Biochemical basis for a cholesterol-lowering activity of 2-[2"-(1",3"-dioxolane)]-2-methyl-4-(2'-oxo-1'-pyrrolidinyl)-6- nitro-2H-1-benzopyran (SKP-450), a novel antihypertensive agent

Administration (p.o.) of SKP-450, 2-[2"-(1",3"-dioxolane)]-2-methyl-4-(2'-oxo-1'-pyrrolidinyl)-6-nitro-2H- 1-benzopyran, a novel antihypertensive agent, to hypercholesterolemic Syrian hamsters led to a significant reduction in plasma lipids in a dose-dependent manner, i.e., a 10.8% to 29% reduction in low-density lipoprotein cholesterol at doses of 0.3 to 10 mg/kg of SKP-450. SKP-450 was found to specifically inhibit the hepatic microsomal lanosterol 14alpha-methyl demethylase (14alpha-DM) in a competitive manner (Ki:2.65 microM). Furthermore, a dose-dependent decrease in the 14alpha-DM activity by SKP-450 parallelled the cholesterol synthetic rate in vitro in both the rat hepatic S10 fractions (supernatants at 10,000 g; IC50:20 microM) and Chinese hamster ovary cells (IC50:23 microM). However, this phenomenon was not seen in AR45 cells, which are deficient in 14alpha-DM, suggesting that 14alpha-DM is the major target for the inhibitory action of SKP-450 in regard to cholesterol biosynthesis.     

Induction of intracellular ceramide by interleukin-1 beta in oligodendrocytes

The sphingomyelin pathway has been implicated in mediating the effect of several extracellular agents leading to important biochemical and cellular changes. The aim of this investigation is to study interleukin-1 beta (IL-1 beta) signaling in oligodendrocytes. For this purpose, the CG4 oligodendrocyte cells were differentiated and incubated with IL-1 beta. This treatment induced a time- and dose-dependent increase of the endocellular ceramide. To mimic the effect of the elevation of endogenous ceramide, the CG4 cells were treated with the ceramide analogue C2-ceramide. Cell survival, measured with the MTT assay, showed that, by increasing the concentration of ceramide, up to 40% of CG4 cells were dying within 6 h, similar data were obtained with the primary differentiated oligodendrocytes. Condensation of chromatin, nuclear fragmentation, and formation of apoptotic bodies indicated that apoptosis was the cause of death. Surprisingly, long-term exposure (72 h) to increasing concentrations of IL-1 beta, which increases intracellular ceramide, did not induce oligodendroglial cell death. These results show that an increase of intracellular ceramide is not sufficient to induce apoptosis in oligodendrocytes and that IL-1 beta signaling through the ceramide pathway in these cells can mediate functions other than programmed cell death.     

Thromboxane A2 synthase inhibition and thromboxane A2 receptor blockade by 2-[(4-cyanophenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15) in rat platelets

The effects of 2-[(4-acetylphenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15), a synthetic 1,4-naphthoquinone derivative, on platelet activity and its mechanism of action were investigated. NQ-Y15 caused a concentration-dependent inhibition of the aggregation induced by thrombin, collagen, arachidonic acid (AA), and A23187. The IC50 values of NQ-Y15 on thrombin (0.1 U/mL)-, collagen (10 microg/mL)-, AA (50 microM)-, and A23187 (2 microM)-induced aggregation were 36.2 +/- 1.5, 6.7 +/- 0.7, 35.4 +/- 1.7, and 93.1 +/- 1.4 microM, respectively. NQ-Y15 also inhibited thrombin-, collagen-, AA-, and A23187-stimulated serotonin secretion in a concentration-dependent manner. However, a high concentration (100 microM) of NQ-Y15 showed no significant inhibitory effect on ADP-induced primary aggregation, which is independent of thromboxane A2 (TXA2) production in rat platelets. In fura-2-loaded platelets, the elevation of intracellular free calcium concentration stimulated by AA, thrombin, and 4-bromo-A23187 was inhibited by NQ-Y15 in a concentration-dependent manner. The formation of TXA2 caused by AA, thrombin, and collagen was inhibited significantly by NQ-Y15. NQ-Y15 inhibited TXA2 synthase in intact rat platelets, since this agent reduced the conversion of prostaglandin (PG) H2 to TXA2. Similarly, NQ-Y15 selectively inhibited the TXA2 synthase activity in human platelet microsomes, whereas it had no effect on activity of phospholipase A2, cyclooxygenase, and PGI2 synthase in vitro. NQ-Y15 inhibited platelet aggregation induced by the endoperoxide analogue U46619 in human platelets, indicating TXA2 receptor antagonism, possibly of a competitive nature. These results suggest that the antiplatelet effect of NQ-Y15 is due to a combination of TXA2 synthase inhibition with TXA2 receptor blockade, and that it may be useful as an antithrombotic agent.     

