The effects of phorbol 12,13-diacetate on responses of guinea-pig isolated trachea to methylxanthines, isoprenaline and ryanodine.

1. Using guinea-pig isolated trachea, we have studied how phorbol 12,13-diacetate (PDA) modulates mechanical responses of the tissue to methylxanthines, isoprenaline and ryanodine. 2. Caffeine (10 microM-5 mM), theophylline (10 microM-5 mM) and isoprenaline (1 nM-1 microM), each inhibited the spontaneous tone of the trachea. Pretreatment with PDA (0.1-10 microM) converted relaxant responses to high concentrations of the methylxanthines into contractions. PDA produced no equivalent effect against isoprenaline. Pretreatment with verapamil (1 or 10 microM), nifedipine (0.1 microM) or incubation with Ca(2+)-free, EGTA (0.1 mM)-containing physiological salt solution (PSS) suppressed the contraction produced by caffeine or theophylline in PDA (5 microM)-treated tissues. 3. The ability of PDA (5 microM) to convert caffeine-induced relaxation into caffeine-induced contraction was retained in tissues pretreated with a combination of atropine (1 microM) and mepyramine (1 microM) and in tissues denuded of the airway epithelium. 4. Caffeine (10 microM-5 mM), theophylline (10 microM-5 mM) and isoprenaline (1 nM-1 microM), each relaxed trachea contracted with histamine (0.1 mM). The relaxation induced by caffeine, theophylline and isoprenaline was markedly reduced in the presence of PDA (5 microM) and the responses to high concentrations of caffeine and theophylline, but not those to isoprenaline, were reversed to contractions. Verapamil (10 microM) prevented the effects of PDA against caffeine- or theophylline-induced relaxation. 5. PDA (1 microM) enhanced the tracheal spasm produced by caffeine (10 mM) and theophylline (10 mM) in indomethacin (2.8 microM)-treated trachea maintained at 20 degrees C. This enhancement was reduced in the presence of verapamil (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Relaxant effects of carvacrol on guinea pig tracheal chains and its possible mechanisms

The bronchodilatory effects of three cumulative volumes (0.02, 0.04, and 0.08 ml) of 1/100 diluted carvacrol were examined by their relaxant effects on tracheal chains of guinea pigs precontracted by 60 mM KCl (group 1) and 10 microM methacholine in two different conditions including: non-incubated tissues (group 2) and incubated tissues with 1 microM propranolol and 1 microM chlorpheniramine (group 3), (for each group, n = 5). The relaxant effects of all three volumes of carvacrol were significantly higher than those of ethanol and even theophylline in all three groups of experiments (p < 0.05 to p < 0.001). There were positive correlations between the relaxant effect of carvacrol and incremental volume (p < 0.05 to p < 0.001). These results indicated that carvacrol has a potent relaxant effect on tracheal chains of guinea pigs which is not due to beta2-adrenergic stimulatory, histamine H1, and muscarinic blocking effect.     

Effect of histamine and histamine analogues on human isolated myometrial strips.

1. The effect of histamine and histamine H1- and H2-receptor agonists on isolated myometrium strips of premenopausal women has been examined. The effect of acetylcholine was also determined. 2. Histamine, 2-pyridylethylamine, 4-methylhistamine and acetylcholine, but not dimaprit, produced a concentration-related contractile response in human isolated myometrial strips. Histamine also produced a further contraction in human isolated myometrial strips precontracted with KCl (55 mM). 3. The contractile response to histamine was antagonized by the histamine H1-receptor antagonist, clemizole (0.1 microM) but was potentiated by the histamine H2-receptor antagonist, ranitidine (10 microM). Clemizole (0.1 nM to 10 nM) competitively antagonized the contractile effect of 2-pyridylethylamine (- log KB = 10.5 +/- 0.5). The concentration-response curve for acetylcholine was displaced to the right by atropine 0.1 microM. 4. Atropine (0.1 microM), propranolol (0.1 microM), prazosin (0.1 microM) and indomethacin (1 microM) failed to modify the contractile response to histamine. 5. In human isolated myometrial strips precontracted with KCl (55 mM), clemizole at 1 microM completely abolished the contractile response to histamine and revealed a concentration-dependent relaxation. Dimaprit alone and 4-methylhistamine (in the presence of clemizole), produced concentration-related relaxation with a magnitude similar to that in response to histamine. The relaxant response to dimaprit was antagonized by ranitidine. 6. It is concluded that human isolated uterine strips possess histamine H1- and H2-receptors: the former mediating contraction and the latter relaxation. The predominant response to histamine in this tissue is contraction.     

