The effects of epithelium removal on the sensitivity of guinea-pig isolated trachealis to bronchodilator drugs.

Mechanical removal of the epithelium increased the sensitivity of tracheal strips to isoprenaline, sodium nitroprusside, and to adenosine (only in the presence of inhibitors of its uptake and metabolism). Epithelium removal was without effect on sensitivity to salbutamol or papaverine. Preincubation of tracheal strips with an inhibitor of extraneuronal uptake, corticosterone (50 microM), had no effect on tissue sensitivity to either salbutamol or papaverine. However, the steroid both increased sensitivity to isoprenaline, and abolished the effect of epithelium removal on sensitivity to this catecholamine. These results suggest that in the guinea-pig, the tracheal epithelium is a major source of extraneuronal uptake for catecholamines. Furthermore, the increase in trachealis sensitivity to isoprenaline following epithelium removal is probably due to loss of these sites of extraneuronal uptake. The fact that sensitivity to salbutamol, papaverine and adenosine (in the absence of metabolic inhibitors) was not increased by denuding the epithelium indicates that loss of a diffusion barrier to drugs is not the mechanism of increased sensitivity. Adenosine (and possibly nitroprusside) may cause the epithelium to release a smooth muscle excitatory factor. Thus, removal of the epithelium attenuates this excitatory influence and enhances smooth muscle responsiveness to adenosine. These results provide further evidence that the epithelium has an important role in modulating the sensitivity of guinea-pig trachealis to drugs.     

Some effects of nifedipine in guinea-pig isolated trachealis.

In trachealis depolarized by a K+-rich medium, nifedipine (0.001-1 mumol 1(-1) caused concentration-dependent antagonism of CaCl2-induced increase in tension, moving the CaCl2 log concentration-effect curve to the right and depressing the maximal response. In trachealis in normal Krebs solution, similar concentrations of nifedipine had marked antispasmogenic activity against the responses to potassium chloride (KCl) and tetraethylammonium (TEA). However, nifedipine had little, if any, antispasmogenic activity against the responses to acetylcholine or histamine. Nifedipine 1 mumol 1(-1) was tested for spasmolytic activity in tissues generating tension in response to the EC50 of acetylcholine, KCl or CaCl2. In producing spasmolysis nifedipine was most effective against CaCl2 and least effective against acetylcholine. Nifedipine (0.01-1 mumol-1) had little or no effect on the tone of trachealis in normal Krebs solution. Intracellular electrophysiological recording showed that nifedipine 1 mumol 1(-1) could abolish spontaneous slow wave activity. This was associated with very minor depolarization and little or no loss of mechanical tone. In tissues treated with TEA (8 mmol 1(-1) nifedipine abolished spike and slow wave discharge and reduced mechanical activity to the pre-TEA level. It is concluded that nifedipine prevents KCl- or TEA-induced spasm by inhibition of Ca2+ influx. Spasm evoked by acetylcholine or histamine and the maintenance of spontaneous tone depend largely on mechanisms for increasing the cytoplasmic concentration of free Ca2+ which are resistant to nifedipine.     

The effects of alterations in electrogenic Na+/K+ -pumping in guinea-pig isolated trachealis: their modulation by the epithelium.

1 An examination was made of the effect of epithelium removal on mechanical responses of guinea-pig isolated tracheal strips after inhibition or activation of electrogenic Na+/K+ -pumping. 2 The Na+/K+ -pump inhibitor ouabain (0.1-10 microM) evoked concentration-dependent contractions which were potentiated by epithelium removal. 3 K+-free solution, which inhibits Na+/K+-pumping, produced a slow, sustained relaxation in intact preparations. In epithelium-free preparations the relaxation was transient and of lesser magnitude. 4 The addition of K+ (10 or 30 mM), which activates Na+/K+-pumping, to preparations bathed in K+-free solution caused a relaxation of preparations under spontaneous tone or contracted with methacholine; the magnitude and duration of relaxation was greater in the epithelium-free preparations. Ouabain (0.1 microM) attenuated the relaxation to K+ in intact preparations and converted the response of epithelium-free preparations to a contraction. In the presence of a higher concentration of ouabain (1 microM), intact preparations contracted in response to K+. 5 In normal K+ solution, ouabain (0.1 microM) increased the sensitivity of intact preparations to methacholine but reduced their sensitivity to K+. Ouabain was without these effects in epithelium-free preparations. 6 Thus, responses of intact preparations to perturbations which affect electrogenic Na+/K+-pumping in trachealis are influenced by an epithelium-derived factor. The production of the factor may be linked to an epithelial Na+/K+-pump, or the factor may modulate the activity of an electrogenic Na+/K+-pump in the muscle.     

