Effects of CIS-19, a novel PAF receptor antagonist,on PAF-induced eosinophil recruitment and enhancement of superoxide anion generation in guinea-pigs

We examined the effects of a novel plateletactivating factor (PAF) receptor antagonist, CIS-19 [cis-2-(3,4-dimethoxyphenyl)-6-isopropoxy-7-methoxyl-1-(N-methylformamido)-1,2,3,4-tetrahydronaphthalene], on PAF-induced inflammatory cells recruitment into airways and enhancement of superoxide anion (O _inf2 ^sup– ) generation from cells retrieved by bronchoalveolar lavage (BAL) in urethane-anesthetized guinea-pigs. Administration of PAF (30 ng/kg, Lv.) produced a selective increase of eosinophils into airways, but no significant increase of the number of macrophages, neutrophils or lymphocytes. CIS-19 (2.5 and 5 mg/kg, Lv.) significantly inhibited the eosinophil recruitment induced by PAF. In vitro, PAF, phorbol 12-myristate 13-acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) directly stimulated generation of O _inf2 ^sup– from BAL cells in a concentration-dependent manner. CIS-19 (10^–7 – 10^–4 M) inhibited production of O _inf2 ^sup– induced by PAF (10^–7 M) in a concentration-dependent manner with an EC₅₀ value of 0.84 M, but not induced by PMA (0.5 g/ml) or FMLP (10^–7 M). Administration of PAF (5 ng/kg, i.v.) enhanced markedly PMA (0.5 g/ml) and FMLP (10^–7 M)-induced generation of O _inf2 ^sup– by 80.2% and 51.3%, respectively. The enhancing effect of PAF was maximal in cells harvested 5 min after the addition of PAF and then declined to baseline level at 60 min. These responses were inhibited by administration of CIS-19 (0.5—2.5 mg/kg, i.v.) or BN 52021(5 mg/kg, i.v.). The results indicate that CIS-19 is potent in inhibition of PAF-induced airway inflammatory response and may have therapeutic potential as an anti-inflammatory drugs.     

Inhibitory Effects of N-acetylcysteine on Superoxide Anion Generation in Human Polymorphonuclear Leukocytes

It has been suggested that reactive oxygen species released by activated polymorphonuclear leukocytes (PMN) in man is one mechanism of tissue injury. Therapeutic action aimed at increasing antioxidant defence mechanisms is still a clinical challenge. This study examines the activity of N-acetylcysteine, a known antioxidant, in the protection of PMN exposed in-vitro to the chemoattractant peptide fMet-Leu-Phe (FMLP), the protein kinase C activator phorbol myristate acetate or the lipid peroxidation promoter t-butyl hydroperoxide. FMLP (3–300 nm) and phorbol myristate acetate (160 pm–160 nm) induced concentration-related superoxide anion generation. Pre-treatment with N-acetylcysteine (33–333 μm) resulted in concentration-related inhibition of superoxide production induced by FMLP (30 nm) or phorbol myristate acetate (16 nm); –log IC50 values were 3.97 ± 0.07 and 3–91 ± 0.10, respectively. Changes in intracellular calcium ion concentration ([Ca²⁺]_i) induced by FMLP (30 nm) were studied in fura-2-loaded human PMN. FMLP produced a transient calcium response, i.e. a peak followed by decay to a residual value above baseline. N-Acetylcysteine (333 μm) did not affect either basal [Ca²⁺]_i values or changes in [Ca²⁺]_i values after treatment with FMLP. Activation by phorbol myristate acetate caused a reduction in glutathione levels from 5.94 ± 0.86 (control) to 1.84 ± 0.51 nmol/3 × 10⁶ cells (P < 0.05 compared with control). Pre-treatment with N-acetylcysteine (333 μm) fully reversed the reduction in glutathione levels induced by phorbol myristate acetate (4.83 ± 0.68 nmol/3 × 10⁶ cells; P > 0.05 compared with control). Exposure to t-butyl hydroperoxide (0.5 mm, 30 min) markedly increased malondialdehyde levels (from 0.03 ± 0.02 to 0.73 ± 0.07 nmol/10⁶ cells), and index of lipid peroxidation. Malondialdehyde levels were significantly reduced in PMN treated with N-acetylcysteine (333 μm; 0.55 ± 0.04 nmol/10⁶ cells; P < 0.05 compared with untreated cells exposed to t-butyl hydroperoxide). In conclusion, N-acetylcysteine reduces superoxide generation in response to FMLP and phorbol myristate acetate and partially protects against lipid peroxidation in PMN from man. The protection afforded by N-acetylcysteine is not related to alteration of the intracellular calcium signal but might be effected by replenishment of the intracellular glutathione levels.     

