Notes: 1. The role of calcium-activated potassium (KCa) channels in bronchodilation produced by a direct adenylyl cyclase activator, forskolin, was investigated. The involvement of intracellular cyclic AMP (cAMP) in the process was also examined.2. The isometric tension records from guinea-pig tracheal smooth muscles indicated that application of charybdotoxin (ChTX), a selective inhibitor of large conductance KCa channels, led to a suppression of the relaxant effect of forskolin in the precontracted tissue by carbachol (CCh). However, the inhibitory action by ChTX had a much greater effect on the relaxation caused by isoproterenol than by forskolin.3. In contrast to the effect of ChTX, glybenclamide, a cromakalim-sensitive K+ channel inhibitor and apamin, a small conductance KCa channel blocker, had no effects on the bronchodilation produced by forskolin.4. The effects of forskolin and nifedipine on tone produced by high K+ was compared. Concentration-inhibition curves in guinea-pig trachealis precontracted by 20 mmol/L K+ solution were similar for forskolin and nifedipine. Conversely, relaxation by forskolin was significantly diminished when tissues were contracted with 40 mmol/ L K+ solution, whereas nifedipine relaxations were unaffected.5. A single channel record from a cell-attached patch in a porcine tracheal myocyte demonstrated that forskolin stimulates reversibly KCa channels without affecting the unitary amplitude.6. The results are consistent with forskolin-induced relaxation occurring at least in part through the opening of ChTX-sensitive KCa channels, by means of a cAMP-dependent channel modulation. The lesser effect of ChTX on forskolin compared with isoproterenol-induced relaxations suggests a tighter coupling between relaxation and channel opening by β-adrenergic receptor stimulation, and is consistent with cAMP-independent mechanisms of receptor-channel coupling.

Notes: 1. For the purpose of clarifying the mechanism of the airways smooth muscle relaxant action of xanthines, cyclic guanosine monophosphate (GMP) phosphodiesterase (PDE) from guinea-pig trachealis muscle was purified with diethylaminoethyl ether (DEAE) cellulose column chromatography.2. Five 3-alkylxanthines (3-methylxanthine, 3-ethylxanthine, 3-n-propylxanthine (enprofylline), 3-n-butylxanthine, and 3-iso-butylxanthine), and five 1-methyl-3-alkylxanthines (1-methyl-3-methyl-xanthine (theophylline), 1-methyl-3-ethylxanthine, 1-methyl-3-n-propylxanthine, 1-methyl-3-n-butyl-xanthine, and 1-methyl-3-iso-butylxanthine (IBMX)) were compared in terms of purified cyclic GMP PDE inhibition. The relationship between the structure and inhibition of cyclic GMP PDE was studied.3. The –log EC50 values for relaxation of spontaneous tone of isolated guinea-pig trachealis preparations by the 3-alkylxanthines and 1-methyl-3-alkylxanthines were determined.4. The five 1-methyl-3-alkylxanthines were each more potent in relaxing isolated trachealis smooth muscle than the corresponding 3-alkylxanthines. The 1-methyl-3-alkylxanthines were also more potent than the corresponding 3-alkylxanthines in their cyclic GMP PDE inhibitory effect. There was a strong positive correlation between the concentration of inhibitor which inhibited hydrolysis by 50% (IC50) values for cyclic GMP PDE inhibition by the xanthine derivatives and their EC50 values for trachealis muscle relaxation.5. It is suggested that the mechanism by which xanthine derivatives relax trachealis smooth muscle involves inhibition of cyclic GMP PDE in addition to inhibition of cyclic adenosine monophosphate PDE.

Notes: 1. The time course of recovery of reduced β-adrenoceptors caused by ovalbumin (OA) challenge was investigated using guinea-pigs.2. The effects of prednisolone on the recovery time course were also evaluated.3. β- and α1-receptor assays were performed using lung membranes. Adenylate cyclase activity was also measured.4. OA challenge reduced the number of β-adrenoceptors by 35%, and a significant decrease (13%) persisted for 7 days. The number of β-adrenoceptors recovered after 14 days.5. OA challenge elevated the number of α1-adrenoceptors. A significant increase (24%) was observed after 7 days, and it took a further 7 days for the recovery.6. After OA challenge there was a significant decrease in adenylate cyclase activity after 7 days, which recovered after a further 7 days.7. Inhalation of prednisolone accelerated the recovery of β-adrenergic responsiveness, though it did not affect the recovery of the number of α1-adrenoceptors. Prednisolone inhalation also elevated β-adrenergic responsiveness in non-asthmatic subjects.8. It is concluded that reduced β-adrenergic responsiveness caused by OA challenge persisted for 7 days and recovered after a further 7 days. Steroid hormone increased β-adrenoceptors.

