Effects of rolipram and siguazodan on the human isolated bronchus and their interaction with isoprenaline and sodium nitroprusside.

1 The effects of the selective inhibitors of cyclic AMP phosphodiesterase type IV (rolipram) and type III (siguazodan) and their interactions with isoprenaline and sodium nitroprusside have been studied in the human isolated bronchus. 2 On bronchi under resting tone rolipram was, in terms of potency (pD2 = 7.77 +/- 0.14, n = 8), very similar to isoprenaline (pD2 = 7.31 +/- 0.12, n = 12) and salbutamol (pD2 = 7.12 +/- 0.17, n = 10) and approximately 10 fold more potent than siguazodan (pD2 = 6.80 +/- 0.12, n = 6). In terms of efficacy (Emax, expressed as percentage of maximal effect induced by theophylline 3 mM), both rolipram and siguazodan were less efficient (Emax = 74 +/- 6.7%, n = 8 and 66 +/- 7.5%, n = 6, respectively) than isoprenaline (Emax = 98 +/- 0.4%, n = 12) and salbutamol (Emax = 83 +/- 2.4%, n = 10). 3 During precontraction induced by methacholine (3 x 10(-7) M) or acetylcholine (10(-3) M), concentration-response curves to rolipram and siguazodan were shifted to the right and maximal effects reduced. Rolipram was more potent than siguazodan and, in terms of efficacy, it was less active. 4 Rolipram 10(-8) and 10(-7) M but not siguazodan potentiated the effects of isoprenaline as shown by the shift to the left of the concentration-response curve to isoprenaline. Sodium nitroprusside-induced relaxation was not modified by either drug.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bronchodilator and anti-inflammatory activities of SCA40: Studies in human isolated bronchus, human eosinophils, and in the guinea-pig in vivo

There is currently interest in the use of inhibitors of cyclic nucleotide phosphodiesterases (PDE) as potential anti-asthma agents. In this study we examined the effects of SCA40 (6-bromo-8-methylaminoimidazol[1,2-a] pyrazine-2-carbonitrile), a preferential inhibitor of PDE 3 also endowed with PDE 4 and 5 inhibitory activities, on isolated bronchus and eosinophil functions and in an animal model of asthma. SCA40 (1 nM–0.1 mM) produced concentration-dependent inhibition of spontaneous and stimulated tone of human isolated bronchus and reached a maximal relaxation similar to that of theophylline (3 mM). The potency (–log EC₅₀ values) of SCA40 against spontaneous tone (6.52 ± 0.10) was greater than against tone raised by equieffective concentrations (∼ 70%) of histamine (5.76 ± 0.06), leukotriene C₄ (5.44 ± 0.11), and acetylcholine (4.98 ± 0.09). In the presence of cytochalasin B, the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 0.5 μM) induced leukotriene C₄ production in human eosinophils isolated in discontinuous metrizamide gradients. The production of leukotriene C₄ was inhibited by SCA40 in a concentration-related fashion (–log IC₅₀ = 6.04 ± 0.20; n = 6). Rolipram, a selective PDE 4 inhibitor, was also effective (–log IC₅₀ = 7.29 ± 0.32) but the selective PDE 3 inhibitor SKF94120 was scarcely effective (< 10% inhibition for 10 μM). In ovalbumin sensitized guinea-pigs, SCA40 (1 mg kg^–1, i.p.) given 30 min before antigen challenge significantly inhibited the acute bronchoconstriction produced by aerosol antigen (5 mg ml^–1, 30 s) (antigen response was 185 ± 13 and 91 ± 21 cmH₂O l^–1 s^–1 in control and SCA40-treated animals, respectively, P < 0.05). Pretreatment with SCA40 (1 mg kg^–1, i.p., 30 min pre- and 3 h post-antigen exposure) prevented airway hyperreactivity to histamine which developed 24 h after exposure of conscious guinea-pigs to aerosol antigen. Eosinophil lung accumulation that accompanied airway hyperreactivity was also inhibited by SCA40 (from 6.15 ± 0.86 in control to 1.27 ± 0.27 in treated animals; expressed as eosinophils × 10⁶; P < 0.05). SCA40 (1 mg kg^–1, i.p.) also inhibited the microvascular leakage produced after inhaled antigen (5 mg ml^–1, 30 s) at all airway levels. The haemodynamic effects of SCA40 (1 mg kg^–1, i.p.) consisted of a rapid decrease (peak at 5 min) in mean arterial blood pressure (–39.4 ± 2.4%) and tracheal mucosal blood flow (–13.5 ± 2.0%) that slowly recovered with time. These data support previous work showing that PDE inhibition results in anti-spasmogenic and anti-inflammatory effects. SCA40 was effective in vitro and in vivo and these effects are probably related to its activity as a mixed PDE inhibitor. Received: 13 March 1997 / Accepted: 28 July 1997     