Serotonergic modulation of neurotransmission in the rat subicular cortex in vitro: a role for 5-HT1B receptors

Summary We have studied the effect of serotonin on synaptic transmission in rat hippocampal subiculum slices. Electrical stimulation of the alveus induced a field potential in the subiculum. The non-NMDA glutamate receptor antagonist, NBQX (3 × 10^–6 mol/l) suppressed the response by 78%, indicating that the signal involves glutamatergic neurons. Application of serotonin suppressed (EC₅₀ = 3.6 × 10^–6 mol/l) the amplitude of he evoked potentials in a reversible, concentration-dependent manner. The responses to 5-HT were not altered after pretreatment with the 5-HT uptake blocker, fluvoxamine (10^–5 mol/l) or a combination of the MAO inhibitor pargyline (10^–5 mol/l) and ascorbic acid (10^–4 mol/l). The responses to 5-HT were also unaffected by pretreatment with the 5-HT_1A selective antagonist NAN-190 (10^–6 mol/l), the 5-HT_2A antagonist ketanserin (10^–6 mol/l) or the 5-HT₃/5-HT₄ antagonist ICS 205–930 (10^–6 mol/l).The 5-HT_1B selective agonist CP 93,129 mimicked the effects of serotonin, but was more potent (EC₅₀ 4.1 × 10^–7 mol/l). The 5-HT_1B receptor antagonist, (±)21-009 (3 × 10^–7 mol/l), antagonized the response to 5-HT and CP 93,129 with a pK_B value of 7.1 and 7.2, respectively. These results suggest that the effect of 5-HT in the rat subiculum is mediated by 5-HT_1B receptors.Correspondence to: H.W.G.M. Boddeke at the above address     

Chemokinetic activity of N-formyl-methionyl-leucyl-phenylalanine on human neutrophils, and its modulation by phenylbutazone

Phenylbutazone (PBZ) is known to inhibit the oriented migration of human polymorphonuclear leukocytes (PMNs) induced by formyl-methionyl-leucyl-phenylalanine (FMLP), and to protect these cells against the deactivation caused by their prior incubation with FMLP. To gain insight into the mechanism of these effects, we measured the oriented PMN migration under agarose induced, in the presence and absence of PBZ, by FMLP, zymosan-activated serum and Klebsiella pneumoniae culture supernatant. The two components of this migration, i.e. the speed (chemokinesis), and direction of locomotion (chemotaxis), were also assessed. At concentrations ranging from 10(-8) to 10(-5) M, FMLP displayed similar chemotactic activity but the speed of PMN locomotion was maximal for 10(-7) M, and lower for concentrations above and below this level. Oriented migration was proportional to the mean cell locomotion speed during the experiments. PBZ inhibited both the oriented migration and locomotion speed induced by 10(-7) M FMLP, but did not affect its chemotactic activity. At concentrations of 10(-6) and 10(-5) M, PBZ increased oriented migration and locomotion speed, again without influencing FMLP chemotactic activity. Oriented migration induced by zymosan-activated serum was not affected by PBZ but the migration induced by Klebsiella pneumoniae culture supernatant diminished slightly. These results demonstrate that PBZ modulates the chemokinetic effect of FMLP on PMNs and thus alters oriented PMN migration.     

EFFECT OF XANTHINE DERIVATIVES ON CHEMOTACTIC POLYPEPTIDE-INDUCED SUPEROXIDE AND ENZYME RELEASE FROM HUMAN POLYMORPHONUCLEAR LEUCOCYTES

1. We investigated the effects of new xanthine derivatives, 1-methyl-3-propyl xanthine (MPX) and 1,3-dipropyl xanthine (DPX), and several other xanthine derivatives on N-formyl-methionyl-leucyl-phenylalanine-induced superoxide and lysozyme release from human polymorphonuclear leucocytes (PMN). 2. MPX and DPX at low concentrations (10^?8-10^?9 mol/L) inhibited superoxide release from PMN by a maximum of 31.2 ± 10.6% and 49.8 ± 10.4% (mean ± s.d.), respectively, and 10^?3 mol/L concentrations completely inhibited the release reactions (4.8 ± 1.2 and 7.6 ± 2.5% of control level). At 10^?5 mol/ L, however, the inhibition did not occur (99.9 ± 7.3 and 110.2 ± 15.8% of control level). When PMN was pre-incubated with adenosine deaminase (ADA, 0.1 U/mL), superoxide release from PMN was inhibited in a dose-dependent manner by MPX and DPX and the interruption of the inhibition at 10^?5 mol/L was not observed. 3. Lysozyme release from PMN was inhibited by MPX at low concentrations (10^?7-10^?6 mol/L) and high concentrations (10^?3 mol/L). However 10^?4 mol/L of MPX facilitated the release (23.7 ± 27.0%). When pretreated with ADA (0.1 U/mL), MPX suppressed lysozyme release in a dose-dependent manner and the facilitation of the release at 10^?4 mol/L was not observed. 4. When comparing effects of some other xanthine derivatives on superoxide release, the interruption of the inhibition of superoxide release at 10^?5 mol/L was commonly observed among xanthine derivatives with adenosine A₂ antagonism. 5. The results suggest that adenosine A₂ antagonism of xanthine derivatives may interfere with their anti-inflammatory effects at therapeutic concentrations (10^?5-10^?4 mol/L).     

