Activities of caffeine, theophylline, and enprofylline analogs as tracheal relaxants

A variety of xanthines cause tracheal relaxation, an activity predictive of antiasthmatic potential. Structural analogs of caffeine, theophylline, and enprofylline were examined for their abilities to relax carbamylcholine-stimulated guinea pig trachea in vitro. All caffeine analogs tested were more potent than caffeine (EC50 = 551 +/- 81 microM) except the 8-p-sulfophenyl analog. 1,3,7-Tripropylxanthine and 1,3,7-tripropargylxanthine were among the more potent analogs with EC50 values of 12 +/- 1.3 and 65 +/- 11 microM respectively. Increasing the polarity at the 1- or 3-position by substituting a propargyl group for an n-propyl group decreased relaxant activity, an effect not observed at the 7-position. The 8-cyclohexyl-, 8-cyclopentyl- and 8-phenyl-derivatives of caffeine were relatively potent (EC 50 = approximately 75 microM). The theophylline analog 1,3-di-n-propylxanthine was approximately two times more active than theophylline (EC50 = 162 +/- 17 microM). 3-Isobutyl-1-methylxanthine (EC50 = 7.1 +/- 1.8 microM) and 1-isoamyl-3-isobutylxanthine (EC50 = 37 +/- 4.2 microM) were among the most potent tracheal relaxants. The 8-substituted theophylline analogs were weak or inactive relaxants except for 8-cyclopentyl- and 8-cyclohexyltheophylline, which were more potent or as potent as theophylline. In contrast to enprofylline (EC50 = 56 +/- 9 microM), 8-substituted enprofylline analogs were weak or inactive as relaxants with the exception of the 8-cyclohexyl analog. The potency of xanthines as tracheal relaxants was unrelated to potency as adenosine receptor antagonists and may reflect activity as phosphodiesterase inhibitors.     

The epithelium and the pharmacology of guinea-pig tracheal tone in vitro.

1. Epithelium removal did not influence the development of spontaneous tone in guinea-pig tracheal smooth muscle mounted as open ring preparations with two adjoining cartilaginous rings in vitro. 2. Epithelium removal did not change the potency of carbachol but tended to reduce the maximal contraction. In the presence of epithelium the EC50 of carbachol was not different in tracheal open ring compared with intact tube preparations (comprising four cartilaginous rings), suggesting that the size of continuous epithelium in vitro was not critical for the potency of carbachol. 3. Substance P produced the same response in intact and rubbed tracheae. The enkephalinase inhibitor thiorphan (0.1 mM) by itself contracted the trachea and appeared to potentiate the substance P response five times more in the absence than in the presence of epithelium. Capsaicin (1 microM)-induced contractions did not differ between intact and rubbed preparations. 4. Arachidonic acid, 22 microM, variably produced small relaxations and contractions in intact as well as in rubbed tracheae. The mean effects of arachidonic acid were not significantly altered by epithelium removal. 5. Adenosine produced small contractions and dose-dependent relaxations in the presence and absence of epithelium. 6. Epithelium removal had no effect on the potency of the relaxant agonists theophylline and enprofylline. The isoprenaline curve was shifted 2 fold to the left and the terbutaline curve 1.5 fold to the right. The maximal relaxations were generally reduced in epithelium-free tissue. The reduction reached statistical significance with theophylline. 7. The present results suggest that epithelium removal is of little consequence for the pharmacology of the guinea-pig tracheal open ring preparation in vitro.     

