One-pot synthesis and cytotoxicity studies of new Mannich base derivatives of polyether antibiotic--lasalocid acid.

Seven Mannich base derivatives of polyether antibiotic Lasalocid acid (2a-2g) were synthesized and screened for their antiproliferative activity against various human cancer cell lines. A novel chemoselective one-pot synthesis of these Mannich bases was developed. Compounds 2a-2c and 2g with sterically smaller dialkylamine substituent, displayed potent antiproliferative activity (IC50: 3.2-7.3 µM), and demonstrated higher than twofold selectivity for specific type of cancer. The nature of Mannich base substituent on C-2 atom at the aromatic ring may be critical in the search for selectivity towards a particular cancer cell.

Enzymatic catalysis of anti-Baldwin ring closure in polyether biosynthesis.

Polycyclic polyether natural products have fascinated chemists and biologists alike owing to their useful biological activity, highly complex structure and intriguing biosynthetic mechanisms. Following the original proposal for the polyepoxide origin of lasalocid and isolasalocid and the experimental determination of the origins of the oxygen and carbon atoms of both lasalocid and monensin, a unified stereochemical model for the biosynthesis of polyether ionophore antibiotics was proposed. The model was based on a cascade of nucleophilic ring closures of postulated polyepoxide substrates generated by stereospecific oxidation of all-trans polyene polyketide intermediates. Shortly thereafter, a related model was proposed for the biogenesis of marine ladder toxins, involving a series of nominally disfavoured anti-Baldwin, endo-tet epoxide-ring-opening reactions. Recently, we identified Lsd19 from the Streptomyces lasaliensis gene cluster as the epoxide hydrolase responsible for the epoxide-opening cyclization of bisepoxyprelasalocid A to form lasalocid A. Here we report the X-ray crystal structure of Lsd19 in complex with its substrate and product analogue to provide the first atomic structure-to our knowledge-of a natural enzyme capable of catalysing the disfavoured epoxide-opening cyclic ether formation. On the basis of our structural and computational studies, we propose a general mechanism for the enzymatic catalysis of polyether natural product biosynthesis.

Mucosal mast cell proteases are involved in colonic permeability alterations and subsequent bacterial translocation in endotoxemic rats.

LPS-induced endotoxemia is associated with gut immune stimulation, mucosal inflammation, colonic paracellular permeability (CPP) alteration, and it promotes bacterial translocation (BT). Gut permeability increase linked to LPS promotes mucosal barrier dysfunction resulting to BT. However, the mechanisms involved in these alterations remain unknown. We aimed to evaluate the role of colonic mucosal mast cells and luminal serine protease activity (PA) in the alterations of CPP and BT induced by LPS. Rats receiving doxantrazole, a mast cell stabilizer, combined or not with LPS from Escherichia coli and CPP as well as BT were evaluated after each treatment. Mucosal mast cell activation was assessed by histological methods and by rat mast cell protease 2 level measurement in colonic content. Colonic luminal PA and mucosal inflammation (myeloperoxidase activity) were biochemically determined. In addition, the ability of luminal contents to act on CPP was evaluated in vitro in Ussing chambers. Peripheral administration of LPS promoted mast cell degranulation and increased CPP, BT, mucosal myeloperoxidase activity as well as rat mast cell protease 2 levels, and PA in colonic content. LPS-induced CPP increase and BT were prevented by doxantrazole. In vitro, exposure of the apical side of colonic tissues with supernatants from colonic contents of LPS-treated rats increased CPP. This effect was blocked by the serine protease inhibitor soybean trypsin inhibitor. Our data bring evidence of a key role of mucosal mast cells in LPS-induced increase of CPP and BT through the release of serine proteases into the colonic lumen.

Secretion of the trefoil factor TFF3 from the isolated vascularly perfused rat colon.

