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R Panizzutti

Publications and source records attributed to R Panizzutti.

3 recordsLinked to original sources

A new strategy to decrease N-methyl-D-aspartate (NMDA) receptor coactivation: inhibition of D-serine synthesis by converting serine racemase into an eliminase.

Serine racemase is a brain-enriched enzyme that synthesizes d-serine, an endogenous modulator of the glycine site of N-methyl-d-aspartate (NMDA) receptors. We now report that serine racemase catalyzes an elimination reaction toward a nonphysiological substrate that provides a powerful tool to study its neurobiological role and will be useful to develop selective enzyme inhibitors. Serine racemase catalyzes robust elimination of l-serine O-sulfate that is 500 times faster than the physiological racemization reaction, generating sulfate, ammonia, and pyruvate. This reaction provides the most simple and sensitive assay to detect the enzyme activity so far. We establish stable cell lines expressing serine racemase and show that serine racemase can also be converted into a powerful eliminase in cultured cells, while the racemization of l-serine is inhibited. Likewise, l-serine O-sulfate inhibits the synthesis of d-serine in primary astrocyte cultures. We conclude that the synthetic compound l-serine O-sulfate is a better substrate than l-serine as well as an inhibitor of d-serine synthesis. Inhibition of serine racemase provides a new strategy to selectively decrease NMDA receptor coactivation and may be useful in conditions in which overstimulation of NMDA receptors plays a pathological role.

Ammonia↗

Sarco/endoplasmic reticulum Ca2+-ATPase isoforms: diverse responses to acidosis.

The effects of acidic pH on the kinetics of Ca2+-ATPase isoforms from intracellular membranes of skeletal muscle, cardiac muscle, cerebellum and blood platelets were studied. At neutral pH, all four Ca2+-ATPase isoforms exhibited similar Ca2+-concentration requirements for half-maximal rates of Ca2+ uptake and ATP hydrolysis. A decrease in the pH from 7.0 to 6.0 promoted a decrease in both the apparent affinity for Ca2+ [increasing half-maximal activation (K0.5)] and the maximal velocity (Vmax) of Ca2+ uptake. With skeletal muscle vesicles these effect were 5 to 10 times smaller than those observed with all the other isoforms. Acidification of the medium from pH 7.0 to 6.5 caused the release of Ca2+ from loaded vesicles and a decrease in the amount of Ca2+ retained by the vesicles at the steady state. With the vesicles derived from skeletal muscle these effects were smaller than for vesicles derived from other tissues. The rate of passive Ca2+ efflux from skeletal and cardiac muscle vesicles, loaded with Ca2+ and diluted in a medium containing none of the ligands of Ca2+-ATPase, was the same at pH 7.0 and 6.0. In contrast, the rate of Ca2+ efflux from cerebellar and platelet vesicles increased 2-fold after acidification of the medium. The effects of DMSO, Mg2+ with Pi and arsenate on the rate of Ca2+ efflux varied among the different preparations tested. The differences became more pronounced when the pH of the medium was decreased from 7.0 to 6.0. It is proposed that the kinetic differences among the Ca2+-ATPase isoforms may reflect different adaptations to cellular acidosis, such as that which occurs during ischaemia.

Acidosis↗

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 Ca 2+ transport measured using 2 mM ATP as substrate was unaffected by GSNO up to 200 microM. GSNO (5-20 microM) inhibited the activity of both soluble and membrane-bound creatine kinase. Oxyhemoglobin (15-40 microM) protected creatine kinase against inactivation by GSNO. The inhibition by 10 microM 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.

Adenosine Triphosphate↗