Relaxation of isolated guinea pig trachea by genistein via inhibition of phosphodiesterase

We investigated the mechanisms of the relaxant action of genistein, an isoflavone, phytoestrogen and non-specific protein tyrosine kinase inhibitor. Changes in tension of guinea pig tracheal segments were isometrically recorded on a polygraph. Genistein concentration-dependently relaxed histamine (30 microM)-, carbachol (0.2 microM)-, KCl (30 mM)- and leukotriene D4 (10 nM)-induced precontractions and inhibited cumulative histamine- and carbachol-induced contractions in a non-competitive manner. Genistein also concentration-dependently and non-competitively inhibited the cumulative, Ca2+-induced contractions in the depolarized (K+, 60 mM) trachealis. The remaining nifedipine (10 microM)-induced tension of the histamine (30 microM)-induced precontraction was further relaxed by genistein, suggesting that regardless of whether voltage-dependent calcium channels are blocked genistein may have other mechanisms of relaxant action. These other mechanisms of the relaxant effect of genistein appeared to be epithelium-independent and were not affected 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), suggesting that the mechanisms are unrelated to activation of the beta-adrenoceptor, of adenylate cyclase, of guanylate cyclase, of adenosine triphosphate-sensitive potassium channel opening, of nitric oxide formation or of neuropeptide release, respectively. However, genistein (17.5-35 microM) produced parallel, leftward shifts in the concentration-response curves of forskolin and nitroprusside and significantly increased the pD2 values of these two agonists. Both genistein and 3-isobutyl-1-methylxanthine at various concentrations (10-300 microM) concentration-dependently and significantly inhibited cAMP- and cGMP-phosphodiesterase (PDE) activities of the trachealis. The -log IC50 values of genistein were estimated to be 4.28 and 4.17, respectively. The above results reveal that the mechanisms of the relaxant action of genistein may be due to its non-selective inhibition of both PDE activities. IBMX:3-ixobutyl-1-methylxanthine VDCCs:voltage-dependent calcium channels cAMP:adenosine 3',5'-cyclic monophosphate cGMP:guanosine 3',5'-cyclic monophosphate ATP:adenosine triphosphate PDE:phosphodiesterase LTD4:leukotriene D4L-NNA:Nomega-nitro-L-arginine DMSO:dimethyl sulfoxide EGTA: N,N,N',N'-tetraacetic acid ANOVA:analysis of variance.     

Relaxant effect of YM976, a novel phosphodiesterase 4 inhibitor,on bovine tracheal smooth muscle

Effects of 4-(3-chlorophenyl)-1,7-diethylpyrido[2,3-d]pyrimidin-2(1H)-one (YM976), a novel and selective phosphodiesterase type 4 inhibitor, on tension and adenosine 3',5'-cyclic monophosphate (cAMP) content of bovine tracheal smooth muscle were compared with those of rolipram and theophylline. YM976, rolipram and theophylline relaxed the tracheal preparations contracted with histamine in a concentration-dependent manner. The relaxant effects of YM976 and rolipram were more potent than those of theophylline. These phosphodiesterase inhibitors-induced relaxations were dramatically diminished when tracheal smooth muscle was contracted with methacholine instead of histamine. Pretreatment of the tracheal preparations with YM976 (10 microM) or rolipram (10 microM), but not with theophylline (1 mM), shifted the concentration-response curves for contractile responses to histamine; however, the same procedure failed to affect concentration-response relationships for methacholine-induced contractions. At 1 and 10 microM, both YM976 and rolipram increased the tissues cAMP content. These results suggest that YM976 relaxes tracheal smooth muscle, probably through the cAMP-dependent mechanism.     

Actions and interactions of adenosine, theophylline and enprofylline on the guinea-pig spirally cut trachea

Adenosine, theophylline and enprofylline induced concentration-dependent relaxations of guinea-pig isolated tracheal spirals whether they had intrinsic tone or were precontracted with carbachol or histamine. The potency order was enprofylline greater than theophylline greater than adenosine and the maximum relaxation in absolute terms was generally less for adenosine. The maximum relaxation (measured in absolute terms of change in tension) induced by the three spasmolytics in preparations with intrinsic tone was generally less than in precontracted tissues. This was attributed to the higher resting tone of precontracted tissues than the intrinsic tone. The adenosine transport inhibitor dipyridamole potentiated adenosine but not theophylline or enprofylline so that the potency order became adenosine greater than enprofylline greater than theophylline. Without dipyridamole, theophylline in a concentration producing 10-30% relaxation of the trachea, failed to antagonize adenosine. However, in the presence of dipyridamole, adenosine was antagonized, indicating that the relaxation by adenosine was mediated via an extracellular P1 receptor. Enprofylline, in a concentration producing equivalent direct effects, failed to antagonize adenosine. It is concluded that the tracheal relaxation by xanthines is independent of adenosine antagonism.     

