The effects of metoclopramide and cloxacepride on human mast cells from adenoidal tissues

Cloxacepride is an amide of the dopamine antagonist metoclopramide and has been reported to possess oral antiallergic properties in the rat PCA model. Both substances have now been tested in isolated mast cell preparations from human adenoidal tissues to determine whether any therapeutic antiallergic potential in man could be expected. Metoclopramide at concentrations 10(-5)-10(-3) M had no inhibitory effect but instead enhanced Con A-induced histamine release at concentrations greater than 10(-4) M. Cloxacepride at concentrations 10(-5)-10(-4) M significantly inhibited Con A-induced histamine release. This inhibitory effect was not diminished by increasing the preincubation time for up to 30 min. In contrast, cloxacepride concentrations greater than 4 x 10(-5) M caused a substantial histamine release. This effect could not be alleviated by an increase in the number of mast cells per sample. These results then suggest a very narrow range of therapeutic potential for cloxacepride.     

Effects of calmodulin antagonists on human adenoidal mast cells.

N-(6-aminohexyl)-1-naphthalenesulfonamide (W5), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) and Triflupromazine (TFPr) are substances with calmodulin antagonistic properties. All of these compounds inhibited the Con A-induced histamine release from human adenoidal mast cells. Maximal inhibition for W5 was observed at 200 microM, for W7 at 50 microM and for TFPr at 20 microM. Higher concentrations of each substance induced a marked histamine release. The kinetics of the mediator release were found to be different for Con A and W7 only after 5 mins incubation. The kinetics of TFPr were found to be biphasic: a slow onset was followed by a fast release reaction.     

The effects of prostaglandins of the E and I type on histamine release from human adenoidal mast cells

(PgE1 (10(-8)-10(-6) M) inhibited dose dependently the Con A-induced histamine release from human adenoidal mast cells. Out of two stable PgI analogues tested, EL 784 had a slight inhibitory effect at 10(-10) M, and SE 63 at 10(-4) M. PgI2 itself was ineffective.     

Histamine release from human adenoidal and mesenteric mast cells induced by bacterial antigens

The histamine-releasing capability of Staphylococcus aureus antigens was examined in human adenoidal and mesenteric mast cells obtained by enzymic dispersion of tissues from non-allergic patients. Both populations of mast cells released histamine after challenge with bacterial protein in concentrations between 5-500 micrograms/ml. The release was dependent on the dose, temperature and metabolic energy. The maximum release was observed at 15 min after challenge. The present results suggest that Staphylococcus aureus antigens release histamine from human adenoidal and mesenteric mast cells via a non-cytotoxic, active secretory process.     

The effects of adenosine and of some adenosine analogues on the concanavalin A-or acetylcholine-induced histamine release from human adenoidal mast cells

Histamine release induced by concanavalin A (Con A) or acetylcholine is enhanced by adenosine or the adenosine analogues PIA and NECA. The enhancement is not affected by preincubation with adenosine deaminase. The degree of the Con A-induced histamine release decreases with increasing incubation time. Under these conditions, the enhancing effect of adenosine on histamine release is either abolished or even reversed to inhibition.     

Properties of the racemic species of verapamil hydrochloride and gallopamil hydrochloride

It is well known that the stereoselective actions associated with the enantiomeric constituents of a racemic drug can differ markedly in their pharmacodynamic or pharmacokinetic properties. Nevertheless, molecular chirality manifests itself in the solid, that is, crystalline state. The aim of this work was to characterize the solid-state properties of verapamil HCl and gallopamil HCl, two well-known chiral calcium channel antagonists. The characterization of the solid state for the single enantiomers and equimolecular mixtures for both the calcium antagonists was performed by solid-state techniques such as Fourier transform infrared (FT-IR spectroscopy), X-ray powder diffractometry (XRD) and differential scanning calorimetry (DSC). The FT-IR spectra and XRD of the single enantiomers are different from those of the corresponding equimolecular mixture owing to their different crystalline structure. The thermal behavior of the racemates and pure enantiomers were examined by DSC, and the resultant experimental and theoretical binary phase diagrams are discussed. Spectroscopic solid-state techniques, such as FT-IR and XRD, are useful in combination with thermal analysis for characterizing the racemic species of chiral drugs. The data obtained prove that the equimolecular mixtures of both verapmil hydrochloride and gallopamil hydrochloride exist as racemic compounds. Determination of the enantiomeric purity of the enantiomers and racemic compounds of both the calcium antagonists analyzed was performed by DSC.     