The trefoil factor TFF3 is a peptide predominantly produced by mucus-secreting cells in the small and large intestines. It has been implicated in intestinal protection and repair. The mechanisms that govern TFF3 secretion are poorly understood. The aim of this study was, therefore, to evaluate the influence of neurotransmitters, hormonal peptides and mediators of inflammation on the release of TFF3. For this purpose, an isolated vascularly perfused rat colon preparation was used. After a bolus administration of 1 ml isotonic saline into the lumen, TFF3 secretion was induced by a 30-min intra-arterial infusion of the compounds to be tested. TFF3 was evaluated in the luminal effluent using a newly developed radioimmunoassay. TFF3 was barely detected in crude luminal samples. In contrast, dithiothreitol (DTT) treatment of the effluent revealed TFF3 immunoreactivity, which amounted to about 0.3 pmol min(-1) cm(-1) in the basal state. Gel chromatography of DTT-treated luminal samples revealed a single peak that co-eluted with the monomeric form of TFF3. TFF3 was not detected in the portal effluent. Bethanechol (10(-6)-10(-4) M), vasoactive intestinal peptide (VIP, 10(-8)-10(-7) M) or bombesin (10(-8)-10(-7) M) induced a dose-dependent release of TFF3. In contrast, substance P evoked a modest release of TFF3, whereas calcitonin gene-related peptide (CGRP), somatostatin, neurotensin or peptide YY (PYY) did not modify TFF3 secretion. The degranulator compound bromolasalocid, 16,16-dimethyl PGE2 (dmPGE2) or interleukin-1-beta (IL-1-beta) also evoked a marked release of TFF3. In conclusion, TFF3 in the colonic effluent is present in a complex. This association presumably involves a disulfide bond. Additionally, the present results suggest a role for enteric nervous system and resident immune cells in mediation of colonic TFF3 secretion.

Systemic lipopolysaccharide influences rectal sensitivity in rats: role of mast cells, cytokines, and vagus nerve.

Intraperitoneal lipopolysaccharide (LPS) produces somatic hyperalgesia, releases interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha), and activates vagal afferents. The aim of this study was to evaluate the effect of peripheral LPS on rectal sensitivity and to specify the mechanisms involved. Abdominal muscle contractions were recorded in conscious rats equipped with intramuscular electrodes. Rectal distension (RD) was performed at various times after LPS or experimental treatments. In controls, RD significantly increased the number of abdominal contractions from a threshold volume of distension of 0.8 ml. At the lowest volume (0.4 ml), this number was increased after administration of LPS (3, 9, and 12 h later), recombinant human IL-1beta (from 3 to 9 h), recombinant bovine TNF-alpha (from 6 to 9 h), and BrX-537A (from 6 to 12 h), a mast cell degranulator. The effect of LPS was reduced by doxantrazole, Lys-D-Pro-Thr, and soluble recombinant TNF receptor. Vagotomy selectively amplified the response to LPS. We conclude that, in vivo, intraperitoneal LPS lowers visceral pain threshold (allodynia) through a mechanism involving mast cell degranulation and IL-1beta and TNF-alpha release and that the vagus nerve may exert a tonic protective role against LPS-induced rectal allodynia.

Effects of neurotransmitters, gut hormones, and inflammatory mediators on mucus discharge in rat colon.

The effect of potential mediators of mucus secretion was investigated in the isolated vascularly perfused rat colon by using a sandwich enzyme-linked immunosorbent assay for rat colonic mucin and by histochemical analysis. Bethanechol (100-200 microM), bombesin (100 nM), and vasoactive intestinal peptide (VIP, 100 nM) provoked a dramatic mucin discharge (maximal response at 900, 900, and 600% of control loops, respectively). VIP-stimulated mucin secretion was abolished by tetrodotoxin, whereas atropine was without effect. In contrast, both tetrodotoxin and atropine significantly decreased mucin release induced by bombesin. Isoproterenol or calcitonin gene-related peptide was without effect. Serotonin (1-5 microM) and peptide YY (10 nM) evoked mucin discharge, whereas glucagon-like peptide-1 did not release mucin. Finally, bromolasalocid (20 microM), interleukin-1beta (0.25 nM), sodium nitroprusside (1 mM), and dimethyl-PGE2 (2.5 microM) induced mucus discharge. The results demonstrated a good correlation between the immunological method and histological analysis. In conclusion, these findings suggest a role for the enteric nervous system, the enteroendocrine cells, and resident immune cells in mediation of colonic mucus release.

Mast cell degranulation induces delayed rectal allodynia in rats: role of histamine and 5-HT.

