Adenosine Inhibits Polymorphonuclear Leukocyte in Vitro Activation: A Possible Role as an Endogenous Calcium Entry Blocker

Adenosine is able to in vitro inhibit FMLP-dependent activation of polymorphonuclear leukocytes as evaluated by enzyme release, superoxide anion generation and chemiluminescence production. The inhibiting effect is more relevant when A23187 is employed as stimulating agent. In this case the effect is significantly reversed by increasing concentrations of extracellular calcium. Since A23187-dependent activation is strictly dependent on Ca⁺⁺ influx into the cell, the hypothesis is suggested that adenosine could act by regulatory mechanisms involving membrane calcium transport.     

Binding Specificity of Mouse Serum Amyloid P-Component for Fibronectin

Binding between purified mouse serum amyloid P-component (SAP) and plasma fibronectin (Fn) occurred when either one of the proteins was immobilized by specific antibody and the second protein was offered in a soluble form. Binding of Fn to immobilized SAP was cooperative and saturable at a molar ratio of SAP/Fn = 7.1. The molar ratio at saturation was 3.7 for SAP/Fn when SAP was allowed to bind to immobilized Fn. The binding required 2 to 3mM amounts of Ca⁺⁺. The binding of SAP to Fn was selectively inhibited by a monoclonal antibody specific for the mid-molecule region of Fn, by soluble gelatin, and by heparin in the presence of 3mM Ca⁺⁺. We conclude that the SAP binding site was localized at the mid-molecule region of Fn that includes the adjacent gelatin-binding domain and the heparin-I binding domain.     

Polymorphonuclear leucocytes as a model of Ca++ and Mg++-dependent cellular activation. Effect of calcium entry blockers

Flunarizine inhibited FMLP- and A23187-induced aggregation, enzyme release and O2- generation from human PMN as a function of its concentration. A23187-dependent PMN aggregation was also studied in media devoid of Ca++ or Mg++. Flunarizine (2.4 X 10(-5)M) significantly affected not only Ca++-supported but also Mg++-sustained PMN aggregation. The inhibiting effect of the drug was reversed by increasing the level of Ca++ (1.2 mM) or Mg++ (2 mM). Nifedipine, another Ca++-entry blocker, was shown to inhibit enzyme release and O2- generation induced by FMLP and A23187 as a function of its concentration, but only slightly affected PMN aggregation at very high concentration (10(-4)M). A role for flunarizine as a specific Mg++-entry blocker is suggested.     

Modulation of Cytoskeleton Assembly Capacity and Oxidative Response in Aged Neutrophils

Several reports have emphasized that aged polymorphonuclear cells (PMN) exhibit an impairment of superoxide anion (O^-₂) generation when triggered with formyl-methionyl-leucine-phenylalanine (FMLP) in comparison to the younger counterpart. Since microfilaments and microtubules are involved in PMN-mediated functions, in a group of old donors we assessed the effects of either actin stabilizing and disrupting agents, i.e. phalloidin and cytochalasin B, or microtubule stabilization or disruption by taxol and colchicine, respectively, on FMLP-triggered neutrophil oxidative responsiveness. Results show that phalloidin treatment, at a concentration ranging from 10^-6 to 10^-8 M, gave rise to an inhibition of O^-₂ release by aged PMN, while the same effect was seen in similarly treated young cells at a concentration of 10^-7 M only. On the contrary, cytochalasin B pretreatment led to an enhancement of O^-₂ generation in both young and aged neutrophils, even if to a lower extent in the latter group. At the same time, taxol at 10^-8 M strength inhibited young cell responsiveness, while no effects were induced by colchicine treatment. Quite interestingly, elderly neutrophil function was negatively modulated by both microtubule affecting compounds.

Alltogether, these findings suggest the possible relevance of cytoskeletal affecting compounds in the modulation of FMLP-stimulated O^-₂ release during senescence.     

In vitro effects of naloxone on T-lymphocyte-dependent antibacterial activity in hepatitis C virus (HCV) infected patients and in inflammatory bowel disease (IBD) patients

Naloxone acts as an opioid antagonist, displacing opioid drugs from cellular receptors. Among opioid substances, β-endorphins are able to bind to several cell receptors, even including those expressed by immune cells. In this respect, evidence has been provided that in the course of viral infections, as well as in patients with ulcerative colitis high levels ofβ-endorphins are detectable. Here, peripheral blood lymphocytes (PBL) from 21 HCV infected patients and 14 patients with IBD, respectively, were incubated with Naloxone and Naloxone + Ca²⁺ in order to evaluate a putative modulation of PBL-mediated antibacterial activity. In fact, previous studies have demonstrated a reduction of this T-cell activity in HCV and IBD patients.