Possible mechanism(s) for relaxant effects of Foeniculum vulgare on guinea pig tracheal chains

In a previous study the relaxant (bronchodilatory) effect of Foeniculum vulgare on isolated guinea pig tracheal chains was demonstrated. To study mechanisms responsible for this effect the present study evaluated the inhibitory effect of this plant on contracted tracheal chains of guinea pig. The relaxant effects of aqueous and ethanol extracts and an essential oil from Foeniculum vulgare were compared to negative controls (saline for aqueous extract and essential oil and ethanol for ethanol extract) and a positive control (diltiazem) using isolated tracheal chains of the guinea pig precontracted by 10 microM methacholine (group 1) and 60 mM KCl (group 2, n = 7 for each group). In the group 1, experiments diltiazem, ethanol extract, and essential oil from Foeniculum vulgare showed a significant relaxant effect on methacholine induced contraction of tracheal chains compared to those of negative controls (p < 0.05 to p < 0.001). In addition the effect of the ethanol extract was significantly greater than that of diltiazem (p < 0.001). However, the aqueous extract did not show any relaxant effect in group 1. In the group 2 experiments, only diltiazem showed a significant relaxant effect on KCl induced contraction of tracheal chains (p < 0.001). The relaxant effects of ethanol extracts and essential oil obtained in the group 2 experiments were significantly lower than those in group 1 (p < 0.05 to p < 0.001). These results confirm the bronchodilatory effects of ethanol extract and essential oil from Foeniculum vulgare. However with regard to the effect of KCl on calcium channels, the results indicated that the inhibitory effect of ethanol extracts and essential oil from Foeniculum vulgare on calcium channels is not contributing to their relaxant (bronchodilatory) effects on guinea pig tracheal chains. However the results suggest a potassium channel opening effect for this plant, which may contribute on its relaxant effect on guinea pig tracheal chains.     

Electrical and mechanical effects of BRL34915 in guinea-pig isolated trachealis.

BRL34915 (0.1-10 microM) suppressed the spontaneous tone of guinea-pig isolated trachealis in a concentration-dependent manner. BRL34915 was not antagonized by propranolol (1 microM). In trachea where spontaneous tone was suppressed by indomethacin (2.8 microM) but subsequently restored to the same level with acetylcholine or histamine, the relaxant potency of BRL34915 was reduced. In Krebs solution containing K+ (120 mM), isolated trachealis muscle developed near-maximal tension. The relaxant effects of BRL34915 were virtually abolished in this medium. Concentration-effect curves for KCl, acetylcholine and histamine were constructed in tissues treated with indomethacin (2.8 microM). BRL34915 (10 microM) depressed the foot of the concentration-effect curve for KCl and caused minor rightward shifts in the concentration-effect curves of acetylcholine and histamine. Four K+-channel inhibitors were tested. Apamin (0.1 microM) did not modify the action of BRL34915. Tetraethylammonium (8 mM) had little effect but procaine (5 mM) and 4-aminopyridine (5 mM) each significantly inhibited the relaxant action of BRL34915. Intracellular electrophysiological recording showed that BRL34915 (0.1 microM) caused very minor relaxation and little, if any, electrical change. Higher concentrations (1-10 microM) evoked relaxation, suppression of spontaneous electrical slow waves and marked hyperpolarization of the trachealis cells. In the presence of TEA (8 mM) or procaine (5 mM) the hyperpolarization induced by BRL34915 was significantly reduced. In trachealis skinned of its plasma membranes, tension development induced by Ca2+ (20 microM) was unaffected either by BRL34915 (10 microM) or by nicorandil (1 mM). In studies of the efflux of 86Rb+ from muscle-rich strips of trachea, BRL34915 (1 and 10 microM) increased the efflux rate constant. It is concluded that BRL34915 evokes relaxation of the trachealis by a mechanism that involves neither beta-adrenoceptor activation nor direct reduction of the sensitivity of the intracellular contractile machinery to cytosolic free Ca2+. The action of BRL34915 may depend on the opening of K+ channels in the plasma membrane which are permeable to 86Rb+. The opening of these channels, or the effects of their opening, may be reduced by K+-channel inhibitors such as 4-aminopyridine, procaine and TEA but not by apamin.     

Evidence that the histamine sensitivity and responsiveness of guinea-pig isolated trachea are modulated by epithelial prostaglandin E2 production.

