Showing papers by "Graeme Nixon published in 2003"

Tumor Necrosis Factor-α–Induced Activation of RhoA in Airway Smooth Muscle Cells: Role in the Ca2+ Sensitization of Myosin Light Chain20 Phosphorylation

Abstract: Tumor necrosis factor-alpha (TNF), an inflammatory cytokine, has a potentially important role in the pathogenesis of bronchial asthma and may contribute to airway hyper-responsiveness Recent evidence has revealed that TNF can increase the Ca(2+) sensitivity of agonist-stimulated myosin light chain(20) (MLC(20)) phosphorylation and contractility in guinea pig airway smooth muscle (ASM) In the present study, the potential intracellular pathways responsible for this TNF-induced Ca(2+) sensitization were investigated In permeabilized cultured guinea pig ASM cells, recombinant human TNF stimulated an increase in Ca(2+)-activated MLC(20) phosphorylation under Ca(2+) "clamp" conditions This increased MLC(20) phosphorylation was inhibited by preincubation with the Rho-kinase inhibitor Y27632 TNF also increased the proportion of GTP-bound RhoA, as measured using rhotekin Rho-binding domain, in a time course compatible with a role in the TNF-induced Ca(2+) sensitization In cultured human ASM cells, recombinant human TNF also activated RhoA with a similar time course In addition, TNF stimulated phosphorylation of the regulatory subunit of the myosin phosphatase, which was inhibited by Y27632 Although human ASM cells expressed both receptor subtypes, TNF-R1 and TNF-R2, the activation of RhoA was predominantly via stimulation of the TNF-R1, although RhoA did not immunoprecipitate with the TNF-R1 In conclusion, the TNF-induced increase in the Ca(2+) sensitivity of MLC(20) phosphorylation is through stimulation of the TNF-R1 receptor and via a RhoA/Rho-kinase pathway leading to inhibition of the myosin light chain phosphatase This intracellular mechanism may contribute to TNF-induced airway hyper-responsiveness