Human endothelial cells bioactivate organic nitrates to nitric oxide: implications for the reinforcement of endothelial defence mechanisms

Abstract

Although in therapeutic use for more than a century, the mode of cellular action of organic nitrates remains incompletely understood. Despite ample experimental evidence from animal studies to show that nitrates are metabolized to NO in the vascular smooth muscle, direct demonstration of such an activity in human vascular cells is still lacking. Moreover, the role of the endothelium in modulating the pharmacodynamic action of nitrates is far from clear. We therefore aimed to investigate whether or not human endothelial cells are capable of bioactivating these drugs to NO and whether the amounts generated are sufficient to elicit any biological effects. Using cultured human umbilical vein endothelial cells (HUVECs) as an established model system a combination of three different methods was used to address this issue: (1) quantification of NO formation upon endothelial nitrate metabolism using the oxyhaemoglobin technique; (2) evaluation of the second messenger response using radioimmunoassay for cGMP; and (3) assessment of mechanism and extent of potentiation of the anti-aggregatory effect of nitrates in the presence of endothelial cells as a relevant bioassay. We now show that superfusion of cultured human endothelial cells on microcarrier beads with either glyceryl trinitrate (GTN) or isosorbide dinitrate (ISDN; both at 0.1-100 mumol L-1) results in a concentration-dependent formation of NO. NO generation from isosorbide 5-mononitrate (IS-5-N) was below the detection limit. The amounts of NO produced (maximally 2.97 +/- 0.98 pmoles NO min-1 x mg protein with 100 mumol L-1 GTN; n = 8) were similar to those elicited upon challenge of the cells with 100 nM bradykinin. NO formation from either organic nitrate was accompanied, in a concentration-dependent and methylene blue-inhibitable manner, by stimulation of endothelial soluble guanylyl cyclase with consequent increases in the intracellular level of cGMP (maximally 32-fold over basal levels with ISDN), a significant portion of which was released into the extracellular space. Upon continuous 30 min superfusion or repeated application of high concentrations of GTN (100 mumol L-1) nitrate bioactivation to NO was subject to partial tachyphylaxis. Co-incubation of washed human platelets with HUVECs potentiated the anti-aggregatory action of nitrates in a cell number dependent and oxyhaemoglobin-sensitive manner and this effect, too, was accompanied by increases in intraplatelet cGMP levels. The potentiating effect was largely inhibited after blockade of sulfhydryl groups by pre-incubation of HUVECs with N-ethylmaleimide and completely abrogated after pretreatment of cells with the tissue fixative glutaraldehyde. These results demonstrate that human endothelial cells are capable of bioactivating organic nitrates to NO by an enzymatic, apparently thiol-sensitive pathway, in quantities sufficient to influence endothelial and platelet function. Besides the well known vasorelaxant action of organic nitrates, which is mainly due to their metabolism in the smooth muscle compartment, these drugs may therefore be endowed with a hitherto underestimated potential to directly influence endothelial functions via the NO/cGMP pathway. Through specific bioactivation in the endothelium itself organic nitrates can thus mimic and reinforce protective functions normally served by a functional endothelium such as the modulation of blood cell/vessel wall interactions and inhibition of cell proliferation.

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ORCID for M. Feelisch: orcid.org/0000-0003-2320-1158

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