Visceral hypersensitivity is a common feature of functional bowel disorders, where an increased number of mast cells have often been described. Thus, we investigated the effect of an experimental mast cell degranulation induced by BrX-537A on somatic (tail heating) and visceral (rectal distension) sensitivity in rats and the involvement of histamine and/or serotonin on this last response. After BrX-537A administration, the latency of tail withdrawal reflex was shortened within the 2- to 8-hr period. Moreover, BrX-537A reduced the distension volume threshold from 0.8 ml to 0.4 ml inducing allodynia, from 6 to 12 hr after its administration. This effect was suppressed by doxantrazole (mast cell stabilizing agent) and WAY 100635 (5-HT1A receptor antagonist), and reproduced by 5-HTP (5-HT precursor) and 8-OH-DPAT (5-HT1A receptor agonist). However, neither granisetron (5-HT3 receptor antagonist) nor H1, H2, or H3 histamine receptor antagonists modified the BrX-537A-induced allodynia. Consequently, mast cell degranulation initiates a delayed somatic and visceral allodynia, with the participation of serotonin, through 5-HT1A receptor activation, on the visceral response.

Role of 5-HT3 receptors and afferent fibers in the effects of mast cell degranulation on colonic motility in rats.

BACKGROUND/AIMS: Mediators released by mast cell degranulation contribute to digestive motility disturbances. According to the role of serotonin and the close proximity of mast cells to nerves, the aim of this study was to assess the role of 5-hydroxytryptamine 3 (5-HT3) receptors, capsaicin-sensitive afferent fibers, and some of their neuropeptides (substance P and calcitonin gene-related peptide) in colonic motor alterations induced by degranulation of mast cells by the compound BrX-537A. METHODS: The effects of BrX-537A (2 mg/kg intraperitoneally) were determined by electromyography in conscious rats implanted with electrodes in the cecocolonic wall. RESULTS: BrX-537A inhibited cecocolonic myoelectric activity for 7-8 hours. A primary and dramatic reduction of spike burst frequency, lasting 30 minutes, was affected by none of the pretreatments tested. The following inhibition was fully antagonized by ketotifen (mast cell stabilizer), granisetron and ondansetron (5-HT3 antagonists), RP-67,580 (NK1 antagonist), and perivagal capsaicin pretreatment. A temporary blockade was observed after administration of CP-96,345 (NK1 antagonist) and in rats systemically treated by capsaicin. The calcitonin gene-related peptide antagonist hCGRP(8-37) did not modify the BrX-537A-induced inhibition. CONCLUSIONS: 5-HT3 receptors, sensory afferent fibers reaching the vagus nerves, and substance P are major components of the colonic motor inhibition induced by mast cell degranulation.

[Role of serotonin and histamine in the effects of degranulation of mast cells on the colonic motility and the transit. Experimental study in rats]. / Rôle de la sérotonine et de l'histamine dans les effets de la dégranulation des mastocytes sur la motricité et le transit coliques. Etude expérimentale chez le rat.

The aim of this work was to describe the alterations of colonic motility and transit induced by an experimental, histologically verified, degranulation of mast cells, provoked by the compound BrX-537A, and to determine the role of serotonin and histamine by specific antagonists, in the rat. Colonic myoelectrical activity was inhibited by BrX-537A (2 mg/kg IP) in a biphasic manner. The initial profound inhibition, lasting 30 min, during which the frequency of spike bursts decreased from 9.2 +/- 1.1 to 1.4 +/- 0.5/10 min, was followed by a sustained (5 h) period of moderate inhibition (5.2 +/- 0.5 spike bursts/10 min). In the same way, BrX-537A increased the mean retention time of a marker injected in the proximal colon (10.8 +/- 1.4 h vs 7.4 +/- 0.4 h). Neither serotoninergic nor histaminergic antagonists, at a dose of 1 mg/kg IP, modified the primary drastic inhibition of colonic motility during the first 20 minutes. After, a selective time-related blockade of this inhibition was observed. Granisetron blocked the inhibition from the 30th minute on, methysergide from the 120th minute on, and chlorpheniramine, between the 20th and 60th minutes. In conclusion, the inhibitory effect of mast cell degranulation depends on serotonin and histamine release, in a time-related manner, and implicates the H1, 5-HT3 and 5-HT1 or 2 receptors.

Relationship between mast cell degranulation and jejunal myoelectric alterations in intestinal anaphylaxis in rats.