Control of the release of newly synthetized 3H-5-hydroxytryptamine by nicotinic and muscarinic receptors in rat hypothalamic slices

Summary The effects of various cholinergic agonists and antagonists on the spontaneous release of newly synthetized ³H-5-HT were examined in rat hypothalamic slices. ³H-5-HT was measured in incubating medium at the end of a 30 min incubation carried out with l-³H-tryptophan in the presence of the various durgs tested. ACh (10^–5 M) in the presence of eserine (2×10^–4 M), and carbachol (10^–5 M) stimulated the release of ³H-5-HT. In contrast, oxotremorine (10^–5 M) reduced the ³H-amine release. The effect of carbachol was blocked by two nicotinic blockers, mecamylamine (10^–6 M) and d-tubocurarine (10^–6 M). It was not reduced by the muscarinic antagonists, atropine (10^–6 M) and scopolamine (10^–6 M). In fact, each of two antagonists added alone to the incubating medium enhanced ³H-5-HT release. The scopolamine (10^–6 M) stimulating effect on ³H-5-HT release was suppressed by d-tubocurarine (10^–6 M). Finally, the inhibiting effect of oxotremorine on ³H-5-HT release was not prevented by d-tubocurarine (10^–6 M) but was in the presence of atropine (10^–6 M) or scopolamine (10^–6 M).In the concentrations used in the release study, the cholinergic agonists and antagonists had no effect on the total formation of ³H-5-HT and ³H-5-HIAA from l-³H-tryptophan and on the accumulation of l-³H-tryptophan in tissues. In these concentrations, except for eserine, they did not affect the uptake of exogenous ³H-5-HT in hypothalamic synaptosomes (P₂ fraction).These results suggest that cholinergic receptors of the muscarinic and nicotinic type are involved in the control of ³H-5-HT release; since the stimulation of the muscarinic and nicotonic cholinergic receptors resulted in an inhibition and an activation of ³H-5-HT release, respectively. As in the case of peripheral noradrenergic and central dopaminergic neurons the cholinergic receptors could be localized on serotoninergic terminals.     

Evidence for two types of 5-hydroxytryptamine receptor on secretomotor neurons of the guinea-pig ileum

Summary Flat sheet preparations of the mucosa plus submucosa from the guinea-pig ileum were placed in Ussing chambers so that short circuit currrent (I _sc), an index of electrolyte movement across the mucosa, could be measured. In these preparations, 5-hydroxytryptamine (5-HT) increasesI _sc indirectly by stimulating both cholinergic and non-cholinergic secretomotor neurons. The 5-HT₃ receptor antagonist, ICS 205–930 (10^–13–10^–5 M), substantially depressed the secretory response due to 5-HT (10^–6 M), but not that produced by direct activation of muscarinic receptors on the mucosal epithelium with carbachol (10^–6 M), or by stimulation of secretomotor neurons with substance P (10^–8 M) or 1,1-dimethyl-4-phenylpiperazinium (10^–5 M). The residual response to 5-HT, after the addition of a maximally effective concentration of ICS 205–930 (10^–6 M), was further reduced by hyoscine (10^–7M). When that part of the 5-HT response attributable to the release of acetylcholine was blocked by hyoscine (10^–7M), ICS 205–930 did not further modify the response to 5-HT. The hyoscine-resistant component was, however, sustantially depressed by tetrodotoxin (3.5 × 10^–7 M). The response remaining after ICS 205–930 and hyoscine was not affected by methysergide (2 × 10^{– 5} M) or cyproheptadine (10^–7 M). We conclude that there are ICS 205–930 sensitive 5-HT receptors on cholinergic secretomotor neurons, and ICS 205–930, methysergide, and cyproheptadine insensitive 5-HT receptors on non-cholinergic secretomotor neurons.     

