Ecto-5'-nucleotidase of cultured rat mesangial cells.
Because adenosine plays a role in the regulation of glomerular filtration rate and of the release of renin, we examined the possibility of a local source for this mediator. We found that rat cultured glomerular mesangial cells converted 5'-AMP into adenosine. The properties of the enzyme involved in the reaction were those of an ecto-5' nucleotidase: (1) the products of the reaction were generated in the extracellular fluid although no 5'-nucleotidase was released by the cells into the medium; (2) identical activities were found for cultured cells in situ and sonicated cells; (3) the diazonium salt of sulfanilic acid which is a nonpenetrating reagent inhibited up to 75% of the enzyme activity. Ecto-5'-nucleotidase activity of intact cells obeyed Michaelis-Menten kinetics. Apparent Km for 5'-AMP was 0.32 mM. 5'-UMP was a strictly competitive inhibitor. ADP exerted a very powerful inhibitory effect and behaved also as a competitive inhibitor. ATP was inhibitory both by increasing Km and by decreasing Vmax. Ecto-5'-nucleotidase was active in the absence of divalent cations. However, Mg2+, Ca2+, Co2+ and Mn2+ were stimulatory. Zn2+ and Cu2+ suppressed the activity. Concanavalin A, a plant lectin, was markedly inhibitory, suggesting that a glycoprotein moiety was necessary to express enzyme activity. Ecto-5'-nucleotidase activity was not modified during phagocytosis of serum-treated zymosan by mesangial cells. Rat cultured glomerular epithelial cells exhibited a 5'-nucleotidase activity which was 4 times lower than that of the mesangial cells in primary culture.(ABSTRACT TRUNCATED AT 250 WORDS)
Stefanovic
V
V
Inserm 64, Hôpital Tenon, Paris, France.
Savic
V
V
Vlahovic
P
P
Ardaillou
N
N
Ardaillou
R
R
D015720:5'-Nucleotidase; D000241:Adenosine / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000502:physiology; D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D002412:Cations; D002478:Cells, Cultured; D003979:Diazonium Compounds / Q000494:pharmacology; D004789:Enzyme Activation; D004847:Epithelial Cells; D005920:Glomerular Mesangium / Q000166:cytology / Q000201:enzymology* / Q000502:physiology; D007202:Indicators and Reagents; D007700:Kinetics; D008264:Macrophages / Q000166:cytology; D008297:Male; D009708:Nucleotidases / Q000032:analysis* / Q000037:antagonists & inhibitors; D010587:Phagocytosis; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D013425:Sulfanilic Acids / Q000494:pharmacology
D002412:Cations; D003979:Diazonium Compounds; D007202:Indicators and Reagents; D013425:Sulfanilic Acids; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009708:Nucleotidases; D015720:5'-Nucleotidase; D000241:Adenosine; C000469:diazobenzenesulfonic acid