Effects of the indolocarbazole 3744W on the tyrosine kinase activity of the cytoplasmic domain of the platelet-derived growth factor beta-receptor.

The cytoplasmic domain of the platelet-derived growth factor (PDGF) beta-receptor was expressed in insect cells by using a baculovirus system. The resulting protein was a constitutively active tyrosine kinase that could phosphorylate both protein and peptide substrates. A recently identified potent and selective inhibitor of intact PDGF receptor autophosphorylation, 3744W, inhibited the autophosphorylation of the cytoplasmic domain both in vitro (IC50 1.8+/-0.12 microM) and within intact insect cells (IC50 2.0 microM). However, under identical assay conditions, 3744W did not inhibit the phosphorylation of the synthetic polymeric peptide poly(Glu4Tyr1) even at concentrations as high as 100 microM. These results suggest that, although 3744W inhibits PDGF receptor autophosphorylation directly, it can discriminate between phosphate acceptor substrates.

Selectivity of protein kinase inhibitors in human intact platelets.

The specificity of commonly used protein kinase inhibitors has been evaluated in the intact human platelet. Protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA) were activated selectively by treating platelets with phorbol dibutyrate (PDBu) or prostacyclin (PGl2). PKC activity was quantitated by measuring PDBu-specific phosphorylation of a 47,000 molecular weight protein, and PKA activity monitored by measuring prostacyclin-dependent phosphorylation of a 22,000 molecular weight protein. Staurosporine and 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7) were found to be non-specific inhibitors in the intact platelet, consistent with their effects on the isolated enzymes. Tamoxifen inhibited PKC activity (IC50 = 80 microM) but increased PKA-dependent protein phosphorylation. These results support the use of human platelets for measuring the specificity of protein kinase inhibitors and indicate that tamoxifen might have value for experimental purposes as a relatively selective PKC inhibitor.

Synthesis and biological evaluation of novel pyrazoles and indazoles as activators of the nitric oxide receptor, soluble guanylate cyclase.

Database searching and compound screening identified 1-benzyl-3-(3-dimethylaminopropyloxy)indazole (benzydamine, 3) as a potent activator of the nitric oxide receptor, soluble guanylate cyclase. A comprehensive structure-activity relationship study surrounding 3 clearly showed that the indazole C-3 dimethylaminopropyloxy substituent was critical for enzyme activity. However replacement of the indazole ring of 3 by appropriately substituted pyrazoles maintained enzyme activity. Compounds were evaluated for inhibition of platelet aggregation and showed a general lipophilicity requirement. Aryl-substituted pyrazoles 32, 34, and 43 demonstrated potent activation of soluble guanylate cyclase and potent inhibition of platelet aggregation. Pharmacokinetic studies in rats showed that compound 32 exhibits modest oral bioavailability (12%). Furthermore 32 has an excellent selectivity profile notably showing no significant inhibition of phosphodiesterases or nitric oxide synthases.

A quantitative investigation into the dependence of Ca2+ mobilisation on changes in inositol 1,4,5-trisphosphate levels in the stimulated neutrophil.

1. The coupling of N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) receptor stimulation to Ca2+ mobilisation has been investigated in the human neutrophil by measuring the concentration-effect curves for inositol 1,4,5-trisphosphate (IP3) formation and Ca2+ mobilisation. 2. fMet-Leu-Phe-dependent mobilisation of intracellular Ca2+ has been monitored in fluo-3-loaded human neutrophils by measuring increases in the cytoplasmic free Ca2+ concentration ([Ca2+]i) in the presence of extracellular EGTA. Fluo-3 was used in preference to fura-2 because it was found to be more sensitive to the high Ca2+ levels seen in stimulated neutrophils. 3. fMet-Leu-Phe induced a rapid mobilisation of intracellular Ca2+ (EC50 = 2.9 +/- 0.1 nM) and increased [Ca2+]i to a maximum of 1286 +/- 184 nM. 4. The amount of IP3 in fMet-Leu-Phe-stimulated neutrophils was determined by competition with [3H]-IP3 for a specific IP3 binding protein isolated from bovine adrenocortical microsomes. Basal IP3 levels of 13.3 +/- 2.0 pmol per 10(7) cells were increased nearly 4 fold by maximally effective concentrations of fMet-Leu-Phe. 5. The EC50 for the IP3 response (95 +/- 18 nM) was much higher than that for mobilisation of intracellular Ca2+, such that only a doubling in the concentration of IP3 was required to fully mobilise intracellular Ca2+. 6. As a result of this relationship IP3 production was more sensitive than Ca2+ mobilisation to inhibition by demethoxyviridin, an inhibitor of phospholipase activation.

