Biomedical subjects
Publications and source records attributed to M Claret.
Ca(2+) movements triggered by noradrenaline were determined in isolated cells and mitochondria from rat livers. It has been shown that these depend on experimental conditions. In cells incubated in 1.8mm-Ca(2+), results suggest that noradrenaline mobilizes Ca(2+) from reticulum before releasing Ca(2+) from mitochondria.
cholesterol depletion alters the apparent affinity of the internal cationic sites and the maximal translocation rate but not the affinity of the external cationic sites of the Na+-K+ pump in human erythrocytes. To test whether these effects were mediated by a direct cholesterol-internal site interaction or by a change in membrane lipid order, the effects of five fluidizing amphiphiles (chlorpromazine, imipramine, benzyl alcohol, sodium oleate and sodium benzenesulphonate) on the kinetic parameters of the Na+-K+ pump were determined. The cholesterol removal and all the agents used induced dose-response decreases in membrane lipid order as measured by fluorescence polarization or ESR. Positive and neutral amphiphiles mimicked the effects of cholesterol removal on the affinity of the internal sites of the pump and to a lesser extent on the maximal translocation rate. Anionic amphiphiles had no effect on internal sites, probably because they distributed preferentially within the outer leaflet on the membrane. These results indicate that cholesterol controls the affinity of the internal sites of the Na+-K+ pump by altering the membrane lipid order. In contrast, neither cholesterol depletion nor the agents used altered the affinity of the external sites of the Na+-K+ pump. This difference in sensitivity to membrane lipids order suggests that internal and external cationic sites, although borne by the same protein, are in different lipid environments.
Cholesterol depletion of erythrocytes, obtained after incubation with phosphatidylcholine vesicles, induces in most of the experiments: (1) a discocytestomatocyte transformation as observed by scanning electron microscopy; (2) a specific decrease in spectrin phosphorylation of intact erythrocytes; (3) an increase in lipid phosphorylation. It is concluded that the effect of cholesterol on erythrocyte shape is probably mediated through its action on the activity o of membrane-bound enzymes, proteases or kinases.
1. K-sensitive electrodes placed in the extracellular fluid have been used to show that ATP and noradrenaline cause a rapid loss of up to 10% of the K content of isolated guinea-pig hepatocytes. 2. The hypothesis tha this response is a consequence of a rise in the K permeability of the hepatocyte membrane triggered by an increase in cytosolic Ca is supported by the finding that the divalent cation ionophore A23187 also initiated K loss, in this instance of up to 20-25% of the amount in the cells. 3. Under similar conditions A23187 caused a transient increase, followed by a larger decrease, in the 45Ca content of guinea-pig hepatocytes equilibrated with this isotope. The decrease alone was seen with ATP and noradrenaline. 4. Quinine (1 mM) and the bee venom neurotoxin apamin (10 nM) greatly reduced the effect of ATP, noradrenaline and A23187 on K content without affecting the changes in 45Ca movement. 5. Apamin (10 nM) also abolished the increase in 42K efflux which follows the application of the alpha-adrenoceptor agonist amidephrine to rabbit liver slices; the concurrent rises in 45Ca efflux and glucose release were unaffected. 6. It was concluded that quinine and apamin are able to block either the Ca-dependent K channels present in guinea-pig and rabbit liver cell membranes or the mechanism that controls them. 7. Surprisingly, rat hepatocytes took up rather than lost K when treated with the concentrations of ATP, noradrenaline or A23187 that initiated K loss from guinea-pig cells. This response was greatly reduced by ouabain. 8. Application of large concentrations of A23187 to rat hepatocytes caused K loss associated with cell death. 9. The influence of apamin (10-1000 nM) and quinine (200-1000 micro M) on the Ca-dependent K permeability of red blood cells and ghosts was also studied. Apamin was without effect even when applied to both sides of the ghost membrane, whereas quinine caused inhibition, as reported by others. 10. The results suggest that Ca-dependent K channels or carriers are present in the membranes of liver cells of the guinea-pig and rabbit, but are either lacking or inactive in rat liver. The finding that apamin blocks this mechanism in hepatocytes but not in erythrocytes may mean that the channels differ in these cells.
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1. Water, K, Na and Cl contents and fluxes of K and Na were determined in isolated rat hepatocytes incubated at 1 degrees C (90 min) then at 38 degrees C (60 min). At 1 degrees C cells progressively gained Na and Cl, lost K and increased their volume by 17%. 2. Rewarming triggered a net loss of K and gain of Na. They were transitory (about 60 sec) being overcome rapidly by movements in the opposite direction until cells recovered their initial K and Na gradients. 3. Determination of time courses of the K rate constant (kappa' K) and net Na influx (phi Na) in cells incubated in ouabain K-free media indicated that these paradoxical movements were due to a temporary shunting of the Na pump by sudden increases in K and Na permeabilities. 4. Increases in kappa' K and phi' Na were not sensitive to inhibitors of Ca-activated K channels such as quinine (10(-3) M) of apamin (10(-8) M), suggesting they were not dependent on internal ionized Ca. 5. In control media containing 1.8 mM-Ca divalent ionophore A23187, though stimulating the Ca pump (Ca efflux), presumably by increasing internal ionized Ca concentration, did not cause substantial and rapid changes in K permeability. This supports the hypothesis that Ca-sensitive K channels are lacking in rat hepatocytes. 6. A 10% increase in cell volume provoked by a hypo-osmotic shock triggered increases in both kappa' K and phi' Na with time courses very similar to those brought about by rewarming. 7. It is proposed that transient changes in K and Na permeabilities are the consequence of the cell swelling, induced by cooling. These volume-dependent permeabilities are blocked at 1 degrees C and revealed by rewarming.