Inhibitory effect of 9-(2-phosphonylmethoxyethyl)-adenine (PMEA) on human and duck hepatitis B virus infection

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) was evaluated for its inhibitory effect on hepadnavirus replication in three different cell systems, i.e., human hepatoma cell lines HepG2 2.2.15 and HB611 (transfected with human hepatitis B virus (HBV)) and primary cultures of duck hepatocytes infected with duck hepatitis B virus (DHBV). PMEA inhibited HBV release from HepG2 2.2.15 cells and HB611 cells at a 50% inhibitory concentration (IC50) of 0.7 and 1.2 microM, respectively. Intracellular viral DNA synthesis was inhibited at concentrations equivalent to those required to inhibit virus release from the cells. DHBV secretion from duck hepatocytes was inhibited by PMEA at an IC50 of 0.2 microM. HBsAg secretion was inhibited by PMEA in a concentration-dependent manner in HB611 cells and DHBV-infected duck hepatocytes but not HepG2 2.2.15 cells. The 50% cytotoxic concentration, as measured by inhibition of [3H-methyl]deoxythymidine incorporation was 150 microM for the two human hepatoma cell lines and 40 microM for the duck hepatocyte cultures. In a pilot experiment PMEA was found to reduce the amounts of DHBV DNA in the serum of Pekin ducks.     

Inhibition of nicotinic responses of bovine adrenal chromaffin cells by the protein kinase C inhibitor, Ro 31-8220

1. The effects of the protein kinase C inhibitor, Ro 31-8220, on the responses of cultured bovine adrenal chromaffin cells to nicotine, phorbol 12, 13-dibutyrate (PDBu) and K+ have been investigated. 2. Tyrosine hydroxylase activity was measured in situ in intact cells by measuring 14CO2 evolved following the hydroxylation and rapid decarboxylation of [14C]-tyrosine offered to the cells. Secretion of endogenous adrenaline and noradrenaline was measured by use of h.p.l.c. with electrochemical detection. Cyclic AMP levels were measured in cell extracts by RIA. 3. Ro 31-8220 produced a concentration-dependent inhibition of 300 nM PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 2 microM and complete inhibition at 10 microM. It had no effect on the responses to forskolin. 4. Ro 31-8220 produced a concentration-dependent inhibition of 5 microM nicotine-stimulated tyrosine hydroxylase activity, adrenaline and noradrenaline secretion and cellular cyclic AMP levels, with an IC50 of about 3 microM and complete inhibition by 10 microM. At concentrations up to 10 microM, Ro 31-8220 had little or no effect on the corresponding responses to 50 mm K+. 5. A structural analogue of Ro 31-8220, bisindolylmaleimide V, that lacks activity as a protein kinase C inhibitor, had no effect up to 10 microM on PDBu-stimulated tyrosine hydroxylase activity or on nicotine-stimulated cyclic AMP levels or noradrenaline secretion and only marginal inhibitory effects on nicotine-stimulated tyrosine hydroxylase activity and adrenaline secretion. 6. A structurally related protein kinase C inhibitor, bisindolylmaleimide I, inhibited PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 1 microM and complete inhibition by 3 microM, but had essentially no effect on nicotine stimulated tyrosine hydroxylase activity or catecholamine secretion. 7. The results suggest that Ro 31-8220 is not only a protein kinase C inhibitor but is also a potent inhibitor of nicotinic receptor responses in adrenal chromaffin cells by a mechanism unrelated to protein kinase C inhibition. The results are consistent with Ro 31-8220 being a nicotinic receptor antagonist.     