Nitric oxide induces acetylcholine-mediated contractions in the guinea-pig small intestine

In longitudinal muscle/myenteric plexus preparations of the guinea-pig ileum, exogenous nitric oxide (NO) induced concentration-dependent relaxations. In tissues at basal tone, NO (3 × 10^–6 M) induced a moderate relaxation followed by a pronounced contraction, consisting of a quick and sustained component. Tetrodotoxin (5 × 10^–7 M) abolished both phases of the contraction. Atropine (5 × 10^–7 M) abolished the quick component and reduced the sustained component of the contraction; the latter was further suppressed by the selective NK₁ receptor antagonist CP 96,345. Hexamethonium (5 × 10^–5 M) failed to affect the contractile response to NO. It is concluded that administration of exogenous NO in the guinea-pig ileum can lead to activation cholinergic and to a lesser degree tachykininergic neurones. Correspondence to: L. Barthó at the above address     

Comparison of AMP579 and adenosine in inhibition of cell–cell interaction between human neutrophil and vascular endothelial cell

The purpose of the present study was to compare inhibitory effects between AMP579 (a new adenosine analog) and adenosine (Ado) in attenuating an interaction between human neutrophils (PMNs) and cultured human umbilical vein endothelial cells (HUVECs). PMN activation was determined by superoxide anion (O₂^–) production and degranulation (myeloperoxidase release). Cell–cell interaction was quantitated by adherence of fluorescent labeled PMNs to HUVECs. AMP579 inhibited O₂^– (nM/5 × 10⁶ PMN) from fMLP‐activated human PMNs (55.3 ± 3.1) in a concentration‐dependent manner ranging from 31.1 ± 2.9 at 10 nM to 11.7 ± 0.9 at 10 ± μM, all P < 0.01 vs. fMLP group. In the same dose range, however, Ado showed significant inhibition only at 1 μM (30.3 ± 4.1) and 10 ± μM (27.5 ± 4.3) vs. the fMLP group. The calculated IC₅₀ value (0.11 ± 0.05 μM) in AMP579 group was significantly less than that in the Ado group (4.1 ± 1.2 μM). Although there was no group difference on PMN myeloperoxidase release (percent inhibition from fMLP) between AMP579 and Ado at concentrations greater than 1 μM (52.9 ± 5.2 vs. 46.4 ± 5.9), AMP579 showed significant attenuation of degranulation compared with Ado at 10 nM (31.7 ± 2.5 vs. 11.6 ± 1.9) and 100 nM (48.2 ± 4.6 vs. 25.6 ± 3.8), respectively, suggesting that AMP579 is more potent in inhibiting PMN activation. AMP579 reduced PMN adherence to TNFα‐stimulated HUVEC (fluorescent units/well) in a concentration‐dependent manner from 472 ± 32 at 10 nM to 214 ± 15 at 10 μM vs. 675 ± 54 in the TNFα group. At 10 nM and 100 nM, adenosine did not attenuate PMN adherence, while it showed significant inhibition at 1 (504 ± 45) and 10 μM (435 ± 50), respectively. The IC₅₀ value (2.8 ± 1 μM) for AMP579 was significantly lower than that (41 ± 8 μM) in the Ado group. The results from the present study suggest that 1) AMP579 directly inhibits adherence‐independent superoxide radical generation and degranulation from activated PMNs and attenuates cell–cell interaction between PMNs and vascular endothelial cells by preventing damage on endothelial cells. 2) AMP579 exerts more potent protective effect compared with adenosine at a lower dose range, indicating its prospect for clinical application. Drug Dev. Res. 49:266–272, 2000. © 2000 Wiley‐Liss, Inc.     

Field stimulation-induced responses of circular smooth muscle from guinea-pig stomach

Summary Field stimulation of circular smooth muscle of guinea-pig stomach from the regions of the cardia and fundus caused contraction responses at low stimulation frequencies (0.25–1 Hz) with relaxation at higher frequencies (1–10 Hz), whilst tissues of the body and antrum responded with contraction throughout the frequency range. Atropine (10^–9–10^–8 M) antagonised the contraction responses of all tissues, with relaxation developing at higher concentrations (except for antral tissue). In contrast, metoclopramide (10^–8–10^–6 M) caused modest (cardia, fundus) or marked (body, antrum) enhancement of contractions to field stimulation, whilst domperidone (10^–8–10^–7 M), haloperidol (10^–8–10^–6 M), prazosin, propranolol and methysergide (10^–8–10^–6 M) failed to modify the contraction responses. However, whilst yohimbine and guanethidine failed to modify the contractions of the cardia, fundus and body tissues, those of the antral preparations were antagonised by nanomolar concentrations of yohimbine and by guanethidine (10^–6–5×10^–5 M). To optimise the relaxation responses for study, atropine was included in the physiological solution. Relaxation to field stimulation of preparations from the body and cardia, but not the fundus, was antagonised by reserpine pretreatment (5 mg/kg i.p., 24h), addition of guanethidine (10^–5–10^–4 M), phentolamine, prazosin or propranolol (10^–7–10^–6 M) (the effects of prazosin and propranolol being additive). Higher concentrations of haloperidol and domperidone antagonised the relaxation responses of the body preparations only. Metoclopramide, yohimbine and methysergide (10^–8–10^–6 M) were ineffective. Thus, it is concluded that the contractile effects of the 4 stomach areas to field stimulation reflects a major cholinergic involvement, with an additional ₂-adrenoceptor contractile component in antral tissue. Relaxation responses of cardia and body tissue involve ₂- and -adrenoceptors plus a further, unidentified, non-adrenergic component; the latter represents the total relaxation response of the fundic preparation.     

The role of presynaptic receptors in the release and synthesis of 3H-dopamine by slices of rat striatum

Summary Striatal slices were continuously superfused with l-3,5-³H-tyrosine (50 Ci/ml) and ³H-H₂O [index of ³H-dopamine (³H-DA) synthesis] and ³H-DA estimated in 0.5 ml (2.5 min) superfusate fractions. Depolarization with 50 mM K⁺ for 7.5 min induced a marked increase in ³H-DA release and a biphasic effect on synthesis (slight increase in the first fraction followed by a significant decrease in the third and fourth fractions). The decrease in the rate of ³H-H₂O formation induced by K⁺ was not related to modifications of the specific activity of tyrosine in tissues. The possibility that the inhibition of synthesis was due to alterations in DA concentration in the synaptic cleft was examined. Benztropine in a concentration which produced inhibition of DA uptake (10^–6 M) increased the K⁺ induced overflow of ³H-DA but failed to alter the inhibition of synthesis. On the other hand, when the powerful neuroleptic fluphenazine was added to the superfusion medium in a concentration which only weakly blocked ³H-DA uptake (10^–6 M) it potentiated ³H-DA release and prevented the inhibition of synthesis both in the absence or presence of benztropine. A similar effect was seen following the in vivo treatment of rats with fluphenazine (2 mg/kg; 1 1/2 h before sacrifice). The addition of exogenous DA (0.6×10^–6 M) or NA (10^–6 M) to the superfusion medium increased ³H-DA outflow and reduced DA synthesis while isoproterenol (10^–6 M) was without effect. The DA inhibitory effect on synthesis was still observed in the presence of benztropine (10^–6 M) while the NA effect was prevented. This concentration of benztropine blocked both DA and NA uptake. The administration of fluphenazine (10^–6 M) significantly prevented the decrease in ³H-DA synthesis induced by exogenous DA and partially prevented the effect of NA. In addition, the effect of exogenous DA on the inhibition of synthesis was still seen in the presence of 2-amino-4-hydroxy-6,7-dimethyl-5,6–7,8-tetrahydropteridine hydrochloride (DMPH₄) (to protect against end-product inhibition). The present results provide direct support for the concept that activation of presynaptic DA receptors located on DA terminals in the striatum of the rat results in an inhibition of synthesis and release of the transmitter.     

Cytotoxic enzyme release and oxygen centered radical formation in human neutrophils are selectively inhibited by E-type prostaglandins but not by PGI2

Summary The action of PGE₁, PGE₂, PGI₂ and iloprost on superoxide anion generation, lysosomal enzyme release, and changes of Ca²⁺ fluxes in human polymorphonuclear leukocytes (PMN) was studied in vitro. Both PGE-type compounds were equipotent inhibitors of FMLP-and PAF-stimulated superoxide anion generation, -glucuronidase release (IC₅₀ 3–5 mol/l) and Ca²⁺ influx while PGI₂ and iloprost were ineffective at concentrations up to 10 mol/l. These inhibitory actions of PGE₁ and PGE₂ were paralleled by an increase in cAMP level of the PMN while no change occurred with PGI₂ and iloprost. None of the prostaglandins affected the initial intracellular Ca²⁺ liberation after challenge with FMLP or PAF. Preincubation of PMN with PGE₁ and PGE₂ but not with iloprost resulted in subsequent desensitization against a second administration of these compounds. None of the compounds affected PMN activation produced by arachidonic acid or calcimycin (A 23187).These data demonstrate that PGE-type compounds are effective inhibitors of receptor-mediated (PAF, FMLP) activation of human PMN while prostacyclins are considerably less potent. This suggests that the inhibitory prostaglandin receptor on human PMN belongs to the E-type being functionally different from the inhibitory prostaglandin receptor on human platelets. These results suggest that compounds, such as PGE₁ and PGE₂ might be superior to prostacyclins to prevent PMN-associated generation of reactive oxygen species and lysosomal enzyme release in situations with endogenous PMN activation, i. e. inflammatory reactions. Send offprint requests to K. Schrör at the above address     

In vivo continuous estimation of 3H-dopamine synthesis and release in the cat caudate nucleus

Summary Synthesis and release of ³H-DA were examined during the continuous superfusion of a limited area of the ventricular surface of the cat caudate nucleus with l-3,5-³H-tyrosine, using a cup technique. ³H–H₂O, an index of the conversion of l-3,5-³H-tyrosine into ³H-Dopa, and ²H-DA were estimated in serial superfusate fractions. Alpha-methyl-paratyrosine (-MpT) (10^–4 M) inhibited rapidly and simultaneously both ³H–H₂O formation and ³H-DA release. Transection of the nigro-striatal dopaminergic pathway immediately blocked ³H-DA release but ³H–H₂O levels were reduced by 30% one hour later.     

