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C Frelin

Publications and source records attributed to C Frelin.

At least 109 records · Page 6Linked to original sources
PubMed1988

Amiloride and its analogs as tools to inhibit Na+ transport via the Na+ channel, the Na+/H+ antiport and the Na+/Ca2+ exchanger.

Amiloride analogs inhibit a number of transmembrane Na+ transport systems: 1) the epithelium Na+ channel, 2) the Na+/H+ exchange system and 3) the Na+/Ca2+ exchange system. Structure--activity relationships using amiloride derivatives with selected modification of each of the functional groups of the molecule indicate that the 3 Na+ transporting systems have distinct pharmacological profiles. 5-N Disubstituted derivatives of amiloride, such as ethylisopropylamiloride are the most potent inhibitors of the Na+/H+ exchange system. Conversely, amiloride derivatives that are substituted on the guanidino moiety, such as phenamil, are potent inhibitors of the epithelium Na+ channel. It is thus possible, by using selected amiloride derivatives to inhibit selectively one or another of the Na+ transport systems.

Amiloride↗
PubMed1987

Properties of the Na+-dependent Cl-/HCO3- exchange system in U937 human leukemic cells.

U937 cell possess two mechanisms that allow them to recover from an intracellular acidification. The first mechanism is the amiloride-sensitive Na+/H+ exchange system. The second system involves bicarbonate ions. Its properties have been defined from intracellular pH (pHi) recovery experiments, 22Na+ uptake experiments, 36Cl- influx and efflux experiments. Bicarbonate induced pHi recovery of the cells after a cellular acidification to pHi = 6.3 provided that Na+ ions were present in the assay medium. Li+ or K+ could not substitute for Na+. The system seemed to be electroneutral. 22Na+ uptake experiments showed the presence of a bicarbonate-stimulated uptake pathway for Na+ which was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate. The bicarbonate-dependent 22Na+ uptake component was reduced by depleting cells of their internal Cl- and increased by removal of external Cl-. 36Cl- efflux experiments showed that the presence of both external Na+ and bicarbonate stimulated the efflux of 36Cl- at a cell pHi of 6.3. Finally a 36Cl- uptake pathway was documented. It was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate (K0.5 = 10 microM) and bicarbonate (K0.5 = 2 mM). These results are consistent with the presence in U937 cells of a coupled exchange of Na+ and bicarbonate against chloride. It operates to raise the intracellular pH. Its pHi and external Na+ dependences were defined. No evidence for a Na+-independent Cl-/HCO3- exchange system could be found. The Na+-dependent Cl-/HCO3- exchange system was relatively insensitive to (aryloxy)alkanoic acids which are potent inhibitors of bicarbonate-induced swelling of astroglia and of the Li(Na)CO3-/Cl- exchange system of human erythrocytes. It is concluded that different anionic exchangers exist in different cell types that can be distinguished both by their biochemical properties and by their pharmacological properties.

Bicarbonates↗
PubMed1987

Differentiation of human promyelocytic HL 60 cells by retinoic acid is accompanied by an increase in the intracellular pH. The role of the Na+/H+ exchange system.

Retinoic acid, which induces the differentiation of HL 60 cells to granulocytes, produces a cell alkalinization from pHi = 7.03 to pHi = 7.37. The half-maximum effect of retinoic acid is observed at 10 nM. The effect of retinoic acid on the pHi develops slowly, and it precedes the differentiation of the cells. A cell alkalinization is also observed after differentiation of the cells by dimethyl sulfoxide. It is not observed using etretinate, a synthetic retinoid that does not promote the differentiation of HL 60 cells. Two pHi regulating mechanisms coexist in HL 60 cells. The Na+/H+ exchange system is the major mechanism that allows HL 60 cells to recover from an intracellular acidosis. A second mechanism is a Na-HCO3 cotransport system. During differentiation of the cells by retinoic acid, a 2-fold increase in the activity of the Na+/H+ exchange system is observed, while the activity of the NaHCO3 cotransport remains constant. The properties of interaction of the Na+/H+ exchanger with internal H+, external Na+, and Li+ as well as with amiloride and its derivatives are defined. The Na+/H+ exchanger of HL 60 cells is characterized by unusually low affinities for alkali cations and a high affinity for amiloride and its derivatives. The pHi dependence of the exchanger is not modified after differentiation by retinoic acid. It is concluded that the mechanism of activation of the Na+/H+ exchanger by retinoic acid is distinct from the short-term effect produced by mitogens and phorbol esters which change the pHi dependence of the system.