Involvement of human CYP1A isoenzymes in the metabolism and drug interactions of riluzole in vitro

Cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) isoenzymes involved in riluzole oxidation and glucuronidation were characterized in (1) kinetic studies with human hepatic microsomes and isoenzyme-selective probes and (2) metabolic studies with genetically expressed human CYP isoenzymes from transfected B-lymphoblastoid and yeast cells. In vitro incubation of [14C]riluzole (15 microM) with human hepatic microsomes and NADPH or UDPGA cofactors resulted in formation of N-hydroxyriluzole (K(m) = 30 microM) or an unidentified glucuroconjugate (K(m) = 118 microM). Human microsomal riluzole N-hydroxylation was most strongly inhibited by the CYP1A2 inhibitor alpha-naphthoflavone (IC50 = 0.42 microM). Human CYP1A2-expressing yeast microsomes generated N-hydroxyriluzole, whereas human CYP1A1-expressing yeast microsomes generated N-hydroxyriluzole, two additional hydroxylated derivatives and an O-dealkylated derivative. CYP1A2 was the only genetically expressed human P450 isoenzyme in B-lymphoblastoid microsomes to metabolize riluzole. Riluzole glucuronidation was inhibited most potently by propofol, a substrate for the human hepatic UGT HP4 (UGT1.8/9) isoenzyme. In vitro, human hepatic microsomal hydroxylation of riluzole (15 microM) was weakly inhibited by amitriptyline, diclofenac, diazepam, nicergoline, clomipramine, imipramine, quinine and enoxacin (IC50 approximately 200-500 microM) and cimetidine (IC50 = 940 microM). Riluzole (1 and 10 microM) produced a weak, concentration-dependent inhibition of CYP1A2 activity and showed competitive inhibition of methoxyresorufin O-demethylase. Thus, riluzole is predominantly metabolized by CYP1A2 in human hepatic microsomes to N-hydroxyriluzole; extrahepatic CYP1A1 can also be responsible for the formation of several other metabolites. Direct glucuronidation is a relatively minor metabolic route. In vivo, riluzole is unlikely to exhibit significant pharmacokinetic drug interaction with coadministered drugs that undergo phase I metabolism.     

Differential mechanism of cytostatic effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, and other antiherpetic drugs on tumor cells transfected by the thymidine kinase gene of herpes simplex virus type 1 or type 2

After they have been transfected with the herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) thymidine kinase (TK) gene murine mammary carcinoma (FM3A) cells become highly sensitive to the growth inhibitory properties of the antiherpetic agents (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), 9(-)[(2-hydroxyethoxy)methyl]guanine (acyclovir, ACV), 9(-)[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, ganciclovir), and 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU). BVDU was 100-fold more potent an inhibitor of HSV TK gene-transfected tumor cell growth (50% inhibitory concentration (IC50), 0.0020-0.0047 microM) than FMAU or DHPG (IC50, 0.051-0.277 microM) and 1000-fold more potent than ACV (IC50, 0.42-4.9 microM). As a rule, the test compounds were more cytostatic to HSV-2 TK than HSV-1 TK gene-transfected FM3A cells. This may be ascribed to the higher phosphorylating capacity (Vmax/Km) of HSV-2 TK than HSV-1 TK and/or to the higher TK enzyme levels of the HSV-2 TK gene-transfected FM3A cells than the HSV-1 TK gene-transfected FM3A cells. Thymidylate synthase of the HSV TK gene-transfected FM3A cells appears to be the target enzyme for the cytostatic action of BVDU, but not FMAU, DHPG, or ACV. Instead, the cytostatic activity of DHPG seems to be correlated with its conversion to the triphosphate form and subsequent incorporation into the DNA of HSV TK gene-transfected FM3A cells.     

