Inhibition of chemiluminescence in granulocytes and alveolar macrophages by azelastine

The effect of azelastine, an orally effective antiasthmatic/antiallergic drug, on the generation of oxygenderived free radicals in phagocytes was investigated using different chemiluminescence-assays. The chemiluminescence (CL) of both human polymorphonuclear granulocytes (PMNL) and guinea-pig alveolar macrophages (AM) was induced either by phorbol myristate acetate (PMA) or zymosan and amplified either by lucigenin or DMNH (7-dimethylamino-naphthalene-1,2-dicarbonic-acidhydrazide). The inhibitory effect of azelastine was dependent on the inducer employed and the condition and type of cells used. Azelastine reduced PMA-induced CL concentration-dependently in both PMNL (IC₃₀=3.9 M) and AM (IC₃₀=9.8 M). In AM zymosan-induced CL was inhibited 21.7% by 10 M azelastine, whereas in PMNL it remained unchanged up to 10 M azelastine. Azelastine has a significantly stronger inhibitory effect (IC₃₀=4.2 M) on oxygen free radical generation in AM primed by fetal calf serum than in unprimed AM. Based on present results it is likely that azelastine inhibitis oxygen-derived free radical generation by interaction with protein kinase C.     

Inhibitory effect of phloretin on histamine release from isolated rat mast cells

The ability of the flavonoid phloretin to inhibit histamine release from rat mast cells varied considerably with the releasing agent investigated. The response to the combination of the ionophore A23187 and the phorbol ester TPA and to suboptimal concentrations of the ionophore (0.5 M) was potently inhibited (IC₅₀ about 5 M), whereas phloretin was less potent against responses to the ionophore (1 M) IC₅₀ of 17 M), to antigen alone and in combination with TPA (IC₅₀ of 30–50 M), to TPA in the absence of calcium (IC₅₀ of 50 M) and to compound 48/80 in the absence and presence of calcium (IC₅₀ of 60–90 M). The inhibition by phloretin at concentrations above 10M was partly counteracted by glucose (5 mM) indicating effects on oxidative metabolism. The flavonoid quercetin was equally potent in inhibiting histamine release induced by antigen, the ionophore at different concentrations and in combination with TPA (IC₅₀ of 20M). Although not conclusive, the results are consistent with an inhibition of protein kinase C by phloretin at concentrations below 10 M. At higher concentrations unspecific actions become apparent and phloretin therefore seems to be of limited utility as a probe for signal-pathways in cell responses.     

Differential effects of malotilate on 5-, 12- and 15-lipoxygenase in human ascites cells

Conclusions These effects of malotilate on eicosanoid formation differ from those of known lipoxygenase inhibitors such as BW 755C (IC₅₀ of 5-lipoxygenase 35 M, 12-lipoxygenase >100 M and 15-lipoxygenase 1.2 M), nordihydroguiaretic acid (IC₅₀ of 5-lipoxygenase 1.4 M, 12-lipoxygenase 26 M and 15-lipoxygenase 1 M) and ketoconazole (5-lipoxygenase 28 M, 12-lipoxygenase not affected and 15-lipoxygenase increased) [5]. The differential effects of malotilate on the 5-, 12- and 15-lipoxygenases and also on the generation of the compounds of the cyclooxygenase, have not previously been reported. The suppression of leukotriene productionin vitro occurred at concentrations found following normal therapeutic dosesin vivo. Inhibition of the production of the chemotactic substance LTB₄ and the vasoconstrictive TxA₂ provide a possible explanation for the useful effects of this drug on liver necrosis and liver fibrosis.     

