Edward F. Labelle

Phosphoinositides in mitogenesis: Neomycin inhibits thrombin-stimulated phosphoinositide turnover and initiation of cell proliferation

Thrombin stimulates 32Pi incorporation into phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bis-phosphate (PIP2), and phosphatidylinositol (PI), and initiates DNA synthesis in hamster (NIL) fibroblasts at a half-maximal concentration of 125 ng/ml. Neomycin, which binds PIP2 and PIP, inhibits both thrombin-stimulated initiation of cell proliferation and 32P pI incorporation into at concentrations above 2 mM without affecting thrombin binding, thymidine uptake, or cellular protein synthesis. At lower concentrations, neomycin inhibits thrombin-stimulated release of inositol 1,4,5-trisphosphate (IP3), by selectively binding PIP2, but does not inhibit 32P incorporation into PI or initiation of DNA synthesis. Phosphoinositide recycling and diacylglycerol release therefore appear necessary for initiation of cell proliferation by thrombin. IP3-stimulated Ca++ mobilization may not be required for thrombin mitogenesis, however, since neomycin can block IP3 release without inhibiting initiation.

A novel dinitrophenylglutathione-stimulated ATPase is present in human erythrocyte membranes.

Vesicles prepared from human erythrocyte membranes were found to catalyze ATP hydrolysis that was stimulated by dinitrophenylglutathione (Dnp‐SG). This activity was dependent on temperature and Me2+ and independent of ion pump ATPases present in erythrocyte membranes. The activity was a linear function of protein and time up to 60 min. The K m values of ATPase for Dnp‐SG and ATP were found to be 49 μM and 1.67 mM, respectively. This suggests that in erythrocytes, the transport of Dnp‐SG requires direct enzymatic hydrolysis of ATP and both Dnp‐SG‐stimulated ATPase activity and the ATP‐dependent efflux of Dnp‐SG from erythrocytes represent different activities of the same protein.

Evidence for different transport systems for oxidized glutathione and S-dinitrophenyl glutathione in human erythrocytes

The effect of oxidized glutathione (GSSG) on the ATP-dependent transport of S-dinitrophenyl glutathione (Dnp-SG) by inside-out vesicles prepared from human erythrocytes and by intact erythrocytes has been studied. It is demonstrated that the transport of Dnp-SG is not inhibited by GSSG in either intact erythrocytes or in inside-out vesicles. These results suggest that Dnp-SG and GSSG are transported out of human erythrocytes by separate systems.

ATP dependent primary active transport of xenobiotic-glutathione conjugates by human erythrocyte membrane

We have demonstrated the presence of a dinitrophenyl glutathione (Dnp-SG) stimulated ATPase in human erythrocyte membranes. This ATPase mediates the active transport of glutathione — xenobiotic conjugate such as Dnp-SG from erythrocytes into the plasma. It is suggested that this transport system is distinct from the system which actively transports oxidized glutathione from the erythrocytes.

Inhibition by amiloride of 22Na+ transport into toad bladder microsomes

The diuretic, amiloride, inhibited 22Na+ accumulation by microsomes from toad (Bufo marinus) bladder by between 40 and 70%. Accumulated 22Na+ was separated from free isotope by ion-exchange chromatography. The amiloride-sensitive process was directly proportional to the protein concentration and a hyperbolic function of the Na+ concentration. The inhibition was competitive. At least 9 times more activity was found in the microsomal fraction than the mitochondrial fraction, and none at all was foumd in the cytosol. Amiloride inhibition could not be reproduced by similar pyrazine and guanidine compounds, such as sulfaguanidine. Amiloride-sensitive Na+ accumulation was totally reversed by nigericin. The intravesicular volume of the lbadder microsomes as determined by the [3H]H2O/[14C]inulin method was shown to be nearly equal to the volume occupied by the amiloride-sensitive portion of the 22Na+ accumulation. The amiloride-sensitive Na+ accumulation most likely represents transport into vesicles. Since amiloride does not affect gramicidin-mediated 22Na+ uptake into phospholipid vesicles, the amiloride inhibition seen with bladder microsomes probably represents a specific channel blockade and not simply interference with cation movement that might be produced by any cationic hydrophobic molecule.

