EFFECT OF NUCLEOTIDES ON THE CYTOSOLIC FREE CALCIUM ACTIVITY AND INOSITOL PHOSPHATE FORMATION IN HUMAN GLOMERULAR EPITHELIAL-CELLS

1 Glomerular epithelial cells (GEC) were cultured from human kidneys and immunologically characterized. 2 The effect of extracellular nucleotides on the cytosolic free calcium activity [Ca2+]i was investigated with the fura-2 microfluorescence method. Extracellular UTP, UDP, UMP, ATP, adenosine 5'-O-(3-thio)-trisphosphate (ATP-gamma-S), inosine-triphosphate (ITP), guanyltriphosphate (GTP), 2-methylthio-ATP, AMP, alpha,beta-methylene-ATP and adenosine led to a rapid, transient, concentration-dependent increase of [Ca2+]i, followed by a plateau above the baseline level. 3 In a calcium-free extracellular solution, the rapid increase of [Ca2+]i was still present, whereas the plateau level was abolished. 4 ATP and UTP (ED, both: 10(-5) M) stimulated inositol trisphosphate (InsP3) formation in GEC. 5 The order of potency for the purine nucleotides in stimulating InSP3 formation was ATP = ATP-gamma-S > ADP > 2-methylthio-ATP > AMP = alpha,beta methylene-ATP = adenosine. 6 The increase of InSP3 induced by ATP (10(-5) M) could be inhibited by the P2 receptor blocker suramin (> 10(-4) M). Reactive blue 2 exhibited a weak stimulating effect on the InsP3 formation and only a weak inhibitory effect at a concentration of 10(-3) m was observed. 7 Protein kinase C activation by preincubation of GEC with phorbol 12-myristate 13-acetate (PMA, 100 ng ml-1, 15 min) abolished the effect of ATP (10(-5) m) on InsP, formation. Downregulation of protein kinase C by long term incubation (18 h) with PMA had no significant effect on the phosphoinositol turnover induced by ATP. 8 The results indicate that an increase of [Ca2+]i and inositol phosphate breakdown can be mediated via activation of a P2 receptor in human GEC.