Red blood cell partitioning of gallopamil, verapamil and norverapamil.

In-vitro binding of calcium-antagonists gallopamil and verapamil (and its main metabolite norverapamil) to human red blood cells (RBCs) was investigated. The drugs are bound reversibly and dose dependent to RBCs in the same order of magnitude, with partition-coefficients of kRBC = 0.12-0.34 for gallopamil, kRBC = 0.10-0.30 for verapamil and kRBC = 0.10-0.27 for norverapamil. The data indicate that, although RBCs may act as subcompartments of the blood for this class of compounds, they may have no influence on therapeutic plasma concentrations, due to their low kRBC.     

Calcium channel antagonists verapamil and gallopamil are powerful inhibitors of acid secretion in isolated and enriched guinea pig parietal cells

In isolated and enriched guinea pig parietal cells the inhibitory effects of the calcium channel antagonists verapamil and gallopamil on 14C-aminopyrine uptake (= H+ secretion) have been analyzed. Both verapamil and gallopamil inhibit acid secretion in a concentration-dependent manner with an IC50 of 12.1 and 10.9 mumol/l respectively. The type of inhibition is noncompetitive in nature. Verapamil inhibits the acid response to histamine, dibutyryl-cAMP, and KCl with IC50 values not significantly different from each other. Exposure of the cells to verapamil and subsequent washing enhances the acid response to histamine for an unknown reason. It is concluded that the calcium channel antagonists verapamil and gallopamil inhibit acid secretion in vitro by interfering with the parietal cell proton pump, the K+/H+-ATPase.     

N-Formylmethionyl-leucyl-phenylalanine: Different releasing effects on human neutrophils and rat mast cells

N-Formylmethionyl-leucyl-phenylalanine (FMLP) is a synthetic chemotactic peptide which induced beta-glucuronidase and lysozyme release from human neutrophils treated with cytochalasin B. FMLP-releasing effects were rapid and dose dependent. Unlike other secretagogues of neutrophils (e.g., zymosan and immune complexes), FMLP secretory activity was not modulated by acetylcholine, which by itself did not release lysosomal enzymes from human neutrophils. Isolated rat mast cells did not respond to FMLP, which has been demonstrated to release histamine from human neutrophils. Two markers of rat mast cell secretory granules, histamine and beta-glucuronidase, were assayed, but the results were negative for both. In the same experimental conditions, 48/80 released histamine and the enzyme: the ratio of the net percentage release of beta-glucuronidase to the net percentage release of histamine was congruent 0.4.     

Uptake and distribution of fullerenes in human mast cells

Fullerenes are carbon cages of variable size that can be derivatized with various side chain moieties resulting in compounds that are being developed into nanomedicines. Although fullerene use in several preclinical in vitro and in vivo models of disease has demonstrated their potential as diagnostic and therapeutic agents, little is known about how they enter cells, what organelles they target, and the time course for their cellular deposition. Fullerenes (C₇₀) that have already been shown to be potent inhibitors of mast cell (MC)–mediated allergic inflammation were conjugated with Texas red (TR) and used in conjunction with confocal microscopy to determine mechanisms of uptake, the organelle localization, and the duration they can be detected in situ. We show that C₇₀-TR are nonspecifically endocytosed into MCs, where they are shuttled throughout the cytoplasm, lysosomes, mitochondria, and into endoplasmic reticulum at different times. No nuclear or secretory granule localization was observed. The C₇₀-TR remained detectable within cells at 1 week. These studies show that MCs endocytose fullerenes, where they are shuttled to organelles involved with calcium and reactive oxygen species production, which may explain their efficacy as cellular inhibitors.From the Clinical EditorFullerenes are carbon cages of variable size that have already been shown to be potent inhibitors of mast cell (MC)-mediated allergic inflammation. These were conjugated with Texas red (TR) and used in conjunction with confocal microscopy to determine mechanisms of uptake, the organelle localization, and duration, demonstrating that MCs endocytose fullerenes, which are shuttled to organelles involved with calcium and reactive oxygen species production. This intracellulac trafficking may explain the efficacy of fullerenes as cellular inhibitors.     

