The activation of nitric oxide synthase by copper ion is mediated by intracellular Ca²⁺ mobilization in human pulmonary arterial endothelial cells

Abstract

1. The aim of the study was to elucidate the vasodilatory mechanism due to Cu²⁺ by assessing nitric oxide (NO) production as determined by NOx (NO, NO₂⁻, and NO₃⁻) that is released from human pulmonary arterial endothelial cell (HPAEC) monolayers using a NO chemiluminescence analyzer, and also to assess Ca²⁺ movement using ⁴⁵Ca and fura 2 in HPAEC.

2. Cu²⁺ (10⁻⁶–10⁻⁴ M) significantly increased NO production in a dose-dependent manner when extracellular Ca²⁺ was present.

3. ⁴⁵Ca influx into the adherent cells was dose-dependently enhanced by Cu²⁺ (10⁻⁶–10⁻⁴ M), but not by Mn²⁺, Zn²⁺ or Fe²⁺.

4. [Ca²⁺]_i, measured by monitoring the fluorescence changes of fura 2, was significantly elevated in the presence of Cu²⁺.

5. The increase in [Ca²⁺]_i induced by Cu²⁺ was inhibited by either diethyldithiocarbamate (DDC) or the depletion of extracellular Ca²⁺.

6. The dihydropyridine receptor agonist, BayK8644, significantly attenuated the Cu²⁺-induced increase in [Ca²⁺]_i in a dose dependent manner and nitrendipine or nifedipine, the dihydropyridine receptor antagonists, dose-dependently inhibited a Cu²⁺-induced increase in [Ca²⁺]_i.

7. These results suggest that Cu²⁺ activates eNOS through the mechanism of [Ca²⁺]_i elevation due to Ca²⁺ influx into HPAEC and that the Cu²⁺-induced [Ca²⁺]_i elevation in HPAEC is likely due to activation of the dihydropyridine-like receptors.

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