Herman Wolosker

INHIBITION OF CREATINE KINASE BY S-NITROSOGLUTATHIONE

The sarcoplasmic reticulum‐bound creatine kinase from rabbit skeletal muscle was inhibited by the nitric oxide donor S‐nitrosoglutathione (GSNO). This led to a decrease in Ca2+ uptake in sarcoplasmic reticulum vesicles when the transport was driven by ATP generated from phosphocreatine and ADP. In contrast, the Ca2+ transport measured using 2 mM ATP as substrate was unaffected by GSNO up to 200 μM. GSNO (5–20 μM) inhibited the activity of both soluble and membrane‐bound creatine kinase. Oxyhemoglobin (15–40 μM) protected creatine kinase against inactivation by GSNO. The inhibition by 10 μM GSNO was reversed by the addition of dithiothreitol (2 mM). The results indicate that nitric oxide (NO, including NO+, NO and NO−) inactivates creatine kinase in vitro by promoting nitrosylation of critical sulphydryl groups of the enzyme.

Regulation of Glutamate Transport into Synaptic Vesicles by Chloride and Proton Gradient

Glutamate uptake into synaptic vesicles is driven by an electrochemical proton gradient formed across the membrane by a vacuolar H-ATPase. Chloride has a biphasic effect on glutamate transport, which it activates at low concentrations (2-8 mM) and inhibits at high concentrations (>20 mM). Stimulation with 4 mM chloride was due to an increase in the V of transport, whereas inhibition by high chloride concentrations was related to an increase in K to glutamate. Both stimulation and inhibition by Cl were observed in the presence of A23187 or (NH)SO, two substances that dissipate the proton gradient (ΔpH). With the use of these agents, we show that the transmembrane potential regulates the apparent affinity for glutamate, whereas the ΔpH antagonizes the effect of high chloride concentrations and is important for retaining glutamate inside the vesicles. Selective dissipation of ΔpH in the presence of chloride led to a significant glutamate efflux from the vesicles and promoted a decrease in the velocity of glutamate uptake. The H-ATPase activity was stimulated when the ΔpH component was dissipated. Glutamate efflux induced by chloride was saturable, and half-maximal effect was attained in the presence of 30 mM Cl. The results indicate that: (i) both transmembrane potential and ΔpH modulate the glutamate uptake at different levels and (ii) chloride affects glutamate transport by two different mechanisms. One is related to a change of the proportions between the transmembrane potential and the ΔpH components of the electrochemical proton gradient, and the other involves a direct interaction of the anion with the glutamate transporter.

Effects of Hg2+ and CH3Hg+ on Ca2+ fluxes in rat brain microsomes.

A permanent increase in cytosolic Ca2+ levels seems to be associated with various pathological situations which may result in cell death. Hg2+ and CH3Hg+ are potent neurotoxic agents, but the precise molecular mechanism(s) underlying their effects are not sufficiently understood. In the present study we investigated the potential role of Ca(2+)-ATPase located in the endoplasmic reticulum as a molecular target for mercury. Hg2+ and CH3Hg+ inhibited Ca(2+)-ATPase and Ca2+ uptake by brain microsomes with similar potencies. However, the inhibitory potency of Hg2+ was higher than that of CH3Hg+, probably reflecting differences in the affinity for the sulfhydryl groups of these compounds. Passive or unidirectional Ca2+ efflux (measured in the absences of Ca(2+)-ATPase ligands) was increased significantly by CH3Hg+ and Hg2+. Again, the potency of Hg2+ was higher than that of CH3Hg+. Blockers of Ca2+ channels (ruthenium red, procaine, heparin) did not affect the increase in passive Ca2+ efflux induced by mercury compounds, possibly indicating that Ca2+ release occurs through Ca(2+)-ATPase. Addition of physiological concentrations of glutathione (GSH) simultaneously with mercury abolished the inhibitory effects of both forms of Hg on ca(2+)-transport. However, if the enzyme was first inhibited with Hg2+ or CH3Hg+ and subsequently treated with GSH, the reversal of inhibition was about 50%, suggesting that part of the cysteinyl residues involved in the inhibitory actions of mercury in Ca(2+)-transport bind to mercury with an extremely high affinity.