Demethoxyviridin and wortmannin block phospholipase C and D activation in the human neutrophil.

1. The fungal metabolite, wortmannin, has recently been shown to inhibit fMet-Leu-Phe-stimulated superoxide production and phospholipase D (PLD) activation in the human neutrophil. 2. We have found that a close structural analogue of wortmannin, demethoxyviridin, has a similar inhibitory profile but in addition blocks phosphatidylinositol 4,5-bisphosphate-specific phospholipase C and hence inositol 1,4,5-trisphosphate (IP3) formation. 3. Inhibition of fMet-Leu-Phe-stimulated PLD by demethoxyviridin was characteristically non-competitive (IC50 = 31 +/- 10 nM). 4. Inhibition of fMet-Leu-Phe-stimulation IP3 formation required concentrations almost 10 times higher (IC50 = 250 +/- 130 nM). 5. Surprisingly, demethoxyviridin only inhibited fMet-Leu-Phe-induced intracellular calcium mobilization at concentrations 100 times greater than those needed to block IP3 formation. 6. Demethoxyviridin also inhibited PLD activation induced by sodium fluoride or phorbol myristate acetate (PMA) but the concentrations required were 100 times those needed to block fMet-Leu-Phe-stimulated PLD. 7. These observations support the contention that PLD plays an important role in signal transduction in the human neutrophil and indicate that wortmannin and demethoxyviridin inhibit PLD activation at a common step in the signalling pathway. 8. Furthermore, these results suggest that demethoxyviridin may block the interaction between the chemotactic peptide receptor and a GTP-binding protein that is intimately involved in PLD activation.

Indolocarbazoles: potent and selective inhibitors of platelet-derived growth factor receptor autophosphorylation.

A quantitative assay for measuring the autophosphorylation of platelet-derived growth factor (PDGF) receptors in intact vascular smooth muscle cells has been developed and used to screen for novel tyrosine kinase (TK) inhibitors. Several novel inhibitors of PDGF receptor autophosphorylation have been identified from the indolocarbazole series, including the 3,9 dimethoxy derivative, 3744W (IC50 = 14.5+/-2 nM). Tested against a panel of tyrosine and serine/threonine kinases, 3744W is at least 1,000 fold selective for the PDGF receptor tyrosine kinase and was found to inhibit autophosphorylation of both the alpha and beta isoforms of the PDGF receptor in human smooth muscle cells. PDGF-BB-stimulated DNA synthesis in quiescent cultures of human smooth muscle cells was blocked in a concentration-dependent manner by 3744W, IC50 = 10 nM. Binding studies showed that 3744W did not block the binding of PDGF-BB to cell surface receptors on human airway smooth muscle cells. Furthermore, inhibition of bone marrow stem cell proliferation by 3744W was only observed at concentrations 100-1,000 times greater than those needed to block PDGF-driven DNA synthesis in human smooth muscle cells. 3744W represents a novel, potent and selective inhibitor of PDGF receptor autophosphorylation and a powerful biochemical probe for investigating PDGF-dependent responses in vitro.

Scanning electron microscopy of cell-surface changes in methylnitrosurea (MNU)-treated rat bladders in vivo and in vitro.

Scanning electron microscopy has been used (1) to characterize epithelial cells of bladders from normal rats and from rats treated with a single initiating but non-carcinogenic dose of 2 mg methylnitrosurea (MNU), 24 h and 6 weeks after treatment; and (2) to compare morphological aspects of epithelial differentiation in organ culture of bladder explants taken from untreated and MNU-treated rats at these time intervals. There are marked differences in vivo between the surface organization of normal urothelium and urothelium undergoing reversible hyperplasia following MNU treatment. Maturation of the normal rat bladder epithelium in vivo is shown to be related to a series of well-defined cell-surface changes readily identified by SEM. By contrast the maturation response is perturbed in the hyperplastic epithelium; the cells lose their ability to differentiate normally and form instead an excess of stubby globular microvilli which project from the cell surface. In organ culture, maturation of normal bladder epithelium (both in re-epithelialized areas of the explant and in areas of epithelial outgrowth over cellulose acetate substrates) can be also related to a series of cell surface changes showing close similarities to those in vivo. However, epithelial maturation remains defective in organ cultures of bladders from MNU-treated animals. The closely parallel behaviour of the bladder epithelium in vivo and in vitro in both normal and treated tissues underlines the potential value of the bladder organ culture system for studying the comparative biology of hyperplastic development produced by a single initiating dose of MNU and suggests it will be useful with which to study carcinogenesis following multiple doses of MNU.