Effects of K+ channel blockers on the relaxant action of dihydralazine, cromakalim and nitroprusside in isolated rabbit femoral arteries.

The relaxant responses to dihydralazine and the influence of different K+ channel blockers were studied in isolated rabbit femoral arteries. The prototype K+ channel opener, cromakalim, and nitroprusside, which does not produce relaxation by K+ channel activation were used for comparison. Dihydralazine was most effective on contractions induced by noradrenaline (EC50 = 1.1 microM; Emax = 95%) and relaxed the contractions elicited by 20 mM K+ (EC50 = 2.0 microM; Emax = 81% in preference to 124 mM K(+)-induced contractions (EC50 = 30.1 microM; Emax = 54%). Cromakalim, but not nitroprusside, also selectively relaxed 20 mM K(+)-induced contractions. In noradrenaline-contracted arteries, glibenclamide (10 microM) completely suppressed the relaxant response to cromakalim but did not influence the vasorelaxation produced by dihydralazine or nitroprusside. Tetraethylammonium (8 mM) and Cs+ (4 mM) shifted the concentration-relaxation curve for dihydralazine 2-fold to the right, whereas Ba2+ (0.1 mM), 4-aminopyridine (5 mM) and procaine (0.1 mM) failed to influence dihydralazine-induced responses. Tetraethylammonium (8 mM) shifted the concentration-relaxation curve for cromakalim and nitroprusside 6-fold to the right and suppressed the maximal relaxant effects by about 30%. It is concluded that dihydralazine produces vascular smooth muscle relaxation by a mechanism different from the opening of glibenclamide- and ATP-sensitive K+ channels.     

Modulation of carbachol-induced inositol phosphate formation in bovine tracheal smooth muscle by cyclic AMP phosphodiesterase inhibitors.

An investigation was made of a range of agents capable of elevating tissue cyclic AMP levels, or acting as a stable analogue of cyclic AMP, upon carbachol induced inositol phosphate responses in bovine tracheal smooth muscle slices. Whereas the beta 2 adrenoceptor agonist salbutamol (1 microM) and the membrane permeable analogue of cyclic AMP, 8-bromo-cyclic AMP (1 mM) were without effect upon total [3H]inositol phosphate formation induced by carbachol, 3-iso-butyl-1-methylaxanthine (IBMX) (EC50 140 microM), the high Km, cyclic AMP selective phosphodiesterase inhibitor rolipram (EC50 41 microM) and theophylline (EC50 76 microM) all inhibited the inositol phosphate response to low (1 microM) concentrations of carbachol. IBMX (IC50 13 microM), rolipram (IC50 4.6 microM) and theophylline (IC50 180 microM) all relaxed bovine tracheal muscle strips precontracted with methacholine (1 microM). The adenylate cyclase activator forskolin (1 microM), produced a much smaller (10% inhibition) effect upon inositol phosphate formation induced by carbachol. Carbachol (1 microM-1 mM) did not inhibit forskolin induced [3H]cyclic AMP formation. An inhibitor of the cyclic GMP preferring phosphodiesterase isozyme, M&B 22948 (1-100 microM), was without effect upon either carbachol induced inositol phosphate formation or trachealis tone. It is concluded that IBMX, rolipram and theophylline inhibit carbachol stimulated inositol phosphate formation, possibly through a cyclic AMP independent mechanism.     

