

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1984

A transient sodium-hydrogen exchange system induced by catecholamines in erythrocytes of rainbow trout, Salmo gairdneri.

The addition of isoprenaline to an isotonic suspension of red blood cells of rainbow trout induces an amiloride-sensitive Na+ transport which is independent of Cl- and insensitive to 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid (DIDS) and furosemide. Na+ uptake is accompanied by amiloride-sensitive H+ release. The H+ efflux is dependent upon the external Na+ concentration, the K0.5 value for Na+ being 16 mM. In the presence of DIDS, when the coupled NaCl entry (NaCl co-transport) induced by catecholamine is blocked, the results provide evidence for a linked movement of Na+ and H+, with a stoicheiometry of 1:1. Exchange of H+ for Na+ induces osmotic swelling of the cells which is due to the replacement of a bound proton by an osmotically active Na+ cation. In the absence of DIDS when the bulk of the Na+ uptake is the result of a coupled entry of Na+ and Cl-, H+ extrusion still occurs and the magnitude of acid excretion is identical to that found in DIDS-treated cells. This suggests that Na+-H+ exchange remains active. Addition of isoprenaline first stimulates the Na+-H+ exchange but only transiently. This is followed by a more permanent stimulation of the NaCl co-transport.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1982

Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds. I. Determination of the flufenamate-binding site by proteolytic dissection of the band 3 protein.

Flufenamate, non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte (I50 = 6 . 10(-7) M). The concentration dependence of the binding to ghosts reveals two saturable components. [14C]Flufenamate binds with high affinity (Kd1 = 1.2 . 10(-7) M) to 8.5 . 10(5) sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity (Kd2 = 10(-4) M) to a second set of sites (4.6 . 10(7) per cell). Pretreatment of cells with 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [14C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport of [14C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [14C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [14C]flufenamate. Thus it appears that 35 kDa peptide is necessary is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1982

Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds. II. Chemical properties of the flufenamate-binding site on the band 3 protein.

Flufenamate is a powerful inhibitor of anion exchange in red blood cells. It binds to the band 3 protein involved in the transport as discussed in the preceding paper (Cousin, J.-L. and Motais, R. (1982) Biochim. Biophys. Acta 687, 147-155). The present study is concerned with the chemical properties of the inhibitory binding site. Structure-activity studies were performed with two sets of compounds derivated from anthranilate (considered as the basic structure of flufenamate). The molar concentrations required to produce 50% inhibition (I50) varied over more than a 10(4) range. The inhibitory activity was quantitatively correlated with the hydrophobic character of the molecules and the electron-withdrawing capacity of the substituents. Comparison between the inhibitory potency of flufenamate analogs made a definition of the contribution of each part of the molecule in the binding to the receptor possible. The results suggest that anionic inhibitors bind to a site which presents a positively charged groups at the water-protein interface whereas the hydrophobic part of the molecule is inserted into an hydrophobic and electron-donor region of the protein. The specificity of amphiphilic compounds towards anion transport is discussed.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1981

Chloride permeability in human red cells: influence of membrane protein rearrangement resulting from ATP depletion and calcium accumulation.

A 15% of band 3 protein, the assumed chloride channel, is associated with spectrin, the major peripheral protein of a lattice located at the red cell membrane-cytosol interface, the present study was undertaken to evaluate whether a rearrangement of the lattice modifies the functional property of band 3 protein. Such a rearrangement was modulated by depletion of cell ATP and/or by accumulation of Ca2+ ions within the cell. ATP depletion induces an inhibition of the electroneutral one-for-one chloride exchanges. Neither the modification of red cell morphology due to ATP depletion (discocyte-echinocyte transformation) nor a direct effect of the decrease in internal ATP level can account for this inhibition. On the other hand, it seems reasonable to consider that inhibition is related to the changes in membrane protein organization (formation of heteropolymers) induced by the decrease in ATP level. But it does not appear that the degree of inhibition is modified when this altered assembly of membrane protein is stabilized by disulfide linkages. Accumulation of Ca2+ ions in the cell at a relatively low concentration (10 micro M range) inhibits chloride exchange without apparent modification of the assembly of membrane proteins. This effect of calcium on chloride exchanges is speculatively denoted as a "direct" effect of calcium. Calcium loading of fresh red cells at higher concentrations (500 to 1000 micro M) obtained by use of the ionophore A23187 induces a very strong inhibition of chloride exchanges. In this case, inhibition can be reasonably accounted for by two simultaneous effects of calcium: a "direct" effect which explains half of the inhibition and an "indirect effect due to the formation of membrane protein complexes stabilized by covalent crosslinkages (activation by Ca2+ ions of a transglutaminase). It is interesting to note that intracellular calcium, whatever the level, inhibits electroneutral exchanges of chloride but increases net chloride movements.