The Ca2+-ATPase Isoforms of Platelets Are Located in Distinct Functional Ca2+ Pools and Are Uncoupled by a Mechanism Different from That of Skeletal Muscle Ca2+-ATPase

Vesicles derived from the dense tubular system of platelets possess a Ca2+-ATPase that can use either ATP or acetyl phosphate as a substrate. In the presence of phosphate as a precipitating anion, the maximum amount of Ca2+ accumulated by the vesicles with the use of acetyl phosphate was only one-third of that accumulated with the use of ATP. Vesicles derived from the sarcoplasmic reticulum of skeletal muscle accumulated equal amounts of Ca2+ regardless of the substrate used. When acetyl phosphate was used in platelet vesicles, the transport of Ca2+ was inhibited by Na+, Li+, and K+; in sarcoplasmic reticulum vesicles, only Na+ caused inhibition. When ATP was used as substrate, the different monovalent cation had no effect on either sarcoplasmic reticulum or platelet vesicles. The catalytic cycle of the Ca2+-ATPase is reversed when a Ca2+ gradient is formed across the vesicle membrane. The stoichiometry between active Ca2+ efflux and ATP synthesis was one in platelet vesicles and two in sarcoplasmic reticulum vesicles. The coupling between ATP synthesis and Ca2+ efflux in sarcoplasmic reticulum vesicles was abolished by arsenate regardless of whether the vesicles were loaded with Ca2+ using acetyl phosphate or ATP. In platelets, uncoupling was observed only when the vesicles were loaded using acetyl phosphate. In both sarcoplasmic reticulum and platelet vesicles, the effect of arsenate was antagonized by thapsigargin (2 μM), micromolar Ca2+ concentrations, Pi (5-20 mM), and MgATP (10-100 μM). Trifluoperazine also uncoupled the platelet Ca2+ pump but, different from arsenate, this drug was effective in vesicles that were loaded using either ATP or acetyl phosphate. Trifluoperazine enhanced Ca2+ efflux from both sarcoplasmic reticulum and platelet vesicles; thapsigargin, Ca2+, Mg2+, or K+ antagonized this effect in sarcoplasmic reticulum but not in platelet vesicles. The data indicate that the Ca2+-transport isoforms found in sarcoplasmic reticulum and in platelets have different kinetic properties.

Alteration of Ca2+ Fluxes in Brain Microsomes by K+ and Na+: Modulation by Sulfated Polysaccharides and Trifluoperazine

Abstract: Rat brain microsomes accumulate Ca2+ at the expense of ATP hydrolysis. The rate of transport is not modulated by the monovalent cations K+, Na+, or Li+. Both the Ca2+ uptake and the Ca2+‐dependent ATPase activity of microsomes are inhibited by the sulfated polysaccharides heparin, fucosylated chondroitin sulfate, and dextran sulfate. Half‐maximal inhibition is observed with sulfated polysaccharide concentrations ranging from 0.5 to 8.0 µg/ml. The inhibition is antagonized by KCl and NaCl but not by LiCl. As a result, Ca2+ transport by the native vesicles, which in the absence of polysaccharides is not modulated by monovalent cations, becomes highly sensitive to these ions. Trifluoperazine has a dual effect on the Ca2+ pump of brain microsomes. At low concentrations (20–80 µM) it stimulates the rate of Ca2+ influx, and at concentrations >100 µM it inhibits both the Ca2+ uptake and the ATPase activity. The activation observed at low trifluoperazine concentrations is specific for the brain Ca2+‐ATPase; for the Ca2+‐ATPases found in blood platelets and in the sarcoplasmic reticulum of skeletal muscle, trifluoperazine causes only a concentration‐dependent inhibition of Ca2+ uptake. Passive Ca2+ efflux from brain microsomes preloaded with Ca2+ is increased by trifluoperazine (50–150 µM), and this effect is potentiated by heparin (10 µg/ml), even in the presence of KCl. It is proposed that the Ca2+‐ATPase isoform from brain microsomes is modulated differently by polysaccharides and trifluoperazine when compared with skeletal muscle and platelet isoforms.

