Prophylactic intravenous tranexamic acid and thromboembolism in non-cardiac surgery: a systematic review, meta-analysis and trial sequential analysis.

Tranexamic acid is an antifibrinolytic drug that is widely used during surgery, but there are concerns about its thromboembolic effects. We aimed to investigate the effect of prophylactic intravenous tranexamic acid on thromboembolic outcomes in patients undergoing non-cardiac surgery. The MEDLINE, EMBASE and Cochrane Central Register of Controlled Trials were searched. Randomised controlled trials comparing intravenous tranexamic acid with placebo or no treatment in patients undergoing non-cardiac surgery were included. The primary outcome was a composite of peri-operative cardiovascular thromboembolic events, defined as any deep vein thrombosis, pulmonary embolism, myocardial ischaemia/infarction or cerebral ischaemia/infarction. A total of 191 randomised controlled trials (40,621 patients) were included in the review. The primary outcome occurred in 4.5% of patients receiving intravenous tranexamic acid compared with 4.9% of patients in the control group. Our analysis showed that there was no difference between groups for composite cardiovascular thromboembolic events (risk ratio 1.02, 95%CI 0.94-1.11, p = 0.65, I2 0%, n = 37,512). This finding remained robust when sensitivity analysis was performed with continuity correction and in studies with a low risk of bias. However, in trial sequential analysis, our meta-analysis only achieved 64.6% of the required information size. There was no association between intravenous tranexamic acid and seizure rate or mortality rate within 30 days. Intravenous tranexamic acid was associated with a reduced blood transfusion rate compared with control (9.9% vs. 19.4%, risk ratio 0.46, 95%CI 0.41-0.51, p < 0.0001). It was encouraging to see the evidence that the administration of intravenous tranexamic in patients undergoing non-cardiac surgery was not associated with an increased risk of thromboembolic outcomes. However, our trial sequential analysis demonstrated that currently available evidence is not yet sufficient to reach a firm conclusion.

Synthesis and biological evaluation of alpha-halogenated bisphosphonate and phosphonocarboxylate analogues of risedronate.

Alpha-halogenated analogues of the anti-resorptive bisphosphonate risedronate (5, Ris) and its phosphonocarboxylate cognate (7, 3-PEHPC) were synthesized and compared with 5, 7, and the corresponding desoxy analogues in bone mineral affinity and mevalonate pathway inhibition assays. The Ris (5e-h) and 3-PEHPC (7e-h) analogues had decreased bone mineral affinity, confirming that the alpha-OH group in 5 and 7 enhances bone affinity. The 5 alpha-halo-analogues potently inhibited farnesyl pyrophosphate synthase (FPPS) with IC50 values from 16 (alpha-F) to 340 (alpha-Br) nM (5, 6 nM). In contrast, 7 alpha-halo-analogues were ineffective versus FPPS (IC50 > 600 microM), but inhibited Rab geranylgeranyl transferase (RGGT) (IC50 = 16-35 microM) similarly to 7 itself (IC50 = 24 microM). The alpha-F analogue 7e was 1-2 times as active as 7 in J774 cell viability and Rab11 prenylation inhibition assays.

Mercuric ion sensing by a film bulk acoustic resonator.

This paper describes a film bulk acoustic resonator (FBAR) mass sensor for detecting Hg2+ ion in water with excellent sensitivity and selectivity. When a thin Au film was deposited on the surface of an FBAR, the resonant frequency shifted to a lower value when the film was exposed to Hg2+ in aqueous solution. The FBAR sensor detected as low as 10(-9) M Hg2+ (0.2 ppb Hg2+) in water. Other ions such as K+, Ca2+, Mg2+, Zn2+, and Ni2+ had little or no effect on the resonant frequency of the FBAR. Coating of the FBAR Au surface with a self-assembled monolayer (SAM) of 4-mercaptobenzoic acid decreased the Hg2+ response.

Effects of second-generation platinum analogs on isolated PM-2 DNA and their cytotoxicity in vitro and in vivo.

