ACTIVATION OF PURINERGIC P2Y2 RECEPTORS INHIBITS INDUCIBLE NO SYNTHASE IN CULTURED RAT MESANGIAL CELLS

Cytokine-induced nitric oxide (NO) is produced on glomerular inflammat ion. Glomerular injury and thrombocyte aggregation result in the relea se of nucleotides, which may regulate induced NO synthesis in cultured rat mesangial cells (MCs). ATP (10(-3) M) inhibited 24-h nitrite prod uction induced by lipopolysaccharide (LPS, 10 mu g/ml)/interferon-gamm a (IFN-gamma, 100 U/ml) by 48.2 +/- 6.3%, as well as induction of indu cible NOS (iNOS) protein and mRNA. Also, coincubation with either 10(- 4) M of UTP, ATP, or ATP gamma S inhibited LPS/IFN-gamma-induced nitri te production by 29.9 +/- 5.8, 36.4 +/- 4.3, and 50.3 +/- 6.5%, respec tively, indicating involvement of purinergic P2Y2 receptors. Correspon dingly, cultured MCs expressed P2Y2 receptor mRNA. Agonists for other purinergic receptors [alpha, beta-methylene-ATP, 3'-O-(4-benzoyl)-benz oyl-ATP, 2-methylthio-ATP, ADP, UDP, adenosine] were ineffective. Trea tment with the protein kinase C (PKC) activator phorbol 12-myristate 1 3-acetate (PMA, 10(-8) M) reproduced the inhibitory effect of ATP on i NOS protein expression and nitrite inhibition (by 46.6 +/- 10.4%). The effect of ATP or PMA was reversed by the PKC inhibitors Re-31-8220 (1 0(-8) M) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (10(-5) M) , indicating that suppression of iNOS is mediated via activation of PK C through stimulated P2Y2 receptors. In conclusion, the release of pur ine mediators may play a critical role for iNOS expression and synthes is of NO during glomerular inflammatory disorders.