The activation of eNOS by copper ion (Cu2+) in human pulmonary arterial endothelial cells (HPAEC).
We have previously reported that Cu2+ endothelium-dependently dilated rat pulmonary arterial rings in a few minutes by increasing NO production via constitutive endothelial nitric oxide synthase (eNOS) activation in rat pulmonary arterial endothelial cells (Eur. J. Pharmacol., 1997). In the present study, using cultured human pulmonary arterial endothelial cells (HPAEC), we assessed the effects of divalent cations (Cu2+, Mn2+, Zn2+, and Fe2+) on NOS activity in crude cell extracts and intact cells. NO synthase activity was measured by monitoring the conversion of L-[14C] arginine to L-[14C] citrulline. The NOS enzyme in crude HPAEC extract showed similar characteristics to previously reported eNOS from other sources. All the divalent cations tested suppressed the NOS activity in crude cell extract by about 50% at 1 x 10(-4) M, but only Cu2+ from 10(-6) M increased eNOS activation dose dependently with a significant elevation in whole-cell assay. Extracellular Ca2+ was prerequisite to the eNOS activation by Cu2+ in intact cells. Furthermore, we measured NO production determined as NOx (NO, .NO2-, and .NO3-) from HPAEC using NO chemiluminescence analyzer. HPAEC monolayers were treated with either buffer alone, Cu2+ (10(-4) M) or thapsigargin (10(-6) M). The amount of .NOx increased from 10.93 (pmoml/ml/10(6) cells) to 41.27 (pmol/ml/10(6) cells) by thapsigargin (10(-6) M) and to 45.24 (pmol/ml/10(6) cells) by Cu2+ (10(-4) M). The increase in NOx by Cu2+ was inhibited by L-NMMA. These results indicated that Cu2+, but not Mn2+, Zn2+, and Fe2+, causes the activation of eNOS, while Cu2+, Mn2+, Zn2+, and Fe2+ directly suppressed eNOS activity extracted from HPAEC. Further, our study showed that extracellular Ca2+ was essential for eNOS activation by Cu2+.
Third Department of Internal Medicine, Faculty of Medicine, Fukui Medical University, Japan. makai@fmsrsa.fukui-med.ac.jp
Y
Y
Demura
S
S
Ameshima
T
T
Ishizaki
S
S
Okamura
I
I
Miyamori
S
S
Matsukawa
D001120:Arginine / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent; D002478:Cells, Cultured; D002956:Citrulline / Q000378:metabolism; D003300:Copper / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008163:Luminescent Measurements; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D011651:Pulmonary Artery / Q000201:enzymology; D019323:omega-N-Methylarginine / Q000494:pharmacology
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D019323:omega-N-Methylarginine; D002956:Citrulline; D009569:Nitric Oxide; D003300:Copper; D001120:Arginine; D004492:Edetic Acid; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D002118:Calcium