Beta-adrenergic-induced CD40 overexpression on gingival fibroblasts: role of PGE2.

CD40, a member of the tumour necrosis factor-alpha 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 beta-AR (beta-adrenoceptor) agonists. Herein, the authors demonstrate that beta-AR subtype activation via their own specific agonists markedly increased CD40 expression on human gingival fibroblasts. This effect was prevented by beta-AR subtype-specific antagonists. In addition, gingival fibroblast beta-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 beta-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 beta-AR-agonist-mediated CD40 overexpression. Our work has revealed an endogenous beta-AR mediator network involving gingival fibroblasts.

Circulating beta(1) Adrenergic Autoantibodies from Patients with Chronic Periodontitis Interact with Gingival Fibroblasts.

OBJECTIVES: To demonstrate the presence of circulating autoantibodies (Abs) from patients with chronic periodontitis (CP) that interacted with human gingival fibroblast membranes activating beta(1) adrenoceptors (beta(1)-AR). METHODS: Sera and purified IgG from 25 patients with CP and 20 age-matched healthy subjects were studied by flow cytometry, ELISA and DNA synthesis. Human gingival fibroblast membranes and/or synthetic peptides with amino acid sequences identical to human beta(1)-AR were used as antigens. RESULTS: By flow cytometry and ELISA procedures, we proved that the serum-purified IgG fraction from patients with CP reacted with the fibroblast surface and to the beta(1) synthetic peptide. The corresponding affinity-purified anti-beta(1) peptide Abs displayed agonist-like activity associated with specific receptor activation, inhibiting the DNA synthesis of human gingival fibroblasts. CONCLUSIONS: This study demonstrates that beta(1)-AR autoantibodies are elevated in patients with CP. These autoantibodies were targeted to the fibroblasts, and specifically to the beta(1)-AR, and has receptor-like activity inhibiting DNA synthesis.

Differential regulation on human skin fibroblast by alpha1 adrenergic receptor subtypes.

Alpha 1 adrenoceptor (alpha1-AR) regulation of DNA synthesis was studied in human neonatal foreskin fibroblast. Saturation assay with a specific radioligand for alpha1 adrenergic [3H]-prazosin revealed two saturated and specific binding sites with high or low affinity. Competitive binding assay with different antagonist subtypes, defined pharmacologically three major types of alpha1-AR. The alpha1-AR agonists (from 1x10(-10) to 1x10(-4) M) triggered a biphasic action on DNA synthesis reaching maximal stimulation at 1x10(-9) M and maximal inhibition at 1x10(-6) M. Prazosin, abolished the stimulatory (pA2: 9.24) and inhibitory (pA2: 8.80) actions of alpha1-AR agonists. The alpha1-AR stimulation resulted in the activation of phosphoinositide turnover (InsP) via phospholipase C (PLC) involving calcium/calmodulin (CaM) and nitric oxide synthase (NOS) that correlates with the DNA synthesis increment; whereas the inhibition resulted in a decrease of cyclic AMP (cAMP) accumulation via adenylate cyclase inhibition. The potency displayed by the specific antagonists tested in binding, DNA synthesis, InsP and NOS at low agonist concentration suggests that they can be elicited by the activation of the same receptor (alpha1B-AR subtype); while the decrement in DNA synthesis and cAMP at high concentration account by the activation of alpha1D-AR coupled to Gi protein. Non-functional alpha1A-AR in neonatal human foreskin fibroblast was observed. Results suggest that the expression of alpha1-AR subtypes on human skin fibroblast may differentially activate signaling pathways that modulate physiological response of the cells.

Nitric oxide synthase and PGE2 reciprocal interactions in rat dental pulp: cholinoceptor modulation.

In this study we determined the effect of cholinoceptor agonist pilocarpine on the stimulation of nitric oxide synthase (NOS) and on prostaglandin E2 (PGE2) generation upon rat dental pulp. By reverse transcriptase/polymerase chain reaction (RT-PCR) we identified several products corresponding to m1, m2, m3, and m4 muscarinic acetylcholine receptors (mAChRs). The stimulation of M1, M2, M3, and M4 mAChRs by pilocarpine increases NOS activity and PGE2 generation. There is a correlation (correlation coefficient=0.05) between NOS activity and PGE2 generation through the activation of phosphoinositide by phospholipase C (PLC), phospholipase A2 (PLA2), and cyclooxygenase 1 (COX-1). Exogenous PGE2 restored NOS activity inhibited by indomenthacin (INDO), whereas nitric oxide (NO) donor restored PGE2 generation inhibited by NG-methyl-L-arginine acetate salt (L-NMMA). These data indicate that both NO and PGE2 interact with their own respective biosynthetic pathways modulating NOS and COX activities. Results could contribute to understanding the involvement of NO and PGE2 in healthy dental pulp given that cellular signals through the parasympathetic system modulate the function of the dentin-pulp complex.

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.

Comparação dos efeitos eletrocardiográficos de doses inotrópicas eqüipotentes de amrinone e de ouabaína em corações isolados de cobaia / Comparison of the electrocardiographic effects of equipotent inotropic doses of amrinone and ouabain in isolated hearts of guinea pigs

Os efeitos eletrocardiográficos de doses de amrinone (A) e de ouabaína (O) capazes de desenvolver açöes inotrópicas eqüipotentes foram estudados em coraçöes isolados de cobaia mantidos em perfusäo de Langendorff, nutridos por soluçäo de Krebs-Henseleit. Um baläo de látex, em comunicaçäo com um transdutor de pressäo, era mantido dentro do ventrículo esquerdo, que contraía isovolumetricamente. Nestas condiçöes, a pressäo desenvolvida durante as contraçöes é o indicador do inotropismo cardíaco. Elétrodos epicárdicos possibilitavam o registro do eletrocardiograma. Foi verificado que a concentraçäo de A (140 mg/l) necessária para elevar a pressäo desenvolvida (PD) em 100% é cerca de 200 vezes maior que a dose de O (0,65 mg/l). Para doses eqüipotentes. A provocou discreta taquicardia (195 + ou - 25 bpm para 220 + ou - 25 bpm; p < 0,05) e raras extra-sístoles. Sob infusäo ocom O, näo houve variaçäo de freqüência cardíaca (183 + ou - 32 bpm para 180 + ou - 27 bpm; ns) e ocorreu retardo da conduçäo A-V do estímulo (PRi passou de 0,08 + ou - 0,03 para 0,12 + ou - 0,04; p < 0,01). Em todas as preparaçöes infundidas com O ocorreram extra-sístoles freqüentes seguidas de arrtmias mais complexas (fibrilaçäo ventricular, taquicardia paroxística supraventricular). Estes resultados estäo de acordo com relatos da literatura de que A pode exercer efeitos inotrópicos equivalentes aos de digitálicos, sem as arrtmias habitualmente provocadas pelos glicosídeos