Cyclic Adenosine Monophosphate Regulates the Expression of the Intercellular Adhesion Molecule and the Inducible Nitric Oxide Synthase in Brain Endothelial Cells

Abstract

The authors studied whether cyclic AMP (cAMP), a widespread regulator of inflammation, modulates the cytokine-mediated expression of the intercellular adhesion molecule, intercellular adhesion molecule-1 (ICAM-1), and the inflammatory nitric oxide synthase 2 (NOS-2), in primary and immortalized brain endothelial cell cultures (GP8.3 cell line). When measured by enzyme-linked immunosorbent assay (ELISA), ICAM-1 was constitutively expressed and was up-regulated twofold by interleukin-1beta, with no effect of interferon-gamma. The NOS-2 activity, assessed by nitrite accumulation, was absent from untreated cultures but was induced by interleukin-1beta and interferon-gamma acting synergistically. Stimulation of cAMP-dependent pathways with forskolin or dibutyryl cAMP decreased ICAM-1 protein expression, whereas it increased NOS-2 protein expression. For both ICAM-1 and NOS-2, mRNA expression correlated with protein expression. Blockade of NOS activity with L-N-monomethylargiuine (L-NMMA) did not alter ICAM-1 expression, indicating that the nitric oxide released by NOS-2 did not cause the down-regulation of ICAM-1. Analysis of NFKB activation indicated that cAMP acted through a mechanism other than inhibition of nuclear translocation of NFKB. The authors conclude that cAMP modulates the expression of proinflammatory molecules in brain endothelium. This suggests that inflammatory processes at the blood-brain barrier in vivo may be regulated by perivascular neurotransmitters via cAMP.

Phosphorylation of GTP dissociation inhibitor by PKA negatively regulates RhoA

“…The cAMP-PKA signaling pathway is important in preventing a wide range of inflammatory activities (4,5,10,26,(32)(33)(34)(35)(36)40). The current results show that a direct target of the cAMP-PKA pathway is Ser 174 of GDI, and this phosphorylation of GDI resulted in the inhibition of RhoA function.…”

JNK/ERK/FAK Mediate Promigratory Actions of Basic Fibroblast Growth Factor in Astrocytes via CCL2 and COX2

“…ELISAs were performed as thoroughly described elsewhere [35] . In brief, the cells were fixed with 4% paraformaldehyde in PBS and incubated overnight with a monoclonal antibody against rat ICAM1 (clone 1A29, Pharmingen; concentration 3 g/ml).…”

“…Electrophoretic mobility shift assays were performed as thoroughly described previously [35] . In brief, the NF-B oligonucleotides corresponding to the palindromic consensus sequences were end-labeled with 10 Ci [ ␥ 32 P-ATP (Amersham) with 10U T4 polynucleotide kinase (Fermentas) and purified in G-25 Sephadex columns (Sigma).…”

Temporal effects of vascular endothelial growth factor and 3,5‐cyclic monophosphate on blood–brain barrier solute permeability in vivo

“…In addition to anti‐VEGF treatment for brain diseases (JuanYin et al, ; Ueta et al, ; MacMillan et al, ; Soffietti et al, ; Batchelor et al, ; Miyatake et al, ), other methods seeking to reinforce microvessel wall integrity to prevent VEGF‐induced BBB hyperpermeability have been investigated. Previous studies have shown that elevated intracellular 3,5‐cyclic monophosphate (cAMP) levels enhance endothelial junction barriers and thus reduce permeability in peripheral microvessels (Adamson et al, ; Fu et al, ; Moore et al, ; Fukuhara et al, ; Mehta and Malik, ; Sayner, ) and in cultured BBB models (Rubin et al, ; Wolburg et al, ; Balyasnikova et al, ). Studies of intact microvessels also have shown that elevation of cAMP levels abolished the increase in the hydraulic conductivity stimulated by adenosine triphosphate in frog and hamster mesenteric microvessels (He and Curry, ) and in the solute permeability induced by VEGF in the same type of microvessels of frogs (Fu et al, ).…”