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Endotoxin-induced desensitization of THP-1 cells is not associated with altered G protein binding or content

Departments of Physiology, Division of Clinical Pharmacology, Medical University of South Carolina, Charleston, South Carolina, USA

S.H. Ashton

Departments of Physiology, Division of Clinical Pharmacology, Medical University of South Carolina, Charleston, South Carolina, USA

M.A. Makhlouf

Departments of Physiology, Division of Clinical Pharmacology, Medical University of South Carolina, Charleston, South Carolina, USA

R. Simmons-Wagner

Departments of Physiology, Division of Clinical Pharmacology, Medical University of South Carolina, Charleston, South Carolina, USA

P.V. Halushka

Pharmacology and Medicine, Division of Clinical Pharmacology, Medical University of South Carolina, Charleston, South Carolina, USA

J.A. Cook

Departments of Physiology, Division of Clinical Pharmacology, Medical University of South Carolina, Charleston, South Carolina, USA

In rats endotoxin tolerance is characterized by decreased endotoxin-stimulated peritoneal macrophage arachidonic acid metabolism and decreased GTP binding protein function. The hypothesis that THP-1 cells can be altered in a similar manner by pretreatment with endotoxin was tested. These studies examined endotoxin's ability to stimulate eicosanoid and tumor necrosis factor (TNF) in control and desensitized THP 1 cells. Additionally, membrane GTP ³⁵S binding and Western blot analyses with specific antisera to G _i1,2, G_i3a, G_common, and the β subunit of G in control and endotoxin-desensitized THP-1 cells were assessed. Endotoxin (10 µg/ml) stimulated thromboxane (Tx) B₂ production in THP-1 cells. Pretreatment with pertussis toxin (PT), resulted in significant inhibition of TxB₂ production at concentrations not inhibited by equimolar concentrations of PT-B protomer. The latter observations suggest a role of PT-sensitive G protein in endotoxin activation of THP-1 cells. Pre-exposure to endotoxin (1 µg/ml) for 18 h desensitized THP-1 cells to endotoxin-stimulated TxB₂ production and endotoxin-stimulated TNF. To determine if endotoxin pretreatment affects G protein function, THP-1 cell membranes were isolated from endotoxin pretreated and control cells for equilibrium binding with GTP³⁵S, a nonhydrolyzable analog of GTP. Neither the total number of binding sites (B_max) nor the dissociation constant (K_d) for GTP³⁵S in desensitized THP-1 cells were significantly different from those of control cells. PT-catalyzed ADP-ribosylation of G proteins in control and LPS-desensitized THP-1 cells demonstrated no difference in the quantity of G protein labelled versus desensitized cells. Immunoblots also showed no difference between control and desensitized cells in the membrane content of specific heterotrimeric G proteins. The data demonstrate that pre-exposure to endotoxin desensitizes the cells subsequent endotoxin stimulation of mediator production. However, unlike the in vivo rat model, this is not associated with a decrease in G protein binding or content.

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