HUMAN ENDOTHELIAL-CELLS BIOACTIVATE ORGANIC NITRATES TO NITRIC-OXIDE - IMPLICATIONS FOR THE REINFORCEMENT OF ENDOTHELIAL DEFENSE-MECHANISMS

Although in therapeutic use for more than a century, the mode of cellu lar action of organic nitrates remains incompletely understood. Despit e ample experimental evidence from animal studies to show that nitrate s are metabolized to NO in the vascular smooth muscle, direct demonstr ation of such an activity in human vascular cells is still lacking. Mo reover, the role of the endothelium in modulating the pharmacodynamic action of nitrates is far from clear. We therefore aimed to investigat e 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 end othelial cells (HUVECs) as an established model system a combination o f three different methods was used to address this issue: (I) quantifi cation of NO formation upon endothelial nitrate metabolism using the o xyhaemoglobin technique; (2) evaluation of the second messenger respon se using radioimmunoassay for cGMP; and (3) assessment of mechanism an d 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 (I SDN; both at 0.1-100 mu mol L(-1)) results in a concentration-dependen t 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 mu mol L(-1) GT N; n = 8) were similar to those elicited upon challenge of the cells w ith 100 nM bradykinin. NO formation from either organic nitrate was ac companied, in a concentration-dependent and methylene blue-inhibitable manner, by stimulation of endothelial soluble guanylyl cyclase with c onsequent increases in the intracellular level of cGMP (maximally 32-f old over basal levels with ISDN), a significant portion of which was r eleased into the extracellular space. Upon continuous 30 min superfusi on or repeated application of high concentrations of GTN (100 mu mol L (-1)) nitrate bioactivation to NO was subject to partial tachyphylaxis . Go-incubation of washed human platelets with HUVECs potentiated the anti-aggregatory action of nitrates in a cell number dependent and oxy haemoglobin-sensitive manner and this effect, too, was accompanied by increases in intraplatelet cGMP levels. The potentiating effect was la rgely inhibited after blockade of sulfhydryl groups by pre-incubation of HUVECs with N-ethylmaleimide and completely abrogated after pretrea tment 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 pat hway, in quantities sufficient to influence endothelial and platelet f unction. Besides the well known vasorelaxant action of organic nitrate s, which is mainly due to their metabolism in the smooth muscle compar tment, these drugs may therefore be endowed with a hitherto underestim ated 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 no rmally served by a functional endothelium such as the modulation of bl ood cell/vessel wall interactions and inhibition of cell proliferation .