Oxidation of beta-phenylethylamine by both types of monoamine oxidase: effects of substrate concentration and pH.

β-Phenylethylamine (PEA) was characterized as substrate for both type A and type B monoamine oxidase (MAO) in rat brain mitochondria at different substrate concentrations and at different pHs of the reaction media. The experiments on sensitivity to clorygline and deprenyl showed that the inhibition patterns with PEA as substrate differed markedly at different substrate concentrations: at 10 μM, PEA acted as a specific substrate for type B MAO, but at 50–1000 μM it became a common substrate for both types of MAO. The inhibition patterns were also affected markedly by a small change in pH of the reaction medium, especially when PEA concentrations were 50 and 100 μM: the change in pH from 7.2 to 7.8 resulted in the incresse in the proportion of type A MAO by 20–30 per cent. To investigate the mechanisms of such changes in substrate specificity of PEA, kinetic analyses were carried out at pH 7.2 and 7.8 with the uninhibited, the clorgyline-treated (type B) and the deprenyl-treated (type A) enzyme. The Lineweaver-Burk plots for the uninhibited MAO showed strong substrate inhibition for both pHs, which is more marked at pH 7.8 than at pH 7.2. Pretreatment of the enzyme with 10^?7 M clorgyline resulted in generally similar K_m values for PEA to those of the uninhibited enzyme, and the substrate inhibition at pH 7.8 was also stronger than that at pH 7.2. After pretreatment with 10^?7 M deprenyl, the K_m values were higher and the V_max values were lower than those of the uninhibited or the clorgyline-treated enzyme; there was no or only slight substrate inhibition in these curves. These results suggest that the remarkable changes in substrate specificity observed at different PEA concentrations and at different pHs may be due to the strong substrate inhibition of type B MAO.     

Inhibition of Tyrosinase Activity and Protein Synthesis in Melanoma Cells by Calcium lonophore A23187

Calcium ionophore A23187 lowers basal levels of tyrosinase and inhibits the MSH-induced increase in tyrosinase in Cloudman S-91 mouse melanoma cell cultures. lonophore at a concentration of 10^–6 g/ml causes a 50% reduction in basal levels of tyrosinase and inhibits the MSH stimulated level of enzyme. lonophore A23187 also inhibits the PGEi mediated stimulation of tyrosinase, as well as the rise in enzyme activity observed in cells exposed to either theophylline (1 mM) or dbcAMP (10^–4M). lonophore does not affect basal levels of cyclic AMP nor the elevated levels produced by either MSH or PGEi, suggesting then, that the antagonistic activity of A23187 is localized to a point in the pathway of tyrosinase activation distal to the formation of cAMP. lonophore causes a rapid and marked (> 50%) inhibition of cellular protein synthesis and it is possible that this calcium mobilizing compound may exert its inhibitory effects on tyrosinase activity by causing a general reduction in cellular translation. Since the inhibition of protein synthesis occurs in cells exposed to ionophore in either the presence or absence of calcium in the medium, it seems, likely that the ionophore may exert its effects by causing the release of calcium from intracellular sites.     

Protein kinase C is required for the disappearance of MPF upon artificial activation in mouse eggs

The aim of the present study was to investigate the implication of protein kinase C (PKC) in the mouse egg activation process. We used OAG (1-oleoyl-2-acetyl-sn-glycerol) as a PKC activator, calphostin C as a specific PKC inhibitor, and the calcium ionophore A23187 as a standard parthenogenetic agent. The exposure of zona-free eggs to 150 μM or 50 μM OAG for 10 min resulted in meiosis II completion in ∼80% of instances. By contrast, at a lower concentration (25 μM), the PKC stimulator was ineffective as parthenogenetic agent. Shortly after the application of 150 μM OAG, the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) increased transiently in all the eggs examined, whereas after the addition of 50 μM OAG, [Ca²⁺]_i remained unchanged for at least 20 min. During this period, the activity of M-phase promoting factor (MPF) dramatically decreased and most of the eggs entered anaphase except when the PKC was inhibited by calphostin C. Similarly, MPF inactivation and meiosis resumption were prevented in calphostin C-loaded eggs following treatment with A23187, even though the ionophore-induced Ca²⁺ signalling was not affected. Taken together, our results indicate that stimulation of PKC is a sufficient and necessary event to induce meiosis resumption in mouse eggs and strongly suggest that, in this species, the mechanism by which a transient calcium burst triggers MPF inactivation involves a PKC-dependent pathway. Mol. Reprod. Dev. 48:292–299, 1997. © 1997 Wiley-Liss, Inc.     