Inhibition of IgE-mediated allergic histamine release from rat peritoneal mast cells by azelastine and selected antiallergic drugs

The ability of azelastine to inhibit IgE-mediated allergic histamine release from the peritoneal mast cells of actively sensitized rats was investigated and compared with selected antiallergic agents. Azelastine added simultaneously with the allergic stimuli (ovalbumin, OA, 10 g/ml + phosphatidylserine, PS, 10 g/ml) or preincubated with cells for 10 min prior to antigen challenge produced similar concentration-dependent inhibition of allergic histamine release. The IC_50s (M) following 10-min preincubation were as follows: azelastine = 4.8; astemizole = 86.3; ketotifen = 112.2; diphenhydramine = 133 and theophylline = 2040.3. At IC₅₀ level azelastine was about 18, 23, 28 and 425 times as effective as astemizole, ketotifen (newer histamine H₁-receptor antagonists), diphenhydramine (a traditional H₁-receptor antagonist), and theophylline (a phosphodiesterase inhibitor), respectively. Sodium cromoglycate in a concentration range or 1–1000 M (0 or 10-min preincubation) failed to exert any inhibitory effect. These data showed that among six drugs tested azelastine is the most potent inhibitor of allergic histamine release from rat peritoneal mast cells.     

Effect of a series of 1-alkyl ether lipids on inhibition of phospholipase A2 activity and PAF responses

Several 1-alkyl ether lipids were studied for their ability to inhibit PLA₂ and antagonize PAF responses. Studies with synthetic micellar substrate (1-stearyl-2-arachidonyl phosphocholine), at concentrations ranging from 0.02 to 1000M, demonstrate that CL 118326 inhibits porcine pancreatic PLA₂ in vitro. As the substrate concentration increases, there is a dose-dependent increase in the IC₅₀ value (IC₅₀ ranges: 1.6–84.6g/ml or 2.6–137M). CL 118326 inhibits mammalian pancreatic PLA₂, but not snake or bee venom PLA₂. CL 118326 inhibits thrombin (IC₅₀ =7.9M), but not Na arachidonate- (IC₅₀ > 100M) induced platelet aggregation, indicative of inhibition of cellular PLA₂. CL 118326 inhibits other PLA₂-dependent processes such as antigen-induced leukotriene (LTC₄) release (IC₅₀=2.3g/ml or 3.8M) and histamine release (IC₅₀=1.4g/ml or 2.2M) in basophil-enriched WBCs. Intradermal coinjection of CL 118326 (10g) with PLA₂ into guinea pig skin inhibits pancreatic PLA₂-induced increase in vascular permeability and leakage, but not snake or bee venom PLA₂-induced leakage. CL 118326 shows no PAF-like agonist activity in stimulating rabbit platelet-rich plasma. It inhibits PAF-induced aggregation (IC₅₀=5.8M), but not ADP-induced aggregation. CL 118326 has greater efficacy as a PLA₂ inhibitor than as a PAF antagonist since the IC₅₀-substrate concentration ratio for PLA₂ inhibition is <- 1.0 at substrate concentrations of 10–1000M while the IC₅₀-agonist ratio for PAF antagonism is > 100. Results for four other compounds related to CL 118326 are also presented.     

Potentiation by adrenaline of human platelet activation and the inhibition by the alpha-adrenergic antagonist nicergoline of platelet adhesion,secretion and aggregation

Adrenaline (1 to 10M) can induce the aggregation of human platelets suspended in citrated plasma but does not induce the aggregation of washed human platelets at doses as high as 1 mM, although these platelets respond normally to ADP, PAF-acether, collagen, arachidonic acid, thrombin, the endoperoxide analog U-46619 and the Ca²⁺ ionophore A23187. Adrenaline (0.5 M) potentiates the aggregation and secretion induced by all the previous agonists in citrated plateletrich plasma (cPRP) or in washed platelets. The activation by adrenaline of human platelets is mediated by alpha 2-adrenergic receptors, as demonstrated by inhibition with a series of adrenergic antagonists. The alpha-adrenergic antagonist nicergoline inhibits the activation of human platelets by adrenaline in the following situations: i) nicergoline inhibits the aggregation and secretion caused by adrenaline in cPRP (IC₅₀ 0.22 M and 0.28 M respectively); ii) nicergoline inhibits the aggregation and secretion induced by the combination of adrenaline and each aggregating agent listed above in cPRP (IC₅₀ ranging from 0.1 to 2.5 M) or in washed platelets (IC₅₀ ranging from 0.1 to 0.8 M); iii) nicergoline inhibits the biding of³H-yohimbine to washed human platelets (IC₅₀ 0.26 M); iv) the intravenous administration of nicergoline (0.5 mg/kg per day) to patients inhibits significantly theex vivo response of their platelets to adrenaline in cPRP. High concentrations of nicergoline also inhibit the aggregation and secretion induced by the aggregating agents listed above in cPRP (IC₅₀ range 108 to 670 M) and in washed platelets (IC₅₀ range 27 to 140 M) and the adhesion of platelets to collagen-coated surfaces. This latter effect is not mediated through blockade of alpha-adrenoceptors. A possible role of adrenaline in platelet activationin vivo could justify the use of nicergoline (Sermion^®), an alpha-adrenergic antagonist in combination therapy to prevent arterial thrombosis.     