Carrier Proteins↗
PubMed1987

Purification and subunit structure of the [3H]phenamil receptor associated with the renal apical Na+ channel.

Sodium crosses the apical membrane of tight epithelia through a sodium channel, which is inhibited by the diuretic amiloride and by analogs such as phenamil. Target size analysis indicated that the functional size of the [3H]phenamil binding sites associated with the epithelial Na+ channel from pig kidney is 92 +/- 10 kDa. The [3H]phenamil receptor was solubilized by using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized material displayed the same properties of interaction with amiloride and its derivatives as the membrane-bound receptor. A two-step purification of the epithelial Na+ channel was achieved by using QAE Sephadex chromatography and affinity chromatography on a Bandeiraea simplicifolia lectin column. It results in an 1100-fold purification of the Na+ channel as compared to pig kidney microsomes with a yield of 15% +/- 5%. The maximal specific activity was 3.7 nmol/mg of protein. NaDodSO4/poly-acrylamide gel electrophoresis of the purified Na+ channel under nonreducing conditions showed the presence of a single major polypeptide chain of apparent molecular mass 185 kDa. Under disulfide-reducing conditions, the purified epithelial Na+ channel migrated as a single band of apparent molecular mass 105 kDa. It is suggested that the epithelial Na+ channel from pig kidney has a total molecular mass of 185 kDa and consists of two nearly identical 90- to 105-kDa polypeptide chains crosslinked by disulfide bridges.

Amiloride↗
PubMed1987

Na+ channels as sites of action of the cardioactive agent DPI 201-106 with agonist and antagonist enantiomers.

This paper shows the interaction of the cardiotonic agent 4-[3-(4-diphenylmethyl-1-piperazinyl)-2-hydroxypropoxy]-1H-indole- 2-carbonitrile (DPI 201-106) and its optic enantiomers R-DPI (205-429) and S-DPI (205-430) with the Na+ channel of a variety of excitable cells. Voltage-clamp experiments show that DPI 201-106 acts on neuroblastoma cells and rat cardiac cells. S-DPI (205-430) increases the peak Na+ current, slows down the kinetics of Na+ channel inactivation, and is cardiotonic on heart cells. Conversely, R-DPI (205-429) reduces the peak Na+ current and blocks Na+ channel activity and cardiac contractions. Binding experiments using radioactively labeled toxins indicate that DPI 201-106 and its enantiomers do not interact with sites already identified for tetrodotoxin or sea anemone and scorpion toxins. DPI 201-106 and its enantiomers inhibit binding of a 3H-labeled batrachotoxin derivative, [3H]batrachotoxinin A 20-alpha-benzoate, to brain membranes. The dissociation constant of the complex formed between the Na+ channel and both R-DPI and S-DPI is Kd congruent to 100 nM. 22Na+ uptake experiments using different cell types have shown that R and S enantiomers of DPI 201-106 are active on the different Na+ channel subtypes with similar IC50 values. These results are discussed in relation with the cardiotonic properties of DPI 201-106 that are not accompanied by cardiotoxic effects.

Animals↗
PubMed1986

The Na+/H+ exchange system in glial cell lines. Properties and activation by an hyperosmotic shock.