Using PM-2 DNA and cytotoxicity assay systems, we have studied several second-generation platinum analogs and compared these to the parent compound cis-diamminedichloroplatinum(II). These results indicate that all planar platinum(II) congeners induced similar effects upon interaction with PM-2 DNA, i.e., alteration of the tertiary DNA conformations. The reactivity of the analogs with DNA depended on the activity of the leaving groups. Octahedral platinum(IV) compounds, however, induced breakage of covalently closed circular PM-2 DNA, and the effects were not inhibited by either chloride or ethylenediaminetetraacetate. This suggests that breakage of isolated PM-2 DNA may be related to the axial trans bonds rather than the equatorial cis bonds of the solvated platinum(IV) compounds, since the activity of the dichloroplatinum(II) compounds has been shown to be inhibited by chloride ions. Studies on the in vitro and in vivo cytotoxicity of the platinum analogs demonstrated that the reactivity of analogs against PM-2 DNA correlated with in vitro and in vivo potencies. The reactivity with PM-2 DNA appeared to depend on the characteristics of the leaving group.

Interaction of cis-diamminedichloroplatinum(II) with PM-2 DNA.

The interaction of cis-diamminedichloroplatinum(II) (CDDP) with PM-2 DNA was studied using two techniques: (a) agarose gel electrophoresis of PM-2 DNA conformation isomers after CDDP binding; and (b) viscometric measurement of different forms of CDDP-bound PM-2 DNA. In both systems, the results indicated that the DNA isomers interacted differently with CDDP. CDDP induced a decrease of viscosity upon interacting with single-strand broken relaxed circular (Form II) and double-strand broken linear (Form III) PM-2 DNA's. These observations are consistent with a "DNA shortening effect" proposed by Cohen et al. [Science (Wash. D. C.), 203: 1014-1016, 1979] and Macquet et al. [Biochimie (Paris), 60: 901-914, 1978] When covalently closed circular (Form I) PM-2 DNA was used, increasing concentrations of CDDP induced an initial slight increase and then decrease of electrophoretic mobility to the degree that it comigrated with CDDP-bound Form II DNA. Further addition of CDDP restored the electrophoretic mobility of Form I DNA. Corresponding changes in the viscosity of CDDP-bound Form I DNA showed an initial decrease, then an increase, and a final prolonged decrease of viscosity. These effects are similar but not identical to those induced by either DNA intercalators (e.g., ethidium bromide) or certain DNA denaturating agents (e.g., formaldehyde, ultraviolet light, alkali trichloroacetate, methylmercuric hydroxide, and carbodiimide). Thus, CDP may induce a DNA superhelix-unwinding process followed either by rewinding or a denaturation process or both. Quantitative analysis of the agarose gel electrophoretic pattern plus sucrose density gradient centrifugation studies also indicated that there was little DNA strand breakage induced by CDDP treatment.

DNA supercoiling, shortening, and induction of single-strand regions by cis-diamminedichloroplatinum(II).

cis-Diamminedichloroplatinum(II) was found to bind to covalently closed circular supercoiled, covalently closed circular nonsupercoiled, and single-strand broken relaxed PM2 DNA and induce different types of DNA conformational changes. Using Kleinschmidt's technique, it was found that binding of cis-diamminedichloroplatinum(II) decreased the DNA length to 75% of the original single-strand broken relaxed DNA without inducing superhelical conformational changes. cis-Diamminedichloroplatinum(II) also shortened the length of covalently closed circular nonsupercoiled DNA before a supercoiled conformation was generated. Single strand-specific nucleases were used to detect drug-induced DNA structural alterations. Local single-strand regions or regions of denaturation were detected by S1 nuclease from Aspergillus oryzae and by BAL-31 nuclease from Alteromonas espejiana BAL-31. The single-strand regions or local denaturation regions do not seem to be related to or caused by DNA superhelical conformational changes since they were detected at drug concentrations at which no significant DNA superhelical turns were found. DNA shortening, superhelical conformational changes, and local denaturation regions can be explained by the previously proposed "DNA intrastrand cross-linking" model (Stone et al., J. Mol. Biol., 104: 793-801, 1976).