Effects of phorbol 12,13-diacetate on human isolated bronchus

Protein kinase C appears to be involved in the regulation of airway contractility. Phorbol 12,13-diacetate (PDA; 0.01-10 microM), a protein kinase C activator, produced a transient relaxation followed by a sustained contraction of human isolated bronchus. Different protein kinase C inhibitors (calphostin C, staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine) (H-7), nifedipine (NIF; 1 microM) or incubation with Ca(2+)-free medium, inhibited the spasmogenic response to phorbol, while ouabain (10 microM) suppressed only the initial relaxation. These results indicate that the initial relaxation, in response to PDA, is related to the activation of Na(+)/K(+)-ATPase, while the ensuing contraction depends on extracellular Ca(2+) entry.Incubation with PDA (1-5 microM) depressed the maximal relaxation to theophylline and caffeine obtained at 37 degrees C but augmented the spasmogenic responses to methylxanthines (10 mM) obtained in cooled preparations. These effects do not result apparently from increased extracellular entry of Ca(2+), but instead, from facilitation of the release of Ca(2+) from intracellular stores.     

Effect of stobadine on human stimulated polymorphonuclear leukocytes.

The effect of stobadine (0.1-100 microM) on human polymorphonuclear (PMN) leukocytes stimulated with N-formyl-methionyl-leucyl-phenylalanine, a specific receptor activator, or with the calcium ionophore, A-23187 (receptor bypassing stimulus) was investigated with respect to: i) superoxide generation, ii) beta-glucuronidase release and iii) 3[H]-arachidonic acid liberation. Stobadine was found to exert an inhibitory effect on N-formyl-methionyl-leucyl-phenylalanine but not on A-23187-stimulated PMN leukocytes. The effect was more intensive on superoxide generation and beta-glucuronidase release than on 3[H]-arachidonic acid liberation. These results indicate that the inhibitory effect of stobadine is most probably via a mechanism dependent on signal transduction across the plasma membrane. This effect may occur through inhibition of arachidonate signal transduction through a regulatory G-protein.     

Effects of cefmetazol, cefoxitin and imipenem on polymorphonuclear leukocytes

1. We have investigated the effects produced in vitro by Cefmetazol, Cefoxitin and Imipenem on the chemotaxis, spontaneous mobility, adherence, phagocytosis and candidicide power of human polymorphonuclear neutrophils (PMNs). 2. The three antibiotics tested significantly stimulated the adherence of neutrophils to nylon fibre at doses either equal (50 mg/l) or superior (500 mg/l) to the therapeutic one. 3. Cefmetazol, Cefoxitin and Imipenem bring about a maximum increase of chemotaxis at the therapeutic dose, whereas the spontaneous mobility diminishes with any one of the doses used. 4. The capacity of the PMNs to phagocytize and produce lysis of Candida albicans is increased in Cefmetazol with therapeutic doses. Cefoxitin produced an increased lysis of candidas.     

Effects of Bay K 8644 on contraction of the human isolated bronchus and guinea-pig isolated trachea.