The influence of H1-, H2- and H3-receptors on the spontaneous and ConA induced histamine release from human adenoidal mast cells.

The effects of the H3-agonist R-alpha-methylhistamine (R-alpha-MeHA) and the H3-antagonist thioperamide on the spontaneous and concanavalin A (ConA) induced histamine release from human mast cells were tested and compared with the effect of some H1- and H2-receptor active substances. R-alpha-MeHA (10(-9)-10(-7) M) exerted no effect on histamine release whereas thioperamide increased the spontaneous release at 10(-6)-10(-4) M but inhibited the ConA induced release in a narrow concentration range (10(-6)-10(-5) M). This enhancement might be taken as an indication of the existence of H3-receptor dependent autoregulation although presently other mechanism cannot be excluded.     

The effects of aspartame on mast cells and basophils

The artificial sweetener aspartame was studied to determine whether it had any direct effects on mast cells and basophils. Aspartame was not shown to be a direct mast cell or basophil secretagogue in vitro, or in vivo as assessed by skin testing. During an acute incubation, aspartame did not affect IgE-mediated histamine release from mast cells. However, mast cells cultured in aspartame for periods of up to 9 days showed enhanced rates of proliferation and decreased responsiveness to releasing stimuli. The effect of aspartame on proliferation of cells in culture could be ascribed to a non-specific enhancing effect of its constituent amino acids.     

Passive sensitization of human intestinal mast cells

Dispersed human intestinal mast cells were used for passive sensitization experiments. Eight biopsies (9.7 +/- 1.2 mg/biopsy) of human duodenum were collected from non-atopic children (5) and adults (5). The tissue was dispersed mechanically and enzymatically to yield single cell suspensions. The method produced 2 x 10(3) mast cells per mg wet weight of tissue in a purity of 2.8%. Passive sensitization of the mast cells was performed with the patients' own plasma and plasma obtained from atopic donors. The non-atopic mast cells were able to bind the allergen-specific IgE. In addition, passive sensitization with atopic donor-plasma enhanced the cell sensitivity and cell reactivity to anti-IgE challenge, but had no effect on the cellular response to the ionophore A23187. The study shows that the enzymatic dispersion of human intestinal mast cells produces functionally intact mast cells with preserved Fc-receptors which can be passively sensitized.     

Isotretinoin-induced effects of mast cells on wound healing

Wound healing is a complex process, and the role of retinoids in wounds is confusing and controversial. In this study, the authors aimed to evaluate the role of oral isotretinoin on mast cells, collagen production and remodeling in the wound healing process. In the first group 2 mg/kg/day isotretinoin dissolved in sunflower oil were administrated for 28 days. In the second group, only sunflower oil was administered. At the end of first week, four incisions were made on rats' back. In the seventh (group 1a and 2a), fourteenth (group 1b and 2b) and twenty-first (group 1c and 2c) days, the skin biopsies were taken from around of the incision areas from both groups. The authors revealed that isotretinoin administration permanently accelerates mast cell accumulation parallel to days in the wound area. The histological features of wound healing in isotretinoin administered rats were faster and better than the group 2.     