The properties of the Na+/H+ exchange system in the glial cell lines C6 and NN were studied from 22Na+ uptake experiments and measurements of the internal pH (pHi) using intracellularly trapped biscarboxyethyl-carboxyfluorescein. In both cell types, the Na+/H+ exchanger is the major mechanism by which cells recover their pHi after an intracellular acidification. The exchanger is inhibited by amiloride and its derivatives. The pharmacological profile (ethylisopropylamiloride greater than amiloride greater than benzamil) is identical for the two cell lines. Both Na+ and Li+ can be exchanged for H+. Increasing the external pH increases the activity of the exchanger in the two cell lines. In NN cells the external pH dependence of the exchanger is independent of the pHi. In contrast, in C6 cells, changing the pHi value from 7.0 to 6.5 produces a pH shift of 0.6 pH units in the external pH dependence of the exchanger in the acidic range. Decreasing pHi activates the Na+/H+ exchanger in both cell lines. Increasing the osmolarity of the external medium with mannitol produces an activation of the exchanger in C6 cells, which leads to a cell alkalinization. Mannitol action on 22Na+ uptake and the pHi were not observed in the presence of amiloride derivatives. Mannitol produces a modification of the properties of interaction of the antiport with both internal and external H+. It shifts the pHi dependence of the system to the alkaline range and the external pH (pHo) dependence to the acidic range. It also suppresses the interdependence of pHi and pHo controls of the exchanger's activity. NN cells that possess an Na+/H+ exchange system with different properties do not respond to mannitol by an increased activity of the Na+/H+ exchanger. The action of mannitol on C6 cells is unlikely to be mediated by an activation of protein kinase C.

Animals↗
PubMed1986

Tetrodotoxin-sensitive and tetrodotoxin-resistant Na+ channels differ in their sensitivity to Cd2+ and Zn2+.

The sensitivity of Na+ channels to inhibition by Cd2+ and Zn2+ was studied in 22Na+ uptake experiments after stabilization of an open conformation of the Na+ channels with different neurotoxins and in voltage clamp experiments. Six different cell types of neuronal, cardiac or skeletal muscle origin were surveyed. Three cell types possess Na+ channels that are highly sensitive to tetrodotoxin (TTX) (Kd = 1-5 nM) and three possess Na+ channels that are resistant to TTX (Kd = 0.3-1 microM). The 22Na+ uptake experiments using veratridine or batrachotoxin to activate Na+ channels indicated that TTX-resistant Na+ channels are more sensitive to the inhibitory action of Cd2+ (IC50(Cd2+) = 0.2 mM) and of Zn2+ (IC50(Zn2+) = 50 microM) than TTX-sensitive Na+ channels (IC50(Cd2+) = 5 mM, IC50(Zn2+) = 2 mM). Electrophysiological experiments showed that high concentrations of Cd2+ (IC50 = 2 mM) are necessary to inhibit both TTX-sensitive and TTX-insensitive Na+ channels when the channels are activated by voltage steps. The results suggest that Cd2+ acts competitively with veratridine or batrachotoxin and that the difference in the effects of Cd2+ and Zn2+ on 22Na+ fluxes in TTX-sensitive and TTX-resistant cells is related to differences at the site of action of alkaloid neurotoxins.

Animals↗
PubMed1986

[3H]phenamil, a radiolabelled diuretic for the analysis of the amiloride-sensitive Na+ channels in kidney membranes.

The interaction of amiloride and amiloride derivatives with the Na+ channels of pig kidney membranes was studied from 22Na+ uptake experiments. The order of potency of the different molecules tested is: phenamil greater than benzamil greater than amiloride, ethylisopropylamiloride. [3H]labelled phenamil was prepared and used to titrate Na+ channels in pig kidney membranes. Kinetics experiments, equilibrium binding studies and competition experiments between [3H]phenamil and unlabelled phenamil indicate that phenamil recognizes a single family of binding sites with a Kd value of 20 nM and a maximum binding capacity of 11.5 pmol/mg of protein. The order of potency of different amiloride analogs tested in [3H]phenamil competition experiments is identical to that found for the inhibition of 22Na+ uptake by apical Na+ channels.

Amiloride↗
PubMed1986

The activity of the Na+/H+ antiporter in cultured cardiac cells is dependent on the culture conditions used.