The effects of Bay K 8644, a dihydropyridine which increases calcium flux through the potential-operated channels were studied on the contractions induced by histamine, acetylcholine, KCl and Ca2+ on human isolated bronchial strips and the results were compared to those obtained on guinea-pig isolated tracheal spirals. Subsequently the contractant effects of Bay K 8644 in K+-enriched medium and in the presence of Ca2+ 0.03 mM were investigated. In Krebs normal calcium medium, Bay K 8644 did not significantly modify the EC50 of acetylcholine or histamine on the human bronchus, but in concentrations of 10(-7)-10(-6)M it potentiated the effects of KCl on that preparation. It did not modify the EC50 of acetylcholine, histamine or KCl on the guinea-pig trachea. In Ca2+-free Krebs medium with additional K+ (30 mM), Ca2+ concentration-response curves were displaced to the left by Bay K 8644 in the two preparations. Shifts were 0.52 +/- 0.11 and 0.72 +/- 0.16 log units respectively with Bay K 8644 10(-8) and 10(-7) M on human bronchus (n = 4) and 0.67 +/- 0.16 and 1.06 +/- 0.19 log units respectively with Bay K 8644 10(-7) and 10(-6) M on the guinea-pig trachea (n = 5). In Krebs medium with Ca2+ 0.03 mM and K+ 30 mM, Bay K 8644 (10(-8) to 10(-6) M) contracted both the human bronchus and the guinea-pig isolated trachea. This effect was competitively antagonized by nicardipine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of human platelet activation by polymorphonuclear leukocytes.

1. The effect of unstimulated human polymorphonuclear leukocytes (PMNs) on platelet activation was examined. 2. Human platelet aggregation and adenosine 5'-triphosphate (ATP) release induced by collagen (1-2 micrograms ml-1); thrombin (0.01-0.02 u ml-1) or arachidonic acid (AA) (0.1-0.2 mM) were markedly inhibited when conducted in the presence of unstimulated PMNs. 3. Platelet inhibition induced by PMNs was dependent on the number of PMNs and on the incubation time of the mixed cell suspension. 4. Platelet inhibition was not reversed in time when PMNs were depleted from the mixed-cell suspension. 5. PMN-mediated platelet-inhibition was not mediated by AA metabolites, oxygen reactive intermediates, nitric oxide or proteases. 6. The factor(s) accounting for the platelet inhibition mediated by PMNs are not yet characterized.     

Effects of non-steroidal anti-inflammatory drugs on isolated human polymorphonuclear leukocytes (PMN): chemotaxis, superoxide production, degranulation and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) receptor binding

1. We have examined the effects of tolmetin and meclofenamate on isolated human PMN functions under FMLP stimulating conditions. 2. In a dose dependent manner, tolmetin and meclofenamate inhibited all PMN functions, except that tolmetin stimulated PMN chemotaxis. 3. Meclofenamate was much more potent than tolmetin as an inhibitory agent. 4. We also conducted competitive receptor binding assays for tolmetin, meclofenamate and ibuprofen on the FMLP receptor. 5. All three NSAID inhibited FMLP binding in a dose dependent manner with the potency order being meclofenamate greater than ibuprofen greater than tolmetin.     

In vitro effects of ureidopenicillins on human polymorphonuclear leukocytes

The in vitro effects of mezlocillin, azlocillin and piperacillin on chemotaxis and adhesivity of human leukocytes were comparatively studied. After incubation with all these antibiotics, chemotactic and adhesivity counts were similar to those of the antibiotic-free cells. Scanning electron microscope examination showed enlargement of surface and length measurements after incubation with azlocillin (P less than 0.005 and P less than 0.001) and mezlocillin (P greater than 0.05 and P less than 0.05), while piperacillin produced no alteration. These findings could provide additional information in the study of leukocyte/antibiotics interactions.     

Effects of acetylsalicylic acid on the phagocytic function of human polymorphonuclear leukocytes in vitro

1. In this paper acetylsalicylic acid (aspirin), an anti inflammatory drug, was studied in vitro at doses of 50, 100, 200 and 500 mg/l to see its effects on adherence, chemotaxis, spontaneous mobility, phagocytosis, candidicide power, nitrobule tetrazolium (NBT) reduction as well as the incorporation and metabolism of arachidonic acid in human polymorphonuclear neutrophils (PMNns). 2. Aspirin significantly stimulated neutrophil adherence to nylon fiber at all the doses used, with a correlation between the doses used and the adherence indices found. 3. At the therapeutic dose (100 mg/l) aspirin brings about a significant increase of chemotaxis, but reduces this property at the highest dose (500 mg/l). On the other hand, spontaneous mobility is not altered except with the 500 mg/l dose of aspirin which produces a significative decrease. 4. The ingestion of Cándida albicans by PMNns is significant at the therapeutic dose; the candidicide power is not modified with any of the doses used with 100 mg/l of aspirin nitroblue tetrazolium (NBT) reduction is significantly increased. 5. No changes are observed in the incorporation of arachidonic acid or in the release of its metabolites.     

