Inhibition of chemotaxis in A7r5 rat smooth muscle cells by a novel panel of inhibitors.
1. Arginine-specific ADP-ribosyltransferase (ART) activity has been implicated in white cell chemotaxis. In this study, we examined the capacity of a panel of structurally unrelated inhibitors and pseudosubstrates of ART to inhibit chemotaxis of A7r5 rat vascular smooth muscle cells in response to PDGF-BB. 2. The IC50 values for nicotinamide (12 mM) and novobiocin (165 microM) were similar to those observed for inhibition of chemotaxis by human polymorphonuclear neutrophil leucocytes (PMN), whereas vitamins K3 (IC50=22 microM) and K1 (IC50=95 microM) were less potent than previously described in PMNs. The pseudo-substrates for the enzyme (DEA-BAG, agmatine and arginine-methylester) also inhibited A7r5 chemotaxis, and in addition inhibited cell adhesion at similar concentrations. Vitamin K3 was unique among the inhibitors of ART, in that it also inhibited cell adhesion. 3. A rat ART1 transcript was amplified by rtPCR from rat skeletal muscle, and was noted to share 94% homology with the mouse ART1 cDNA sequence. No such transcript could be detected in A7r5 cells by Northern blot analysis or rtPCR. 4. Evidence for ART activity on the surface of A7r5 cells was investigated using 32P-NAD+ as substrate, and labelled membrane proteins were observed with MWt values of 116, 100, 90 and 70 kDa. Exposure of the labelled proteins to phosphodiesterase yielded 32P-AMP, and hydrolysis with NaOH yielded 32P-NAD+. These results indicated that the labelled proteins were adducts with NAD+, and not the products of ART activity. The absence of ART catalytic activity in A7r5 cells was confirmed in protocols designed to show ADP-ribosylation of agmatine. 5. We conclude that the chemotactic activity of A7r5 cells is independent of ART activity, and the mechanism whereby the novel panel of inhibitors reduced cell migration remains undefined.
Division of Medicine, Imperial College School of Medicine, Hammersmith Hospital, London.
B A
BA
Saxty
M
M
Yadollahi-Farsani
P
P
Kefalas
S
S
Paul
J
J
MacDermot
D000818:Animals; D001013:Aorta, Thoracic; D000077214:Becaplermin; D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000201:enzymology / Q000502:physiology; D008565:Membrane Proteins / Q000032:analysis; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D010761:Phosphorus Radioisotopes; D010982:Platelet-Derived Growth Factor / Q000502:physiology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D020574:Proto-Oncogene Proteins c-sis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
D008565:Membrane Proteins; D010761:Phosphorus Radioisotopes; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D000077214:Becaplermin; D011065:Poly(ADP-ribose) Polymerases