Notes: 1. In order to examine the mechanisms of cGMP-induced relaxation in airway smooth muscle, the effects of atrial natriuretic peptide (ANP) and 8-brom cGMP on muscle tone were studied by measuring isometric tension, while the effects on cytosolic Ca2+ concentrations were studied by measuring the spectra of fura-2 loaded in guinea-pig tracheal strips.2. Atrial natriuretic peptide and 8-brom cGMP caused a concentration-dependent inhibition of spontaneous tone in the guinea-pig trachea. The relaxant effects of these agents on spontaneous tone were markedly suppressed in the presence of iberiotoxin (IbTX), a selective inhibitor of large-conductance Ca2+-activated K+ (BKca) channels. Iberiotoxin (30 nmol/L) markedly affected the maximal effect induced by ANP and 8-brom cGMP and augmented EC70 values for ANP and EC50values for 8-brom cGMP approximately 27- and 17-fold, respectively. The inhibitory effects of IbTX on relaxation induced by these agents were diminished in the presence of 1 μmol/L nifedipine, an antagonist of voltage-operated Ca2+channels (VOCC).3. The inhibitory action of ANP and 8-brom cGMP on spontaneous tone was not affected by the presence of 10 μmol/L glibenclamide, an inhibitor of ATP-sensitive K+ channels, and 100 nmol/L apamin, an inhibitor of small-conductance Ca2+-activated K+ channels. When these agents were applied to tissues precontracted by high (40mmol/L) K+, the relaxant effects of these agents markedly diminished.4. The extracellular Ca2+-dependent contraction was inhibited in the presence of 0.3 μmoI/L ANP or 0.1 mmol/L 8-brom cGMP. Concentration—response curves to extracellular Ca2+ (0.03—2.4 mmol/L) were markedly diminished by exposure to these agents. The maximal effect induced by extracellular Ca2+ was affected by these agents.5. Atrial natriuretic peptide caused an inhibition of spontaneous tone accompanied by a reduction in the intracellular Ca2+ concentration. In the presence of IbTX, the elimination of both muscle tone and cytosolic Ca2+ by ANP was suppressed.6. We conclude that ANP and 8-brom cGMP activate BKca channels and that the inhibition of Ca2+ influx through VOCC, mediated by BKca channel activation, may be involved in cGMP-dependent bronchodilation.

Notes: 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).

Notes: 1. To determine the inhibitory effects of agents that pass through and bypass β-adrenoceptors under conditions of tolerance to β-adrenoceptor agonists, we examined the inhibition by the β-adrenoceptor agonists forskolin and theophylline against contraction induced by methacholine (MCh) after exposure to higher concentrations of a β-adrenoceptor agonist for a long time in guinea-pig tracheal smooth muscle, using isometric tension records.2. After exposure to procaterol (0.0003–3 μmol/L) for 45 min, the inhibitory effect of 0.03 μmol/L procaterol on 1 μmol/L MCh-induced contraction was attenuated in a concentration-dependent manner, whereas after exposure to isoprenaline (0.0003–3 μmol/L) for an equivalent time, the inhibitory effect of isoprenaline was markedly attenuated at each concentration. However, after exposure to 3 μmol/L procaterol for 45 min, the inhibitory actions of forskolin and theophylline were, conversely, augmented.3. These phenomena were observed under conditions whereby the response to MCh returned to control levels 6 h after removal of 3 μmol/L procaterol. The percentage inhibition produced by 0.1 μmol/L forskolin against 1 μmol/L MCh after exposure to normal bathing solution or 3 μmol/L procaterol for 45 min was 9.8 ± 5.5 and 82.8 ± 6.5%, respectively (P 〈 0.001). These values for 100 μmol/L (18 μg/mL) theophylline on MCh resposnes were 9.9 ± 8.5 and 88.0 ± 4.4% (n = 6 for both), respectively (P 〈 0.001).4. The inhibitory action of agents that bypass β-adrenoceptors was markedly augmented under conditions of β-adrenoceptor desensitization in airway smooth muscle.5. In conclusion, procaterol is less potent in causing desensitization of β-adrenoceptors than isoprenaline. The activity of adenylyl cyclase may be enhanced after exposure to a high concentration of β-adrenoceptor agonists.

Notes: 1. The combined effects of the macrolide antibiotics erythromycin, josamycin, clarithromycin and YM17K (3,4′-dideoxy mycaminosyl tylonolide hydrochloride) on in vitro intracellular accumulation of vinblastine or cyclosporine (Cs)A and on the in vivo antitumour activity of vinblastine were investigated using mouse leukaemia P388 cells (P388/S) and anticancer drug-resistant (P388/ADR) cells. These effects were compared with those of a calcium antagonist (verapamil) or immunosuppressants (FK506 and CsA).2. All tested macrolide antibiotics increased the accumulation of both vinblastine and CsA in P388/ADR cells in a dose- dependent manner, but their potency was lower than that of verapamil, CsA or FK506.3. When vinblastine (200 μg/kg) was administered intraperitoneally with each of the macrolide antibiotics (10 or 100 mg/kg) or with verapamil (25 mg/kg) once a day for 10 days in P388/ADR-bearing mice, combined effects of vinblastine with the macrolide antibiotics (erythromycin, clarithromycin and YM17K) or verapamil were observed.4. The present study suggests that macrolide antibiotics may overcome anticancer drug resistance by inhibiting the binding of vinblastine or CsA to P-glycoprotein in P388/ADR cells.5. We believe that these results are encouraging for combination chemotherapy to overcome P-glycoprotein-dependent anticancer drug-resistant tumours in clinical practice.

Notes: 1. To clarify the role of platelet-activating factor (PAF) in the development of adult respiratory distress syndrome (ARDS), we performed bronchoalveolar lavage (BAL) in 19 patients with ARDS and examined cell populations, albumin concentrations and PAF levels. PAF levels were measured by a newly developed radioimmunoassay.2. In the BAL fluid of ARDS patients, neutrophil percentages and albumin concentrations markedly increased compared with control subjects.3. PAF was detected in 14 of 19 patients with ARDS, whereas it did not exist in the control subjects.4. Furthermore, we investigated the priming effect of recombinant human tumour necrosis factor-α (TNFα), which is known to be one of the most important mediators in the development of ARDS, on PAF production induced by the calcium ionophore in neutrophils.5. Pre-incubation with TNFα dose-dependently increased both extracellular and intracellular PAF production in neutrophils.6. These results suggest that PAF might play an important role in the development of ARDS.