P305Endothelial cells are able to synthesize and release catecholamines both in vitro and in vivo

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Abstract

Recently it has been demonstrated that catecholamines are produced and used by macrophages and mediate immune response. The aim of this study was to verify if endothelial cells (EC), that are of myeloid origin, can produce catecholamines. We demonstrated by Real Time PCR that genes coding for TH, DDC, DβH and PNMT, enzymes involved in the synthesis of catecholamines, are all expressed in basal conditions in bovine aorta EC (BAEC) and their expression is enhanced in response to 16 hours of hypoxia (fold of basal: TH: 4.7 ± 0.15; DDC: 3.9 ± 0.21; DβH: 4.8 ± 0.23; PNMT: 5.01 ± 0.01). This result was confirmed by western blot and immunohistochemical analysis. Moreover, hypoxia enhances norepinephrine (NE) and epinephrine (EPI) release respect to basal conditions (NE: + 44,7 ± 11,3; EPI: + 51,6 ± 6% of basal). In order to assess the signal transduction pathway that regulates catecholamines synthesis in EC, we overexpressed in BAEC either PKA or the transcription factor CREB, since PKA/CREB activation induces TH transcription and activity in response to stress. In BAEC, CREB overexpression enhances gene expression of DβH (CREB: 2.1 ± 0.09 fold of basal) and PNMT (CREB: 9.8 ± 0.3 fold of basal) and regulates NE (CREB: + 56,7 ± 0.7% vs basal) and EPI (CREB: + 51,2 ± 5.3% vs basal) release. PKA overexpression increases hypoxia-induced DβH (16 ± 0.6 fold of hypoxia) and PNMT (9.6 ± 0.2 vs hypoxia) gene expression and enhances cathecolamines release (EPI: 10 ± 1.9; NE: 16 ± 0.19 % vs hypoxia). On the contrary, H89, inhibitor of PKA, exerts the opposite effect (DβH: -73.7 ± 0.03% and PNMT: -75.2 ± 0.04% vs hypoxia; EPI: -70,8 ± 5.9 and NE: -39,6 ± 0.19% vs hypoxia). These data indicate that PKA/CREB transduction pathway regulates catecholamines release in BAEC. Finally, we evaluated by immunohistochemistry the expression of TH, DDC, DβH and PNMT in superficial femoral arteries from hindlimbs of C57/Black mice one week after removal of the common femoral artery to provoke chronic ischemia. Ischemia induces DDC, DβH and PNMT expression in the endothelium, while TH is maximally expressed and not induceble any further. In conclusion, our study demonstrates for the first time the ability of endothelial cells to synthesize and release cathecolamines in response to ischemia, both in vitro and in vivo.

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