Pharmacological modulation of plasminogen activator secretion by P388D1 cell line

P388D1 is a murine macrophage cell line which spontaneously secretes plasminogen activator (PA; activated function) and lysozyme (LYS; constitutive function). Compounds which decrease PA secretion without affecting LYS secretion have potential as down-regulators of macrophage function and, hence, of the immune system. Glucocorticoids (e.g., dexamethasone, IC₅₀<0.01 M) and auranofin (IC₅₀=1 M) are positive in this model. In contrast, cyclooxygenase inhibitors (indomethacin, ibuprofen and piroxicam, all at 1 M) boost PA secretion; lipoxygenase inhibitors (REV-5901, NDGA and piriprost, all at 10 M) have little or no effect.Dexamethasone, but not auranofin, induces a urokinase-inhibitory activity which elutes between 0.13 and 0.19M NaCl upon anion exchange HPLC (TSK-DEAE-5-PW). Fibrin overlay following SDS-PAGE of the HPLC peak reveals a urokinase-inhibitory band at 90 Kd.     

Different inhibitory effect of etomidate and ketoconazole on the human adrenal steroid biosynthesis

Summary The narcotic agent etomidate and the antimycotic drug ketoconazole are known to block steroid biosynthesis in man. To study the different effects of these imidazole derivatives on human adrenal steroid biosynthesis we incubated slices of human adrenal glands with 3H-labeled precursors and increasing concentrations of etomidate or ketoconazole (0-2000 M). After extraction the labeled metabolites were separated by thin-layer chromatography and quantified by scintillation counting. Etomidate inhibited most potently 11-hydroxylase activity by suppressing the formation of corticosterone from 11-deoxycorticosterone to 1 % of control [50% inhibitory concentration (IC₅₀) 0.03 M] while ketoconazole suppressed 11-hy-droxylase to only 39% of control activity (IC₅₀ 15 M). Ketoconazole however, most potently blocked the conversion of 17-hydroxy-proges-terone to androstenedione by C17,20-desmolase to about 15% of control activity (IC₅₀ 1 M) while etomidate showed a much weaker effect on this enzyme with a suppression to 50% of C17,20-desmolase control activity at a concentration of 380 M. Both imidazole drugs showed a similar strong inhibitory effect on the activity of 17-hy-droxylase (IC₅₀ 6-18 M) and 16-hydroxylase (IC₅₀ 4–8 M) and did not affect 21-hydroxylase. These in vitro data indicate a predominant inhibitory effect of etomidate on corticosteroid biosynthesis by relative selective inhibition of 11-hydroxylase and of ketoconazole on the adrenal androgen biosynthesis by a predominant inhibition of C17,20-desmolase. This differential inhibitory effect of etomidate and ketoconazole on human steroid biosynthesis may be of clinical importance for a possible therapeutic use of these imidazole derivatives in endocrine disorders.Abbreviations IC₅₀ 50% inhibitory concentration Dedicated to Prof. Dr. G. Paumgartner on the occasion of his 60th birthday     

Topical anti-inflammatory properties of flutrimazole,a new imidazole antifungal agent