Influence of fentanyl,levorphanol and pethidine on the release of noradrenaline from rat brain cortex slices

Summary In slices of rat brain cortex preincubated with (–)-³H-noradrenaline, the influence of fentanyl, levorphanol and pethidine on the efflux of tritium was investigated. The spontaneous outflow of tritium was not changed by low, and was accelerated by high concentrations of the drugs. The overflow of tritium evoked by electrical stimulation at 3 Hz was diminished by 10^–8–10^–7 M fentanyl and by 10^–7–10^–6 M levorphanol, but was augmented by 10^–5 M levorphanol. Naloxone prevented the inhibitory effect of fentanyl and levorphanol. In contrast to fentanyl and levorphanol, pethidine did not decrease, but at concentrations of 10^–6–10^–5 M greatly increased the stimulation-induced overflow of tritium. However, the increase was abolished, and the stimulation-evoked overflow slightly reduced, after the re-uptake of noradrenaline had been blocked by cocaine. It is concluded that fentanyl, levorphanol and pethidine share with morphine the ability to inhibit the release of transmitter from cerebrocortical noradrenaline neurones evoked by nerve impulses.     

Influence of morphine and naloxone on the release of noradrenaline from rat brain cortex slices

Summary In slices of rat brain cortex preincubated with (–)-³H-noradrenaline, the influence of morphine and naloxone on the efflux of tritium was investigated. The spontaneous outflow of tritium was not changed by 10^–7–10^–5 M morphine and by 10^–6–10^–4 M naloxone, but was accelerated by 10^–4 M morphine. Electrical field stimulation augmented tritium outflow. The overflow evoked per ppulse decreased as the frequency of stimulation was increased from 0.3 to 3 Hz, but remained approximately constant when it was further increased to 10 Hz. At frequencies of 0.3, 1, and 3 Hz, but not at 10 Hz, morphine in concentrations of 10^–7–10^–5 M depressed the stimulation-induced overflow of tritium. 10^–4 M morphine did not influence the overflow induced by stimulation at 0.3 and 1 Hz and increased that evoked by stimulation at 10 Hz. Naloxone (10^–6–10^–4 M) did not change the response to stimulation. In the presence of 10^–4 M naloxone, 10^–6 M morphine did not diminish, and 10^–5 M morphine even enhanced the stimulation-induced overflow of tritium. The inhibitory effect of 10^–6 M morphine was not reduced, after tyrosine hydroxylase had been blocked by -methyltyrosine-methylester. It is concluded that morphine through an action on specific opiate receptors inhibits the release of transmitter from cerebrocortical noradrenergic neurones evoked by nerve impulses. By an action unrelated to opiate receptors, morphine at high concentrations increases the stimulation-induced overflow of noradrenaline, presumably by inhibiting its re-uptake into nerve endings.     

Diclofenac binding to human polymorphonuclear neutrophils: effect on respiratory burst and N-formylated peptide binding

The respiratory burst of human polymorphonuclear neutrophils (PMN) induced by particle or soluble stimuli was measured in the presence of the nonsteroidal anti-inflammatory drug, diclofenac sodium (Voltaren). Diclofenac (25-100 micrograms/ml) inhibited the oxygen consumption of PMN stimulated by 5 X 10(-7) M of N-formyl-methionyl-leucyl-phenylalanine (FMLP). The inhibition was linearly correlated to diclofenac concentration. By contrast, diclofenac did not affect the rate of heat-killed Klebsiella pneumoniae ingestion of PMN, or the PMN O2-uptake induced by (0.67 microgram/ml) serum-opsonized zymosan or (1 microgram/ml) phorbol myristate acetate (PMA). The PMN production of superoxide anion induced by various FMLP concentrations (10(-7), 10(-6) and 10(-5) M) was also decreased by diclofenac. However, this inhibition declined when the formylated peptide concentration was raised suggesting that diclofenac could alter FMLP binding to the PMN membrane. Binding experiments of tritiated FMLP to intact PMN performed at 22 degrees and 4 degrees showed high- and low-affinity FMLP sites with dissociation constant (Kd) values of approximately 2 X 10(-8) M and 10(-5) M respectively. Diclofenac did not significantly alter the low-affinity component but induced modifications of the high-affinity component which were different at 22 degrees and 4 degrees. At 22 degrees only the dissociation constant value was enhanced by diclofenac (competitive inhibition) whereas at 4 degrees both binding parameters (i.e. dissociation constant and number of available binding sites) were modified (mixed inhibition). Diclofenac was also shown to bind to PMN with a low affinity. This binding was not diminished at 4 degrees by various concentrations of FMLP which even increased the number of diclofenac binding sites on PMN at 22 degrees. These data suggest that diclofenac binding to PMN may decrease FMLP-induced PMN respiratory burst by interfering with the peptide recognition by specific FMLP receptors.     

