Inhibition of platelet-activating factor (PAF)-induced platelet aggregation by fatty acids from human saliva.

Experiments were undertaken to identify the nature of a previously identified inhibitor of PAF-induced platelet aggregation (PA) in human saliva. Human saliva fractionated by preparative thin layer chromatography (TLC) yielded a fraction that co-migrated with fatty acids (FAs) and inhibited PAF-induced aggregation of platelets. Synthetic FAs tested for their capacities to inhibit 0.1 nM PAF-induced PA showed that only the cis-unsaturated compounds were inhibitory with activities of some of the polyunsaturated FAs (PUFA) reaching almost 100% at 20 µM. Eicosapentanoic acid (EPA) and 8,11,14-eicosatrienoic acid also deaggregated the PAF-induced aggregates. With the exception of oleic acid (OLA), cis-monounsaturated FAs, and elaidic acid, the trans isomer of OLA, were poor inhibitors. In a direct comparison with other platelet agonists, ADP, thrombin, and ionophore A23187, the active saliva fraction and selected individual FAs inhibited, to greater or lesser extent, PA induced by each of the agonists. EPA, OLA, linoleic acid (LNA), and the active saliva fraction were potent inhibitors of ADP-induced PA, EPA completely inhibited thrombin-induced PA and the saliva fraction showed only weak - moderate inhibitory activity to both thrombin- and ionophore A23187-induced PA. Other reports of endogenous PAF inhibitors in mammalian tissues are compared to the present results. PAF can trigger and amplify inflammatory cascades suggesting a possible modulation role for cis-unsaturated FAs in some diseases.

Calcium ionophore, A23187 and its amino acid complexes: spectroscopic and molecular modeling studies.

The circular dichroism, fluorescence, Nuclear Magnetic Resonance and BLM conductance studies indicate that A23187 forms a stable complex with amino acids at low ionophore concentrations (< 10(-4)M). However, A23187 prefers to be in a dimeric structure with no significant binding to amino acids, at concentrations higher than 10(-4)M. It was also observed that at lower concentrations, at which the amino acids bind to the ionophore, the affinity for calcium ions was several orders of magnitude lower than that at higher ionophore concentrations. We have also conducted molecular modeling studies to examine the structure of the A23187 dimer and its amino acid complexes. The results of these modeling studies strongly support our experimental results and validate the formation of a hydrogen bonded and energetically stable A23187 dimer and its amino acid complexes.

Studies on the mechanism of embryonic frog nerve orientation in a small applied electric field.

The mechanism of nerve orientation in an applied electric field has been investigated using a number of pharmacological agents. Galvanotropism may depend on redistribution within the plasma membrane of integral membrane proteins (IMP); blocking this with concanavalin A inhibited orientation. Orientation may depend also on an influx of Ca2+; Co2+ and La3+ blockade of calcium channels inhibited turning in an electric field. Organic blockers of calcium channels did not influence orientation, suggesting that L-type Ca2+ channels may not be present at the growth cone. Procedures that may induce asymmetric entry of Ca2+ on the anodal side of cells caused a reversal of normal galvanotropism, with growth directed towards the anode. This may implicate local levels of cytoplasmic Ca2+ within the growth cone in controlling turning behaviour. An asymmetric distribution of filopodia precedes and may predict the direction of nerve growth in an electric field. Various pharmacological agents perturbed the distribution of filopodia in such a way that this did not reflect subsequent orientation. It is suggested that, normally, local Ca2+ increases and an asymmetry of filopodia operate together in determining orientation, but that filopodial activity is subordinate to and can be overriden by local Ca2+ levels in the growth cone. In addition, two of the drug treatments markedly increased rates of nerve growth, which may be of importance in nerve regeneration.

Identification of tumor promoters by their inhibitory effect on intercellular transfer of lucifer yellow.

The effect of the tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein, teleocidin, anthralin, the Ca2+-ionophore A23187, butylated hydroxytoluene (BHT), dichlorodiphenyltrichloroethane (DDT) and phenobarbital (PB) on lucifer yellow transfer in cultures of SV-40-transformed Djungarian hamster fibroblasts was studied. TPA, mezerein, teleocidin, A23187, DDT and BHT exerted a strong inhibitory effect on cell-to-cell dye transfer. Anthralin uncoupled cells in 3 experiments out of 6. PB appeared to enhance lucifer yellow transfer. Sodium nitrite, a substance with unknown promoting activity, effectively uncoupled cells. All the promoters investigated had a reversible effect on the dye transfer. The value of the dye transfer method for promoter screening is discussed.

Microbial transformation of A23187, a divalent cation ionophore antibiotic.

A23187 is an ionophore antibiotic that forms dimeric complexes with divalent cations such an Mn2+ and Ca2+. Over 200 randomly selected soil microorganisms were incubated with A23187. None of these cultures was capable of transforming this compound. In contrast, many microorganisms were able to modify the methyl ester of A23187. The transformation products produced by one culture, Streptomyces chartreusis, were isolated and identified as 16-hydroxy-N-demethyl A23187 methyl ester, 16-hydroxy-A23187 methyl ester, and N-demethyl A23187 methyl ester. These ester derivatives lack most of the ionophore properties of the acids and cannot readily form dimeric complexes with divalent cations. However, they could be hydrolyzed by a mild treatment with ethanolic KOH to free acids that possess good ionophore activity. The use of the ester substrate in conjunction with the hydrolysis procedure is, at the present time, the only known method for microbiologically producing A23187 derivatives.

Semiautomated turbidimetric bioassay for the ionophore A23187.

The inhibitory effect of A23187 against Staphylococcus aureus was used to quantitate the activity of this ionophore in fermentation samples and in isolation and crystallization samples. The assay was shown to be rapid and reproducible.