Mechanisms of relaxant action of 3-O-methylquercetin in isolated guinea pig trachea

We investigated the mechanisms of action of 3-O-methylquercetin (3-MQ), isolated from Rhamnus nakaharai (Hayata) Hayata (Rhamnaceae) which is used as a folk medicine for treating constipation, inflammation, tumors and asthma in Taiwan. The tension changes of tracheal segments were isometrically recorded on a polygraph. 3-MQ 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. 3-MQ also concentration-dependently and non-competitively inhibited cumulative Ca(2+)-induced contractions in depolarized (K(+), 60 mM) guinea-pig trachealis. The nifedipine (10 microM)-remaining tension of histamine (30 microM)-induced precontraction was further relaxed by 3-MQ, suggesting that no matter whether VDCCs were blocked or not, 3-MQ may have other mechanisms of relaxant action. The relaxant effect of 3-MQ 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), N(omega)-nitro-L-arginine (20 microM), or alpha-chymotrypsin (1 U/ml). However, 3-MQ (7.5 - 15 microM) and IBMX (3 - 6 microM), a positive control, produced parallel and leftward shifts of the concentration-response curve of forskoline (0.01 - 3 microM) or nitroprusside (0.01 - 30 microM). 3-MQ 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 3-MQ were estimated to be 13.8 and 14.3 microM, respectively. The inhibitory effects of 3-MQ on both enzyme activities were not significantly different from those of IBMX, a non-selective PDE inhibitor. The above results reveal that the mechanisms of relaxant action of 3-MQ may be due to its inhibitory effects on both PDE activities and its subsequent reducing effect on [Ca(2+)]i of the trachealis.3-MQ:3-O-methylquercetinIBMX:3-isobutyl-1-methylxanthineVDCCs:voltage dependent calcium channelscAMP:adenosine 3',5'-cyclic monophosphatecGMP:guanosine 3',5'-cyclic monophosphatePDE:phosphodiesteraseWe investigated the mechanisms of action of 3-O-methylquercetin (3-MQ), isolated from Rhamnus nakaharai (Hayata) Hayata (Rhamnaceae) which is used as a folk medicine for treating constipation, inflammation, tumors and asthma in Taiwan. The tension changes of tracheal segments were isometrically recorded on a polygraph. 3-MQ 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. 3-MQ also concentration-dependently and non-competitively inhibited cumulative Ca(2+)-induced contractions in depolarized (K(+), 60 mM) guinea-pig trachealis. The nifedipine (10 microM)-remaining tension of histamine (30 microM)-induced precontraction was further relaxed by 3-MQ, suggesting that no matter whether VDCCs were blocked or not, 3-MQ may have other mechanisms of relaxant action. The relaxant effect of 3-MQ 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), N(omega)-nitro-L-arginine (20 microM), or alpha-chymotrypsin (1 U/ml). However, 3-MQ (7.5 - 15 microM) and IBMX (3 - 6 microM), a positive control, produced parallel and leftward shifts of the concentration-response curve of forskoline (0.01 - 3 microM) or nitroprusside (0.01 - 30 microM). 3-MQ 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 3-MQ were estimated to be 13.8 and 14.3 microM, respectively. The inhibitory effects of 3-MQ on both enzyme activities were not significantly different from those of IBMX, a non-selective PDE inhibitor. The above results reveal that the mechanisms of relaxant action of 3-MQ may be due to its inhibitory effects on both PDE activities and its subsequent reducing effect on [Ca(2+)]i of the trachealis.3-MQ:3-O-methylquercetinIBMX:3-isobutyl-1-methylxanthineVDCCs:voltage dependent calcium channelscAMP:adenosine 3',5'-cyclic monophosphatecGMP:guanosine 3',5'-cyclic monophosphatePDE:phosphodiesterase     

Positive inotropic and chronotropic effects and coronary vasodilation in vitro by two antiasthmatic xanthines with different abilities to antagonize adenosine