Chloride-dependent inhibition of vesicular glutamate uptake by α-keto acids accumulated in maple syrup urine disease

Maple syrup urine disease is a metabolic disorder caused by mutations of the branched chain keto acid dehydrogenase complex, leading to accumulation of alpha-keto acids and their amino acid precursors in the brain. We now report that alpha-ketoisovaleric, alpha-keto-beta-methyl-n-valeric and alpha-ketoisocaproic acids accumulated in the disease inhibit glutamate uptake into rat brain synaptic vesicles. The alpha-keto acids did not affect the electrochemical proton gradient across the membrane, suggesting that they interact directly with the vesicular glutamate carrier. Chloride anions have a biphasic effect on glutamate uptake. Low concentrations activate the uptake (0.2 to 8 mM), while higher concentrations are inhibitory. The alpha-keto acids inhibited glutamate uptake by a new mechanism, involving a change in the chloride dependence for the activation of glutamate uptake. The activation of glutamate uptake by low chloride concentrations was shifted toward higher concentrations of the anion in the presence of alpha-keto acids. Inhibition by alpha-keto acids was abolished at high chloride concentrations (20 to 80 mM), indicating that alpha-keto acids specifically change the stimulatory effect of low chloride concentrations. High glutamate concentrations also reduced the inhibition by alpha-keto acids, an effect observed both in the absence and in the presence of low chloride concentrations. The results suggest that in addition to their possible pathophysiological role in maple syrup urine disease, alpha-keto acids are valuable tools to study the mechanism of vesicular transport of glutamate.

LIGAND-GATED CHANNEL OF THE SARCOPLASMIC RETICULUM CA2+ TRANSPORT ATPASE

In resting muscle, cytoplasmic Ca2+ concentration is maintained at a low level by active Ca2+ transport mediated by the Ca2+ ATPase from sarcoplasmic reticulum. The region of the protein that contains the catalytic site faces the cytoplasmic side of the membrane, while the transmembrane helices form a channel-like structure that allows Ca2+ translocation across the membrane. When the coupling between the catalytic and transport domains is lost, the ATPase mediates Ca2+ efflux as a Ca2+ channel. The Ca2+ efflux through the ATPase channel is activated by different hydrophobic drugs and is arrested by ligands and substrates of the ATPase at physiological pH. At acid pH, the inhibitory effect of cations is no longer observed. It is concluded that the Ca2+ efflux through the ATPase may be sufficiently fast to support physiological Ca2+ oscillations in skeletal muscle, that occur mainly in conditions of intracellular acidosis.

Modulation of the low affinity Ca2+-binding sites of skeletal muscle and blood platelets Ca2+-ATPase by nordihydroguaiaretic acid.

The antioxidant nordihydroguaiaretic acid (NDGA) inhibited the different sarco/endoplasmic reticulum Ca2+-ATPase isoforms found in skeletal muscle and blood platelets. For the sarcoplasmic reticulum, but not for the blood platelets Ca2+-ATPase, the concentration of NDGA needed for half-maximal inhibition was found to vary depending on the substrate used and its concentration in the assay medium. The phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase by ATP and by Pi were both inhibited by NDGA. In leaky vesicles, measurements of the ATP ↔ Pi exchange showed that NDGA increases the affinity for Ca2+ of the E2 conformation of the enzyme, which has low affinity for Ca2+. The effects of NDGA on the Ca2+-ATPase were not reverted by the reducing agent dithiothreitol nor by the lipid-soluble antioxidant butylated hydroxytoluene.