s-nitrosocysteine and mastoparan

Previously, we proposed that S-nitroso-cysteine (SNC) was incorporated via the L-type-like amino acid transporters in rat brain slices. In PC12 cells (rat neuronal cell line), SNC inhibited [(3)H]arachidonic acid (AA) release induced by mastoparan (wasp venom peptide). We investigated the involvement of amino acid transporters on SNC-induced inhibition of [(3)H]AA release in PC12 cells. SNC inhibited mastoparan-stimulated [(3)H]AA release in a concentration-dependent manner in normal Na(+)- and low Na(+)-containing buffer. The inhibitory effect of 0.6 mM SNC in low Na(+) buffer decreased by 10 mM L-leucine, L-phenylalanine, L-methionine and L-cysteine. In contrast, L-alanine, L-threonine, L-valine or L-isoleucine showed very limited effects. Addition of L-leucine and L-phenylalanine, but not L-alanine or L-valine, also decreased the inhibitory effect of SNC on ionomycin/Na(3)VO(4)-stimulated [(3)H]AA release in normal Na(+) buffer. These findings suggest that SNC is incorporated via the amino acid transporters and inhibits AA release in PC12 cells.

Topics: Amino Acid Transport Systems; Amino Acids; Animals; Arachidonic Acid; Cysteine; Intercellular Signaling Peptides and Proteins; Ionomycin; Ionophores; Neurons; Nitric Oxide Donors; PC12 Cells; Peptides; Rats; S-Nitrosothiols; Tritium; Vanadates; Wasp Venoms

Arachidonic acid and nitric oxide (NO) act as retrograde and intercellular messengers in the nervous system. Regulation of cyclooxygenase is well established, but regulation of phospholipase A(2), the enzyme responsible for the liberation of arachidonic acid, by NO has not been thoroughly investigated. Using the PC12 cell line as a neuronal model, we studied the effects of exogenous NO compounds on arachidonic acid release. Incubation with Ca(2+) ionophores or mastoparan (wasp venom peptide) stimulated [3H]arachidonic acid release from prelabeled PC12 cells. [3H]Arachidonic acid release was inhibited by cytosolic phospholipase A(2) inhibitors, but not by dithiothreitol. A cytosolic phospholipase A(2) protein band with a molecular mass of approximately 100 kDa was detected by immunoblotting. S-Nitroso-cysteine inhibited basal and stimulated [3H]arachidonic acid release in concentration-dependent manners. Other NO compounds such as sodium nitroprusside and S-nitroso-N-acetylpenicillamine did not affect [3H]arachidonic acid release. N-Ethylmaleimide also inhibited [3H]arachidonic acid release. The inhibitory effects of S-nitroso-cysteine and N-ethylmaleimide were irreversible, because [3H]arachidonic acid release from PC12 cells preincubated with S-nitroso-cysteine or N-ethylmaleimide was much lower than that from nontreated cells. These findings suggest (a) cytosolic phospholipase A(2) is activated by Ca(2+) or mastoparan, and inhibited by S-nitroso-cysteine in a cyclic GMP-independent manner, (b) N-ethylmaleimide also inhibits cytosolic phospholipase A(2) and arachidonic acid release in PC12 cells. S-Nitroso-cysteine can regulate the production of other retrograde messenger arachidonic acid.

Topics: Animals; Arachidonic Acid; Cyclic GMP; Cysteine; Cytosol; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Ethylmaleimide; Intercellular Signaling Peptides and Proteins; Nitroso Compounds; PC12 Cells; Peptides; Phospholipases A; Rats; S-Nitrosothiols; Tritium; Wasp Venoms