The direct actions and the ability to antagonize adenosine of theophylline (1,3-dimethylxanthine) and enprofylline (3-propylxanthine) were examined in isolated guinea pig heart preparations. Enprofylline was approximately six times as potent as theophylline in reducing coronary perfusion pressure, and between three and five times more potent in increasing rate and force of contraction in isolated perfused hearts, spontaneously beating right atrias, and electrically stimulated left atrias. Adenosine reduced the coronary perfusion pressure and had negative inotropic and chronotropic actions. Theophylline (5-75 microM) concentration-dependently antagonized the coronary vasodilation and the negative chronotropic and inotropic actions of adenosine, whereas enprofylline (2.5-75 microM) did not antagonize adenosine. Since adenosine may be a protective autacoid in the heart under select stress conditions, the possibility that adenosine nonblocking xanthines, like enprofylline, may offer therapeutic advantages over theophylline should be investigated.     

The actions of some beta-receptor agonists and xanthines on isolated muscle strips from the human oesophago-gastric junction

Isolated preparations from the circular muscle layer of the human oesophago-gastric junction were mounted in organ baths and isometric tension recorded. During an equilibration period, active resting tension developed suggesting that the preparations were representing the lower oesophageal sphincter. Active tension was abolished by exposing the preparations to Ca(++)-free medium. The two xanthines theophylline and enprofylline almost equipotently relaxed the preparations in a concentration-dependent manner (10(-7)-10(-3) M). Within therapeutic concentrations, theophylline inhibited active resting tension by 30-60%, while enprofylline lowered tension by less than 20%. Inhibitory actions of adenosine were demonstrated, and this suggests that adenosine antagonism is not the mechanism of action for xanthines in the oesophagus. Non-selective beta-receptor stimulation with isoprenaline inhibited active tension by 70% (10(-7) M), while beta 2-receptor stimulation with terbutaline inhibited tension by 47% (10(-5) M). Dobutamine, believed to preferentially stimulate beta 1-receptors, inhibited active tension in a concentration-dependent manner (10(-7)-10(-4) M). Metoprolol (10(-6) M), a selective beta 1-receptor antagonist, shifted the concentration-response curve for isoprenaline to the right, but left the maximal response unchanged. It is concluded that xanthines and beta-receptor agonists have inhibitory actions on circular muscle from the human oesophagogastric junction. The experimental data suggest the presence of beta 1- as well as beta 2-receptors, both mediating inhibition of active resting tension.     

Relaxant effects of xanthines, a beta 2-receptor agonist and Ca2+ antagonists in guinea-pig tracheal preparations contracted by potassium or carbachol

Potassium (124 mM K+ Krebs) produced a biphasic contractile response in the guinea-pig isolated trachea. An initial phasic contraction was followed by a larger and sustained contraction. Repeated potassium-induced contractions in spontaneously contracted guinea-pig tracheas were not reproducible. However, reproducible K+ responses were obtained in the presence of indomethacin (10(-6) M) that almost abolished the spontaneous tone. This suggested that endogenous cyclooxygenase products were variably released by K+ and interfered with its contractile effects. Both phases of K+-induced contractions were inhibited in Ca2+-free/EGTA Krebs. In contrast, about 80% of the contractile response to carbachol persisted in this medium. Tracheas contracted by potassium (indomethacin present) were completely relaxed by theophylline and enprofylline but only partly relaxed by terbutaline. All bronchodilators completely relaxed carbachol-contracted preparations. Each bronchodilator was 2-3 times less potent to relax K+- than carbachol-induced contractions. In sharp contrast, two Ca2+ antagonists, verapamil and nimodipine, preferentially relaxed K+-induced contractions. The results obtained with Ca2+ antagonists, which are poorly effective in asthma, compared to the established antiasthma drugs, xanthines and beta 2-receptor agonists, may indicate that depolarization-induced mechanisms contribute little to bronchoconstriction in asthma.     

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.     

