Clinical and Experimental Pharmacology and Physiology zyxwvut (1 zyxwvut 995) 22,452-454 PROCEEDINGS OF THE HBPRCA PAF ANTAGONISTS BLOCK INDUCTION OF NITRIC OXIDE SYNTHASE IN CULTURED MACROPHAGES AND VASCULAR SMOOTH MUSCLE CELLS Jane F. Arthur, Susan Shahin and Gregory J. Dusting Department zyxwvut of Physiology, University of Melbourne, Parkville, Victoria, Australia SUMMARY 1. Nitric oxide (NO) synthase inhibitors and Paf antagonists abrogate hypotension in septic shock. The latter may act by blocking intracellular transduction mechanisms in vascular smooth muscle cells and inflammatory cells. We examined the effect of Paf antagonists on expression of inducible NO synthase. 2. A murine macrophage cell line (5774.2) and rat vascular smooth muscle cells (VSMC) were stimulated with lipopoly- saccharide (LPS), either alone or in combination with Paf or Paf antagonists, BN 50739 or E-6123. 3. NO synthase activity in 5774.2 was measured by the conversion of [3H&-arginineto [3H]~-citrulline. Nitrite accumu- lation was measured in the culture medium of 5774.2 and VSM. 4. BN 50739 (lOpmol/L) and E-6123 (1 pmol/L) both re- duced the expression of calcium-independent NO synthase activity and nitrite accumulation, while Paf alone had no effect. zyxwvutsrqp 5. Inhibition of NO synthase induction by Paf antagonists might afford therapeutic benefits in the management of septic shock and possibly other cardiovascular disorders. Key words: lipopolysaccharide, macrophages, nitric oxide synthase, platelet-activating factor, Paf antagonists, vascular smooth muscle. INTRODUCTION Platelet-activating factor (Pa0 is a potent phospholipid mediator that is synthesized by inflammatory cells, such as macrophages, neutrophils and platelets, as well as by endothelial cells. Paf exhibits an extremely diverse range of actions, with roles in both inter- and intracellular cell activation (Stewart et al. 1990). Paf has been implicated in the pathology of endotoxic shock which is characterized by activation of many neuro- humoral systems including the kinin, coagulation, complement and endorphin systems, leading to a dramatic fall in blood Correspondence: Dr G. J. Dusting, Department of Physiology, University of Melbourne, Parkville, Victoria 3052, Australia. Received 22 December 1994; accepted 3 February 1995. Presented at the High Blood Pressure Research Council of Australia meeting on 8-9 December 1994. pressure, fall in peripheral resistance and multiple organ failure. Increased levels of Paf have been measured in experimental models of endotoxic shock (Doebber et al. 1985) and the intra- venous administration of Paf mimics many of the cardio- vascular features of shock (Bessin et al. 1989; Szabo et al. 1993). More recently, nitric oxide (NO), a potent vasodilator released from many cells including the vascular endothelium, inflammatory cells and vascular smooth muscle, has been found to have a key role in the profound hypotension associ- ated with endotoxic shock. NO is produced enzymatically from L-arginine by constitutive and inducible isoforms of NO syn- thase (NOS). The inducible isoform (iNOS) is expressed in many cell types in response to stimulation by lipopolysaccharide and cytokines. Excess production of NO by iNOS is thought to be the principal cause of the hypotension and hyporeactivity to vasoconstrictor agents observed in endotoxic shock (Wright et al. 1992). Inhibitors of NO have been found not only to raise blood pressure in shock but also to improve the survival of experimental animals (Wright et al. 1992). The aim of the present study was to determine whether there is a relationship between Paf and the induction of NOS in a mouse macrophage cell line (5774.2) and cultured rat vascular smooth muscle cells (VSMC). METHODS Cell culture Under aseptic conditions, cells from the mouse macrophage cell line, 5774.2, were cultured in 250 mL tissue culture flasks in complete Dulbecco's Modified Eagle Medium (DMEM), con- taining Hepes (358 mg/L) and Na2HC03 (370 mg/L), supplem- ented with 10% (v/v) fetal calf serum (FCS), 2mmol/L L- glutamine and antibiotics, streptomycin (100 pg/mL) and benzylpenicillin (100 units/ mL). The flasks were incubated at 37" C in 5% COz until the cells became confluent (approximately 3 days). Before experiments, the cells were plated in 6-well culture plates at a density of 1 zyxwv X 106 cells/well. Cell viability was > 90% as determined by Trypan blue exclusion. The plates were incubated at 37O C in 5% C02 overnight to allow time for the cells to adhere. Cells were then stimulated for 6 h with Escherichia coli lipopolysaccharide (LPS; zyx 1 pg/ mL) alone or in combination with Paf or Paf antagonists, E6123 or BN 50739. Following stimulation, the cells were washed with phos-