The effects of two degranulators of mast cells and intestinal anaphylaxis on jejunal myoelectric activity were compared in rats fasted for 15 hours. Attempts to antagonize the motility changes were performed using antagonists of histamine and serotonin and a cyclooxygenase and lipoxygenase inhibitor. Hooded Lister rats were chronically fitted with electrodes implanted in the jejunal wall. A group of rats was sensitized to egg albumin and challenged 14 days later by intraduodenal infusion of antigen. Sensitized animals had serum titers greater than or equal to 1:64. The other group was administered with mast cells degranulators. Both 48/80 (1 mg/kg), a degranulator of connective mast cells, and bromolasalocid (2 mg/kg), acting on connective and mucosal mast cells, induced a phase of total spiking inhibition followed by a progressive irregular spiking activity until the recovery of migrating myoelectric complex pattern (about 3 hours after injection). In contrast, antigen challenge disrupted the migrating myoelectric complex pattern, which was replaced by a peculiar pattern characterized by propagated spike burst, lasting 98 +/- 11.3 minutes. Chlorpheniramine (1 mg/kg) antagonized only the inhibitory phase induced by degranulators and was ineffective on the intestinal anaphylaxis-induced motor changes. Methysergide (1 mg/kg) and indomethacin (5 mg/kg) significantly reduced the degranulator effects as well as the anaphylaxis-induced alterations of intestinal motility. It is concluded that anaphylaxis-induced motor disturbances are relevant to mucosal mast cell degranulation involving 5-hydroxytryptamine and arachidonic acid derivative products, whereas histamine release appears to be a minor component.

Involvement of 5-hydroxytryptamine in the intestinal motor disturbances induced by mast cell degranulation in rats.

Fasted rats with chronically implanted electrodes were used for investigation of the effects of mast cell degranulation induced by compound 48/80 and BrX-537A and their antagonism by previous administration of 5-hydroxytryptamine (5-HT) antagonists on duodenal and jejunal myoelectric activity. Administered i.p., both 48/80 (1 mg/kg i.p.) and BrX-537A (2 mg/kg i.p.) abolished the intestinal spiking activity of duodeno-jejunum with a progressive recovery, BrX-537A being less active. These effects were dose-related. Injected prior to 48/80, methysergide (1 mg/kg) reduced by about 80% both duodenal and jejunal inhibition of spiking activity with early recovery of a normal pattern. In contrast, ketanserin (1 mg/kg) had selective reducing effects on the duration of the spiking inhibition induced by 48/80 and BrX-537A on the duodenum only. Zacopride (1 mg/kg) and ICS 205-930 (50 micrograms/kg) shortened and suppressed, respectively, the inhibition of intestinal spiking activity with early restoration of intestinal motility in both duodenum and jejunum. We conclude that, in fasted rats (i) the degranulation of peritoneal mast cells induces alterations in intestinal myoelectric activity through the release of 5-HT (ii) these effects are mainly mediated through both 5-HT1 and 5-HT3 receptors.

Cluster significance analysis contrasted with three other quantitative structure-activity relationship methods.

Cluster significance analysis (CSA), a new statistical method to analyze structure-activity relationships in graphically displayed data, is contrasted with linear discriminant analysis, SIMCA, and the method of "relative odds". The data sets evaluated are as follows: antibacterial lasalocid derivatives, antimalarial naphthoquinones, and carcinogenic polycyclic aromatic hydrocarbons. CSA gives results comparable to these other methods, involves fewer assumptions, can be more reliable, and in general is easier to understand.

Bromolasalocid (Ro 20-0006) antihypertensive ionophore.

Bromolasalocid (Ro 20-0006) is a calcium ionophore with antihypertensive activity that does not belong to any known class of antihypertensive agents. Bromolasalocid produces a relatively flat systolic blood pressure dose-response effect in the spontaneously hypertensive rat. An intensive cardiovascular evaluation of bromolasalocid at the highest dose used in the dose-response study showed full hemodynamic compensation; there was a significant decrease in both mean arterial blood pressure and peripheral resistance without a significant decrease in cardiac index. The antihypertensive action of bromolasalocid lasts many days after termination of dosing. Bromolasalocid is specifically antihypertensive and does not decrease arterial blood pressure in normotensive animals or in animal models of hypertensive cardiovascular disease with normal pulse pressures. Bromolasalocid is not a vasodilator and appears to mediate its antihypertensive action by restoring compliance of the large conduit arteries. Both the derived arterial compliance index and the blood pressure-pressor response to the carotid occlusion reflex are enhanced in the dog perinephritis model of hypertensive cardiovascular disease treated with bromolasalocid. Bromolasalocid appears to reverse the damage to cardiovascular tissue caused by prolonged hypertension via an action on calcium perturbations in large artery smooth muscle cells.