Distinctive pharmacological profile of a nonadrenergic inhibitory system in bullfrog lung

1 Bullfrog hemilungs, pretreated with atropine, are markedly relaxed on addition of carbachol. Since the relaxant effect is inhibited by tetrodotoxin or hexamethonium, it is neurally mediated and involves stimulation of nicotinic receptors with release of an unknown inhibitory transmitter.2 Carbachol-induced relaxation is nonadrenergic since: (a) it considerably exceeds the maximal effects of isoprenaline or the effect of 10(-3)M adrenaline or noradrenaline; (b) it elicits marked further relaxation in preparations already relaxed by high concentrations of catecholamines; (c) it is not attenuated by low concentrations of propranolol (10(-6) and 3 x 10(-6)M) that competitively antagonize isoprenaline-induced relaxation.3 Carbachol-induced relaxation has multiple distinguishing characteristics, which serve as a fingerprint for the unknown inhibitory transmitter. These include an exceptionally rapid onset of action, a ceiling effect at 50% of maximal relaxation, and minimal retardation by concentrations of procaine that block or markedly retard relaxant responses to all other agonists.4 This distinctive pharmacological profile cannot be reproduced by addition of exogenous catecholamines, 5-hydroxytryptamine, adenosine triphosphate (ATP) or adenosine, or by addition of ATP or adenosine following pretreatment with indomethacin. Furthermore, addition of carbachol to preparations previously relaxed with 10(-3)M concentrations of these agents produced marked, additional relaxation.5 Maximally effective concentrations of vasoactive intestinal peptide produced a barely detectable relaxant response equivalent to 8% of maximal relaxation. The response was totally prevented by pretreatment with procaine.6 Carbachol-induced relaxation was not impaired by pretreatment with 10(-4)M indomethacin.7 Carbachol-induced relaxation of bullfrog lung therefore involves a postganglionic inhibitory transmitter that in nonadrenergic, non-5-hydroxytryptaminergic, and nonpurinergic, and whose effects are not dependent on prostaglandin synthesis. Although a peptide may function as the inhibitory transmitter, it is not vasoactive intestinal peptide.     

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.     

Different spasmolytic effects of smooth muscle relaxants on the guinea-pig esophageal muscularis mucosae contracted by carbachol or high potassium in vitro

The responsiveness of the guinea-pig esophageal muscularis mucosae to smooth muscle relaxants was examined in vitro during the contractile state induced by carbachol (3 microM) or high potassium (60 mM). In the presence of phentolamine (3 microM), all catecholamines tested (10 nM-30 microM) relaxed the carbachol-induced tone more effectively than the high potassium-induced tone, and the maximum relaxations reached about 90-95% for carbachol but only about 40% for high potassium. Verapamil produced a concentration-dependent relaxation of the muscularis mucosae precontracted with either spasmogen; the mean EC50 values (-log M) were 6.73 for high potassium and 4.65 for carbachol. Methylene blue (1-300 microM) relaxed the carbachol-contracted muscularis mucosae in a concentration-dependent manner but relaxed the high potassium-contracted one less potently. Forskolin (1-300 microM), papaverine (1-100 microM), aminophylline (10-300 microM), trifluoperazine (1-300 microM), 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (10-300 microM), quinacrine (1-300 microM) and dibutyryl cyclic AMP (100 microM-3 mM) produced relaxation almost equipotently in both contractile states whereas adenosine (10 microM-1 mM), sodium nitroprusside (10-300 microM) and dibutyryl cyclic GMP (100 microM-3 mM) were virtually ineffective. The present results indicate that a variety of smooth muscle relaxants have different spasmolytic effects on the guinea-pig isolated esophageal muscularis mucosae which was precontracted with carbachol or high potassium and that catecholamines and methylene blue may produce relaxation independent of the changes in intracellular cyclic nucleotides, calmodulin or phosphoinositides.     

