6-Nitrodopamine potently increases intracellular calcium (Ca2+i) and potentiates catecholamine-induced Ca2+i release in human aortic smooth muscle and human aortic endothelial cells

Abstract Background/Introduction Mammalian vascular tissues basally release of 6-nitrodopamine (6-ND), which vasorelaxes pre-contracted vascular rings via D2-like receptor antagonism and exerts potent inotropic and chronotropic effects in the heart. In human isolated vas deferens and seminal vesicles, 6-ND enhances contractions induced by noradrenaline (NA), adrenaline (ADR) and dopamine (DA). Purpose We compared the effect of 6-ND on intracellular calcium (Ca2+i) levels in human aortic smooth muscle (HASMC) and endothelial cells (HAEC) with that induced by classical catecholamines. We also investigated whether 6-ND potentiates catecholamine-induced Ca2+i increase in these cell types. Methods HASMC or HAEC at passage 3-6 were cultured in smooth muscle cell medium or endothelial muscle cell medium respectively, containing 5% fetal bovine serum and 20 units/mL penicillin/streptomycin, and seeded at a density of 5x105 cells/mL followed by incubation for 48 h at 37°C in a 95% air/5% CO2 atmosphere. Cells were then loaded with 1 µM fura-2 AM for 45 min at 37 °C, followed by washing, addition of 100 µL Hank's balanced salt solution, and excitation at 340 and 380 nm in a plate reader with emission detection at 510 nm, at 37 °C. Some experiments were performed with calcium free Hank’s solution. Results 6-ND, NA, ADR and DA all induced concentration-dependent increase in Ca2+i in HASMC (Figures 1A and 1B). 6-ND showed significantly higher potency (pEC50: 10.60 ± 0.41 for area under curve (AUC), 9.95 ± 0.33 for peak) compared to NA, ADR, and DA (7.39 ± 0.16, 6.97 ± 0.34, and 8.71 ± 0.25 respectively for AUC, and 6.89 ± 0.17, 6.53 ± 0.28, and 8.81 ± 0.18, respectively for peak increase; n = 5 each). Emax for AUC was comparable between 6-ND (92.85 ± 5.74), NA (98.56 ± 6.14) and ADR (81.45 ± 11.87), while DA (169.77 ± 14.65) elicited the highest response. For peak increase, 6-ND (0.83 ± 0.06) had a similar maximal effect to ADR (1.49 ± 0.13), whereas NA (1.24 ± 0.22) and DA (1.45 ± 0.08) induced greater responses. Pre-incubation with 6-ND (1pM) potentiated the effects of NA, ADR, and DA at 30 pM, as shown in Figure 1C for AUC and Figure 1D for peak increase. In preparations with calcium-free Hanks' solution, 6-ND exhibited a reduced effect at concentrations of 10 and 30 nM compared to the response in Hanks' solution containing Ca²⁺. In HAEC, 6-ND induced increase in Ca²⁺i only at concentrations ranging from 30 nM to 1 μM, with a pEC50 of 7.71 ± 0.36 for AUC and 7.30 ± 0.32 for peak increase, as shown in Figures 2A and 2B respectively. Additionally, absence of external calcium gave rise to a reduction in the Ca²⁺i increase at concentrations of 30 nM and 100 nM, as shown in Figures 2C and 2D respectively. Conclusion 6-ND modulates intracellular Ca²⁺i in HASMC and HAEC, exhibiting higher potency than the classical catecholamines and synergizing with them.Figure 01 Figure 02