Activation of β3 Adrenergic Receptor Decreases DNA synthesis in Human Skin Fibroblasts Via Cyclic GMP/Nitric Oxide Pathway

Abstract

Background: Evidences have shown that β₁ and β₂ adrenoceptors co-exist in human fibroblasts, but it is not yet clear the functional expression of β₃ adrenoceptor in these cells. The aim of this study was to investigate the expression and biological effect of β₃ 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 (β₃ agonist) on cell DNA synthesis, radioligand binding assay, cyclic GMP and cyclic AMP accumulation and nitric oxide synthase (NOS) activity were evaluated. Results: ³H-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 β₃adrenoceptor subtypes. ZD 7114 stimulation of β₃ 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 β₃ 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 β₃ 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: β₃ 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.