Effects of DNA superhelical changes induced by ethidium bromide on the DNA-degrading activity of two antitumor antibiotics, bleomycin and phleomycin.

The effects of changes in the conformational state of DNA on the single-strand and double-strand breakage activity of two antitumor antibiotics, bleomycin (BLM) A2 and phleomycin D1, have been studied by the gel electrophoretic analysis of the drug-degraded PM2 phage superhelical DNA pretreated with an intercalating agent, ethidium bromide (EB). Both the single-strand and double-strand breakage activities of BLM A2 increased as the negatively superhelical turns of native PM2 DNA were gradually removed by intercalation with increasing EB concentrations. The activities peaked when DNA was completely relaxed and gradually decreased as the higher concentrations of EB twisted DNA into the positively superhelical form. The decrease in breakage activity was not due to any inhibitory effect of EB at higher concentrations, since treatment of the relaxed Form I0 DNA with low EB concentrations also reduced the activity. In contrast to BLM A2, phleomycin D1 responded minimally to DNA conformational changes, which suggested further that the two drugs may react with DNA differently. The differential responses of BLM A2 activity towards different DNA conformational states may have biological implications, since DNA in cells may exist in different conformational states relating to various gene functions. The current study may serve as a model for studying combined effects of intercalative and nonintercalative antitumor antibiotics which are used frequently in combination treatments of cancer.

Intermolecular cross-linking of DNA through bifunctional intercalation of an antitumor antibiotic, luzopeptin A (BBM-928A).

A bifunctional intercalator may intercalate with DNA in at least two ways. Both intercalating moieties may intercalate with the same DNA molecule (type I, intramolecular cross-linking) or with two separate DNA molecules (type II, intermolecular cross-linking). Production of type I is often assumed. Type II biintercalation has been suggested, but no direct evidence has been reported. In the present study, endonuclease-restricted PM2 phage or pBR322 plasmid DNA fragments were treated with the bifunctional intercalative antitumor antibiotics, luzopeptin A (BBM-928A) and echinomycin, and analyzed by agarose gel electrophoresis. Luzopeptin A treatment produced additional DNA bands which were the products of type II biintercalation. The types of restriction fragments involved were identified. Maximal type II biintercalation occurred at a luzopeptin A/DNA range of 0.14 to 0.18, at which more than 50% of the total DNA molecules were involved. Type II products were converted gradually to type I products upon prolonged incubation at 37 degrees, probably due to the tendency for intermolecular bonds to disrupt. Echinomycin treatment failed to produce type II products, probably because of a DNA-binding affinity weaker than that of luzopeptin A. Thus, it is possible to use the present gel system to demonstrate the type II biintercalation for strong biintercalators, but milder systems are needed for weak biintercalators.

Bleomycin and talisomycin sequence-specific strand scission of DNA: a mechanism of double-strand cleavage.

Computer analyses of DNA sequencing data obtained using various restriction fragments of pBR 322 DNA indicate that a trinucleotide sequence (-Pyr-G-C-) is the most preferred site for cleavage by the antitumor antibiotic bleomycin A2. Talisomycin A, a structurally related bleomycin analog, cleaved at the sequences -G-T/A- most preferentially. However, the presence of a pyrimidine at the 5' side of guanine at the cleavage site did not increase the probability of that site being cleaved by talisomycin. Using denaturing and nondenaturing polyacrylamide gel analyses of the drug-DNA reaction products. The sites of both single- and double-strand breaks have been localized and differentiated. The results indicate that a major determinant for location of a site-specific double-strand break is the production of two closely spaced sequence-specific single-strand breaks by the drugs on opposite strands of the DNA. A four-base pair sequence is proposed for the optimal sequence for bleomycin-induced double-strand breaks.

In vitro and intracellular inhibition of topoisomerase II by the antitumor agent merbarone.