The topical anti-inflammatory properties of flutrimazole, a new imidazole antifungal, have been evaluated. Flutrimazole inhibited mouse ear oedema induced by arachidonic acid, tetradecanoylphorbolacetate and dithranol, with IC₅₀ values of 3.32, 0.55 and 2.42 mols/ear, respectively. Ketoconazole showed similar potency in arachidonic acid and dithranol models (IC₅₀=3.76 and 2.41 mols/ear) whereas it was less active against tetradecanoylphorbol acetate (IC₅₀=1.96 mols/ear). The standard anti-inflammatory sodium diclofenac was overall slightly more potent than antifungals (IC₅₀=2.23, 0.57 and 0.57 mols/ear against arachidonic acid, tetradecanoylphorbol acetate and dithranol, respectively). Both 2% flutrimazole and 2% ketoconazole creams, applied topically, inhibited carrageenan-induced rat paw oedema by about 40%. Under the same conditions, 1% flutrimazole and diclofenac creams inhibited by 26 and 54%, respectively. Flutrimazole may work through the inhibition of 5-lipoxygenase, as it inhibited LTB₄ production by human granulocytes with an IC₅₀ value of 11 M (IC₅₀ value for ketoconazole was 17 M), whereas ram seminal vesicle cyclooxygenase was only inhibited by 16% at a concentration of 25 M. Drugs such as flutrimazole, with dual anti-inflammatory/antifungal activity, may be advantageous in the treatment of topical fungal infections with an inflammatory component.accepted by I. Ahnfelt-Rønne     

Inhibitory effects ofγ-interferon on bradykinin-induced bone resorption and prostaglandin formation in cultured mouse calvarial bones

The effects of mouse recombinant-interferon (-IFN) and indomethacin on bone resorption stimulated by bradykinin, Lys-bradykinin, Met-Lys-bradykinin, des-Arg⁹-bradykinin and prostaglandin E₂ (PGE₂) have been studied using cultures of neonatal calvarial bones and analyzing the release of⁴⁵Ca from prelabelled bones as a paramenter of bone resorption. In addition, the effects of-IFN and indomethacin on formation of PGE₂ in bone cultures stimulated by bradykinin was analyzed. Indomethacin (1 mol/l) totally abolished bradykinin (1 mol/l) induced⁴⁵Ca release. The inhibitory effect of indomethacin could be fully reversed by addition of PGE₂ (1 mol/l).-IFN (1000 U/ml) almost totally inhibited⁴⁵Ca release stimulated by bradykinin (1 mol/l), but the inhibitory effect could only be partially overcome by PGE₂.-IFN and indomethacin also inhibited the stimulatory effects of Lys-bradykinin, Met-Lys-bradykinin and des-Arg⁹-bradykinin (1 mol/l) on⁴⁵Ca release. The stimulatory effects of PGE₂ (1 mol/l) on radioactive calcium mobilization was partially inhibited by-IFN (1000 U/ml), whereas indomethacin (1 mol/l) was without effect. The inhibitory effect of-IFN on⁴⁵Ca release stimulated by bradykinin and PGE₂ was dose-dependent with threshold for action at 3–30 U/ml. Comparative dose-response curves showed that-IFN was most potent as inhibitor of bradykinin induced⁴⁵Ca release. Bradykinin (1 mol/l) significantly stimulated PGE₂ formation by a mechanism that was completely inhibited by indomethacin (1 mol/l).-IFN (1000 U/ml) partially inhibited the stimulatory effect of bradykinin on PGE₂ formation. These data show that i)-IFN is a potent inhibitor of bone resorption induced by bradykinin and bradykinin analogues and ii) that the mechanism of action can be mainly explained by an inhibition of kinin induced prostaglandin biosynthesis. The results, however, also show that-IFN can inhibit bone resorption by mechanisms unrelated to prostaglandin formation.     

Interaction of azelastine with adenosine receptors in guinea pig trachea

The effects of azelastine, 8-phenyltheophylline, NDGA, atropine and mepyramine on PIA-induced contraction and relaxation of isolated guinea pig tracheal chains were investigated. Atropine (1 nM) and mepyramine (1 M) had no effect on PIA-induced relaxation whereas 8-phenyltheophylline (5 M) caused strong inhibition of PIA-induced relaxation, indicating that the latter effect is mediated by stimulation of extracellular adenosine receptors. NDGA (0.5 M) caused potentiation of PIA-induced relaxation. Azelastine (10 nM–1 M) caused dose-dependent potentiation of PIA-induced relaxation. In another model for investigation of extracellular adenosine receptors, namely the negative inotropic effect in the electrically driven isolated guinea pig atrium, the action of PIA was fully reversed by the addition of 8-phenyltheophylline. In contrast, the negative inotropic effect of azelastine was not reversed by 8-phenyltheophylline, indicating that azelastine does not act on extracellular adenosine receptors. The negative inotropic effect of azelastine can be reversed by addition of calcium as for verapamil. It is concluded that the calcium-antagonistic and perhaps antiallergic properties of azelastine are responsible for the potentiation of extracellular adenosine receptor mediated relaxation by azelastine. Since asthmatics show increased hyperreagibility (bronchospasm) to inhalation of adenosine, the inhibition of PIA-induced contraction by azelastine indicates that the drug may be worthwhile in the treatment bronchial hyperreactivity in asthmatic patients.     

Control of recombination within and between DNA plasmids of Saccharomyces cerevisiae

Summary [2 m⁺ and [2m°] yeast were transformed to stable leucine prototrophy with the hybrid yeast — E. coli plasmid, pJDB219. This plasmid contains the entire sequence of the endogenous 2 m yeast DNA plasmid in addition to the yeast nuclear LEU2 ⁺ gene and the Co1E1 derivative, pMB9. In the [2 m⁺] transformants, a new wholly yeast LEU2 ⁺ plasmid, pYX, was generated, probably by a recombination event between pJDB219 and 2 m DNA. The plamid, pYX, in the absence of 2 m DNA, was found to exist in equimolar amounts of two forms, A and B, which probably arise by intramolecular recombination across the inverted repeat sequences of the 2 m DNA portion of the plasmid. pJDB219 was found to require the presence of 2 m DNA to undergo this intramolecular recombination. The results suggest that 2, m DNA and pYX code for a gene product required in this recombination event which pJDB219 cannot produce.     

Electrophysiological characterization of histamine receptor subtypes in sheep cardiac Purkinje fibers

The histamine-receptor-subtype-mediated effects on action potentials of electrically driven and spontaneously active isolated sheep cardiac Purkinje fibers were investigated using H₁-and H₂-selective agonists and antagonists.In electrically stimulated Purkinje fibers, histamine (3 mol/l) increased the action potential plateau height, decreased the action potential duration measured at a repolarization level of –60 mV and enhanced the pacemaker activity. These effects were abolished by the H₂-selective antagonist cimetidine (30 mol/l), but were not impaired by the H₁-selective antagonist dimetindene (0.3 mol/l).In spontaneously active Purkinje fibers, histamine (10 mol/l) increased the spontaneous rate by 24%, the slope of diastolic depolarization by 45% and shortened the duration of the diastole by 32% of the respective control measurements. These effects were blocked by 30 mol/l cimetidine, but remained unchanged in the presence of 0.3 mol/l dimetindene.Concentration-response curves of histamine were shifted to the right by approximately 2 logarithmic units in the presence of 30 mol/l cimetidine, but were not influenced in the presence of 0.3 mol/l dimetindene. The H₂-selective agonist impromidine (0.001–0.3 mol/l) had similar actions as histamine on spontaneously active Purkinje fibers, while the H₁-selective agonist 2-(2-pyridyl-)ethylamine was ineffective. It is concluded that the pronounced stimulatory action of histamine on spontaneous activity in sheep cardiac Purkinje fibers is exclusively mediated by H₂ receptors.Dedicated to Prof. Dr. E. Mutschler on the occasion of his 60th birthday.Supported by Ministerium für Wissenschaft und Forschung, Nordrhein-Westfalen, Projekt-Nr. 40008786.     

Inhibition of interleukin-2 production and lymphocyte responsiveness by the cell activation inhibitor,CI-959

CI-959 (5-methoxy-3-(1-methylethoxy)-N-1H-tetrazol-5-yl-benzo[b]-thiophene-2-carboxamide), an antiallergy compound, blocked release of IL-2 from Con A stimulated rat splenocytes and human lymphocytes with respective IC₅₀s of 19.1 and 23.1 M. Inhibition of IL-2 production required the presence of CI-959 in culture medium for the first 9 hr. CI-959 also inhibited Con A-stimulated rat and human lymphocyte proliferation with IC₅₀s of 4.7 and 5.4 M, respectively. Inhibition of the Con A proliferative response could not be overcome by exogenous recombinant human IL-2 (300 units/ml) in either the rat or human systems. Although potent in the human mixed lymphocyte reaction (IC₅₀=3.5 M), CI-959 was less effective in blocking the PHA response (IC₅₀=43.9 M), and had minimal effect on the release of IL-1 and TNF from LPS-stimulated human monocytes. These findings suggest that CI-959 selectively inhibits some lymphocyte functions, as opposed to monocyte functions, and that among these is the production of IL-2.     

Mycoplasma fermentans—Induced Inflammatory Response of Astrocytes: Selective Modulation by Aminoguanidine, Thalidomide, Pentoxifylline and IL-10

Exposure of primary rat glial cells, mostly astrocytes, to heat-inactivated Mycoplasma fermentans triggers the production of tumor necrosis factor (TNF) nitric oxide (NO) and prostaglandin E₂ (PGE₂). To attenuate the production of these proinflammatory mediators, four agents: aminoguanidine, pentoxifylline, thalidomide and IL-10 were added to astrocyte cultures. Aminoguanidine (1 and 3 mM), an inhibitor of inducible nitric oxide synthase (iNOS), suppressed the production of the three mediators. TNF was the most sensitive to thalidomide, showing dose-response inhibition at concentrations of 20 g/ml, 50 g/ml and 250 g/ml. PGE₂ was affected only by concentrations of 50 g/ml and 250 g/ml, whereas NO responded solely to the highest amount of this inhibitor. The cytokine IL-10, at 10 U and 50 U, inhibited only TNF production. Our results imply that selective suppression of proinflammatory mediators by various agents may prove feasible for amelioration of central nervous system inflammatory diseases.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Efficient selection of μ m − mutants from μm-expressing myeloma cells by treatment with ricin A-conjugated anti-μ antibody

We have developed an efficient system for obtaining myeloma mutants defective intrans-acting factors required for immunoglobulin (Ig) gene expression. The system consists of a myeloma cell line designed for this purpose and an efficient method for selecting mutants from it. The cell line is X63.653 transfected with the gene, whose tailpiece sequence was replaced with the transmembrane sequence of human EGF receptor to hold on the cell surface and whose CH1 sequence was removed to prevent from being retained in the endoplasmic reticulum. It efficiently and stably expressed chains of IgM on the cell surface ( _m ⁺ ) without light chains. To obtain mutants lacking _m ( _m ^– ) from the _m ⁺ cell line by selectively killing _m ⁺ cells, a method with ricin A-conjugated anti- antibody was more reliable than complement lysis mediated by anti- antibody. Applying the system, we obtained a variety of _m ^– mutants.     

Activation of nucleotide receptors inhibits M-type K current [I K(M)] in neuroblastoma × glioma hybrid cells