The effects of the dihydropyridine Bay K 8644 in guinea-pig isolated trachealis.

In trachea bathed by Krebs solution containing indomethacin 0.8 mumol l-1, Bay K 8644 (0.01-1 mumol l-1) evoked mild spasm. Peak tension was achieved after 10 min and was generally less than 20% of an acetylcholine (ACh) maximum. The effect of Bay K 8644 was not potentiated by addition of 2.5 mmol l-1 potassium chloride (KCl) to the Krebs solution. Bay K 8644 (1 mumol l-1) caused a small potentiation of KCl and tetraethylammonium (TEA). In contrast it did not modify the actions of ACh or histamine. Bay K 8644 (1 mumol l-1) caused a small potentiation of the effect of calcium chloride (CaCl2) tested in trachea bathed by a K+-rich, Ca2+-free, MOPS-buffered physiological salt solution. Organic inhibitors of calcium influx such as nifedipine (0.1 mumol l-1), verapamil (1 mumol l-1) or diltiazem (10 mumol l-1) each caused marked depression of concentration-effect curves to KCl. Bay K 8644 (0.01-1 mumol l-1) provided concentration-dependent protection against this effect in all three cases. Estimation of calcium influx by the lanthanum technique revealed that Bay K 8644 (1 mumol l-1) was able to promote the cellular influx of Ca2+. Intracellular electrophysiological recording showed that Bay K 8644 (1 mumol l-1) caused no change in the resting membrane potential of trachealis cells and no change in the properties of the spontaneous electrical slow waves. However, Bay K 8644 was able to delay the slow wave suppression evoked by 1 mumol l-1 nifedipine. The ability of Bay K 8644 to promote Ca2+ influx and its ability to protect against the effects of several structurally-unrelated inhibitors of Ca2+ influx are consistent with Bay K 8644 acting as an agonist at the dihydropyridine receptor associated with the voltage-operated Ca2+ channel (VOC) of trachealis muscle. By this action it potentiates those spasmogens (KCl, TEA) which act by permitting Ca2+ influx through VOCs. In contrast it has no effect on those spasmogens (ACh, histamine) which principally act to liberate Ca2+ from intracellular sites of sequestration.     

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.     

Cromakalim-induced relaxation of guinea-pig isolated trachealis: antagonism by glibenclamide and by phentolamine.