Influence of membrane viscosity on the lateral and transverse mobility of carboxylic ionophores.

The rate of 45Ca or 22Na exchange-diffusion in multilamellar liposomes formed of dipalmitoyl-phosphatidylcholine (DPPC) and cholesterol and containing the ionophore A23187 or Br-X537A was dramatically increased when the temperature and, hence, fluidity of the lipid bilayer were increased. In the case of 45Ca transport, i.e. when each Ca2+ ion binds to two molecules of ionophore, the relative increment in transport velocity in response to a given increase in temperature or fluidity was much more marked in the high range of temperature (30-40 degrees C) than in the low range of temperature (22-28 degrees C). In the case of 22Na transport, however, i.e. when each Na+ ion binds to only one ionophoretic molecule, the temperature-dependency of the transport process followed a single pattern throughout the entire range of temperature. In the latter case, the slope of the temperature-dependent line was the same as that seen for 45Ca transport by the same ionophore at high temperatures. A decrease in the ionophore content of the liposomes shifted to a higher temperature the transition point between the flat and steep lines characterizing the temperature dependency of 45Ca transport. It is concluded that the membrane viscosity affects both the lateral mobility of the ionophoretic molecules and the transverse mobility of the cation-ionophore complex.

Determination of bromo-lasalocid in plasma by high-performance liquid chromatography with fluorimetric detection.

A rapid, sensitive, and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of bromo-lasalocid in plasma. The compound was extracted into isooctane-ethyl acetate (90:10) from plasma saturated with potassium chloride and adjusted to strongly alkaline pH. The residue of this extract was dissolved in methanol-2-methoxyethanol (95:5) and analyzed by HPLC on a 10-micrometer C18 column [mobile phase of methanol-water-2-methoxyethanol-1 M potassium phosphate buffer, pH 3.0 (90:10:2.5:0.2)] using fluorescence detection with excitation at 215 nm and emission at wavelengths greater than 370 nm. The overall recovery of the assay was 65%, with a limit of sensitivity of 0.1 microgram/ml. The method was used to obtain plasma concentration-time profiles in the dog following oral administration of bromo-lasalocid-ethanolate.

Surface potential and conductance induced in lipid bilayers by the negatively charged ionophore Br-X537A.

The adsorption of Br-X537Z and its effect on the surface potential of monoolein lipid bilayers was measured using the nonactin conductances as a probe to determine the surface charge density. Because of the pH dependence of this adsorption, it was concluded that not only the negatively charged molecules X- could induce a surface charge but also a dimer HX2- made from X- and the neutral molecule HX. Also an important bilayer conductance was induced by Br-X537A. From the BrX537A concentration dependence of this conductance, the effect of pH, and the induced surface potential, it was found that two charged complexes are transported across the bilayer depending on pH. At pH greater than or equal to 7 the conducting molecule is X- and at pH less than or equal to 5 the complex is H2X3-. A quantitative model is obtained to calculate both the induced surface potential and the conductance.

Cation and local anesthetic conductance induced by the negatively charged ionophore Br-X537A in lipid bilayers.

The cation conductance induced by Br-X537A was measured for K+ and Ca2+ at different pH and different Br-X537A concentrations. The K+-concentration dependence was different depending on the pH of the solution. At pH 4.3, the conductance depended linearly on [K+] while at pH 7 it depended on [K+]2. About the same dependence on [Br-X537A] was found at these pH values. From these results it was concluded that two complexes were transporting K+; at pH greater than or equal to 7 the complex is mostly K2X3- and at pH less than or equal to 5 the complex is mostly KHX3-. From the conductance dependence on [Ca2+], pH, and [Br-X537A] it was found that the only conducting complex is CaX3-. It was also observed that Br-X537A could transport tetracaine. Taking into account the surface charge induced by tetracaine, the conductance depended linearly on the tetracaine concentration. Correcting for the surface charged induced by Br-X537A, the tetracaine conductance was found to depend on [Br-X537A]2. Consequently, the conducting complex is TX2- at pH greater than or equal to 7. No other conducting complexes are formed at the pH is decreased and at pH 4 tetracaine is not conducted. It was found that procaine was not conducted between pH 8 and 4.