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C Frelin

Publications and source records attributed to C Frelin.

At least 91 records · Page 5Linked to original sources
PubMed1990

Human kidney amiloride-binding protein: cDNA structure and functional expression.

Phenamil, an analog of amiloride, is a potent blocker of the epithelial Na+ channel. It has been used to purify the porcine kidney amiloride-binding protein. Synthetic oligonucleotides derived from partial sequences have been used to screen a human kidney cDNA library and to isolate the cDNA encoding the human amiloride-binding protein. The primary structure was deduced from the DNA sequence analysis. The protein is 713 residues long, with a 19-amino acid signal peptide. The mRNA was expressed in 293-S and NIH 3T3 cells, yielding a glycoprotein (i) that binds amiloride and amiloride analogs with affinities similar to the amiloride receptor associated with the apical Na+ channel in pig kidney membranes and (ii) that is immunoprecipitated with monoclonal antibodies raised against pig kidney amiloride-binding protein.

Amiloride↗
PubMed1990

Astrocytes are target cells for endothelins and sarafotoxin.

Endothelin-1, endothelin-3, and the snake venom toxin sarafotoxin S6b stimulate the hydrolysis of phosphatidylinositol by phospholipase C with similar potencies in primary cultures of astrocytes prepared from rat brain cortex. In indo 1-loaded cells, endothelin-1, endothelin-2, endothelin-3, and sarafotoxin induce the rapid mobilization of intracellular Ca2+ stores and promote a more slowly developing influx of Ca2+. These responses were insensitive to pertussis toxin and to inhibitors of cyclooxygenase and lipoxygenase. Similar actions of endothelins and sarafotoxin were observed using astrocytes from the cerebellum and glioma cells from the C6 and NN cell lines. The endothelin receptor of astrocytes differs from the receptor previously characterized in endothelial cells from brain microvessels in that it has a high affinity for endothelin-3. Thus, brain endothelin-1 and endothelin-3 have different target cells in the brain and may have different functions.

Animals↗
PubMed1990

Mechanism of the cardiotoxic action of palytoxin.

Palytoxin (PTX) is a non-12-O-tetradecanoylphorbol-13-acetate-type tumor promoter that has potent cardiotoxic properties. In embryonic chick ventricular cells, PTX increased [Ca2+]i (K0.5 = 5 nM) in a manner that was dependent on the presence of extracellular Ca2+. The action of PTX was not consequent to its depolarizing action, to the opening of voltage-dependent Ca2+ channels, to an intracellular Na+ load, or to intracellular acidification. Flow cytometric analysis of the [Ca2+]i distribution in PTX-treated cells showed that only the largest ventricular cells responded to the toxin. All ventricular cells responded to PTX by intracellar acidification. PTX also increased 22Na+ uptake by cardiac cells (K0.5 = 100 nM) via a pathway that was sensitive to 3,4-dichlorobenzamil (K0.5 = 8 microM), suggesting a possible involvement of the Na+/Ca2+ antiporter. We conclude that the action of PTX in chick cardiac cells is distinct from that in erythrocytes or in fibroblasts and that it likely involves several distinct mechanisms. A primary action of PTX could be to open a Ca2+ uptake pathway in the plasma membrane, which would then trigger 22Na+ uptake by the Na+/Ca2+ antiporter.

Acrylamides↗
PubMed1989

Phorbol esters and chemotactic factor induce distinct changes in cytoplasmic Ca2+ and pH in granulocytic like HL60 cells.

Differentiation of HL60 cells into neutrophil-like cells after exposure to dimethylsulfoxide is accompanied by an increase in intracellular pH (pHi) which results from an increased activity of the Na+/H+ antiporter at physiological pHi value, but not at acidic pHi values. The functional responses of differentiated HL60 cells to the chemotactic peptide, N-formylmethionylleucylphenylalanine (fMLP), and to phorbol myristate acetate (PMA) were studied. In differentiated cells fMLP produced a large increase in cytosolic Ca2+ levels and a small biphasic change in pHi, whereas PMA produced cellular acidification, which was potentiated by ethylisopropylamiloride and no change in [Ca2+]i. In undifferentiated HL60 cells, PMA produced the opposite effect on pHi, i.e., a cellular alkalinization. The extent of the acidification produced by PMA in differentiated HL60 cells correlated with the production of reactive oxygen species.

Amiloride↗
PubMed1989

The inotropic effect of endothelin-1 on rat atria involves hydrolysis of phosphatidylinositol.