In general terms, the above treatment led to a recovery of the depressed antibacterial activity. In some cases, increase in T lymphocyte function was obtained with Naloxone alone, while in other cases the combination Naloxone + Ca²⁺ gave rise to a restorative effect. Of note, in some instances, lymphocytes were unresponsive to pharmacological modulation.

The overall results suggest that β-endorphins may down modulate T-cell antibacterial response in HCV and in IBD patients by saturating peripheral receptors on immune cells. Therefore, it is likely that Naloxone and/or Naloxone + Ca²⁺ may displace opioid drugs, thus antagonizing their effects.     

Modulation of Human T-Lymphocyte Plasma Membrane Ca +2 Permeability by Imidazole Antimycotics

The role of cytochrome P-450 in the regulation of plasma membrane Ca⁺² permeability of human peripheral T-lymphocytes by intracellular Ca⁺² was examined. We assessed the effect of imidazole inhibitors of cytochrome P-450 on the intracytoplasmic free Ca⁺² ([Ca⁺²]i) response generated using the microsomal ATPase inhibitor thapsigargin (THG) to deplete the intracellular Ca⁺² stores. Econazole, miconazole and clotrimazole dramatically inhibited the THG mediated increase in [Ca⁺²]i and indud an increase in [Ca⁺²]i themselves. This inhibitory effect was previously observed in other cell systems and was attributed to inhibition of cytochrome P-450 by these agents. However, we evaluated a variety of structurally dissimilar P-450 inhibitors and found that none affected [Ca⁺²]i, indicating that the mechanism of imidazole action does not involve P-450.     

Inhibitoty Effects of Local Anesthetics on Migration, Extracellular Release of Lysosomal Enzyme, and Superoxide Anion Production in Human Polymorphonuclear Leukocytes

This study examined the effects of four typical local anesthetics, lidocaine, prilocaine, procaine and tetracaine, on the functioning of human polymorphonuclear leukocytes (PMN). PMN were stimulated by fMet-Leu-Phe (FMLP) or phorbol myristate acetate (PMA) to elicit chemotaxis, extracellular release of beta-glucuronidase (BGL) and superoxide anion (SOA) production. the four agents inhibited chemotaxis efficiently and in a concentration-dependent manner but had only weak effects on the release of BGL. the effect of tetracaine was strongest, followed by lidocaine, then prilocaine, whereas the effect of procaine was blunt. the 50% inhibitory concentrations (IC₅₀ in molarity) of the four local aesthetics for chemetaxis were as follows: tetracaine=4.1×10^-4, lidocaine=3.2×10^-3, prilocaine=3.6×10, procaine=4.9×10^-3, those for SOA production induced by FMLP were : tetraaine=3.1×10^-4, lidocine=5.9×10^-3, prilocaine=1.9×10^-2, procaine=1.2×10^-2, those for SOA production indced by PMA were : tetracaine=1.1×10^-3, lidocine=1.2×10^-2, prilocaine=1.5×10-2, procaine=2.5×10^-2, and those for rlease of BGL were : tetracaine=1.6×10^-, lidocaine=5.3×10^-3, prilocaine=2.8×10^-2, procaine=1.2×10^-1. the IC₅₀ seemed to relate to the anesthetic's chemical structures and their inhibitory properties on PMN functions, as lidocaine and prilocaine, which are aminoamide type anesthetics, preferentially inhibited chemotaxis, whereas tetracaine and procaine, aminoester type anesthetics, inhibited SOA production induced by FMLP. the results suggest that the inhibitory effects of local anesthetics on human PMN functions are also correlated with local anesthetic potency and vary according to differences in their chemical structures.     

Calcium Uptake During Mitogenic Stimulation of Human Lymphocytes: Characterization of Intracellular Calcium Compartments and Demonstration of the Presence of Immunoreactive Calrecticulin

Phytohemagglutinin (PHA) stimulation of human peripheral blood lymphocytes (HPBL) rapidly increases ⁴⁵Ca²⁺ uptake into intracellular pools. Detectable increase in ⁴⁵Ca²⁺ uptake occurred only on exposure to mitogenic lectins but not with non-mitogenic lectins. However, intracellular free Ca²⁺ concentration [(Ca²⁺)i] increased comparably on exposure to either mitogenic or non-mitogenic lectins.

Permeabilization of ⁴⁵Ca²⁺ loaded cells revealed distinct pools of Ca²⁺ uptake. The highly digitonin sensitive pool #1 (permeabilized by 0.02% digitonin) exchanged slowly and included a part that represented endoplasmic reticulum. Pool II was defined by lower digitonin sensitivity, had a much faster initial uptake. Pool III was digitonin-resistant and predominantly non-vesicular. During the first 120 min of PHA stimulation, significant increase in ⁴⁵Ca²⁺ uptake occurred only into pool II. Progressive increase in uptake into pool I then occurred so that by 24 hours, this pool constituted the major fraction of PHA induced increment in total ⁴⁵Ca²⁺ uptake. Using specific antibody to the calcium binding protein calreticulin, an analogous immunoreactive protein was detectable in resting HPBL. PHA stimulation led to a striking increase in abundance of immunoreactive calreticulin so that 24 hrs after PHA stimulation, there was a 28 and 3.4 fold increase in the amount of immunoreactive calreticulin present in the non-particulate fraction and the total particulate membrane fraction, respectively. A major part (72%) of the total cellular immunoreactive calreticulin in PHA stimulated cells at 24 hrs was released into the medium after permeabilization of lymphocytes with 0.02% digitonin, corresponding to the location of calcium uptake pool I.     

Anti-inflammatory effect of lemon mucilage: in vivo and in vitro studies

The mucilage extracted from a lemon juice centrifugation pulp was studied for its anti-inflammatory effect in rat. In vivo the lemon mucilage significantly inhibited carrageenan-induced edema in rat paw from 59% to 73.5% showing the highest effect at the third hour. In vitro, at the doses of 10^-8, 10^-6, 10^-4 or 10^-2 mg/mL the lemon mucilage stimulated the superoxide anion production in rat testing neutrophils in whole blood but inhibited it in FMLP stimulated cells at the dose of 10^-2 mg/mL. The neutrophils of rats receiving p.o. the lemon mucilage for 21 days showed a significant decrease of 45.5% in O₂^- generation after FMLP stimulation, and a not-significant increase after phorbol-12-myristate-13-acetate (PMA) or zymosan stimulation. Since the activity on zymosan- and PMA-induced O₂^- production was not significant, the inhibition exerted by FMLP in rat neutrophils occurred mainly through the blockade of phospholipase D.     

Activity and preliminary characterization of Branchiostoma lanceolatum agglutinin

The physical and chemical properties of a hemagglutinin from whole-body homogenates of the cephalochordate, Branchiostoma lanceolatum, are described. The hemagglutinin is proteinaceous since it is precipitated by trichloroacetic acid and ammonium sulphate, and all activity is lost at 60°C or by treating with proteases. Carbohydrate moieties are probably also present since activity is lost after incubation with sodium metaperiodate. Activity is stable over pH 6–10. The agglutinin does not require Ca⁺⁺ or Mg⁺⁺, and a reduced titer after treatment with 2-mercaptoethanol and urea suggests the presence of both disulphide bonds and noncovalent linkages. Haemagglutination inhibition experiments with 17 saccharides and glycoproteins failed to show clear-cut carbohydrate specificity, with only mucin and fetuin having any inhibitory effect, so that the binding may be complex. Finally, cross-adsorption experiments suggest that only one lectin is present. The function of this lectin, especially in an immunobiological context, remains speculative.     

Analysis of Ejaculates from Patients with Cystic Fibrosis

Ejaculates from two men with cystic fibrosis (CF) were examined. Both had azoospermia. A considerable decrease in volume and fructose content was noted. The absolute amounts of calcium, magnesium, and zinc per ejaculate showed normal values in one of the patients but were two to three times increased in the other compared to mean values of a control group. Thus the concentrations of these cations were increased at least fivefold in both patients.

The amount of Mg²⁺- and Ca²⁺dependent ATPase was comparable to that of controls, but values were higher than in men with oligospermia. Both the divalent cations and the Mg²⁺- and Ca²⁺ -dependent ATPase curve profiles of split ejaculate fractions were atypical. Secretory granules and vesicles were plentiful in the seminal plasma of both patients while amorphous substance was practically absent.

The present findings agree with a less affected function of the prostate gland and a dysfunction of the seminal vesicles in these patients.     

Characterization and Mitogenic Activity of Haemophilus Influenzae Type B Capsular Polysaccharide

Studies were conducted on the characterization of Haemophilus influenzae type b polysaccharide (HITB-PS) and its mitogenic activity upon peripheral lymphocytes. This capsular polysaccharide was found to contain hexosamines and hexoses in addition to the main components of ribose and ribitol phosphate. The molecular weight of HITB-PS was determined as 585,000. The affinity constant of HITB-PS to unfraction-ated lymphocytes was 3.13 × 10³ M^-1 with 1.11 × 10⁴ binding sites per cell.

HITB-PS was found to be mitogenic for both numan T and B lymphocytes. At optimum doses, a three to five fold increase in ³H-thymidine incorporation into T and B cells was observed. Higher than optimum doses resulted in suppression of this mitogenicity. The effect of concanavalin A (Con A) mitogenicity was detected in T and B cells treated with effective as well as suppressive doses of HITB-PS; the mitozenic activities of Con A and HITB-PS were found to be independent of each other.     

Defibrotide in vitro inhibits neutrophil activation by a Ca++-involving mechanism

Defibrotide, a polydeoxyribonucleotide provided with a pro-fibrinolytic and prostacyclin-like activity, was studied as an inhibitor of polymorphonuclear leucocyte activation in vitro. It was found capable of dose-dependently (1-8 X 10(-5) M) inhibiting FMLP-induced activation, as shown by a decrease of enzyme release and free-radical formation (superoxide anion generation and chemiluminescence). A similar inhibiting activity was observed on A23187-induced activation. An increase in extracellular Ca++ concentration significantly prevented the effect of defibrotide on ionophore stimulation. When PMA was employed as stimulating agent, the drug did not show any inhibiting effect. Finally the pre-treatment of cells with theophylline markedly reduced the inhibition by defibrotide of FMLP- and A23187-dependent activation. Since the stimulation of neutrophils by FMLP and A23187 depends on the increase of cytoplasmic free-calcium availability or extracellular calcium entrance respectively, whereas PMA activation is completely independent from any Ca++ change, the inhibiting effect of defibrotide could be attributed to a Ca++-involving mechanism.     

Developmentally regulated changes of glutamate binding sites in mouse deep cerebellar nuclei

The expression of L-[³H]glutamate binding sites of different ionic and pharmacological sensitivities was studied in mouse deep cerebellar nuclei during early postnatal development by means of in vitro autoradiography. Ca²⁺/Cl⁻-dependent, quisqualate/AMPA/ibotenate-sensitive, and APB-insensitive binding sites are present at high density in the deep cerebellar nuclei of young animals, but greatly decrease between the 10th and 25th postnatal day and remain low in the adult. The density of Ca²⁺/Cl⁻-independent binding sites remains low and constant during the whole of postnatal development. The possible involvement of the Ca²⁺/Cl⁻-dependent binding sites in brain development is discussed.     

Intracellular Ca antagonist TMB-8 blocks catecholamine secretion evoked by caffeine and acetylcholine from perfused cat adrenal glands in the absence of extracellular Ca

Unlike acetylcholine, caffeine was much more effective in releasing catecholamine in the absence of extracellular Ca²⁺ than in its presence in perfused cat adrenal glands. The intracellular Ca²⁺ antagonist, TMB-8 (10⁻⁴ M), inhibited reversibly the catecholamine secretion evoked by caffeine (40 mM) and that induced by acetylcholine (10⁻⁴ M) in the presence of hexamethonium (10⁻³ M) during perfusion with Ca²⁺-free Locke solution containing EGTA (10⁻⁵ M). These results support our view that muscarinic receptor activation causes catecholamine secretion by mobilizing Ca²⁺ from an intracellular pool just as caffeine does.     

Modulation of Ca channels by activation of adenosine A1 receptors in rat striatal glutamatergic nerve terminals

We determined that activation of adenosine A₁ receptors in striatal synaptosomes with 100 nM N⁶-cyclopentyladenosine (CPA) inhibited both the release of endogenous glutamate and the increase of intracellular free Ca²⁺ concentration ([Ca²⁺]_i), due to 4-aminopyridine (4-AP) stimulation, by 28 and 19%, respectively. Furthermore, CPA enhanced the inhibition of endogenous glutamate release due to ω-conotoxin GVIA (ω-Cgtx GVIA), ω-Cgtx MVIIC or ω-Cgtx GVIA plus ω-Cgtx MVIIC. Similar effects were observed in the [Ca²⁺]_i signal. The inhibitory effects of CPA and ω-Cgtx GVIA were additive, but the effects of CPA and ω-Cgtx MVIIC were only partially additive. These results suggest that P/Q-type Ca²⁺ channels and other type(s) of Ca²⁺ channel(s), coupled to glutamate release, are inhibited subsequently to activation of adenosine A₁ receptors.     

The Effects of Cytochalasins on Lymphocytes: V. Interaction of Trifluoperazine and Cytochalasin B in Inhibition of Human Lymphocyte Proliferation

Trifluoperazine (TFP), a phenothiazine derivative, is known to inhibit calmodulin-mediated phenomena. We report here that TFP reversibly inhibited lymphocyte proliferative responses to mitogenic lectins. This inhibition was observed only when TFP was added during the early stages of exposure of lymphocytes to the stimulus. Furthermore, at sub optimally inhibitory concentrations of each compound, effects of TFP on lymphocyte proliferation were additive to those of cytochalasin B (CB). Incubation of lymphocytes in TFP (10^-5 -10^-4 M) markedly inhibited cytochalasin B binding to the actin associated, low affinity binding site without affecting its binding to the high affinity site or to the medium affinity site. This effect developed gradually during incubation with TFP, becoming demonstrable after 30 minutes reaching maximum after 30-60 min of incubation at 37.

The findings suggest the occurrence of an interaction of TFP with the lymphocyte cytoskeleton, which may play a role in the impairment in the transmission of the mitogenic signal.     

Heparin inhibits FMLP and Con-A dependent activation of human polymorphonuclear leucocytes in vitro

The effects in vitro of heparin on different functions of human neutrophilic leucocytes were assessed. Granulocyte aggregation, enzyme release and superoxide anion generation induced by FMLP (10(-6) M), ConA (30 micrograms/ml), and A 23187 (5 microM) were evaluated. Heparin was able to inhibit, in a dose-dependent way, FMLP-mediated and ConA-mediated granulocyte activation. Heparin inhibited cellular aggregation at the final concentration of 100-500 micrograms/ml, while the inhibiting effect on enzyme release and superoxide anion production was present at lower concentrations (10-100 micrograms/ml). No activity was observed on A 23187-induced granulocyte stimulation. The inhibiting effect of heparin on FMLP-induced granulocyte activation proved to be time-dependent. The hypothesis that an interaction of heparin with cells may possibly initiate the inhibitory effect is proposed.     

Aging does not alter cytosolic calcium levels of cortical synaptosomes in Fischer 344 rats

Several lines of experimental evidence support an association between altered Ca²⁺ regulation and aging. It has been supposed that free cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) may decrease or increase in aged animals. In this study, both resting and KCl-stimulated [Ca²⁺]_i were measured in purified cortical synaptosomes from young (3 mo.), middle-aged (12 mo.), and old (24 mo.) Fischer 344 rats. Two additional groups of rats were included, one middle-aged and one old which were trained on a treadmill for 6 months prior to experimentation. The [Ca²⁺]_i was determined using the fluorescent Ca²⁺ chelator fura-2. Net KCl-dependent changes (ΔK) in [Ca²⁺]_i were determined by the difference between stimulatory (100 μM Ca²⁺/60 mM KCl) and resting (100 μM Ca²⁺/5 mM KCl buffer) conditions among the 3 age groups. Significant increases in [Ca²⁺]_i were observed in each age group upon depolarization with 60 mM KCl. However, there were no significant age-dependent differences in either resting [Ca²⁺]_i or KCl-stimulated [Ca²⁺]_i.     

Effect of tea polyphenols on histamine release from rat basophilic leukemia (RBL-2H3) cells: the structure–inhibitory activity relationship

We studied the effect of tea polyphenols on histamine release from rat basophilic leukemia (RBL-2H3) cells. Among tea polyphenols, (-)-epigallocatechin gallate (EGCG) most strongly and dose-dependently inhibited histamine release from cells stimulated with a calcium ionophore, A23187. (-)-Epigallocatechin (EGC) and (-)-epicatechin gallate (ECG) with a triphenol residue moderately inhibited histamine release, whereas diphenolic (+)-catechin (C) and (-)-epicatechin (EC) did not. The magnitude of the inhibitory effect was in the order EGCG > ECG > EGC. Among simple polyphenols, the triphenol compounds, pyrogallol (PG) and gallic acid (GA) exerted inhibitory activity, but the diphenols, pyrocatechol, hydroquinone, and resorcinol did not. In addition, the mixture of PG and GA inhibited histamine release as strongly as EGCG with two triphenol residues. Similarly, they inhibited histamine release induced by IgE-antigen complex stimulation more efficiently than that induced by A23187 stimulation. EGCG did not inhibit the increase of intracellular Ca²⁺ in RBL-2H3 cells stimulated with A23187 or IgE antigen. These results indicate that the triphenol structure plays an important role in the inhibitory activity of tea polyphenols. Their activity seemed to be exerted through the metabolic events occurring after the elevation of intracellular Ca²⁺ concentration.