Adenosine Triphosphate↗

PubMed1980

Inhibition of anion and glucose permeabilities by anesthetics in erythrocytes. The mechanisms of action of positively and negatively charged drugs.

(1) The mode of action of anesthetics as inhibitors of Cl- and glucose transports in human red cells was studied. The term anesthetic is taken in its broad meaning as defined by Seeman (Seeman, P. (1972) Pharmacol. Rev. 24, 583-655) and covers anionic and cationic liposoluble compounds which reversibly block the rising phase of the action potential, without effect on the resting membrane potential. (2) Phenothiazine derivatives were chosen as prototypes of anesthetics because they represent a set of compounds having the same basic chemical structure, the phenothiazine ring, but with either a positive or a negative charge. (3) The Cl- self-exchange is inhibited by both cationic and anionic derivatives. However, to obtain the same level of inhibition, it is necessary to use a concentration 10-100 times higher with cationic than with anionic drugs. (4) At a concentration which inhibits Cl- permeability, cationic derivatives induce a very strong morphological change (cup-shaped cells: stomatocytes or spherostomatocytes) and protect erythrocytes against osmotic hemolysis, signifying that the membrane is fully expanded. Conversely, with anionic derivatives, inhibition occurs at a concentration which does not induce any apparent shape change or protect against osmotic hemolysis: there is no significant membrane expansion. (5) Glucose permeability, measured by glucose exit, is inhibited by cationic and anionic phenothiazine, but always at a concentration which fully expands the membrane as indicated by morphological changes and anti-hemolytic effects. It is interesting to point out that whilst glucose exit shows inhibition by cationic derivatives, glucose exchange flux is scarcely altered. (6) It is concluded that cationic and anionic anesthetics are general inhibitors of transmembrane solute movements involving a facilitated-diffusion process. However, the mechanism of inhibition is not identical for all: inhibition of glucose permeability by anionic and cationic anesthetics, as well as inhibition of Cl- permeability by cationic anesthetics may be of a non-specific nature and result from their interaction with the bilayer (this indirect effect is discussed); on the other hand, inhibition of Cl- permeability by anionic anesthetics may result from a specific perturbation of the transport mechanism according to recent evidence in some cases (Cousin, J.L. and Motais, R. (1979) J. Membrane Biol. 46, 125-153; Zaki, L., Ruffing, W. Gärtner, E.M., Fasold, H., Motais, R. and Passow, H. (1977) 11th FEBS Meeting, Copenhagen, A4 17-671.

Anions↗

PubMed1979

Inhibition of anion permeability by amphiphilic compounds in human red cell: evidence for an interaction of niflumic acid with the band 3 protein.

In human erythrocyte, permeability to the anion is instantaneously, reversibly, and noncompetitively inhibited by the nonsteroidal anti-inflammatory drug, niflumic acid. The active form of this powerful inhibitor (I50 = 6 X 10(-7) M) is the ionic form. We demonstrated that: (i) The binding of niflumic acid to the membrane of unsealed ghosts show one saturable and one linear component over the concentration range studied. The saturable component vanishes when chloride transport is fully inhibited by covalently bound 4-acetamido-4'-isothiocyano stilbene-2,2'-disulfonic acid (SITS). Our estimate of these SITS protectable niflumate binding sites (about 9 x 10(5) per cell) agrees with the number of protein molecules per cell in band 3. These sites are half-saturated with 10(-6) M niflumic acid, a concentration very close to I50. (ii) Niflumic acid inhibits the binding reaction of SITS with anion controlling transport sites. These results indicate that niflumic acid and SITS are mutually exclusive inhibitors, suggesting that niflumic acid interacts with the protein in band 3. Niflumic acid also decreases glucose and ouabain-insensitive sodium permeabilities. However, these effects are produced at a very high concentration of niflumic acid (in millimolar range), suggesting unspecific action, possibly through lipid phase.

