Activation of β 3 adrenergic receptor decreases DNA synthesis in human skin fibroblasts via cyclic GMP/nitric oxide pathway

Background: Evidences have shown that β 1 and β 2 adrenoceptors co-exist in human fibroblasts, but it is not yet clear the functional expression of β 3 adrenoceptor in these cells. The aim of this study was to investigate the expression and biological effect of β 3 adrenoceptor activation in human skin fibroblast and the different signaling pathways involved in its effect. Methods: For this purpose in vitro cultures of human skin fibroblast were established from human foreskin and grown in Dulbecco's modified Eagle's medium. The effect of ZD 7114 (β 3 agonist) on cell DNA synthesis, radioligand binding assay, cyclic GMP and cyclic AMP accumulation and nitric oxide synthase (NOS) activity were evaluated. Results: 3 H-CGP binding to human fibroblast membranes was a saturable process to a single class of binding site. The equilibrium parameters were: Kd 20±3 pM and Bmax 222±19 fmol/mg protein. Ki values showed that these cells express a high number of β 3 adrenoceptor subtypes. ZD 7114 stimulation of β 3 adrenoceptor exerts a concentration-dependent inhibition of DNA synthesis and cAMP accumulation with parallel increase in NOS activity that led to cGMP accumulation. All these effects were blocked by the β 3 adrenoceptor antagonist (SR 59230A). The effect of ZD 7114 on DNA synthesis significantly correlated with its action either on cAMP or NOS-cGMP signaling system. Inhibitors of NOS activity and NO-sensitive guanylate cyclase prevented the inhibitory effect of ZD 7114 on DNA synthesis. In addition, the β 3 adrenoceptor-dependent increase in cGMP and activation of NOS were blocked by the inhibition of phospholipase C (PLC), calcium/calmodulin (CaM), endothelial NOS activity and cGMP accumulation. Conclusions: β 3 adrenoceptor activation exerts inhibitory effect on human fibroblast DNA synthesis as a result of the activation of NO-cGMP pathway and the inhibition of adenylate cyclase activity. The mechanism appears to occurs secondarily to stimulation of PLC and CaM. This in turn triggers cascade reaction leading to increase production of NO-cGMP with decrease in cAMP accumulation.Fil: Furlán, César. Universidad de Buenos Aires; ArgentinaFil: Sterin Borda, Leonor. Universidad de Buenos Aires; ArgentinaFil: Borda, Enri Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Odontología; Argentin

β-Adrenergic-induced CD40 overexpression on gingival fibroblasts: role of PGE2

CD40, a member of the tumour necrosis factor-α receptor family, is constitutively expressed by cells of haematopoietic and non-haematopoietic origin, including fibroblasts. Signalling through this receptor molecule regulates inflammatory mediator secretion by many cell types. The work has been performed in healthy subjects and the authors studied, by cellular culture, flow cytometric analysis and ELISA assay, the expression of CD40 and PGE2 (prostaglandin E2) generation on gingival fibroblasts stimulated by β-AR (β-adrenoceptor) agonists. Herein, the authors demonstrate that β-AR subtype activation via their own specific agonists markedly increased CD40 expression on human gingival fibroblasts. This effect was prevented by β-AR subtype-specific antagonists. In addition, gingival fibroblast β-AR stimulation resulted in an increase in PGE2 generation. The inhibition of PLA2 (phospholipase A2) and COX-1 (cyclo-oxygenase-1) but not COX-2 impaired β-AR increase of PGE2, an effect that was restored by the addition of low concentrations of PGE2, suggesting that PGE2 generation is implicated in the mechanism underlying β-AR-agonist-mediated CD40 overexpression. Our work has revealed an endogenous β-AR mediator network involving gingival fibroblasts.Fil: Furlán, César. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; ArgentinaFil: Sterin, Leonor Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; ArgentinaFil: Borda, Enri Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentin