Chemotactic selection with insulin, di-iodotyrosine and histamine alters the phagocytotic responsiveness of Tetrahymena

Chemotactic selection is a method by which populations of cells exposed to ligands can be isolated and subsequently cultivated. We used Tetrahymena pyriformis GL cultures selected by chemotactic selection to insulin (10 nM), histamine (0.1 nM) and di-iodotyrosine (T2, 10 nM) to study the phagocytotic capacity under the induction of selector hormones. Our results show a long-lasting link between chemotactically selected cultures and phagocytotic activity. Cells selected to histamine produced the highest phagocytotic activity upon a second exposure to the selector hormone. T2 selection was also strongly effective, however, the phagocytosis stimulation was not specific to the hormone given later. Insulin selected sub-populations had different phagocytotic responses to the control substance itself, whereas histamine selected sub-populations seem to be heterogeneous in the phagocytotic response to histamine. For insulin, the increased endocytotic or metabolic activity was demonstrated by the lack of non-phagocytotic cells. These experiments call attention to the evolutionary role of selection in the later developing receptor-hormone relationship.     

Hormone receptor studies on frog macrophage cells by means of histamine, serotonin and indoleacetic acid.

The phagocytotic activity of frog macrophage cells could be increased by 50% with histamine and by 24% with serotonin. These hormones have a similar effect at the various stages of phylogenetic development, to judge from the respective responses of the unicellular Tetrahymena which showed roughly the same percentual increases of phagocytosis as frog macrophages at roughly the same hormone concentrations. It is concluded that cytoplasmic membrane receptor patterns for a given function do not change in the course of phylogenetic development and the receptors have a capacity for selection, preferring histamine to serotonin, and the latter to the chemically closely related plant hormone indoleacetic acid.     

Effects of H1 and H2 receptor antagonists on Tetrahymena.

In Tetrahymena pyriformis the phagocytotic rate increases in response to histamine, but neither the H1 antagonist phenindamine nor the H2 antagonist metiamide stimulate phagocytosis. The H1 antagonist counteracts the effect of histamine, whereas the H2 antagonist does not. The histamine receptor of Tetrahymena is of H1-type, since it cannot distinguish between histamine and antagonists which are closely related to it chemically. It does, however, distinguish between histamine and the chemically unrelated H1 antagonist, phenindamine. The H2 antagonist does not interact with the receptor.     

Characterization of histamine H1-receptor on rat hepatocytes

The binding sites for [3H]pyrilamine in isolated rat hepatocytes were characterized. Scatchard analysis revealed two kinds of binding sites in hepatocytes, a high-affinity site and a low-affinity one. The rates of binding of the radioligand with the high-affinity binding site and its dissociation were rapid. The specificity of the sites for various histamine antagonists indicated that the high-affinity [3H]pyrilamine binding site is representative of the histamine H1 receptor. Treatment of hepatocytes with protease or phospholipase A2 significantly decreased the maximum binding capacity of the high-affinity site without affecting its dissociation constant, suggesting that the binding site is proteinaceous and is sensitive to a change in the lipid moiety of the membrane. Hepatocytic cyclic AMP and cyclic GMP were not significantly modulated by incubating hepatocytes with histamine. Thus, the action of histamine on hepatocytes might not be mediated by the cyclic nucleotides.     

Flow cytometric quantitative determination of ingestion by phagocytes needs the distinguishing of overlapping populations of binding and ingesting cells.

BACKGROUND: The use of flow cytometry with fluorescently labeled particles provides the means to examine quantitatively the phagocytotic capacity of an individual phagocyte. This report describes an improved flow cytometric method of analysis for kinetic measurement of phagocytosis of fluorescein isothiocyanate (FITC)-labeled zymosan particles by human leukocytes. METHODS: FITC-labeled zymosan was incubated with leukocyte suspension, and at selected time intervals fluorescence positive neutrophils were divided by phagocytotic gates into three subpopulations: neutrophils that were neither binding nor ingesting particles, neutrophils that were only binding particles (binding cells), and neutrophils that were binding and ingesting particles (ingesting cells). For the distinction between internalized and surface-bound FITC-labeled zymosan, trypan blue (1.2 mg/ml) was used to quench surface-bound fluorescence. RESULTS: The technical challenges related to settings of phagocytotic gates and derivation of phagocytotic equations were presented. From 28 control samples, numerical values of mean fluorescence intensities and percentages of phagocytotic subpopulations inside phagocytotic gates before and after quenching were inserted into phagocytotic equations and corrected phagocytotic parameters were calculated. Calculated parameters were surprisingly constant across individuals. CONCLUSIONS: Essential elements of the present method appeared to be partial quenching of extracellular fluorescence with trypan blue and distinguishing between overlapping populations of binding and ingesting cells. Corrections using derived phagocytotic equations proved necessary for accurate kinetic phagocytotic measurements. Corrections were less necessary when the ingestion process was finished.     

Comparing binding of histamine and H2-antagonist with their effects on gastric acid secretion

A microsomal fraction from isolated frog gastric mucosa was used to study the binding of labeled histamine, labeled metiamide (a histamine H2-antagonist), and competition between labeled histamine and unlabeled metiamide. The separation of free from bound ligand was done by gel chromatography. The acid secretion was studied in frog gastric mucosa in vitro by a pH-stat method. The binding data could be interpreted in terms of two independent binding sites for both histamine and metiamide. However, the competition between histamine and metiamide does not support the independence of the sites. Moreover, the dissociation kinetics of labeled metiamide in the presence of unlabeled metiamide is non-monotone and, thus, indicates cooperativity. In the physiological studies, the dependence of the rate of acid secretion on histamine stimulation occurs within very narrow limits, which is the result of characteristics other than related to binding. However, the total amount of acid secreted caused by a pulse of histamine does indicate two sites, of which the high-affinity site is the more effective. Metiamide inhibition of acid secretion can be interpreted as an interaction between high-affinity sites of histamine and metiamide. Overall, studies involving physiological effects provide less precise data than the direct binding studies.     

Binding of histamine to the H1 receptor—a molecular dynamics study

Binding of histamine to the G-protein coupled histamine H₁ receptor plays an important role in the context of allergic reactions; however, no crystal structure of the resulting complex is available yet. To deduce the histamine binding site, we performed unbiased molecular dynamics (MD) simulations on a microsecond time scale, which allowed to monitor one binding event, in which particularly the residues of the extracellular loop 2 were involved in the initial recognition process. The final histamine binding pose in the orthosteric pocket is characterized by interactions with Asp107^3.32, Tyr108^3.33, Thr194^5.43, Asn198^5.46, Trp428^6.48, Tyr431^6.51, Phe432^6.52, and Phe435^6.55, which is in agreement with existing mutational data. The conformational stability of the obtained complex structure was subsequently confirmed in 2 μs equilibrium MD simulations, and a metadynamics simulation proved that the detected binding site represents an energy minimum. A complementary investigation of a D107A mutant, which has experimentally been shown to abolish ligand binding, revealed that this exchange results in a significantly weaker interaction and enhanced ligand dynamics. This finding underlines the importance of the electrostatic interaction between the histamine ammonium group and the side chain of Asp107^3.32 for histamine binding.     

Evidence that tamoxifen is a histamine antagonist

Recently we reported that both the triphenylethylene antiestrogen tamoxifen, and the novel compound N,N-diethyl-2-[(4 phenylmethyl)-phenoxy]-ethanamine. HCl (DPPE), which is selective for the antiestrogen binding site, may be histamine antagonists and have suggested that the antiestrogen binding site may be a growth-promoting histamine receptor different from H1 and H2 (?H3). We now show that along with established H1-antagonists, tamoxifen and DPPE specifically block the histamine-induced (H1) contraction of canine tracheal smooth muscle in the order: pyrilamine = hydroxyzine greater than tamoxifen = 4-hydroxytamoxifen greater than DPPE. The H1-antagonist hydroxyzine, which competes about equally with DPPE for the antiestrogen binding site, is up to 10(3) times stronger than DPPE in blocking histamine-induced muscle contraction. This shows that H1 antagonism is distinct from binding to the antiestrogen binding site and suggests that if the latter is a histamine receptor, it is not H1; presumably tamoxifen and DPPE compete for this novel site in addition to, and with greater affinity than, H1.     

An attempt to differentiate selection and amplification in hormone receptor development: the unicellular model.

Earlier experiments demonstrated that a membrane pattern of Protozoa behaves as a receptor with respect to hormones of higher organisms. This raised the possibility that some selection or strengthening of this unspecific patter is involved in the evolution of the specific membrane patterns of the individual cells of higher organisms. In the present experiments, as a result of continual histamine treatment, the phagocytotic ability of a population of Tetrahymena was increased more intensely than after a single histamine treatment. The phagocytotic rate remained high for some time after the animals were returned from histamine-containing to normal medium. Thus, from the experimental data, it appears likely that as the hormone appears, selection becomes involved in the proliferation of cells possessing receptors. This observation might not only be of phylogenetic interest, but could also be relevant in receptor maturation as manifested in ontogenetic membrane differentiation.     

An Attempt to Differentiate Selection and Amplification in Hormone Receptor Development

Earlier experiments demonstrated that a membrane pattern of Protozoa behaves as a receptor with respect to hormones of higher organisms. This raised the possibility that some selection or strengthening of this unspecific pattern is involved in the evolution of the specific membrane patterns of the individual cells of higher organisms. In the present experiments, as a result of continual histamine treatment, the phagocytotic ability of a population of Tetrahymena was increased more intensely than after a single histamine treatment. The phagocytotic rate remained high for some time after the animals were returned from histamine-containing to normal medium. Thus, from the experimental data, it appears likely that as the hormone appears, selection becomes involved in the proliferation of cells possessing receptors. This observation might not only be of phylogenetic interest, but could also be relevant in receptor maturation as manifested in ontogenetic membrane differentiation.     

Alpha-thrombin stimulation of heparin secretion by the peritoneal mast cells in rats

The interaction of alpha-thrombin with connective tissue-type mast cells (CTMC) purified by Ficoll density gradient centrifugation has been examined. It was demonstrated that exposure of CTMC to polymixin (widely used histamine liberator) (3 mg/ml) induced the release of heparin and histamine. Exposure of CTMC to 10(-11) M alpha-thrombin resulted in increase of heparin secretion by 75.5% in relation to basal level. CTMC which were stimulated by very low concentrations of alpha-thrombin (10(-11)-10(-8) M) can release high level of heparin, but not histamine. We have a suggestion that the thrombin specificity is connected with the additional recognition binding site for high molecular substrates (HMS) distinct from the active centre. Unlike alpha-thrombin which has both the active centre and the recognition site for HMS, beta/gamma-thrombin with catalytic activity but with disrupted recognition site induced the heparin release from mast cells only at higher concentrations than alpha-thrombin. It was revealed that DIP-alpha-thrombin without proteolytic activity was unable to activate mast cells in contrast to alpha-thrombin. We consider that alpha-thrombin induced release of heparin by CTMC account for proteolytic and hormone-like activity enzyme by means of both the active centre and the additional recognition site for HMS.     

Tick histamine-binding proteins: lipocalins with a second binding cavity

Tick histamine-binding proteins (HBPs) are lipocalins with two binding pockets. One of these binds histamine with a high affinity and is found at the position expected from other lipocalins, adjacent to the omega-loop at the open-end of the beta-barrel. A second binding cavity, which is a low-affinity site for histamine in one of the HBPs, is located at the end of the barrel that is closed off in other lipocalins. In order to create the second site, the 'closed-end' region has undergone a major reconstruction. Typical lipocalin characteristics, such as the 3(10) helix and a structural cluster of highly conserved residues, have been lost, while an alpha-helix now shields the cavity from the exterior. The prominence of acidic residues in the binding pockets is another distinctive characteristic of HBPs. Whereas most lipocalins have highly hydrophobic binding cavities designed to bind lipophilic compounds, HBPs have evolved to trap cationic, hydrophilic molecules.     

A new model for the agonistic binding site on the histamine H2-receptor: The catalytic triad in serine proteases as a model for the binding site of

The historical model for the agonistic binding site on the histamine H₂-receptor is based on a postulated activation mechanism: it has been suggested that the histamine monocation binds to the histamine H₂-receptor via the formation of three hydrogen bonds. The cationic ammonium group in the side chain and the —NH— group in the π-position of the imidazole act as proton donors, whereas the N— atom in the π-position of the imidazole acts as a proton acceptor. Participation of the ammonium group in H-bonding with a presumed negative charge on the receptor leads to a decrease in positive charge, which is thought to induce a tautomeric change in the imidazole ring system from N^τ-H to N^π-H. A consequence of this tautomeric shift is the donation of a proton from the receptor to the agonist on one side, while on the other side a proton is donated from the agonist to the receptor. The proposed tautomeric shift has been suggested to trigger the H₂-stimulating effect.However, this model for the constitution of the agonistic binding site and the accessory activation mechanism cannot explain the weak histamine H₂-activity of β-histine and the activity of several other recently synthesized H₂-agonists. Based on a thorough literature study and with the aid of molecular electrostatic potentials (MEPs) we demonstrate that the sulphur atom present in histamine H₂-agonists as dimaprit and 2-amino-5-(2-aminoethyl)thiazole does not function as a proton acceptor, which implicitly means that a tautomeric shift is not a prerequisite for H₂-stimulation. As a consequence, the model for the agonistic binding site is adjusted, resulting in a strong resemblance to the nature and orientation of the amino acids constituting the catalytic triad in serine proteases. Within this concept, the N^π-H tautomer of histamine is the biologically active form, in contrast with the existing model in which the N^τ-H tautomer is the active form.     

Cholinergic and glutamatergic activation reverses working memory failure by hippocampal histamine H1 receptor blockade in rats

Intrahippocampal administration of the histamine H1 receptor antagonist pyrilamine (3.2-32 ug/ side) but not the histamine H2 receptor antagonist cimetidine (1.0-10 microg/side) increased the number of errors in the working memory task with a three-panel runway setup. The increase in working memory errors induced by intrahippocampal 32 microg/side pyrilamine was significantly reduced by concurrent infusion of the histamine H1 receptor agonist 2-pyridylethylamine (3.2 and 10 microg/side). The cholinesterase inhibitor physostigmine ( 1.0 and 3.2 microg/side) and D-cycloserine (0.32 and 1.0 microg/side), the partial agonist at the glycine binding site on the NMDA receptor/channel complex, reduced the increase in working memory errors induced by intrahippocampal 32 microg/side pyrilamine. These results suggest that the hippocampal histaminergic activity via histamine H1 receptor is necessary for normal working memory processes and that the septohippocampal cholinergic activation and positive modulation of the NMDA receptor/channel through activation of the glycine site can alleviate dysfunction of hippocampal histamine H1 receptor-mediated neurotransmission involved in working memory function.     