Merbarone has previously been shown to have antitumor activity of unknown mechanism in P388 and L1210 tumor models (A. D. Brewer et al., Biochem. Pharmacol., 34:2047-2050, 1985) and is currently undergoing Phase I clinical trials. Here we report that merbarone is an inhibitor of topoisomerase II. Merbarone inhibited purified mammalian topoisomerase II with a 50% inhibitory concentration of 20 microM, as assessed by ATP-dependent unknotting of P4 phage DNA or relaxation of supercoiled pBR322 plasmid. In contrast to the type II enzyme, inhibition of catalytic activity of topoisomerase I required about 10-fold higher concentrations of merbarone, with a 50% inhibitory concentration of approximately 200 microM. Unlike epipodophyllotoxin analogues and certain DNA intercalative agents which stabilize the topoisomerase II-DNA "cleavable complex," merbarone did not cause detectable topoisomerase II-induced DNA cleavage. Furthermore, merbarone inhibited the production by amsacrine or teniposide of topoisomerase II-associated DNA strand breaks; under identical conditions novobiocin did not decrease these breaks, setting merbarone apart from a novobiocin-like class of topoisomerase II inhibitor. In L1210 cells, merbarone produced only small numbers of protein-associated DNA strand breaks, and only at very high concentrations. Merbarone reduced in a concentration-dependent manner the number of amsacrine- or teniposide-stimulated protein-associated DNA strand breaks in L1210 cells or their isolated nuclei. The data suggest that merbarone represents a novel type of topoisomerase II inhibitor.

Relationship between the intracellular effects of camptothecin and the inhibition of DNA topoisomerase I in cultured L1210 cells.

Results of filter elution assays of lesions produced in the DNA of cultured L1210 cells by the antineoplastic alkaloid camptothecin support the notion that topoisomerase I is an intracellular target of this drug. One to 10 microM camptothecin induced DNA single-strand, but not double-strand, breaks when incubated with intact cells or with their isolated nuclei. Approximately one half of the strand breakage was protein concealed, as judged by filter elution. Camptothecin-induced, protein-concealed DNA strand breaks disappeared rapidly after drug removal. DNA-protein cross-links were generated by camptothecin with frequencies approximately equal to those of protein-concealed DNA strand breaks. It is likely that camptothecin can inhibit topoisomerase I in intact cells in a manner similar to that in which other antineoplastic agents such as amsacrine or teniposide inhibit topoisomerase II. DNA-breaking lesions other than those resulting from trapped topoisomerase I-DNA complexes may also be generated by camptothecin. The yields of DNA strand breaks induced by camptothecin, amsacrine, or teniposide were approximately doubled when cells were incubated for 16 h with 3-aminobenzamide, an inhibitor of poly(ADP ribosylation) of proteins, prior to 1-h exposure to the antineoplastic compounds. 3-Aminobenzamide also enhanced the cytotoxic action of camptothecin, amsacrine, and teniposide. These results suggest that protein-concealed strand breaks can be lethal lesions and that intracellular topoisomerase I and II activity may be regulated coordinately through poly(ADP ribosylation).

Normocalcemic effect of gallium nitrate in a hypercalcemic rat model.

An established rat hypercalcemia model was used to study the effects of gallium nitrate on elevated serum calcium levels. Gallium nitrate was administered by i.v. or i.p. injection at daily doses of 0.07-0.45 mmol/kg for 5 days to the hypercalcemic rats beginning 1 day following surgery. A dose-correlated normocalcemic response was observed. Gallium nitrate administered late after the induction of the hypercalcemic state was also effective in reducing serum calcium levels. The p.o. administration, however, even at doses as high as 0.45 mmol/kg, did not reduce serum calcium to normal levels. The values of area under the concentration versus time curve (0-24 h) of gallium in normal rats were comparable after i.v. [49.2 (micrograms/ml)h] or i.p. [57.0 (micrograms/ml)h] injections. In contrast, the p.o. route achieved only 15% bioavailability, which may explain the ineffectiveness of p.o. administered gallium nitrate at that dose level. This study suggests that daily i.v. bolus injections of gallium nitrate for managing hypercalcemia may be potentially as effective as the current regimen of continuous i.v. infusion.