Taurine uptake by cultured human lymphoblastoid cells

Cultured human lymphoblastoid cells take up taurine from the medium by two processes: 1) a temperature-dependent, Na⁺-dependent, saturable “active”-transport system and 2) diffusion. The active transport has properties similar to those reported for taurine transport by other tissues. Apparent K_m is about 25 μM and V_max about 7.2 pmol/min/10⁶ cells; saturation occurs at 100 μM taurine. Uptake is competitively inhibited by the β-amino acids hypotaurine (50% inhibition at 44 μM) and β-alanine (50% at 152 μM), as measured at 50 μM taurine. Taurocyamine inhibits 50% at 260 μM. Chlorpromazine and imipramine are strong uncompetitive inhibitors, giving 50% inhibition at 26 μM and 115 μM, respectively; at these concentrations cellular viability per se is not affected. Ouabain inhibits 40–50% over a concentration range of 4–500 μM. Diffusion of taurine into the cells is proportional to concentration up to 20 mM. However, at the concentration of taurine in human plasma, 40–100 μM, active transport would provide 90% of the taurine taken up.     

Transport parameters and stoichiometry of active calcium ion extrusion in intact human red cells

Ca²⁺-transport and its energy consumption were studied in intact human red cells loaded with Ca²⁺ by the aid of the ionophore A23187.After the complete elimination of the ionophore the passive Ca²⁺-permeability of the membrane returned to its normal low value, except when the intracellular Ca²⁺-concentration was higher than 3 mM or the ATP level fell below 100 μM. Within these limits the rate of Ca²⁺-extrusion was independent of the cellular ATP content but was greatly enhanced by increasing [Ca²⁺]_i and reached a plateau at about 1 mM intracellular Ca²⁺-concentration. The maximum rate of Ca²⁺-efflux was about 85 μmol/l of cells per min at 37°C, pH 7.4. The activation energy of active Ca²⁺-extrusion was found to be 15 200 cal/mol, and the optimum pH in the suspension was 7.7.Ca²⁺-efflux was not connected with the counter-transport of cations.The Ca²⁺-pump was not affected by ouabain or oligomycin and only partial inhibition could be achieved by the SH-reagents: ethacrynic acid, $\text{N-}\text{ethylmaleimide}$ and $\text{p-}\text{chloromercuribenzoate}$ or with propranolol and ruthenium red. An 80 to 95% inhibition of the active Ca²⁺-extrusion was brought about by 50–250 μM lanthanum, which in the above concentrations caused no aggregation or haemolysis. The inhibition of the Ca²⁺-pump by lanthanum was found to be reversible, the site of inhibition being at the external surface of the cell membrane.To examine the energy consumption of the Ca²⁺-extrusion, ATPase activity was assessed by measuring inorganic phosphate liberation in Ca²⁺-loaded red cells the metabolism of which was inhibited by iodoacetamide + Na⁺-tetrathionate. Ca²⁺-activated ATPase activity connected with the Ca²⁺-pump was distinguished from other Ca²⁺-ATPase by using the non-penetrating inhibitor, lanthanum. The molar ratio of Ca²⁺-transported per ATP split was found to be $\text{2 : 1}$.     

Rapid changes in the activities of the enzymes of cyclic AMP metabolism after addition of A23187 to macrophages

The ionophore A23187 stimulated adenylate cyclase activity in intact macrophages within 1 min. This action was blocked by pretreatment with indomethacin (25 μmol/l) suggesting the involvement of a prostaglandin (PG). PGE₂ (500 nmol/l) also stimulated adenylate cyclase activity in intact cells, but this was not prevented by indomethacin pretreatment. Colchicine (100 μmol/l) potentiated the increases in macrophage cyclic AMP production seen after addition of PGE₂ or A23187. The high affinity form of cyclic AMP phosphodiesterase (PDE) was activated within 1 min of the addition of A23187 to intact macrophages. The data suggest that the increase in macrophage cyclic AMP production after A23187 is a consequence of adenylate cyclase activation and not PDE inhibition. The endogenous production of a prostaglandin probably mediates this effect of A23187, emphasizing the importance of arachidonic acid metabolites in the regulation of macrophage functions.     