Platelets enhance contractility in perfused rat mesenteric arteries: Involvement of endothelin-1

We investigated the effects of platelet supernatant on pressor responses to norepinephrine in isolated perfused rat mesenteric arteries. Perfusion of the arteries with platelet supernatant for 2 h markedly enhanced the pressor responses to norepinephrine (10⁻⁶ and 3×10⁻⁶ M). This enhancement was significantly inhibited by phosphoramidon (10⁻⁴ M), an endothelin converting enzyme inhibitor. Both BQ788 [N-cis-2,6-dimethylpiperidinocarbonyl-

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-γ-methylleucyl-

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-1-methoxycarbonyltryptophanyl-

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-norleucine] (10⁻⁶ M), an endothelin ET_B receptor antagonist, and bosentan (Ro47-0203, 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2,2´-bipyrimidin-4-yl]-benzenesulfonamide) (10⁻⁵ M), a nonselective endothelin receptor antagonist, also prevented the potentiation of responses to norepinephrine evoked by platelet supernatant, but FR139317 ((R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carbonyl]amino-4-methyl-pentanoyl] amino-3-[3-(1-methyl-1H-indoyl)]propionyl]amino-3-(2-pyridyl) propionic acid) (10⁻⁶ M), an endothelin ET_A receptor antagonist, had little effect. Suppressor doses of endothelin-1 (3×10⁻¹⁰ M) or sarafotoxin S6c (S6c) (3×10⁻¹⁰ M) potentiated significantly the norepinephrine-induced vasoconstriction, in the same preparation. Moreover, supernatant-induced enhancement of pressor responses to norepinephrine was markedly suppressed by TGF-β1 neutralizing antibody. Transforming growth factor-β1 (TGF-β1) (40 pM) also significantly enhanced the pressor responses to norepinephrine (10⁻⁶ M) and this enhancement was significantly inhibited by phosphoramidon. These results suggest that platelet-derived TGF-β1 stimulates the vascular production of endothelin-1 and thereby enhances vasoconstrictor responses to norepinephrine. Platelet-induced enhancement of vasoconstrictor responses to norepinephrine seems to be mainly mediated by endothelin ET_B receptor, in rat mesenteric arteries.     

Influence of calcium on the effects of okadaic acid and its interaction with caffeine and theophylline in rat myometrium

The effects of okadaic acid (OA), a monocarboxylic acid produced by marine dinoflagellates belonging to the genera Dinophysis and Prorocentrum, and their interactions with theophylline and caffeine were studied on the rat-isolated uterus in a calcium-containing medium and a calcium-free medium in the presence of 10^–3 M EGTA.Okadaic acid (5 × 10^–6 to 5 × 10^–5 M) induced a concentration-dependent contraction of the rat-isolated uterus corresponding, with 5 × 10^–5M, to 142.3±6.1% (n = 7) of the contraction induced by oxytocin 10^–6 M. The time to peak tension was inversely proportional to the maximum effect produced. The contraction was not sustained and was followed by a concentration-dependent decrease in tone. In a Ca²⁺-free medium containing 10^–3 M EGTA the contractile effects of OA were significantly inhibited or reduced. A 30 min pretreatment with theophylline (3 × 10^–3 M) or caffeine (2 × 10^–2 M) significantly reduced, in a Ca²⁺-containing medium, the maximum contractile effect of OA 10^–5 and/or 2 × 10^–5 M and shortened the relative time to peak tension. In a Ca²⁺-free medium containing 10^–3 M EGTA, only the second effect was observed. With a 1 min pretreatment and in a Ca²⁺-containing medium, theophylline 3 × 10^–3 M and caffeine 10^–2 M did not modify the maximum effect of OA 10^–5 M but shortened the time to peak tension. The same concentrations of the xanthines potentiated the E_max of OA 5 × 10^–6 M in the Ca²⁺-containing medium or in a Ca²⁺-free medium containing 10^–3 M EGTA. Okadaic acid 10^–6 M used as 30 min pretreatment versus OA 10^–5 M and 2 × 10^–5 M behaved like caffeine or theophylline.These results suggest that the OA-induced contraction of the rat uterine smooth muscle is partly effected by transmembrane calcium movements which can be inhibited in an O-Ca²⁺–10^–3 M EGTA solution or by theophylline or caffeine. This contraction also involves mobilization of Ca²⁺ from an intracellular pool which is also xanthine-sensitive. The latter effect seems to be important in inducing the contractile effect. This study does not exclude the possibility of other mechanisms being involved in the contraction induced by OA. Correspondence to: M. L. Candenas at the above address in Burjassot-València     