Reconstituted amiloride-inhibited sodium transporter from rabbit kidney medulla is responsible for Na+-H+ exchange

Microsomes formed from rabbit kidney medulla and reconstituted proteoliposomes formed from these microsomes were capable of amiloride-inhibited Na+ transport that was insensitive to valinomycin either with or without K+. This indicated that the Na+ transport process was electroneutral. This Na+ transport process was insensitive to extravesicular Cl- or HCO-3 and not stimulated by high intravesicular gradients of K+, Ca2+ or Mg2+, which indicated that the process did not require NaCl or NaHCO3 co-transport or Na+/K+, Na+/Ca2+ or Na+/Mg2+ counter-transport. Na+ uptake into microsomes or proteoliposomes was inhibited by extravesicular K+, Ca2+, Mg2+ or La3+, which indicated that these ions interacted with the Na+-binding site on the transport protein. Na+ uptake into microsomes was stimulated by intravesicular protons and inhibited by extravesicular protons. This suggested that microsomes were capable of Na+-H+ exchange and this was confirmed when Na+ was shown to stimulate H+ efflux from microsomes. The amiloride-inhibited Na+ transporter from medulla microsomes which has been reconstituted into proteoliposomes is most likely a Na+-H+ exchanger. This Na+ transporter was totally insensitive to the uncoupler 1799, either in the presence or absence of valinomycin plus K+ and less sensitive to NH3 than to amiloride. This indicated that amiloride inhibited Na+ transport not merely by acting as a weak-base uncoupler but by directly interacting with the protein responsible for Na+-H+ exchange.

The interaction of amiloride analogues with the Na+/H+ exchanger in kidney medulla microsomes

The effects of ten amiloride analogues on Na+-H+ exchange in rabbit kidney medulla microsomes have been examined. Most of the analogues appeared to inhibit Na+ uptake into the microsomes more effectively than did amiloride either in the presence or absence of a pH gradient. However, the analogues were also capable of stimulating Na+ efflux from the microsomes at concentrations somewhat higher than the concentrations at which they inhibited Na+ influx. The concentrations at which the analogues stimulated Na+ efflux were about 2-4-times higher than the concentrations at which they blocked influx. This suggested that the two processes were related. The analogues that stimulated efflux most effectively (the 5-N-benzyl-amino analogue of amiloride and the 5-N-butyl-N-methylamino analogue) were shown to induce completely reversible effects. These analogues did not stimulate L-[3H]glucose efflux from medulla microsomes which ruled out nonspecific vesicle destruction or reversible detergent effects. These analogues also induced Na+ efflux from microsomes in the presence of high concentrations of added buffer, which ruled out weak-base uncoupling effects. The possibility exists that these analogues are carried into the microsomes via the Na+-H+ exchange protein and that this permits them to both block Na+ influx into the microsomes and stimulate Na+ efflux as well.

Amiloride-inhibited Na+ uptake into toad bladder microsomes is Na+-H+ exchange

Amiloride-inhibited Na+ transport into toad urinary bladder microsomes is sensitive to a pH gradient across the vesicular membrane. The magnitude of the gradient was measured directly with acridine orange. Also Na+ could stimulate amiloride-sensitive proton efflux from the microsomes. These results indicated that the transport process was Na+-H+ exchange.

Inhibition by amiloride of sodium transport into rabbit kidney medulla microsomes

Sodium transport into rabbit kidney medullar microsomes was 50% inhibited by amiloride. This Na+ uptake was shown to represent transport when the uptake process was reserved by the ionophore nigericin. The transport was complete within 60 min and proportional to the microsomal protein concentration. The effect of amiloride on transport was specific since the similar compound sulfaguanidine failed to affect microsomal Na+ transport. Amiloride-sensitive Na+ transport into microsomes was inhibited 70% by decreasing the pH (from 7.0 to 5.9), but was unaffected by the presence of a pH gradient. The kinetics of Na+ transport could be explained by a simple model, assuming that amiloride lowered the rate of Na+ entrance into the vesicles but had not effect on the rate of efflux. The failure of amiloride to effect efflux from the vesicles was also demonstrated directly.

Sulfhydryl reagents affect Na+ uptake into toad bladder membrane vesicles

SummaryThe effect of sulfhydryl reagents on the Na+ permeability mechanisms of toad urinary bladder vesicles was examined. The reagents 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), iodosobenzoate, and ethylenimine were able to decrease amiloride-inhibited sodium uptake into vesicles when used at low concentrations. When used at higher concentrations these reagents were able to induce large increases in vesicle Na+ permeability that were not sensitive to amiloride. The reagentp-chloro-mercuribenzene sulfonate was able to induce such leaks even at low concentrations. The reagent N-ethylmaleimide was incapable of substantially affecting vesicle Na+ transport in any way. All of the effects observed could be reversed by removing the reagents from the solution surrounding the vesicles. Our results help explain the varied actions of sulfhydryl reagents on intact epithelial tissue.