The hamster adipocyte adenylate cyclase system I. Regulation of enzyme stimulation and inhibition by manganese and magnesium ions

In hamster adipocyte ghosts, ACTH stimulates adenylate cyclase by a GTP-dependent process, whereas prostaglandin E E₁, α-adrenergic agonists and nicotinic acid inhibit the enzyme by a mechanism which is both GTP- and sodium-dependent. The influence of the divalent cations Mn²⁺ and Mg²⁺, was studied on these two different, apparently receptor-mediated effects on the adipocyte adenylate cyclase. At low Mn²⁺ concentrations, GTP (1 μM) decreased enzyme activity by about 80%. Under this condition, ACTH (0.1 μM) stimulated the cyclase by 6- to 8-fold, and NaCl (100 mM) caused a similar activation. In the presence of both GTP and NaCl, prostaglandin E₁ (1 or 10 μM) and nicotinic acid (30 μM) inhibited the enzyme by about 70–80% and epinephrine (300 μM, added in combination with a β-adrenergic blocking agent) by 40–50%. With increasing concentrations of Mn²⁺, the GTP-induced decrease and the NaCl-induced increase in activity diminished, with a concomitant decrease in prostaglandin E_1^?, nicotinic acid- and epinephrine-induced inhibitions as well as in ACTH-induced stimulation. At 1 mM Mn²⁺, inhibition of the enzyme was almost abolished and stimulation by ACTH was largely reduced, whereas activation of the enzyme by KF (10 mM) was only partially impaired. The uncoupling action of Mn²⁺ on hormone-induced inhibition was half-maximal at 100–200 μM and appeared not to be due to increased formation of the enzyme substrate, Mn · ATP. It occurred without apparent lag phase and could not be overcome by increasing the concentration of GTP. Similar but not identical findings with regard to adenylate cyclase stimulation and inhibition by hormonal factors were obtained with Mg²⁺, although about 100-fold higher concentrations of Mg²⁺ than of Mn²⁺ were required. The data indicate that Mn²⁺at low concentrations functionally uncouples inhibitory and stimulatory hormone receptors from adenylate adenylate cyclase in membrane preparations of hamster adipocytes, and they suggest that the mechanism leading to uncoupling involves an action of Mn²⁺ on the functions of the guanine nucleotide site(s) in the system.     

Dual action of ionophore A23187 on intact chloroplasts

1. Ionophore A23187 induces uncoupling of potassium ferricyanide-dependent O₂ evolution by envelope-free chloroplasts and oxaloacetate-dependent O₂ evolution by intact chloroplasts. The half maximal concentration ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for stimulation of oxygen evolution in both cases is approximately 4 μM · 100 μg chlorophyll · ml^?1.2. Ionophore A23187 also induces inhibition of CO₂ and 3-phosphoglycerate-dependent O₂ evolution by intact chloroplasts in the presence of 3 mM MgCl₂. The half maximal concentrations ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for inhibition of O₂ evolution are 3 μM and 5 μM respectively · 100 μg^?1 chlorophyll · ml^?1.3. A very high concentration of ionophore A23187 (10 μM · 20 μg^?1 chlorophyll · ml^?1) plus 0.1 mM EDTA lowers the fluorescence yield of intact chloroplasts suspended in a cation-free medium in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, indicating loss of divalent cation from the diffuse double layers of the thylakoid membranes.4. These results are discussed in relation to ionophore A23187-induced divalent cation/proton exchange at both the thylakoid and the envelope membranes of intact chloroplasts.     

