Inhibition of inositol 1,4,5-trisphosphate 5-phosphatase by micromolar concentrations of disulfiram and its analogues.

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 microM [3H]Ins(1,4,5)P3 by Ins(1,4,5)P3 5-phosphatase from GH3 cells, with IC50 values (in microM), 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 microM)-stimulated inositol phospholipid breakdown in prelabelled GH3 cells was inhibited by disulfiram (IC50 values 63/52 microM respectively), FLA 46 (89/110 microM), EWP 815 (83/71 microM) and EWP 840 (220/200 microM), 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 microM). [3H]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 microM, 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.

GH4ZD10 cells expressing rat 5-HT1A receptors coupled to adenylyl cyclase are a model for the postsynaptic receptors in the rat hippocampus.

1. Vasoactive intestinal polypeptide (VIP) stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) production by cultured GH4ZD10 cells with an EC50 value of about 7 nM. The extracellularly recovered cyclic AMP predominated, and was reduced by co-incubation with 8-hydroxy-2-(di-n-propyl-amino) tetralin (8-OH-DPAT) and 5-hydroxytryptamine (5-HT), whereas dopamine (0.1-30 microM) did not reduce VIP-stimulated cyclic AMP production. 2. The responses to 5-HT and 8-OH-DPAT were blocked by (-)-alprenolol and NAN 190. The antagonism by (-)-alprenolol was competitive in nature with a pA2 value of 7.0. 3. The responsiveness of the cells to 5-HT agonists was highly dependent upon the culturing conditions used. Thus, 8-OH-DPAT inhibition of VIP (30 nM)-stimulated cyclic AMP production decreased with increasing passage number of the cells. Reduction of the zinc concentration used to promote expression of the 5-HT1A receptor gene produced a greater sensitivity of the cells to 5-HT agonists. 4. Under such conditions, the following efficacies (5-HT = 100) were found: lisuride 106, (+)-lysergic-acid diethylamide 100, 5-methoxy-N,N-dimethyltryptamine 98, RU 24949 98, 5-carboxamidotryptamine 97, (+/-)-8-OH-DPAT 90, (+)-8-OH-DPAT 87, 1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)-piperazine 86, flesinoxan 79/88, (-)-8-OH-DPAT 62, buspirone 43/50, ipsapirone 46. Spiroxatrine and spiperone had a low intrinsic activity, but reduced the response to 5-HT. These efficacies are similar to those reported in the literature for post-synaptically localized 5-HT1A receptors in the rat hippocampus. Thus, the GH4ZD10 cells serve as a useful in vitro model system for these receptors.

Antagonism by 8-hydroxy-2(di-n-propylamino)tetraline and other serotonin agonists of muscarinic M1-type receptors coupled to inositol phospholipid breakdown in human IMR-32 and SK-N-MC neuroblastoma cells.

IMR-32 and SK-N-MC cells were found to contain [3H]quinuclidinyl benzilate specific binding sites inhibited by pirenzepine in a manner suggesting the presence of both M1-type and M2-type muscarinic receptor recognition sites. Neither cell had detectable [3H]8-OH-DPAT binding sites. Carbachol stimulated the rate of inositol phospholipid breakdown in IMR-32 and SK-N-MC human neuroblastoma cells with an EC50 value of about 50 microM in both cases. Pirenzepine inhibited the carbachol (100 microM)-stimulated inositol phospholipid breakdown in both cells with Hill slopes of unity and IC50 values of 15 nM (IMR-32) and 12 nM (SK-N-MC). The 5-HT1A receptor agonist 8-OH-DPAT competitively inhibited carbachol-stimulated inositol phospholipid breakdown with pA2 values of 5.78 (IMR-32) and 5.61 (SK-N-MC). These values are consistent with the inhibitory potency of 8-OH-DPAT towards [3H]quinuclidinyl benzilate binding in these cells. The 5-HT agonists 5-MeODMT and buspirone at micromolar concentrations inhibited carbachol-stimulated breakdown in IMR-32 cells. The inhibition by 8-OH-DPAT and 5-MeODMT was not affected by preincubation with (-)alprenolol. 5-HT (10-100 microM) was without effect on either basal or carbachol-stimulated breakdown. It is concluded that IMR-32 and SK-N-MC neuroblastoma cells express muscarinic M1-type but not serotoninergic receptors coupled to phosphoinositide-specific phospholipase C. 8-OH-DPAT acts as a weak antagonist at these muscarinic receptors.