Role of endothelial AADC in cardiac synthesis of serotonin and nitrates accumulation

Abstract

Serotonin (5-HT) regulates different cardiac functions by acting directly on cardiomyocytes, fibroblasts and endothelial cells. Today, it is widely accepted that activated platelets represent a major source of 5-HT. In contrast, a supposed production of 5-HT in the heart is still controversial. To address this issue, we investigated the expression and localization of 5-HT synthesizing enzyme tryptophan hydroxylase (TPH) and L-aromatic amino acid decarboxylase (AADC) in the heart. We also evaluated their involvement in cardiac production of 5-HT. TPH1 was weakly expressed in mouse and rat heart and appeared restricted to mast cells. Degranulation of mast cells by compound 48/80 did not modify 5-HT cardiac content in mice. Western blots and immunolabelling experiments showed an abundant expression of AADC in the mouse and rat heart and its co-localization with endothelial cells. Incubation of cardiac homogenate with the AADC substrate (5-hydroxy-L-tryptophan) 5-HTP or intraperitoneal injection of 5-HTP in mice significantly increased cardiac 5-HT. These effects were prevented by the AADC inhibitor benserazide. Finally, 5-HTP administration in mice increased phosphorylation of aortic nitric oxide synthase 3 at Ser (1177) as well as accumulation of nitrates in cardiac tissue. This suggests that the increase in 5-HT production by AADC leads to activation of endothelial and cardiac nitric oxide pathway. These data show that endothelial AADC plays an important role in cardiac synthesis of 5-HT and possibly in 5-HT-dependent regulation of nitric oxide generation.

Figure 1. Expression and localization of TPH1 in heart.

(A) TPH1 mRNA expression in cardiac tissue was defined by RT-PCR (MW: molecular weight) (left panel) and Western blot (right panel). Immunoblots showed a faint immunoreactive band in heart lysates of 129/SvJ mice but not in those from 129/SvJ TPH1 KO mice. (B) TPH1 immunostaining localized within vesicular granules in rat heart (right and upper panel). Metachromatic granules of mast cells stained with toluidine blue; magnification 400X (right panels). Few mast cells stained with toluidine blue were also present in heart of 129/SvJ mice, magnification 100X (left panel).

Figure 2. Expression and localization of AADC in cardiac tissue.

(A) AADC mRNA expression in mouse cardiac tissue was defined by RT-PCR (left panel) and protein expression by Western blot (right panel) (MW: molecular weight). (B) Immunofluorescent double staining of heart frozen sections with antibodies directed against AADC (a,d green) and CD31 (b,e red). The merged pictures (c, f) show co-localization of AADC and CD31 proteins in microvascular (c) and coronary endothelium (d). Magnification 200X.

Figure 3. AADC activity in cardiac tissue.

(A) Enzyme activity was performed by measuring the conversion of different concentration of 5-hydroxytryptophan (5-HTP) (10–100 µM) into 5-HT in homogenates of mouse cardiac tissue. Values are means ± SEM of 5-HT concentration obtained from three different heart homogenates performed in triplicate. (B) Synthesis of 5-HT from 20 µM 5-HTP in presence or not of 50 µM of AADC inhibitor benserazide. Values are means ± SEM (n = 3 to each group) ***P<0.0001.

Figure 4. Cardiac and blood 5-HT level after 5-HTP administration.

(A) 5-HT and 5-HTP were measured in heart of 129/SvJ mice treated with 5-HTP (5 to 20 mg/kg ip) or saline 30 min before euthanasia. (B) 5-HT concentration in blood of 5-HTP treated mice. Values are means ± SEM (n = 3–7 to each group). *P<0.05; **P<0.001; ***P<0.0001 vs. saline.

Figure 5. Inhibition of cardiac 5-HT synthesis in vivo in presence of the AADC inhibitor benserazide.

(A) Mice were pretreated with benserazide (100 mg/kg ip) or saline 30 min before 5-HTP administration (20 mg/kg ip) and they were sacrified 30 min after the last injection. 5-HT and 5-HTP were measured in heart. (B) Mice were treated only with benserazide (100 mg/kg ip) or saline 30 min before euthanasia. 5-HT and 5-HTP were measured in heart and 5-HTP in plasma. Values are means ± SEM (n = 8–10 to each group). *P<0.05; **P<0.001 vs. saline.

Figure 6. Cardiac NOx content and phosphorylation level of aortic NOS3.

(A) Cardiac NOx content in 129/SvJ mice treated with 5-HTP (20 mg/kg ip) or saline 5, 10 or 30 min before euthanasia. A group of mice were pretreated with benserazide (100 mg/kg ip) 30 min before 5-HTP injection. Values are means ± SEM (n = 8–10 to each group). *P<0.05 vs. saline, § P<0.05 vs. benserazide. (B) Immunodetection of aortic NOS3 phosphorylation 10 min after 5-HTP administration by Western blot. Representative Western blot of aortic endothelial NOS3 phosphorylation levels. Densitometric analysis of Western blot (right panel) (mean ± SEM, n = 4 to each group). *P<0.05 vs. saline; § P<0.05 vs 5-HTP.