INHIBITION OF INOSITOL 1,4,5-TRISPHOSPHATE 5-PHOSPHATASE BY MICROMOLAR CONCENTRATIONS OF DISULFIRAM AND ITS ANALOGS

Following a preincubation period of 10 min, disulfiram and its analogues FLA 46, FLA 63, FLA 99. EWP 815 and EWP 840 inhibited the breakdown of 10 muM [H-3]Ins(1,4,5)P3 by Ins(1,4,5)P3 5-phosphatase from GH, cells, with IC50 values (in muM), for soluble/particulate enzymes respectively, of: disulfiram, 24/24; FLA 46, 23/30; FLA 63, 24/6; FLA 99. 50/48; EWP 815, 8 6; EWP 840, 11/8. The inhibition produced by FLA 99 was time-dependent in nature, although inhibition was found in the absence of a preincubation period. EWP 815 and EWP 840 were more potent inhibitors of Ins(1,4)P2 phosphatase than of Ins(1,4,5)P3 5-phosphatase. Thyrotropin-releasing hormone (TRH; 3/100 muM)-stimulated inositol phospholipid breakdown in prelabelled GH, cells was inhibited by disulfiram (IC50 values 63/52 muM respectively), FLA46 (89/110/ muM), EWP 815 (83/71 muM) and EWP 840 (220/200 muM), without affecting basal breakdown rates. FLA 99 did not inhibit either basal or TRH-stimulated activity at any of the concentrations tested (30, 100 and 300 muM). [H-3]Ins(1,4,5)P3 binding to its cerebellar receptor was not inhibited by any of the compounds over a concentration range of 3-300 muM, although an increased level of binding was seen at high concentrations. FLA 99 and EWP 840 increased the basal intracellular Ca2+ concentration in GH3 cells, but with no corresponding effect on the Ca2+ response to TRH stimulation. These compounds did not increase the cellular permeability to Trypan Blue. but did affect cell proliferation. It is concluded that disulfiram and related compounds produce dramatic effects on Ins(1,4.5)P3 metabolism in GH3 cells.