Endothelin-1 induces a positive inotropic response in isolated left atria of the rat with an IC50 value of 20 nM. The contractile effect of endothelin is larger than that of other inotropic hormones such as phenylephrine and epinephrine and smaller than that of Bay K8644. In the spontaneously active right atria, endothelin induces a positive inotropic effect with no chronotropic effect. Endothelin does not modify intracellular levels of cAMP under basal conditions or after stimulation with isoproterenol but stimulates the formation of inositol phosphates. Mobilization of inositol phospholipids is observed in the same range of concentrations as for the contractile action of endothelin. The contractile action of endothelin is not mediated by protein kinase C. It is antagonized by blockers of L-type Ca2+ channels, low external Ca2+ concentrations and drugs such as caffeine and ryanodine that interfere with Ca2+ release by the sarcoplasmic reticulum.

Animals↗
PubMed1989

A new type of amiloride-sensitive cationic channel in endothelial cells of brain microvessels.

Endothelial cells from brain microvessels form the blood-brain barrier. Brain microvessels and endothelial cells isolated from rat brain microvessels express an amiloride-sensitive cationic channel that was characterized using [3H]phenamil binding and patch-clamp experiments. [3H]Phenamil, a labeled amiloride analog, recognizes a single family of binding sites with a dissociation constant of 20-30 nM and a maximum binding capacity of 8-15 pmol/mg protein. The pharmacological profile of the channel (phenamil greater than benzamil greater than amiloride) is very similar to that of the epithelium Na+ channel of mammalian kidney and of frog epithelia. Long-lasting currents were observed in patch-clamp experiments using excised outside-out patches. Application of amiloride or phenamil first produced a rapid flickering of channel activity and then its complete blockade. The mean unit channel conductance at 140 mM Na+ was 23 picosiemens. The selectivity of Na+ over K+ was estimated from reversal potentials to be 1.5:1. Properties of the channel in microvessels are clearly distinct from those of the Na+ channel of the kidney, suggesting the existence of several isoforms of cationic channels that are sensitive to amiloride and its derivatives. The low selectivity cationic channel of endothelial cells in brain microvessels might be important for controlling both Na+ and K+ movements across the blood-brain barrier.

Amiloride↗
PubMed1989

Biochemical identification of two types of phenamil binding sites associated with amiloride-sensitive Na+ channels.

The existence of distinct forms of the epithelium Na+ channel that differ in their sensitivity to amiloride has been repeatedly suggested by physiological data. The biochemical basis for these differences was analyzed by using phenamil, the most potent inhibitor known so far for the epithelium Na+ channel. [3H]Phenamil of high radioactive specific activity (30 Ci/mmol) was prepared and used to titrate [3H]phenamil binding sites in pig kidney membranes. Kinetic experiments, equilibrium binding studies, and competition experiments indicated the presence in crude membrane preparations of two classes of independent binding sites. A first binding site was characterized by a high affinity for phenamil (Kd1 = 0.4 nM) and for amiloride (Kd1 = 0.1 microM). A second binding site recognized phenamil and amiloride with lower affinities [Kd2(phenamil) = 28 nM, Kd2(amiloride) = 4 microM]. The ratio of the respective amounts of low- and high-affinity binding sites was 14 +/- 2 in different membrane preparations (range: 6-22). The two types of binding sites for [3H]phenamil copurified and were still observed after purification of the epithelium Na+ channel to homogeneity. These results indicate that at least two types of pharmacologically distinguishable Na+ channels exist in the kidney. They correspond either to two isoforms of the apical Na+ channel or to one single type of channel under two different states of covalent regulation.

Amiloride↗
PubMed1989

Guanabenz, guanochlor, guanoxan and idazoxan bind with high affinity to non-adrenergic sites in pig kidney membranes.

[3H]Idazoxan is a labelled ligand that is frequently used to study alpha 2-adrenoceptors in the central nervous system. In pig kidney membranes, [3H]idazoxan labelled high-affinity binding sites (Kd = 1.5 nM) that were not alpha 2-adrenoceptors and which recognized clonidine with low affinity. This new class of binding sites was recognized by amiloride derivatives; however, it is not likely that these sites are the well-known targets of amiloride in the kidney: the Na+/H+ exchanger and the epithelium Na+ channel. These binding sites may be the normal target of a series of imidazolidines derivatives (guanabenz, guanochlor, guanoxan), which are known for their antihypertensive properties.

Amiloride↗
PubMed1989

Molecular mechanism of endothelin-1 action on aortic cells.