Aggregation of human polymorphonuclear leukocytes by endothelin: role of platelet-activating factor.

The mechanisms by which endothelin-1 (ET-1) acts on polymorphonuclear leukocytes (PMN) are insufficiently known. In this study, we assessed the hypotheses that ET-1 is a PMN-aggregating agent, and that platelet-activating factor (PAF) is the principal mediator of ET-1-induced PMN aggregation. ET-1 induced dose-related PMN aggregation, which started 1 min after ET-1 exposure. Two different specific PAF receptor antagonists blocked the effect of ET-1 on PMN aggregation. In addition, ET-1 induced a significant increase in the production of PAF by PMN after 2 to 5 min of ET-1 incubation. ET-1 induced PAF release from PMN rather than accumulation. This PAF production was dependent on intra- and extracellular Ca2+. In this regard, the PAF receptor antagonists significantly blunted the ET-1-induced peak in cytosolic free Ca2+ ([Ca2+]i). Our results, therefore, indicate that ET-1 is effective in causing aggregation of human PMN and that its action appears to be mediated by PAF production via a Ca(2+)-dependent mechanism.     

Effects of S 9977-2 on the guinea pig isolated trachea and the human isolated bronchus

The effects of S 9977-2, a new compound which belongs to the trimethylxanthine family and has shown in vivo promnesic activity and in vitro acetylcholinesterase activities inhibition, were studied on the guinea pig isolated trachea and on human isolated bronchi. On the guinea pig isolated trachea, S 9977-2 in concentrations of 10^?7–10^?4 M potentiated the contractile effect of acetylcholine. The potency of acetylcholine was increased 3.39, 4.26, 9.54, and 13.18 fold with concentrations of 10^?7, 10^?6, 10^?5, and 10^?4M, respectively. The maximum effect of acetylcholine was not modified. S 9977-2 did not potentiate the effects of carbachol or pilocarpine. Under similar conditions, eserine (10^?8 and 10^?6M) and tacrine (10^?8–10^?6M) also potentiated the effects of acetylcholine. The potentiating effect of these two substances was stronger than that of S 9977-2, with a 44.7 fold increase for eserine (10^?7M) and a 64.6 fold increase for tacrine (10^?6M). However, both eserine and tacrine had a contractile effect of their own on the guinea pig isolated trachea, whereas S 9977-2 had no such effect. On the human isolated bronchus, S 9977-2 at a concentration of 10^?5M produced a 19.1 fold increase of acetylcholine effects. On the guinea pig isolated trachea, the effects of S 9977-2 vs. acetylcholine were not modified by epithelium removal. They were increased by pretreatment with indomethacin 10^?6M. In very high concentrations (10^?4 and/or 10^?3M), S 9977-2 reduced the effects of histamine and those of serotonin on the guinea pig isolated trachea. S 9977-2 had no effect on the contractile action of potassium chloride or on the relaxant action of adenosine. The results suggest that in the airway smooth muscle S 9977-2 partially inhibits acetylcholinesterase and/or pseudocholinesterase activity. © 1992 Wiley-Liss, Inc.     

Modulation of vasodilatation to levcromakalim by hypoxia and EDRF in the rabbit isolated ear: a comparison with pinacidil, sodium nitroprusside and verapamil.

