Activation of beta3 adrenergic receptor decreases DNA synthesis in human skin fibroblasts via cyclic GMP/nitric oxide pathway.
BACKGROUND
Evidences have shown that beta1 and beta2 adrenoceptors co-exist in human fibroblasts, but it is not yet clear the functional expression of beta3 adrenoceptor in these cells. The aim of this study was to investigate the expression and biological effect of beta3 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 (beta3 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 beta(3)adrenoceptor subtypes. ZD 7114 stimulation of beta3 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 beta3 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 beta3 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
beta3 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.
Furlán
César
C
Pharmacology Unit, School of Dentistry, University of Buenos Aires, Buenos Aires, Argentina.
Sterin-Borda
Leonor
L
Borda
Enri
E
D058667:Adrenergic beta-3 Receptor Agonists; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008297:Male; D008954:Models, Biological; D009569:Nitric Oxide / Q000378:metabolism*; D010642:Phenoxyacetates / Q000494:pharmacology; D050198:Phenoxypropanolamines / Q000494:pharmacology; D022702:Receptors, Adrenergic, beta-3 / Q000378:metabolism*; D015398:Signal Transduction; D012867:Skin / Q000166:cytology / Q000378:metabolism
D058667:Adrenergic beta-3 Receptor Agonists; D000318:Adrenergic beta-Agonists; D010642:Phenoxyacetates; D050198:Phenoxypropanolamines; D022702:Receptors, Adrenergic, beta-3; C071909:ICI D7114; D009569:Nitric Oxide; D004247:DNA; D006152:Cyclic GMP