Relationship between biotransformation of glyceryl trinitrate and cyclic GMP accumulation in various cultured cell lines

We assessed glyceryl trinitrate (GTN) biotransformation and cyclic GMP accumulation in cultured rat lung fibroblasts (RLF), porcine kidney epithelial (PK1), bovine aortic endothelial (BAE) and bovine aortic smooth muscle (BASM) cells. Biotransformation of 0.1 μM GTN was linear over 30 min and the percentage of glyceryl dinitrate (GDN)/10⁶ cells for BAE, BASM, RLF and PK1 at 30 min was 3.1, 2.3, 5.8 and 21.7%, respectively. At low GTN concentration (0.01-0.1 μM) there was a highly selective formation of 1,2-GDN, whereas at higher GTN concentration (>1 μM) this selectivity was lost. Cyclic GMP accumulation did not occur in BAE or BASM at any GTN concentration, whereas for RLF and PK1 it was highly correlated to the rate of GDN formation. Upon re-exposure to GTN after treatment of RLF or PK1 cells for 3 hr with 0.1 mM GTN, there was an almost complete loss of the cyclic GMP response, GTN biotransformation was attenuated markedly and the selective formation of 1,2-GDN at low GTN concentration was absent. However, when GTN-treated cells were incubated for 18 hr in GTN-free media, there was a recovery of the cyclic GMP response, GTN biotransformation and selective 1,2-GDN formation toward control values. We conclude that: 1) the rate of GTN biotransformation correlates with the magnitude of the cyclic GMP response, 2) the concentration-dependent regioselectivity for denitration of GTN suggests at least two pathways for GTN biotransformation and 3) the loss of selective 1,2-GDN formation in cells made tolerant to the effect of GTN on cyclic GMP accumulation suggests that this biotransformation pathway may play a central role in GTN-induced activation of guanylate cyclase.