On the isolation of mast cells from human adenoids and tonsils

Human adenoids and tonsils were disintegrated mechanically and the cells dispersed by passage through a stainless-steel screen in EDTA-containing buffer. Collagenase digestion did not increase the yield of adenoidal cells. The mast cell content of the cell suspensions was in the range of 1-10 mast cells/10(4) cells with an estimated mean of 1-2 mast cells/10(4) cells, a value considerably below previous reports on adenoidal cell suspensions. The mast cell content was determined by staining with toluidine blue at low pH (to prevent interference by phagocytes). The mast cell count as assessed by alcian blue staining and by fluorescence microscopy after FITC-anti-human IgE binding was similar. Various attempts to enrich the cell suspension (i.e. by differential centrifugation, by gradient centrifugation on Ficoll or Ficoll-Hypaque and by velocity sedimentation at unit gravity) all gave negative results.     

Inhibitory effects of verapamil and diltiazem on simvastatin metabolism in human liver microsomes

AIMS: To determine the effects of verapamil and diltiazem on simvastatin metabolism in human liver microsomes and to compare their inhibitory potencies and CYP3A4 inactivation parameters with those reported previously for mibefradil. METHODS: Simvastatin metabolism was investigated in human liver microsomes in the presence and absence of verapamil or diltiazem (0.1-250 microM). Kinetics of CYP3A4 inactivation by verapamil and diltiazem were determined using testosterone as the substrate. RESULTS: When verapamil was coincubated with simvastatin, IC50 values ranged from 23 to 26 microM for all major metabolites. The IC50 values ranged from 4.8 to 5.6 microM on preincubation of verapamil for 30 min in the presence of an NADPH-generating system. Corresponding IC50 values for diltiazem ranged from 110-127 microM and from 21-27 microM, respectively. Verapamil and diltiazem inhibited testosterone 6beta-hydroxylation in a time- and concentration-dependent manner, key features of mechanism-based inactivation. Values for the inactivation parameters kinact and KI were 0.15 +/- 0.04 min-1 (mean +/- s.d.) and 2.9 +/- 0.6 microM, respectively, for verapamil and 0.07 +/- 0.01 min-1 and 3.3 +/- 1.5 microM, respectively, for diltiazem. CONCLUSIONS: The IC50 values for coincubation of verapamil and diltiazem were 46- and 220-fold higher, respectively, than those reported previously for mibefradil, and 16- and 71-fold higher, respectively, for preincubation. Thus, the results of this study suggest that verapamil and diltiazem are less likely than mibefradil to cause acute drug interactions with simvastatin in vivo. However, verapamil and diltiazem are moderate mechanism-based inhibitors of CYP3A4 and therefore may still cause significant inhibition of simvastatin metabolism in vivo during chronic therapy.     

In vitro effects of gallopamil on platelet aggregation and thromboxane generation

The in vitro effects of gallopamil, a highly specific calcium-channel blocker, on platelet function were investigated. Gallopamil caused a dose-dependent inhibition of platelet aggregation. In fact, the drug in concentrations as low as 0.5 microgram/ml inhibited platelet activating factor and epinephrine-induced platelet aggregation. Similarly, the inhibition of adenosine diphosphate and epinephrine-induced thromboxane generation was demonstrated. Since platelet activation and thromboxane release may be related to myocardial ischemia, it is conceivable that some of the antianginal effects of gallopamil may in part be due to platelet inhibitory effects.     

Tolerance to monovalent dextran of human mast cells

In the present study it was investigated if monovalent dextran (Dx 1, HR 573, Promit) possibly involving activated complement is capable of inducing mediator (histamine) release from isolated human mast cells. Dx 1 in the absence or in the presence of 20% fresh human serum did not induce a histamine release from human mast cells. The "non-release" could be established statistically using a new sequential test which provides a substantial reduction of the average sample size required to reach a prescribed test power. In contrast, concanavalin A (Con A) or ionophore A 23187 caused a marked histamine release in the absence of human serum. In the presence of human serum the spontaneous histamine release was increased but the Con A- or ionophore-induced release was inhibited.