1. Guinea-pig isolated tracheal preparations in which the epithelium had been removed exhibited a greater contractile response to histamine (intact: 1.91 +/- 0.12 g; n = 6 and rubbed: 2.76 +/- 0.15 g; n = 11; P less than 0.001). The histamine sensitivity (pD2 value) of these preparations was also significantly greater (intact: 4.80 +/- 0.04 and rubbed: 5.40 +/- 0.08; P less than 0.01). 2. Indomethacin suppressed the basal tone of both intact and rubbed preparations but was more effective in the former tissues (intact: -0.70 +/- 0.14 g; n = 22 and rubbed: -0.17 +/- 0.05 g; n = 12; P less than 0.02). 3. Arachidonic acid (AA; 10 microM) suppressed the basal tone of intact tissues but contracted such preparations following indomethacin treatment (1.7 microM; 30 min). However, in rubbed tissues AA (10 microM) induced a contraction which was attenuated following indomethacin treatment. 4. Prostaglandin E2 (PGE2; 0.01 and 0.1 microM) suppressed the basal tone of intact preparations and always evoked contraction of rubbed tissues. Following indomethacin treatment PGE2 (0.01 and 0.1 microM) generally evoked spasm of intact and rubbed tissues while at higher concentrations (1 microM) relaxant effects were observed. 5. Removal of the epithelium did not alter the relaxant effect of PGE2 (pD2 value) on histamine (50 microM)-contracted tissues (intact: 6.86 +/- 0.08 and rubbed: 7.10 +/- 0.3; n = 4; P greater than 0.1). 6. In rubbed preparations treated with indomethacin, PGE2 (0.01 and 0.1 microM) evoked spasm. However, when added to preparations contracted with 5 microM histamine, PGE2 always caused relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Ca(2+) channel blocker-like effect of dehydrocurdione on rodent intestinal and vascular smooth muscle

Effects of dehydrocurdione, a zedoary-derived sesquiterpene, on smooth muscle were investigated by recording the mechanical activity of intestines and aorta from guinea pigs and rats. Dehydrocurdione (0.1-3 mM) induced a sustained relaxation of rat duodenum and inhibited spontaneous motility. Dehydrocurdione (0.1-1 mM) inhibited the contractile response of guinea pig ileum induced by acetylcholine (0.01-10 microM), histamine (0.03-10 microM) and substance P (0.1-30 nM) in a non-competitive manner. Acetylcholine (0.5 microM) elicited a transient contraction followed by a sustained contraction of guinea pig ileum, and dehydrocurdione pretreatment inhibited the sustained component, which depends on Ca(2+) entry from the extracellular space. The high K(+)-induced contraction of rat aortic ring is reported to be blocked by Ca(2+) channel blockers, while the norepinephrine-induced contraction includes a Ca(2+) channel blocker-resistant component. Dehydrocurdione (1 mM) blocked the high K(+) (60 mM)-induced contraction of rat aortic ring by 81%, while it inhibited the norepinephrine (1 microM)-induced contraction by only 28%. Dehydrocurdione (1 mM) significantly reduced the high K(+)-stimulated increase in cytosolic Ca(2+) level of Fura-2-loaded mesenteric artery from rats. These results suggest that the inhibitory effects of dehydrocurdione on intestinal and vascular smooth muscle are mediated by blockade of Ca(2+) entry from the extracellular space.     

Modulation of agonist-induced phosphoinositide metabolism, Ca2+ signalling and contraction of airway smooth muscle by cyclic AMP-dependent mechanisms.

1. The effects of increased cellular cyclic AMP levels induced by isoprenaline, forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cyclic AMP) on phosphoinositide metabolism and changes in intracellular Ca2+ elicited by methacholine and histamine were examined in bovine isolated tracheal smooth muscle (BTSM) cells. 2. Isoprenaline (pD2 (-log10 EC50) = 6.32 +/- 0.24) and forskolin (pD2 = 5.6 +/- 0.05) enhanced cyclic AMP levels in a concentration-dependent fashion in these cells, while methacholine (pD2 = 5.64 +/- 0.12) and histamine (pD2 = 4.90 +/- 0.04) caused a concentration-related increase in [3H]-inositol phosphates (IP) accumulation in the presence of 10 mM LiCl. 3. Preincubation of the cells (5 min, 37 degrees C) with isoprenaline (1 microM), forskolin (10 microM) and 8-Br-cyclic AMP (1 mM) did not affect the IP accumulation induced by methacholine, but significantly reduced the maximal IP production by histamine (1 mM). However, the effect of isoprenaline was small (15.0 +/- 0.6% inhibition) and insignificant at histamine concentrations between 0.1 and 100 microM. 4. Both methacholine and histamine induced a fast (max. in 0.5-2 s) and transient increase of intracellular Ca2+ concentration ([Ca2+]i) followed by a sustained phase lasting several minutes. EGTA (5 mM) attenuated the sustained phase, indicating that this phase depends on extracellular Ca2+. 5. Preincubation of the cells (5 min, 37 degrees C) with isoprenaline (1 microM), forskolin (10 microM) and 8-Br-cyclic AMP (1 microM) significantly attenuated both the Ca(2+)-transient and the sustained phase generated at equipotent IP producing concentrations of 1 microM methacholine and 100 microM histamine (approx. 40% of maximal methacholine-induced IP response), but did not affect changes in [Ca2+]i induced by 100 microM methacholine (95.2 +/- 3.5% of maximal methacholine-induced IP response). 6. Significant correlations were found between the isoprenaline-induced inhibition of BTSM contraction and inhibition of Ca2+ mobilization or influx induced by methacholine and histamine, that were similar for each contractile agonist. 7. These data indicate that (a) cyclic AMP-dependent inhibition of Ca2+ mobilization in BTSM cells is not primarily caused by attenuation of IP production, suggesting that cyclic AMP induced protein kinase A (PKA) activation is effective at a different level in the [Ca2+]i homeostasis, (b) that attenuation of intracellular Ca2+ concentration plays a major role in beta-adrenoceptor-mediated relaxation of methacholine- and histamine-induced airway smooth muscle contraction, and (c) that the relative resistance of the muscarinic agonist-induced contraction to beta-adrenoceptor agonists, especially at (supra) maximal contractile concentrations is largely determined by its higher potency in inducing intracellular Ca2+ changes.     

Terpinen-4-ol: mechanisms of relaxation on rabbit duodenum

The effect of terpinen-4-ol was studied on rabbit duodenum in-vitro. Terpinen-4-ol induced relaxation of the basal tonus (IC50 170.2 (95% confidence interval, 175-204) microM) with a maximal relaxant response of 180.4+/-3.9% (n=6) of the contraction induced by 60 mM [K(+)]. The maximal relaxation induced in control conditions was not affected (P>0.05) by pretreatment of the tissues with phentolamine (50 microM) or propranolol (10 microM), N(G) nitro-L-arginine methyl ester (L-NAME; 1 mM), 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 100 microM), hexamethonium (1 mM), tetrodotoxin (1 microM), the mixture charybdotoxin-apamin (1 microM), glibenclamide (10 microM), 4-aminopyridine (10 microM) or tetraethyl-ammonium (100 microM). In addition, terpinen-4-ol completely relaxed tissues precontracted with 60 mM [K(+)] solutions (IC50 325.9 (245.1-433.1) microM) and also blocked (IC50 154.7 (117.7-191.7) microM) the phasic component of this contraction. At a concentration of 195 and 650 muM it reduced by 41.3+/-3.4% and 75.4+/-3.1%, respectively the maximal contractile response to Ca(2+) in depolarized duodenum. Terpinen-4-ol completely blocked the component of carbachol-induced contraction, which was resistant to nifedipine (100 microM) pretreatment or to a Ca(2+)-free solution. These data show that terpinen-4-ol relaxes intestinal smooth muscle and suggest that this effect is myogenic in nature and depends on calcium antagonism.     

Role of K+ channels in N-acetylprocainamide-induced relaxation of bovine tracheal smooth muscle

We examined the relaxant effects of N-acetylprocainamide, the major hepatic metabolite of procainamide, on bovine tracheal smooth muscle, focusing on the possible involvement of K+ channels. N-acetylprocainamide produced a concentration-dependent and full inhibition of the tension development elicited by methacholine (0.3 or 1 microM). The potency of N-acetylprocainamide in diminishing methacholine-elicited tension development was one-half of that of procainamide. By comparison, N-acetylprocainamide inhibited high-K+ (40 mM)-induced contraction more potently than procainamide though both inhibitions were largely reduced when compared to those against methacholine-induced contraction. Iberiotoxin (30 nM), Ba(2+) (1 mM) or a combination of both agents significantly attenuated the relaxant effect of N-acetylprocainamide on methacholine-induced contraction, whereas apamin (100 nM), 4-aminopyridine (300 microM), and glibenclamide (10 microM) did not affect it. These results suggest that N-acetylprocainamide, similar to procainamide, elicits tracheal smooth muscle relaxation mainly through the activation of plasma membrane K+ channels.     

Effect of nifedipine on constriction of human tracheal strips in vitro

1 Autopsy specimens of human trachealis muscle were used to investigate the effect of the calcium channel blocker, nifedipine, on airway smooth muscle constriction. With these tracheal strips, two consecutive cumulative concentration-effect curves to histamine (0.1-100 microM) obtained at a 60 min interval were highly reproducible. 2 We examined the effects of adding nifedipine (2.9 microM) to the incubation medium before the second histamine response. The concentration-effect relationships determined after nifedipine incubation were significantly different from control: the response to 100 microM histamine was reduced by approximately one-half (to 2.53 +/- 0.6 g; P less than 0.025), and the concentration of histamine causing 40% of the maximal control contraction (EC40) increased nearly ten fold (to 36.9 +/- 10.5 microM; P less than 0.02). 3 In two additional tracheal strips submaximally constricted with 10 microM histamine, nifedipine 2.9 microM caused complete relaxation to resting tension or below. 4 These results indicate a direct inhibitory effect of nifedipine on airway smooth muscle constriction and partial dependence of human trachealis muscle on calcium ion fluxes for initiation and maintenance of contraction. In addition, the results suggest a potential mechanism for the inhibitory effects of calcium channel blocking drugs in exercise-induced asthma.     

