Role of L-arginine in the vascular actions and development of tolerance to nitroglycerin

Abstract

The goal of this work was to test the role of nitric oxide synthase (NOS) and its substrate L-arginine in development of tolerance to nitroglycerin's (GTN) vasodilator actions. GTN's effects on NOS activity and NO formation were tested in cultured bovine aortic endothelial cells (BAECs). The arginine to citrulline conversion assay showed that GTN stimulated NOS basal activity in BAECs by approximately 40%, comparable with acetylcholine (ACh)-treated controls. Both effects were blocked by L-NMMA. Photometric assays showed that both GTN and ACh-stimulated NO formation. Both effects were potentiated by L-arginine and inhibited by L-NAME. L-NAME inhibited ACh responses approximately 80% compared with approximately 40% for GTN responses. The aortic ring assay showed that 2 h pretreatment with GTN caused substantial tolerance to GTN's vasodilating effects as evidenced by a 38 fold rightward shift of the concentration-relaxation curve. In contrast to D-arginine, addition of L-arginine substantially inhibited this effect, reducing the rightward shift to 4.4 fold of control values. GTN tolerance was associated with a 40% reduction in L-arginine tissue levels. GTN had a biphasic effect on BAEC uptake of L-arginine, stimulating uptake at 5 and 15 min, and suppressing uptake after 1 and 4 h In summary, acute GTN treatment stimulates endothelial NOS activity in producing NO and increases cellular uptake of L-arginine. Prolonged GTN exposure reduces GTN's vasodilator actions, decreases L-arginine tissue levels and depresses BAECs uptake of L-arginine. Supplementation of L-arginine reduces development of GTN tolerance. These data indicate that GTN tolerance depends in part on activation of the NOS pathway.

Figure 1

Effect of ACh and GTN (1 μM each) on eNOS activity in BAECs as measured by the conversion of [³H]-L-arginine into [³H]-citrulline. The basal activity of eNOS was 4.11±0.6 pmol mg protein⁻¹ min⁻¹. Treatment with L-NMMA (10⁻³ M) reduced basal ³H-citrulline formation by 71.7±5.4%. This treatment reduced the effects of ACh and GTN by over 90%. Data are presented as a mean±s.e.mean of six experiments run in duplicates. ^*Indicates a significant difference from the baseline value.

Figure 2

Effects of acetylcholine and nitroglycerin (10⁻⁶ and 10⁻⁵ M) on NO production. Responses to drugs are transient elevations in NO production above basal levels of NO production in control buffer with 10⁻⁵ M L-arginine. Subsequent perfusion of the cell/bead system with 10⁻³ M L-NAME in buffer, and then with 10⁻³ M of L-arginine in the buffer system. Production of NO is displayed in nmol min⁻¹ (mean±s.e.mean). #Denotes difference between responses in L-NAME buffer vs both basic and L-arginine added buffers (P<0.05). ^*Indicates difference vs control basic buffer responses (P<0.05).

Figure 3

Induction of tolerance to GTN in isolated rat aortic segments and the effects of L-arginine and D-arginine thereon. Tissues were incubated for 2 h with GTN (5×10⁻⁴ M) without or with 5×10⁻⁴ M of L-arginine or D-arginine (n=8–20). The Initial pD₂ value for GTN before tolerance is 7.75±0.08. After incubation for 2 h with GTN, the pD₂ values were 7.13±0.12 and 6.17±0.22, respectively in the presence and absence of L-arginine (P<0.05). The presence of 5×10⁻⁴ M of D-arginine did not significantly affect the development of tolerance of GTN (pD₂ value is 5.71±0.3).

Figure 4

ACh-induced vasorelaxant responses of GTN-tolerant and non-tolerant aortic segments. The pD₂ value for ACh in non-tolerant vessels is similar to the tolerant vessels (7.72±0.31 vs 7.63±0.33, respectively). However, the curves are significantly different at concentrations up to 10⁻⁸ M of ACh (P<0.05). Data are represented as the mean of eight experiments±s.e.mean.

Figure 5

L-arginine content in rat aortic segments pre-constricted with phenylephrine. The figure shows L-arginine levels in control buffer initial (n=11), presence of GTN for 2 h (5×10⁻⁴ M, n=7), and control buffer for 2 h (n=8). ^*Indicates a difference from values for both initial control buffer and control buffer after 2 h (P<0.05).

Figure 6

The time course of effects of GTN (10⁻⁶ M) on [³H]-L-arginine uptake in isolated bovine aortic endothelial cells. Basel rates of L-arginine uptake are 44.8±10.3, 113.8±34, and 344±68 pmol L-arginine mg protein⁻¹ min⁻¹, respectively after 5, 15 and 60 min, respectively. For the 240 min exposure to GTN, uptake of [³H]-L-arginine was measured during the fourth hour. ^*Indicates difference from basal uptake of L-arginine at specific time points (P<0.05).