1. We have used an isolated buffer-perfused preparation of the rabbit ear to investigate the effects of hypoxia and inhibition of endothelium-derived relaxing factor (EDRF) synthesis on the vasodilator responses to the potassium channel opener, levcromakalim (the active (-)-enantiomer of cromakalim). The results obtained with levcromakalim have been compared with those for pinacidil, sodium nitroprusside and verapamil. 2. Levcromakalim relaxed preconstricted preparations with an EC50 = 343 +/- 41 nM and Rmax = 80.3 +/- 6.4%. Under hypoxic conditions the concentration-response curve was significantly (P < 0.01) shifted to the left with an EC50 = 118 +/- 16 nM and Rmax = 89.9 +/- 2.7%. Hypoxia did not influence relaxation to either pinacidil, sodium nitroprusside or verapamil. 3. Inhibition of EDRF synthesis with 100 microM NG-nitro-L-arginine methyl ester (L-NAME) also significantly (P < 0.001) increased the vasodilator potency of levcromakalim (EC50 = 56 +/- 5 nM), and caused a similar shift in the concentration-response curve to sodium nitroprusside. It did not influence vasodilation to either verapamil or pinacidil. The potentiation of vasodilator responses to levcromakalim by L-NAME was reversed by an excess of L-arginine. 4. Impairment of oxidative phosphorylation with 400 nM carbonyl cyanide m-chlorophenylhydrazone significantly (P < 0.05) increased the potency of levcromakalim (EC50 = 120 +/- 20 nM) but did not influence vasodilation to pinacidil or endothelium-dependent relaxations to acetylcholine. 5. Vasodilatation to levcromakalim was augmented both by hypoxia and by inhibition of EDRF activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cadmium on metallothionein levels in human peripheral blood leukocytes: a comparison with zinc

Metallothioneins (MT) are low-molecular-weight, cysteine-rich proteins that are induced in response to a variety of chemical stresses and therefore can be used to assess human exposure to environmental agents. In the current study, flow cytometry was used to characterize the basal and cadmium-induced expression of MT in the three major leukocyte populations of human peripheral blood. In the analysis, monocytes were the most sensitive leukocytes to this toxic metal, with significant increases in cellular MT levels being detected at concentrations of cadmium as low as 0.1 microM (24 h). The lymphocyte population also exhibited pronounced treatment-associated elevations in cellular MT, while the granulocyte population was found to be nonresponsive. Although both CdCl2 (3 microM) and ZnCl2 (50 microM) induced MT expression in monocytes to a similar degree and did not affect the expression of this protein in granulocytes, cadmium but not zinc treatment induced dramatic increases in MT levels of lymphocytes. Our results indicate that cellular MT protein levels, as determined by this flow cytometric method, may be used to characterize the differential responsiveness of the major human leukocyte subpopulations to transitional metals. It is evident from the current work that the responsiveness of all peripheral blood leukocyte populations should be analyzed in exposure assessment studies.     

The effect of cyanide on vitamin C uptake by human polymorphonuclear leukocytes.

1. Cyanide inhibited the uptake of vitamin C by human polymorphonuclear leukocytes (PMNs). 2. Preincubation of PMNs with cyanide had no effect on cytochalasin B-inhibitable uptake of dehydroascorbic acid (DHA) (the reversibly oxidized and transportable form of vitamin C). 3. Preincubation of DHA with cyanide resulted in inhibition of DHA uptake. 4. Vitamin C uptake was decreased by cyanide to the same degree as it was by glutathione (GSH), which effectively reduces DHA to ascorbic acid. The effects of cyanide and GSH were not additive. 5. The data are consistent with the hypothesis that cyanide inhibition of vitamin C uptake represents the chemical elimination of extracellular DHA rather than the inhibition of active transport in these cells.     

Influence of human plasma kallikrein on lysosomal enzyme release from polymorphonuclear leukocytes.

Stimulation of polymorphonuclear leukocytes with kallikrein demonstrated that enzyme acts selectively on the release of lysosomal enzymes of these cells. The release of collagenase, similarly to the release of lysozyme into the incubation medium increased proportionally to kallikrein concentration and the duration of incubation. Kallikrein had a small effect on beta-glucuronidase secretion. No effect on cytoplasm lactate dehydrogenase release was detected. These results suggest that kallikrein, as a soluble stimulus, predominantly induces degranulation of specific granules containing collagenase capable of degrading the connective tissue. Secretion of lysozyme and collagenase requires the presence of active kallikrein. Soybean trypsin inhibitor diminished the enzyme release.