Further analysis of the mechanisms underlying the tracheal relaxant action of SCA40.

1. SCA40 (1nM-10 microM), isoprenaline (1-300 nM) and levcromakalim (100 nM-10 microM) each produced concentration-dependent suppression of the spontaneous tone of guinea-pig isolated trachea. Propranolol (1 microM) markedly (approximately 150 fold) antagonized isoprenaline but did not antagonize SCA40. The tracheal relaxant action of SCA40 was unaffected by suramin (100 microM) or 8-(p)-sulphophenyltheophylline (8-SPT; 140 microM). 2. An isosmolar, K(+)-rich (80 mM) Krebs solution increased tracheal tone, antagonized SCA40 (approximately 60 fold), antagonized isoprenaline (approximately 20 fold) and very profoundly depressed the log concentration-effect curve for levcromakalim. Nifedipine (1 microM) did not itself modify the relaxant actions of SCA40, isoprenaline or levcromakalim. However, nifedipine prevented the rise in tissue tone and the antagonism of SCA40 and isoprenaline induced by the K(+)-rich medium. In contrast, nifedipine did not prevent the equivalent antagonism of levcromakalim. 3. Charybdotoxin (100 nM) increased tracheal tone, antagonized SCA40 (approximately 4 fold) and antagonized isoprenaline (approximately 3 fold). Nifedipine (1 microM) prevented the rise in tissue tone and the antagonism of SCA40 and isoprenaline induced by charybdotoxin. 4. Quinine (30 microM) caused little or no change in tissue tone and did not modify the relaxant action of isoprenaline. However, quinine antagonized SCA40 (approximately 2 fold). Nifedipine (1 microM) prevented the antagonism of SCA40 induced by quinine. 5. Tested on spontaneously-beating guinea-pig isolated atria SCA40 (1 nM-10 microM) increased the rate of beating in a concentration-dependent manner. Over the concentration-range 1 microM-10 microM, SCA40 also caused an increase in the force of atrial contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the relaxant effects of AH 21-132 in guinea-pig isolated trachealis.

1. Experiments have been performed with the dual intent of analysing the mechanism by which AH 21-132 relaxes airways smooth muscle and determining whether the effects of this compound can be distinguished from those of theophylline. 2. AH 21-132 (0.25-8 microM) and theophylline (1-1000 microM) each caused concentration-dependent suppression of the spontaneous tone of guinea-pig isolated trachealis. The maximal effect of AH 21-132 was equivalent to that of theophylline. No evidence was obtained that the tissue became sensitized or desensitized to the action of AH 21-132. 3. Propranolol (1 microM) profoundly antagonized the tracheal relaxant action of isoprenaline but not that of AH 21-132. 4. In indomethacin (2.8 microM)-treated tissues, tone was induced by K+-rich (120 mM) Krebs solution, acetylcholine (ACh, 1 mM) or histamine (200 microM). Log concentration-relaxation curves for AH 21-132, isoprenaline and theophylline were all moved to the right in the presence of the spasmogens, the smallest rightward shift being induced by histamine and the greatest by ACh. While maximal effects of AH 21-132 and theophylline were unaffected by the spasmogens, that of isoprenaline was reduced by KCl and ACh. 5. In tissues treated with indomethacin (2.8 microM), AH 21-132 (0.1-100 microM) inhibited the spasmogenic effects of potassium chloride (KCl), ACh and histamine in a concentration-dependent manner. The inhibition was characterized by rightward shifts in the spasmogen concentration-effect curves with depression of their maxima.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that tachykinins relax the guinea-pig trachea via nitric oxide release and by stimulation of a septide-insensitive NK1 receptor.