The (8-thiotheophyllinate) (triphenylphosphine) gold (I), (tTAuP): a new gold complex as an anticancer agent

In continuous Friend leukemia cell exposure to tTAuP, the IC50 was 0.2 microM whereas in cells exposed 15 or 60 min to tTAuP followed by 72 h in drug-free medium the IC50 was 2.2 and 1.3 microM respectively. A combination of tTAuP and cisplatin (CDDP) is shown to be more active than either agent alone. Intracellular accumulation studies analysed by SXRF have shown that to achieve a cytotoxic effect, large concentrations of gold are necessary to accumulate in both the nuclear and cytoplasmic fraction. However, at cytostatic doses (1 microM), tTAuP has no effect on the cell cycle but does affect DNA synthesis. At a higher dose, greater than that necessary to induce cytotoxicity, it produces DNA damage, as observed by alkaline elution method. When cells were exposed to toxic doses of tTAuP (10 microM) in the presence of albumin, cytotoxicity was significantly reduced. Similar results were obtained when cells were co-treated with L-cysteine, dithiothreitol or reduced gluthatione. Reduced cytotoxic effect can be related to the interaction with free thiol groups. According to these data it is concluded that tTAuP is highly effective in vitro; whether it is active in vivo remains to be determined.     

Ceramide accumulation during oxidant renal tubular injury: mechanisms and potential consequences

Ceramide is an important signaling molecule that is typically generated via sphingomyelinase (SMase)-mediated sphingomyelin (SM) hydrolysis. Although diverse forms of renal injury elicit ceramide accumulation, the molecular determinants of this change and its contribution to tissue damage are poorly defined. The present study uses iron (Fe/hydroxyquinoline)-mediated injury of cultured human proximal tubular (HK-2) cells to gain additional insights into these issues. A 4-h Fe exposure doubled ceramide levels in the absence of cell death. This was independent of de novo synthesis, since ceramide synthase inhibition (with fumonisin B1) had no effect. Oxidant stress directly suppressed, rather than stimulated, SMase activity by: (1) decreasing SMase levels; (2) depleting SMase-stimulating glutathione; and (3) increasing SM resistance to SMase attack. Fe suppressed cell sphingosine levels (3 to 4 times ceramide/sphingosine ratio increments), suggesting a possible ceramidase block. Fe did not directly affect HK-2 ceramidase levels. However, arachidonic acid (C20:4) accumulation, a consequence of oxidant-induced phospholipase A2 (PLA2) activation, markedly suppressed ceramidase and stimulated SMase activity. Exogenous C20:4, as well as PLA2 (in doses simulating Fe-induced deacylation) recapitulated Fe's ceramide-generating effect. Because C20:4 is directly cytotoxic, it was hypothesized that ceramide might offset some of C20:4's adverse effects. Supporting this possibility were the following: (1) C20:4 exacerbated Fe toxicity; (2) this was abrogated by ceramide treatment; and (3) ceramide blunted Fe-mediated cell death. Conclusions: (1) ceramide accumulation during acute cell injury can be an adaptive response to PLA2 activation/C20:4 generation; (2) C20:4-induced ceramidase inhibition, coupled with SMase stimulation, may trigger this result; and (3) these ceramide increments may exert a "biostat" function, helping to offset C20:4/PLA2- and "catalytic" iron-mediated tubular cell death.     

CVT-313, a specific and potent inhibitor of CDK2 that prevents neointimal proliferation

The activity of cyclin-dependent kinase 2 (CDK2) is essential for progression of cells from G1 to the S phase of the mammalian cell cycle. CVT-313 is a potent CDK2 inhibitor, which was identified from a purine analog library with an IC50 of 0.5 microM in vitro. Inhibition was competitive with respect to ATP (Ki = 95 nM), and selective CVT-313 had no effect on other, nonrelated ATP-dependent serine/threonine kinases. When added to CDK1 or CDK4, a 8.5- and 430-fold higher concentration of CVT-313 was required for half-maximal inhibition of the enzyme activity. In cells exposed to CVT-313, hyperphosphorylation of the retinoblastoma gene product was inhibited, and progression through the cell cycle was arrested at the G1/S boundary. The growth of mouse, rat, and human cells in culture was also inhibited by CVT-313 with the IC50 for growth arrest ranging from 1.25 to 20 microM. To evaluate the effects of CVT-313 in vivo, we tested this agent in a rat carotid artery model of restenosis. A brief intraluminal exposure of CVT-313 to a denuded rat carotid artery resulted in more than 80% inhibition of neointima formation. These observations suggest that CVT-313 is a promising candidate for evaluation in other disease models related to aberrant cell proliferation.     