Lack of an effect of saxitonin on the contractility of isolated guinea-pig trachea, lung parenchyma and aorta

The effects of saxitonin were investigated in guinea-pig tracheal rings, lung parenchymal strips and aorta rings. Tracheal rings were used both with epithelium present and with it removed. Aorta rings were used both with endothelium present and with it removed. Saxitoxin, 1 pM to 0.1 microM, did not alter the resting tension of either airway tissues or aorta. Also 0.1 microM saxitonin did not reduce tension of tracheal rings contracted by 10 microM carbachol, parenchymal strips contracted by 100 microM acetylcholine or by 10 microM histamine, or aorta rings contracted by 10 microM norepinephrine. Responses of tracheal rings to 0.03-10 microM carbachol added cumulatively were not altered by 0.1 microM saxitoxin (EC50 with epithelium: 1.11 +/- 0.48 microM control, 2.01 +/- 0.71 microM with saxitoxin; EC50 without epithelium: 2.83 +/- 0.55 microM control, 2.05 +/- 0.58 microM with saxitoxin). Also, 1 microM saxitoxin did not alter contractions of parenchymal stirps to cumulatively added acetylcholine (EC50: 3.47 +/- 1.06 microM, control; 3.98 +/- 1.19 microM with saxitoxin), histamine (EC50: 0.83 +/- 0.24 microM, control; 0.60 +/- 0.13 microM with saxitoxin); or of aorta strips to norepinephrine with endothelium (EC50: 1.78 +/- 0.80 microM, control; 0.74 +/- 0.21 microM with saxitoxin) or without endothelium (EC50: 2.18 +/- 0.78 microM, control; 1.42 +/- 0.62 microM with saxitoxin). Thus, saxitoxin did not significantly alter contractile activity of airways of large arteries in vitro.     

Theophylline antagonizes some effects of purines in the intestine but not those of intramural inhibitory nerve stimulation.

1 Hyoscine- and guanethidine-treated preparations of longitudinal muscle of rabbit duodenum, guinea-pig taenia caeci and fundic strip relaxed when exposed to noradrenaline, adenosine, adenosine triphosphate (ATP) or to field stimulation of their intramural nerves. 2 In guinea-pig taenia caeci and fundus, theophylline 100 mumol/l had no effect on responses to noradrenaline, adenosine, ATP and intramural nerve stimulation. 3 In rabbit duodenum, theophylline 100 mumol/l antagonized some responses to adenosine but had no effect on responses to noradrenaline, ATP and intramural nerve stimulation. 4 Theophylline 1 mmol/l itself relaxed the intestinal tissues and in the fundic strip and taenia caeci, these relaxant effects were associated with abolition of spike activity and cellular hyperpolarization. In the taenia caeci, the amplitude of inhibitory post-junctional potentials was reduced. 5 Theophylline 1 mmol/l antagonized the twitch suppression produced by adenosine and ATP in the transmurally-stimulated guinea-pig ileum but not that evoked by noradrenaline. 6 It is concluded that theophylline can selectively antagonize some actions of purines in the intestine but that it does not specifically antagonize the effect of intramural inhibitory nerve stimulation.     

Mechanism of vanadate-induced contraction of airways smooth muscle of the guinea-pig.

