Stereoselectivity at the β 2 -adrenoceptor on macrophages is a major determinant of the anti-inflammatory effects of β 2 -agonists

Abstract

Previous research has shown that beta-adrenoceptor (beta-AR) agonists have potent anti-inflammatory capabilities, e.g. represented by suppression of release of the proinflammatory cytokines. Aim of this research was to determine whether the effects of beta-agonists on LPS-induced TNFalpha and IL-10 release are influenced by their different stereochemistry. In addition, the role of the beta-AR subtypes was studied. The effect of two stereoisomers of the selective beta2-AR agonist TA2005 [(R,R)- and (S,S)-] on the LPS-induced TNFalpha and IL-10 release by U937 macrophages was compared. The (R,R)-stereoisomer was 277 times more potent in inhibiting the TNFalpha release than the (S,S)-form. The (R,R)-stereoisomer also appeared to be more potent in increasing the IL-10 release. In radioligand binding studies the affinity of (R,R)-TA2005 for the beta-adrenoceptor was 755 times higher than the (S,S)-TA2005 stereoisomer. In addition, the elevation of intracellular cAMP in U937 cells appeared to be stereoselective: (R,R)-TA2005 was more potent in elevating intracellular cAMP. The effect of both stereoisomers on the LPS-induced TNFalpha release could almost completely be antagonized by preincubation with the selective beta2-AR-antagonist ICI-118551. Further evidence that the effect of the beta-agonists is mediated via the beta2-adrenoceptor subtype exclusively was acquired by incubation of U937 cells with selective beta1- and beta3-agonists. None of these receptor subtype agonists showed significant suppressive effect on TNFalpha release. This study provides additional proof that the anti-inflammatory effects of beta2-agonists are mediated via the beta2-adrenoceptor and indicates that these effects are highly dependent on the stereoselectivity of the ligand.

Endotoxin-induced liver damage in rats is minimized by � 2 -adrenoceptor stimulation

“…This observation clearly indicated that the effects of clenbuterol on systemic cytokine concentrations are mediated via the b 2 -AR, which is in accordance with previous in vitro findings [15,17,19].…”

“…In vitro studies on the mechanism of action at cellular level, not only showed a selective effect through the b 2 -AR but also an increase in intra-cellular cAMP, comparable to levels induced by phosphodiesterase-IV inhibitors. Based on comparative studies between b 2 -AR agonists in our laboratories we selected clenbuterol as the most active and potent agent in the current experimental setup [e. g. 17,18,19].…”

“…Based on these previous findings the present study was designed to test the hypothesis that the release of major actors in the inflammatory cascade, such as the cytokines TNFa , IL-1b , IL-6 and IL-10, is modulated by b 2 -AR agonists in vivo in the same way as has been demonstrated in vitro [17,19]. In addition, timing of pharmacological intervention is regarded as an important aspect in the clinical management of acute inflammatory disorders.…”

Evidence for Bidirectional Endocannabinoid Transport across Cell Membranes

“…AEA, N-(2-hydroxyethyl-1,1,2,2-d 4 )-(5Z,8Z,11Z,14Z)-eicosatetraenamide, 2-AG, (5Z,8Z,11Z,14Z)-eicosatetraenoic acid, 2-glycerol-1,1,2,3,3-d 5 ester, palmitoylethanolamide (PEA), Cell Culture-Human monocytic leukemia U937 cells were purchased from American Type Culture Collection (Manassas, VA) and were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1 g/ml fungizone (amphotericin B), 100 units/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine (all from Invitrogen). The U937 monocytic cells were differentiated into macrophages using 2 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma) for 48 h, according to standard procedures (42)(43)(44). Cells were grown in a humidified incubator at 37°C and 5% CO 2 at 37°C in humidified 5% CO 2 atmosphere.…”

Different action of IBMX, isoproterenol and rutin on orthovanadate-induced nitric oxide release in mouse macrophage cells

“…ISO and IBMX; Safrany and Shears, 1998) have been tested on orthovanadate-induced NO production in J774A.1 cells. As macrophages bear β2-adrenergic receptors (Izeboud et al, 2000), and isoproterenol is a potent superoxide anion and hydrogen peroxide scavenging agent, we expected the inhibition of NO production of VO 4 or rutin + VO 4 induced J774A.1 cells. (Koncz and Horváth, 2000).…”