Leopoldo de Meis

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.

Uncoupled ATPase Activity and Heat Production by the Sarcoplasmic Reticulum Ca2+-ATPase REGULATION BY ADP

Sarcoplasmic reticulum vesicles of rabbit skeletal muscle accumulate Ca2+ at the expense of ATP hydrolysis. The heat released during the hydrolysis of each ATP molecule varies depending on whether or not a Ca2+ gradient is formed across the vesicle membrane. After Ca2+accumulation, a part of the Ca2+-ATPase activity is not coupled with Ca2+ transport (Yu, X., and Inesi, G. (1995)J. Biol. Chem. 270, 4361–4367). I now show that both the heat produced during substrate hydrolysis and the uncoupled ATPase activity vary depending on the ADP/ATP ratio in the medium. With a low ratio, the Ca2+ transport is exothermic, and the formation of the gradient increases the amount of heat produced during the hydrolysis of each ATP molecule cleaved. With a high ADP/ATP ratio, the Ca2+ transport is endothermic, and formation of a gradient increased the amount of heat absorbed from the medium. Heat is absorbed from the medium when the Ca2+ efflux is coupled with the synthesis of ATP (5.7 kcal/mol of ATP). When there is no ATP synthesis, the Ca2+ efflux is exothermic (14–16 kcal/Ca2+mol). It is concluded that in the presence of a low ADP concentration the uncoupled ATPase activity is the dominant route of heat production. With a high ADP/ATP ratio, the uncoupled ATPase activity is abolished, and the Ca2+ transport is endothermic. The possible correlation of these findings with thermogenesis and anoxia is discussed.

Conformational fluctuations of the Ca2+-ATPase in the native membrane environment. Effects of pH, temperature, catalytic substrates, and thapsigargin.

Digestion with proteinase K or trypsin yields complementary information on conformational transitions of the Ca2+-ATPase (SERCA) in the native membrane environment. Distinct digestion patterns are obtained with proteinase K, revealing interconversion of E1 and E2 or E1∼P and E2-P states. The pH dependence of digestion patterns shows that, in the presence of Mg2+, conversion of E2 to E1 pattern occurs (even when Ca2+ is absent) as H+ dissociates from acidic residues. Mutational analysis demonstrates that the Glu309 and Glu771 acidic residues (empty Ca2+-binding sites I and II) are required for stabilization of E2. Glu309 ionization is most important to yield E1. However, a further transition produced by Ca2+ binding to E1 (i.e. E1·2Ca2+) is still needed for catalytic activation. Following ATP utilization, H+/Ca2+ exchange is involved in the transition from the E1∼P·2Ca2+ to the E2-P pattern, whereby alkaline pH will limit this conformational transition. Complementary experiments on digestion with trypsin exhibit high temperature dependence, indicating that, in the E1 and E2 ground states, the ATPase conformation undergoes strong fluctuations related to internal protein dynamics. The fluctuations are tightly constrained by ATP binding and phosphoenzyme formation, and this constraint must be overcome by thermal activation and substrate-free energy to allow enzyme turnover. In fact, a substantial portion of ATP free energy is utilized for conformational work related to the E1∼P·2Ca2+ to E2-P transition, thereby disrupting high affinity binding and allowing luminal diffusion of Ca2+. The E2 state and luminal path closure follow removal of conformational constraint by phosphate.

Control of energy fluxes by the sarcoplasmic reticulum Ca2+‐ATPase: ATP hydrolysis, ATP synthesis and heat production

The experiments described indicate that heat is released when Ca2+ leaks through the Ca2+‐ATPase of sarcoplasmic reticulum vesicles. In the presence of a transmembrane Ca2+ concentration gradient, agents that modify the amount of ATP synthesized from ADP and Pi also modify the amount of heat produced by the hydrolysis of each ATP molecule. Thus, in the presence of heparin, less ATP is synthesized and more heat is produced. Conversely, with dimethyl sulfoxide more ATP is synthesized and less heat is produced. The data indicate that between limits (−10 to −30 kcal/mol) the Ca2+‐ATPase can regulate the interconversion of energy in such a way as to vary the fraction of energy derived from ATP hydrolysis which is converted into heat and that which is converted into other forms of energy.

Inhibition of glutamate uptake and proton pumping in synaptic vesicles by S-nitrosylation

Abstract: Nitric oxide (NO; including NO•, NO+, and NO−) was found to inhibit glutamate uptake by isolated synaptic vesicles of rat brain. This was observed when two unrelated NO donors, S‐nitrosogluthathione and S‐nitroso‐N‐acetylpenicillamine, were used. The primary target of NO is the H+‐ATPase found in the synaptic vesicles, which leads to dissipation of the electrochemical proton gradient and inhibition of glutamate uptake. Oxyhemoglobin (12 µM) and, to a much lesser extent, methemoglobin protected the vacuolar H+‐ATPase from inhibition. Inhibition of H+ pumping by NO was reversed by addition of 0.5 mM dithiothreitol. The results indicate that the vacuolar H+‐ATPase from synaptic vesicles is inhibited by NO by a mechanism that involves S‐nitrosylation of critical sulfhydryl groups in the enzyme. The interaction of NO with synaptic vesicles might be of importance for the understanding of the multiple effects of NO in neurotransmission.