The vasoconstricting action of endothelin-1 (ET-1) is partly mediated by voltage-dependent L-type Ca2+ channels. Activation of the Ca2+ channels is indirect. ET-1 action involves (i) the hydrolysis of phosphatidylinositol and the release of Ca2+ from internal stores and (ii) the opening of a nonselective cation channel in the plasma membrane. The resulting depolarization triggers the activity of L-type Ca2+ channels.

Animals↗
PubMed1988

Molecular mechanism of action of the vasoconstrictor peptide endothelin.

Endothelin, one of the most potent vasoconstrictor known, has been suggested to act as an endogenous agonist of L-type Ca2+ channels. In this paper we show that endothelin stimulates the metabolism of inositol phosphates and induces the mobilization of intracellular Ca2+ stores. The transient activation of Ca2+-sensitive K+ channel provokes an hyperpolarization of the membrane. It is followed by a sustained depolarization which is due to the opening of a non-specific cation channel which is permeable to Ca2+ and Mg2+. The depolarization then activates L-type Ca2+ channels. This mechanism of action explains why part of the endothelin-induced vasocontriction is eliminated by L-type Ca2+ channel blockers.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1988

Dual control of the intracellular pH in aortic smooth muscle cells by a cAMP-sensitive HCO3-/Cl- antiporter and a protein kinase C-sensitive Na+/H+ antiporter.

Two mechanisms are involved in the regulation of the intracellular pH (pHi) of aortic smooth muscle cells: the Na+/H+ antiporter and a Na+-independent HCO3-/Cl- antiporter. The Na+/H+ antiporter acts as a cell alkalinizing mechanism. It is activated by vasopressin and by phorbol esters when cells are incubated in the presence of bicarbonate but is not affected in the absence of bicarbonate. The HCO3-/Cl- antiporter acts as a cell acidifying mechanism. Agents such as forskolin, 8-Br-cAMP, and isoproterenol which raise intracellular cAMP levels inhibit the HCO3-/Cl- antiporter by shifting its pHi dependence in the alkaline direction. Thus, within the same cell type, different hormones control pHi variations by acting on different pHi regulating systems. An increase in pHi can be achieved either by a stimulation of a cell alkalinizing mechanism or by inhibition of a cell acidifying mechanism. A change of the activity of one pHi regulating mechanism modifies the responsiveness of the other to regulatory agents. Bicarbonate turns on the HCO3-/Cl- antiporter, decreases pHi and allows its regulation by protein kinase C through the Na+/H+ antiporter. Inhibition of the HCO3-/Cl- antiporter by cAMP increases the pHi and switches off the protein kinase C-mediated regulation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

The regulation of the cytoplasmic free Ca2+ concentration in aortic smooth muscle cells (A7r5 line) after stimulation by vasopressin and bombesin.

The characteristics of intracellular Ca2+ transient induced by vasopressin and bombesin in aortic smooth muscle cells were studied using flow cytometric analysis of indo-1 loaded cells. The two hormones induced a rapid and transient rise in [Ca2+]i. This Ca2+ transient was independent of the presence of extracellular Ca2+. Addition of bombesin to cells that have already been stimulated by vasopressin (or conversely the addition of vasopressin to bombesin-stimulated cells) results in a second Ca2+ transient that has a smaller amplitude. This transient is the same when the external Ca2+ concentration is lowered from 1.8 mM to 50 nM, suggesting that the agonist-sensitive pool reloaded using the Ca2+ that has been previously released into the cytoplasm. Intracellular Ca2+ pools that have been depleted by a prolonged incubation of the cells in a low Ca2+ medium can be refilled by shifting cells to a high Ca2+ medium. The reloading was analyzed in detail and found to be a slow process. It is hardly affected by Ni2+ or by (-)D888, a potent inhibitor of the voltage-dependent Ca2+ channels. It is accelerated when Ca2+ uptake by the Na+/Ca2+ exchange system is stimulated. The results suggest that Ca2+ homeostasis in aortic smooth muscle cells is achieved using mechanisms that are distinct from those operating in various acini and in striated muscles.

Animals↗
PubMed1988

Single-cell analysis of the intracellular pH and its regulation during the monocytic differentiation of U937 human leukemic cells.

