P2U-Purinergic Receptor Activation Mediates Inhibition of cAMP Accumulation in Cultured Renal Mesangial Cells

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Abstract
Extracellular ATP has been reported to exert mitogenic and contractile effects on cultured renal mesangial cells (MCs). Since it is possible that these actions involve changes in the cAMP second messenger system, we examined the effect of extracellular nucleotides on the accumulation of cAMP in rat MCs. ATP, UTP and adenosine 5’-0-(3-thio)triphosphate (ATPΓS) (100 µM) had no significant effects on baseline cAMP levels, but inhibited forskolin-stimulated accumulation of cAMP by 21-75% in the presence of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX). Maximal inhibitory effects were observed at 100 µM of ATPΓS with a threshold dose of 1 µM. ATPΓS, ATP and UTP were the most potent inhibitors indicating stimulation of the P2u receptor. The P2x agonists adenosine 5’-(Α,Β-methylene) triphosphate and adenosine 5’-(Β,Γ-methylene) triphosphate, and the P2y agonist 2-methylthio-ATP did not affect cAMP accumulation. Treatment with the P2 receptor antagonist suramin (200 µM) reduced the inhibition by 58%. The inhibitory effects of the nucleotides were significantly attenuated by preincubation with pertussis toxin (10-100 ng/ml). Inhibition of phospholipase C and protein kinase C did not prevent the inhibitory effect of the nucleotides. Inhibitors of forskolin-stimulated cAMP accumulation had different effects on DNA synthesis in cultured MCs as measured by 3H-thymidine uptake at 48 h: ATP, ATPΓS and the inhibitor of adenylyl cyclase, SQ 22536, stimulated DNA synthesis in MCs, while UTP showed no significant mitogenic effect. Agents which increased baseline levels of intracellular cAMP (forskolin, IBMX, dibutyryl-cAMP) significantly diminished DNA synthesis in MCs. The results indicate that the P2u-purinergic receptor mediates inhibition of forskolin-induced cAMP accumulation which is likely due to inhibition of adenylyl cyclase. This effect appears to be partially mediated by PTX-sensitive G proteins. While the increase in cAMP accumulation is anti-mitogenic, inhibition of cAMP accumulation by P2u receptors is not correlated with MC growth control. Thus, additional mechanisms other than inhibition of cAMP accumulation by P2u receptors are likely to be involved in the mitogenesis of extracellular ATP.

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