Reduction by inhibitors of mono(ADP-ribosyl)transferase of chemotaxis in human neutrophil leucocytes by inhibition of the assembly of filamentous actin.
1. Chemotaxis of human neutrophils is mediated by numerous agents [e.g. N-formyl-methionyl-leucyl-phenylalanine (FMLP) and platelet activating factor (PAF)] whose receptors are coupled to phospholipase C. However, the subsequent transduction pathway mediating cell movement remains obscure. We now propose involvement of mono(ADP-ribosyl)transferase activity in receptor-dependent chemotaxis. 2. Human neutrophils were isolated from whole blood and measurements were made of FMLP or PAF-dependent actin polymerization and chemotaxis. The activity of cell surface Arg-specific mono(ADP-ribosyl)transferase was also measured. Each of these activities was inhibited by vitamin K3 and similar IC50 values obtained (4.67 +/- 1.46 microM, 2.0 +/- 0.1 microM and 4.7 +/- 0.1 microM respectively). 3. There were similar close correlations between inhibition of (a) enzyme activity and (b) actin polymerization or chemotaxis by other known inhibitors of mono(ADP-ribosyl)transferase, namely vitamin K1, novobiocin, nicotinamide and the efficient pseudosubstrate, diethylamino(benzylidineamino)guanidine (DEA-BAG). 4. Intracellular Ca2+ was measured by laser scanning confocal microscopy with two fluorescent dyes (Fluo-3 and Fura-Red). Exposure of human neutrophils to FMLP or PAF was followed by transient increases in intracellular Ca2+ concentration, but the inhibitors of mono(ADP-ribosyl)transferase listed above had no effect on the magnitude of the response. 5. A panel of selective inhibitors of protein kinase C, tyrosine kinase, protein kinases A and G or phosphatases 1 and 2A showed no consistent inhibition of FMLP-dependent polymerization of actin. 6. We conclude that eukaryotic Arg-specific mono(ADP-ribosyl)transferase activity may be implicated in the transduction pathway mediating chemotaxis of human neutrophils, with involvement in the assembly of actin-containing cytoskeletal microfilaments.
Allport
J R
JR
Department of Clinical Pharmacology, Royal Postgraduate Medical School, London.
Donnelly
L E
LE
Hayes
B P
BP
Murray
S
S
Rendell
N B
NB
Ray
K P
KP
MacDermot
J
J
D036002:ADP Ribose Transferases; D000199:Actins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D000328:Adult; D002118:Calcium / Q000378:metabolism*; D002634:Chemotaxis, Leukocyte / Q000187:drug effects* / Q000502:physiology; D005260:Female; D006490:Hemostatics / Q000494:pharmacology; D006801:Humans; D008297:Male; D008875:Middle Aged; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000037:antagonists & inhibitors; D009504:Neutrophils / Q000187:drug effects*; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014812:Vitamin K / Q000494:pharmacology
D000199:Actins; D006490:Hemostatics; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D014812:Vitamin K; D009240:N-Formylmethionine Leucyl-Phenylalanine; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; D002118:Calcium