Evaluation of inhibitors of eicosanoid synthesis in leukocytes: Possible pitfall of using the calcium ionophore A23187 to stimulate 5′ lipoxygenase

The effect on arachidonate metabolism of two compounds (BW755C and benoxaprofen) which have been reported to inhibit 5′ lipoxygenase in leukocytes has been evaluated in human polymorphonuclear leukocytes (PMN) stimulated with the calcium ionophore A23187 and serum-treated zymosan (STZ). The syntheses of leukotriene B₄ (LTB₄) and thromboxane B₂ (TXB₂) from endogenous substrate were determined by specific radioimmunoassays as indicators of 5′ lipoxygenase and cyclo-oxygenase activity in the PMN respectively. Benoxaprofen inhibited the synthesis of leukotriene B₄ by human PMN stimulated with the calcium ionophore A23187, but it was approximately 5 times less potent than BW755C. However, benoxaprofen (IC₅₀ 1.6 × 10⁻⁴M) was approximately 100 times less potent than BW755C (IC₅₀ 1.7 × 10⁻⁶M) at inhibiting leukotriene B₄ synthesis induced by serum-treated zymosan. Both drugs inhibited thromboxane synthesis by leukocytes stimulated with A23187 or serum-treated zymosan at similar concentrations (approximately 5 × 10⁻⁶M). The data obtained using STZ as stimulus are consistent with previous studies and indicate that benoxaprofen is a relatively selective inhibitor of cylco-oxygenase. However, this selectivity was far less apparent when A23187 was used as a stimulus to release the eicosanoids which suggests that this inhibition could be via an indirect mechanism and therefore A23187 should be used with caution as a stimulus of 5′ lipoxygenase for evaluating inhibitors of eicosanoid synthesis.     

Nonselective Inhibition of Monoamine Oxidases A and B by Activators of Soluble Guanylate Cyclase

Several activators of soluble guanylate cyclase were investigated as potential inhibitors of rat liver mitochondrial monoamine oxidases (MAO) A and B. They all fitted into the previously designed molds of substrate–inhibitor binding sites of these enzymes. However, only two of them, NO donors (7-nitro-benzotetrazine-1,3-dioxide (7-NBTDO) and benzodifuroxan), caused nonselective inhibition of MAO A and MAO B with IC ₅₀ values of 1.3-1.6 and 6.3-6.8 M, respectively. The inhibitory effect on both MAO A and MAO B was reduced by mitochondria wash suggesting reversible mode of the enzyme inhibition. There was no correlation between potency of MAO inhibition and activation of human platelet soluble guanylate cyclase. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) had no effect on the manifestation of MAO inhibition by benzodifuroxan and 7-NBTDO; however, at 50 M concentration carboxy-PTIO caused potent inhibition of MAO A with minor effect on MAO B activity. The data suggest that nonselective inhibition of MAO A and MAO B by benzodifuroxan and 7-NBTDO can be attributed to the properties of the chemical structures of these compounds. The results of the present study demonstrate a real possibility for the development of a new generation of effective reversible nonselective MAO inhibitors exhibiting equal inhibitory activity with respect to both MAO A and MAO B.     

Antagonistic effect of magnesium chloride on the nickel chloride–induced inhibition of DNA replication in chinese hamster ovary cells

The degree of inhibition of semiconservative DNA replication induced by nickel chloride (NiCl₂) was analyzed by radiolabeled-thymidine incorporation alone or with cesium chloride (CsCl) density gradient centrifugation. The onset and duration of this Ni²⁺-induced inhibition was time- and concentration- dependent, but the degree of inhibition was not. A maximal reduction in the rate of DNA synthesis was observed within the first hour of treatment with 2.5 mM NiCl₂, which was the highest noncytotoxic concentration utilized. After six hours, 500 μM and 1 mM as well as 2.5 mM NiCl₂ all produced the same 50% to 60% reduction in [³H]-thymidine incorporation into DNA. The inhibitory effect of nickel ions on DNA synthesis was reversible. The rate of DNA synthesis following a 500 μM or 1 mM NiCl₂ treatment began to increase after washout of nickel, but a six-hour exposure of cells to 2.5 mM NiCl₂ produced a sustained 50% to 60% suppression of DNA synthetic activity for at least 36 hours. At all concentrations of NiCl₂ used in this study, some inhibition of DNA synthesis persisted for at least 48 hours, but by 72 hours after treatment, the rate of [³H]-thymidine incorporation was actually 10% above the control. Examination of autoradiographic slides of cells treated with 2.5 mM NiCl₂ for six hours demonstrated a 60% reduction of silver grains, but there was no preferential reduction in the quantity of grains in the nucleolus or any other region. Cesium chloride density gradient analysis of the replication of nucleolar DNA in cells treated with 2.5 mM nickel supported the autoradiographic findings. The inhibitory effect of NiCl₂ on DNA replication was prevented by the addition of magnesium chloride (MgCl₂) to cells maintained in a simple salts/glucose medium (SGM). This effect did not appear to be due to an antagonism of the cellular uptake of nickel by Mg²⁺, since the maximally effective dose of Mg²⁺ reduced ⁶³Ni²⁺ uptake by no more than 25% while the inhibition of replication was completely reversed.     