Role of the Sarcoplasmic Reticulum Ca2+-ATPase on Heat Production and Thermogenesis

The sarcoplasmic reticulum of skeletal muscle retains a membrane bound Ca2+-ATPase which is able to interconvert different forms of energy. A part of the chemical energy released during ATP hydrolysis is converted into heat and in the bibliography it is assumed that the amount of heat produced during the hydrolysis of an ATP molecule is always the same, as if the energy released during ATP cleavage were divided in two non-interchangeable parts: one would be converted into heat, and the other used for Ca2+ transport. Data obtained in our laboratory during the past three years indicate that the amount of heat released during the hydrolysis of ATP may vary between 7 and 32 kcal/mol depending on whether or not a transmembrane Ca2+ gradient is formed across the sarcoplasmic reticulum membrane. Drugs such as heparin and dimethyl sulfoxide are able to modify the fraction of the chemical energy released during ATP hydrolysis which is used for Ca2+ transport and the fraction which is dissipated in the surrounding medium as heat.The sarcoplasmic reticulum of skeletal muscle retains a membrane bound Ca2+-ATPase which is able to interconvert different forms of energy. A part of the chemical energy released during ATP hydrolysis is converted into heat and in the bibliography it is assumed that the amount of heat produced during the hydrolysis of an ATP molecule is always the same, as if the energy released during ATP cleavage were divided in two non-interchangeable parts: one would be converted into heat, and the other used for Ca2+ transport. Data obtained in our laboratory during the past three years indicate that the amount of heat released during the hydrolysis of ATP may vary between 7 and 32 kcal/mol depending on whether or not a transmembrane Ca2+ gradient is formed across the sarcoplasmic reticulum membrane. Drugs such as heparin and dimethyl sulfoxide are able to modify the fraction of the chemical energy released during ATP hydrolysis which is used for Ca2+ transport and the fraction which is dissipated in the surrounding medium as heat.

ATP-dependent calcium accumulation in brain microsomes. Enhancement by phosphate and oxalate.

1. ATP-dependent calcium uptake by a rabbit brain vesicular fraction (microsomes) was studied in the presence of phosphate or oxalate. These anions, which are known to form insoluble calcium salts, increased the rate of calcium uptake and the capacity of the vesicles for calcium accumulation. 2. The degree of activation depended on the concentration of phosphate or oxalate. Under optimal conditions, phosphate promoted a 5-fold increase in the amount of calcium stored at steady state. This level was 200-250 nmol Ca-2+/mg protein. 3. Initial rate of calcium uptake followed Michaelis-Menten kinetics with an apparent Km for calcium of 6.7-10-minus 5 M and a V of 44 nmol/min per mg protein. Optimal pH was 7.0. With 2 mM ATP, optimal Mg-2+ concentration was 2 mM. 4. Dintrophenol and NaN3 inhibited calcium uptake in a mitochondria-enriched fraction but not in the microsomal fraction. 5. Calcium uptake activity was compared in the six subfractions prepared from the whole microsomal fraction by means of a sucrose density gradient fractionation. 6. The Mg-2+-dependent ATPase activity of brain microsomes was activated by calcium. Maximal activation was attained with 100 muM CaCl2. Greater calcium concentrations caused a progressive inhibition. 7. The data suggest that the ATP-dependent calcium uptake in brain microsomes, as in muscle microsomes, is brought about by an active transport process, calcium being accumulated as a free ion inside the vesicles.

Cold Tolerance in Hypothyroid Rabbits: Role of Skeletal Muscle Mitochondria and Sarcoplasmic Reticulum Ca2+ ATPase Isoform 1 Heat Production

Brown adipose tissue (BAT) is involved in rat and mice thermoregulation, and heat produced by BAT depends on the concerted action of thyroid hormones and catecholamines. Little is known about cold-induced thermogenesis in mammals that have little or no BAT, such as rabbits. In these animals, thermogenesis primarily occurs in skeletal muscle. In this work, we have studied the effect of cold acclimation (4 C for 10 d) in normal and hypothyroid rabbits. It is known that hypothyroid rats die after a few hours of cold exposure. We now show that, different from rats, hypothyroid rabbits sustain their body temperature and survive after 10 d cold exposure. When compared with rabbits kept at room temperature, the muscles of cold-exposed rabbits showed a dark red color characteristic of oxidative muscle fibers. According to this pattern, we observed that in both normal and hypothyroid rabbits, cold exposure promotes an increase in oxygen consumption by skeletal muscle mitochondria. Moreover, in red muscle, cold acclimation induces an increase in the expression and activity of sarcoplasmic reticulum Ca(2+) ATPase isoform 1 (SERCA1), one of the muscle enzymes involved in heat production. We conclude that rabbit cold tolerance is probably related to increased muscle oxidative metabolism and heat production by SERCA1 and that these changes are not completely dependent on normal thyroid function.

Hyperthyroidism increases the uncoupled ATPase activity and heat production by the sarcoplasmic reticulum Ca2+-ATPase

The sarcoplasmic reticulum Ca2+-ATPase is able to modulate the distribution of energy released during ATP hydrolysis, so that a portion of energy is used for Ca2+ transport (coupled ATPase activity) and a portion is converted into heat (uncoupled ATPase activity). In this report it is shown that T4 administration to rabbits promotes an increase in the rates of both the uncoupled ATPase activity and heat production in sarcoplasmic reticulum vesicles, and that the degree of activation varies depending on the muscle type used. In white muscles hyperthyroidism promotes a 0.8-fold increase of the uncoupled ATPase activity and in red muscle a 4-fold increase. The yield of vesicles from hyperthyroid muscles is 3-4-fold larger than that obtained from normal muscles; thus the rate of heat production by the Ca2+-ATPase expressed in terms of g of muscle in hyperthyroidism is increased by a factor of 3.6 in white muscles and 12.0 in red muscles. The data presented suggest that the Ca2+-ATPase uncoupled activity may represent one of the heat sources that contributes to the enhanced thermogenesis noted in hyperthyroidism.

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.