Turnover of endogenous ceramide in cultured normal and Farber fibroblasts

De novo synthesis and turnover of endogenous ceramide in cultured skin fibroblasts from patients affected with Farber lipogranulomatosis were studied by biosynthetical labeling of cellular sphingolipids with [14C]serine. The cellular uptake of [14C]serine and incorporation into de novo synthesized ceramide was similar in normal and Farber fibroblasts, with a half life of newly synthesized ceramide of 2.7 h in normal and diseased cells. Newly synthesized ceramide was found to be channeled directly into biosynthesis of complex sphingolipids rather than contributing to the pool of accumulated ceramide in Farber fibroblasts. The degradation of ceramide generated by the catabolism of complex sphingolipids in Farber cells was greatly delayed compared with control fibroblasts, with differences in the amount of radiolabeled cellular ceramide becoming evident after 6 h chase time. Individual Farber cell lines differed from each other in the amount of accumulated ceramide; however, no correlation was found between ceramide accumulation and residual acid ceramidase activity as determined in vitro. In addition, the amount of radiolabeled sphingomyelin was significantly increased in Farber fibroblasts suggesting a delayed degradation of this compound in this ceramide storage disorder. We propose biosynthetical labeling of endogenous ceramide with [14C]serine, in addition to other established methods, as a highly sensitive and reliable method for the diagnosis of Farber disease, allowing semiquantitative measurement of ceramide accumulation in cultured skin fibroblasts of patients affected with Farber lipogranulomatosis.     

Synthesis and calcium antagonistic activity of 8-[N-[2-(3,4-dimethoxy- phenyl)ethyl]-beta-alanyl]-5,6,7,8-tetrahydrothieno[3,2-b][1,4]thiazepi ne fumarate

KT-362 is an antiarrhythmic and antihypertensive agent with vasodilating activity. Since it carries a homoveratryl group in the side chain, an obvious relation exists to the verapamil-type calcium antagonists. Replacement of the fused aromatic moiety in KT-362 with thiophene provided 8-[N-[2-(3,4-dimethoxyphenyl) ethyl]-beta-alanyl]-5,6,7,8-tetrahydrothieno[3,2-b][1,4] thiazepine (1). Compound 1 shows a negative chronotropic activity in spontaneously beating right atria (IC50 = 23 microM, n = 7), and a negative inotropic effect in papillary muscles (IC50 = 2.7 microM, n = 7) and left atria (IC50 = 4 microM, n = 6) of the guinea-pig heart. The decrease of contractility in papillary muscles could be antagonized by increasing the extracellular calcium concentration. Compound 1 was found to affect high (IC50: 70 +/- 5 microM) and low (IC50: 129 +/- 34 microM) voltage-activated calcium channel currents as well as voltage-activated sodium channel currents (IC50: 80 +/- 13 microM) in chick dorsal root ganglion neurons. In addition nicotine-induced currents were potently inhibited (IC50: 6 +/- 0.7 microM) in bovine chromaffin cells.     