The characteristics of vanadate-induced contraction of airways smooth muscle are described in isolated preparations of guinea-pig central and peripheral airways. Vanadate (1-1000 microM) induced sustained contractions of trachea and lung parenchymal strips within 1 min of challenge. It was more potent (P less than 0.001) on the lung strip (EC50 = 63 microM) than on the trachea (EC50 = 123 microM). The lung strip also developed greater maximum isometric tension (P less than 0.001) than the trachea. The efficacy on the lung strip was 2 and the trachea 0.6, relative to the response to acetylcholine (efficacy = 1). Vanadate-induced contractions of the trachea were not inhibited by atropine, mepyramine, phentolamine or indomethacin, nor after mast cell depletion by compound 48/80, showing that contractions were not mediated via specific receptors or by release of endogenous mediators of tone. Inorganic phosphate specifically inhibited vanadate responses in a dose-dependent and reversible manner, suggesting a common site of action. Contractions could be elicited in depolarized muscle and after treatment with ouabain plus propranolol, showing that membrane depolarization and inhibition of the Na, K-ATPase system were not involved in the contractile action of vanadate. Pretreatment of tracheal smooth muscle with verapamil had no influence on contractions elicited by vanadate. After removal of extracellular calcium, vanadate-induced contractions declined slowly with time, indicating that influx of extracellular calcium was not giving rise to contractions elicited by vanadate. Vanadate markedly increased the rate of calcium efflux from trachealis muscle loaded with 45Ca into both Ca2+-free and normal Krebs solutions; this is compatible with vanadate mobilizing an intracellular store of Ca2+. Such a store involving sites with Ca-ATPase activity would be consistent with the action of vanadate in isolated membrane preparations. Membrane-skinned tracheal fibres contracted by micromolar Ca2+ were relaxed by vanadate in a reversible dose-related manner, indicating that the contractile action of vanadate was not related to its interaction with proteins at the cross-bridge level.     

Enprofylline and theophylline on small human placental arteries: studies of in vitro effects and mode of action

Vascular effects of theophylline and enprofylline, a novel xanthine derivative lacking adenosine receptor antagonism, were studied comparatively in tubular preparations of small human placental arteries mounted in an isometric myograph. Both xanthines produced concentration-dependent (10(-7)-3 X 10(-3) M) relaxation of arteries contracted by PGF2 alpha or PGE2 in both normal Ca2+-medium and in Ca2+-depleted medium. Enprofylline was about three times more potent than theophylline. Also in vasopressin-contracted arteries enprofylline was a more potent vasodilator in both media. In contrast the xanthines were equally potent in relaxation of the tonic as well as the phasic part of a K+-induced contraction, but less potent than in relaxation of PG-induced contractions. Propranolol, phentolamine, atropine, indomethacin or tetrodotoxin did not influence the xanthine relaxations. It is concluded that the theophylline-induced relaxation of small human placental arteries is not due to adenosine receptor antagonism. A common important mechanism of action, in which enprofylline is more potent than theophylline, seems to be interference with intracellular Ca2+-binding/mobilisation processes. Some decrease in cellmembrane Ca2+-permeability produced by the xanthines seems to take part in the mechanism of relaxation.     

BRONCHODILATORY ACTIVITY AND PHARMACOKINETICS OF NEW XANTHINES IN GUINEA-PIGS

1. The in vitro biological activities and the effect of protein binding on the relaxant effects in vivo of N-3-alkylxanthine and N-3-alkyl-N-1-methylxanthine derivative were investigated in guinea-pigs. 2. A significantly positive correlation was observed among the in vitro muscle relaxant activity, the cyclic adenosine monophosphate (cAMP) phosphodiesterase (PDE) inhibitory activity and the protein-binding potency of xanthine derivatives. However, there was a weak relationship between these activities and affinity for adenosine receptors. 3. When theophylline, enprofylline and 1-methyl-3-propylxanthine (MPX) were injected intravenously in guinea-pigs, their ED50 values were 6.1, 3.3 and 1.0 mg/kg, respectively. Plasma concentrations of these drugs obtained following the intravenous injection of the ED50 approximated the theoretically effective concentration (EC50) predicted from both the relaxant effects in vitro and the protein binding parameters. A good linear correlation was observed between bodyweight in four species (rats, guinea-pigs, rabbits and humans) and certain pharmacokinetic parameters of enprofylline and theophylline. 4. The present study indicates that differences in the relaxant effects of these drugs in vitro and in vivo can be explained in part by protein binding, and that the protein binding of these xanthine bronchodilators is an important determinant for their pharmacological activity. Guinea-pigs provide a useful model for studying pharmacodynamic-pharmacokinetic relationships of new bronchodilators.