1. Tested against the spontaneous tone of guinea-pig isolated trachealis, cromakalim (0.1-100 microM), isoprenaline (1 nM-1 microM) and theophylline (1 microM-1 mM) each produced concentration-dependent relaxation. 2. Glibenclamide (0.1-10 microM) did not itself alter the spontaneous tone of the trachea nor did it modify the relaxant actions of isoprenaline or theophylline. In contrast, glibenclamide (0.1 and 1 microM) caused a concentration-dependent rightward shift of the log concentration-effect curve of cromakalim. Glibenclamide (10 microM) reduced the slope of the log concentration-effect curve of cromakalim and moved the foot of the curve back towards the control position. 3. Phentolamine (1, 10 and 100 microm) did not itself alter the spontaneous tone of the trachea nor did it modify the relaxant actions of isoprenaline or theophylline. In contrast phentolamine caused concentration-dependent depression of the log concentration-effect curve of cromakalim. 4. Neither prazosin (1 microM) nor yohimbine (10 microM) modified the spontaneous tone of the trachea. Prazosin and yohimbine each failed to antagonise the effects of cromakalim, isoprenaline and theophylline. 5. Intracellular electrophysiological recording showed that glibenclamide (1 microM) and phentolamine (100 microM) caused minor change in the resting membrane potential of trachealis cells. Slow wave activity was slightly depressed by these agents. In contrast tetraethylammonium (TEA; 8 mM) caused marked depolarisation, and promoted the conversion of slow waves into regenerative action potentials. These electrical changes were accompanied by tonic tension development. 6. Phentolamine (100 microM) and glibenclamide (1 microM) reduced and reversed both the relaxation and the hyperpolarisation induced by cromakalim (10 microM). 7. It is concluded that glibenclamide and phentolamine each provide selective antagonism of the relaxant action of cromakalim in guinea-pig trachealis. These agents also inhibit the plasmalemmal hyperpolarisation induced by cromakalim. The effect of phentolamine is unrelated to the blockade of alpha 1- or alpha 2-adrenoceptors. If either glibenclamide or phentolamine act to block the K+ channels opened by cromakalim, then such channels are not identical to those which endow the trachealis plasmalemma with its powerful rectifying behaviour.     

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)     

Mechanism of xanthine-induced relaxation of guinea-pig isolated trachealis muscle.

1. Four 3-alkylxanthines (3-methylxanthine, 3-n-propylxanthine (enprofylline), 3-n-butylxanthine and 3-iso-butylxanthine) and four 1-methyl-3-alkylxanthines (1-methyl-3-methylxanthine (theophylline), 1-methyl-3-n-propylxanthine, 1-methyl-3-n-butylxanthine and 1-methyl-3-iso-butylxanthine (IBMX], were compared in terms of cyclic AMP phosphodiesterase (PDE) inhibition and trachealis muscle relaxation. The relationship between xanthine structure and cyclic AMP PDE inhibition was also studied. 2. Xanthine induced relaxation of guinea-pig isolated trachealis muscle was measured against spontaneous tone. 3. The four 1-methyl-3-alkylxanthines were each significantly more potent than the corresponding 3-alkylxanthines in relaxing the isolated trachealis muscle. The 1-methyl-3-alkylxanthines were similarly more potent than the corresponding 3-alkyl derivatives in inhibiting low Km cyclic AMP PDE. There was a strong positive correlation between low Km cyclic AMP PDE inhibition and the tracheal smooth muscle relaxation evoked by the xanthine derivatives. 4. Since methylation of the 1-position of each 3-alkylxanthine increased the potency of the derivative in inhibiting low Km cyclic AMP PDE and in relaxing trachealis muscle and since a strong positive correlation was observed between the relaxant EC50 and the Ki value of each xanthine derivative, it is suggested that low Km cyclic AMP PDE inhibition by xanthines plays an important role in their tracheal relaxant effect.     

The relaxant action of nicorandil in guinea-pig isolated trachealis.

Nicorandil (1-1000 mumol l-1) caused concentration-dependent relaxation of guinea-pig isolated trachealis. Propranolol (1 mumol l-1) did not modify the relaxant action of nicorandil but antagonized isoprenaline. Among K+-channel inhibitors tested, apamin (0.1 mumol l-1) and procaine (5 mmol l-1) did not modify the relaxant action of nicorandil. In contrast, tetraethylammonium (TEA, 8 mmol l-1) caused five fold antagonism. Trachealis exposed to K+-rich (120 mmol l-1) Krebs solution developed near-maximal tension. Nicorandil relaxed the K+-depolarized tissue though its concentration-effect curve was shifted markedly to the right. In tissues in which tone was induced by histamine, methylene blue (100 mumol l-1) antagonized nicorandil and sodium nitroprusside but did not modify the relaxant action of aminophylline. Intracellular electrophysiological recording showed that nicorandil (1 mumol l-1) could evoke some relaxation in the absence of electrical changes. Higher concentrations (10-1000 mumol l-1) reduced the amplitude and frequency of spontaneous electrical slow waves. Nicorandil also caused concentration-dependent hyperpolarization and relaxation. When the hyperpolarization was sufficiently pronounced slow wave activity was abolished. TEA (8 mmol l-1) induced slow waves which were surmounted by a spike potential. TEA slightly reduced the maximal hyperpolarization induced by nicorandil and increased the time required for nicorandil to abolish slow wave discharge. Procaine (5 mmol l-1) induced slow waves of relatively low frequency. Sometimes these were surmounted by a spike potential Procaine markedly reduced the hyperpolarization induced by nicorandil and increased the time required for abolition of slow waves. In studies of the efflux of 86Rb+ from muscle-rich strips of trachea, nicorandil (1000 mumol l-1) increased the efflux rate constant, whereas isoprenaline (1 mumol l-1) was without effect. It is concluded that nicorandil-induced relaxation does not involve the activation of beta-adrenoceptors but is partly attributable to the formation of nitric oxide from the nitrate moiety in its molecular structure. Nicorandil can evoke relaxation in the absence of membrane potential change but towards the upper end of its effective concentration range, nicorandil increases membrane K+ conductance and thereby evokes hyperpolarization of trachealis cells. The K+ channels opened by nicorandil are permeable to 86Rb, insensitive to apamin and TEA but may be inhibited by procaine.     