In vivo antitumor activity and in vitro cytotoxic properties of bis[1,2-bis(diphenylphosphino)ethane]gold(I) chloride.

We have previously reported the cytotoxicity and antitumor activity of bis(diphenylphosphino)ethane (DPPE) and a variety of its transition metal complexes. During studies of the chemistry of a gold complex of this group [(AuCl)2(DPPE)], it was observed that this complex readily underwent ring closure on reaction with DPPE to form the tetrahedral complex [Au(DPPE)2]+. Various counterion forms (e.g., Cl-) of this cation were isolated and were found to exhibit a remarkably high stability in solution. Evaluation of [Au(DPPE)2]Cl in mice bearing i.p. P388 leukemia demonstrated that the compound produced an average of 87% increase in life span at its maximally tolerated dose (2-3 mumol/kg/day for 5 days). Activity was also seen in i.p. M5076 reticulum cell sarcoma (60% increase in life span) and s.c. mammary adenocarcinoma 16/c. Modest activity was evident in i.p. B16 melanoma and L1210 leukemia. A subline of P388 leukemia resistant to cisplatin was not cross-resistant to [Au(DPPE)2]Cl. In addition, combination therapy of [Au(DPPE)2]Cl and cisplatin against i.p. P388 demonstrated an advantage over single-agent therapy. In vitro studies of [Au(DPPE)2]Cl showed that the compound: is cytotoxic to tumor cell lines; is only minimally inhibited in its cytotoxic activity by the presence of serum; produces DNA protein cross-links and DNA strand breaks in cells; and inhibits macromolecular synthesis with a preferential inhibitory effect on protein synthesis relative to DNA and RNA synthesis. 31P nuclear magnetic resonance spectroscopy indicated that the compound is stable in the presence of serum proteins, thiols, or disulfides and that it reacts with Cu(II) resulting in the formation of a Cu(I)DPPE complex. The results of these in vivo and in vitro experiments suggest that the contrasting pharmacological profile of [Au(DPPE)2]Cl with respect to other gold(I) phosphine complexes may be related to both the kinetic stability of the complex and its stability in the presence of thiols.

Gallium nitrate inhibits bone resorption and collagen synthesis in neonatal mouse calvariae.

Gallium nitrate (GN) is an agent used in the treatment of hypercalcemia. To more fully characterize the direct actions of GN on bone, we examined its effects on medium calcium, medium beta-glucuronidase (beta-GLU), and collagen synthesis in control and hormone-stimulated neonatal (4-6 days) mouse calvariae in vitro. GN (10 micrograms/ml) inhibited parathyroid hormone-stimulated (PTH; 1 nM) calcium release. A 24 h preincubation with 10 micrograms/ml of GN was required for complete inhibition; partial inhibition was seen with 12 h preincubation; 1, 3, or 6 h was inadequate. A dose-response study showed that with 24 h preincubation, 5, 3, and 1 microgram/ml of GN inhibited 81, 62, and 0% of PTH-induced calcium release. The effects of GN on the release of beta-GLU generally paralleled those on the release of calcium except that 10 micrograms/ml of GN stimulated beta-GLU release. Collagen synthesis was inhibited 50% by 3 micrograms/ml of GN, whereas noncollagen protein synthesis was unaffected. With PTH + GN no further decrease was observed. When GN was withdrawn from the medium after 24 h of preincubation, the inhibitory effect on calcium release and beta-GLU activity, but not on collagen synthesis, persisted through the 72 h of culture. GN also inhibited the resorption elicited by thyroxine (1 microM) and interleukin-1 beta (10 nM) but not by 1,25-dihydroxyvitamin D3 (30 pM). Our results indicate that GN is a powerful inhibitor of bone resorption in neonatal mouse calvariae even at low doses.(ABSTRACT TRUNCATED AT 250 WORDS)

cis-4-Carboxy-6-(mercaptomethyl)-3,4,5,6-tetrahydropyrimidin-2(1 H)-one , a potent inhibitor of mammalian dihydroorotase.

