Effects of LDL on intracellular free calcium and nitric oxide-dependent cGMP formation in porcine endothelial cells.

ABSTRACT

Nitric oxide (NO)-mediated, endothelium-dependent vasodilation is reduced in atherosclerotic arteries. A number of in vivo studies suggest that infusion of the substrate of NO synthase, L-arginine, partly counteracts this effect. We studied the potential inhibitory effects of native and of oxidized low density lipoproteins (n-LDL, ox-LDL) on NO-dependent cyclic guanidine monophosphate (cGMP) formation in porcine aortic endothelial cells and the role of extracellular L-arginine in counteracting this process. NO-dependent cGMP production in the cells (passage 1; preincubated in L-arginine-free medium for 24 h) was stimulated for 3 min with bradykinin (BK 1 nM) or the calcium-ionophore A23187 (100 nM) or by a 20 min incubation with L-arginine (L-Arg 0.1 mM, 20 min). The endothelium-independent NO-donor, sodium nitroprusside (SNP 1 microM) was used as control stimulus. Experiments were performed in the presence of LDL which was kept as much as possible under antioxidative conditions, further referred to as n-LDL (1 mg/ml), or LDL which was oxidized by incubation with copper (ox-LDL 0.1 mg/ml). The NG-nitro-L-arginine (L-NNA, inhibitor of NO-synthase) -sensitive intracellular cGMP concentration was taken as a measure of endothelial NO formation and determined by radioimmunoassay. BK-induced changes of intracellular free Ca2++ were measured immediately after washout of LDL using the FURA-2 method. n-LDL reduced the cGMP-levels in unstimulated cells as well as the cGMP increase in response to bradykinin (-10%) and the calcium-ionophore A23187 (-80%). The SNP-induced cGMP-increase was, however, not affected. L-arginine increased the intracellular cGMP concentration under both conditions by a similar amount, without affecting intracellular free calcium. Uptake of 3H-L-arginine was not significantly altered by n-LDL treatment. Ox-LDL significantly reduced SNP-induced cGMP-increases but did not alter bradykinin-induced cGMP increases. The BK-induced increase of intracellular free calcium was even enhanced after exposure of the cells to ox-LDL. L-arginine increased cGMP by a similar amount as in untreated cells. It is concluded that both n-LDL and ox-LDL can reduce the NO-dependent cGMP formation in cultured endothelial cells, albeit by different mechanisms. However, a limitation of the uptake or availability of extracellularly applied L-arginine does not appear to be a causal factor, at least not after 2 h exposure.