Anions↗

PubMed1978

Uncouplers of oxidative phosphorylation. A structure-activity study of their inhibitor effect on passive chloride permeability.

Uncoupling agents inhibit chloride transport in red blood cells, which is a metabolism-independent process. An analysis of the molecular requirements shows that this inhibitory activity is closely correlated with the electronic and the hydrophobic bonding properties of phenols: the more lipophilic and the more electron-attracting the substituent groups are, the greater the activity they confer on the parent molecule. A recent structure-activity study concerning various classes of reversible inhibitors of chloride transport led to the same conclusion (Motais, R. and Cousin, J.L. (1977) in International Conference on Biological Membranes: Drugs, Hormones and Membranes (Bolis, L., Hoffman, J.F. and Straub, R.W., eds.), Raven Press, New York, in the press). The effects of substituents on the activity of phenols as uncouplers have been recently examined (Stockdale, M. and Selwyn, M.J. (1971) Eur. J. Biochem. 21, 565). The comparison of these results with our data shows that uncoupling depends more on electronic properties of phenols than does choloride inhibition.

Biological Transport↗

PubMed1977

The chloride transport induced by triaklyl-tin compound across erythrocyte membrane.

The effect of tripropyl-tin chloride on anion permeability was studied using red cells previously treated with a covalent binding inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) to inhibit completely and irreversibly the natural anion transport system. It was demonstrated that the tin compound can mediate chloride-hydroxide and chloride-chloride exchanges across the "impermeabilised" erythrocyte membrane. In the non hemolytic range, the rate of exchange increased with the concentration of the tin compound in a non linear fashion, and no saturation effect was seen. The temperature profile of the chloride self exchange induced by tripropyl-tin was studied and the apparent activation energy found was 29 Kcal/mol. The tripropyl-tin chloride cannot mediate a chloride-bicarbonate exchange. Because of this discriminatory effect between hydroxide and bicarbonate, the tin compound can be useful in certain experimental conditions as seen for the study of the anion "carrier" of the red cell membrane ("cousin, J.L., Motais, R. and Sola, F. (1975) J. Physiol. Lond. 253, 385-399).

Bicarbonates↗

PubMed1976

[Demonstration of the inhibitory effect of ethacrynic acid on permeability to chlorine].

Ethacrynic acid specifically inhibits chloride permeability in Ox erythrocyte. The I50 is 7 x 10(-6) M. The inhibitory effect is instantaneous and completely reversed by washing the cells with a Ringer solution. Dihydroethacrynic acid, a derivative that lacks the ability to combine with SH groups, also inhibits chloride permeability and the characteristics of inhibition are strictly identical. It is a proof that ethacrytic acid does not act by its reactivity with thiol groups.

Animals↗

PubMed1976

The inhibitor effect of probencid and structural analogues on organic anions and chloride permeabilities in ox erythrocytes.

Probenecid inhibits anion movements (organic anions and chloride) in ox erythrocytes. The I50 is 4. 10(-5) M. Structural analogues such as carinamide, p-carboxybenzene sulfonamide and p-carboxy N,N-diethyl benzene sulfonamide, which are drugs of the sulfonamide class, were also found to inhibit anion transport. These results reinforce the previously discussed view based on structural considerations, that sulfonamides act on the red cell membrane as competitors of anion transport. It is possible that probenecid and carinamide act in a similar way in the kidney.

Animals↗

PubMed1976

The role of carbonic anhydrase inhibitors on anion permeability into ox red blood cells.