1. This study investigated the possibility that tachykinins relax the guinea-pig isolated trachea by releasing nitric oxide (NO) from the epithelium. The types of tachykinin receptor mediating both relaxation and contraction of the trachea were also studied. Isometric tension was recorded in isolated tracheal tube preparations precontracted with acetylcholine (10 microM) in which compounds were administered intraluminally in the presence of phosphoramidon and indomethacin (both 1 microM) and the tachykinin NK2 receptor antagonist, SR 48,968 ((S)-N-methyl-N[4-(4-acetyl amino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide), 0.1 microM). 2. In the presence of the inactive enantiomer of an NO-synthase inhibitor, NG-monomethyl-D-arginine (D-NMMA, 100 microM), substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and the selective NK1 receptor agonist, [Sar9, Met(O2)11]-SP, (0.1-10 nM) relaxed tracheal tube preparations. This relaxation was changed into a contraction by pretreatment with the NO-synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA, 100 microM). The effect of L-NMMA on SP- and [Sar9, Met(O2)11]-SP-induced responses was reversed by L-arginine (L-Arg, 1 mM), but not by D-Arg (1 mM). After removal of the epithelium SP, NKA and NKB and [Sar9, Met(O2)11]-SP (0.1-10 nM) evoked contractile responses in the presence of either L-NMMA (100 microM) or D-NMMA (100 microM). The effects of SP and [Sar9, Met(O2)11]-SP obtained in the presence of another NO-synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) or its inactive enantiomer, NG-nitro-D-arginine methyl ester (D-NAME, 100 microM) were similar to those observed with L-NMMA or D-NMMA, respectively. 3. The selective NK1 receptor agonist, [pGlu6, Pro9]-SP(6-11) (septide, 0.1-10 nM) evoked contractile responses of tracheal tube preparations in the presence of either D-NMMA (100 microM) or L-NMMA (100 microM). The log concentration-response curve to septide obtained in the presence of L-NMMA was similar to that obtained in the presence of D-NMMA. [Sar9, Met(O2)11]-SP (0.1-10 nM) relaxed tracheal tube preparations precontracted with septide (1 microM), whereas septide (0.1 nM-1 microM) further contracted tracheal tube preparations precontracted with [Sar9, Met(O2)11]-SP (1 microM). 4. Relaxant and contractile responses evoked by SP, NKA, NKB and by [Sar9, Met(O2)11]-SP (0.1-10 nM) were not affected by a combination of the histamine H1 (pyrilamine, 1 microM) and H2 (cimetidine, 1 microM) receptor antagonists, but were abolished by the tachykinin NK1 receptor antagonist, CP-99,994 ((2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine, 1 microM), though not by its inactive enantiomer CP-100,263 (1 microM). Contractile responses evoked by septide (10 nM and 1 microM) were also abolished by CP-99,994 (1 microM) but not by CP-100,263 (1 microM). 5. These results demonstrate that tachykinins relax guinea-pig tracheal tube preparations by releasing NO via the stimulation of epithelial NK1 receptors by a mechanism independent of histamine release. The NK1 receptor type involved is sensitive to SP, NKA, NKB and [Sar9, Met(O2)11]-SP but not to septide, and is pharmacologically distinct from the NK1 receptor that mediates contraction, which is stimulated by all the agonists, including septide.     

GABA-mediated inhibition of the anaphylactic response in the guinea-pig trachea.

1. In sensitized guinea-pigs, the effects of gamma-aminobutyric acid (GABA) and GABAmimetic drugs have been investigated on tracheal segments contracted by cumulative application of an allergen (ovoalbumin, OA) and on serosal mast cells. The same drugs have also been tested on activation of alveolar macrophages isolated from unsensitized guinea-pigs. 2. Superfusion with GABA (1-1000 microM) reduced the contraction intensity of tracheal strips. The effect of GABA (100 microM) was not affected by the carrier blockers, nipecotic acid and beta-alanine (300 microM each). It was mimicked by the GABAB agonist (-)-baclofen (100 microM) but not 3-aminopropanephosphinic acid (100 microM, 3-APA). The GABAA agonist, isoguvacine (100 microM) did not exert any effect. GABA (10 microM)-induced inhibition of tracheal contractions was reduced by the GABAB antagonist, 2-hydroxysaclofen (100 microM, 2-HS), but not by the GABAA antagonist, bicuculline (30 microM). 3. The reduction in contraction intensity induced by GABA (100 microM) was prevented by a 40 min preincubation of tracheal strips with capsaicin (10 microM), but not tetrodotoxin (TTX, 0.3 microM). The effect of GABA (1000 microM) was absent after preincubation with indomethacin (2.8 microM) but unmodified when nordihydroguaiaretic acid (NDGA, 3.3 microM) was used. Finally, removal of the epithelium prevented the GABA effect. 4. Anaphylactic histamine release from serosal mast cells isolated from sensitized animals was not affected either by GABA (10-1000 microM) or the selective receptor agonists (-)-baclofen (0.1-1000 microM) and isoguvacine (10-1000 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of relaxant action of S-petasin and S-isopetasin, sesquiterpenes of Petasites formosanus, in isolated guinea pig trachea