A series of cis- and trans-4-carboxy-3,4,5,6-tetrahydropyrimidin-2(1H)-ones possessing either a carboxy, hydroxymethyl, or mercaptomethyl substituent at C-6 were prepared and tested for their ability to inhibit mammalian dihydroorotase. Of these compounds, only the cis-6-mercaptomethyl compound, cis-1, was found to be a potent competitive inhibitor of the enzyme (Ki = 140 nM at pH 7.4 and 8.5) when assayed in the direction of dihydro-L-orotate hydrolysis. These results suggest that the inhibition arises from the ligation of the thiolate to the zinc atom which is thought to be located in the enzyme's active site. Although analysis of cis-1 with 2,2'-dithiobis(5-nitrobenzoic acid) revealed significant loss of the free thiol group under enzymatic assay conditions, the addition of the reducing agent, dithiothreitol, to the enzymatic reaction mixtures afforded cis-1 complete protection against this chemical decomposition, as evidenced by lowering of the inhibition constant in the presence of dithiothreitol. Compound cis-1 had no significant antiproliferative activity against B16 melanoma cells in tissue culture, possibly due to the rapid decomposition of the compound or poor permeability into cells.

Discovery of TBC11251, a potent, long acting, orally active endothelin receptor-A selective antagonist.

Previously we reported the discovery of amidothiophenesulfonamides as endothelin receptor-A antagonists with high potency and selectivity. Replacement of an amide group in this class of compounds with an acetyl group maintained the in vitro binding affinity and in vivo activity while providing a compound with oral bioavailability and longer duration of action. The optimal compound discovered during these studies, 15q (TBC11251), binds competitively to human ETA receptors with a Ki of 0.43 +/- 0.03 nM and an IC50 of 1.4 nM (IC50 for ETB = 9800 nM). This compound inhibits ET-1-induced stimulation of phosphoinositide turnover with a Ki of 0.686 nM and a pA2 of 8.0. The compound has a serum half-life in the rat and the dog of 6-7 h and 60-100% oral bioavailability. This compound is one of the most selective ETA antagonists reported and therefore is suitable for additional pharmacological and clinical investigation of the role of ETA receptors in diseases.

Synthesis, tubulin binding, antineoplastic evaluation, and structure-activity relationship of oncodazole analogues.

In an attempt to identify a soluble oncodazole analogue that could be easily formulated, a series of substituted oncodazoles was synthesized and evaluated for tubulin binding affinity, in vitro cytotoxicity against cultured mouse B-16 cells, and ability to prolong lifespan at the maximally tolerated dose in the P388 mouse leukemia model. Biological evaluation of all the isomeric methyloncodazoles demonstrated the thiophene 4'-position to be the only site of significant bulk tolerance, although substitution of this position with polar or charged functional groups abolished biological activity. Simple esters of the 4'-carboxymethyloncodazole were shown to have enhanced antitumor activity and tubulin binding affinity relative to oncodazole. Despite a failure of this study to identify a water-soluble oncodazole with antitumor activity, the structure-activity relationship developed led to a derivative with enhanced activity in the P388 leukemia model and facilitated the preparation of a biologically active photolabile analogue.

Modification of the hydroxy lactone ring of camptothecin: inhibition of mammalian topoisomerase I and biological activity.

Several camptothecin derivatives containing a modified hydroxy lactone ring have been synthesized and evaluated for inhibition of topoisomerase I and cytotoxicity to mammalian cells. Each of the groups of the hydroxy lactone moiety, the carbonyl oxygen, the ring lactone oxygen, and the 20-hydroxy group, were shown to be critical for enzyme inhibition. For example the lactol, lactam, thiolactone, and 20-deoxy derivatives did not stabilize the covalent DNA-topoisomerase I complex. With a few exceptions, those compounds that did not inhibit topoisomerase I were not cytotoxic to mammalian cells. Two cytotoxic derivatives that did not inhibit topoisomerase I were shown to produce non-protein-associated DNA single-strand breaks and are likely to have a different mechanism of action. One of these compounds was tested for antitumor activity and was found to be inactive. The present findings, as well as other reports that the hydroxy lactone ring of camptothecin is critical for antitumor activity in vivo, correlate with the structure-activity relationships at the level of topoisomerase I and support the hypothesis that antitumor activity is related to inhibition of this target enzyme.