Regulation of erythrocyte membrane shape by Ca2+

In the presence of 1.0 mM ATP and MgCl₂, the specific viscosity of suspensions of human erythrocyte ghosts decreases 35% in 20 minutes at 22°C. The changes in viscosity are a sensitive index of Mg-ATP dependent shape changes in these membranes. Low concentrations of Ca²⁺ (1 to 5 μM) inhibit Mg-ATP dependent viscosity changes. If ghosts were preincubated with 1 mM Mg-ATP and 20 μM A23187 to produce a maximal decrease in viscosity, addition of 10 μM Ca²⁺ to the preincubated ghosts increased the viscosity to levels observed in ghosts preincubated without ATP. Ca²⁺ (1 to 5 μM) also inhibited Mg²⁺ dependent phosphorylation 30% and stimulated dephosphorylation 25% in ghost membranes. These effects of Ca²⁺ on viscosity and phosphorylation may be due to a membrane bound Ca²⁺ phosphatase activity which dephosphorylates membranes phosphorylated by a Mg²⁺ dependent kinase activity.     

Calcium and lymphocyte activation.

The role of ionized calcium in the early phases of activation of human peripheral blood lymphocytes was evaluated by stimulating the cells with a calcium ionophore A23187 (Lilly) or with mitogenic lections over a broad range of extracellular calcium concentrations (< 1 to > 1000 μM). A number of biochemical parameters shown previously to be altered during stimulation of these cells by mitogenic lectins were studied including: 1) amino acid transport, 2) phosphatidylinositol turnover, 3) cyclic nucleotide accumulation, and 4) calcium uptake. The ionophore (0.1–0.5 μg/ml) was shown to produce stimulatory effects in all of these systems with the changes closely simulating those produced by the lectins themselves both in regard to time course and magnitude. A23187 also produced 5–10 fold increases in DNA synthesis as measured at 48–72 hr after exposure of the cells to this agent. The responses to A23187 were shown to be almost completely dependent on the presence of ionized calcium. Since mitogenic lectins are known to stimulate calcium uptake and DNA synthesis appears to require extracellular calcium, the early responses to A23187 suggested that calcium was important both during the early and later phases of lymphocyte activation. However, short time course studies of amino acid transport, cyclic AMP accumulation, and phosphatidylinositol turnover in calcium deficient media failed to provide convincing evidence of calcium dependency in lectin stimulation since the three responses were well preserved (<25% inhibition) in “calcium free” medium containing 1–3 mM ethylene bis (ethylene oxynitrilo) tetraacetic acid (EGTA) (an estimated final Ca²⁺ concentration of <1 μM). Greater than 50% inhibition of the lectin response was seen only when the cells were incubated in calcium free, EGTA-containing medium for 30 min prior to stimulation with lectin. Thus despite the striking ability of A23187 complexed with calcium to mimic the action of mitogenic lectins, its effects may involve more than simple transport of calcium into the cell. A23187 may also exert a direct membrane action as suggested by its ability to produce rapid increases in cAMP and the occurrence of cytotoxicity at 5–10 fold higher concentrations (2–4 μg/ml). However, these data do not entirely exclude a mechanism of ionophore action whereby: 1) mobilization of intracellular stores of calcium and 2) diminished intracellular transport of ionized calcium at extracellular concentrations less than or equal to 1 μM combine to provide an effective stimulus for cellular activation.     