Antagonism of calcium by zinc in guinea-pig isolated taenia caeci and trachealis muscle.

1 In guinea-pig isolated taenia caeci and trachealis bathed in a K+-rich, Ca2+-free medium, CaCl2 (0.01-10 mM) produced a concentration-dependent contraction. Zn2+ (0.01-1 mM), Cd2+ (0.01-1 mM), verapamil (0.01-100 microM) and trifluoperazine (1-100 microM) were effective antagonists of CaCl2-induced responses. 2 Zn2+ and Cd2+ in concentrations from 0.01 to 1 mM were without effect on the tone of taenia and trachea in normal Tris solution. Conversely, Zn2+ and Cd2+, in concentrations of 1 mM, caused contraction of these tissues in a K+-rich, Ca2+-free medium. Zn2+ (1 mM)-induced contractions of taenia and trachea were completely inhibited by verapamil (10 microM). 3 In taenia and trachea skinned of their plasma membranes, tension development induced by Ca2+ (10 microM or 1 microM, respectively) was unaffected by verapamil (100 microM), whereas trifluoperazine (100 microM) depressed the maximal tension produced by Ca2+. Segments of skinned preparations contracted in response to low concentrations of Zn2+ (10 microM) or Cd2+ (10 microM). 4 It is concluded that Zn2+ may suppress Ca2+-induced spasm by a direct action on the binding sites of the Ca2+ channel.     

The relaxant and spasmogenic effects of some xanthine derivatives acting on guinea-pig isolated trachealis muscle.

1. Caffeine (10 mM)-induced relaxation of guinea-pig isolated trachealis was attenuated and converted to a small spasmogenic response on cooling to 22 degrees C. The relaxant response was restored on rewarming to 37 degrees C and was abolished by indomethacin (2.8 microM). Cooling to 22 degrees C in the presence of indomethacin revealed spasmogenic responses to caffeine which were abolished on rewarming to 37 degrees C. 2. Trachealis treated with indomethacin (2.8 microM) was repeatedly dosed with acetylcholine (ACh, 10 microM). Caffeine (1 or 10 mM), added as each ACh-induced spasm reached equilibrium, transiently augmented but then suppressed the spasm. On cooling from 37 degrees C to 12 degrees C, the increment in spasm evoked by caffeine increased relative to the spasm evoked by ACh. 3. Trachealis treated with indomethacin (2.8 microM) was repeatedly dosed with caffeine (10 mM). At 37 degrees C caffeine had little effect but it caused spasm when the tissue was cooled to 32 degrees C. Spasm amplitude increased as cooling progressed to 12 degrees C. Similar results were obtained with caffeine (1 mM). 4. At 37 degrees C, caffeine, enprofylline, 1,3,7,9-tetramethylxanthinium (TMX), theobromine, theophylline, xanthine and forskolin each caused concentration-dependent suppression of tracheal tone. Among the xanthine derivatives the rank order of potency was enprofylline greater than theophylline greater than caffeine greater than theobromine greater than xanthine greater than TMX. 5. In trachealis treated with indomethacin (2.8 microM) and maintained at 12 degrees C, the xanthines each caused concentration-dependent spasm. The rank order of potency was theobromine greater than or equal to theophylline greater than or equal to caffeine greater than or equal to enprofylline greater than xanthine greater than TMX. Forskolin was devoid of spasmogenic activity. 6. Trachealis treated with indomethacin (2.8 microM) and maintained at 12 degrees C, was repeatedly dosed with either caffeine (10 mM) or potassium chloride (KCl, 40 mM). Caffeine-induced spasm was attenuated in a Ca2+-free medium containing EGTA (2 mM), modestly at first but subsequently more profoundly. KCl did not evoke spasm at 12 degrees C but at 37 degrees C the KCl-induced spasm was virtually abolished at its first trail in the Ca2+-free, EGTA-containing medium. 7. It is concluded that caffeine, other alkylated xanthines and xanthine itself share a spasmogenic action in guinea-pig isolated trachealis which is best observed when the tissue is treated with indomethacin (2.8 microM) and maintained at 12 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ca2+ ion sequestration by guinea-pig tracheal cartilage: its influence on trachealis reactivity to KCl.