Phagocytotic activity of glial cells in culture

The phagocytotic activity of glial cells was tested in primary cultures of astrocytes and C6 glioma cultures. Latex bead uptake served as an index of the respective phagocytotic activity. The content of latex beads in glial cells continued to increase for 24 h and this increase could be inhibited by incubation in ice bath or treatment with cytochalasin B (CB) or D (CD) at 37 °C. Addition of brain extract reduced latex bead uptake, whereas the phagocytotic activity was not markedly influenced by serum withdrawal and/or db-cAMP addition, both of which are usually used to induce certain characteristics of differentiated glial cells. Our results support the hypothesis that astroglial cells may act as phagocytes in situations where ‘professional’ phagocytes are not so numerous. In addition, the results imply that the phagocytotic activity of glial cells also depends on environmental conditions.     

Carbonic anhydrase activators: kinetic and X-ray crystallographic study for the interaction of D- and L-tryptophan with the mammalian isoforms I-XIV

An activation study of mammalian carbonic anhydrase (CA, EC 4.2.1.1) isoforms I-XIV with D- and L-tryptophan has been performed both by means of kinetic and X-ray crystallographic techniques. These compounds show a time dependent activity against isozyme CA II, with activation constants of 1.13 microM for L-Trp and 0.37 microM for D-Trp, respectively, after 24 h of incubation between enzyme and activator. The high resolution X-ray crystal structure of the hCA II-D-Trp adduct revealed the activator to bind in a totally unprecedented way to the enzyme active site as compared to histamine, L-/D-Phe, L-/D-His or L-adrenaline. D-Trp is anchored at the edge of the CA II active site entrance, strongly interacting with amino acid residues Asp130, Phe131 and Gly132 as well as with a loop of a second symmetry related protein molecule from the asymmetric unit, by means of hydrogen bonds and several weak van der Waals interactions involving Glu234, Gly235, Glu236 and Glu238. Thus, a second activator binding site (B) within the CA II cavity has been detected, where only D-Trp was shown so far to bind, in addition to the activator binding site A, in which histamine, L-/D-Phe, and L-/D-His are bound. These findings explain the strong affinity of D-Trp for CA II and may be useful for designing novel classes of CA activators by using this compound as lead molecule.     

Hexasaccharides from the histamine-modified depolymerization of porcine intestinal mucosal heparin

Specific sequences in heparin are responsible for its modulation of the biological activity of proteins. As part of a program to characterize heparin-peptide and heparin-protein binding, we are studying the interaction of chemically discrete heparin-derived oligosaccharides with peptides and proteins. We report here the isolation and characterization, by one- and two-dimensional 1H NMR spectroscopies, of ten hexasaccharides, one pentasaccharide, and one octasaccharide serine that were isolated from depolymerized porcine intestinal mucosal heparin. Hexasaccharides were chosen for study because they fall within the size range, typically tetra- to decasaccharide in length, of heparin sequences that modulate the activity of proteins. The depolymerization reaction was catalyzed by heparinase I (EC 4.2.2.7) in the presence of histamine, which binds site specifically to heparin. Histamine increases both the rate and extent of heparinase I-catalyzed depolymerization of heparin. It is proposed that oligosaccharides produced by heparinase I-catalyzed depolymerization can inhibit the enzyme by binding to the imidazolium group of histidine-203, which together with cysteine-135 forms the catalytic domain of heparinase I. The increased rate and extent of depolymerization are attributed to competitive binding of the oligosaccharides by histamine.     

Presynaptic localization of histamine H3-receptors in rat brain.

The localization of histamine H3-receptors in subcellular fractions from the rat brain was examined in a [3H] (R) alpha-methylhistamine binding assay and compared with those of histamine H1- and adrenaline alpha 1- and alpha 2-receptors. Major [3H](R) alpha-methylhistamine binding sites with increased specific activities ([3H]ligand binding vs. protein amount) were recovered from the P2 fraction by differential centrifugation. Minor [3H](R)alpha-methylhistamine binding sites with increased specific activities were also detected in the P3 fraction. Further subfractionation of the P2 fraction by discontinuous sucrose density gradient centrifugation showed major recoveries of [3H](R)alpha-methylhistamine binding in myelin (MYE) and synaptic plasma membrane (SPM) fractions. A further increase in specific activity was observed in the MYE fraction, but the SPM fraction showed no significant increase in specific activity. Adrenaline alpha 2-receptors, the pre-synaptic autoreceptors, in a [3H] yohimbine binding assay showed distribution patterns similar to histamine H3-receptors. On the other hand, post-synaptic histamine H1- and adrenaline alpha 1-receptors were closely localized and distributed mainly in the SPM fraction with increased specific activity. Only a negligible amount was recovered in the MYE fraction, unlike the histamine H3- and adrenaline alpha 2-receptors.     