Role of ceramide in cellular senescence

Recently the sphingomyelin cycle, involving the hydrolysis of membrane sphingomyelin by an activated sphingomyelinase to generate ceramide, has emerged as a key pathway in cell differentiation and apoptosis in leukemic and other cell types. Here we investigate a role for this pathway in the senescence of WI-38 human diploid fibroblasts (HDF). We found that endogenous levels of ceramide increased considerably (4-fold) and specifically (compared with other lipids) as cells entered the senescent phase. Investigation of the mechanism of increased ceramide led to the discovery that neutral sphingomyelinase activity is elevated 8-10 fold in senescent cells. There were no changes in sphingomyelinase activity or ceramide levels as HDF entered quiescence following serum withdrawal or contact inhibition. Thus, the activation of the sphingomyelinase/ceramide pathway in HDF is due to senescence and supports the hypotheses that senescence represents a distinct program of cell development that can be differentiated from quiescence. Additional studies disclosed the ability of ceramide to induce a senescent phenotype. Thus, when exogenous ceramide (15 microM) was administered to young WI-38 HDF, it produced endogenous levels comparable to those observed in senescent cells (as determined by metabolic labeling studies). Ceramide concentrations of 10-15 microM inhibited the growth of young HDF and induced a senescent phenotype by its ability to inhibit DNA synthesis and mitogenesis. These concentrations of ceramide also induced retinoblastoma dephosphorylation and inhibited serum-induced AP-1 activation in young HDF, thus recapitulating basic biochemical and molecular changes of senescence. Sphingomyelinase and ceramide may thus be implicated as mediators of cellular senescence.     

Lobeline and nicotine evoke [3H]overflow from rat striatal slices preloaded with [3H]dopamine: differential inhibition of synaptosomal and vesicular [3H]dopamine uptake

Lobeline is currently being developed as a substitution therapy for tobacco smoking cessation. Activation of CNS dopamine (DA) systems results in the reinforcing properties of nicotine. The present study compared the effects of lobeline and nicotine on rat striatum. Both lobeline and nicotine evoked [3H]overflow from striatal slices superfused in the presence of pargyline and nomifensine in the buffer. Marked DA depletion (42-67%) and a concomitant 2-fold increase in dihydroxyphenylacetic acid (DOPAC) in slices superfused with high concentrations (30-100 microM) of lobeline were observed. The effect of nicotine (10 microM) was inhibited in a concentration-dependent manner by mecamylamine (1-100 microM). However, lobeline (0.1-100 microM)-evoked [3H]overflow was calcium-independent, and was not antagonized by mecamylamine (1-100 microM), suggesting a mechanism of action other than stimulation of nicotinic receptors. Lobeline inhibited [3H]DA uptake into synaptosomes (IC50 = 80 +/- 12 microM) and vesicles (IC50 = 0.88 +/- 0.001 microM), whereas nicotine (< or =100 microM) did not inhibit synaptosomal or vesicular [3H]DA uptake. In the absence of pargyline and nomifensine in the buffer, endogenous DA was detected in superfusate only in those slices exposed to the highest concentration (100 microM) of lobeline. However, endogenous DOPAC concentration was increased in a concentration-dependent manner, indicating that lobeline exposure resulted in increased cytosolic DA which was rapidly metabolized to DOPAC. Under these conditions, lobeline (10-100 microM) also significantly depleted (66-85%) DA content; however, no change in DOPAC content was observed. The results suggest that, unlike nicotine, lobeline increases DA release by potent inhibition of DA uptake into synaptic vesicles, and a subsequent alteration in presynaptic DA storage.     

(R,R)-2,2'-[1,2-ethanediylbis[imino(1-methyl-2,1-ethanediyl)]]- bis[5-nitro-1H-benz[de]isoquinoline-1,3-(2H)-dione] dimethanesulfonate (DMP 840), a novel bis-naphthalimide with potent nonselective tumoricidal activity in vitro