We investigated the mechanisms of action of S-petasin and S-isopetasin, from Petasites formosanus Kitamura which is used as a folk medicine for treating hypertension, tumors, and asthma in Taiwan. The tension changes of tracheal segments were isometrically recorded on a polygraph. S-Petasin and S-isopetasin non-competitively inhibited cumulative histamine-, and carbachol-induced contractions with an exception that S-isopetasin produced a parallel, rightward shift of the concentration-response curve of carbachol in a competitive manner. S-Petasin also non-competitively inhibited cumulative Ca(2+)-induced contractions in depolarized (K+, 60 mM; histamine, 100 microM; or carbachol, 10 microM) guinea-pig tracheas. S-Isopetasin did in depolarized (K+, 60 mM) trachea too. The nifedipine (10 microM)-remaining tension of carbachol (0.2 microM)-induced precontraction was further relaxed by S-petasin or S-isopetasin, suggesting that no matter whether either blocked VDCCs or not, S-petasin or S-isopetasin may have other mechanisms of relaxant action. The relaxant effect of S-petasin or S-isopetasin was unaffected by the presence of propranolol (1 microM), 2',5'-dideoxyadenosine (10 microM), methylene blue (25 microM), glibenclamide (10 microM), N omega-nitro-L-arginine (20 microM), or alpha-chymotrypsin (1 U/ml). However, S-petasin (100-300 microM), but not S-isopetasin, significantly inhibited cAMP-, but not cGMP-dependent PDE activity of the trachealis. The above results reveal that the mechanisms of relaxant action of S-petasin and S-isopetasin may be primarily due to its non-specific antispasmodic and antimuscarinic effects, respectively.     

Induction of apoptosis by esculetin in human leukemia cells

Esculetin, a coumarin compound, has been shown to exhibit antioxidant and anti-inflammatory effects. In the present study, esculetin was found to inhibit the survival of human promyelocytic leukemia HL-60 cells in a concentration-dependent and time-dependent manner. HL-60 cells underwent internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after a 24-h treatment with esculetin (100 microM). Flow cytometric analysis showed that the hypodiploid nuclei of HL-60 cells were increased to 40.93% after a 36-h treatment with esculetin (100 microM). Further investigation showed that esculetin induced the release of cytochrome c from mitochondria into cytosol in a time-dependent and concentration-dependent manner. Moreover, esculetin application reduced Bcl-2 protein expression to 58% after 9 h as compared with that time at 0. Cysteine protease 32 kDa proenzyme (CPP32), a caspase 3, was activated and its substrate, poly (adenosine diphosphate-ribose) polymerase, was cleaved after a 24-h treatment of HL-60 cells with esculetin. These data suggest that esculetin induces apoptosis in human leukemia cells by increasing cytosolic translocation of cytochrome c and activation of CPP32.     

Mechanisms of relaxant action of luteolin in isolated guinea pig trachea

We have investigated the mechanisms of action of luteolin, a flavone found in Perilla frutescens, a Chinese herbal medicine for treating asthma. In fact, luteolin occurs mostly as a glycoside in many plant species. The tension changes of tracheal segments were isometrically recorded on a polygraph. Luteolin concentration-dependently relaxed histamine (30 microM)-, carbachol (0.2 microM)- and KCl (30 mM)-induced precontractions, and inhibited cumulative histamine- and carbachol-induced contractions in a non-competitive manner. Luteolin also concentration-dependently and non-competitively inhibited cumulative Ca2+-induced contractions in depolarized (K+, 60 mM) guinea-pig trachealis. The nifedipine (10 microM)-remaining tension of histamine (30 microM)-induced precontractions was further relaxed by luteolin, suggesting that no matter whether VDCCs were blocked or not, luteolin may have other mechanisms of relaxant action. The relaxant effect of luteolin was unaffected by the removal of epithelium or by the presence of propranolol (1 microM), 2',5'-dideoxyadenosine (10 microM), methylene blue (25 microM), glibenclamide (10 microM), Nomega-nitro-L-arginine (20 microM), or alpha-chymotrypsin (1 U/mL). However, luteolin (10-20 microM) produced parallel and leftward shifts of the concentration-response curve of forskolin or nitroprusside. Luteolin or IBMX at various concentrations (10-300 microM) concentration-dependently and significantly inhibited cAMP- and cGMP-PDE activities of the trachealis. The IC50 values of luteolin were estimated to be 32.4 and 34.6 microM, respectively. IBMX at various concentrations (10-300 microM) selectively inhibited neither cAMP-, nor cGMP-PDE activity. In contrast to IBMX, luteolin at 100 and 300 microM more potently (P < 0.05) inhibited cGMP-, than cAMP-PDE activity. The above results indicate that the mechanisms of relaxant action of luteolin may be due to its inhibitory effects on both PDE activities and its reduction on [Ca2+]i of the trachealis.     