Effects of treatment with N-methyl-N-nitrosourea, artificial sweeteners, and cyclophosphamide on adult rat urinary bladder in vitro.

The effect of N-methyl-N-nitrosourea (MNU), sodium saccharin, sodium cyclamate and cyclophosphamide on rat bladder explants in vitro was studied. MNU administered as a single dose or in multiple treatments induced concentration-dependent changes in urothelial ultrastructure and cell surface topography. In a single treatment protocol, extensive cytotoxicity was observed in both the urothelium and stroma at concentrations of 500 to 1000 micrograms/ml, establishing a toxic threshold within this range. In a multiple treatment protocol, repeated doses of low concentrations of carcinogen (7 or 8 x 50 micrograms/ml, 6 x 100 micrograms/ml) induced hyperplastic and dysplastic changes in the urothelium with no cytotoxicity, but cytotoxic effects were observed following treatments of 4 x 200 micrograms/ml or 2 x 400 micrograms/ml. Sodium saccharin, sodium cyclamate, and cyclophosphamide induced changes in urothelial cell surface topography consistent with hyperplasia and preneoplasia. Prolonged exposure to saccharin or cyclamate followed by a single dose of MNU elicited more extensive abnormalities in the urothelium than either saccharin or cyclamate alone, suggesting that these artificial sweeteners have initiating activity in a multistage process. The ultrastructural changes induced by in vitro treatment showed a good correlation with the pathological changes observed in vivo in rats treated with MNU or fed either with saccharin or cyclamate.

The temporal relationship between phospholipase activation, diradylglycerol formation and superoxide production in the human neutrophil.

Fluctuations in the amounts of choline, inositol 1,4,5-trisphosphate (IP3) and diradylglycerol have been used to monitor phospholipase activation in the human neutrophil. Stimulation of human neutrophils by formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) resulted in a rapid activation of both phosphatidylinositol 4,5-bisphosphate breakdown by phospholipase C and phosphatidylcholine breakdown by phospholipase D. Diradylglycerol accumulation occurred more slowly than that of either choline or IP3 and was inhibited by 30 mM-butanol, suggesting that the bulk was derived from the phospholipase D pathway via phosphatidate phosphohydrolase. Consistent with this is the observation that choline and diradylglycerol are produced in similar amounts. 1,2-Diacylglycerol (DAG) and 1-O-alkyl-2-acyl-sn-glycerol species accumulated with different time courses, indicating that one or more steps in the phospholipase D pathway was selective for the diacyl species. Superoxide production by fMet-Leu-Phe-stimulated neutrophils paralleled DAG accumulation over the first 5 min, but thereafter this production stopped, despite the fact that DAG remained elevated. We conclude that DAG derived from the phospholipase D pathway is only one of the second messengers important in controlling this functional response.

Tyrosine phosphorylation is involved in receptor coupling to phospholipase D but not phospholipase C in the human neutrophil.

The tyrosine kinase inhibitors ST271, ST638 and erbstatin inhibited phospholipase D (PLD) activity in human neutrophils stimulated by fMet-Leu-Phe, platelet-activating factor and leukotriene B4. These compounds did not inhibit phorbol ester-stimulated PLD, indicating that they do not inhibit PLD per se, but probably act at a site between the receptor and the phospholipase. In contrast, the protein kinase C inhibitor Ro-31-8220 inhibited phorbol 12,13-dibutyrate- but not fMet-Leu-Phe-stimulated PLD activity, arguing against the involvement of protein kinase C in the receptor-mediated activation of PLD. ST271 did not inhibit Ins(1,4,5)P3 generation, but did inhibit protein tyrosine phosphorylation stimulated by fMet-Leu-Phe. The phosphotyrosine phosphatase inhibitor pervanadate increased tyrosine phosphorylation and stimulated PLD. These results suggest that tyrosine kinase activity is involved in receptor coupling to PLD but not to PtdIns(4,5)P2-specific phospholipase C in the human neutrophil.