(R,R)-2,2'-[1,2-ethanediylbis[imino(1-methyl-2,1-ethanediyl)]]- bis[5-nitro-1H-benz[de]isoquinoline-1,3-(2H)-dione] dimethanesulfonate (DMP 840), is a bis-naphthalimide anticancer tumoricidal agent currently in phase I clinical trials. DMP 840 exhibits curative activity in human tumor xenografts, where it shows selectivity for human solid tumors over murine leukemias. In contrast to the selectivity found for DMP 840 in vivo, DMP 840 exhibits potent antiproliferative activity in vitro against a variety of human and murine leukemia and solid tumor cell lines in culture, with inhibitory doses that reduce the number of treated cells to one half (IC50) values ranging from 2.3 to 53 nM. DMP 840 was growth inhibitory to three doxorubicin-resistant cell lines with IC50 values also in the nanomolar range. Clonogenic survival experiments showed that DMP 840 was equally cytotoxic to both exponentially growing and quiescent human clone A colon carcinoma cells. A 1-h incubation of DMP 840 (1.22-12 microM) caused 5-log cell kill in KB-3-1 human epidermoid carcinoma, clone A human colon carcinoma, and L1210 murine leukemia cell lines. The rapid cell killing by DMP 840 in clonogenic survival experiments and initial mechanism of action studies was consistent with a DNA-interactive mechanism for DMP 840 cytotoxicity. Mechanism of action studies in L1210 leukemia cells demonstrated that DMP 840 inhibited the incorporation of thymidine and uridine into DNA and RNA with IC50 values of 0.55 and 0.08 microM, respectively. DMP 840 produced DNA single-strand breaks in a dose-dependent manner. Double-strand breaks were not observed with DMP 840 treatment, even at higher concentrations of compound. Chinese hamster ovary (CHO) and P388 cells resistant to camptothecin and containing a mutant form of topoisomerase I were also used to evaluate whether DMP 840 was cross-resistant with agents active against topoisomerase I. While the CHOR line was 163-fold resistant to camptothecin, the CHOR line was only 1.7-fold resistant to DMP 840. In summary, DMP 840 is a DNA-interactive agent that demonstrates excellent antiproliferative activity in vitro against cultured tumor cells from both human and murine sources. Its mechanism of tumoricidal activity may be novel.     

UA(1) breaking and scalar mesons in the Nambu and Jona-Lasinio model

The effects of the nicotinic agonists acetylcholine, (+)-anatox in-a, carbachol, cytisine, dimethylphenylpiperazinum, (+)-epibatidine, (-)-epibatidine, methylcarbachol, D-nicotine, L-nicotine, and tetramethylammonium on 86Rb+ efflux from mouse thalamic synaptosomes were investigated. All 11 agonists evoked a concentration-dependent stimulation of 86Rb+ efflux as well as a time- and concentration-dependent reduction of response (desensitization). The agonists varied widely in potency, efficacy and rate of desensitization. (+)-Epibatidine was the most potent agonist (EC50 = 10 nM), whereas tetramethylammonium was the least potent (EC50 = 65 microM). The agonists containing a quaternary ammonium group were generally more efficacious than the other agonists, except for both of the enantiomers of epibatidine, which stimulated 86Rb+ efflux at least as well as acetylcholine. Cytisine was the least efficacious compound tested with a maximal response approximately 10% that of (-)-epibatidine. Exposure of the thalamic synaptosomes to agonist concentrations that generally stimulated little or no efflux reduced in a concentration-dependent manner a subsequent response to 10 microM nicotine. The IC50 values for this functional blockade (desensitization) were highly correlated with the Ki values for the inhibition of [3H]nicotine binding. Furthermore, exposure of the thalamic synaptosomes to 300 nM L-nicotine reduced the responses evoked by a subsequent exposure to a stimulating concentration of all 11 agonists. The observation of desensitization by both stimulating and substimulating concentrations of each agonist is consistent with the predictions of the two-state model of Katz and Thesleff.     

ZD1542, a potent thromboxane A2 synthase inhibitor and receptor antagonist in vitro