1. The contractile response of guinea-pig isolated trachealis to KCl has been studied in the presence and absence of cartilage. 2. Dissection of cartilage from the trachealis resulted in both a rightward displacement of the concentration-response curve to KCl (EC50 value: intact strip, 26.9 +/- 3.7 mM n = 5; dissected strip, 38.7 +/- 2.6 mM n = 5; P less than 0.05), and a reduction in the contractile response to KCl (30 mM) observed in a nominally Ca(2+)-free medium. 3. Removal of cartilage from the trachealis did not alter the responsiveness of the tissue to CaCl2 (2.5 mM) when added to K+ depolarized tissues. 4. Muscle-denuded cartilage rings were prepared by surgical removal of the trachealis muscle. Autoradiographic studies, and a direct comparison of Ca2+ (2.5 mM) uptake with that of sorbitol (2.5 mM) showed that cartilage per se had a high capacity to accumulate Ca2+ ions by a process which was resistant to iodoacetate (100 microM), diflunisal (100 microM) and boiling. 5. The uptake of 45Ca into isolated cartilage was unaltered by the addition of orthovanadate (500 microM), verapamil (10 microM), diltiazem (10 microM) or Bay K 8644 (10 microM), but was significantly reduced (P less than 0.05) in the presence of LaCl3 (1-10 mM). 6. We conclude, like previous studies, that cartilage may supply a pool of Ca2+ ions to airway smooth muscle during the generation of tension in a noninally Ca(2+)-free medium, and that LaCl3 may provide an experimental tool to elucidate further the role of non-muscle Ca(2+)-depots in smooth muscle contraction.     

Electrical slow waves and tone of guinea-pig isolated trachealis muscle: effects of drugs and temperature changes

1 Simultaneous recordings of electrical and mechanical activity have been made from guinea-pig isolated trachealis muscle. Electrical activity was recorded both by extracellular and intracellular techniques.

2 Extracellular studies showed that the spontaneous development of tone was accompanied by electrical slow waves which frequently exhibited pronounced waxing and waning. Intracellular recording confirmed the discharge of these slow waves in individual cells. Extracellularly-recorded slow waves were often of greatest amplitude while the tissue was developing rather than maintaining tension. Some tissues became electrically quiescent on reaching peak tone.

3 Cooling to 27.5°C caused some relaxation. Slow wave amplitude and frequency fell, slow waves eventually being abolished. Subsequent rapid rewarming initially evoked a more profound relaxation. An intense discharge of slow waves then occurred as the tension rapidly rose again towards the pre-cooling value.

4 Sodium nitrite, (-)-isoprenaline, adenosine and adenosine triphosphate (ATP) each evoked relaxation and reduced the frequency and amplitude of slow waves. High concentrations of these agents often abolished slow waves. The actions of these drugs were reversible.