Lack of effect of opioid peptides,morphine and naloxone on superoxide formation in human neutrophils and HL-60 leukemic cells

Summary There are controversial reports in the literature concerning the effects of opioids on superoxide (O ₂ ^– ) formation in phagocytes, these agents being either inhibitory or stimulatory. We re-examined this issue and compared the effects of the Chemotactic peptide, N-formyl-l,-methionyl-l-leucyl-l-phenylalanine (fMet-Leu-Phe), phorbol myristate acetate (PMA), ATP, platelet activating factor (PAF), cytochalasin B (CB) and prostaglandin E₁ (PGE₁) with those of various opioids on O ₂ ^– formation in human neutrophils and HL-60 leukemic cells under defined experimental conditions. In the presence of CB, fMet-Leu-Phe and PAF concentration-dependently activated O ₂ ^– formation in neutrophils with EC₅₀ values of 20 nM and 100 nM, respectively. In the absence of CB, fMet-Leu-Phe and PAF were much less effective. PAF synergistically enhanced O ₂ ^– formation induced by fMet-Leu-Phe. ATP at a concentration of 100 M and the opioids, methionine enkephalin, -endorphin, dynorphin, [d-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin, [d-Ala²-d-Leu⁵]-enkephalin and morphine at concentrations between 10 pM to 1 M did not activate O ₂ ^– formation. ATP but not \-endorphin potentiated fMet-Leu-Phe-induced O ₂ ^– formation. O ₂ ^– formation induced by a maximally stimulatory concentration of PMA (100 ng/ml) was enhanced by fMet-Leu-Phe but was unaffected by methionine enkephalin or PGE₁. PMA at a non-stimulatory concentration (2 ng/ml) potentiated the effect of fMet-Leu-Phe but did not induce responsiveness to PAF, ATP or -endorphin. PGE₁ strongly inhibited fMet-Leu-Phe-induced O ₂ ^– formation, whereas morphine, methionine enkephalin and the opioid antagonist, naloxone, were without effect. In HL-60 cells differentiated with dibutyryl cAMP, fMet-Leu-Phe, PAF and ATP but not -endorphin activated O ₂ ^– formation. Our results show that O ₂ ^– formation is differentially regulated by various classes of intercellular signal molecules and that opioids do not play a role in the regulation of O ₂ ^– formation. The precise definition of the experimental conditions and control experiments with established modulators of O ₂ ^– formation are essential to evaluate the role of opioids in the regulation of this effector system.Send offprint requests to R. Seifert at the above address     

Role of adenosine in functional hyperemia in skeletal muscle as indicated by pharmacological tools

Summary The hypothesis that adenosine mediates blood flow increments in contracting skeletal muscle was evaluated by intravital microscopy of the microcirculation in the tenuissimus muscle of anesthetized rabbits. Motor nerve stimulation elicited muscle contractions and frequency-dependent arteriolar dilatation, particularly in terminal arterioles. The pulse duration (0.05 ms) and voltage (1.5–5 V) precluded activation of vasoconstrictor fibers, as also indicated by the lack of effect of phentolamine on resting vascular tone and on the hyperemic response to nerve stimulation. The specific adenosine receptor antagonist, 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX; 10^–5 M), attenuated the hyperemic response to muscle contractions. The adenosine uptake inhibitor dipyridamole (10^–8–10^–6 M) dose-dependently dilated microvessels, an effect prevented by DPSPX (10^–5 M). Moreover, dipyridamole (10^–7 M) augmented contraction-induced hyperemia. The enhancement by dipyridamole was reversed by DPSPX (10^–5 M). The effects of adenosine uptake inhibitor and antagonist were invariably more marked in terminal than in transverse arterioles, and also more pronounced at higher stimulation frequencies. Motor nerve stimulation failed to induce alterations in vascular diameters when the neuromuscular junction was blocked by pancuronium. Thus, our observations indicate that functional hyperemia after motor nerve-induced contractions of the skeletal muscle was of postjunctional origin. Apparently, activation of adenosine receptors was responsible for a part of the evoked vasodilation. Send offprint requests to M. G. Persson at the above address