A phospholipase-C-linked nucleotide receptor, sensitive to both uridine and adenosine triphosphate (UTP and ATP) has been cloned from NG108-15 neuroblastoma × glioma hybrid cells. We have tested whether activation of this receptor could inhibit the voltage-dependent K⁺ current [I _K(M) or M-current] in NG108-15 cells recorded using whole-cell patch-clamp methods. Both UTP and ATP inhibited I _K(M) by 44% and 42%, respectively, at 100 M. Mean IC₅₀ values were: UTP, 0.77±0.27 M; ATP, 1.81±0.82 M. The order of nucleotide and nucleoside activity at 100 M was: UTP = ATP > ATP[S] = ITP > 2 MeSATP > ADP = GTP AMP-CPP, adenosine, where ATP[S] is adenosine 5-O-(3-thiotriphosphate), ITP is inosine 5-triphosphate, 2-MeSATP is 2-methylthio ATP and AMP-CPP is , methylene ATP. This rank order accords with their activities at the cloned P_2U receptor. Effects were not inhibited by suramin (up to 500 M) or by pre-incubation for 12 h in 500 ng·ml^–1 Pertussis toxin. Inhibition of I_K(M) was frequently preceded by a transient outward current, probably a Ca²⁺-activated K⁺ current, responding to Ca²⁺ mobilization. No effect on the delayed rectifier K⁺ current was observed. These observations match those expected from stimulating other phospholipase-C-linked receptors in NG108-15 cells.Shemyakin Institute of Bio-organic Chemistry, on leave from the Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia     

Effect of calmidazolium (R24571) on histamine release from isolated rat mast cells

The effects of selected calmodulin-antagonists, i.e. calmidazolium (R24571), trifluoperazine, cis- and transflupenthixol, chlorpromazine, and imipramine, on rat mast cells and on mast cell histamine release were investigated. The drugs induced histamine release, apparently by cytotoxic effects, with a rank order of potency in accordance with their lipid solubility and with maximal release at calmidazolium (5 mol/l), trifluoperazine (40 mol/l), cis- and trans-flupenthixol (50 mol/l), chlorpromazine (100 mol/l), and imipramine (500 mol/l). Inhibition of the histamine release induced by antigen, compound 48/80, and the ionophore A23187 was only observed with some of the drugs tested, with maximal inhibition at calmidazolium (2 mol/l), chlorpromazine (25–50 mol/l), and imipramine (100–250 mol/l). The concentration-response curve for histamine release induced by calmidazolium was significantly shifted to the right by antigen (i.e. horse serum) in the medium and the addition of antigen was capable of immediately stopping the release induced by calmidazolium, indicating binding of calmidazolium by antigen. Similar effects on the actions of calmidazolium were observed with phosphatidylserine. The inhibition by calmidazolium of the histamine release induced by antigen, compound 48/80, and the ionophore A23187 was significantly counteracted by glucose in the medium. The findings do not confirm an involvement of calmodulin in the histamine release process in rat mast cells. The ability of calmidazolium to bind to proteins and phospholipids in the medium indicates multiple cellular targets for calmidazolium, and the observations with glucose suggest an impaired mitochondrial function to be of major significance. The present investigation, in accordance with other recent studies, indicates that the calmodulin-antagonists are of limited utility as probes for an involvement of calmodulin in cellular responses.     

Effect of lysolecithin on blood vessel reactivity and of some ethers and esters of glycerophosphocholine and phosphocholine,respectively, on aggregation of rat platelets

Infusion of lysolecithin (LPC; e.g. 88 g/ml for 0.5–1.0 min) did not significantly impair the vasopressor action of norepinephrine (NE), prostaglandin F₂ (PGF₂) and extract of posterior pituitary (EPP) in the isolated perfused hind legs of rats. In other words, vascular smooth muscle behaves differently from the smooth muscle of the guinea-pig small intestine, since, in the latter, contractions evoked by acetylcholine, prostaglandins etc., are inhibited by LPC. Triton X 100 which, by comparison, was used as a detergent effective on the guinea-pig small intestine, depressed the vasopressor effect of NE, PGF₂ and EPP.LPC, at low concentrations (40 mol/l), potentiated (15% max.) ADP-induced platelet aggregation (PA) in rat PRP but, at high concentrations, inhibited PA (IC₅₀=390 mol/l). 2-Hexadecylglycerophosphocholine and its short-chain 1-alkyl ethers, which are structurally related to platelet-activating factor, as well as some long-chain alkanol phosphocholine esters, were somewhat more active than LPC. Dipalmitoyllecithin (4–700 mol/l) was without any effect.