1. Organic anion permeability in ox red blood cell was measured by studying steady-state self-exchange of oxalate, chosen as a prototypical substrate of the organic anion transport system previously described; chloride self-exchange measured the inorganic anion permeability. 2. Carbonic anhydrase inhibitors of the sulphonamide class inhibit both organic anion self-exchange (A-/A-) and chloride self-exchang (CL-/CL-) although carbonic anhydrase plays no role in these exchanges. These results confirm the conclusions already published that sulphonamides can act directly on the cellular membrane as specific inhibitors of anion transport. 3. There is a correlation between the chemical structure of the sulphonamides and their capacity for inhibiting transmembrane anionic exchange. It is of significance that N-sulphamyl substitution, which abolishes the carbonic anhydrase inhibitory potency, does not destroy anionic inhibitory capacity and may even increase it. 4. For each sulphonamide the capacities for inhibiting chloride transport and oxalate transport are strictly identical. Inhibition appears non-competitive. 5. The temperature sensitivity of oxalate self-exchange is exactly the same as that of chloride self-exchange. From this, and from the nature of their inhibition by sulphonamides, it is proposed that chloride and organic anions share the same transport mechanism. 6. In the light of the present results the chloruretic action of sulphonamides in various tissues, in particular the kidney, is discussed.

Animals↗

PubMed1976

Inhibitory effect of ethacrynic acid on chloride permeability.

Ethacrynic acid inhibits anion movements in ox red blood cells. The I50 for chloride is 7 X 10(-6) M. The inhibitory effect is instantaneous and completely reversed by washing the cells with a Ringer solution, suggesting that reaction with a membrane SH group is not involved in this process. Direct proof that ethacrynic acid does not act by its reactivity with thiol groups is given by experiments with dihydroethacrynic acid, a derivative that lacks the ability to combine with SH groups: the characteristics of inhibition are strictly identical (instantaneous and reversible; I50 equals 9 X 10(-6) M). All the phenoxyacetic derivatives tested were also more or less inhibitory. The relative activity of all the derivatives was highly correlated with their liposolubility, indicating that hydrophobic interaction is important in determining drug effect and influence of steric factors is minimal. The data suggest that inhibition essentially results from a hydrophobic interaction between ethacrynic acid and apolar regions of the membrane protein allowing chloride transport.

Animals↗

PubMed1975

[Transmembrane exchange of C1- and HCO3- in erythrocytes: direct action of carbonic anhydrase inhibitors on the transport mechanism].

The only currently recognised role of unsubstituted sulphonamides is their specific inhibition of carbonic anhydrase. The results presented here show that they inhibit the HCO3/Cl- and Cl-/Cl- exchanges across the membrane of the ox erythrocyte. Carbonic anhydrase is not involved in this inhibition which is the result of a direct and specific action on the anion transfer mechanism at membrane level. This newly-recognised role of sulphonamides satisfactorily explains and integrates many experimental results concerning secretory tissues and the interpretation of the action of diuretics.

Animals↗

PubMed1975

Molecular features of organic anion permeablity in ox red blood cell.

1. The penetration of organic anions into bovine red blood cells has been studied under experimental conditions where it could be distinguished from the penetration of undissociated acids which proceeds by diffusion through lipid zones of the membrane. 2. Several lines of evidence suggest that the entry of organic anions cannot be ascribed to simple diffusion across aqueous channels limited by positive charges but needs a specific interaction of the penetrating anion with a component of the membrane. 3. The structural requirements allowing for ionic transfer is a strong polar head for the smallest molecules and in addition an amphiphilic structure for acids with chain length greater than C4. Interaction between substrate and receptor requires at least a three point attachment involving three oxygen atoms in the substrate which react with complementary loci on the receptor to form ionic and hydrogen bonds. Such a three point attachment can be made by a sulphonic group or with carboxylic acid by alpha ketosubstitution, alpha hydroxysubstitution, addition of an amidegroup or addition of a second carboxyl group spatially close to the first. 4. As suggested by the behaviour of the formate anion, in such a transport system any carboxylic acid could interact transiently with the receptor and therefore interfere with the transport of an organic anion even though such ionic interaction with the receptor were insufficient to produce transport of the acid itself.

Animals↗