Biomedical subjects

J L Cousin

Publications and source records attributed to J L Cousin.

31 records · Page 2Linked to original sources

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↗

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

Transmembrane exchange of chloride with bicarbonate ion in mammalian red blood cells: evidence for a sulphonamide-sensitive "carrier".

1. It is well known that red blood cells suspended in isotonic NH4Cl solution swell because penetration of NH3 induces a transmembrane exchange between Cl-o and OH-i(or HCO3-i). The rate of swelling thus depends on the speed of the transmembrane exchanges and on the amount of anions available for exchange. 2. It has been demonstrated in experiments carried out in a CO2-free medium that OH-ions are poorly permeating whereas the permeability for HCO3-is very high. Thus the rate of swelling is largely dependent on the intracellular HCO3-concentration. In this context the well-known inhibitory effect of sulphonamides upon swelling can be interpreted, and always has been until now, as being due to the inhibitory action of the drug on the intracellular carbonic anhydrase. However, this inhibitory effect could also result from a direct action of the drug on the transmembrane exchange; it would explain why under conditions of total carbonic anhydrase inhibition we have shown that the inhibition of swelling is far from maximal. 3. A direct experimental evidence of such an effect of carbonic anhydrase inhibitors on the transmembrane exchange of Cl-with HCO3- was obtained with benzolamide (Cl 11,366), Cl 13,580 and ethoxzolamide. Surprisingly enough, however, acetazolamide (Diamox) does not affect the transmembrane exchange process. 4. The inhibitory effect of sulphonamides on HCO3-transport process is discussed in terms of an interaction of the drug with a transport system common to HCO3- and organic anions.

Acetazolamide↗

PubMedDate not supplied

Pertussis toxin-induced mitogenesis in human T lymphocytes.

Pertussis toxin (PT) has previously been shown to affect a wide variety of immune responses and to cause lymphocyte proliferation. We have investigated the biochemical basis for the mitogenic activity of PT by using human peripheral blood lymphocytes. PT was found to induce a rapid rise in cytosolic free calcium concentration and an alkalinization of the cytosol through the Na+/H+ antiporter. The toxin was also found to induce expression of IL-2-receptor on CD3+ cells and to stimulate IL-2 production. PT induced proliferation of both CD4+ and CD8+ T cells in the presence (but not in the absence) of accessory cells. PT also stimulated IL-1 production by monocytes but neither IL-1, IL-6 alone nor a combination of the two lymphokines could replace accessory cells suggesting that cell:cell contact is required. Low doses of PT induced ADP-ribosylation of G proteins but this treatment did not affect significantly PHA-induced [Ca2+]i increase and IL-2-induced DNA synthesis suggesting that the substrates of the ADP-ribosyltransferase activity of PT are not involved in the signalling pathways leading to DNA replication.

Adenosine Diphosphate Ribose↗

PubMedDate not supplied

Calcium-dependent regulation of phosphatidylserine synthesis in control and activated Jurkat T cells.

Activation of Jurkat T cells with phytohemagglutinin (PHA), CD3 or CD2 monoclonal antibodies (mAbs) results in a marked inhibition of phosphatidylserine (PS) synthesis. Activation of Jurkat T cells with PHA in a Ca(2+)-free medium resulted in an arrest of PS synthesis which was not reversed by the addition of Ca2+. The use of BAPTA to chelate Ca2+ ions released from intracellular stores prevented PHA-induced inhibition of PS synthesis. In addition, it was found that during activation, in the presence of BAPTA, a net Ca2+ influx paralleled an increase in PS synthesis, demonstrating that Ca2+ uptake caused an enhanced PS synthesis rather than an inhibition. The use of a CD2 mAb, D66, able to mobilize exclusively Ca2+ from intracellular stores, resulted in 51% inhibition of PS synthesis. N-Ethylmaleimide (NEM), which inhibits both the release of Ca2+ from internal stores and the influx of Ca2+, totally prevents the inhibition of PS synthesis induced by PHA, anti-CD3 or anti-CD2 mAbs. The presence, in the incubation medium, of either NDGA, TPCK or TPA, three drugs able to markedly inhibit Ca2+ influx without modifying the release of Ca2+ from internal stores, did not modify the inhibition of PS synthesis induced by PHA. Moreover all the drugs known to interact with calmodulin were also found to prevent the PHA-induced inhibition of this phospholipid. Taken together, these results show that the inhibition of PS synthesis induced by T cell activators is regulated by both calmodulin and Ca2+ ions recruited from intracellular compartments.

Antibodies, Monoclonal↗