When chick cardiac cells are maintained in serum-free NCTC-135 medium, the pHi dependence of the Na+/H+ exchanger is much more acidic (pK = 7.0) than when a serum-free M199 medium is used (pK greater than 7.5). Phorbol esters activate the Na+/H+ exchanger and produce a cell alkalinization in cells maintained in NCTC-135 medium by shifting the pHi dependence of the system to more alkaline values. They have no action on cells maintained in M199 medium. The alkaline pHi dependence of the Na+/H+ exchanger and the loss of responsiveness of phorbol ester action in M199 medium correlate with increased rates of phosphatidylinositol turnover.

Amiloride↗
PubMed1986

Interaction of guanidinium and guanidinium derivatives with the Na+/H+ exchange system.

Guanidinium, a small organic monovalent cation that is permeant through voltage-dependent cationic channels cannot be transported by the cardiac Na+/H+ exchange system. Yet it recognizes the exchanger and is able to block its activity (K0.5 = 30 mM). Guanidinium derivatives that do not belong to the amiloride series and which possess potent antihypertensive properties also block the activity of the Na+/H+ exchange system in various cell types with a greater potency than unsubstituted guanidinium. The most potent compound found, guanochlor, has an affinity for the exchanger ranging between 0.5 microM and 6 microM in different systems and is more potent than amiloride in all systems studied. Guanochlor has the same action as amiloride derivatives on the cardiac cells; it prevents intracellular pH recovery in cardiac cells that have been acidified and also antagonizes the effect of ouabain on 45Ca2+ uptake by chick cardiac cells. Guanochlor does not compete with [3H]ethylpropylamiloride for its binding to the Na+/H+ exchange system of rabbit kidney brush border membrane. It is suggested that guanochlor recognizes a binding site on the Na+/H+ exchanger that is distinct from the amiloride binding site.

Amiloride↗
PubMed1986

Biochemical characterization of the Na+/K+/Cl- co-transport in chick cardiac cells.

Cultured chick cardiac cells possess a Na+K+Cl-co-transport system that is inhibited by the "loop diuretics" benzmetanide (IC50 = 0.3 microM), bumetanide (IC50 = 0.6 microM), piretanide (IC50 = 1.5 microM) and furosemide (IC50 = 5 microM). The K0.5 values for Cl- and Na+ activation of the bumetanide-sensitive 86Rb+ uptake are 59 mM and 40mM respectively. Bumetanide also inhibits a 22Na+ uptake component that is suppressed when external Cl- or K+ are substituted by impermeant ions. The ratio of bumetanide-sensitive 86Rb+ to 22Na+ uptake is close to 1. The cardiac Na+/K+/Cl- cotransport is a major uptake pathway for Na+ and K+. It accounts for 50% of the initial rate of 86Rb+ uptake and 17% of the initial rate of 22Na+ uptake by chick cardiac cells. It is activated two-fold by an hyperosmotic shock produced with 200 mM mannitol.

Animals↗
PubMed1986

The Na+/H+ antiport of eukaryotic cells: relationship between the kinetic properties of the system and its physiological function.

The Na+/H+ antiport is present in the plasma membrane of virtually all vertebrate cells and it plays a central role in cell homeostasis. The pharmacological properties and the characteristics of the interaction of extracellular Na+, Li+, H+ and of intracellular H+ with the Na+/H+ antiport are reviewed herein. The kinetic properties of the system are shown to be essential for defining its four main physiological functions: transepithelial ion transport, control of the pHi, control of the intracellular Na+ concentration, and control of the cell volume. The activity of the Na+/H+ antiport can be modulated by a large number of effectors which are thought to act via protein kinases. At least three mechanisms of activation of the Na+/H+ exchanger are defined from the analysis of the kinetic properties of the system. Activation of the Na+/H+ antiport leads to very different consequences, depending upon the activity of other ion transporting systems in the membrane.

Animals↗
PubMed1985

[3H]Ethylpropylamiloride, a ligand to analyze the properties of the Na+/H+ exchange system in the membranes of normal and hypertrophied kidneys.