Effects of Ba2+ on F&F 96365-sensitive sustained contraction of rat pulmonary artery

The effects of Ba2+ on receptor-mediated sustained contraction of rat pulmonary artery and guinea-pig oesophageal muscularis mucosae were studied in-vitro. In rat isolated pulmonary artery, sustained contraction induced by noradrenaline (1 microM) was resistant to nicardipine (1 microM), but this same sustained contraction was completely inhibited by SK&F 96365 (30 microM), a blocker of voltage-dependent L-type Ca2+ channels and receptor-activated Ca2+ influx. The SK&F 96365-sensitive sustained contraction induced by noradrenaline (1 microM) may be due primarily to Ca2+ influx through receptor-activated Ca)+ channels resistant to nicardipine. Cumulatively applied BaCl2 (0.1 - 10 mM) increased the noradrenaline (1 microM)-induced sustained contraction of the pulmonary arterial preparation in the absence of nicardipine, but in the presence of nicardipine (1 microM), BaCl2 (0.1 - 3 mM) did not affect this contraction. A higher concentration of BaCl22 (10 mM), however, weakly inhibited the noradrenaline (1 microM)-induced tone. In addition, BaCl2 (3-10 mM) increased the tone induced by KCl (60 mM), and the BaCl2-elevated KCl tone was markedly inhibited by nicardipine (1 microM) treatment. In the guinea-pig isolated oesophageal muscularis mucosae, sustained contraction induced by acetylcholine (3 microM) was resistant to nicardipine (1 microM) but was returned to its basal level by SK&F 96365 (30-60 microM). The SK&F 96365-sensitive, acetylcholine-induced sustained contraction of the oesophageal muscularis mucosae is also likely to link with receptor-activated Ca2+ channels resistant to nicardipine. In contrast to the rat pulmonary artery, cumulatively applied BaC12 (0.3 - 10 mM) inhibited the acetylcholine (3 microM)-induced sustained contraction of the oesophageal muscularis mucosae in a concentration-dependent manner in the presence of nicardipine (1 microM). In conclusion, Ba2+ presumably activates voltage-dependent Ca2+ channels by depolarizing plasma membrane and also passes through voltage-dependent Ca2+ channels to contract the pulmonary artery in the absence of nicardipine, and Ba2+ also has a minor effect on the nicardipine-resistant, SK&F 96365-sensitive sustained contraction induced by noradrenaline in rat isolated pulmonary artery.     

Mechanisms involved in the relaxant action of the ethanolic extract of propolis in the guinea-pig trachea in-vitro

This study examines the mechanisms by which the standardised ethanolic extract of propolis induces relaxation of the guinea-pig trachea in-vitro. In guinea-pig trachea with or without epithelium and contracted by histamine, the propolis extract caused reproducible and graded relaxation, with a mean EC50 value of 3.8 or 10.5 microg mL(-1) and Emax of 100%, respectively. The propolis extract-induced relaxation was markedly reduced (26+/-9 and 96+/-3%) when guinea-pig tracheas were exposed to Krebs solution containing elevated K+ in the medium (40 or 80 mM). Pre-incubation of guinea-pig tracheas with tetraethylamonium (100 mM) or with 4-aminopyridine (10mM) reduced the propolis extract-induced relaxation by 31+/-10% and 28+/-2%. Likewise, apamin (0.1 microM), charybdotoxin (0.1 microM) or iberiotoxin (0.1 microM) caused marked inhibition of propolis extract-mediated relaxation in guinea-pig trachea (percentage of inhibition: 65+/-3%, 60+/-5% and 65+/-9%, respectively). Also, glibenclamide (1 microM) inhibited the relaxant response caused by the propolis extract by 57+/-4%. Omega-conotoxin GIVA (0.1 microM) or capsaicin (1 microM) produced small but significant inhibition (30+/-5% or 47+/-7%, respectively) of the propolis extract-induced relaxation. The vasoactive intestinal peptide (VIP) antagonist D-p-Cl-Phe6,Leu17[VIP] porcine (0.1 microM) inhibited relaxation by 55+/-5%, while propranolol (1 microM) induced a parallel rightward displacement (about 20 fold) of the propolis extract concentration-response curve. Finally, the propolis extract-induced relaxation was inhibited by the nitric oxide synthase inhibitor L-N(G)-nitroarginine (L-NOArg, 100 microM) (48+/-6%), and by the soluble guanylatecyclase inhibitormethylene blue (10 microM) (37+/-6%), whilethe moreselectivesoluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolol[4,3-alquinoxalin-1-one (ODQ, 1 microM) produced only a parallel (about 3 fold) rightward displacement of the propolis extract concentration-response curve. Collectively, these results support the notion that the propolis extract-mediated relaxation in the guinea-pig trachea involves the release of nitric oxide, probably from sensory neurons, besides the activation of soluble guanylate cyclase and activation of Ca2+- and ATP-sensitive K+ channels. Furthermore, the stimulation of beta2-adrenergic and VIP receptors also seems to account for its relaxant action.