Effects of some calcium antagonists on aggregation by adrenalin and serotonin and on alpha-adrenoceptor radioligand binding in human platelets

The inhibitory effects of verapamil, nifedipine and diltiazem, representatives of different classes of calcium antagonists, were studied on aggregation of human platelets induced by adrenalin and serotonin (5-HT). For references, the alpha-adrenoceptor-antagonists phentolamine (alpha 1 and alpha 2) and rauwolscine (alpha 2), and the 5-HT 2-receptor-antagonist ketanserin were included. Verapamil in the concentration range 10(-6) 10(-4) M inhibited both adrenalin- and serotonin-induced aggregation in a concentration-dependent manner, whereas nifedipine and diltiazem had little or no effect. Phentolamine and rauwolscine were clearly weaker than verapamil as antagonists of serotonin, and ketanserin lacked effect on adrenalin-induced aggregation. Binding studies with [3H]dihydro-alpha-ergocryptine and [3H]rauwolscine on human platelet membranes showed equal numbers of binding sites, suggesting that only alpha 2-adrenoceptors were present. In the same concentration range as inhibition of aggregation was obtained, verapamil inhibited binding of either radioligand. Nifedipine, diltiazem and 5-HT were all poor inhibitors of radioligand binding. The results suggest that verapamil at high concentrations not only has alpha-adrenoceptor antagonistic properties but also exerts 5-HT-receptor blocking effects. This was not found with the other calcium channel blockers examined (nifedipine, diltiazem).     

The effect of amiloride on sodium and potassium fluxes in red cells

1. The effect of amiloride on the influx and efflux of (24)Na and (42)K in red cells was studied. The drug was added to the bathing Ringer or else incorporated in resealed ghosts.2. Amiloride does not inhibit the active or the passive (ouabain insensitive) extrusion of (24)Na.3. Amiloride inhibits the influx of (24)Na into red cells by 70%.4. Whether added to the inside or to the outside of the cells amiloride has no effect on the efflux of (42)K.5. Amiloride does not modify the uptake of (42)K from control Ringer. This uptake is strongly inhibited by the removal of Na. Amiloride has no effect on the extent of this inhibition.6. It is concluded that amiloride specifically inhibits the passive penetration of Na, and has no effect on the Na-K-pumping mechanism. However, at the concentration which inhibits 70% of the influx, amiloride fails to produce an observable effect on the ouabain-insensitive Na-efflux.7. On the basis that the information obtained could be extrapolated to other membranes, the effect of amiloride on epithelial membranes is discussed.     

Alpha fetoprotein inhibits aggregation of human platelets

The effect of human alpha-fetoprotein (AFP) on platelet aggregation induced by physiological (ADP, PAF, collagen, arachidonic acid) and aphysiological (ionophore A 23,187) activators was investigated. It was found that AFP at the concentrations of 60-750 micrograms/ml, i.e. far below that observed in human blood, inhibits platelet aggregation induced by physiological activators. Platelet aggregation induced by arachidonic acid was two-four times more sensitive to inhibition by AFP than that induced by other physiological agonists, whereas aggregation induced by A 23,187 was not affected by AFP even at concentration of 750 micrograms/ml.     

Effect of benzydamine on exocytosis and respiratory burst in human neutrophils and mononuclear phagocytes