Monocytic differentiation of U937 cells induced by retinoic acid is accompanied by a 0.2-pH-unit cell alkalinisation. The effect of retinoic acid on intracellular pH (pHi) develops slowly and it precedes the differentiation of the cells by 24 h. Heterogeneity in cellular pHi values was assessed using flow cytometry. It was higher at the differentiated stage than at the undifferentiated stage. It was reduced under conditions of clamped pHi values. Two membrane mechanisms allow U937 cells to recover from an intracellular acidosis. These are the Na+/H+ exchange system and a Na+-dependent HCO3-/Cl- exchange system. The increase in the pHi observed after monocytic differentiation resulted from a twofold increase in the maximum activity of the Na+/H+ exchange system with no change in the activity of the bicarbonate-dependent system. The properties of interaction of the Na+/H+ exchanger of U937 cells with Na+, Li+, amiloride and its derivatives were defined and appeared to be unique to human leukemic cells.

Amiloride↗
PubMed1988

An increase in intracellular pH is a general response of promonocytic cells to differentiating agents.

Intracellular pH (pHi) was measured in HL60 and U937 cells before and after differentiation into monocyte-macrophage like cells. 12-O-Tetradecanoyl phorbol-13-acetate (PMA), butyrate, interferon, retinoic acid and 1,25-dihydroxyvitamin D3 all increased pHi. The increases elicited were rapid with PMA, much slower with retinoic acid and interferon and still slower with 1,25-dihydroxyvitamin D3. Increases in pHi are due to an activation of the Na+/H+ exchange system. High pHi values are unlikely to serve as an early intracellular signal for initiating monocytic differentiation.

Butyrates↗
PubMed1988

The Na+/Ca2+ antiporter in aortic smooth muscle cells. Characterization and demonstration of an activation by phorbol esters.

The Na+/Ca2+ antiporter is present in aortic smooth muscle cells of the A7r5 cell line. Imposing an outward Na+ gradient to the cells promoted a 45Ca2+ uptake component which was sensitive to amiloride derivatives and insensitive to blockers of the voltage-dependent Ca2+ channel. The Ca2+ uptake system was dependent on intracellular Na+ concentration; it was inactive when Li+ replaced intracellular Na+ and it was electrogenic. Flow cytometric analysis of cells that had been loaded with the Ca2+ indicator indo-1 showed that all conditions that promoted Ca2+ influx led to corresponding increases in the free cytoplasmic Ca2+ concentration. Treatment of the A7r5 cells with phorbol myristate acetate, a known activator of protein kinase C (Ca2+/phospholipid-dependent enzyme), led to a two-fold activation of the system and to larger intracellular Ca2+ transients when cells were shifted to Na+-free solutions. Activation was observed at all intracellular Na+ concentrations. Changing the activity of the Na+/Ca2+ system did not affect the size and duration of intracellular Ca2+ transients elicited by the Ca2+ mobilizing hormone vasopressin. It is concluded that the Na+/Ca2+ antiporter in smooth muscle cells is a target for protein kinase C but that the system is not involved in the regulation of Ca2+ transients induced by vasopressin.

Animals↗
PubMed1988

The Na+/K+/Cl- cotransport in C6 glioma cells. Properties and role in volume regulation.

The role of the Na+/K+/Cl- cotransporter in the regulation of the volume of C6 astrocytoma cells was analyzed using isotopic fluxes and cell cytometry measurements of the cell volume. The system was inhibited by 'loop diuretics' with the following order of potency: benzmetanide greater than bumetanide greater than piretanide greater than furosemide. Under physiological conditions of osmolarity of the incubation media, equal rates of bumetanide-sensitive inward and outward K+ fluxes were observed. Blockade of the Na+/K+/Cl- cotransporter with bumetanide did not lead to a modification in the mean cell volume. When C6 cells were incubated in an hyperosmotic solution, a cell shrinkage was observed. It was accompanied by a twofold increase in the activity of the Na+/K+/Cl- cotransport, which then catalyzed the net influx of K+. In spite of this increased activity, no cell swelling could be measured. Incubation of the cells in an iso-osmotic medium deprived of either Na+, K+ or Cl- also produced cell shrinkage. Large activations (up to tenfold) of the Na+/K+/Cl- cotransport together with a cell swelling back to the normal volume were observed upon returning ion-deprived C6 cells to a physiological solution. This cell swelling was completely prevented in the presence of bumetanide. It is concluded that the Na+/K+/Cl- cotransport system is one of the transport systems involved in volume regulation of glial cells. The system can either be physiologically quiescent or active depending on the conditions used. A distinct volume regulating mechanism is the Na+/H+ exchange system.

Biological Transport↗