Direct Measurement of Nitric Oxide Release from Cultured Endothelial Cells Stimulated by Bradykinin or Ramiprilat

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Abstract
Stimulation of endothelial nitric oxide production by bradykinin or the angiotensin converting enzyme inhibitor “ramiprilat” has been indirectly assessed in terms of intracellular cyclic GMP accumulation. However, direct measurement of endothelial nitric oxide synthesis and release has not been analyzed. Using a selective porphyrinic microsensor, nitric oxide release was detected from single primary cultured bovine aortic endothelial cells. Maximal nitric oxide release of 540 ± 38 or 122 ± 10 n mol/L was achieved by bradykinin at 5 × 10−8 mol/L or by ramiprilat at 10−7 mol/L respectively. The time course of nitric oxide release by bradykinin showed a rapid initial production rate (22.5 ± 1.6 nmol/L/sec, 4.2% increase per second) and was transient within 15 min (decay rate 0.60 ± 0.05 nmol/L/sec). In contrast, the initial production rate of nitric oxide release by ramiprilat was slow (0.34 ± 0.03 nmol/L/sec, 0.28% increase per second) and reached a plateau level after 6 min, which remained stable for at least 14 min. Maximal nitric oxide release induced by bradykinin was immediately blocked by the kinin receptor antagonist “icatibant” (Hoe 140). The subsequent decay rate of nitric oxide was 8.0 ± 1.4 nmol/L/sec. In comparison, the inhibitory effect of icatibant on ramiprilat-induced nitric oxide release revealed a decay rate of nitric oxide which was 1/6 as fast. These data, especially the different kinetics of both compounds, which are quite similar to previously reported data for intracellular cyclic GMP accumulation in the same cells provide strong evidence that endothelial cyclic GMP can be considered as an index for nitric oxide generation.