The effect of benzydamine on stimulus-dependent respiratory burst activity and enzyme release was tested in human neutrophils, monocytes and monocyte-derived macrophages. Establihsed anti-inflammatory compounds, indomethacin, phenylbutazone and bufexamac, were tested for comparison. Care was taken to avoid cytotoxic or cytolytic concentrations of the test compounds, and their effect on release of lactate dehydrogenase was also tested.Release of specific and azurophil granules contents were induced in human neutrophils by A23187, PMA and fMLP with and without cytochalasin B pretreatment. Benzydamine inhibited stimulus-dependent release of vitamin B₁₂-binding proteins, a marker for the specific granules, in a concentration-dependent fashion. By contrast, phenylbutazone and bufexamac were practically inactive. The effect of benzydamine on exocytosis of azurophil granules was tested in cytochalasin B-pretreated neutrophils. Benzydamine, again in contrast to the two reference anti-inflammatory compounds, inhibited release concentration-dependently also under these conditions. The concentration of the compound which inhibited exocytosis by 50% was 30–100 M in normal and 3–10 M in cytochalasin B-treated neutrophils.The effect of benzydamine and reference compounds on the respiratory burst was tested by assaying for superoxide formation in neutrophils and H₂O₂ formation in mononuclear phagocytes. Benzydamine was inactive on neutrophils and inhibited slightly the burst response of monocytes and macrophages. Two reference compounds, bufexamac and phenylbutazone, were generally more active. The strongest inhibitory effect was that of phenylbutazone on fMLP-stimulated cells. Benzydamine lacked activity receptor of formylated chemotactic peptides.The profile of activity of benzydamine shown in these experiments on human phagocytes suggest that this compound may act therapeutically by decreasing the release of enzymes and other granule constitutents from stimulated neutrophils.     

Aggregating and prostanoid-releasing effects of platelet-activating factor and leukotrienes on human polymorphonuclear leukocytes and platelets

Aggregating and prostanoid-releasing properties of the inflammatory mediators, platelet-activating factor (PAF) and leukotrienes B4, C4 and D4 were studied in human polymorphonuclear leukocytes (PMNL) and in human platelet-rich plasma. Leukotriene B4 (LTB4) and PAF both induced a reversible aggregation of human PMNL with concomitant stimulation of PGE2 formation, whereas LTC4 had no effect on human PMNL. Arachidonic acid (AA) caused an irreversible aggregation of PMNL which was accompanied by formation of both PGE2 and TXB2. Inhibition of TXB2 synthesis by indomethacin or by OKY-1581, a thromboxane synthetase inhibitor, had no effect on the PMNL aggregation induced by LTB4, PAF or AA. Leukotrienes B4, C4 and D4 caused neither aggregation nor TXB2 release in human platelet-rich plasma. PAF, on the other hand, induced a dose-dependent, reversible platelet aggregation which was not accompanied by TXB2 formation nor inhibited by OKY-1581. The present study indicates that in addition to inducing PMNL aggregation, LTB4 is capable of releasing arachidonate metabolites from human PMNL but not from human platelets. Also the responses of PMNL and platelets to PAF seem to differ as the PAF-induced PMNL aggregation was accompanied by increased prostanoid formation whereas the PAF-induced reversible platelet aggregation was obviously independent from arachidonate metabolism.     

Platelet release protein which inhibits plasminogen activators.

An inhibitor of plasminogen activator has been identified in human platelets by the technique of sodium dodecyl sulphate polyacrylamide gel electrophoresis and zymography. The inhibitor has a molecular weight of about 40 000 and is distinct from known plasma protease inhibitors. It is associated almost exclusively with platelets, with only trace amounts in platelet free plasma. The inhibitor is released during platelet aggregation or in vitro coagulation. This inhibitor inhibits both tissue type plasminogen activator and urokinase but has no effect on plasmin. It forms a 1:1 complex with tissue type plasminogen activator, which retains activity detectable under the analytical conditions used. A similar complex with urokinase either forms less readily or retains less activity.     

Effects of some calcium antagonists on aggregation by adrenalin and serotonin and on α-adrenoceptor radioligand binding in human platelets

The inhibitory effects of verapamil, nifedipine and diltiazem, representatives of different classes of calcium antagonists, were studied on aggregation of human platelets induced by adrenalin and serotonin (5-HT). For references, the α-adrenoceptor-antagonists phentolamine (α₁ and α₂) and rauwolscine (α₂), and the s-HT₂-receptor-antagonist ketanserin were included. Verapamil in the concentration range 10^-6- 10^-4 M inhibited both adrenalin- and serotonin-induced aggregation in a concentration-dependent manner, whereas nifedipine and diltiazem had little or no effect. Phentolamine and rauwolscine were clearly weaker than verapamil as antagonists of serotonin, and ketanserin lacked effect on adrenalin-induced aggregation. Binding studies with [³H]dihydro-α-ergocryptine and [³H]rauwolscine on human platelet membranes showed equal numbers of binding sites, suggesting that only α₂-adrenoceptors were present. In the same concentration range as inhibition of aggregation was obtained, verapamil inhibited binding of either radioligand. Nifedipine, diltiazem and 5-HT were all poor inhibitors of radioligand binding. The results suggest that verapamil at high concentrations not only has α-adrenoceptor antagonistic properties but also exerts 5-HT-receptor blocking effects. This was not found with the other calcium channel blockers examined (nifedipine, diltiazem).     

A structural approach to understanding the iron-binding properties of phylogenetically different frataxins

Friedreich's ataxia (FRDA), an autosomal recessive cardio- and neurodegenerative disease, is caused by low expression of frataxin, a small mitochondrial protein, encoded in the nucleus. At the biochemical level, the lack of frataxin leads to dysregulation of mitochondrial iron homeostasis and oxidative damage, which eventually causes neuronal death. It is, however, still unclear whether frataxin is directly involved in iron binding, since the yeast orthologue, but not the human protein, has been shown to form large aggregates in the presence of large iron excess. We have compared the properties of three proteins from the frataxin family--the bacterial CyaY from Escherichia coli, the yeast Yfh1 and human frataxin--as representative of organisms of increasing complexity. We show that the three proteins have the same fold but different thermal stabilities and iron-binding properties. While human frataxin has no tendency to bind iron, CyaY forms iron-promoted aggregates with a behaviour similar to that of yeast frataxin. However, aggregation can be competed by chelator agents or by ionic strength. At physiological salt conditions, almost no aggregation is observed. The design of mutants produced to identify the protein surface involved in iron-promoted aggregation allows us to demonstrate that the process is mediated by a negatively charged surface ridge. Mutation of three of these residues is sufficient to convert CyaY in a protein with properties similar to those of human frataxin. On the other hand, mutation of the exposed surface of the beta sheet, which contains most of the conserved residues, does not affect aggregation, suggesting that iron binding is a non-conserved part of a more complex cellular function of frataxins.     

Intramuscular ketorolac inhibits activation of rat peripheral NMDA receptors

The nonsteroidal anti-inflammatory drug (NSAID) diclofenac has local anesthetic-like and peripheral N-methyl-d-aspartate (NMDA) receptor antagonist characteristics when administered at higher concentrations to masticatory muscle. It is not known if the ability to inhibit NMDA receptors is unique to diclofenac or shared by other NSAIDs. This study was undertaken to determine whether intramuscular injection of ketorolac or naproxen at concentrations that do not induce local anesthetic-like effects could attenuate jaw-closer muscle nociceptor discharge in anesthetized Sprague-Dawley rats. It was found that ketorolac (5 mM) inhibited hypertonic saline-evoked nociceptor discharge, which suggests that at this concentration, ketorolac has local anesthetic-like properties. A lower concentration of ketorolac (0.5 mM), which did not affect hypertonic saline-evoked discharge, did inhibit nociceptor discharge evoked by NMDA. In contrast, naproxen (5 mM) did not alter hypertonic saline- or NMDA-evoked nociceptor discharge. Subsequent experiments revealed that ketorolac (0.5 mM) had no effect on nociceptor discharge evoked by αβ-methylene ATP, 5-hydroxytryptamine, or AMPA. The inhibitory effect of ketorolac did not appear to be related to cyclooxygenase inhibition, because the concentration of prostaglandin E(2) in the masticatory muscles 10 min after injection of either NSAID was not significantly decreased. The present study indicates that in vivo, ketorolac, but not naproxen, can antagonize NMDA-evoked nociceptor discharge similarly to diclofenac. We speculate that structural similarities between ketorolac and diclofenac could account for the ability of these NSAIDs to inhibit NMDA-evoked nociceptor discharge. These properties may partly explain the analgesic effect of intramuscularly injected ketorolac in the clinic.     

Benzydamine: an alternative nonsteroidal anti-inflammatory drug in patients with nimesulide-induced urticaria

BACKGROUND: Cutaneous adverse reactions to nonsteroidal anti-inflammatory drugs (NSAIDs), in particular urticaria/angiedema syndrome, represent a frequent problem in clinical practice. To date laboratory tests for the diagnosis of these adverse reactions are not available. A patient with an adverse drug reaction to NSAIDs needs an alternative drug to assume if necessary. Nimesulide is a highly prescribed nonsteroidal anti-inflammatory drug (NSAID) world-wide. It is also described as one of the most tolerated NSAID. In this paper we present data on the tolerability of benzydamine in nimesulide-sensitive patients. PATIENTS AND METHODS: One hundred and thirty-seven patients with nimesulide-induced urticaria were submitted to a single-blind, placebo-controlled peroral challenge with increasing doses of benzydamine. RESULTS: One hundred and thirty-four out of 137 (98%) patients tolerated benzydamine without adverse effects, only three (2%) experienced immediate systemic urticaria (1 at the first dose and 2 at the second dose). CONCLUSION: Benzydamine is a well tolerated drug in patients with nimesulide-induced urticaria and it may represent a valid alternative NSAID in nimesulide-sensitive patients.     

Anti-inflammatory,analgesic, antipyretic and related properties of meloxicam,a new non-steroidal anti-inflammatory agent with favourable gastrointestinal tolerance

The anti-inflammatory, analgesic and antipyretic properties of the new non-steroidal anti-inflammatory agent, meloxicam, were investigated in a variety of animal models and compared with the properties of piroxicam, diclofenac, indomethacin and several other NSAIDs.With respect to the total effect of a single oral dose, the anti-exudative effect of meloxicam on carrageenaninduced oedema in the rat exceeded that of all the NSAIDs included in the comparison. Additionally, meloxicam showed the greatest potency of all the compounds examined with respect to adjuvant-induced arthritis in the rat, the granuloma pouch model and the cotton pellet test in the rat. Unlike indomethacin, in the carrageenan pleurisy model in the rat, meloxicam caused both a dose-dependent reduction in exudate volume and also inhibition of leucocyte migration.Meloxicam showed a strong and lasting effect on inflammatory pain in the rat. Like other NSAIDs, but unlike dipyrone, meloxicam had no effect in the hot plate and tail clamp tests, which are used to identify weak central analgesic effects. Unlike dipyrone and like indomethacin, meloxicam had no effect in a model of visceral distention pain.In common with other NSAIDs, meloxicam had no influence on the body temperature of normothermic rats in the anti-inflammatory dose range, but did reduce yeastinduced fever in the rat in a dose-dependent manner. Like piroxicam, meloxicam had a uricosuric effect on rats treated with oxonic acid.Low-dose meloxicam inhibited both bradykinin-induced and PAF-induced bronchospasm in the guinea-pig, but had no effect on acetylcholine-induced bronchospasm.Piroxicam had greater ulcerogenic effects in the rat stomach than meloxicam.The therapeutic range of meloxicam in the rat, with regard to inhibition of adjuvant arthritis, was several times greater than that of piroxicam, indomethacin, diclofenac and naproxen.     

Immunization by aerosol with La Sota strain of Newcastle disease virus

The respiratory symptoms observed after aerosol vaccination with the La Sota strain of Newcastle disease virus were eliminated by mixing the anti-inflammatory compound benzydamine with the vaccine prior to aerosol administration. Benzydamine had no effect on the vaccine virus or on the development of haemagglutination-inhibiting antibodies.     

Development of predictive retention-activity relationship models of non-steroidal anti-inflammatory drugs by micellar liquid chromatography: comparison with immobilized artificial membrane columns

The predictive and interpretative capability of quantitative chromatographic retention-biological activity models is supported by the fact that under adequate experimental conditions the solute partitioning into chromatographic system can emulate the solute partitioning into lipid bilayers of biological membranes, which is the basis for drug and metabolite uptake, passive transport across membranes and bioaccumulation. The use of micellar solutions of Brij35 as mobile phases in reversed-phase liquid chromatography has proven to be valid to predict some biological activities of different kinds of drugs. In this study, quantitative retention-activity relationship (QRAR) models to describe some of the biological activities and pharmacokinetic properties of non-steroidal anti-inflammatory drugs (NSAIDs) with predictive and interpretative ability are obtained. These models are compared with those obtained using immobilized artificial membrane (IAM) column data taken from the literature. For NSAIDs, the statistical characteristics of the micellar liquid chromatography (MLC) QRAR models were better than or at least comparable to those of the IAM-QRAR models.     

Inhibition of the assembly of Newcastle disease virus by monensin

Monensin inhibits the intracellular transport of the glycoproteins of Newcastle disease virus between cis and trans Golgi stacks of infected BHK cells, as evidenced by its effect upon their post-translational modifications such as fatty acid acylation, glycosylation and proteolytic cleavage. Thus the drug has markedly altered the subcellular distribution of the glycoproteins so that they accumulate in the internal smooth membranes but are virtually absent in the plasma membrane. These glycoproteins that accumulated in intracellular membranes have a cytoplasmic domain susceptible to protease digestion and thus are transmembranous. Under such conditions, the behavior of M protein, which plays a crucial role in virus assembly (Y. Nagai et al., 1976, Virology 69, 523-538), has been analyzed. It has been found that the M protein can neither associate with the internal membranes nor bind to the plasma membrane. Thus no virus budding has been observed, either at the plasma membranes or at internal membranes. These results substantiate the view that the interaction between M and glycoproteins is of great importance for virus assembly and suggest further that this interaction is possibly only when the glycoproteins have been incorporated into the plasma membrane.     

Properties of the potassium conductances of principal cells of rat cortical collecting ducts

In this study we examined by impalement techniques properties of the macroscopic K⁺ conductances in the luminal and basolateral membrane of principal cells from isolated perfused cortical collecting ducts (CCD) of the rat. Both membranes possess a dominating K⁺ conductance. Compared to their behaviour with K⁺, both membranes appear much less permeable to NH ₄ ⁺ and Rb⁺, and the K⁺ conductances of both membranes are inhibited by these cations. In light of these findings, it is very unlikely that significant amounts of NH ₄ ⁺ , which is secreted in the CCD, cross the principal cells as NH ₄ ⁺ . Several inhibitors with known effects on K⁺ channels in patch-clamp studies have been examined. Tetraethylammonium, which inhibits the excised K⁺ channels of these cells, has no effect on the macroscopic K⁺ conductances of either membrane. Verapamil, which inhibits the K⁺ channels in the luminal membrane, acts predominantly on the basolateral membrane K⁺ conductance in the intact tubule. Therefore, some of the macroscopic properties of the K⁺ conductances are distinct from those of single channels thus far observed in patchclamp studies.Supported by DFG Schl 277/2-1 and Gr 480/10     

Comparison of the effect of heparin and citrate on platelet aggregation

When whole blood was passed through a column of glass beads, heparinized and native blood gave similar results. With citrated blood far fewer platelets were lost on the column. Thus citrate inhibits this test and heparin has no effect.Platelet-rich plasma (PRP) was prepared from citrated and heparinized blood and the responses in a number of aggregation tests were compared. With added adenosine diphosphate and adrenaline (first wave), the response in heparin PRP was greater than that in citrate PRP and was proportional to the amount of free calcium ions. With added collagen and adrenaline (second wave) the response was worse in heparin than in citrate platelet-rich plasma. It is concluded that heparin has no effect on the adhesive forces but inhibits the release mechanism. This may be of therapeutic importance.     

Production of monoclonal antibodies against glucose oxidase, alkaline phosphatase and peroxidase. Their application in a highly sensitive antigen spot microassay

The production of monoclonal antibodies against peroxidase, alkaline phosphatase and glucose oxidase and the use of the respective enzyme monoclonal anti-enzyme complexes in immunoassays are described. A micromethod using nitrocellulose membranes as solid phase was developed. This microassay has the advantage of being a rapid and simple test procedure, using only 0.2 microliter of antigen solution and 25 microliter of test reagents. A high sensitivity was achieved by repeated incubation of monoclonal enzyme anti-enzyme complexes bridged by anti-mouse immunoglobulin. As little as 200 pg human IgG and 400 pg human IgM could be detected. The glucose oxidase assay together with the alkaline phosphatase assay displays the highest sensitivity and has significant advantages including easy evaluation due to high color contrast of the enzymatic reaction, availability of non-toxic substrate reactions, and no interference with endogenous enzyme activity in mammalian antigen preparations.     

NSAIDs: Learning new tricks from old drugs

Nonsteroidal anti‐inflammatory drugs (NSAIDs) comprise a heterogeneous group of pharmacological agents used for the symptomatic treatment of fever, pain, and inflammation. Although the main mechanism of action of NSAIDs consists of inhibiting prostaglandin synthesis by blocking the enzyme cyclooxygenase (COX), clinical, and experimental data strongly indicate the existence of additional mechanisms. Some of the COX‐independent effects are related to the ability of NSAIDs to penetrate biological membranes and disrupt important molecular interactions necessary for a wide array of cellular functions, including cell adhesion. These effects, in particular those that interfere with l ‐selectin function in neutrophils during the inflammatory response, may contribute to the anti‐inflammatory properties that NSAIDs exert in vivo. Recent contributions in this field have shown that the anti‐l ‐selectin effect of NSAIDs is related to the NADPH‐oxidase‐dependent generation of superoxide anion at the plasma membrane. These findings might represent a novel approach for developing new and effective anti‐inflammatory compounds with a better safety profile than the currently available NSAIDs.     

CD29 integrin- and LIMK1/cofilin-mediated actin reorganization regulates the migration of haematopoietic progenitor cells underneath bone marrow stromal cells

Migration and successive homing of haematopoietic stem/progenitor cells (HS/PCs) into haematopoietic microenvironments are critical to their proliferation and differentiation. To investigate molecular mechanisms underlying HS/PC migration, we used a human erythroleukaemia (HEL) cell line which has been characterized as a haematopoietic progenitor cell line and displays high migratory properties underneath the haematopoietic-supportive stromal cell line, HESS-M28. HEL cell migration is mediated by the adhesion of the CD29 integrin on HEL cells to HESS-28 cells which leads to the localization of filamentous actin and formation of cell polarity at membrane protrusions via actin cytoskeleton reorganization. HEL cell migration is inhibited by both dominant negative forms of the Rho-GTPase family members and a cell permeable inhibitor of LIMK1, S3 peptide. Expression of constitutively active- or inactive-forms of cofilin also inhibits HEL cell migration and phosphorylated cofilin is localized to the front protrusions of HEL cells. These results suggest that cytoskeleton reorganization mediated by a Rho-GTPase/LIMK1/cofilin pathway plays a critical role in the migration of HEL cells underneath HESS-M28 cells.     

Study on Paradoxical Effects of NSAIDs on Platelet Activation

We recently described a stimulatory effect of high doses (>100 mol/L) diclofenac on platelet adhesion. In this study we extend our research to the possible biochemical mechanisms of the observed effects, to other non steroidal anti-inflammatory drugs (NSAIDs) (flurbiprofen, indomethacin, acetylsalicylic acid, ibuprofen, nitrofenac and nitroflurbiprofen) and to the effect of high doses diclofenac and flurbiprofen on platelet aggreation. We observed that high doses of diclofenac and of flurbiprofen, but not of the other tested NSAIDs, increased platelet adhesion at doses ranging from 100 to 500 mol/L, an effect completely removed by the 12-lipoxygenase-inhibitor nordihydroguaiaretic acid. Moreover, they had no pro-aggregating effect, inhibiting platelet aggregation induced by 10 mol/L arachidonic acid and dose-dependently increasing the [Ca²⁺]_i. Finally, whereas no basal nitric oxide release by washed platelets was detected, when platelets were incubated by 500 mol/L diclofenac or flurbiprofen, the production of nitric oxide, as measured by amounts of nitrite released, was 4.4 ± 0.5 and 3.8 ± 0.4 pmol/5 × 10⁸ platelets/min, respectively. Our data indicate that high doses diclofenac and flurbiprofen are promoters of the early phases of platelet activation, probably through the 12-lipoxygenase pathway.