Inhibition of Human Immunodeficiency Virus Type-1 (HIV-1) Replication by Immunor (Im28), a New Analog of Dehydroepiandrosterone

Abstract

The inhibition of' HIV-I replication in vitro by Immunor 28 (IM²⁸), an analog of dehydroepiandrosterone (DHEA), was monitored using the HIV- 1 laboratory wild-type strain IIIB. Evaluation of the 50% inhibitory dose (IC₅₀) revealed a decrease in HIV-1 replication giving an IC, value around 22 μM. The toxicity of the drug has been determined also, in MT2 cells and PBMCs. 60 μM of IM²⁸ provoked a 50% decrease in cell viability while DHEA caused the same decrease at 75 μM in MT2 cells. These values are 125 μM for IM²⁸ in PBMCs and 135 μM for DIIBA. Thus, DHEA is less toxic than IM²⁸, but IM²⁸ has a higher antiviral activity.     

Inhibition of rabbit monoamine oxidase by doxepin and related drugs.

The psychotherapeutic agent, doxepin, inhibits both the B and A forms of rabbit lung mitochondrial monoamine oxidase (MAO). The K_i values for doxepin inhibition of phenylethylamine (PEA) and 5-hydroxytryptamine (5-HT) deamination is 3 × 10⁻⁵ and 2 × 10⁻⁴M, respectively. Doxepin is, thus, similar to other tricyclic antidepressant drugs in that it has a greater affinity for the B form of the rabbit oxidase. The ability of doxepin and imipramine and its mono and didesmethyl derivatives to inhibit rabbit MAO is decreased as the pH is raised above 8.0. However, results suggest that the basicity of the propylamine side chain has little or no influence on the ability of these drugs to inhibit the rabbit oxidase. In addition, it is demonstrated that 7 × 10⁻⁶M doxepin inhibits PEA deamination by human platelet MAO approximately 50%.     

Reciprocal regulation of fructose 1,6-bisphosphatase and phosphofructokinase by fructose 2,6-bisphosphate in swine kidney

The effect of fructose 2,6-P₂, AMP and substrates on the coordinate inhibition of FBPase and activation of PFK in swine kidney has been examined. Fructose 2,6-P₂ inhibits the activity of FBPase and stimulates the activity of PFK in the presence of inhibitory concentrations of ATP. Under similar conditions 2.2 μM fructose 2,6-P₂ was required for 50% inhibition of FBPase and 0.04 μM fructose 2,6-P₂ restored 50% of the activity of PFK. Fructose 2,6-P₂ also enhanced the allosteric activation of PFK by AMP and it increased the extent of inhibition of FBPase by AMP. Fructose 2,6-P₂, AMP and fructose 6-P act cooperatively to stimulate the activity of PFK whereas the same latter two effectors and fructose 1,6-P₂ inhibit the activity of FBPase. Taken collectively, these results suggest that an increase in the intracellular level of fructose 2,6-P₂ during gluconeogenesis could effectively overcome the inhibition of PFK by ATP and simulataneously inactivate FBPase. When the level of fructose 2,6-P₂ is low, a glycolytic state would be restored, since under these conditions PFK would be inhibited by ATP and FBPase would be active.     

[3H]HARMALINE AS A SPECIFIC LIGAND OF MAO A—I. PROPERTIES OF THE ACTIVE SITE OF MAO A FROM RAT AND BOVINE BRAINS

A high-affinity (K_d= 5.9 nM) specific binding site for [³H]harmaline was detected in membranes from rat and bovine brains. Studies of the regional and subcellular distributions of this binding indicated its close association with monoamine oxidase type A activity (MAO A) measured with [³H]serotonin ([³H]5-HT) as the substrate. Maximal binding capacity and MAO A activity were found in mitochondrial enriched fractions. Mitochondria of synaptosomal or extra-synaptosomal origin exhibited very similar properties with respect to [³H]harmaline binding characteristics and MAO A activity. Among psychoactive drugs, only monoamine oxidase inhibitors (MAO I) prevented the specific binding of [₃H]harmaline. Logit-log inhibition curves of binding by MAO I gave only one slope which was not significantly different from 1.0, suggesting the existence of only 1 category of specific sites for [³H]harmaline in the membrane preparations from rat and bovine brains. Consistent with the preferential inhibition of MAO A by harmaline, other MAO I of this class, i.e. clorgyline and Lilly 51641, were 10²-2 × 10³ times more efficient than deprenyl and pargyline, two inhibitors of MAO type B, in displacing [³H]harmaline from its specific binding site. K_i and IC₅₀ values for the inhibition of [³H]harmaline binding by MAO I and MAO substrates (tryptamine, 5-HT, norepinephrine) were almost identical with those characterizing their action on MAO A activity with [³H]5-HT as the substrate. In conclusion, the specific binding site for [³H]harmaline exhibited all the expected properties of the active site of MAO A. Like the technique of precipitation with a specific antibody, binding of [³H]harmaline should be of great help for studying the structural characteristics of the active site of MAO A and determining the number of MAO molecules in tissues under various physiological conditions.     