Isolation, purification and characterization of regulatory properties of adenylate cyclase from rabbit heart]

Rabbit heart membranes possessing the adenylate cyclase activity were isolated and purified by extraction with high ionic strength solutions and centrifugation in the sucrose density gradient. It was shown that the membranes are characterized by a high percentage of cholesterol (molar ratio cholesterol/phospholipids is 0.24) and an increased activity of Na, K-ATPase, which suggests the localization of adenylate cyclase in the sarcolemma. During centrifugation in the sucrose density gradient the activities of andenylate cyclase and Na,K-ATPase are not separated. Treatment of heart sarcolemma with a 0.3% solution of lubrol WX results in 10--20% solubilization of adenylate cyclase. Purification of the enzyme in the membrane fraction is accompanied by a decrease in the activity of phosphodiesterase; however, about 2% of the heart diesterase total activity cannot be removed from the sarcolemma even after its treatment with 0.3% lubrol WX. Epinephrine and NaF activate adenylate cyclase without changing the pH dependence of the enzyme. The alpha-adrenergic antagonist phentolamine has no effect on the adenylate cyclase activation by catecholamines, glucagon and histamine; the beta-adrenergic antagonist alprenolol competitively inhibits the effects of isoproterenol, epinephrine and norepinephrine, having no effect on the enzyme activation by glucagon and histamine. There is no competition between epinephrine, glucagon and histamine for the binding site of the hormone; however, there may occur a competition between the hormone receptors for the binding to the enzyme. A combined action of several hormones on the membranes results in the averaging of their individual activating effects. When the hormones were added one after another, the extent of adenylate cyclase activation corresponded to that induced by the first hormone; the activation was insensitive to the effect of the second hormone added. It is assumed that the outer membrane of myocardium cells contains a adenylate cyclase and three types of receptors, each being capable to interact with the same form of enzyme. The activity of adenylate cyclase is determined by the type of the receptor, to which it is bound and by the amount of the enzyme-receptor complex.     

Evidence of an adriamycin binding site in the secretory granules of the mast cell

Adriamycin induced significant non-cytotoxic histamine release from rat peritoneal mast cells to which the drug showed a very high affinity. The relationship between adriamycin-induced exocytosis and its uptake by purified rat peritoneal mast cells was studied. Adriamycin induced histamine release and was highly concentrated in mast cells at 37 degrees C but not at 0 degrees C. However, if exocytosis was provoked by other secretagogues like compound 48/80, protamine, concanavalin A, and ionophore A23187, and cells were then treated with adriamycin at 0 degrees C, the concentrations of the antineoplastic drug significantly increased. Adriamycin binding to purified granular material was similar to that of intact cells treated at 37 degrees C, but was not modified by metabolic inhibitors, extremes of temperature (0 or 45 degrees C) or by the carboxylic ionophore monensin. On the contrary, sodium cromoglycate limited adriamycin binding to granular materials as well. In addition, sodium cromoglycate, but not monensin, displaced the antineoplastic drug from mast cells, even when added after adriamycin. We conclude that the high affinity of adriamycin for mast cells is ascribable to the externalization of a granular binding site, as a consequence of the exocytotic process. The experiments with sodium cromoglycate suggest that this binding site could be in common with the antiallergic drug.