5 Treatment with methoxyverapamil (D600) 1 μmol/l for 15 min abolished slow wave activity but only evoked partial relaxation of the tissue.

6 Acetylcholine, histamine and tetraethylammonium (TEA) each evoked contraction, but TEA was unique in consistently promoting slow waves and (in high concentration) spike activity. Spasm evoked by acetylcholine and histamine did not usually involve the initiation or promotion of slow waves. Indeed in appropriate concentration these two agents always suppressed slow wave activity. The actions of the spasmogens were reversible.

7 It is concluded that the smooth muscle cells of the trachealis are electrically coupled. While co-ordinated slow wave activity is associated with the spontaneous development of tension in trachealis, it may not be necessary for the maintenance of the major part of the spontaneous tension exhibited by the tissue or for the spasm evoked by histamine or acetylcholine. Slow wave promotion by TEA suggests that the tissue may have a high resting potassium conductance which normally attenuates the slow waves. Slow waves may be suppressed by a variety of drugs acting by different mechanisms. Since D600 suppresses slow waves of the trachealis the mechanisms underlying the waves may be similar to those underlying spike activity in other smooth muscles.

     

Electrophysiological and other aspects of the relaxant action of isoprenaline in guinea-pig isolated trachealis.

In guinea-pig isolated trachealis isoprenaline (0.001-0.1 mumol l-1) caused concentration-dependent relaxation. Propranolol (1 mumol l-1) antagonized the effects of isoprenaline by more than 100 fold but did not modify the relaxant action of sodium nitrite. The tracheal relaxant actions of isoprenaline and ATP were unaffected by apamin (0.1 mumol l-1) but apamin profoundly antagonized the effects of noradrenaline and ATP on guinea-pig isolated taenia caeci. Tetraethylammonium (TEA; 8 mmol l-1) and procaine (5 mmol l-1) each evoked tracheal spasm but neither agent antagonized the isoprenaline-evoked relaxation of the trachealis. Trachealis exposed to K+-rich (120 mmol l-1) Krebs solution developed near-maximal tension. Both isoprenaline and sodium nitrite relaxed the K+-depolarized tissue though concentration-effect curves for both relaxants were moved to the right compared to those obtained in non-depolarized tissues. The maximal effect of sodium nitrite was markedly reduced. Intracellular electrophysiological recording showed that isoprenaline (0.01-1 mumol l-1) caused hyperpolarization and reduced or abolished slow wave discharge in trachealis muscle. These effects were accompanied by relaxation. Propranolol (1 mumol l-1) virtually abolished both the electrical and mechanical responses to isoprenaline (0.1 mumol l-1). Apamin (0.1 mumol l-1) did not alter the spontaneous electrical activity of trachealis cells or their electrical and mechanical responses to isoprenaline (0.1 mumol l-1). TEA (8 mmol l-1) caused depolarization and often increased slow wave amplitude and induced spike discharge. Isoprenaline (0.01 mumol l-1) failed to hyperpolarize TEA-treated trachealis cells. Higher concentrations of isoprenaline suppressed TEA-induced spasm, caused hyperpolarization and thereby increased slow wave or spike amplitude. Slow wave or spike frequency decreased as the hyperpolarization progressed but abolition of slow waves or spikes sometimes required more than 4 min exposure to isoprenaline. Procaine (5 mmol l-1) increased the amplitude of slow waves and induced spike discharge. Procaine markedly reduced the hyperpolarization induced by isoprenaline (0.1 and 1 mumol l-1) but had little effect on isoprenaline-induced relaxation. It is concluded that isoprenaline activates beta-adrenoceptors in guinea-pig trachealis and thereby evokes relaxation and hyperpolarization of the smooth muscle. The hyperpolarization does not involve the opening of apamin-sensitive K+-channels and it probably plays a supportive rather than a crucial role in the process by which isoprenaline-induced relaxation is achieved.     

Some features of the spasmogenic actions of acetylcholine and histamine in guinea-pig isolated trachealis.