1. The thromboxane A2 synthase (TXS) inhibitory activity and the thromboxane A2 (TP)-receptor blocking action of ZD1542 (4(Z)-6-[2S,4S,5R)-2-[1-methyl-1-(2-nitro-4-tolyloxy)ethyl]-4-(3- pyridyl)-1,3-dioxan-5-yl]hex-4-enoic acid) has been evaluated in vitro on platelets and whole blood from a range of species including man. Antagonist activity has also been investigated in vascular and pulmonary smooth muscle preparations in vitro. 2. ZD1542 caused concentration-dependent inhibition of human platelet microsomal thromboxane B2 (TXB2) production in vitro (IC50 = 0.016 microM); this inhibition was associated with an increase in prostaglandin E2 (PGE2) and PGF2 alpha formation. 3. ZD1542 also inhibited collagen-stimulated TXS in human, rat and dog whole blood giving IC50 values of 0.018, 0.009 and 0.049 microM respectively. The drug did not modify platelet cyclo-oxygenase activity as inhibition of TXB2 formation was associated with a concomitant increase in the levels of PGD2, PGE2 and PGF2 alpha. ZD1542 had little if any effect against cultured human umbilical vein endothelial cell (HUVEC) cyclo-oxygenase (IC50 > 100 microM) and prostacyclin (PGI2) synthase (IC50 = 18.0 +/- 8.6 microM). 4. ZD1542 caused concentration-dependent inhibition of U46619-induced aggregation responses of human, rat and dog platelets yielding apparent pA2 values of 8.3, 8.5 and 9.1 respectively. The drug was selective as, at concentrations up to 100 microM, it did not modify 5-hydroxytryptamine (5-HT) or the primary phases of adenosine diphosphate (ADP) and adrenaline-induced aggregation. Furthermore, ZD1542 (100 microM) modified only weakly the platelet effects of PGD2, PGE1 and PGI2. 5. ZD1542 also caused concentration-dependent inhibition of U46619-mediated contractions of rat thoracic aorta, guinea-pig trachea and lung parenchyma preparations giving apparent pA2 values of 8.6,8.3 and 8.5 respectively. At concentrations approaching three orders of magnitude greater than those required to block U46619-mediated contractions, the drug did not affect the actions of non-prostanoid agonists or exhibit agonist activity in any of the smooth muscle preparations employed; neither did it interact at EP- or FP-receptors.6. In conclusion, the present study demonstrates that ZD1542 is a drug that exhibits both potent,selective TXS inhibition and TXA2 receptor antagonism.     

Controlled and automatic memory process in Alzheimer''s disease

Treatment of cultured rat hepatocytes with cyclosporin A (0.01-1 microM) for 24, 48, or 72 h in the presence of insulin and epidermal growth factor induced an inhibition on cell proliferation in a time- and concentration-dependent manner, with an IC50 = 0.05 microM CsA corresponding to 48-h treatment. The inhibitory effect of CsA at < or = 0.1 microM doses for 48 h on [3H]thymidine uptake was reversed after withdrawal of the drug and subsequent addition of insulin plus EGF or serum; however, at 1 microM CsA the effect was irreversible and numerous bright small vesicles were observed. The molecular mechanism involved in CsA action in hepatocytes seems to be independent on cAMP and pertussis-toxin sensitive G proteins.     

Electric field-induced changes in agonist-stimulated calcium fluxes of human HL-60 leukemia cells

The mechanism of biological effects of extremely-low-frequency electric and magnetic fields may involve induced changes of Ca2+ transport through plasma membrane ion channels. In this study we investigated the effects of externally applied, low-intensity 60 Hz electric (E) fields (0.5 V/m, current density 0.8 A/m2) on the agonist-induced Ca2+ fluxes of HL-60 leukemia cells. The suspensions of HL-60 cells received E-field or sham exposure for 60 min and were simultaneously stimulated either by 1 microM ATP or by 100 microM histamine or were not stimulated at all. After E-field or sham exposure, the responses of the intracellular calcium levels of the cells to different concentrations of ATP (0.2-100 microM) were assessed. Compared with control cells, exposure of ATP-activated cells to an E-field resulted in a 20-30% decrease in the magnitude of [Ca2+]i elevation induced by a low concentration of ATP (<1 microM). In contrast, exposure of histamine-activated HL-60 cells resulted in a 20-40% increase of ATP-induced elevation of [Ca2+]i. E-field exposure had no effect on non-activated cells. Kinetic analysis of concentration-response plots also showed that compared with control cells, exposure to the E-field resulted in increases of the Michaelis constant, Km, value in ATP-treated cells and of the maximal [Ca2+]i peak rise in histamine-treated HL-60 cells. The observed effects were reversible, indicating the absence of permanent structural damages induced by acute 60 min exposure to electric fields. These results demonstrate that low-intensity electric fields can alter calcium distribution in cells, most probably due to the effect on receptor-operated Ca2+ and/or ion channels.