Structure–activity relationship studies of 3-dodecanoylindole-2-carboxylic acid inhibitors of cytosolic phospholipase A2α-mediated arachidonic acid release in intact platelets: variation of the keto moiety

Recently we found that 1-methyldodecanoylindole-2-carboxylic acid (1) and 1-[2-(4-carboxyphenoxy)ethyl]-3-dodecanoylindole-2-carboxylic acid (4) were inhibitors of the cytosolic phospholipase A₂α (cPLA₂α)-mediated arachidonic acid release in calcium ionophore A23187-stimulated human platelets with IC₅₀-values of 4.8 μM (1) and 0.86 μM (4). We have now replaced the 3-acyl residue of these compounds by alkylated sulfinyl-, sulfony-, sulfinamoyl-, sulfamoyl-, carbonylamino-, or carbonylaminomethyl-substituents. Structure–activity relationship studies revealed that the pronounced cellular activity of 4 strongly depends on the presence of the 3-acyl moiety. Surprisingly, when testing 4 and its derivatives in an assay with the isolated cPLA₂, none of these compounds showed an inhibitory potency at 10 μM indicating that they do not inhibit cPLA₂α in the cells by a direct interaction with the active site of the enzyme.     

Thrombin, collagen and A23187 stimulated endogenous platelet arachidonate metabolism: Differential inhibition by PGE1, local anesthetics and a serine-protease inhibitor

The formation of malondialdehyde (MDA) and rabbit aorta contracting substance (RCS) induced by treatment of platelets with thrombin and collagen, but not that produced from exogenous arachidonic acid, is inhibited by prostaglandin E₁ (10⁻⁸ − 10⁻⁷M), the local anesthetics tetracaine, SKF 525-A and dibucaine (1 mM), and the serine-protease inhibitor phenylmethanesulfonyl fluoride (PMSF). The burst in oxygen consumption which accompanies platelet stimulation by thrombin and collagen in the presence of antimycin A, known to be due to the oxidation of endogenous arachidonate, is also markedly suppressed by PGE₁, tetracaine and PMSF. The inhibitory effect of PGE₁ is strongly potentiated by theophylline (1.0 mM).Addition of the Ca²⁺ ionophore A23187 to platelet suspensions overcomes PGE₁ and PMSF inhibition of MDA and RCS formation, and induces a vigorous increase in O₂ consumption. Tetracaine and dibucaine, however, block the responses to A23187.Formation of MDA and RCS (a mixture of PG endoperoxides and TXA₂) due to stimulation by thrombin and collagen depends upon activation of Ca²⁺-dependent phospholipase A₂ (PLA₂) to supply free arachidonate from specific membrane phospholipids. These experiments therefore indicate that increased cellular cAMP, induced by PGE₁, antagonizes the mobilization of the Ca²⁺ which is normally required for PLA₂ activity. Thrombin-stimulated platelets exhibit enhanced ⁴⁵Ca uptake which probably reflects exchange of extracellular Ca²⁺ with an increased available pool of exchangeable intracellular Ca²⁺. PGE₁ strongly suppresses this ⁴⁵Ca uptake, providing more direct evidence supporting the view that cAMP prevents the rise in free cytoplasmic Ca²⁺ induced by thrombin. Under conditions which make sufficient free cytoplasmic Ca²⁺ available (i.e., A23187), despite high cellular cAMP, formation of RCS and MDA, and O₂ uptake are nearly normal indicating that activation of PLA₂ can occur. Local anesthetics on the other hand since they abolish the response to A23187 as well, appear to directly antagonize the ability of Ca²⁺ to activate PLA₂. The effect of PMSF suggests that stimulus-specific proteases may be involved in the thrombin and collagen-induced activation of PLA₂ activity.