Intracellular electrophysiological recording showed that acetylcholine (1 mumol l-1) and histamine (2 mumol l-1) depolarized trachealis cells and often increased the frequency of slow waves. Higher concentrations of these agents caused greater depolarization and abolition of slow waves. Marked depolarization was often associated with the appearance of electrical 'noise'. These electrical phenomena were accompanied by tonic tension development in a contiguous segment of trachea. Electrical 'noise' and tension evoked by high concentrations of acetylcholine or histamine could be dissipated by washing the agonist from the tissue. Acetylcholine-induced 'noise' was resistant to tetrodotoxin (3 mumol l-1) and to hexamethonium (1 mmol l-1). Neither acetylcholine (10-1,000 mumol l-1) nor histamine (2-200 mumol l-1) increased the lanthanum-resistant calcium fraction of muscle-containing strips of trachea. It is concluded that, while developing tension under the influence of acetylcholine or histamine, trachealis cells depolarize markedly but there is relatively little cellular influx of Ca2+.     

Mechanical and electrical aspects of the relaxant action of aminophylline in guinea-pig isolated trachealis.

Aminophylline (1-1000 mumol l-1) suppressed the spontaneous tone of guinea-pig isolated trachealis in a concentration-dependent manner. In Krebs solution containing acetylcholine (1 mmol l-1), histamine (200 mumol l-1) or K+ (120 mmol l-1) isolated trachealis muscle developed near-maximal tension. The log concentration-effect curve for aminophylline was shifted 20 fold, 3 fold and 4 fold to the right, respectively, in the presence of these spasmogens. Three K+-channel inhibitors were tested: tetraethylammonium (TEA, 8 mmol l-1) did not modify the action of aminophylline, procaine (5 mmol l-1) shifted the log concentration-effect curve for aminophylline 2 fold to the left and 4-aminopyridine (5 mmol l-1) shifted the curve 2.5 fold to the right. Intracellular electrophysiological recording showed that aminophylline 10 mumol l-1 could cause relaxation in the absence of electrical changes. Higher concentrations of aminophylline (100-1000 mumol l-1) suppressed spontaneous slow waves and hyperpolarized the trachealis cells. In the presence of procaine (5 mmol l-1) or TEA (8 mmol l-1), the hyperpolarization induced by aminophylline (1000 mumol l-1) was significantly reduced but its relaxant effect was unchanged. In trachealis skinned of its plasma membranes, tension development induced by Ca2+ (20 mumol l-1) was unaffected either by aminophylline (1000 mumol l-1) or by isoprenaline (1 mumol l-1). In studies of the efflux of 86Rb+ from muscle-rich strips of trachea, aminophylline (100-1000 mumol l-1) was without effect whereas nicorandil (100 and 1000 mumol l-1) increased the efflux rate constant. It is concluded that aminophylline does not directly reduce the sensitivity of the contractile proteins to cytosolic Ca2+. In low concentration (1-10 mumol l-1) its relaxant action is not accompanied by membrane potential change but towards the upper end of its effective concentration range, aminophylline evokes hyperpolarization. This hyperpolarization may involve the opening of K+-channels which are inhibited by procaine and (to a lesser extent) by TEA. These K+-channels may be impermeable to 86Rb+.     

Effects of leukotriene D4 on the mechanical and electrical properties of guinea-pig isolated trachealis.

1. The effects of leukotriene D4 (LTD4) on mechanical and electrical activity were examined in guinea-pig isolated trachealis muscle and compared with two other bronchoconstrictors, methacholine and potassium chloride (KCl). 2. LTD4 elicited concentration-dependent increases in tension in trachealis muscle which were slower in time course than responses induced by either methacholine or KCl. The maximum response to LTD4 was approximately 85% of the methacholine maximum. 3. At a concentration close to the EC50 for tension changes, LTD4 had no significant effect on either transmembrane potential or slow wave activity recorded in single trachealis cells. 4. At a concentration close to the EC90 for tension changes, LTD4 caused significant membrane depolarization, transiently reduced the amplitude and increased the frequency of slow wave discharge and ultimately abolished slow wave discharge. LTD4-induced depolarization was less marked, and developed more slowly, than that evoked by either methacholine or KCl. 5. These results show that LTD4 can elicit substantial increases in tension without altering transmembrane potential and are consistent with the view that LTD4 initiates contraction mainly through potential-independent mechanisms. However, at high concentrations the depolarization evoked by LTD4 allows the possibility that potential-dependent mechanisms may contribute to the spasm.     

