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. 2022 Apr 2;23(7):3982.
doi: 10.3390/ijms23073982.

Trimethylamine N-Oxide (TMAO) Impairs Purinergic Induced Intracellular Calcium Increase and Nitric Oxide Release in Endothelial Cells

Giulia Querio  1 , Susanna Antoniotti  1 , Federica Geddo  1 , Renzo Levi  1 , Maria Pia Gallo  1
Affiliations

Trimethylamine N-Oxide (TMAO) Impairs Purinergic Induced Intracellular Calcium Increase and Nitric Oxide Release in Endothelial Cells

Giulia Querio et al. Int J Mol Sci. .

Abstract

Trimethylamine N-oxide (TMAO) is a diet derived compound directly introduced through foodstuff, or endogenously synthesized from its precursors, primarily choline, L-carnitine, and ergothioneine. New evidence outlines high TMAO plasma concentrations in patients with overt cardiovascular disease, but its direct role in pathological development is still controversial. The purpose of the study was to evaluate the role of TMAO in affecting key intracellular factors involved in endothelial dysfunction development, such as reactive oxygen species, mitochondrial health, calcium balance, and nitric oxide release using bovine aortic endothelial cells (BAE-1). Cell viability and oxidative stress indicators were monitored after acute and prolonged TMAO treatment. The role of TMAO in interfering with the physiological purinergic vasodilatory mechanism after ATP stimulation was defined through measurements of the rise of intracellular calcium, nitric oxide release, and eNOS phosphorylation at Ser1179 (eNOSSer1179). TMAO was not cytotoxic for BAE-1 and it did not induce the rise of reactive oxygen species and impairment of mitochondrial membrane potential, either in the basal condition or in the presence of a stressor. In contrast, TMAO modified the purinergic response affecting intracellular ATP-induced calcium increase, nitric oxide release, and eNOSSer1179. Results obtained suggest a possible implication of TMAO in impairing the endothelial-dependent vasodilatory mechanism.

Keywords: TMAO; calcium; endothelial dysfunction; endothelial nitric oxide synthase; nitric oxide; trimethylamine N-oxide; vasodilatory mechanism.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
TMAO does not impact BAE-1 viability. BAE-1 were treated with different concentrations of TMAO that showed no cytotoxic effect after (a) 24 h, (b) 48 h, and (c) 72 h (n = 3 independent experiments).
Figure 2
Figure 2
TMAO does not impact mitochondrial membrane potential. Bar graphs of 568/488 nm ratio related to control condition (CTRL) showing that TMAO was not able to induce mitochondrial membrane depolarization after (a) 1 h and (b) 24 h of treatment and it acted as a neutral factor in presence of a stressor, as MEN, that on the contrary induced a reduction of 568/488 nm ratio. Number of cells for each condition in 1 h treatment: CTRL: 180; TMAO: 180; MEN: 180; TMAO + MEN: 180 (n = 3 independent experiments). Number of cells for each condition in 24 h treatment: CTRL: 83; TMAO: 83; MEN: 83; TMAO+MEN: 83 (n = 3 independent experiments). * p < 0.05, ** p < 0.01 (vs. CTRL); # p < 0.05, ## p < 0.01, ### p < 0.001 (vs. TMAO). (c) Representative confocal microscopy images of BAE-1 treated for 1 h with TMAO 100 μM, MEN 100 μM, MEN 100 μM + TMAO 100 μM. Merged images at 568 nm and 488 nm (magnification 60X).
Figure 3
Figure 3
TMAO does not impact reactive oxygen species (ROS) increase. Bar graphs of mean fluorescence (Ex/Em 485 nm/520 nm) related to control condition (CTRL) showing that TMAO 100 μM was not able to induce ROS production in basal condition and it acted as a neutral factor in presence of a stressor, MEN, after (a) 1 h and (b) 24 h of treatment (n = 3 independent experiments). * p < 0.05 (vs. CTRL); # p < 0.05, ## p < 0.01 (vs. TMAO).
Figure 4
Figure 4
TMAO treatment alters intracellular calcium in purinergic response to ATP. (a) Bar graph representing the Fmax-Ft/2 fluorescence after ATP stimulation in basal condition (ATP) or after treatment with TMAO 100 μM for 1 h or 24 h. Number of cells for each experimental condition: ATP: 47; TMAO 1 h + ATP: 47; TMAO 24 h + ATP: 47 (n = 4 independent experiments). * p < 0.05 (b) Mean fluorescence variations of normalized curves aligned with respect to the peak value (Fmax) after ATP stimulation in a single representative experiment. Dotted line indicates the t/2 at which curve differences were considered.
Figure 5
Figure 5
TMAO influences nitric oxide (NO) release in purinergic response to ATP. (a) Bar graph showing fluorescence variations before and after ATP stimulation related to basal treatment (CTRL). Treatment with TMAO for 24 h reduced NO release during purinergic stimulation. Number of cells for each experimental condition: CTRL: 63; TMAO 1 h + ATP: 63; TMAO 24 h + ATP: 57 (n = 4 independent experiments). Statistical differences were evaluated with ANOVA and Kruskal–Wallis test. * p < 0.05. (b) Normalized traces of fluorescence variations (568 nm) of single cells stimulated with ATP in basal condition and after 1 h or 24 h of TMAO pretreatment.
Figure 6
Figure 6
TMAO impacts on eNOS phosphorylation at Ser1179 in purinergic stimulation with ATP. (a) Basal treatment with TMAO for 1 h and 24 h did not induce any variation in eNOS phosphorylation, while treatment for 24 h induced a reduction of eNOSSer1179/eNOS ratio in purinergic response to ATP. (n = 4 independent experiments). **** p < 0.0001; ** p < 0.01. (b) Representative Western blot showing the effect of TMAO treatment for 1 h and 24 h in eNOS phosphorylation at Ser1179 after ATP stimulation.
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