Apamin is a neurotoxic polypeptide of known structure isolated from bee venom. Shuba and coworkers have recently shown that it abolishes the hyperpolarising action of externally-applied ATP on visceral smooth muscle (guinea pig stomach and taenia coli) as well as the hyperpolarisation (inhibitory junction potential) that follows stimulation of the non-adrenergic inhibitory nerve supply to these tissues. As it has been proposed that ATP is the neurotransmitter involved in the latter response, Vladimirova and Shuba tentatively concluded that apamin is a specific postsynaptic blocking agent of this non-adrenergic, possibly 'purinergic', inhibition. We have confirmed the important observation that nanomolar concentrations of apamin reduce inhibition by ATP and by non-adrenergic nerve stimulation, but further experiments suggest that, rather than acting as a specific blocker of ATP receptors, apamin inhibits the increase in potassium permeability caused by a number of agents, including ATP.
Isolated hepatocytes incubated at 1 degrees C loss K, gain Na and water. Rewarming results in a transient increase in K and Na permeabilities. These are insensitive to quinine, suggesting that they are not caused by an intracellular Ca accumulation.
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1. Human red blood cells (R.B.C.) were incubated with phosphatidylcholine vesicles to obtain partial depletion of their membrane cholesterol. The kinetic parameters of the Na pump (affinities and maximal fluxes) were determined in these cells and compared with controls. 2. In the presence of internal K, progressive cholesterol depletion induces gradually (i) an increase of the maximal flux (ii) a reduction of the apparent affinity for internal Na. 3. In the absence of internal K, cholesterol depletion has no apparent effect, indicating that the observed changes were mediated by the internal K. Cholesterol depletion acts by increasing these two K effects: (i) activation of the maximal fluxes, and (ii) reduction of the internal Na affinity. 4. The cholesterol effect (on the K-pump interaction) is specific; it occurs without any change (i) in the affinities for internal Na and inorganic phosphate and for external Na and K, and (ii) in the number of pumping sites. 5. Under physiological conditions, cholesterol reduces the cation translocation rate and increases the selectivity of the pump for internal Na.
Isolated hepatocytes behave like osmometers immediately after an incubation in hypo-osmotic media. During long-term incubation periods they recover their normal volume either in the absence or in the presence of ouabain. The volume regulation is parlty due to a decrease in intracellular potassium and amino acid contents.
1. Fluxes and distribution of Ca were studied in the perfused rat liver. Kinetic analysis of (45)Ca exchange revealed three compartments with time constants of 4, 14 and 223 min, pool sizes of 250, 385 and 670 mumole.kg(-1) wet wt. respectively, and one non-exchangeable compartment of 400 mumole.kg(-1).2. (45)Ca uptake by in situ mitochondria followed, as a function of cell loading time, a mono-exponential function with a time constant of 16 min. This suggests that the second compartment may be identified as the intracellular pool of Ca. The calculated cell transmembrane flux of Ca was 28 mumole.kg(-1) min(-1) or 0.17 p-mole.cm(-2).sec(-1).3. The maximum (45)Ca uptake by in situ mitochondria was 2.3 n-mole.mg(-1) of protein which represents, on the basis of 50 mg of mitochondrial protein per g of fresh liver, 115 mumole.kg(-1) or 30% of the cytoplasmic pool. A pool of 10.8 n-mole.mg(-1) protein (or 540 mumole.kg(-1)) of non-exchangeable Ca (at steady state) was probably in the form of Ca phosphate precipitated in the mitochondrial matrix.4. Extracellular Ca pools were studied using competitor of Ca binding (La) or Ca chelators (EGTA). La displaced specifically a homogeneous pool of Ca (tau = 5.1 min) which represented a fraction (55 mumole.kg(-1)) of the rapidly exchangeable Ca (first compartment) without perturbing other external pools. On the other hand, EGTA displaced completely that compartment, and about 85% of the Ca of the third compartment. These results suggest that the first and the major fraction of the third compartments are extracellular. They account for 63% of total exchangeable Ca.5. A model of distribution and exchange of Ca in hepatocytes is proposed.
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The loading and loss kinetics of cellular 45Ca were studied in rat liver cells. The data are described by a three compartment system. Six arrangements of the exchange pathways of these pools are presented. Taking into account the Ca stocking properties of cell organels, two models may be emphasized.