Evaluation of the relaxant effects of SCA40, a novel charybdotoxin-sensitive potassium channel opener, in guinea-pig isolated trachealis.

1. Experiments have been performed in order to analyse the mechanism whereby SCA40, a new imidazo[1,2-a]pyrazine derivative relaxes airway smooth muscle. 2. SCA40 (0.01-10 microM) caused a complete and concentration-dependent relaxation of guinea-pig isolated trachea contracted with 20 mM KCl but failed to inhibit completely the spasmogenic effects of 80 mM KCl. 3. Quinine (30 microM) antagonized the relaxant activity of SCA40 in 20 mM KCl-contracted guinea-pig isolated trachea. The ATP-sensitive K(+)-channel blocker, glibenclamide (3 microM), did not antagonize the relaxant activity of SCA40 in either 20 mM KCl or 1 microM carbachol-contracted isolated trachea. 4. SCA40 (0.01-10 microM) and isoprenaline (0.1 nM-10 microM) caused a complete and concentration-dependent relaxation of guinea-pig isolated trachea contracted with carbachol 1 microM. 5. The large-conductance Ca(2+)-activated K(+)-channel blocker, charybdotoxin (60-180 nM), non-competitively antagonized the relaxant activity of isoprenaline on 1 microM carbachol-contracted trachea. The inhibition was characterized by rightward shifts of the isoprenaline concentration-relaxation curves with depression of their maxima. 6. The relaxant activity of SCA40 in 1 microM carbachol-contracted trachea was antagonized by charybdotoxin (60-600 nM) in an apparently competitive manner. The concentration-relaxation curves to SCA40 were shifted to the right with no significant alteration in the maximum response. 7. It is concluded that SCA40 is a novel potassium channel opener which is a potent relaxant of guinea-pig airway smooth muscle in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antagonism of Ca2+ and other actions of verapamil in guinea-pig isolated trachealis.

In trachealis bathed by a K+-rich, Ca2+-free physiological salt solution, calcium chloride (CaCl2) at 0.01 to 10 mmol l-1 evoked concentration-dependent spasm. Verapamil (0.1 to 10 mumol l-1) was an effective antagonist of CaCl2. Spasm evoked by acetylcholine, histamine, potassium chloride (KCl) and tetraethylammonium (TEA) was studied in trachealis bathed by normal Krebs solution. Verapamil (0.1 to 10 mumol l-1) markedly suppressed spasm evoked by KCl and TEA. In contrast the actions of acetylcholine and histamine were much less affected by verapamil. Spasm evoked by prostaglandin E2 was studied in trachealis bathed by Krebs solution containing indomethacin (2.8 mumol l-1). Verapamil (0.1 to 10 mumol l-1) had little or no effect against prostaglandin E2-induced spasm. Verapamil (0.1 to 10 mumol l-1) had relatively little effect on the tone of trachealis bathed by normal Krebs solution. In contrast bathing in Krebs solution lacking CaCl2 caused almost complete tone loss. Extracellular electrophysiological recording showed that verapamil (10 mumol l-1) suppressed not only TEA-evoked spasm but also TEA-evoked slow waves and spike potentials. Verapamil also abolished the transient period of slow wave activity associated with the spasm evoked by KCl. Intracellular electrophysiological recording showed that TEA-induced spike activity was resistant to tetrodotoxin (3 mumol l-1). However, verapamil (10 mumol l-1) abolished the tetrodotoxin-resistant spikes without increasing the resting membrane potential. It is concluded that verapamil suppresses TEA- or KCl-induced spasm, slow waves or spikes by inhibition of Ca2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)