Activation of brain nitric oxide synthase in depolarized human temporal cortex slices: differential role of voltage-sensitive calcium channels

摘要

class="enumerated" style="list-style-type:decimal">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.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 μM) 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.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.The 35 mM K⁺ response was insensitive to ω-conotoxin GVIA (1 μM) and nifedipine (100 μ class="small-caps">M), but could be prevented in part by ω-agatoxin IVA (0.1 and 1 μ class="small-caps">M). The inhibition caused by 0.1 μ class="small-caps">M ω-agatoxin IVA (∼30%) was enhanced by adding ω-conotoxin GVIA (1 μ class="small-caps">M) or nifedipine (100 μ class="small-caps">M). 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 μ class="small-caps">M) the K⁺-evoked NO synthase activation. This effect of sFTX was further enhanced (up to 70%) by adding 1 μ class="small-caps">M ω-conotoxin GVIA plus 100 μ class="small-caps">M nifedipine. No further inhibition could be observed upon addition of MK-801 or/and NBQX.It was concluded that elevation of extracellular [K⁺] causes NO synthase activation by external Ca⁺ 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.