Identification and characterization of leukotriene D4 receptors in adult and fetal human lung.

Leukotriene D4 (LTD4) receptors were identified and characterized in adult and fetal human lung membranes. Macroscopically normal adult lung tissue was selected from seventeen surgical biopsy specimens, and twenty-seven fetal lung samples were obtained from therapeutic abortions. Binding assays were performed using pooled adult or fetal human lung membranes at 30 degrees under conditions which prevented metabolism of [3H]LTD4. Specific binding reached equilibrium within 30 min, remained constant for 60 min, was enhanced by Mg2+, and was inhibited by Na+ and guanyl-5'-yl-imidodiphosphate. Computer-assisted analyses of saturation binding data showed a single class of binding sites with similar apparent Kd (0.15 +/- 0.09 and 0.12 +/- 0.003 nM) and Bmax (68 +/- 29 and 62 +/- 14 fmoles/mg protein) values for adult and fetal samples respectively. Competition binding studies with [3H]LTD4 showed the same rank order potency for adult and fetal lung receptors (5S, 6R-LTD4 greater than 5S, 6R-LTD1 greater than 5R, 6S-LTD4 greater than 5S,6R-LTE4 greater than FPL 55712). A comparison of the receptor binding affinities of these compounds with their smooth muscle contractile agonist (pD2) and antagonist (-log[KB]) activities in guinea pig lung and trachea showed a good correlation (r = 0.88), suggesting that the saturable, high-affinity, stereoselective [3H]LTD4 specific binding sites identified in human lung may be physiologically relevant receptor moieties.

Differential inhibition of histamine release from mast cells by protein kinase C inhibitors: staurosporine and K-252a.

Pretreatment of rat peritoneal mast cells with either staurosporine or an analog K-252a [(8R*,9S*,11S*)-(-)-9-hydroxyl-9-methoxycarbonyl-8-methyl-2,3, 9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11-atrizadibenzo- [a,g]cycloocta[cde]trinden-1-one] led to a concentration-related inhibition of histamine release when the cells were stimulated with anti-IgE (IC50: staurosporine = 110 nM; K-252a = 100 nM). In contrast, the two protein kinase C (PKC) inhibitors (1-1000 nM) partially (less than 15%) inhibited histamine release induced by compound 48/80 (0.5 to 1 micrograms/mL). Furthermore, prostaglandin E2 (PGE2) synthesis mediated by anti-IgE from rat peritoneal mast cells was also inhibited by staurosporine and K-252a (IC50 = 100 nM). Exposure of anti-arsenate IgE (anti-Ars-IgE) sensitized mouse bone marrow derived mast cells to arsenate-bovine serum albumin (Ars-BSA) led to the release of both histamine (510 +/- 12.6 ng/10(6) cells) and immunoreactive leukotriene C4 (LTC4) (27.0 +/- 2.6 ng/10(6) cells). Both histamine and LTC4 release was inhibited by staurosporine and K-252a with an IC50 of 50 nM for both compounds. We also characterized a 45K molecular weight protein which is phosphorylated by PKC after Ars-BSA or phorbol, 12-myristate, 13-acetate (PMA) stimulation. This protein is phosphorylated in a broken cell preparation in which PKC is activated by phosphatidylserine/Diolein and Ca2+. Peptide mapping by V8 protease of the phosphorylated 45K protein revealed that the 45K protein phosphorylation patterns induced by IgE or PMA or in the broken cell preparation are identical. Pretreatment of 32P-labeled mouse bone marrow derived mast cells with either staurosporine or K-252a led to a concentration-related inhibition of 45K protein phosphorylation induced by PMA or Ars-BSA. This inhibition of protein phosphorylation correlated well with the inhibition of histamine and leukotriene release in bone marrow derived mast cells.