nitroarginine and 2-amino-3-phosphonopropionic-acid

1. Nitric oxide (NO) synthase activity was studied in slices of human temporal cortex samples obtained in neurosurgery by measuring the conversion of L-[3H]-arginine to L-[3H]-citrulline. 2. Elevation of extracellular K+ to 20, 35 or 60 mM concentration-dependently augmented L-[3H]-citrulline production. The response to 35 mM KCl was abolished by N(G)-nitro-L-arginine (100 microM) demonstrating NO synthase specific conversion of L-arginine to L-citrulline. Increasing extracellular MgCl2 concentration up to 10 mM also prevented the K+ (35 mM)-induced NO synthase activation, suggesting the absolute requirement of external calcium ions for enzyme activity. 3. However, the effect of high K+ (35 mM) on citrulline synthesis was insensitive to the antagonists of ionotropic and metabotropic glutamate receptors dizocilpine (MK-801), 6-nitro-7-sulphamoylbenzo(f)-quinoxaline-2-3-dione (NBQX) or L-2-amino-3-phosphonopropionic acid (L-AP3) as well as to the nicotinic receptor antagonist, mecamylamine. 4. The 35 mM K+ response was insensitive to omega-conotoxin GVIA (1 microM) and nifedipine (100 microM), but could be prevented in part by omega-agatoxin IVA (0.1 and 1 microM). The inhibition caused by 0.1 microM omega-agatoxin IVA (approximately 30%) was enhanced by adding omega-conotoxin GVIA (1 microM) or nifedipine (100 microM). Further inhibition (up to above 70%) could be observed when the three Ca2+ channel blockers were added together. Similarly, synthetic FTX 3.3 arginine polyamine (sFTX) prevented (50% at 100 microM) the K+-evoked NO synthase activation. This effect of sFTX was further enhanced (up to 70%) by adding 1 microM omega-conotoxin GVIA plus 100 microM nifedipine. No further inhibition could be observed upon addition of MK-801 or/and NBQX. 5. It was concluded that elevation of extracellular [K+] causes NO synthase activation by external Ca2+ entering cells mainly through channels of the P/Q-type. Other Ca2+ channels (L- and N-type) appear to contribute when P/Q-channels are blocked.

Topics: Alanine; Arginine; Calcium Channel Blockers; Calcium Channels; Citrulline; Dizocilpine Maleate; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Humans; In Vitro Techniques; Mecamylamine; Nicotinic Antagonists; Nitric Oxide Synthase; Nitroarginine; Potassium; Potassium Chloride; Quinoxalines; Temporal Lobe