[3H]Ethylpropylamiloride is a useful radioactive label to identify the Na+/H+ exchange system (Vigne, P., Frelin, C., Audinot, M., Borsotto, M., Cragoe, E. J., and Lazdunski, M. (1984) EMBO J. 3, 2647-2651). This paper extends the analysis of the properties of interaction of [3H]ethylpropylamiloride with the exchanger and describes its use with hypertrophied kidneys. [3H]Ethylpropylamiloride-binding sites copurify with the luminal membrane marker alkaline phosphatase but not with the basolateral membrane marker (Na+,K+)ATPase, thus indicating an asymmetric distribution of the Na+/H+ exchanger. Specific [3H]ethylpropylamiloride binding is dependent on pH. The pH dependency indicates that an ionizable function with a pKapp of 7.0 is essential in the association of the amiloride derivative. H+ acts competitively on [3H]ethylpropylamiloride binding; Na+, Li+, or cholinium ions have no effect on the association. Compensatory adaptation of the kidney to chronic reduction of renal mass is accompanied by a 1.7-fold increase in the activity of the Na+/H+ exchange system. Properties of interaction of internal and external pH with the Na+/H+ exchanger of normal and hypertrophied kidneys are identical. Titration of [3H]ethylpropylamiloride-binding sites in normal and hypertrophied kidneys suggests that the increased activity of the Na+/H+ exchange system is not accompanied by an increased concentration of exchangers.

Amiloride↗
PubMed1985

Biochemical properties of the Na+/H+ exchange system in rat brain synaptosomes. Interdependence of internal and external pH control of the exchange activity.

Properties of the Na+/H+ exchange system in synaptosomes have been studied primarily by using acridine orange fluorescence to follow H+ efflux. Results obtained from 22Na+ uptake experiments and [3H]ethylpropylamiloride binding experiments are also presented for comparison. The basal properties of the Na+/H+ antiport in synaptosomes are similar to those found in other systems; (i) the stoichiometry of Na+/H+ exchange is 1:1; (ii) Li+ can be successfully substituted for Na+; its affinity for the exchanger (KLi+ = 3 mM) is higher than that of Na+ (KNa+ = 12 mM), but the maximal rate of H+ efflux in the presence of Li+ is about 3 times lower than the maximal rate of H+ efflux in the presence of Na+; and (iii) the Na+/H+ antiport is inhibited by amiloride derivatives with the rank order:ethylisopropylamiloride greater than ethylpropylamiloride greater than amiloride greater than benzamil. The most important finding of this paper is that the external pH dependence of the synaptosomal Na+/H+ antiport is controlled by the value of internal pH and vice versa. For example apparent pHo values for half-maximum activation of the Na+/H+ exchanger are pHo = 7.12 when pHi = 6.4 and pHo = 7.95 when pHi = 7.3. Therefore, a 0.9 pH unit increase in internal pH produces a shift of at least a 0.83 pH unit in the external pH dependence. In addition, changing pHo from 7.75 to 8.50 also shifts the half-maximum pHi value for activation of the Na+/H+ antiport from 6.67 to 7.54.

Acridine Orange↗
PubMed1985

The Na+/H+ antiport is activated by serum and phorbol esters in proliferating myoblasts but not in differentiated myotubes. Properties of the activation process.

The properties of the Na+/H+ exchange system have been studied with 22Na+ uptake techniques at two stages of muscle development: proliferating myoblasts and differentiated myotubes. The characteristics of the interactions of the exchanger with external H+, with external Na+, and with amiloride or its more potent analogs are the same at both stages of development. Differences between the two stages of development concern: (i) the internal pH (pHi) dependence of the Na+/H+ exchanger, and (ii) the activation of the Na+/H+ exchanger by serum and phorbol ester which is observed in myoblasts but not in myotubes. Properties of the Na+/H+ exchanger in myoblasts after serum activation seem to be identical to those observed in myotubes with or without serum as if myotube formation stabilized a fully activated state of the exchanger. The activation of the myoblast Na+/H+ exchange system by serum is due to a shift of the pHi dependence towards alkaline pHi values and to an increase in the maximal activity of the Na+/H+ exchange system at acidic pH. Phorbol esters which are well-known activators of protein kinase C can only partially mimic the effects of serum on the Na+/H+ exchanger: they produce a shift of the pH dependence, but they do not increase the maximal activity at acidic pH.

Amiloride↗